hypothesis on photosynthesis

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Development of the idea

Overall reaction of photosynthesis.

• Basic products of photosynthesis
• Evolution of the process
• Light intensity and temperature
• Carbon dioxide
• Internal factors
• Energy efficiency of photosynthesis
• Structural features
• Light absorption and energy transfer
• The pathway of electrons
• Evidence of two light reactions
• Photosystems I and II
• Quantum requirements
• The process of photosynthesis: the conversion of light energy to ATP
• Elucidation of the carbon pathway
• Carboxylation
• Isomerization/condensation/dismutation
• Phosphorylation
• Regulation of the cycle
• Products of carbon reduction
• Photorespiration
• Carbon fixation in C 4 plants
• Carbon fixation via crassulacean acid metabolism (CAM)
• Differences in carbon fixation pathways
• The molecular biology of photosynthesis

Why is photosynthesis important?

What is the basic formula for photosynthesis, which organisms can photosynthesize.

photosynthesis

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• Table Of Contents

Photosynthesis is critical for the existence of the vast majority of life on Earth. It is the way in which virtually all energy in the biosphere becomes available to living things. As primary producers, photosynthetic organisms form the base of Earth’s food webs and are consumed directly or indirectly by all higher life-forms. Additionally, almost all the oxygen in the atmosphere is due to the process of photosynthesis. If photosynthesis ceased, there would soon be little food or other organic matter on Earth, most organisms would disappear, and Earth’s atmosphere would eventually become nearly devoid of gaseous oxygen.

The process of photosynthesis is commonly written as: 6CO 2 + 6H 2 O → C 6 H 12 O 6 + 6O 2 . This means that the reactants, six carbon dioxide molecules and six water molecules, are converted by light energy captured by chlorophyll (implied by the arrow) into a sugar molecule and six oxygen molecules, the products. The sugar is used by the organism, and the oxygen is released as a by-product.

The ability to photosynthesize is found in both eukaryotic and prokaryotic organisms. The most well-known examples are plants, as all but a very few parasitic or mycoheterotrophic species contain chlorophyll and produce their own food. Algae are the other dominant group of eukaryotic photosynthetic organisms. All algae, which include massive kelps and microscopic diatoms , are important primary producers.  Cyanobacteria and certain sulfur bacteria are photosynthetic prokaryotes, in whom photosynthesis evolved. No animals are thought to be independently capable of photosynthesis, though the emerald green sea slug can temporarily incorporate algae chloroplasts in its body for food production.

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photosynthesis , the process by which green plants and certain other organisms transform light energy into chemical energy . During photosynthesis in green plants, light energy is captured and used to convert water , carbon dioxide , and minerals into oxygen and energy-rich organic compounds .

It would be impossible to overestimate the importance of photosynthesis in the maintenance of life on Earth . If photosynthesis ceased, there would soon be little food or other organic matter on Earth. Most organisms would disappear, and in time Earth’s atmosphere would become nearly devoid of gaseous oxygen. The only organisms able to exist under such conditions would be the chemosynthetic bacteria , which can utilize the chemical energy of certain inorganic compounds and thus are not dependent on the conversion of light energy.

Energy produced by photosynthesis carried out by plants millions of years ago is responsible for the fossil fuels (i.e., coal , oil , and gas ) that power industrial society . In past ages, green plants and small organisms that fed on plants increased faster than they were consumed, and their remains were deposited in Earth’s crust by sedimentation and other geological processes. There, protected from oxidation , these organic remains were slowly converted to fossil fuels. These fuels not only provide much of the energy used in factories, homes, and transportation but also serve as the raw material for plastics and other synthetic products. Unfortunately, modern civilization is using up in a few centuries the excess of photosynthetic production accumulated over millions of years. Consequently, the carbon dioxide that has been removed from the air to make carbohydrates in photosynthesis over millions of years is being returned at an incredibly rapid rate. The carbon dioxide concentration in Earth’s atmosphere is rising the fastest it ever has in Earth’s history, and this phenomenon is expected to have major implications on Earth’s climate .

Requirements for food, materials, and energy in a world where human population is rapidly growing have created a need to increase both the amount of photosynthesis and the efficiency of converting photosynthetic output into products useful to people. One response to those needs—the so-called Green Revolution , begun in the mid-20th century—achieved enormous improvements in agricultural yield through the use of chemical fertilizers , pest and plant- disease control, plant breeding , and mechanized tilling, harvesting, and crop processing. This effort limited severe famines to a few areas of the world despite rapid population growth , but it did not eliminate widespread malnutrition . Moreover, beginning in the early 1990s, the rate at which yields of major crops increased began to decline. This was especially true for rice in Asia. Rising costs associated with sustaining high rates of agricultural production, which required ever-increasing inputs of fertilizers and pesticides and constant development of new plant varieties, also became problematic for farmers in many countries.

A second agricultural revolution , based on plant genetic engineering , was forecast to lead to increases in plant productivity and thereby partially alleviate malnutrition. Since the 1970s, molecular biologists have possessed the means to alter a plant’s genetic material (deoxyribonucleic acid, or DNA ) with the aim of achieving improvements in disease and drought resistance, product yield and quality, frost hardiness, and other desirable properties. However, such traits are inherently complex, and the process of making changes to crop plants through genetic engineering has turned out to be more complicated than anticipated. In the future such genetic engineering may result in improvements in the process of photosynthesis, but by the first decades of the 21st century, it had yet to demonstrate that it could dramatically increase crop yields.

Another intriguing area in the study of photosynthesis has been the discovery that certain animals are able to convert light energy into chemical energy. The emerald green sea slug ( Elysia chlorotica ), for example, acquires genes and chloroplasts from Vaucheria litorea , an alga it consumes, giving it a limited ability to produce chlorophyll . When enough chloroplasts are assimilated , the slug may forgo the ingestion of food. The pea aphid ( Acyrthosiphon pisum ) can harness light to manufacture the energy-rich compound adenosine triphosphate (ATP); this ability has been linked to the aphid’s manufacture of carotenoid pigments.

General characteristics

The study of photosynthesis began in 1771 with observations made by the English clergyman and scientist Joseph Priestley . Priestley had burned a candle in a closed container until the air within the container could no longer support combustion . He then placed a sprig of mint plant in the container and discovered that after several days the mint had produced some substance (later recognized as oxygen) that enabled the confined air to again support combustion. In 1779 the Dutch physician Jan Ingenhousz expanded upon Priestley’s work, showing that the plant had to be exposed to light if the combustible substance (i.e., oxygen) was to be restored. He also demonstrated that this process required the presence of the green tissues of the plant.

In 1782 it was demonstrated that the combustion-supporting gas (oxygen) was formed at the expense of another gas, or “fixed air,” which had been identified the year before as carbon dioxide. Gas-exchange experiments in 1804 showed that the gain in weight of a plant grown in a carefully weighed pot resulted from the uptake of carbon, which came entirely from absorbed carbon dioxide, and water taken up by plant roots; the balance is oxygen, released back to the atmosphere. Almost half a century passed before the concept of chemical energy had developed sufficiently to permit the discovery (in 1845) that light energy from the sun is stored as chemical energy in products formed during photosynthesis.

This equation is merely a summary statement, for the process of photosynthesis actually involves numerous reactions catalyzed by enzymes (organic catalysts ). These reactions occur in two stages: the “light” stage, consisting of photochemical (i.e., light-capturing) reactions; and the “dark” stage, comprising chemical reactions controlled by enzymes . During the first stage, the energy of light is absorbed and used to drive a series of electron transfers, resulting in the synthesis of ATP and the electron-donor-reduced nicotine adenine dinucleotide phosphate (NADPH). During the dark stage, the ATP and NADPH formed in the light-capturing reactions are used to reduce carbon dioxide to organic carbon compounds. This assimilation of inorganic carbon into organic compounds is called carbon fixation.

Van Niel’s proposal was important because the popular (but incorrect) theory had been that oxygen was removed from carbon dioxide (rather than hydrogen from water, releasing oxygen) and that carbon then combined with water to form carbohydrate (rather than the hydrogen from water combining with CO 2 to form CH 2 O).

By 1940 chemists were using heavy isotopes to follow the reactions of photosynthesis. Water marked with an isotope of oxygen ( 18 O) was used in early experiments. Plants that photosynthesized in the presence of water containing H 2 18 O produced oxygen gas containing 18 O; those that photosynthesized in the presence of normal water produced normal oxygen gas. These results provided definitive support for van Niel’s theory that the oxygen gas produced during photosynthesis is derived from water.

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Article Contents

Origins of photosynthesis, photosynthetic pigments, reaction centers, electron transport chains, antenna systems, carbon fixation pathways, transition to oxygenic photosynthesis, literature cited.

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Early Evolution of Photosynthesis

This work was supported by the Exobiology Program from the U.S. National Aeronautics and Space Administration (grant no. NNX08AP62G).

E-mail [email protected] .

www.plantphysiol.org/cgi/doi/10.1104/pp.110.161687

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Robert E. Blankenship, Early Evolution of Photosynthesis, Plant Physiology , Volume 154, Issue 2, October 2010, Pages 434–438, https://doi.org/10.1104/pp.110.161687

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Photosynthesis is the only significant solar energy storage process on Earth and is the source of all of our food and most of our energy resources. An understanding of the origin and evolution of photosynthesis is therefore of substantial interest, as it may help to explain inefficiencies in the process and point the way to attempts to improve various aspects for agricultural and energy applications.

A wealth of evidence indicates that photosynthesis is an ancient process that originated not long after the origin of life and has evolved via a complex path to produce the distribution of types of photosynthetic organisms and metabolisms that are found today ( Blankenship, 2002 ; Björn and Govindjee, 2009 ). Figure 1 shows an evolutionary tree of life based on small-subunit rRNA analysis. Of the three domains of life, Bacteria, Archaea, and Eukarya, chlorophyll-based photosynthesis has only been found in the bacterial and eukaryotic domains. The ability to do photosynthesis is widely distributed throughout the bacterial domain in six different phyla, with no apparent pattern of evolution. Photosynthetic phyla include the cyanobacteria, proteobacteria (purple bacteria), green sulfur bacteria (GSB), firmicutes (heliobacteria), filamentous anoxygenic phototrophs (FAPs, also often called the green nonsulfur bacteria), and acidobacteria ( Raymond, 2008 ). In some cases (cyanobacteria and GSB), essentially all members of the phylum are phototrop2hic, while in the others, in particular the proteobacteria, the vast majority of species are not phototrophic.

Small subunit rRNA evolutionary tree of life. Taxa that contain photosynthetic representatives are highlighted in color, with green highlighting indicating a type I RC, while purple highlighting indicates a type II RC. The red arrow indicates the endosymbiotic event that formed eukaryotic chloroplasts. Tree adapted from Pace (1997) .

Overwhelming evidence indicates that eukaryotic photosynthesis originated from endosymbiosis of cyanobacterial-like organisms, which ultimately became chloroplasts ( Margulis, 1992 ). So the evolutionary origin of photosynthesis is to be found in the bacterial domain. Significant evidence indicates that the current distribution of photosynthesis in bacteria is the result of substantial amounts of horizontal gene transfer, which has shuffled the genetic information that codes for various parts of the photosynthetic apparatus, so that no one simple branching diagram can accurately represent the evolution of photosynthesis ( Raymond et al., 2002 ). However, there are some patterns that can be discerned from detailed analysis of the various parts of the photosynthetic apparatus, so some conclusions can be drawn. In addition, the recent explosive growth of available genomic data on all types of photosynthetic organisms promises to permit substantially more progress in unraveling this complex evolutionary process.

While we often talk about the evolution of photosynthesis as if it were a concerted process, it is more useful to consider the evolution of various photosynthetic subsystems, which have clearly had distinct evolutionary trajectories. In this brief review we will discuss the evolution of photosynthetic pigments, reaction centers (RCs), light-harvesting (LH) antenna systems, electron transport pathways, and carbon fixation pathways. These subsystems clearly interact with each other, for example both the RCs and antenna systems utilize pigments, and the electron transport chains interact with both the RCs and the carbon fixation pathways. However, to a significant degree they can be considered as modules that can be analyzed individually.

We know very little about the earliest origins of photosynthesis. There have been numerous suggestions as to where and how the process originated, but there is no direct evidence to support any of the possible origins ( Olson and Blankenship, 2004 ). There is suggestive evidence that photosynthetic organisms were present approximately 3.2 to 3.5 billion years ago, in the form of stromatolites, layered structures similar to forms that are produced by some modern cyanobacteria, as well as numerous microfossils that have been interpreted as arising from phototrophs ( Des Marais, 2000 ). In all these cases, phototrophs are not certain to have been the source of the fossils, but are inferred from the morphology or geological context. There is also isotopic evidence for autotrophic carbon fixation at 3.7 to 3.8 billion years ago, although there is nothing that indicates that these organisms were photosynthetic. All of these claims for early photosynthesis are highly controversial and have engendered a great deal of spirited discussion in the literature ( Buick, 2008 ). Evidence for the timing of the origin of oxygenic photosynthesis and the rise of oxygen in the atmosphere is discussed below. The accumulated evidence suggests that photosynthesis began early in Earth’s history, but was probably not one of the earliest metabolisms and that the earliest forms of photosynthesis were anoxygenic, with oxygenic forms arising significantly later.

Chlorophylls are essential pigments for all phototrophic organisms. Chlorophylls are themselves the product of a long evolutionary development, and can possibly be used to help understand the evolution of other aspects of photosynthesis. Chlorophyll biosynthesis is a complex pathway with 17 or more steps ( Beale, 1999 ). The early part of the pathway is identical to heme biosynthesis in almost all steps and has clearly been recruited from that older pathway. The later steps include the insertion of magnesium and the elaboration of the ring system and its substituents. The earliest version of the pathway (and that used by most modern anoxygenic photosynthetic organisms) almost certainly was anaerobic, both not requiring and not tolerating the presence of O 2 . However, all modern oxygenic photosynthetic organisms now require O 2 as an oxidant at several steps in the pathway. This has been explained in terms of gene replacement of the genes coding for the enzymes at these steps, with the result that the overall pathway is unchanged but the enzymes at key steps are completely different in different groups of phototrophs ( Raymond and Blankenship, 2004 ).

A key concept in using chlorophyll biosynthesis pathways to infer the evolution of photosynthesis is the Granick hypothesis, which states that the biosynthetic pathway of chlorophyll recapitulates the evolutionary sequence ( Granick, 1965 ). This is an appealing idea and probably at least partly true. However, in some cases, in particular the situation of chlorophyll and bacteriochlorophyll, it has been argued that the strict version of the Granick hypothesis is misleading and other interpretations are more likely ( Blankenship, 2002 ; Blankenship et al., 2007 ).

All photosynthetic organisms contain carotenoids, which are essential for photoprotection, usually also function as accessory pigments, and in many cases serve as key regulatory molecules. Carotenoids, unlike chlorophylls, are also found in many other types of organisms, so their evolutionary history may reflect many other functions in addition to photosynthesis ( Sandman, 2009 ).

The RC complex is at the heart of photosynthesis; so much attention has been paid to understand the evolution of RCs. A wealth of evidence, including structural, spectroscopic, thermodynamic, and molecular sequence analysis, clearly segregates all known RCs into two types of complexes, called type I and type II ( Blankenship, 2002 ). Anoxygenic phototrophs have just one type, either type I or II, while all oxygenic phototrophs have one of each type. The primary distinguishing feature of the two types of RCs are the early electron acceptor cofactors, which are FeS centers in type I RCs and pheophytin/quinone complexes in type II RCs. The distribution of RC types on the tree of life is shown in Figure 1 and a comparative electron transport diagram that compares the different RCs in different types of organisms is shown in Figure 2 , with type I RCs color coded green and type II RCs color coded purple.

Electron transport diagram indicating the types or RCs and electron transport pathways found in different groups of photosynthetic organisms. The color coding is the same as for Figure 1 and highlights the electron acceptor portion of the RC. Figure courtesy of Martin Hohmann-Marriott.

Further analysis strongly suggests that all RCs have evolved from a single common ancestor and have a similar protein and cofactor structure. This is clearly seen when structural overlays of both type I and II RCs are made, showing a remarkably conserved three-dimensional protein and cofactor structure, despite only minimal residual sequence identity ( Sadekar et al., 2006 ). These comparisons have been used to derive structure-based evolutionary trees that do not rely on sequence alignments. Figure 3 shows a schematic evolutionary tree of RCs that is derived from this sort of analysis. It proposes that the earliest RC was intermediate between type I and II (type 1.5) and that multiple gene duplications have given rise to the heterodimeric (two related yet distinct proteins that form the core of the RC) complexes that are found in most modern RCs.

Schematic evolutionary tree showing the development of the different types of RC complexes in different types of photosynthetic organisms. This tree is based on structural comparisons of RCs by Sadekar et al. (2006) . Blue color coding indicates protein homodimer, while red indicates protein heterodimer complexes. Red stars indicate gene duplication events that led to heterodimeric RCs. Helio, Heliobacteria; GSB, green sulfur bacteria; FAP, filamentous anoxygenic phototroph.

A second important issue that relates to RC evolution is the question of how both type I and II RCs came to be in cyanobacteria, while all other photosynthetic prokaryotes have only a single RC. The various proposals that have been made to explain this fact can all be divided into either fusion or selective loss scenarios or variants thereof ( Blankenship et al., 2007 ). In the fusion hypothesis, the two types of RCs develop separately in anoxygenic photosynthetic bacteria and are then brought together by a fusion of two organisms, which subsequently developed the ability to oxidize water. In the selective loss hypothesis, the two types of RCs both evolved in an ancestral organism and then loss of one or the other RC gave rise to the organisms with just one RC, while the ability to oxidize water was added later. Both scenarios have proponents, and it is not yet possible to choose between them.

The primary photochemistry and several of the early secondary electron transfer reactions take place within the RC complex. However, additional electron transfer processes are necessary before the process of energy storage is complete. These include the cytochrome bc   1 and b   6 f complexes. These complexes oxidize quinols produced by photochemistry in type II RCs or via cyclic processes in type I RCs and pumps protons across the membrane that in turn contribute to the proton motive force that is used to make ATP. All phototrophic organisms have a cytochrome bc   1 or b   6 f complex of generally similar architecture, with the exception of the FAP phylum of anoxygenic phototrophs ( Yanyushin et al., 2005 ). This group contains instead a completely different type of complex that is called alternative complex III. The evolutionary origin of this complex is not yet clear. While the cytochrome bc   1 and b   6 f complexes are similar in many ways, the cytochrome c   1 and f subunits are very different and are almost certainly of distinct evolutionary origin ( Baniulis et al., 2008 ).

All photosynthetic organisms contain a light-gathering antenna system, which functions to collect excitations and transfer them to the RC where the excited state energy is used to drive photochemistry ( Green and Parson, 2003 ). While the presence of an antenna is universal, the structure of the antenna complexes and even the types of pigments used in them is remarkably varied in different types of photosynthetic organisms. This very strongly suggests that the antenna complexes have been invented multiple times during the course of evolution to adapt organisms to particular photic environments. So while evolutionary relationships are clear among some categories of antennas, such as the LH1 and LH2 complexes of purple bacteria and the LHCI and LHCII complexes of eukaryotic chloroplasts, it is not possible to relate these broad categories of antennas to each other in any meaningful way. This is in contrast to the RCs, where all available evidence clearly points to a single origin that has subsequently undergone a complex evolutionary development.

Most phototrophic organisms are capable of photoautotrophic metabolism, in which inorganic substrates such as water, H 2 S, CO 2 , or HCO 3   − are utilized along with light energy to produce organic carbon compounds and oxidized donor species. However, there are some groups of phototrophs that cannot carry out photoautotrophic metabolism and there are at least three entirely separate autotrophic carbon fixation pathways that are found in different types of organisms ( Thauer, 2007 ). By far the dominant carbon fixation pathway is the Calvin-Benson cycle, which is found in all oxygenic photosynthetic organisms, and also in most purple bacteria. The GSB use the reverse tricarboxylic acid cycle, and many of the FAPs use the 3-hydroxypropionate cycle ( Zarzycki et al., 2009 ). The Gram-positive heliobacteria lack any known autotrophic carbon fixation pathway and usually grow photoheterotrophically ( Asao and Madigan, 2010 ). Similarly, the aerobic anoxygenic phototrophs, which are closely related to the purple bacteria, lack any apparent ability to fix inorganic carbon. In the latter case, it seems most likely that the ancestor of this group contained the Calvin-Benson cycle but lost the genes because of their obligate aerobic lifestyle ( Swingley et al., 2007 ).

The carbon fixation machinery is thus similar to the antennas, in that several entirely separate solutions have been adopted by different classes of phototrophic organisms. This would be consistent with the idea that the earliest phototrophs were photoheterotrophic, using light to assimilate organic carbon, instead of being photoautotrophic. The ability to fix inorganic carbon was then added to the metabolism somewhat later during the course of evolution, possibly borrowing carbon fixation pathways that had developed earlier in autotrophic nonphotosynthetic organisms.

Perhaps the most widely discussed yet poorly understood event in the evolution of photosynthesis is the invention of the ability to use water as an electron donor, producing O 2 as a waste product and giving rise to what is now called oxygenic photosynthesis. The production of O 2 and its subsequent accumulation in the atmosphere forever changed the Earth and permitted the development of advanced life that utilized the O 2 during aerobic respiration. Several lines of geochemical evidence indicate that free O 2 began to accumulate in the atmosphere by 2.4 billion years ago, although the ability to do oxygenic photosynthesis probably began somewhat earlier ( Buick, 2008 ). In order for O 2 to accumulate, it is necessary that both the biological machinery needed to produce it has evolved, but also the reduced carbon produced must be buried by geological processes, which are controlled by geological processes such as plate tectonics and the buildup of continents. So the buildup of O 2 in the atmosphere represents a coming together of the biology that gives rise to O 2 production and the geology that permits O 2 to accumulate.

Oxygen is produced by PSII in the oxygen evolving center, which contains a tetranuclear manganese complex. The evolutionary origin of the oxygen evolving center has long been a mystery. Several sources have been suggested, but so far no convincing evidence has been found to resolve this issue ( Raymond and Blankenship, 2008 ). The possibility that functional intermediate stages existed that connect the anoxygenic type II RCs to PSII seems likely ( Blankenship and Hartman, 1998 ).

The process of photosynthesis originated early in Earth’s history, and has evolved to its current mechanistic diversity and phylogenetic distribution by a complex, nonlinear process. Current evidence suggests that the earliest photosynthetic organisms were anoxygenic, that all photosynthetic RCs have been derived from a single source, and that antenna systems and carbon fixation pathways have been invented multiple times.

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Observing earthworm locomotion

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Investigating factors affecting the rate of photosynthesis, class practical.

In this experiment the rate of photosynthesis is measured by counting the number of bubbles rising from the cut end of a piece of Elodea or Cabomba .

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The work could be carried out individually or in groups of up to 3 students (counter, timekeeper and scribe).

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Elodea ( Note 1 ) or other oxygenating pond plant ( Note 2 )

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Normal laboratory safety procedures should be followed. There is a slight risk of infection from pond water, so take sensible hygiene precautions, cover cuts and wash hands thoroughly after the work is complete.

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1 Elodea can be stored in a fish tank on a windowsill, in the laboratory or prep room. However it is probably a good idea to replace it every so often with a fresh supply from an aquarist centre or a pond. (It’s worth finding out if any colleague has a pond.) On the day of the experiment, cut 10 cm lengths of Elodea , put a paper-clip on one end to weigh them down and place in a boiling tube of water in a boiling tube rack, near a high intensity lamp, such as a halogen lamp or a fluorescent striplight. Check the Elodea to see if it is bubbling. Sometimes cutting 2–3 mm off the end of the Elodea will induce bubbling from the cut end or change the size of the bubbles being produced.

2 Cabomba (available from pet shops or suppliers of aquaria – used as an oxygenator in tropical fish tanks) can be used as an alternative to Elodea , and some people find it produces more bubbles. It does, though tend to break apart very easily, and fish may eat it very quickly.

3 If possible, provide cardboard to allow students to shield their experiment from other lights in the room.

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The effect of light quality on plant physiology, photosynthetic, and stress response in Arabidopsis thaliana leaves

Nafiseh yavari.

1 Department of Bioresource Engineering, McGill University–Macdonald Campus, Sainte-Anne-de-Bellevue, Quebec, Canada

Rajiv Tripathi

2 Department of Plant Science, McGill University–Macdonald Campus, Sainte-Anne-de-Bellevue, Quebec, Canada

Sarah MacPherson

Jaswinder singh, mark lefsrud, associated data.

All relevant data are within the manuscript and its Supporting Information files.

The impacts of wavelengths in 500–600 nm on plant response and their underlying mechanisms remain elusive and required further investigation. Here, we investigated the effect of light quality on leaf area growth, biomass, pigments content, and net photosynthetic rate (Pn) across three Arabidopsis thaliana accessions, along with changes in transcription, photosynthates content, and antioxidative enzyme activity. Eleven-leaves plants were treated with BL; 450 nm, AL; 595 nm, RL; 650 nm, and FL; 400–700 nm as control. RL significantly increased leaf area growth, biomass, and promoted Pn. BL increased leaf area growth, carotenoid and anthocyanin content. AL significantly reduced leaf area growth and biomass, while Pn remained unaffected. Petiole elongation was further observed across accessions under AL. To explore the underlying mechanisms under AL, expression of key marker genes involved in light-responsive photosynthetic reaction, enzymatic activity of antioxidants, and content of photosynthates were monitored in Col-0 under AL, RL (as contrast), and FL (as control). AL induced transcription of GSH2 and PSBA , while downregulated NPQ1 and FNR2 . Photosynthates, including proteins and starches, showed lower content under AL. SOD and APX showed enhanced enzymatic activity under AL. These results provide insight into physiological and photosynthetic responses to light quality, in addition to identifying putative protective-mechanisms that may be induced to cope with lighting-stress in order to enhance plant stress tolerance.

Introduction

Among various environmental factors, light is one of the most important variables affecting photosynthesis as well as plant growth and development [ 1 ]. Plants require light not only as an energy source but also as a clue to adjust their development to environmental conditions. During photosynthesis, absorbed energy is transferred to the photosynthetic apparatus, which is comprised of Photosystem I (PSI), Photosystem II (PSII), electron transport ‬carriers (cytochrome b6f (cytb6f), plastoquinone (PQ), plastocyanin (PC)), and ATP synthase. The light-responsive photosynthetic process is driven by the released electrons through the water-splitting reaction on the PSII side, followed by NADP + reduction to NADPH, and proton flow into the lumen in order to generate ATP. Generated NADPH and ATP serve as an energy source for the carbon fixation process [ 2 ].‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬

‬‬‬‬‬‬‬‬‬‬‬‬ Both quality and quantity of incident light can have drastic impacts on photosynthetic activity and photosystem adaption to changing light quality [ 3 , 4 ]. Earlier studies on photosynthetic activity reported that photosynthesis is a wavelength-dependent response, in which amber light (AL; 595 nm) induces higher photosynthetic rates than blue light (BL; 450 nm) or red light (RL; 650 nm) [ 3 , 5 , 6 ]. These studies have become the foundation for our plant lighting research as light emitting diodes (LEDs) are proven to be ‬an ‬optimal and effective ‬tool to study the effect of ‬‬‬‬wavelength on plant physiological and biochemical responses [ 7 – 10 ]. Prior research has demonstrated that the wavelength range from 430–500 nm is effective at simulating pigmentation, metabolism of secondary metabolites, photosynthetic function, and development of chloroplasts [ 11 – 14 ]. The wavelength range of 640–670 nm was found effective in promoting photosynthetic activity, plant biomass and leaf area growth [ 3 , 15 ] while playing critically important roles in the development of photosynthetic apparatus, net photosynthetic rate (Pn) and primary metabolism [ 12 , 16 ]. Growing research on the wavelength range 500–600 nm have highlighted its important physiological and morphological impact on growth, chlorophyll content, and photosynthetic function [ 8 , 17 – 19 ]. However, conflicting results on the impact of AL were reported [ 3 , 20 ]. Although AL results in high photosynthetic activity, poor plant growth responses such as elongation and growth suppression have been reported [ 20 , 21 ], and this underlying mechanism remains unknown. In addition to this, AL is weakly absorbed by the photosynthetic pigments [ 22 ]. At the current state, further investigation of AL impact is required to better understand the photoactivity of the photosystems.‬ ‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬

Recent studies reported that light quality and quantity can have drastic impacts on imbalanced excitation of either PSII or PSI, resulting in energy imbalance between photosystems and triggering stoichiometric adjustments of photosynthetic complexes [ 23 , 24 ]. This imbalance between the two photosystems can result in generation of harmful reactive intermediates, mainly reactive oxygen species (ROS) [ 25 , 26 ]. Generation of ROS can result in oxidative damage to the chloroplasts, leading to photosystem photo-inhibition that strongly limits plant growth [ 27 ].‬‬‬‬ To maintain steady state photosynthetic efficiency and prevent ROS accumulation, plants activate the buffering mechanisms, including cyclic photosynthetic electron flow (CEF) and non-photochemical quenching (NPQ) [ 28 , 29 ]. To scavenge ROS, plants further stimulate antioxidative mechanisms via enhanced activity of associated enzymes such as glutathione synthetase (GSH), ascorbate peroxidase (APX), and superoxide dismutase (SOD) [ 30 ]. These studies and their findings allow us to understand the impact of light within photosystems; however, the wavelength that can induce such stress responses and their physiological consequence on plants remain poorly studied.

Therefore, to better understand the effect of light quality on plant growth and photosynthetic performance, we studied three narrow-wavelength LEDs of blue light (BL; 450 nm), amber light (AL; 595 nm), and red light (RL; 650 nm), and compared them with fluorescent light (FL; 400–700) as the control. We chose light quality of BL and RL as leaf pigments have absorption peaks at these wavelengths [ 31 ]. AL was chosen due to the conflicting results between high photosynthetic activity and poor plant growth responses [ 3 , 5 ]. Furthermore, to assess whether light quality-induced changes in plant growth and photosynthesis are mediated by the genotype, we investigated the light quality response in three A . thaliana accessions Col-0, Est-1, and C24. These accessions show different geographical distributions and hence are adopted to different environments [ 32 – 34 ]. Congruently, they show a high degree of divergence in their photosynthetic response to the light environment [ 35 , 36 ]‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬. Two experiments were designed to‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬ ‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬assess the impact of light quality on the plant. First, we investigated the physiological and photosynthetic response of A . thaliana to BL, AL, and RL lights compared it to FL by measuring leaf area growth, biomass content, Pn, and pigments content. Second, we tested whether changes in plant response to light quality is genotype specific by conducting the experiments across three A . thaliana accessions. Third, we investigated the potential induction of stress responses under AL by testing whether there are light quality-specific changes in the expression of marker genes involved in light-responsive photosynthetic process and enzymatic activity of antioxidants, as well as photosynthates content. Our findings expand the current understanding on physiological and photosynthetic responses of plants to light quality, in addition to identifying putative protective-mechanisms that may be induced to cope with lighting-stress in order to enhance plant stress tolerance.

Materials and methods

Plant materials and growth condition.

Seeds of A . thaliana accessions Col-0, Est-1, and C24 were obtained from the Arabidopsis Biological Resource Center (ABRC; Columbus, OH, US). Seeds were placed in rockwool cubes (Grodan A/S, DK-2640, Hedehusene, Denmark) and incubated at 4°C for 2 days. White broad-spectrum light (FL; 4200 K, F72T8CW, Osram Sylvania, MA, US) were used as light sources for seed germination. Seedlings were hydroponically grown under FL for 21 days with the environmental condition of 24 h photoperiod, 23°C, 50% relative humidity, and ambient CO 2 in a growth chamber (TC30, Conviron, Winnipeg, MB, Canada). Seed density was adjusted to limit treated plants from shadowing each other. FL was placed over the plant-growing surface area (49 cm × 95 cm) at a low photosynthetic photon flux density (PPFD) of 69 to 71 μmol·m -2 ·sec -1 . PPFD was measured at the conjunction of a grid (square area 3 cm 2 ) placed over the growing area. After 21 days, plants formed rosettes with nine (C24) and eleven (Col-0 and Est-1) leaves. To reach the same growth stage as Col-0 and Est-1 plants, C24 plants were allowed to grow for 23 days [ 37 ]. Fresh half-strength Hoagland nutrient solution [ 38 ] was provided every other day.

Light treatment

After day 21 (Col-0 and Est-1) or 23 (C24), plants were transferred to their respective light treatment for 5 days, each with the same environmental conditions: 24 h photoperiod, 23°C, 50% relative humidity, and ambient CO 2 . 21-day old plants were randomly divided into four experimental groups and received treatments using light emitting diodes (LED) (VanqLED, Shenzhen, China) of BL (peak wavelength: 450 nm), AL (peak wavelength: 595 nm), and RL (peak wavelength: 650 nm). The fourth group was treated with FL (400–700 nm), as the control. The light spectra and PPFD were monitored daily by using a PS-300 spectroradiometer (Apogee, Logan, UT, US). PPFD was maintained at 69 to 71 μmol·m -2 ·sec -1 throughout the whole plant growth period. Fresh half-strength Hoagland nutrient solution [ 38 ] was provided every other day. Biological replicates were grown at different time points under the same environmental settings.

Physical and biochemical analyses

Leaf area growth determination.

Three plants per biological replicate were randomly selected for each measurement. Leaves from the selected plants were collected for the determination after treatment (5 days). Digital images of leaves were taken with a window size of 640 x 480 pixels and a camera-object distance of approximately 80 cm. The digital images were next used to determine leaf area growth using Image J software with default settings (Bethesda, MD, US), as described previously [ 39 ].

Biomass content determination

Three plants per biological replicate were randomly selected for each determination. Leaf samples from the selected plants were collected for the dry mass determination before (0 h) and after treatment (5 days). Leaves were dried at 80°C for 2 days until a constant mass was achieved (less than < 5% mass difference over a 2 h period).

Pigment content determination

Five plants per biological replicate were randomly selected for each assay. Leaf samples from the selected plants were collected for the determination after treatment (5 days). Methods and equations described by [ 40 – 42 ] were used to estimate the content of chlorophyll (Chl a and Chl b), carotenoids, and anthocyanin, respectively. Briefly, chlorophylls and carotenoids were extracted with 5 ml of 80% acetone at 4°C overnight, before centrifugation at 13,000 g for 5 min. Total anthocyanins were determined by extracting with 5 ml 80% methanol containing 1% HCl solvent at 4°C overnight, before centrifugation at 13,000 g for 5 min. The absorbance of the extraction solution was determined for Chl a (664 nm), Chl b (647 nm), carotenoids (440 nm), and anthocyanins (530 nm and 657 nm) using a UV–VIS spectrophotometer (UV-180, Shimadzu, Japan).

Net photosynthetic rate determination

Net photosynthetic rate was monitored before (0 h) and after treatment (5 days) using the LI-6400XT Portable Photosynthesis System (LI-COR Biosciences, Lincoln, NE, US) equipped with a 6400–17 Whole Plant Arabidopsis Chamber (LI-COR Biosciences). To reduce potential measurement errors, three plants were grouped as a single sample for determinations [ 43 ]. To avoid mismatch between the light quality used by the LI-6400XT Portable Photosynthetic System, and the LED lights used for the treatments [ 44 ], measurements were taken inside the controlled-chamber, in which whole plants (still embedded in rockwool) were placed and illuminated with LEDs. As a precaution, parafilm was placed on top of the rockwool cube to maintain moisture within the root zone while measurements were recorded. The environmental conditions of the chamber were set as: 400 ppm CO 2 , 50% relative humidity, 23°C, and 400 μl min -1 flow rate. Each measurement was taken over 20 min, including 5 min in the dark and 10–15 min under a light treatment at 69–71 μmol·m -2 ·sec -1 . A stable Pn reading was reached 10 min after illumination. Leaf area growth was determined to normalize Pn per unit leaf area growth. Measurements for three replicates (three plants per replicate, three replicates per treatment) were performed.

Photosynthate content determination

Previous studies have reported that the diurnal cycle and developmental stage of plants, along with the stress response can affect the plant metabolism [ 45 , 46 ]. Thus, we performed a time course assessment of 0, 1, 3, 5, and 7 days to determine the content of leaf photosynthates (proteins, starches, and lipids). Five plants per biological replicate were randomly selected for each measurement. Leaf samples from selected plants were collected for the determination prior (0 h) and after light treatments (1, 3, 5, and 7 days). Samples were immediately frozen in liquid nitrogen and stored at -80 ∘ C, before they were used for determination. Protein : Total protein content was measured using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Rockford, IL, USA). As a standard, the absorbance of the bovine serum albumin was determined at UV/Vis: λ max 562 nm. Starch : A previously described method [ 47 ] was used to estimate total starch content. Lipids : Previously described methods [ 48 , 49 ] were used (with minor modifications) to estimate the total lipid content. Briefly, each sample was homogenized with (CHCl 3 /MeOH, 70:30 v/v), before centrifuged at 1000 rpm for 5 min. The collected supernatant was incubated for 30 min at 70°C in a boiling water bath. Next, (H 2 SO 4 : 1 ml) was added and heated for 20 min. Following 2 min cooling on ice, (H 3 PO 4 : 1.5 ml) was added and incubated for 10 min until a pink color developed.

Antioxidative enzyme activity estimation

Five plants per biological replicate were randomly selected for each measurement. Leaf samples from selected plants were collected for the determination after treatment (5 days). Samples were immediately frozen in liquid nitrogen and stored at -80 ∘ C, before they were used for determination. Methods described by [ 50 , 51 ] were used to monitor the activity of SOD and APX antioxidative enzymes, respectively. Enzymatic activity was measured for 5 min at room temperature. The protein content in the supernatant was determined by the Pierce™ BCA Protein Assay Kit. The activity of SOD and APX was expressed as unit min −1 mg −1 protein.

Gene expression analysis

Cdna synthesis.

Changes in transcription of the interested genes were analyzed in A . thaliana Col-0 treated for 24 h under AL, RL, and FL. Leaf samples from selected plants were collected for the determination prior to treatment (0 h) and after treatment (2 h, 4 h, and 24 h). Samples were immediately frozen in liquid nitrogen and stored at -80 ∘ C, before they were used for determination. Four biological replicates were examined. For each biological replicate, five A . thaliana plants were selected, and their leaves were pooled together to represent a biological replicate. Plants in each biological replicate were grown independently, and at different times. Total RNA was extracted from (100 mg) leaves using the Sigma Spectrum Plant Total RNA Kit (STRN50; Sigma, Seelze, Germany) according to the manufacturer’s protocol. A total of (2 μg) RNA per sample was treated with amplification grade DNase I (Invitrogen, Carlsbad, CA, USA) to remove any traces of genomic DNA contamination. RNA concentrations were measured before and after DNase I digestion with a NanoDrop ND-1000 UV-Vis spectrophotometer (NanoDrop Technologies, Wilmington, Delware, USA). The cDNA was synthesized using AffinityScript QPCR cDNA Synthesis Kit (Agilent, Tech., Santa Clara, USA).

Primer design

Primers for genes of interest ( S1 Table ) were designed using IDT software ( https://www.idtdna.com/calc/analyzer ) with the following criteria: Tm of 58–60°C and PCR amplicon lengths of 70 to 120 bp, yielding primer sequences 20 to 25 nucleotides in length with G-C contents of 40% to 50%. Specificity of the resulting primer pair sequences was examined using Arabidopsis transcript database with TAIR BLAST ( http://www.arabidopsis.org/Blast/ ). Specificity of the primer amplicons was further confirmed by melting-curve analysis (30 amplification cycles by PCR and subsequent gel-electrophoretic analysis). Primer amplicons were resolved on (agarose gels, 2% w/v) run at 110 V in Tris-borate/EDTA buffer, along with a 1Kb + DNA-standard ladder (Invitrogen, Carlsbad, CA, USA).

Quantitative real time-PCR (qRT-PCR) analysis

Real-time qRT-PCR was performed with a MX3000P qPCR System (Agilent, Tech., Santa Clara, CA, USA) using three biological and two technical replicates, as described previously [ 52 ]. Relative expression was conducted following the manufacturer’s recommendations with two reference genes gamma tonoplast intrinsic protein 2 ( TIP2 ; AT3g26520) and actin 2 ( ACT2 ; AT3g18780) and the Brilliant III SYBR Green QPCR master mix (Agilent, Tech., Santa Clara, CA, USA). Amplification was performed in a (20 μL) reaction mixture containing (160 nmol) for each primer, 1x Brilliant III SYBR Green QPCR master mix, (15μM) ROX reference dye, and (0.3 μL) of cDNA template. Amplification conditions were 95°C for 10 min (hot start), followed by 40 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s. Fluorescence readings were taken at 72°C, at the end of the elongation cycle.

Data analysis

Ct values were calculated with CFX-Manager and MX-3000P software. Relative expression changes (delta-delta Ct) were calculated according to [ 53 ] using A . thaliana TIP2 (AT3g26520) and ACT2 (AT3g18780) as reference genes. To avoid multiple testing, the p-values were only considered for 0 h with 24 h (a total of 12 genes and two light conditions). A gene was considered differentially expressed if p < 0.05 and the fold change pattern at 24 h was consistent with those observed at 2 and 4 h.

Statistical analysis

Differences between light treatments were tested using the two-tailed Student’s t-test. A two-way ANOVA was used to assess the effects of accession and different light treatments on leaf area growth, biomass content, Pn value, and pigments content. We observed similar patterns using the non-parametric tests of Wilcoxon-Mann-Whitney and Kruskal-Wallis tests (data not shown). ‬

Effect of light quality and natural genotype variation on leaf area growth, biomass content, net photosynthetic rate, and pigment content in A . thaliana

To assess the effect of light quality, 21-days-old plants (11 leave plants) of three A . thaliana accessions Col-0, Est-1, and C24 were randomly divided into groups and treated under narrow-spectrum light (BL, AL, and RL), along with FL as control (baseline), for 5 days at approximately 70 μmol m -2 sec -1 ( Fig 1A and 1B ). Summary of light quantity compositions emitted from FL and LEDs light sources are shown in Table 1 . After 5 days of narrow-spectrum light treatments, leaf area growth, leaf biomass (dry mass), net photosynthetic rate, and pigment contents were measured across three A . thaliana accessions and compared with the baseline FL treatment ( Fig 1C–1E and Table 2 ).

(A) Light emission spectra of LED light sources and FL. (B) Eleven-leaves stage A . thaliana accessions Col-0, Est-1, and C24 were grown hydroponically and treated for 5 days under narrow-spectrum BL, AL, and RL lights, as well as FL as control. (C) Leaf area growth. (D) Leaf biomass (dry mass). (E) Net photosynthetic rate (Pn) measured at 69–71 μmol m -2 sec -1 . Data are expressed as mean values ± standard deviation (n = 3). Statistical analysis was performed against FL using a two-tailed Student’s t-test (n.s.: not statistically significant; *: P < 0.05; **: P < 0.01).

Data are expressed as mean values ± standard deviation (μg g -1 dry mass) (n = 5). Statistical analysis was performed against FL using a two-tailed Student’s t-test (*, P < 0.05 and **, P < 0.01).

Under RL, the leaf area growth was significantly increased across accessions ( P < 0.05; Fig 1C ). Under BL, leaf area growth was significantly increased in C24 and Col-0 ( P < 0.05; Fig 2C ), but the increase in the leaf area growth was not significant in Est-1. Under AL, leaf area growth showed a severe reduction in Col-0 and C24 ( P < 0.05; Fig 1C ), while Al showed no change in Est-1. Petioles were noticeably elongated under AL ( Fig 1B ).

The leaf biomass significantly increased under RL across the three accessions ( P < 0.05; Fig 1D ). Under BL, the leaf biomass was significantly decreased in Est-1 and C24 but increased in Col-0 ( P < 0.01; Fig 1D ). Under AL, the leaf biomass was significantly lower in Col-0 and C24 ( P < 0.01), while it showed no change in Est-1 ( Fig 1D ).

As for the net photosynthetic rates (Pn), it significantly increased under RL across the accessions ( P < 0.05; Fig 1E ). In contrast, there was no significant difference in Pn under AL ( Fig 1E ). Under BL, Pn significantly increased in Col-0 and Est-1 ( P < 0.05; Fig 1E ) but remained unchanged in C24.

There was no significant difference in contents of chlorophyll a (Chl a) and chlorophyll b (Chl b) in Col-0 and C24 under the light quality of BL, AL, and RL ( Table 2 ). In contrast, Chl a content significantly increased in Est-1 under RL ( P < 0.05; Table 2 ). Across accessions, Chl a: b content significantly increased, remained unchanged, and decreased under RL, BL, and AL, respectively ( Table 2 ). Moreover, there was no significant difference in carotenoid and anthocyanin contents across the accessions under AL and RL. However, BL significantly stimulated carotenoids content in Est-1 and Col-0 ( P < 0.05; Table 2 ). Additionally, anthocyanins content significantly increased under BL in Est-1 and C24 ( P < 0.01; Table 2 ).

The two-way ANOVA analysis indicated significant effects of the light treatments for the determined parameters, except Chl b. Also, the interaction between light treatments and genotype was significant for leaf area growth and leaf biomass ( P < 0.01; Table 3 ).

Shown are p-values for each set of tests.

*, Significant effects (* P < 0.05

** P < 0.01

*** P < 0.001).

Changes in transcription of photosynthetic marker genes, content of photosynthates, and activity of antioxidant in A . thaliana Col-0 under AL and RL

The severe reduction in leaf area growth and biomass, along with unchanged levels of Pn in Col-0 and C24 under AL suggested that amber light has mismatched effects on photosynthetic activity and photomorphology. Further to this, although chlorophyll contents under AL were 10–20% lower than the FL, both light treatments triggered similar photosynthetic activity, which implies that amber light has unidentified mechanisms in the photosynthetic process. To identify the mechanisms that amber light triggers within plants, we next explored transcriptional changes in marker genes associated with the photosynthetic light reaction and photo-protective mechanisms, photosynthates content and antioxidant enzymatic activity in Col-0 under AL ( Fig 2 ). Among three accession, Accession Col-0 was chosen for the transcription analysis, as it is the most common A . thaliana accession in conducting biological analysis. In addition to AL and FL (as control), changes were investigated under RL, as RL-treated plants showed opposing changes in leaf physiological phenotypes compared to AL.

Gene expression analysis indicated a significant increase in transcription level of ATP synthase gamma chain 1 ( ATPC1 ;member of ATP synthase complex) and proton gradient regulation Like 1 ( PGRL1B ;member of CET complex), after 24 h treatment under AL ( P < 0.05; Fig 3B ). ATPC1 transcription significantly increased after 24 h treatment under RL ( P < 0.05; Fig 3B ). No significant difference, after 24 h treatment, was observed in the transcription level of the selected marker genes associated with linear photosynthetic electron transfer (i.e., ferredoxin-2 ( Fd2) , plastocyanin (PETE1) , and cytochrome b6f complex ( PETC ) under both AL and RL ( Fig 3B ). After the 24 h treatment, transcription of ferredoxin-NADP+-oxidoreductase (FNR2) was significantly decreased under AL ( P < 0.05; Fig 3B ), while it remained unchanged under RL ( Fig 3B ). The transcription level of ribulose bisphosphate carboxylase small chain (RBCS1A) was significantly reduced at 2 h and 4 h treatment under both AL and RL ( P < 0.05; Fig 3B ). The RBCS1A transcription level significantly was downregulated under AL ( P < 0.05; Fig 3B ). However, RBCS1A transcription level recovered after the 24 h treatment under RL ( Fig 3B ).

(A) Genes of interest are highlighted in green. (B) Transcription of genes implicated in the light-responsive photosynthetic process that is located within the thylakoid membrane. A time course assessment prior to treatment (0 h), and after treatment (2, 4, and 24 h) of AL and RL was performed, compared to FL. Four biological replicates were examined. For each biological replicate, five A . thaliana plants were selected, and their leaves were pooled together to represent a biological replicate. Plants in each biological replicate were grown independently, and at different time.

All data were normalized to the housekeeping genes; gamma tonoplast intrinsic protein 2 ( TIP2 ; AT3g26520) and actin 2 ( ACT2 ; AT3g18780). Red borders represent significant changes in expression ( P < 0.05). Studied genes include: ATP synthase gamma chain 1, ATPC1 (AT4g04640); fatty acid desaturase 6, FAD6 (AT4g30950); ferredoxin-2, Fd2 (AT1g60950); ferredoxin-NADP+-oxidoreductase, FNR2 (AT1g20020); (Fdx)-thioredoxin (Trx)-reductase, FTRB (AT2g04700); glutathione synthetase, GSH2 (AT5g27380); PSII nonphotochemical quenching, NPQ1 (AT1g08550); cytochrome b6f complex (Cyt b6f), PETC (AT4g03280); plastocyanin, PETE1 (AT1g76100); proton gradient regulation Like 1, PGRL1B (AT4g11960); photosystem II protein D1, PSBA (ATCG00020) and ribulose bisphosphate carboxylase small chain, RBCS1A (AT1g67090).

To confirm changes in the ATP synthase and CET complex under AL, we leveraged available proteomics data where eleven-leaves plants of A . thaliana Col-0 were grown under AL and RL for 5 days. Consistent with the observed transcriptomic data, a significant increase in the level of protein abundance was observed for both CET complex ( P < 1.3 x 10 −12 ; S1A Fig ) and ATP synthase ( P < 2 x 10 −4 ; S1B Fig ) under AL compared to RL.

Regulation patterns of PSBA , NPQ1 , GSH2 and FAD6 transcripts in A . thaliana Col-0 under AL and RL

The transcription level of photosystem II protein D1 ( PSBA) was significantly upregulated at 4 h and 24 h treatment under RL ( P < 0.05; Fig 3B ). Under AL, the transcription level of PBSA showed a similar increase after the 4 h treatment ( P < 0.05; Fig 3B ); However, its transcription level was reduced to a comparable level with FL after the 24 h treatment under AL. After the 24 h treatment, the transcription level of PSII nonphotochemical quenching (NPQ1) was significantly downregulated under AL ( P < 0.05; Fig 3B ), while it remained steady under RL. Between the 2 h and 4 h treatment, the transcription level of GSH2 gradually increased under both AL and RL ( P < 0.05; Fig 3B ) but reduced to a comparable level with FL after the 24 h treatment under RL. No significant difference was observed in the transcription level of fatty acid desaturase 6 ( FAD6) after the 24 h treatment under either AL or RL ( Fig 3B ).

Photosynthates content in A . thaliana Col-0 under AL and RL

Photosynthates accumulation was probed in Col-0 treated under AL, RL, and FL. Total lipid, protein and starch were measured at days 0, 1, 3, 5, and 7 ( Fig 4 ). The lipid content gradually increased under AL and RL ( P < 0.05; Fig 4A ). The content level of proteins and starches increased under RL but decreased under AL ( P < 0.05; Fig 4B and 4C ).

(A) Lipid; (B) Protein; (C) Starch. Data are expressed as mean values ± standard deviation (n = 5). Statistical analysis was performed against FL using a two-tailed Student’s t-test.

Antioxidative enzyme activity in A . thaliana Col-0 under AL and RL

We examined the antioxidative activity of superoxide dismutase (SOD) and ascorbate peroxidase (APX) enzymes in Col-0 treated under AL, RL, and FL ( Fig 5 ). After the 24 h treatments, activity of both antioxidants was significantly increased under AL ( P < 0.05; Fig 5 ), while no significant changes were observed for either of these enzymes when plants were treated under RL.

(A) Superoxide dismutase (SOD) activity. One unit of SOD activity was defined as the amount of enzyme required to result in a 50% inhibition of the rate of reduction at 550 nm in 1 min. (B) Ascorbate peroxidase (APX) activity. One unit of APX activity was defined as the amount of enzyme required to oxidize 1 μmol of ascorbate at 290 nm in 1 min. Enzymatic activity was measured for 5 min at room temperature and data are expressed as mean values ± standard deviation (n = 5). Statistical analysis was performed against FL using a two-tailed Student’s t-test (n.s., not statistically significant; *, P < 0.05; **, P < 0.01).

In this work, we investigated the impact of light quality BL, AL, and RL on leaf growth and photosynthetic response across three A . thaliana accessions Col-0, Est-1, and C24. The analyses clearly demonstrate the significant impact of light quality on leaf area growth, biomass content, and pigments accumulation (chlorophylls, carotenoid, and anthocyanin). The results indicate that light quality significantly influences Pn across accessions, consistent with the reported results that leaf photosynthetic reaction is wavelength-dependent in higher plants [ 54 ].

Importance of geographic habitats on light quality response of leaf growth and biomass

The selected accessions Col-0, Est-1, and C24 have different geographic habitats; C24 originated from a part of Europe (Portugal), Est-1 from Northern Asia (Russia), and Col-0 from United States (Columbia). Therefore, we took into account differences in geographical range for these accessions resulted in a high degree of divergence in photosynthetic characteristics to light [ 35 ]. The most extreme responder in leaf area growth and leaf biomass analyses was Est-1 from Russia. It is worth pointing out that the two Col-0 and C24 accessions highlighted here as weak responders, they elongated very quickly under AL and thus may not be true candidates for weak responders. Previous studies have found negative correlations between hypocotyl height and latitude of accession origin in European Arabidopsis accessions [ 55 , 56 ], suggesting that this natural variation in light sensitivity could be a result of adaptation to the north-south gradient in ambient light intensity.

The results of the study described here emphasize the strength of explicitly incorporating LxG interactions into the leaf area growth and leaf biomass content across the accessions. Importantly, as further elaborated below, the genotype-specific responses in leaf area growth and biomass content were observed exclusively under AL and BL, while the three accessions exhibited similar patterns of changes under RL. Our findings are consistent with previous reports on different accessions and light quality treatments, and underscores the importance of considering the natural habitat effect in characterizing the impact of light quality on leaves [ 57 ].

Leaf development varied between accessions such that the overall dynamic of growth and biomass were different. For example, we took efforts in synchronizing leaf growth stage in the accessions, resulting in the C24 plants being grow for 23 days to reach the same leaf stages of the plant. Some of the observed variation in leaf growth response could be simply a manifestation of the different time-course between accessions. These differences between accession can be significant and have the potential to enhance our understanding of the ecological role of specific adaptations.

Findings on BL supports its role on activation of protective pigments

BL induced higher leaf area growth across three accessions. However, its impact on biomass production is accession-dependent, and may be caused by accessory pigment accumulation (anthocyanins). Under BL, only Col-0 showed an increase in biomass, as opposed to Est-1 and C24, which showed a decrease in biomass. It was observed that BL induced a significantly higher concentration of anthocyanins in Est-1 and C24 than Col-0. These results imply that the impact of wavelength on accessory pigment accumulation is accession-dependent, and that this difference in accessory pigment accumulation consequently leads to differences in biomass production across accessions. Anthocyanin is a photo-protective pigment, which protects plant and its chloroplast membrane by absorbing blue light and against photo-oxidation [ 58 , 59 ]. Higher concentration of anthocyanin accumulating in a plant results in lower BL interception, which consequently lead to lower biomass production over the long term. Further to this, in this study, we found the ratio of Chl a:b is similar under BL and FL across accessions. This consistency in Chl a:b, suggests a lack of photosystems reconfiguration under BL [ 60 , 61 ]. Our results confirm the role of BL in stimulating anthocyanin content in plants and protecting them from light stress [ 62 ]. Plants activate photo-protective mechanisms under BL to cope with a potential induced-light stress, resulting in an increased accumulation of photo-protective pigments [ 58 , 59 ]. Notably, we found different patterns in content of anthocyanin accumulation in the accessions. Results showed that anthocyanin accumulation can be triggered at low BL (~70 μmol m -2 sec -1 ), which suggests that this protective mechanism against BL can vary based on the accession (i.e. natural adaptations) and can be triggered under low light. Further investigation on these two accessions on BL with a wide range of BL intensity is required. Our results thus encourage future studies analyzing this trait using BL with a wide range of BL intensity to further advance our understanding of the underlying mechanisms.

Plants showed high antioxidative and photo-protective under AL

AL had no impact on the photosynthetic activity across the three accessions compared to FL; yet it induced the poorest morphological traits. Col-0 and C24 showed a severe reduction ‬‬in leaf area growth and biomass, while Est-1 was unaffected. These two accessions (Col-0 and C24) showed a clear elongation of petioles under AL, which suggests that leaf resources are redirected from leaves to petioles as insufficient lighting conditions under AL were performed in this study [ 63 ]. However, the results on transcriptional changes and photosynthates content showed the opposite responses to the morphological traits.

The photosynthates, including proteins and starches, showed lower content in leaves of plants treated under AL. A downregulation of RBCS1A (small subunit of Rubisco) transcription was also observed in the leaves treated under AL. A lower accumulation of proteins was previously observed under AL [ 64 ], suggesting a positive contribution of downregulated Rubisco genes, as it is the main protein in leaves. A lower content of carbohydrates under stress conditions has been observed before in A . thaliana [ 65 ]. Future work is needed to explore if a reduced conversion of light energy into chemical energy has occurred in the photosynthesis process under AL.

High capacity for lipids accumulation was observed for plants treated under AL. Lipid accumulation had been previously linked to oxidative stress [ 66 ]. suggesting an increase in lipophilic antioxidants content such as tocopherols, which play an important role in the scavenging of singlet oxygen [ 67 ]. Moreover, we found a significant increase in both expression and enzymatic activity of antioxidants under AL. Plants stimulate antioxidative mechanisms to protect the photosynthetic apparatus from incurring damage via ROS detoxification [ 30 , 68 ]. Our results on photosynthates thus suggest that plants tried to cope with a potential ROS stress condition under AL.

A significant upregulation in glutathione biosynthesis, transcription level of PGRL1B , ATPC1 , and marker genes associated with ATP synthase and CET complex was observed. In agreement with this result, a significant increase in the expression of ATPC1 at the protein level was recently reported in A . thaliana Col-0 treated with 595 nm light [ 69 ]. CET plays an important role to protect plants under high light and other stress environments [ 70 ]. During CET, electrons are cycled around PSI and protons are translocated to generate a proton gradient across the thylakoid membranes [ 71 ]. In addition to contributing ATP synthesis, another function of a generated proton gradient is to dissipate excess energy as heat from the PSII antennae [ 72 ]. Further to this, an upregulation of CET and ATP synthase suggests of an accelerated rate of PSII repair through elevated ATP synthesis [ 73 , 74 ]. As such, the results on photosynthates and at the transcription level under AL both suggest that AL, even at low light, induces protective mechanisms of photosystems related to light stress, which consequently triggers low protein and starch accumulation and result in poor morphological traits.

One possible hypothesis for the conflicting AL responses can be explained by the detour effect [ 75 , 76 ], where a major part of AL transmitted into the leaf is reflected within leaf tissues and re-absorbed by unsaturated chlorophylls multiple times, which leads to an observed light stress response. Due to the nature of the high absorbing efficiency of the chloroplast, nearly 90% of BL and RL are absorbed at the leaf surface and their detour effect is small [ 76 , 77 ]. While for the wavelength within 500–600 nm [i.e. green light (GL) and AL] that are less absorbed by chloroplast, its light path can increase by several folds and this results in its increased/overexpressed photosynthetic activity through light absorption by unsaturated chloroplast. Although there is no study reporting the underlying mechanisms triggered by AL, several studies have observed the impact of supplemented GL and AL on photosynthetic activity and plant productivity in horticultural plants, which reinforces our hypothesis on the increased photosynthetic activity under AL. Further to this, the aggressive suppression responses on morphological traits in A . thaliana under AL, opposed to the positive impact on plant development, is expected as A . thaliana is a low light plant. They are more sensitive to the change in light properties. Overall, our results suggest AL as a potential light source in activating the potential of increased plant productivity efficiently, but it requires high control on its intensity. This study clarifies why AL alone induces overexpressed high photosynthetic activity yet results in poor plant development.

RL modulated plant adaptation and energy assimilation

The leaf area growth was significantly increased under RL across all accessions, which in turn enabled a greater light interception by the leaves [ 78 ]. This agrees with the increased Pn that was observed across accessions. These observations along with a significant increase of leaf biomass suggests proper plant adaptation under RL across accessions. We found a significant increase in the Chl a: b under RL across accession. Chl a is mainly concentrated around PSI and PSII, whereas Chl b is most abundant in light-harvesting complexes [ 79 ]. An increase in Chl a: b can increase the likelihood of an efficient electron transfer system within the chloroplast membrane [ 80 ]. This, in turn, could positively influence the photosynthetic performance in plants under RL. Considering that timely synthesis of D1 protein is key to maintain the PSII function and consequently, photosynthetic performance in leaves [ 25 ]. An increasing trend of PSBA expression was observed in plants under RL. The PSBA gene is critical for the de novo synthesis of the D1 protein during PSII repairs [ 81 , 82 ]. Therefore, upregulated transcription of PSBA gene could play an important role in accelerating the process of D1 protein turnover under RL. Plants showed that leaf photosynthates (starches, lipids, proteins) increased under RL. Overall, our results present RL as an efficient light source in helping the leaf energy assimilation process, resulting in an increased leaf growth, photosynthetic performance, and photosynthates content in plants.

Supporting information

In this experiment, eleven-leaves plants were grown under AL and RL for 5 days (three biological replicates per light condition). A) The expression pattern of protein members involved in Cyclic electron transfer (CET) complex. B) The expression pattern of protein members involved in ATP synthase complex. Expression levels for each protein is normalized to have mean of zero and standard deviation of one. Yellow or blue color indicates upregulation or downregulation, respectively.

Funding Statement

This work was supported by the “Natural Sciences and Engineering Research Council of Canada (NSERC)”. The specific grant number is RGPIN 355743-13, CRDPJ418919-11. It is “all” the funding and/or financial sources of support (whether external and/or internal to our organization) that were received during this study. And there was no additional external and/or internal funding received for this study. ML is the author, who received this award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability

ENCYCLOPEDIC ENTRY

Photosynthesis.

Photosynthesis is the process by which plants use sunlight, water, and carbon dioxide to create oxygen and energy in the form of sugar.

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Learning materials, instructional links.

• Photosynthesis (Google doc)

Most life on Earth depends on photosynthesis .The process is carried out by plants, algae, and some types of bacteria, which capture energy from sunlight to produce oxygen (O 2 ) and chemical energy stored in glucose (a sugar). Herbivores then obtain this energy by eating plants, and carnivores obtain it by eating herbivores.

The process

During photosynthesis, plants take in carbon dioxide (CO 2 ) and water (H 2 O) from the air and soil. Within the plant cell, the water is oxidized, meaning it loses electrons, while the carbon dioxide is reduced, meaning it gains electrons. This transforms the water into oxygen and the carbon dioxide into glucose. The plant then releases the oxygen back into the air, and stores energy within the glucose molecules.

Chlorophyll

Inside the plant cell are small organelles called chloroplasts , which store the energy of sunlight. Within the thylakoid membranes of the chloroplast is a light-absorbing pigment called chlorophyll , which is responsible for giving the plant its green color. During photosynthesis , chlorophyll absorbs energy from blue- and red-light waves, and reflects green-light waves, making the plant appear green.

Light-dependent Reactions vs. Light-independent Reactions

While there are many steps behind the process of photosynthesis, it can be broken down into two major stages: light-dependent reactions and light-independent reactions. The light-dependent reaction takes place within the thylakoid membrane and requires a steady stream of sunlight, hence the name light- dependent reaction. The chlorophyll absorbs energy from the light waves, which is converted into chemical energy in the form of the molecules ATP and NADPH . The light-independent stage, also known as the Calvin cycle , takes place in the stroma , the space between the thylakoid membranes and the chloroplast membranes, and does not require light, hence the name light- independent reaction. During this stage, energy from the ATP and NADPH molecules is used to assemble carbohydrate molecules, like glucose, from carbon dioxide.

C3 and C4 Photosynthesis

Not all forms of photosynthesis are created equal, however. There are different types of photosynthesis, including C3 photosynthesis and C4 photosynthesis. C3 photosynthesis is used by the majority of plants. It involves producing a three-carbon compound called 3-phosphoglyceric acid during the Calvin Cycle, which goes on to become glucose. C4 photosynthesis, on the other hand, produces a four-carbon intermediate compound, which splits into carbon dioxide and a three-carbon compound during the Calvin Cycle. A benefit of C4 photosynthesis is that by producing higher levels of carbon, it allows plants to thrive in environments without much light or water. The National Geographic Society is making this content available under a Creative Commons CC-BY-NC-SA license . The License excludes the National Geographic Logo (meaning the words National Geographic + the Yellow Border Logo) and any images that are included as part of each content piece. For clarity the Logo and images may not be removed, altered, or changed in any way.

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Practical: Investigating Factors Affecting the Rate of Photosynthesis ( OCR A Level Biology )

Revision note.

Biology Lead

Practical: Investigating Factors Affecting the Rate of Photosynthesis

• Investigations to determine the effects of light intensity, carbon dioxide concentration and temperature on the rate of photosynthesis can be carried out using aquatic plants , such as Elodea or Cabomba (types of pondweed )
• Light intensity – change the distance ( d ) of a light source from the plant (light intensity is proportional to 1/ d 2 )
• Carbon dioxide concentration – add different quantities of sodium hydrogencarbonate (NaHCO 3 ) to the water surrounding the plant, this dissolves to produce CO 2
• Temperature (of the solution surrounding the plant) – place the boiling tube containing the submerged plant in water baths of different temperatures
• For example, when investigating the effect of light intensity on the rate of photosynthesis, a glass tank should be placed in between the lamp and the boiling tube containing the pondweed to absorb heat from the lamp – this prevents the solution surrounding the plant from changing temperature
• Distilled water
• Aquatic plant, algae or algal beads
• Sodium hydrogen carbonate solution
• Thermometer
• Test tube plug
• This will ensure oxygen gas given off by the plant during the investigation form bubbles and do not dissolve in the water
• This will ensure that the plant contains all the enzymes required for photosynthesis and that any changes of rate are due to the independent variable
• Ensure the pondweed is submerged in sodium hydrogen carbonate solution (1%) – this ensures the pondweed has a controlled supply of carbon dioxide (a reactant in photosynthesis)
• Cut the stem of the pondweed cleanly just before placing into the boiling tube
• Measure the volume of gas collected in the gas-syringe in a set period of time (eg. 5 minutes)
• Change the independent variable (ie. change the light intensity, carbon dioxide concentration or temperature depending on which limiting factor you are investigating) and repeat step 5
• Record the results in a table and plot a graph of volume of oxygen produced per minute against the distance from the lamp (if investigating light intensity), carbon dioxide concentration, or temperature

The effect of light intensity on an aquatic plant is measured by the volume of oxygen produced

Results - Light Intensity

• The closer the lamp, the higher the light intensity (intensity ∝ 1/ d 2 )
• Therefore, the volume of oxygen produced should increase as the light intensity is increased
• This is when the light stops being the limiting factor and the temperature or concentration of carbon dioxide is limiting the rate of photosynthesis
• The effect of these variables could then be measured by increasing the temperature of water (by using a water bath) or increasing the concentration of sodium hydrogen carbonate respectively
• Rate of photosynthesis = volume of oxygen produced ÷ time elapsed

Limitations

• Immobilised algae beads are beads of jelly with a known surface area and volume that contain algae, therefore it is easier to ensure a standard quantity
• Immobilised algae beads are easy and cheap to grow, they are also easy to keep alive for several weeks and can be reused in different experiments
• The method is the same for algae beads though it is important to ensure sufficient light coverage for all beads

Practical: Measuring the rate of the light-dependent stage of photosynthesis

• The light-dependent reactions of photosynthesis take place in the thylakoid membrane and involve the release of high-energy electrons from chlorophyll a molecules
• These electrons are picked up by the electron acceptor NADP in a reaction catalysed by the enzyme dehydrogenase
• However, if a redox indicator (such as DCPIP or methylene blue ) is present, the indicator takes up the electrons instead of NADP
• DCPIP: oxidised ( blue ) → accepts electrons → reduced ( colourless )
• Methylene blue: oxidised ( blue ) → accepts electrons → reduced ( colourless )
• The colour of the reduced solution may appear green because chlorophyll produces a green colour
• When light is at a higher intensity, or at more preferable light wavelengths, the rate of photoactivation of electrons is faster, therefore the rate of reduction of the indicator is faster

The light activates electrons from chlorophyll molecules during the light-dependent reaction. Redox indicators accept the excited electrons from the photosystem, becoming reduced and therefore changing colour.

• Isolation medium
• Pestel and mortar
• Aluminium Foil
• DCPIP or methylene blue indicator
• Buffer solution

Method – Measuring light as a limiting factor

• This produces a concentrated leaf extract that contains a suspension of intact and functional chloroplasts
• The medium must have the same water potential as the leaf cells so the chloroplasts don’t shrivel or burst and contain a buffer to keep the pH constant
• The medium should also be ice-cold (to avoid damaging the chloroplasts and to maintain membrane structure)
• The room should be at an adequate temperate for photosynthesis and maintained throughout, as should carbon dioxide concentration
• If different intensities of light are used, they must all be of the same wavelength (same colour of light) – light intensity is altered by changing the distance between the lamp and the test tube
• If different wavelengths of light are used, they must all be of the same light intensity – the lamp should be the same distance in all experiments
• DCPIP or methylene blue indicator is added to each tube, as well as a small volume of the leaf extract
• A control that is not exposed to light (wrapped in aluminium foil) should also be set up to ensure the affect on colour is due to the light
• This is a measure of the rate of photosynthesis
• A graph should be plotted of absorbance against time for each distance from the light
• This is because the lowered light intensity will slow the rate of photoionisation of the chlorophyll pigment, so the overall rate of the light dependent reaction will be slower
• This means that less electrons are released by the chlorophyll, hence the DCPIP accepts less electrons. This means that it will take longer to turn from blue to colourless
• A higher rate of decrease, shown by a steep gradient on the graph, indicates that the dehydrogenase is highly active.
• This experiment is not measuring the rate of dehydrogenase activity directly (through measuring the rate of substrate use or product made) but is instead predicting what the rate would be by measuring the rate of electron transfer from the photosystems
• It is therefore important to control the amount of leaf used to produce the chloroplast sample and also how much time is spent crushing the leaf to release the chloroplast
• It is also a good idea to measure a specific wavelength absorption by each sample on the colorimeter before and after the experiment so you can get a more accurate change in oxidised DCPIP concentration
• Results should also be repeated and the mean value calculated
• The time taken to go colourless is subjective to each person observing and therefore one person should be assigned the task of deciding when this is
• Light intensity – the distance of the light source from the plant (intensity ∝ 1/ d 2 )
• Temperature - changing the temperature of the water bath the test tube sits in
• Carbon dioxide - the amount of NaHCO 3 dissolved in the water the pondweed is in

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Lára graduated from Oxford University in Biological Sciences and has now been a science tutor working in the UK for several years. Lára has a particular interest in the area of infectious disease and epidemiology, and enjoys creating original educational materials that develop confidence and facilitate learning.

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Science Projects > Life Science Projects > Test for Starch in Plants  

Test for Starch in Plants

Photosynthesis is the process in which green plants (primarily) convert energy from the sun’s light into usable, chemical energy. Plants require energy for growth, reproduction, and defense. Excess energy, created from photosynthesis, is stored in plant tissue as starch. Starch is a white and powdery substance. It houses glucose, which plants use for food. The presence of starch in a leaf is reliable evidence of photosynthesis. That’s because starch formation requires photosynthesis.

( Adult supervision required. )

Starch Testing Experiment

What you need:.

• Beaker or glass jar
• Saucepan on the stove
• Ethyl alcohol
• Iodine solution

Test for starch in plants:

1. Place one of the plants in a dark room for 24 hours; place the other one on a sunny windowsill.

2. Wait 24 hours.

3. Fill the beaker or jar with ethyl alcohol.

4. Place the beaker or jar in a saucepan full of water.

5. Heat the pan until the ethyl alcohol begins to boil.

6. Remove from the heat.

7. Dip each of the leaves in the hot water for 60 seconds, using tweezers.

8. Drop the leaves in the beaker or jar of ethyl alcohol for two minutes (or until they turn almost white).

9. Set them each in a shallow dish.

10. Cover the leaves with some iodine solution and watch.

What Happened:

The hot water kills the leaf and the alcohol breaks down the chlorophyll, taking the green color out of the leaf. When you put iodine on the leaves, one of them will turn blue-black and the other will be a reddish-brown. Iodine is an indicator that turns blue-black in the presence of starch. The leaf that was in the light turns blue-black, which demonstrates that the leaf has been performing photosynthesis and producing starch.

Try the test again with a variegated leaf (one with both green and white) that has been in the sunlight. A leaf needs chlorophyll to perform photosynthesis — based on that information, where on the variegated leaf do you think you would find starch?

Buy Testing For Starch Experiment Kit

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•   Make a Butterfly Feeder
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•   Make Spider Web Art

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• Science is LIT

Explore How Light Affects Photosynthesis

Algae are aquatic, plant-like organisms that can be found in oceans, lakes, ponds, rivers, and even in snow. But don’t worry, if you’re not near a waterway, it can easily be ordered from Amazon or Carolina Biological. Algae range from single-celled phytoplankton (microalgae) to large seaweeds (macroalgae). Phytoplanktons can be found drifting in water and are usually single-celled. They can also grow in colonies (group of single-cells) that are large enough to see with the naked eye. The specific types of algae that can be used in this experiment are  Scenedesmus, Chlamydomonas, or  Chlorella , all of which are phytoplanktons or microalgae. 

Experimental variables

• Color filter paper
• Table/desk lamp
• Light bulbs (varying intensities and colors)

Laboratory Supplies

• Transfer pipettes
• Vials with caps
• Freshwater Algae ( Scenedesmus , Chlorella , or Chlamydomonas )
• Small beakers or cups

Laboratory Solutions

• 2% Calcium Chloride
• 2% Sodium alginate
• Cresol red/thymol blue pH indicator solution

Solution Preparations

2% calcium chloride (cacl 2 ).

• 20 g of CaCl 2
• Fill to 1000 mL with water

2% CaCl 2 is stable at room temperature indefinitely.

2% Sodium alginate (prepared in advance)

• 2 g sodium alginate
• Fill to 100 mL with water

It takes a while for the alginate to go into solution. We recommend to dissolve by stirring using a magnetic stir bar overnight at room temperature. Store at 4 °C for up to 6 months or use immediately.

Cresol red/Thymol blue pH indicator solution (10x)

• 0.1 g cresol red
• 0.2 g thymol blue
• 0.85 g sodium bicarbonate (NaHCO 3 )
• 20 mL ethanol
• Fill to 1L with fresh boiled water

Measure indicators and mix with ethanol. Measure sodium bicarbonate and mix with warm/hot water. Mix the solutions together and fill with remaining freshly boiled water up to 1L final solution. The 10x stock solution is stable for at least a year.

In preparation for doing the experiment, prepare 1x indicator solution by diluting the 10x indicator solution with distilled water (e.g. 20 ml 10x into 200 mL final solution).

Experimental Bench Set-Up

• ~10 mL of 2% CaCl 2 in a cup or beaker
• ~3-5 mL of sodium alginate in cup or beaker
• Cup with ~10 mL of water
• Empty cup or beaker that holds a minimum of 30 mL

Preparing Algae for Experiment

• Prepare a concentrated suspension of algae. Without centrifuge : leave ~50 mL of algae suspension to settle (preferably overnight), then carefully pour off the supernatant to leave ~3-5 mL of concentrated algae. With centrifuge : Centrifuge ~50 mL of algae suspension at low speed for 10 minutes and then carefully pour off the supernatant, leaving behind ~3-5 mL of concentrated algae.
• In a small beaker, add equal volumes of sodium alginate and then add in the concentrated algae. Gently mix algae and sodium alginate together using a transfer pipette until its evenly distributed.
• Using the transfer pipette, carefully add single drops of the algae/sodium alginate mixture into the CaCl 2 to make little “algae balls”
• Once all of the “algae balls” are in the CaCl 2 solution, allow them to harden for 5 minutes
• Place the strainer over the empty cup or beaker, and pour over the entire solution of “algae balls” and CaCl 2 into the strainer allowing the CaCl 2 to pass through, leaving just the algae in the strainer
• Keeping the strainer over the container, pour the water over the “algae balls” to rinse the remain CaCl 2
• Transfer your newly made “algae balls” to a new cup or beaker

Setting up Photosynthesis Experiment

• Distance from light (using ruler) – group can set up vials different distances from one light source
• Different color lights (using color filter paper or different color light bulbs) – group can set up by covering the vials with different colored films and arrange them the same distance away from the light source or set up 1 vial in front of a different colored lamp same distance away.
• With or without light – group places 1 vial in front of an illuminated lamp and another has the vial or lamp covered with black paper the same distance away

• When starting your experiment, be sure to take note of the time that you placed your vial in front of the light source. Vials should be left for ~1-2 hours.

What would happen if the algae photosynthesizes (increase O2) in a solution that started at pH8.2?

Analyzing photosynthesis results

• After 1-2 hours, return to the experiment. Without disturbing the vials, analyze and take pictures of results. Have students write down the time that their experiment ended.
• Using the color chart above, determine which pH matches your sample the closest.
• Have students determine if they got what they expected and discuss amongst their group members.

Explain how the rate of photosynthesis is affected by their different variables.

What were your conclusions from this experiment? If you were to repeat the experiment, what would you change and why? What’s the relationship with O2 and CO2 during the process of photosynthesis? Is there a “best” source of light that allowed the algae to photosynthesize better?

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July 10, 2024

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Photosynthesis powers our world, but what fuels this fundamental process?

by Carnegie Institution for Science

It's hard to overstate the importance of photosynthesis, the biochemical pathway by which plants, algae, and certain bacteria convert the sun's energy into the organic material that feeds the entire living biosphere. But there are still aspects of the photosynthetic function that scientists are working to understand. Expanding their knowledge could help improve agriculture and fight climate change.

Photosynthesis provides the foundation for life on Earth by making our atmosphere oxygen rich; it also sequesters carbon pollution from human activity and forms the basis of the food chain.

"On a molecular level , photosynthesis has two components," explained Carnegie Science algae expert Adrien Burlacot. "There's the sunlight-powered splitting of water molecules, which produces the energy molecules that are used by all cells, and the fixation of carbon dioxide from the atmosphere into organic material , or biomass, which consumes chemical energy."

He added, "But there's a disconnect that has caused decades of debate. This is because the chemical energy needed to power the second half of this process—the transformation of carbon dioxide into biomass—is different than the energy currency created by splitting water molecules. And the processes involved in converting the basic energy molecules into the energy used to synthesize sugars are still mysterious."

When it comes to photosynthetic efficiency , organisms have wide variation in their capacity to transform sunlight into biomass. While a tree or a grass would typically be able to use between 0.5% and 1% of the sun's energy, microalgae are able to use up to 5% of that energy.

"Photosynthesis in plants is very inefficient," Burlacot added. "Because algae are much better at it, they hold important potential for understanding how to improve this fundamental process that underpins nearly every aspect of life on our planet."

Part of the secret lies in algae harboring a special biochemical system for concentrating carbon dioxide within the photosynthetic apparatus. For the last couple of years, Burlacot's lab has been investigating how algae power this carbon-concentrating ability by studying Chlamydomonas, a group of photosynthetic algae that are found around the globe in fresh and saltwater, moist soils, and even at the surface of snow.

Recently, they expanded this to examine how the basic energy currency is converted to the chemical energy necessary for the carbon-fixation process itself in algae.

In their paper published in The Plant Cell , Burlacot's team revealed that three biochemical energy circuits power carbon fixation in Chlamydomonas. Their work demonstrated that all three pathways can sustain high rates of sugar production. However, the three circuits were not all equally efficient.

"Two out of three pathways are wasting twice more energy than the most efficient one," Burlacot said. "And—interestingly—the two most efficient pathways are not present in crop plants ."

Looking ahead, the group wants to elucidate the contributions of each of the three mechanisms and to build connections between these and similar pathways in other photosynthetic organisms. A big question remains on whether differences in photosynthetic efficiency between species could be related to the energy circuits that they are using.

"We are attempting to understand biochemical and biophysical steps of how algae capture carbon dioxide , which could enable us to improve the efficiency of important crop plants and to enhance carbon capture solutions," Burlacot concluded. "More work is needed, and we are revealing the full story of how carbon fixation is fueled."

Journal information: Plant Cell

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• Published: 03 July 2024

Effect of silicon spraying on rice photosynthesis and antioxidant defense system on cadmium accumulation

• Hongxing Chen 1 , 2 ,
• Xiaoyun Huang 1 , 2 ,
• Hui Chen 1 , 2 ,
• Song Zhang 1 , 2 ,
• Chengwu Fan 3 ,
• Tianling Fu 2 , 4 ,
• Tengbing He 1 , 2 &
• Zhenran Gao 1 , 2  

Scientific Reports volume  14 , Article number:  15265 ( 2024 ) Cite this article

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• Environmental sciences
• Plant sciences

Cadmium (Cd) pollution is a serious threat to food safety and human health. Minimizing Cd uptake and enhancing Cd tolerance in plants are vital to improve crop yield and reduce hazardous effects to humans. In this study, we designed three Cd concentration stress treatments (Cd1: 0.20 mg·kg −1 , Cd2: 0.60 mg·kg −1 , and Cd3: 1.60 mg·kg −1 ) and two foliar silicon (Si) treatments (CK: no spraying of any material, and Si: foliar Si spraying) to conduct pot experiments on soil Cd stress. The results showed that spraying Si on the leaves reduced the Cd content in brown rice by 4.79–42.14%. Si application increased net photosynthetic rate (Pn) by 1.77–4.08%, stomatal conductance (Gs) by 5.27–23.43%, transpiration rate (Tr) by 2.99–20.50% and intercellular carbon dioxide (CO 2 ) concentration (Ci) by 6.55–8.84%. Foliar spraying of Si significantly increased the activities of superoxide dismutase (SOD) and peroxidase (POD) in rice leaves by 9.84–14.09% and 4.69–53.09%, respectively, and reduced the content of malondialdehyde (MDA) by 7.83–48.72%. In summary, foliar Si spraying protects the photosynthesis and antioxidant system of rice canopy leaves, and is an effective method to reduce the Cd content in brown rice.

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Introduction.

Cadmium (Cd) is a major pollutant affecting the quality of farmland soil, and its biological toxicity is significant 1 . Because of its high solubility and fluidity, the toxic effects of Cd on plants are manifested in various metabolic activities. A large amount of Cd accumulates in plants, leading to a reduction in plant photosynthetic rate, inhibition of plant antioxidant enzyme activity, and suppression of cell division, thereby impacting plant growth 2 , 3 , 4 . Moreover, it enters the human body through the food chain and causes harm 5 . Rice ( Oryza sativa L.) is the main food crop in China and also the main source of food for over 50% of the global population. However, because of soil pollution, the accumulation of Cd in rice has led to “Cd rice” 6 , and long-term consumption of food contaminated by Cd often induces cancer, pain, kidney toxicity and hypertension 7 . Therefore, the monitoring and control of Cd pollution need to be strengthened to ensure human health and food security.

Many measures have been taken to reduce the accumulation of Cd in rice, including soil remediation works, agricultural practices, and phytoremediation 8 . These remediation techniques are complex and often expensive on a site scale 9 . As the most important foreign nutrient organ of rice, leaves can absorb foreign substances and transport nutrients to other organs of rice 10 . Compared with other agronomic control measures, foliar spraying barrier agent has the characteristics of consistent farming time, convenient application, economical and efficient, and has been widely used in farmland production. It has a good effect on improving crop stress resistance, enhancing crop heavy metal tolerance and increasing crop yield 11 .

Silicon (Si) is not only a beneficial element 12 , the application of Si can promote the absorption of nutrients by plants, significantly enhance their biological and abiotic resistance, which is conducive to plant growth, but also reduce the toxic effect of Cd on plants 13 . Research has found that under Cd stress, Si application increased chlorophyll content (SPAD), carotenoid content and photosynthetic rate of maize leaves 14 . The application of exogenous Si reduced the malondialdehyde (MDA) content of cotton, increased the activity of antioxidant enzymes, alleviated the adverse effects of Cd stress on the growth and photosynthetic characteristics of cotton, and improved the quality of cotton 2 . Foliar Si application significantly increased rice yield, reduced the bioavailability of Cd in soil, inhibited the migration and transformation of Cd in soil and plants, slow down the content of Cd in rice, and improved the quality of rice 15 .

The above researches mainly focus on acid soil or hydroponic experiments, but there are few researches on neutral paddy soil in southern China. Therefore, a pot experiment was conducted to study the effects of foliar Si spraying on growth, photosynthetic characteristics and antioxidant system of Cd-stressed rice in southern rice soil. We proposed the following hypothesis: that foliar Si spraying treatment suppresses the migration and transportation of Cd by rice plants, thereby reducing the Cd content in brown rice, increasing rice yield, improving rice light utilization efficiency, and enhancing antioxidant effects, ultimately enhancing the alleviating effect on Cd toxicity. Therefore, this study aimed to: (1) explore the effect and mechanism of foliar Si application on the migration and accumulation of Cd in rice plants; (2) explore the potential and mechanism of foliar Si application on improving rice photosynthesis and stress resistance; and (3) explore the effect and mechanism of foliar Si application on enhancing the antioxidant capacity of rice. The results will provide a valuable reference for reducing the accumulation of Cd in rice, improving its safety as food, and ensuring human health.

Materials and methods

Experimental design.

The experiment was conducted at the Guiyang Comprehensive Experimental Station of the Guizhou Academy of Agricultural Sciences in China from March to October 2021 (106°39′20″E, 26°29′59″N). The pot experiment was conducted under natural sunlight and temperature (from March to October, 2022). The air temperature ranged between 10.5 ± 5.6 and 28.6 ± 2.5 °C. The relative humidity varied from 77 ± 2.8 to 93 ± 0.5%. The basic physical and chemical indicators of soils are shown in Table 1 .

The background value of soil Cd was 0.20 mg·kg −1 , and 0.20 mg·kg −1 was the risk screening value for Cd in the rice fields. The experiment set up three exogenous Cd concentration addition treatments: Cd1 (0), Cd2 (0.20 mg∙kg −1 ), and Cd3 (0.40 mg∙kg −1 ). The rice variety used was “Jingliangyou 534” (Guoshen Rice 20,176,004). Each box was uniformly inserted in four holes as a treatment, with two plants per hole, and each treatment was replicated six times for a total of 18 pots. Before transplanting the rice seedlings, base fertilizer was applied: 450 kg·hm −2 rice-specific compound fertilizer. Tillering fertilizer (urea 120 kg·hm −2 ) was sprayed onto the plants during the tillering stage, and potassium chloride fertilizer (112.5 kg·hm −2 ) was sprayed onto the plants during the booting stage. The soil type was yellow loamy paddy soil. Adding of exogenous Cd to the soil involved mixing Cd chloride (CdCl 2 ) (Shanghai Aladdin biochemical technology Co., Ltd., Shanghai, China) in solution with air-dried soil, injecting water to saturation, and equilibrating for 4–5 weeks. The actual values after addition were Cd1 (0.20 mg·kg −1 ), Cd2 (0.60 mg·kg −1 ), and Cd3 (1.60 mg·kg −1 ). When the rice was mature, the SPAD, Cd content, photosynthetic parameters, and enzyme activity of rice canopy leaves under different Cd concentrations were measured. Two foliar spraying treatments: CK (no spraying of any material) and Si (foliar spraying of Si) were used. In the spraying of “Jianggeling” rice with foliar Si fertilizer (Foshan Ironman Environmental Technology Co., Ltd., Foshan, China), the active ingredients were primarily high-purity SiO 2 sols (Si ≥ 85 g·L −1 , pH = 5.0–7.0) at a concentration of 2.5 g·L −1 . One spray was administered at the jointing stage and one at the heading stage, at 17:00–18:00 p.m.

Measurement of indicators

Determination of cd content.

An inductively coupled plasma optical emission spectrometer (ICP-OES, Thermo Fisher Scientific, Waltham, MA, USA) was used to measure the Cd content 16 . At the maturity stage of rice, we handpicked three robust and evenly developed specimens from every group. Subsequently, these specimens underwent multiple cleansings using faucet water, followed by a deionized water purge. Post-washing, the samples were subjected to a brief heat exposure at 105 °C for half an hour and subsequently desiccated in a heating chamber regulated at 75 °C to achieve a stable mass. Afterward, the samples were methodically segmented into the roots, stems, leaves, husks, and brown rice parts, and were finely pulverized with the assistance of a high-velocity FW-100 grinder (Tianjin Taist Instrument Co., Ltd.). Subsequently, a 200 mg portion of the rice specimen was measured out, and to this, we introduced 5 mL of nitric acid (HNO 3 ). The digestion process for the specimen was carried out with a graphite digestion device at a temperature of 120 °C for a duration of two hours, proceeding until no residual sediment remained within the digestion chamber. The temperature was adjusted to 150 °C to evaporate the acid. The sample was removed and allowed to cool, diluted to a volume of 50 mL in a volumetric flask, filtered, and analyzed via ICP–OES.

Measurement of photosynthetic parameters

Portable photosynthetic apparatus (GFS-3000, Heinz Walz GmbH, Bavaria, Germany) was used to measure the photosynthetic parameters of rice at the heading stage 17 . Between 10:00–11:00 a.m. on a clear, cloudless day, the carbon dioxide (CO 2 ) concentration was set to 400 µ mol·mol −1 , the light intensity was set to 1200 µ mol·m −2 ·s −1 , the air velocity was set to 0.5 L·min −1 , leaf temperature was 25 °C, and relative humidity was set to 70%. We handpicked three robust and evenly developed specimens from every group to measure the net photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), and intercellular CO 2 concentration (Ci).

Measurement of fluorescence parameters

A portable fluorometer (Junior-PAM, Heinz Walz GmbH, Bavaria, Germany) was used to measure chlorophyll fluorescence parameters 18 . We selected rice leaves with consistent growth conditions, subjected to fully adapt to darkness for 30 min, and measured the maximum photochemical quantum yield of photosystem II (PS II) of the leaves, maximum photochemical efficiency (Fv/Fm), actual photochemical efficiency (Y(II)), initial fluorescence (Fo), and non-photochemical quenching coefficient (NPQ).

Measurement of MDA and antioxidant enzymes

We cleaned rice leaves with distilled water and weighed 100 mg of fresh rice leaves, ground them into a homogenate using liquid nitrogen in a mortar and pestle, and then transferred the homogenate to a 4 mL centrifuge tube. We added 1 mL of 0.05 mol·L −1 phosphate buffer (pH 7.8) to the tube and fixed the volume to 4 mL. This was mixed well using a vortexer, and was put into a frozen high-speed centrifuge at 4 °C and 10,000 r·min −1 for 10 min, The supernatant was then placed in a refrigerator at 4 °C as a backup. The activities of superoxide dismutase (SOD) and peroxidase (POD) in rice leaves and the MDA content were determined using the nitroblue tetrazolium (NBT) photoreduction method 19 , guaiacol method, and thiobarbituric acid (TBA) method, respectively. All measurements were performed using enzyme activity assay kits from Wuhan PureBiochemical Co., Ltd.

Determination of relative chlorophyll content

We used a portable chlorophyll meter (SPAD-502 Plus, Minolta, Tokyo, Japan) to measure the SPAD value of leaves in situ 20 . When they had been measured, we selected three rice plants with uniform growth conditions and, for each plant, selected an intact leaf and measured the SPAD value at the central position six times, and took the average value as the SPAD value for that point. When taking measurements, we avoided areas with concentrated veins and used appropriate shading to block direct sunlight, to ensure the accuracy of the measurement.

Data processing and analysis

The bioconcentration factor (BCF) of Cd in rice was calculated (1) as 21 :

The transport factor (TF) of Cd in rice was calculated (2) as 22 :

where TF refers to the ratio of heavy metal concentration in part A to that in part B of the rice plant.

Data processing and statistical analysis were carried out using SPSS 24.0 (IBM Corp., Armonk, NY, USA). Single factor analysis of variance (ANOVA) was used to tested the same treatment under different Cd concentrations. The treatment effects at different Cd concentrations were compared by using the least significant different test with the P value < 0.05. The effects with Si treatment and CK treatment were tested by t test. Plots were generated using Origin.

Effect of foliar spraying Si on Cd content in various organs and on rice yield

The impact of foliar spraying Si on the Cd content in various organs and on rice yield are shown in Table 2 .

Under the three Cd concentrations, the Cd content in each organ of rice increased with the increase in Cd concentration. At Cd1 concentration, compared with CK, Si treatment increased the Cd content in roots, leaves, and cobs by 112.99%, 30.00%, and 51.85%, respectively, and reduced the Cd content in husks and brown rice by 29.17% and 40.91%, respectively. At Cd2 concentration, compared with CK, Si treatment significantly increased the Cd content in the stems and cobs by 145.07% and 61.36%, respectively, and significantly decreased the Cd content in husks by 41.67%, respectively. At Cd3 concentration, compared with CK, Si treatment increased the Cd content in leaves and cobs by 26.47% and 62.80%, respectively, and decreased the Cd content in husks and brown rice by 33.90% and 12.57%, respectively. Rice yield decreased with increasing Cd concentration. At the three Cd concentrations, spraying Si on the leaves increased rice yield by 4.18%, 2.36%, and 6.14% compared to CK, respectively. It can be seen that Cd stress will increase the Cd content in various organs and reduce rice yield. In contrast, spraying Si on the leaves can change the accumulation of Cd in various organs, and increase rice yield.

Effect of foliar spraying Si on the accumulation and transport of Cd in rice

The effect of foliar spraying Si on the BCF in various organs are shown in Fig.  1 . The accumulation of Cd in rice showed a trend of root > stem > leaf > brown rice. Under CK treatment, the enrichment coefficients of roots, stems, leaves, and brown rice were 1.65–2.16, 0.18–1.00, 0.28–0.49, and 0.22–0.28, respectively. Compared with CK, under treatments Cd1, Cd2, and Cd3, foliar application of Si enhanced rice BCFroot by 112.91%, 0.41%, and 11.03%; BCFstem by 17.78%, 146.33%, and 1.60%; BCFleaf by 32.25%, 8.18%, and 26.41%; and decreased rice BCFbrown rice by 42.27%, 4.78%, and 12.59%, respectively. It can be seen that foliar application of Si can increase the enrichment coefficients of rice roots and leaves, which decreased the Cd enrichment coefficients of brown rice.

The effect of foliar Si spraying on the BCF of various organs in rice: BCFroot ( a ), BCFstem ( b ), BCFleaf ( c ), BCFbrown rice ( d ). Cd1, Cd2, Cd3: Three Cd concentration stress treatments (Cd1: 0.20 mg·kg −1 , Cd2: 0.60 mg·kg −1 , and Cd3: 1.60 mg·kg −1 ). CK, Si: two spraying treatments (CK: no spraying of any material, Si: foliar Si spraying). Data present the mean ± standard deviation of three replicates. Capital letters indicate significant differences ( p  < 0.05) between CK and Si treatments at the same Cd concentration. Lowercase letters indicate significant differences ( p  < 0.05) between CK or Si treatments at different Cd concentrations.

The effect of foliar spraying Si on the TF in various organs are shown in Fig.  2 . Foliar spraying of Si decreased TFleaf-brown rice, and TFstem-brown rice. Compared with CK, foliar spraying of Si decreased rice TFleaf-brown rice by 57.27%, 11.43%, and 30.90%, and of TFstem-brown rice by 51.44%, 61.15%, and 15.90%. It can be seen that foliar application of Si can decrease the transfer coefficient of rice leaf to brown rice and stem to brown rice, which decreased the Cd content of brown rice.

The effect of foliar Si spraying on the TF of various organs in rice: TFroot-stem ( a ), TFstem-leaf ( b ), TFleaf-brown rice ( c ), TFstem-brown rice ( d ). Cd1, Cd2, Cd3: Three Cd concentration stress treatments (Cd1: 0.20 mg·kg −1 , Cd2: 0.60 mg·kg −1 , and Cd3: 1.60 mg·kg −1 ). CK, Si: two spraying treatments (CK: no spraying of any material, Si: foliar Si spraying). Data present the mean ± standard deviation of three replicates. Capital letters indicate significant differences ( p  < 0.05) between CK and Si treatments at the same Cd concentration. Lowercase letters indicate significant differences ( p  < 0.05) between CK or Si treatments at different Cd concentrations.

Effect of foliar spraying Si on the SPAD values of rice leaves

The effect of foliar spraying Si on the SPAD values of rice leaves are shown in Fig.  3 . The SPAD values of leaves under three Cd concentration treatments all decreased with the increase in Cd concentration. Compared with the control Cd1 treatment, the decrease in Cd2 and Cd3 treatments was 4.31% and 7.22%, respectively. Under the treatment of spraying Si, the decrease in Cd2 and Cd3 treatments was 3.97% and 6.75%, respectively, compared with the treatment Cd1. Foliar spraying of Si resulted in an increase of 2.02–2.53% in SPAD values compared to the control group. Thus it can be seen that Cd reduces the SPAD values of rice leaves, whereas foliar spraying of Si enhances these values.

The effects of foliar Si spraying on the SPAD values of rice leaves. Cd1, Cd2, Cd3: Three Cd concentration stress treatments (Cd1: 0.20 mg·kg −1 , Cd2: 0.60 mg·kg −1 , and Cd3: 1.60 mg·kg −1 ). CK, Si: two spraying treatments (CK: no spraying of any material, Si: foliar Si spraying). Data present the mean ± standard deviation of six replicates. Capital letters indicate significant differences ( p  < 0.05) between CK and Si treatments at the same Cd concentration. Lowercase letters indicate significant differences ( p  < 0.05) between CK or Si treatments at different Cd concentrations.

Effect of foliar spraying Si on photosynthetic parameters of rice leaves

The impact of foliar spraying Si on the photosynthetic parameters of rice leaves are shown in Fig.  4 a–d. As Cd concentration increased, under CK and Si treatment, the Pn, Gs, Tr, and Ci values all decreased. Under CK treatment, compared with Cd1 concentration, the Pn, Gs, Tr, and Ci at Cd2 concentration decreased by 6.68%, 19.97%, 4.65%, and 6.13%, respectively. In contrast, at the concentration of Cd3, the decrease amplitude was 12.86%, 40.54%, 31.88%, and 15.25%, respectively. Compared to CK, foliar spraying Si resulted in a 1.77–4.08% increase in Pn, a 5.27–23.43% increase in Gs, a 2.99–20.50% increase in Tr, and a 6.55–8.84% increase in Ci, respectively. Therefore, Cd diminished the photosynthetic attributes of rice, whereas foliar spraying Si can mitigate the toxic impact of Cd on rice, enhance its the photosynthetic parameters, and foster photosynthesis.

The effects of foliar Si spraying on the photosynthetic parameters of rice leaves: net photosynthetic rate (Pn) ( a ), stomatal conductance (Gs) ( b ), transpiration rate (Tr) (c), intercellular CO 2 concentration (Ci) (d). Cd1, Cd2, Cd3: Three Cd concentration stress treatments (Cd1: 0.20 mg·kg −1 , Cd2: 0.60 mg·kg −1 , and Cd3: 1.60 mg·kg −1 ). CK, Si: two spraying treatments (CK: no spraying of any material, Si: foliar Si spraying). Data present the mean ± standard deviation of three replicates. Capital letters indicate significant differences ( p  < 0.05) between CK and Si treatments at the same Cd concentration. Lowercase letters indicate significant differences ( p  < 0.05) between CK or Si treatments at different Cd concentrations.

Effect of foliar spraying Si on the fluorescence parameters of rice leaves

The influence of foliar spraying Si on the fluorescence parameters of rice leaves are shown in Fig.  5 a–d. where Y(II) and Fv/Fm decreased with increasing Cd concentration, whereas Fo and NPQ increased with increasing Cd concentration. At the three concentrations of Cd1, Cd2, and Cd3, compared to CK, foliar Si spraying resulted in an increase in Y(II) and Fv/Fm by 0.38–5.98% and 1.55–2.78%, respectively, while simultaneously reducing Fo and NPQ by 3.11–9.67% and 7.48–16.47%, respectively. Under Cd stress, foliar application of Si can effectively maintain high photosynthetic characteristics.

The effects of foliar Si spraying on the chlorophyll fluorescence parameters of rice leaves: actual photochemical efficiency: Y (II) ( a ), non photochemical quenching coefficient: (NPQ) ( b ), initial fluorescence (Fo) ( c ), maximum photochemical efficiency (Fv/Fm) ( d ). Cd1, Cd2, Cd3: Three Cd concentration stress treatments (Cd1: 0.20 mg·kg −1 , Cd2: 0.60 mg·kg −1 , and Cd3: 1.60 mg·kg −1 ). CK, Si: two spraying treatments (CK: no spraying of any material, Si: foliar Si spraying). Data present the mean ± standard deviation of three replicates. Capital letters indicate significant differences ( p  < 0.05) between CK and Si treatments at the same Cd concentration. Lowercase letters indicate significant differences ( p  < 0.05) between CK or Si treatments at different Cd concentrations.

Effect of foliar spraying Si on MDA, POD, and SOD content in rice leaves

The impact of foliar spraying Si on the levels of MDA, POD, and SOD activity are shown in Fig.  6 a–c. Under CK treatment, as the concentration of Cd increased, the content of MDA rose, whereas the activities of POD and SOD decreased. Compared to CK, foliar spraying of Si significantly reduced the content of MDA in rice leaves by 7.83–48.72%. Additionally, compared to CK, foliar spraying of Si significantly increased the activities of SOD and POD in rice leaves by 9.84–14.09% and 4.69–53.09%, respectively. It can be seen that foliar Si application can improve the antioxidant enzyme activity of rice leaves.

The effects of foliar Si spraying on MDA, POD and SOD content in rice leaves: malondialdehyde: MDA ( a ), peroxidase: (POD) ( b ), superoxide dismutase (SOD) ( c ). Cd1, Cd2, Cd3: Three Cd concentration stress treatments (Cd1: 0.20 mg·kg −1 , Cd2: 0.60 mg·kg −1 , and Cd3: 1.60 mg·kg −1 ). CK, Si: two spraying treatments (CK: no spraying of any material, Si: foliar Si spraying). Data present the mean ± standard deviation of three replicates. Capital letters indicate significant differences ( p  < 0.05) between CK and Si treatments at the same Cd concentration. Lowercase letters indicate significant differences ( p  < 0.05) between CK or Si treatments at different Cd concentrations.

The excessive accumulation of Cd in plants not only significantly impacts their growth, development, quality, and yield, but also poses a potential threat to human health through the food chain because of its concealed presence within the plants 23 . The half-life of Cd in the human body is up to 30 years and it has a cumulative effect 24 . Excess Cd accumulation in the human body will cause calcification of kidney and bone, metabolic dysfunction, bone pain, hypertension, diabetes, emphysema, and other diseases. It can lead to deoxyribonucleic acid (DNA) oxidative damage and inhibition of the repair path, and induce the generation of cancer cells 25 . Research has shown that Si can effectively reduce the harm of Cd to plants and its inhibitory effect on plant growth, thereby improving the beneficial effects of food safety and human health 26 . In this study, foliar Si application had no significant effect on rice yield, but there was a trend of increasing yield under different soil Cd concentrations (Table 2 ). This is consistent with previous research findings 27 . The application of Si under metal stress can improve plant growth by increasing nutrient elements, SPAD, root volume, organic acid secretion, and histological characteristics 28 , 29 , 30 . Cd was highest in the roots of rice plants (Table 2 ), consistent with previous research findings 31 , 32 . The root system is the first plant organ to sense adversity. Excessive Cd can cause a decrease in the number of roots, a shortening or browning of roots, a decrease in root area, and can affect the division of root tip cells, inducing stress Chromosome aberrations and other factors can significantly reduce the absorption capacity of roots for water and nutrients in the soil 33 . Overall, the high retention of Cd in roots is considered a defense mechanism for plants to alleviate metal stress 27 . The Cd content in brown rice is a major health risk of significant concern 33 . The results showed that Si application could significantly reduce the Cd content in brown rice (Table 2 ) ( P  < 0.05). Under Cd stress in brown rice, Si application significantly reduced the transfer and enrichment coefficients of Cd, and reduced the Cd content 34 . The application of Si on the leaves mainly reduces the accumulation of Cd in brown rice by inhibiting the migration of Cd from the stem to the rice grains (Fig.  1 ). The stem is the main organ that restricts the transport of Cd to rice 35 . The stem nodes of Poaceae plants are the hub for the distribution of mineral elements to different organs, and the upward transport of Cd is significantly restricted at these nodes 36 . The high Cd content in the cell wall of stems and leaves is due to the presence of a large number of negatively charged functional groups in the cell wall 37 . These functional groups are precipitated and complexed with positively charged heavy metal ions, allowing most of the Cd to bind to the cell wall. Rice can alleviate the toxic effects of Cd by combining Cd and Si in the cell wall to alter the redox potential 38 . Combining with Si in the form of negatively charged hemicellulose can inhibit the absorption of Cd by rice cells. Therefore, foliar spraying of Si fertilizer is a feasible method to control Cd accumulation in rice grains, thereby reducing its risk to human health through the food chain.

In higher plants, Cd affects photosynthesis mainly by reducing the SPAD values, causing a decrease in the content of photosynthetic pigments, disrupting the position of matrix layers and grana within chloroplasts, leading to a decrease in the photosynthetic capacity of chloroplasts 39 . Cd can also inhibit the enzymes related to photosynthesis and affect plant growth by altering transpiration, respiration, and stomatal switch, thereby inhibiting crop photosynthesis 40 . This study found that, under Cd stress, the SPAD in rice leaves decreased (Fig.  3 ), this is consistent with the previous studies 39 . On the one hand, this is because Cd accumulates in rice leaves, altering the ultrastructure of chloroplasts, severely damaging the thylakoid membrane and chloroplasts, and leading to a decrease in SPAD 41 . On the other hand, because the peroxidation reaction produces a large amount of hydrogen peroxide (H 2 O 2 ), this enters the chloroplast through the plasma membrane, attacks the chloroplast pigment protein complex, and inhibits the activity of plant chlorophyll ester reductase, and SPAD reduction is caused by factors such as chlorophyll degradation 39 , 42 . The gas exchange parameters (Ci and Tr) are limiting factors for CO 2 diffusion and immobilization, and are related to the activities of CO 2 immobilized enzymes, ribulose diphosphate carboxylase, and oxygenase (RuBisCO) 43 . The toxicity of Cd can be mediated by increasing the carboxylation efficiency of RuBioCO 44 . In this study, the photosynthetic parameters Pn, Gs, Tr, and Ci of rice decreased with increasing Cd concentration (Fig.  4 ). This is consistent with previous reports on Cd inhibiting plant photosynthesis 45 , where low Cd concentrations significantly inhibited plant growth and photosynthesis in rice and mustard 45 , 46 . The inhibition of photosynthesis induced by Cd is usually attributed to the inhibition of key enzyme activities in the Calvin cycle and photosynthetic electron transport chain 47 , and this negative effect can be alleviated through the supply of Si. When Si is sprayed on the leaves, the plant toxicity of Cd is reduced, and the inhibition of Cd on photosynthesis is reduced, thereby improving the performance of photosynthesis. Pn is a determining factor in plant growth 48 . In this study, the increase in Pn value after foliar Si spraying treatment may be attributed to the increase in Gs and Tr, which accelerates the effective carbon assimilation period of rice leaves and thus accelerates the accumulation of photosynthetic products. Therefore, Si can increase the SPAD value of rice leaves and improve photosynthesis 39 .

The changes in chlorophyll fluorescence can reflect biotic or abiotic stress 49 . The decrease in Fv/Fm and Y (II) indicates that the toxicity of Cd inhibits the photoactivation of PSII, which is due to the destruction of antennal pigments, limited electron transfer from PSII to photosystem I (PSI), and disruption of the integrity of the thylakoid membrane structure. The decrease in Fo (Fig.  5 c) means that the potential efficiency of PSII has undergone a negative change, and an decrease in NPQ (Fig.  5 b) indicates an improvement in the efficiency of photochemical reactions. In this experiment, foliar application of Si resulted in more light energy absorbed by rice plants being used for photochemical reactions and energy or carbohydrate synthesis, thereby increasing quantum yield and protecting the photosynthetic system from damage. These findings are also consistent with the results of rice photosynthetic parameters.

Cd does not participate in redox reactions in cells, but can induce the formation of reactive oxygen species (ROS) in plants 50 . Although the increase in ROS synthesis in cells poses a threat to cellular biomolecules, ROS also acts as a signaling molecule, activating stress response and defense-related genes through signaling pathways 51 . In this study, the increase in ROS production level under Cd stress was manifested as an increase in MDA content (Fig.  6 a), a decrease in SPAD (Fig.  3 ), and a decrease in leaf photosynthetic gas exchange (Fig.  4 ). These findings are consistent with previous studies 52 , 53 . In previous research findings, Cd toxicity was found to have a negative impact on various physiological, biochemical, and metabolic processes in plants 2 , 54 . Research has shown that the toxicity of Cd to maize can induce the production of H 2 O 2 and MDA 55 . However, the mediation by Si can reduce the final product of lipid peroxidation, namely the MDA content, which helps to reduce membrane permeability and maintain its integrity 56 . Under Cd toxicity, various enzymatic and non-enzymatic antioxidant defense systems are activated to control the production of ROS. Enzyme antioxidants, including SOD , POD, and catalase (CAT), are another defense system. SOD converts superoxide radicals into H 2 O 2 , which appears in plant tissues as a result of Cd stress. H 2 O 2 is a powerful oxidant that accumulates in plant tissues through SOD channelization reactions. It is blocked by the circulation of ascorbic acid glutathione. In addition to H 2 O 2 , another toxic oxide is heme oxygenase-1 (OH-1), which can react with all large molecules. SOD can prevent the formation of OH-1 in plant tissues 57 . POD can alter ROS levels in plants due to its role in consuming and clearing H 2 O 2 . Unlike SOD, POD has a high affinity for H 2 O 2 . However, POD can convert H 2 O 2 into H 2 O and oxygen (O 2 ) 58 . In this study, Si application significantly increased the activity of POD and SOD (Fig.  6 b and c) ( P  < 0.05). This is consistent with previous research results, which showed that Si application increased SOD activity in wheat and sorghum plants 50 , 54 . Si application increases POD activity in wheat leaves under Cd stress 59 . Similarly , Si treatment reduces the production of ROS and promotes enzymatic and non-enzymatic antioxidants for ROS clearance 60 .

Cd exposure can cause a range of harmful effects on organisms, including humans. Therefore, understanding the mechanisms of Cd uptake, translocation and accumulation in rice is important for strengthening strategies to effectively reduce Cd. In the future, the effect of foliar Si spraying on Cd accumulation at stem nodes and internodes of rice must be further enhanced. It can both efficiently control the transfer of Cd to the critical part of the grain and reduce the Cd contamination of rice in the soil.

This study demonstrated that Cd stress increased the Cd content in rice roots, stems, and leaves, decreased the SPAD of rice leaves and photosynthetic efficiency of rice leaves, inhibited the activities of SOD and POD in rice leaves, increased the MDA content in rice leaves, and inhibited rice growth. After applying Si, the Cd content of brown rice can be reduced, the SPAD of rice leaves can be increased, the photosynthetic characteristics of rice leaves can be improved, the SOD and POD activities of rice leaves can be increased, the MDA content of rice leaves can be reduced, and rice yield can be promoted.

Experimental research and feld studies on plants statement

In the study only cultivated plants were used which are neither endangered nor at risk of extinction. We confrm that their handling was performed in compliance with relevant institution, national and international guidelines and legislation.

Data availability

Data is provided within the manuscript or supplementary information files.

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Acknowledgements

We thank Guizhou University, China for providing the funding and facilities to carry out the experimental work presented in this study.

This study was supported by the National Natural Science Foundation of China (4216070281); Key Laboratory of Molecular Breeding for Grain and Oil Crops in Guizhou Province (Qiankehezhongyindi (2023) 008); Key Laboratory of Functional Agriculture of Guizhou Provincial Higher Education Institutions (Qianjiaoji (2023) 007).

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H.C. (Hongxing Chen) and Z.G. wrote the main manuscript text, X.H. prepared Figs.  1 – 3 , H.C. (Hui Chen) conducted a format analysis, S.Z. and C.F. prepared Figs.  4 – 6 , H.C. (Hongxing Chen), C.F., T.F., T.H. and Z.G. conducted supervision. All authors reviewed the manuscript.

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Chen, H., Huang, X., Chen, H. et al. Effect of silicon spraying on rice photosynthesis and antioxidant defense system on cadmium accumulation. Sci Rep 14 , 15265 (2024). https://doi.org/10.1038/s41598-024-66204-9

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• 1 Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czechia
• 2 Department of Plant Origin Foodstuffs Hygiene and Technology, Faculty of Veterinary Hygiene and Ecology, University of Veterinary Sciences, Brno, Czechia
• 3 Department of Pharmacognosy, Faculty of Pharmacy, Delta University for Science and Technology, Gamasa, Egypt
• 4 School of Pharmacy and Biomolecular Sciences, Royal College of Surgeons in Ireland, Dublin, Ireland
• 5 Archaea Physiology & Biotechnology Group, Department of Functional and Evolutionary Ecology, Universität Wien, Wien, Austria

The bacterial light-dependent energy metabolism can be divided into two types: oxygenic and anoxygenic photosynthesis. Bacterial oxygenic photosynthesis is similar to plants and is characteristic for cyanobacteria. Bacterial anoxygenic photosynthesis is performed by anoxygenic phototrophs, especially green sulfur bacteria (GSB; family Chlorobiaceae ) and purple sulfur bacteria (PSB; family Chromatiaceae ). In anoxygenic photosynthesis, hydrogen sulfide (H 2 S) is used as the main electron donor, which differs from plants or cyanobacteria where water is the main source of electrons. This review mainly focuses on the microbiology of GSB, which may be found in water or soil ecosystems where H 2 S is abundant. GSB oxidize H 2 S to elemental sulfur. GSB possess special structures—chlorosomes—wherein photosynthetic pigments are located. Chlorosomes are vesicles that are surrounded by a lipid monolayer that serve as light-collecting antennas. The carbon source of GSB is carbon dioxide, which is assimilated through the reverse tricarboxylic acid cycle. Our review provides a thorough introduction to the comparative eco-physiology of GSB and discusses selected application possibilities of anoxygenic phototrophs in the fields of environmental management, bioremediation, and biotechnology.

1 Introduction

In bacteria the mechanisms of photosynthesis differ from those of eukaryotes ( Kolber et al., 2000 ). Cyanobacteria are the only bacteria that use oxygenic photosynthesis, in which water acts as an electron donor being oxidized to molecular oxygen (O 2 ) ( Percival and Williams, 2014 ; Sánchez-Baracaldo and Cardona, 2020 ). An advantage is that H 2 O is already abundantly available in many ecosystems, but this H 2 O-dependent photosynthetic reaction is energetically very demanding, because water is a stable compound. However, it also means a large yield of energy can be obtained. These advantages led to a significant expansion of oxygenic phototrophs in radiation events during evolution ( Percival and Williams, 2014 ; Lazar et al., 2022 ). The differences between oxygenic and anoxygenic phototrophs are summarized in Figure 1 .

Figure 1 . Differences between oxygenic and anoxygenic phototrophs.

Both of these groups can be present in soil or freshwater and are Gram-negative bacteria. In anaerobic phototrophic bacteria, different mechanisms for energy conservation are known ( Hanada, 2016 ). Different electron donors can be used, for example molecular hydrogen (H 2 ) or reduced metal ions ( Unden, 2013 ). A specific group of anaerobic organisms are phototrophic sulfur bacteria. These organisms use various reduced forms of sulfur, most often hydrogen sulfide (H 2 S), as an electron donor ( Madigan and Jung, 2009 ; Swingley et al., 2009 ). Anoxygenic sulfur bacteria can be divided into two main groups according to their pigmentation: green sulfur bacteria (GSB) and purple sulfur bacteria (PSB) ( Kushkevych et al., 2021b ). Macroscopic and light microscopic images of cultures of GSB and PSB are shown in Figure 2 .

Figure 2 . Cultures of GSB [ Cbi. limicola (A,B) ] and PSB [ Thiocapsa sp. (C,D) ], with light microscopic images at a magnification of 1,000 times (B,D) . These cultures were enriched and purified in the authors’ lab but have not yet been microbiologically characterized.

GSB and PSB occur lower in the water column compared to oxygenic phototrophs ( Kushkevych et al., 2021a ). Overall, it can be asserted that GSB are adapted to the deeper regions of the water column or soil than PSB ( van Niel, 1932 ; Manske et al., 2005 ). GSB can grow better under low light intensity, exhibit greater sensitivity to oxidizing environments compared to PSB, and, conversely, demonstrate tolerance to higher concentrations of H 2 S ( Hunter et al., 2009 ). Another difference between the two types of sulfur bacteria concerns their way to store sulfur, which can be a product of H 2 S oxidation. H 2 S can be toxic for other group of microbial communities ( Černý et al., 2018 ; Dordević et al., 2020 ; Kushkevych et al., 2020 ). PSB store sulfur in intracellular globules, while GSB deposit sulfur extracellularly ( Woese et al., 1985 ; Bryant and Frigaard, 2006 ; Frigaard and Dahl, 2008 ).

The biotechnological use of anoxygenic phototrophs has only been the topic of few studies ( Struk et al., 2023 ). However, H 2 S is a frequent contaminant of wastewater ( Kushkevych et al., 2017 , 2018a ), whether industrial or municipal, and of natural gas or biogas ( Struk et al., 2019 ). Thus, biotechnological removal of H 2 S through microbial oxidation could be a suitable alternative to physico-chemical cleaning methods ( Struk et al., 2020 ). The product is most often elemental sulfur, which is insoluble in water, so it can be easily separated ( Saeid and Chojnacka, 2014 ). Another interesting feature for putative biotechnological applications is the accumulation of glycogen during photosynthesis, e.g., known from Chlorobium limicola , a member of the GSB. Moreover, the use of captured light energy to generate electrical energy through microbial electrochemical cells is also being considered ( Figueras et al., 2006 ).

This review aims to summarize knowledge about the microbiology and physiology of GSB, especially the genus Chlorobium . First, a general introduction of physiology, phylogeny, and taxonomy is provided. Then the process of anoxygenic photosynthesis from the capture of a light quantum through the transport of an excited electron and the replenishment of electrons from H 2 S or other electron donors is discussed. Finally, examples of practical applications of these anaerobic phototrophs will be presented, especially focusing on H 2 S removal, to evaluate the current status of their biotechnological potential.

2 General characteristics of green sulfur bacteria

2.1 taxonomy.

The initial system of GSB taxonomy was based on morphological features: color (brown or green, or the content of the carotenoids isorenieratene and chlorobactane), the formation of gas pockets and the ability to use thiosulfate as an electron donor for photosynthesis. The phylogenetic trees of GSBs ( Figure 3 ) were constructed and discussed using various literature papers of Overmann (2015) , Imhoff and Thiel (2010) , and Bello et al. (2022) .

Figure 3 . Phylogenetic trees of GSB: panel (A) is constructed using data by Overmann (2015) , panel (B) is created from data by Imhoff and Thiel (2010) , and panel (C) is based on results of Bello et al. (2022) .

A phylogenetic tree based on 16S rRNA gene sequences of GSBs was generated by Overmann and Tuschak (1997) . Overmann (2015) who added Chloroherpethon thalassium to this tree ( Figure 3A ), also compared the tree to other biochemical and cytological traits such as the GC pair content, growth on saline medium, fatty acid composition, carotenoid composition, and gas vesicles presence. Their conclusion was that the GC pair content and fatty acid composition are similar for closely related species based on 16S rRNA gene sequence and therefore might give us information about the phylogeny. However, carotenoids and gas vesicles did not correlate with the results from 16S rRNA gene sequencing ( Overmann, 2015 ).

Imhoff and Thiel (2010) revised the phylogeny of GSB based on the sequences of genes of 16S rRNA and of the FMO protein (gene fmoA ) by taking the evolutionary significance of individual positions into account. An additional criterion was the content of GC pairs ( Imhoff and Thiel, 2010 ). Although GSB are an ecologically defined group, they also belong to the same phylogenetic group, referred to as the Chlorobi . Only one chemotrophic representative, Ignavibacterium album has been found in this phylum.

However, within the class Chlorobia , which could be considered as a synonym for GSB, only phototrophic representatives are known. This class is phylogenetically distant from other bacteria (<83% similarity in the 16S rRNA gene sequence), the mutual relationship of its representatives (>88% similarity in the 16S rRNA gene sequence) corresponds to the fact that it includes a single family: Chlorobiaceae . Within this family, the existence of four genera was confirmed: Chlorobaculum , Chlorobium , Chloroherpethon , and Prosthecochloris . The type species were selected for these genera: Chlorobium limicola (at the same time it is the type species for the entire family), Cba. tepidum , Ptc. aestuarii , and Chp. thalassium . The only studied representative of the genus Chloroherpethon is a phylogenetically basal species of obligate marine bacteria ( Imhoff and Thiel, 2010 ).

Imhoff and Thiel introduced the following changes ( Imhoff and Thiel, 2010 ). Chlorobium limicola f. thiosulfatophilum was reclassified as Cba. thiosulfatophilum (strains with a GC pair content of around 58%) and Cbi. limicola (strains with a GC pair content of around 52%) (In some of the studies cited in their work, it is not possible to distinguish which of these species it is, so the invalid name corresponding to the relevant study has not been consiered). Chlorobium vibriofirme was divided into five species: Cbi. luteolum , Cbi. phaeovibrioides , Ptc. vibrioformis , and Chlorobaculum sp.; Cbi. vibriofirme f. thiosulfatophilum was reclassified as Cba. parvum . Pelodictyon clathratiforme was reclassified as Cbi. clathratifirme ; since it was the type species of the genus Pelodictyon , this means the extinction of this genus and the reclassification of its other species: Pld. luteolum → Cbi. luteolum , Pld. phaeoclathratiforme → Cbi. clathratiforme . Selected strains of Cbi. phaeobacteroides were reclassified as Cbi. limicola or as Cba. limnaeum sp. nov. Chlorobium tepidum was reclassified as Chlorobaculum tepidum . Chlorobium chlorovibrioides was reclassified as Cba. Chlorovibrioides . Figure 3B shows the phylogenetic relationships of selected described species. Several different isolates from some species were examined, all mentioned species appeared as monophyletic in the analysis. Three representatives of the genus Flexibacter were used as an outgroup ( Imhoff and Thiel, 2010 ). In Figure 3C , an updated phylogenetic tree of 282 conserved proteins is shown. This phylogenetic tree was then compared with phylogenetic trees constructed according to DNA gyrases A and B ( GyrA , GyrB ), DNA polymerase A (PolA), DNA helicase UvrD and 16S rRNA. Several members of the Bacteroidetes tribe were used as an outgroup. Furthermore, suitable sequences for the determination of GSB, the so-called conservative signature indels (CSIs), were selected. Chlorobiaceae were confirmed to be uniformly non-motile, anaerobic, photoautotrophic bacteria, using reduced forms of sulfur as electron donors for photosynthesis; in the genus Chloroherpethon it is bioinformatically predicted that the organisms ought to grow photoheterotrophically. Moreover, it lacks the genes of the DSR and Sox groups. Unexpectedly, the study recognizes Pld. phaeoclathratiforme as a valid name, even though it phylogenetically classifies this species within the genus Chlorobium . The phylogenetic relationships of some species are slightly different compared to the previous study, for example the relationship of Cba. limnaeum , Cba. tepidum , and Cba. thiosulphatophilum , or the position of Cbi. limicola within the tree. However, the described species and genera remain valid. Probably the biggest change introduced in their study is the allocation of the genus Chloroherpethon to the new family Chloroherpethonaceae together with the new genus Candidatus thermochlorobacter ( Bello et al., 2022 ).

Based on the sequence similarity of the operational genes, the Bacteroidetes phylum was identified as the closest relative of the phylum Chlorobi . An unusually high number of proteins (about 12%) are similar to archaeal proteins. Since Chlorobi are considered one of the basal groups within bacteria, they may have retained genes from the common ancestor of bacteria and archaea that phylogenetically later evolved groups have lost. Another possibility is the horizontal transfer of these genes from archaea ( Eisen et al., 2002 ).

2.2 Genomic studies

The thermophilic GSB Cba. tepidum was the first GSB with a sequenced genome harboring a genome size is 2,154,946 bp ( Bryant, 2019 ). The organism is predicted to harbor 20 genes for regulation of transcription and 19 genes encode for aminoacyl-tRNA synthetases ( Rubio Gomez and Ibba, 2020 ). The gene for the asparagine synthetase is missing and the gene for the glutamine synthetase is encoded but the holoenzyme is putative nonfunctional due to the absence of another necessary enzyme. Cba. tepidum probably uses aspartate and glutamate instead of asparagine and glutamine and converts them to the corresponding amides posttranslationally. A gene for the enzyme GatABC, which could transfer the amide group from glutamine, was found ( Eisen et al., 2002 ), which indicated that this gene is essential for the amino acid metabolism of this organism. Although GSB are found in reducing and light-reduced environments, where DNA damage does not occur very often, Cba. tepidum has several DNA repair mechanisms: two UvrA endonucleases, DNA polymerase B, and a class II photolyase homolog. Genes for antioxidant enzymes are also present: superoxide dismutase, rubredoxin, oxygen oxidoreductase, and cytochrome bd quinol oxidase. Enzymes for the assimilation of organic substances are generally absent; only one such operon was found with genes that could enable import, phosphorylation and further processing of maltose and other maltooligosaccharides. Some components of the phosphotransferase system were also found, but incomplete; these may rather have a regulatory function. The usual source of nitrogen is NH 4 + , but genes for molecular nitrogen (N 2 ) fixation have also been detected. If this species is able to fix N 2 it might assist them to thrive in habitats that lack other forms of nitrogen. Furthermore, many genes for the transport of various metal ions and maintenance of their concentration, specifically six homologs of the ArsA enzyme, for ATP-dependent arsenite out-port have been identified ( Eisen et al., 2002 ).

In a study by Davenport et al. (2010) , comparative genomics of several species of all four genera of GSB were examined ( Eisen et al., 2002 ). All genomes consisted of a single circular DNA molecule, which was 1.9–3.3 Mbp long. The GC pairs constituted a median of 50% of the content, with 87% of sequences being coding, aligning with the typical characteristics observed in bacteria. However, the share of regulatory sequences and therefore the size of the genome is smaller than in Firmicutes or Pseudomonadota . Moreover, CRISPR elements were detected. The study also dealt with pseudogenes created by displacement mutation. Due to the high content of coding sequences, not many pseudogenes were found. Furthermore, a map of tetranucleotide use and frequent 8–14 oligomers was constructed. Adhesins were among the largest genes that were identified. The arrangement of genes on the chromosome is not conserved except for closely related taxa ( Davenport et al., 2010 ).

In another study, the genomes of populations of GSB from various locations in Europe and North America were examined. A total of 509 genomes were obtained. Based on average nucleotide identity (ANI), the genomes were arranged into 71 metagenomic operational taxonomic units and a phylogenetic tree was constructed. As expected, the presence of genes for glycolysis, gluconeogenesis, the reverse tricarboxylic acid cycle, and the synthesis of chlorophylls and bacteriochlorophylls was conserved. Nitrogenase genes were highly prevalent as well, especially NifH (nitrogenase iron protein). Several genes for H 2 S oxidation and for other photosynthetic electron donors were found. Sometimes more than one of these genes was present per genome. The vast majority of sulfur related genes that were found are sulfide: quinone reductases, reverse dissimilation reductases of sulfite and flavocytochrome c . Moreover, thiosulfohydrolases were present only in a small part of units from the genus Chlorobium . They were far more represented in the genus Chlorobaculum . Hydrogenases were also detected, however the ecological significance of H 2 oxidation in GSB has generally not yet received much attention. Homologs of cyc2 , which encodes cytochrome c —capable of oxidizing iron ions—were unexpectedly frequent in the genus Chlorobium . It is therefore possible that GSB play a significant role in the geobiochemical cycle of iron. Other putatively confirmed pathways were the pentose phosphate and ornithine cycles ( Garcia and Kim, 2021 ).

2.3 Cell structure and chlorosomes

The cells of different GSB possess different morphologies: they appear as cocci (often in chains), rods, curved or spiral cells. Some species produce gas vesicles. The genus Chloroherpethon has a special structure: the cells are shaped like long flexible fibers and can move by gliding. Other GSB are listed as immobile ( van Niel, 1932 ; Overmann, 2015 ). Some representatives of GSB, such as Cbi. thiosulfatophilum , are able to form polyphosphate granulae. Up to three electron-dense granules per cell were observed using electron microscopy. The formation of polyphosphate granules was monitored during growth. In the lag phase, polyphosphate was rather consumed. During the exponential and stationary phases, polyphosphate was stored ( Hughes et al., 1963 ).

Green sulfur bacteria of the genus Prosthecochloris may form appendages, which are termed prosthecae. Prosthecae are protrusions of the cytoplasmic membrane that provide a larger area for photosynthetic processes. Unlike prosthecae of some heterotrophs, which help them to adapt to an oligotrophic environment, the formation of prosthecae is light-dependent: when the light flux is low, cells produce more prosthecae to capture more photons. The morphological changes of Prosthecochloris aestuarii depending on illumination were studied. These changes were observed using scanning and transmission electron microscopy. At a light quanta flux intensity of 0.5 μmol m −2 s −1 , the average cell length was 900 nm and the average diameter was 231.8 nm; at an intensity of 100 μmol m −2 s −1 , the average cell length was 1,300 nm, but the average diameter was only 98 nm ( Guyoneaud et al., 2001 ).

Chlorosomes are an unusual type of light-harvesting antennae that are found only in GSB and green non-sulfur bacteria ( Chloroflexi ) ( Chen et al., 2020 ). Chlorosomes are the most efficient light-harvesting complexes known, capable of working even at very low light intensity (less than 4 μEinst m −2 s −1 ), which enables to thrive deep in the water column or in sediments ( Overmann et al., 1992a ). Chlorosomes have a unique organization: instead of interactions between proteins and pigments, they are held together by interactions directly between pigment molecules, which, in addition, are assembled into supramolecular units by themselves without any protein stabilization. Chlorosomes are quite large since they contain up to hundreds of thousands of bacteriochlorophyll molecules, have an ellipsoidal shape with the longest diameter up to 1,800 Å and the shortest up to 500 Å. They are found attached beneath the cytoplasmic membrane. In Ptc. aestuarii the density of chlorosomes and the ratio of the membrane perimeter to its area reached the highest values at mean light intensity of 5 μmol m −2 s −1 . The main pigments in Ptc. aestuarii were identified using HPLC as bacteriochlorophyll c , chlorobactane and hydroxychlorobactane. The specific content of bacteriochlorophyll c and carotenoids increased with decreasing light intensity as well as the development of new prosthecae in order to provide sufficient rate of photosynthesis. The ratio of the content of bacteriochlorophyll c to carotenoids had a value of around 12 at light intensity of 2.5 μmol m −2 s −1 and less, and increased up to fourfold at higher intensities to avoid photodamage of chlorosomes ( Guyoneaud et al., 2001 ).

The internal organization of chlorosomes of Cba. tepidum was studied using X-ray scattering and electron microscopy ( Pšenčík et al., 2004 ). This study disproved the theory that pigments are organized into rods: such rods would have to be compressed to an unimaginable density. Instead, a lamellar model of pigment organization was created. The results indicate that the lamellae wave is perpendicular to the longest axis of the chlorosome. The rod-like elements, previously observed with electron microscopes, may have been created by splitting the lamellae along these waves during the sample preparation. The lamellae consist of bacteriochlorophylls and the hydrophobic space between them is filled by carotenoids. There are various hypotheses regarding the particular relative arrangement of the pigment molecules; X-ray crystallography is usually used to evaluate the structure, but it is not suitable for chlorosomes due to their size and diversity. The chlorosomes are attached to the cytoplasmic membrane via the basal plate which contains bacteriochlorophyll a , bound by the CsmA protein into a paracrystalline structure. The basal plate allows light-excited electrons to leave the chlorosome and be transferred to the photosynthetic reaction center. The rest of the chlorosome is enveloped in a lipid monolayer with proteins ( Pšenčík et al., 2004 ). The structure of chlorosomes can be influenced by the environment. The influence of the carbon source (bicarbonate alone, with acetate or with pyruvate) and temperature (30°C, 50°C, increase from 30 to 50°C, decrease from 50 to 30°C) on growth of Cba. tepidum cultures was monitored. In the case of the growth of bicarbonate with pyruvate and at decreasing temperatures, the slowest growth was observed, accompanied by the reduction of chlorosomes. A further study of these chlorosomes showed more frequent ethylation of positions 8 and 12 of bacteriochlorophyll c (which leads to blue-shift of absorption maxima) or a smaller number of reaction centers in the chlorosome (15–25 compared to the usual 25–45). Under stress conditions, cells likely lack the energy to synthesize complete chlorosomes ( Tang et al., 2013 ). Later, surface-rendered representation and intracellular chlorosome organization of Cba. tepidum cells was performed using cryo-electron tomography to reveal the distribution of chlorosomes in 3D in an unperturbed cell. Furthermore, the connecting elements between chlorosomes and the cytoplasmic membrane as well as the distribution of reaction centers in the cytoplasmic membrane has been revealed ( Kudryashev et al., 2014 ).

2.4 Pigments

The main pigments of the light-collecting antennas in chlorosomes are bacteriochlorophylls c , d or e . These are the so-called chlorobial bacteriochlorophylls ( Figure 4 ), known only in organisms bearing chlorosomes, i.e., in GSB and members of the Chloroflexi phylum. GSB usually contain only one of these pigments. Figure 4A shows the structure of these pigments. R 8 is ethyl, propyl, isobutyl or neopentyl, R 12 is methyl or ethyl. The differences between them are these:

• bacteriochlorophyll c : R 7  = –CH 3 , R 20  = –CH 3

• bacteriochlorophyll d : R 7  = –CH 3 , R 20  = –H

• bacteriochlorophyll e : R 7  = –CHO, R 20  = –CH 3

• bacteriochlorophyll f (not yet observed): R 7  = –CHO, R 20  = –H

Figure 4 . Chemical structure of the chlorobial bacteriochlorophylls: panel (A) indicates bacteriochlorophylls c , d , e , and f , and panel (B) shows bacteriochlorophyll a .

Otte et al. (1993) studied bacteriochlorophylls in Ptc. vibrioformis (strain 6030, DSM 260 T ) and Cbi. phaeovibrioides (strain 2631). Bacteriochlorophyll c and bacteriochlorophyll d were found in Ptc. vibrioformis . Cbi. phaeovibrioides was found to possess bacteriochlorophyll e . Only 1–2% of the pigments are bacteriochlorophyll a ( Figure 4B ). Its primary function is not the capture of the light quantum, but the transport of the captured energy through the basal plate (see chapter 2.1) ( Orf and Blankenship, 2013 ).

After the release of the Cba. tepidum genome, orthologs of all genes for the synthesis of bacteriochlorophyll a from protoporphyrin IX were found, which shows that this species is not auxotrophic for this compounds ( Eisen et al., 2002 ). In the synthesis of bacteriochlorophyll c , only three of the enzymes that are also part of the pathway for the synthesis of bacteriochlorophyll a are apparently used: magnesium chelatase, vinyl reductase, and protochlorophyllide reductase, whereof the latter three variants are known. The remaining genes for the bacteriochlorophyll c synthetic pathway were identified as paralogs of various other genes for tetrapyrrole metabolism. Two genes for cobalt chelatases and other genes for enzymes that should be able to synthesize vitamin B 12 were also found. In laboratory conditions, however, this vitamin is usually added to the growth medium as a source for the tetrapyrrole ring. A metaproteo-genomic study of a GSB from Antarctica showed that a complete synthetic pathway for glutamate-derived tetrapyrroles that are present in this bacterium ( Ng et al., 2010 ).

At reduced light intensity, alkylation of bacteriochlorophyll in positions 18 and 20 is increased, which leads to a shift of the absorbance to higher wavelengths. The carbon source for this alkylation is S-adenosylmethionine. Five genes for proteins from the BchE/P-methylase family, which are possible catalysts of this alkylation, were found in the genome of Cba. tepidum ( Eisen et al., 2002 ).

In addition to tetrapyrroles, GSB also contain carotenoids. These have the highest absorbance in wavelengths 400–550 nm and transmit the captured energy toward the photosynthetic reaction center. They also protect the cell from the harmful effects of radiation and free radicals ( Frank and Cogdell, 1996 ; Maresca et al., 2008 ). Furthermore, carotenoids can have a structural function. More than 90% of carotenoids in Cba. tepidum cells are located in chlorosomes. Chlorobaculum limnaeum , a brown colored representative is adapted to lower light intensity, mainly contains isorenieratene or β-isorenieratene. In Cba. tepidum , the most abundant carotenoid is chlorobactane, or its derivatives, the structure of which is shown in Figure 5 : 1′,2′-dihydrochlorobactane, hydroxychlorobactane, hydroxychlorobactane glucoside, and hydroxychlorobactane glucoside laurate ( Ng et al., 2010 ). The biosynthesis of carotenoids was also described in a GSB that has been isolated from Antarctica. Their precursors, isopentenyl diphosphate, and dimethylallyl diphosphate, are formed in the MEP pathway (also known as the methylerythritol phosphate pathway or the non-mevalonate pathway) from pyruvate and glyceraldehyde-3-phosphate. Other intermediates are geranyl diphosphate, farnesyl diphosphate, geranylgeranyl diphosphate, phytoene, ζ-carotene, lycopene and γ-carotene, precursor of the most important carotenoid chlorobactane ( Figure 5 ) and its derivatives with different R groups: –H (1′,2′-dihydrochlorobactane), –OH (hydroxychlorobactane); glucose can also be attached to the hydroxyl group, with or without laurate. Chlorobactane is also a precursor of β-isorenieratene, from which isorenieratene can be formed ( Figure 5 ). These two pigments are only present in brown members of the class Chlorobia , adapted to lower light intensity and higher wavelengths. It is not without interest that the conversion of γ-carotene to chlorobactane is catalyzed by the same enzyme (CruB) as the conversion of β-isorenieratene to isorenieratene—chemically it is a reaction of an identical functional group ( Ng et al., 2010 ).

Figure 5 . Chemical structures of some carotenoids that may occur in GSB.

3 Photosynthesis and carbon assimilation

3.1 light-dependent processes.

The capture of the light quantum and excitation of the electron occur on bacteriochlorophyll c , d , or e molecules, which are deposited in the chlorosome. The excited electron relaxes back with simultaneous excitation of another chlorobial bacteriochlorophyll molecule. These molecules transfer the energy to the basal plate to bacteriochlorophyll a -795 molecules. It then moves on to the FMO complex, a designation derived from the names of the scientists Fenna, Matthews, and Olson. It is a complex of a pigment (bacteriochlorophyll a ) and a protein with an absorption maximum at 808 nm. The energy is transferred from the FMO complex to the reaction center ( Hauska et al., 2001 ).

The FMO protein is a trimer, each of its subunits binds seven molecules of bacteriochlorophyll a . It is water soluble, which is unusual for protein complexes with bacteriochlorophylls. It is only found in GSB. The efficiency of energy transfer from chlorosomal carotenoids to the reaction center in Cba. tepidum was 23%; the major limiting step was the transfer from the FMO complex to the reaction center with the efficiency of 35%. Based on sequence similarity, it was suggested that the FMO protein is evolutionionary related to the reaction center protein PscA ( Olson, 2004 ). It has already been shown how the FMO trimer associates with the photosynthetic reaction center ( Chen et al., 2020 ). The reaction center is considered to be a homolog of photosystem I (PS I) in oxygenic phototrophs, but PS I is a heterodimer, whereas the reaction center of GSB is a homodimer with protein subunit composition [(FMO) 3 (PscA) 2 PscBCD] 2 ( Figure 6 ), containing eight molecules of bacteriochlorophyll a and two molecules of chlorophyll a for each molecule of PscA ( Hauska et al., 2001 ). The reaction center of GSB has a homodimeric architecture with a symmetric distribution of pigments, suggesting two identical branches of electron transport chains ( Gorka et al., 2021 ). The photosystem P840 is formed by a pair of bacteriochlorophyll a molecules. The excitation energy generally comes to it from bacteriochlorophyll a -837. Excited P840 is capable of charge separation: as the primary donor, it passes an electron to the carrier A 0 (primary acceptor) and acquires a positive charge itself. Energy transfer to P840 and charge separation takes 25 ps; it is not clear whether the limiting step is the transfer of energy from bacteriochlorophyll a -837 to P840 or charge separation. The electron is then transferred to the secondary acceptor A 1 , then to the Fe–S center F X , and finally to PscB with the Fe–S centers F A and F B . All of these three Fe-S centers can reduce ferredoxin, which can further reduce various metabolites, either directly or with an intermediate electron carrier such as NADH, NADPH, or FADH 2 . The sequence of the electron transport chain is schematically illustrated in Figure 6 , including indicative values for the electrochemical potential of each compound and the times of individual reactions (or of groups of consecutive reactions in cases where partial reaction times could not be determined). The A 0 carrier in GSB has been identified as chlorophyll a -670 ( Hauska et al., 2001 ).

Figure 6 . Electron transport chain: P840 is photosystem with maximum absorbance at 840 nm, A 0 , A 1 means carriers of electrons, F X , F A , and F B means iron–sulfur clusters, Fd means ferredoxins, and Cyt is cytochrome.

Baymann et al. (2001) reported that chlorophyll has a higher reducing power than bacteriochlorophyll and thus contributes to efficient electron transport toward the Fe-S centers with lower potential. The A 1 carrier is phylloquinone. In other groups of phototrophic organisms, other molecules with the same function can be found. The localization of the individual members of the electron transport chain in the reaction center ( Figure 7 ) is following: the PscC protein binds to cytochrome c -551, the PscA protein binds to P840, A 0 , A 1 and F X , and the PscB protein binds to F A and F B .

Figure 7 . Schematic illustration of the photosyntetic reaction center of GSB: FMO is the abbreviation for Fenna, Matthews, and Olson complex, Psc depicts photosynthetic center proteins.

The description above refers to non-cyclic electron transport. Cyclic electron transport has only been predicted to date. The possible mechanism is the following: phyloquinone is sometimes reduced to phylloquinol, which may reduce menaquinone to menaquinol. Menaquinol can be reoxidized by Rieske Fe–S cluster, from which are the electrons transferred to cytochrome c -551 and back to P840 in the reaction center. This mechanism can only work if phylloquinol is mobile ( Hauska et al., 2001 ).

The structure of the intact reaction center-FMO apparatus from Cba. tepidum became recently available from cryogenic electron microscopy at a resolution of 2.5 Å ( Chen et al., 2020 ). Furthermore, the photosynthetic supercomplex consisting of the reaction center proteins and FMO was purified from Cba. tepidum and its high-resolution structure has been determined using single-particle cryogenic electron microscopy ( Puskar et al., 2022 ). The purified supercomplexes revealed different stoichiometries of reaction center and FMO proteins. Moreover, the cryogenic electron microscopy reconstructions suggest that the reaction center can host at most two FMO trimer complexes on its cytoplasmic surface ( Puskar et al., 2022 ). A cryogenic electron microscopy-based reconstruction of the photosynthetic supercomplex from Cba. tepidum is shown in Figure 8 .

Figure 8 . Reconstruction models of the reaction center-FMO 2 photosynthetic supercomplex of Cba. tepidium . Three-dimensional cryogenic electron microscopy density map of the reaction center-FMO 2 assembly. The color codes are: FMO1—dark green; FMO2—forest green; PscA1—blue; PscA2—light blue; PscB—yellow; PscC—light pink; PscD—purple; PscE—magenta; and PscF—cyan. A 6 Å −1 -filtered surface envelope (1.0σ) is overlaid over the density of the protein supercomplex (3.6σ). Horizontal dashed lines (orange) indicate membrane boundaries. The text was adapted from and the image was taken from Puskar et al. (2022) .

Recently the asymmetrical arrangement of the two FMO trimers has been revealed ( Xie et al., 2023 ). A model of the asymmetrical arrangement of the two FMO complexes above the reaction center is shown in Figure 9 .

Figure 9 . The reaction center [two PscA subunits are related by a 2-fold symmetry axis (blue line) (C2)] perpendicular to the membrane plane. The threefold symmetry axes (C3) of the first and second FMO trimer are indicated as green and purple lines, respectively. From this side view, the C3 axes of the first and second FMO trimer form a 3.5° and 3.0° angle on the cytoplasmic membrane normal. Text modified from and figure taken from Xie et al. (2023) .

Ferredoxin was successfully reoxidized by ferredoxin-NADP + reductase from spinach. However, genes for such a reductase were not found in Cba. tepidum , so the mechanism of its reoxidation was further investigated. Seo and Sakurai successfully isolated ferredoxin-NAD(P) + reductase from Cba. tepidum and studied its activity ( Seo and Sakurai, 2002 ). It was able to reduce the artificial electron acceptor DPIP (2,6-dichlorophenol-indophenol) using both NADP + and NAD + . At carrier concentrations below 0.5 mmol L −1 , it was more active with NADP + , but at higher concentrations, it was more active with NAD + ; thus, high concentrations of NADP + appear to inhibit the reaction. Based on the sequence of the first 25 N-terminal amino acids, the corresponding ORF was found in the genome. The reductase was named TRLP (thioredoxin reductase-like protein) based on its sequence similarity to thioredoxin reductases from various bacteria (e.g., Escherichia coli , Rickettsia prowazekii , Bacillus subtilis , and Bacillus halodurans ; Seo and Sakurai, 2002 ).

It has been proposed that GSB use a ferredoxin: NAD + oxidoreductase of the RNF type, which works with the energy of the membrane gradient of Na + concentration. Such enzymes are known from various heterotrophic bacteria, for example in connection with anaerobic fermentation (gradient generation) or with nitrogen assimilation and synthesis of Fe–S centers (gradient consumption). When the gradient is consumed, flavodoxin, which is a variant of ferredoxin with a reduced potential, tends to be reduced. In this study, the complete operon carrying rnf genes was found in some GSB, e.g., Cbi. limicola , Cbi. luteolum , Cbi. phaeovibrioides , and Chp. thalassium , Prosthecochloris spp., most of them being marine representatives. The operon showed 46–81% identity with the corresponding operon in the heterotrophic bacterium Acetobacterium woodii , known for having RNF enzymes. On the contrary, this operon was not found at all in the GSB Cba. tepidum , Cba. limnaeum , and others. Transcription of the mentioned operon was confirmed by RT-PCR. Thanks to a sufficient level of transcription, it was possible to observe the activity of RNF directly during ferredoxin (or, respectively, its low-potential form—flavodoxin) oxidation and NAD + reduction. Previous experiments confirmed that this event is light-dependent. In addition to RNF, a cytoplasmic ferredoxin:NAD + oxidoreductase, independent of the membrane gradient of Na + concentration, was found in Cbi. phaeovibrioides . In Cba. limnaeum , only this cytoplasmic form was found. The gene coding is not part of the rnf operon. RNF ensures a high representation of reduced ferredoxin compared to its oxidized form, which leads to increased oxidative stress in aerobic environments. Other enzymes also use the energy of the membrane gradient of Na + . In Cbi. phaeovibrioides , this is the case of oxaloacetate decarboxylase, methylmalonyl-CoA decarboxylase and pyrophosphatase. The membrane gradient of Na + can also be used for transport processes. If a Na + gradient can be used to drive ATP synthesis in these organisms is not yet known. However, it is possible that the membrane gradient of Na + helps to conserve energy and cover fluctuations in light intensity during the day ( Bertsova et al., 2019 ).

Baymann et al. (2001) studied the evolutionary relationship between the reaction centers of different phototrophs. Cyanobacteria and photosynthetic eukaryotes, which are equipped with plastids derived from cyanobacteria have two different reaction centers: photosystem I and II ( Baymann et al., 2001 ). The reaction centers of other phototrophic prokaryotes can be divided into type I (analogous to photosystem I) and type II (analogous to photosystem II). Besides cyanobacteria, type I reaction centers can be found in GSB and heliobacteria, and type II reaction centers in PSB and green filamentous bacteria ( Chloroflexi ). Type I reaction centers are generally homodimeric with two domains (PscA in GSB); the exception is the heterodimeric photosystem I with one domain PsaA and the other PsaB. Another common feature of these reaction centers is that they consist of an outer (antenna) and an inner domain. Electron transport occurs through chlorophyll a and phylloquinone to the Fe–S centers, the first of which F X lies on the axis of symmetry, is present in a single copy and is followed by F A and F B . Consequently, the electron is transferred to ferredoxin. Based on multiple sequence attachments for selected helices of the antennal and nuclear domains of the reaction centers, it appears that the reaction center of GSB is the most evolutionary distant of all others, including the type II reaction centers. This is consistent with the phylogenetic remoteness of the Chlorobi phylum from other bacteria ( Gupta, 2004 ; Gupta and Lorenzini, 2007 ). It can be speculated that the two different reaction center variants arose by gene duplication in an ancestor close to the separation of a group of heliobacteria from the others and subsequent divergent evolution ( Sattley and Swingley, 2013 ), with each group of phototrophic bacteria except cyanobacteria losing one of them. Another possibility is that the genes for type II reaction centers have been transferred laterally between groups, but the exact direction of transfer remains unresolved ( Baymann et al., 2001 ).

3.2 Sulfur metabolism

The main electron donor for GSB is H 2 S, which is oxidized to elemental sulfur ( Holkenbrink et al., 2011 ). The ability to oxidize sulfur to sulfate by some GSB was also reported. Besides that, Cbi. limicola f. thiosulfatophilum and Cba. parvum also oxidize thiosulfate and tetrathionate ( Di Nezio et al., 2021 ; Han et al., 2022 ). These bacterial species also have an unique ability of photochemical sulfur disporportionation: they break down sulfur into H 2 S and sulfite in the presence of light and without access to carbon dioxide (CO 2 ), then the resulting sulfite and part of the H 2 S synproportionate to form thiosulfate ( Van Vliet et al., 2021 ). Thiosulfate was even observed in these organisms as an intermediate in the oxidation of H 2 S to sulfur. The use of sulfite as an electron donor has not been described in GSB. It is considered that polysulfides, e.g., S 3 2− , are also formed during the oxidation of H 2 S ( Brune, 1989 ).

Various enzymatic options for sulfur oxidation are known among the GSB. However, none of GSB harbor all these sulfur metabolization pathways. The basic enzyme for H 2 S oxidation in GSB is sulfide:quinone oxidoreductase (SQR). Based on their structure, these enzymes can be divided into six classes (I–VI). The SQR in GSB usually belong to classes IV and VI, sometimes also to classes III or V. Using H 2 S, they reduce quinones, in the case of GSB it is menaquinone, which is reduced to menaquinol. Another enzyme capable of oxidizing sulfide is flavocytochrome c , which transfers electrons to cytochrome c . Flavocytochrome c has two subunits, FccA (type c cytochrome) and FccB (flavoprotein). They can also use DSR (dissimilatory sulfite reduction) systems, which use menaquinone as an electron acceptor. DSR systems are also known to occur in sulfate-reducing bacteria, where they play a crucial role in directing reduction reactions. Based on the comparison of the phylogeny of the studied GSB according to rRNA and DSR systems, it can be concluded that the genes for DSR were obtained by horizontal gene transfer—either from other sulfur-oxidizing bacteria or from sulfate-reducing bacteria. Finally, the Sox enzyme system can be used for sulfur oxidation. It was originally described in Paracoccus pantotrophus , but its homologs are present in all thiosulfate-oxidizing GSB. One of its components, the SoxCD complex, is absent in GSB, therefore the DSR system is also involved in the oxidation of thiosulfate by the Sox system. Electrons are transferred from the Sox system to cytochrome c ( Gregersen et al., 2011 ).

Menaquinol, which is produced by the SQR and DSR systems, is reoxidized to menaquinone by Rieske’s Fe–S cluster, which transfers electrons to cytochrome c . From cytochrome c , electrons are returned to the reaction center at P840 ( Hauska et al., 2001 ). In order to use extracellular sulfur globules as an electron donor, close contact of cells and globules is usually required. Chlorobaculum tepidum cells were observed to move toward sulfur globules, attaching to the globules, and detaching from the globules. Based on the comparison with the same culture in the dark and with a terminated culture, it was concluded that Cba. tepidum is motile. Genetic analysis determined that type IV pili are responsible for its motility. In a phase contrast microscope, it was further observed that the globules do not have to form attached to the cells, they most often formed at a distance of around 4 μm, the largest observed distance was 8 μm. The formation rate of the globules did not depend on the distance from the cell. The globule size was typically 0.5–1 μm. Based on observations using a scanning electron microscope, the globules were smooth and round, only slightly deformed when in contact with the cells. There were fewer globules in suspension than cells, so more cells are thought to contribute to the formation of one globule. Cells also divide during cultivation, while globules tend to grow. By having the condensation core located outside the cell, it is ensured that the cell is not surrounded by sulfur ( Marnocha et al., 2016 ).

During the formation and degradation of globules, the presence of polysulfides was recorded, which are apparently an important intermediate in the metabolism of elemental sulfur ( Loka Bharathi, 2008 ; Kushkevych et al., 2018a , b ). Polysulfides are produced in the periplasm (in case of Fcc, SQR, and Sox systems) or in cytoplasm (when using DSR systems). The mechanism of their export through membranes remains still unknown ( Gregersen et al., 2011 ; Marnocha et al., 2016 ). Polysulfides can form cycles, which is consistent with the detected presence of cyclic S 8 molecules in biogenic sulfur. These cycles aggregate rapidly, the critical size of the condensation nucleus is only 30 nm. Contrary to original assumptions, it was observed that cell growth and consumption of sulfur globules are not dependent on close cell-globule contact. Some cells grew without being in contact with the globules and some globules degraded even without cell contact. One plausible explanation is as follows: cells that adhere to globules break them down into polysulfides. They import part of the polysulfides and use them as electron donors, but some of them diffuse. These polysulfides can serve as a substrate for cells that are not attached to the sulfur globules ( Maki, 2013 ). However, at least part of the cells must be attached to the globules: if the cells and globules are separated by a membrane, the cells cannot grow on this sulfur. Extracellular storage of sulfur globules enables the sharing of sulfur between different cells as well as species of sulfur-oxidizing bacteria ( Overmann and van Gemerden, 2000 ). Sulfur in this form is also more available to sulfur-reducing bacteria, which convert it back to H 2 S, the preferred electron donor for GSB ( Jørgensen and Nelson, 2004 ; Marnocha et al., 2016 ).

3.3 Metabolic pathways used for carbon assimilation

The reductive pentose phosphate cycle, also known as the Calvin–Benson–Bassham (CBB), or as Calvin cycle, was described as the first CO 2 fixation pathway that allows phototrophic organisms to assimilate CO 2 . The CBB cycle occurs in aerobic phototrophic, autotrophic organisms. Evans et al. described a completely different pathway for CO 2 fixation that occurs was discovered in the GSB Cbi. thiosulfatophilum . Experimentally, Na 2 14 CO 3 was added to the Cbi. thiosulfatophilum culture. Then, the cell suspension was gradually removed over time, then it was dissolved in ethanol (80% final concentration) and analyzed by two-dimensional paper chromatography and radioautography. The content of labeled glutamate, glutamine, aspartate, succinate and phosphoric acid esters was evaluated. Similar tests (extended to include other intermediates of the reductive TCA cycle) were also performed with cell extract and with isolated enzymes that should participate in the cycle. The detected labeled metabolites allowed the conclusion that CO 2 is processed in another aerobic CO 2 fixation pathway. This cycle is now referred to as reductive citric acid cycle [sometimes also referred to as the reductive tricarboxylic acid (TCA) cycle or Arnon-Buchanon cycle]. Figure 10 illustrates the reductive TCA cycle ( Evans et al., 1966 ). The cycle starts and ends with oxaloacetate. In the first part of the cycle, operating from oxaloacetate to citrate, 2 mol CO 2 are fixed and 1 ATP is used. With the consumption of one additional ATP and coenzyme A, citrate is further converted to oxaloacetate and acetyl-CoA. The latter may be carboxylated twice more: first into pyruvate, then into oxaloacetate with the consumption of another ATP. However, there is also the possibility for a carboxylation of pyruvate to oxaloacetate using ATP (not shown in Figure 10 ).

Figure 10 . Schematic representation of the reductive tricarboxylic acid cycle.

Thus, during 1 cycle, four molecules of CO 2 are fixed and two molecules of oxaloacetate are formed from one molecule of oxaloacetate, while one NADH, one FADH 2 , one NADPH, and two ATP are consumed. Intermediates of the mentioned cycle enter other anabolic pathways. For example, the proteogenic amino acid aspartate may be produced from oxaloacetate, glutamate from α-ketoglutarate, and alanine from pyruvate. The carboxylation of acetyl-CoA to pyruvate and the carboxylation of succinyl-CoA to α-ketoglutarate deserve special attention. Analogous reactions in the Krebs cycle are irreversible ( Buchanan and Arnon, 1990 ). The reductive TCA acid cycle ( Kumari, 2018 ) uses different enzymes and different co-factors: the fixation of CO 2 requires the reduced form of ferredoxin ( Li et al., 2021 ).

ATP citrate lyase (EC 4.1.3.8) catalyzes the conversion of citrate to oxaloacetate and acetyl-CoA in the reverse TCA cycle. This enzyme from Cbi. limicola underwent partial purification. It was observed that the utilization of substrates and the generation of products occurred in a balanced manner, and citrate breakdown followed the si-type mechanism. The enzyme activity was hindered by ADP and oxaloacetate, with the latter also impeding the growth of Cbi. limicola ( Antranikian et al., 1982 ).

Later, a functional ribulose-1,5-diphosphate carboxylase was isolated from Cbi. limicola , upon which the reductive TCA cycle was contradicted. Catalysis of the reaction between ribulose-1,5-diphosphate and 14 C labeled sodium bicarbonate was monitored in Tris–HCl and MgCl 2 . In contrast to the study by Evans et al. the assimilation was evaluated in a shorter time (first point already after 5 s) and it was found that the first labeled product is 3-phosphoglycerate. In addition, citrate ATP lyase, one of the enzymes of the reductive TCA cycle, could not be detected. These results would be more consistent with the use of the Calvin cycle ( Sharma et al., 2020 ) rather than the reductive TCA cycle. The relative molecular weight of the studied enzyme was calculated as 3.61·10 5 based on sedimentation comparison with other known proteins. The quaternary structure was evaluated by SDS-PAGE; thus, a single fraction with a relative molecular weight of 53,000 was obtained, leading to the conclusion that the enzyme has six subunits. Unlike other ribulose-1,5-diphosphate carboxylases, it does not contain any smaller subunit ( Tabita et al., 1974 ).

However, later studies lean toward assimilation by the reductive TCA cycle in Cbi. limicola . The organism was cultured with 14 C-labeled pyruvate and unlabeled CO 2 . In the cells, 20% of the carbon originated from pyruvate. No oxidation of pyruvate to CO 2 was observed. Labeled compounds, including alanine, aspartate, glutamate, and glucose, were identified. Since pyruvate is not an intermediate in the Calvin cycle, this observation and the measured radioactivity support the theory that Cbi. limicola does not use the Calvin cycle ( Fuchs et al., 1980b ). 3-phosphoglycerate was not experimentally studied but it probably arises from pyruvate through gluconeogenesis. This conclusion was supported by another similar experiment, where labeled propionate was used instead of pyruvate. The latter was assimilated, probably via propionyl-CoA, methylmalonyl-CoA, and succinyl-CoA. Labeling of the individual carbon atoms in alanine, aspartate, and glutamate depend on the labeled position in the propionate also indicates the use of the reductive TCA cycle rather than the Calvin cycle ( Fuchs et al., 1980a ). Similar experiments were also performed using Cbi. thiosulfatophilum with non-radioactively labeled 13 CO 2 , while the results were evaluated using NMR spectroscopy. This method provides signals for a selected nuclide with an odd number of nucleons, in this case 13 C. The intensity of the signal depends on the 13 C content and its position (so-called chemical shift) on the chemical environment of the respective atom, thanks to which one can distinguish which of the carbons in the molecule are labeled. Cbi. thiosulfatophilum was cultured on Larsen’s medium containing both Na 2 S and Na 2 S 2 O 3 as sulfur sources and NaH 13 CO 3 (enriched to 69% 13 C) as carbon source; a second carbon source, unlabeled sodium acetate, was also added. Harvested cells were fractionated and selected metabolites were characterized by 13 C NMR. Specifically, they were alanine, glycine, aspartate, glutamate, tyrosine, uridine, and guanosine. The enrichment of their individual carbons was calculated and compared to that predicted based on known metabolic pathways. These data were in agreement with the assumption that the assimilation takes place in the reductive TCA cycle, and the synthesis of other substances then in the assumed pathways, e.g., the Krebs cycle. On the contrary, it was obvious from the measured spectrum for ribose that this molecule could not have been formed in the Calvin cycle ( Portis and Parry, 2007 ). This can be regarded as evidence indicating the exclusive utilization of the reductive TCA cycle for assimilation ( Paalme et al., 1982 ). After sequencing the genome of Cba. tepidum , the gene for ribulose-1,5-diphosphate carboxylase was found, or only for the ortholog of its large subunit. However, CO 2 fixation was not observed for the purified enzyme, and mutants in which this gene was knocked out had a standard phenotype ( Eisen et al., 2002 ).

3.4 Assimilation of organic substances

In addition to photolithoautotrophic growth, mixotrophic growth has also been observed in GSB. The carbon source may be an organic molecule, for example acetate, but it is still necessary to supply the carbon source CO 2 , an electron donor (most often H 2 S) and light energy ( Manske et al., 2005 ). Most of the fixed CO 2 is directed to glucose-based polysaccharide and organic acids production. The addition of fluoroacetate to a culture of Cbi. thiosulfatophilum (strain 8346) generally inhibited CO 2 fixation, but at the concentration lower than 1 mmol L −1 it increased the accumulation of α-ketoglutarate. CO 2 fixation was completely inhibited by the addition of 0.1 mmol L −1 arsenite. Malonate, which inhibits the oxidative variant of the tricarboxylic acid cycle, caused a slight increase in CO 2 uptake and a large increase in polysaccharide accumulation in Cbi. thiosulfatophilum ( Cole and Hughes, 1965 ).

Furthermore, the assimilation of several other substances was tested in the presence of thiosulfate or H 2 as an electron donor. Pyruvate was assimilated. In case of succinate, assimilation varied greatly in different bacterial suspensions. N 2 did not affect metabolism in any way, but 0.05% ammonium chloride completely inhibited acid accumulation without affecting CO 2 consumption. After the addition of fluoroacetate, which inhibits aconitase, there was no accumulation of citrate, which may indicate the absence of the oxidative tricarboxylic acid cycle. Accumulation of α-ketoglutarate instead of isocitrate can be explained by a shift in the balance in favor of α-ketoglutarate, possibly also through the inhibition of isocitrate dehydrogenase by fluoroacetate ( Sirevåg and Ormerod, 1970 ).

Although GSB use the reductive TCA cycle to assimilate carbon, they can also operate the oxidative variant, Krebs cycle, which was proved by the following experiments. In Cba. tepidum , it was observed that the addition of pyruvate improves its growth by 20% and the addition of acetate by up to 50%. Neither acetate nor pyruvate excretion was observed during autotrophic growth, whereas 0.2 mmol L −1 acetate was excreted in mixotrophic growth on 20 mmol L −1 pyruvate. During mixotrophic growth on pyruvate or acetate labeled at various positions with 13 C, the occurrence of labeled bacteriochlorophyll c was observed. Labeling was evaluated on the basis of MALDI–TOF. Furthermore, experiments were carried out with fluoroacetate, which successfully inhibited autotrophic growth and mixotrophic growth on pyruvate (in the latter case, however, weak growth was observed after increasing the concentration of pyruvate). In contrast, the addition of fluoroacetate had no significant effect during mixotrophic growth on acetate. Acetate is thought to be assimilated by acetyl-CoA synthetase. Since the assimilation of fluoroacetate practically did not occur, it is probably an unsuitable substrate for this enzyme. On the basis of the proportion of labeled carbon in bacteriochlorophyll c , a hypothesis was created that acetate is assimilated both by the reductive and oxidative tricarboxylic acid cycles, while pyruvate only by the reductive TCA cycle. This hypothesis is also confirmed by the experiment with fluoroacetate. If the overwhelming majority of pyruvate is assimilated by the reductive TCA cycle, then acetyl-CoA is formed by the breakdown of citrate with the enzyme aconitase. However, fluoroacetate inhibits aconitase, making it impossible for the cell to form acetyl-CoA. The activity of all enzymes specific for the reductive TCA cycle was observed, while the genes for enzymes specific for the oxidative cycle are either completely absent (pyruvate dehydrogenase, α-ketoglutarate dehydrogenase) or have a reduced level of expression (citrate synthase, succinate dehydrogenase). Thus, it can be concluded that in Cba. tepidum only a branch of the citric acid cycle oxidatively operates to form α-ketoglutarate. All other intermediates are only formed by the reductive TCA cycle, and therefore generated by consuming CO 2 . This is consistent with the already known fact that GSB do not grow heterotrophically ( Tang and Blankenship, 2010 ).

3.5 Carbohydrate metabolism

The growth of GSB on glucose has not been observed. Accordingly, neither hexokinase nor glucose transporter genes were found in the genome of Cba. tepidum . Glucose is synthesized only inside the cells by the process of gluconeogenesis ( Eisen et al., 2002 ; Tang et al., 2011 ). GSB accumulate polysaccharides during phototrophic growth, that are either of autotrophic or mixotrophic origin. However, more polysaccharide accumulates during mixotrophic growth. This polysaccharide was identified as a polymer of glucose in the genus Chlorobium . During subsequent cultivation in the dark, the cells consume the polysaccharide and release organic acids into the medium. When a culture of Cbi. thiosulfatophilum strain 8327 was incubated with NaHCO 3 , acetate, and thiosulfate for 3.5 h, an increase in polysaccharide concentration from 70 to 122 μg mL −1 was observed. Ultrathin sections were then made from these cells for electron microscopy and stained specifically for polysaccharide. In the electron microscope, dozens of colored granules were observed in each cell, which had an approximately uniform size (less than 30 μm) and consisted of smaller grains. The increase in their cell number after the incubation was in accordance with the results of the biochemical analysis of the polysaccharide consumption. The size of the individual granules did not change. In another experiment, cells were first incubated with 14 CO 2 in phototrophic mode for 4.5 h, then washed and incubated in the dark in the presence of unlabeled CO 2 and thiosulfate for 12.5 h resulting in a degradation of polysaccharide. The vast majority of labeled carbon was found in the supernatant after centrifugation, of which 85% was in the form of lower fatty acids (80% of which was acetate, 17.5% capronate and 2.5% propionate). Unlike the bacterium Rhodospirillum rubrum , there was no formation of labeled formate. As for the remaining 15% of labeled carbon, the most abundant form was succinate. Last but not least, cells with accumulated labeled polysaccharide were cultured in the absence of both thiosulfate and any other common electron donor, either in the light or in the dark. Less polysaccharide (23%) was consumed during incubation in light conditions. However, the radioactivity of the supernatant was three times higher in the case of cells cultured in the dark. Polysaccharide may not only serve as an energy source for heterotrophic nutrition, but also as an emergency electron donor for photosynthesis ( Sirevåg and Ormerod, 1970 ).

The storage polysaccharide of Cbi. limicola was later identified as glycogen, which is a homopolymer of glucose with α-(1 → 4) bonds and frequent branching using α-(1 → 6) bonds, soluble in water. As is typical for prokaryotes, 1-ADP-glucose is used to initiate glycogen synthesis ( Levytska and Gudz, 2010 ). During cultivation in light in a medium containing H 2 S, NaHCO 3 acetate, and pyruvate, the glycogen and glucose content in dry matter was measured. The glycogen content of the cells was almost always slightly higher than the glucose content, with approximately a 3-fold increase in both values between days 21 and 30. In addition, the cultivation of Cbi. limicola in wastewater from a distillery was studied. The wastewater was highly polluted with organic substances, but it did not contain glucose nor glycogen. Glycogen content in dry matter reached more than twice larger values than in the control. During heterotrophic growth in the dark, the glucose content in dry matter was similar in control and in wastewater, but the glycogen content decreased to 0.2% in wastewater. In any case, light was an effective method of bioremediation of wastewater, reducing its biological O 2 consumption and H 2 S content ( Saeed et al., 2022 ).

4 Cultivation conditions

4.1 culture media.

To cultivate GSB as phototrophic microorganisms, the main components of the culture medium are various minerals. Considering the mechanism of their photosynthesis, the most important medium components are H 2 S and CO 2 . Other important elements are primarily nitrogen, usually supplied as an ammonium salt, and phosphorus, supplied as hydrogen phosphate or dihydrogen phosphate. Van Niel used a basic mineral medium of the following composition for both PSB and GSB: NH 4 Cl, K 2 HPO 4 , MgCl 2 , NaHCO 3 , and Na 2 S·9H 2 O, all to a final mass fraction of 0.1% and dissolved in distilled water. With this medium, experiments were performed to enrich a mixed culture with either GSB or PSB. The temperature was approx. 25°C. At pH lower than 8, an increase in GSB was observed within 3 weeks of inoculation with an environmental sample containing both PSB and GSB. The cultivation success of GSB from the mixed culture could be improved by increasing the Na 2 S concentration (up to 0.25%) and decreasing the pH (up to 7.5). Higher Na 2 S concentrations or lower pH were not investigated in this study ( van Niel, 1932 ). The GSB also grew faster than the PSB, so their capture is easier if the grown culture is collected early ( Van Gemerden, 1986 ; Oren and Garrity, 2021 ).

Some GSB species, historically referred to as Cbi. thiosulphatophilum , are capable of utilizing thiosulfate as an electron donor. During their cultivation, 0.1–0.2% thiosulfate can be added to the medium described above. In this way, they can be enriched from the mixed culture with thiosulfate utilizing GSB at the expense of the others ( van Niel, 1971 ).

Other authors use van Niel’s medium as a basis, but adjust the concentrations and sometimes add other components. The Handbook of Microbiological Media ( Atlas, 2010 ) recommends a modified Pfennig medium prepared by mixing the following solutions for GSB: solution A: basic mineral solution, contains CaCl 2 , KH 2 PO 4 , NH 4 Cl, KCl, and MgSO 4 ; solution B: distilled water only; solution C: vitamin B 12 ; solution D: trace elements, contains iron, boron, cobalt, manganese, zinc, molybdenum, nickel and copper; solution E: NaHCO 3 (1.5 g L −1 of the resulting medium); solution F: Na 2 S·9H 2 O (2 g L −1 of the resulting medium). Another study recommends the addition of ferric citrate and resazurin to the basic mineral solution ( Malik, 1984 ).

The solutions are sterilized either by autoclaving or by filtration (this applies to solutions C and E). To reduce the redox potential, it is recommended to remove dissolved O 2 from all solutions by bubbling with N 2 (or CO 2 in the case of solution E). The resulting pH should be 6.8 ± 0.2 at 25°C, adjusted with HCl and Na 2 CO 3 . There are several variants with different proportions of mineral salts. For marine representatives, the addition of NaCl to a final concentration of 10 g L −1 is recommended ( Atlas, 2010 ). MgCl 2 is also sometimes used as a magnesium source ( Kobayashi et al., 1983 ).

4.2 Physical conditions

For the cultivation of GSB, a temperature of 25–30°C and lighting with an intensity of 700–2,000 Lx with a classic light bulb are recommended. Under these conditions, at pH of 6.6 and Na 2 S·9H 2 O concentration in the range of 0.1–0.2%, GSB grow significantly better than PSB, which can be used to enrich GSB cultures obtained from the environment. The absorption maxima of chlorophylls are most often in the range of 720–760 nm, the author assumes that illumination with these wavelengths could improve cultivation results. In natural conditions, the light spectrum is mainly influenced by the height of the water column above the bacteria, as water absorbs mainly higher wavelengths, and the presence of other phototrophs in it. At specific locations, absorption spectra were measured at varying water depths, extending to a point where no discernible amount of light penetrated. Pigments were extracted from the collected water samples to determine their concentration and mutual representation, which provided information on the ecological groups of phototrophs present. Furthermore, the rate of H 2 S consumption was studied depending on the light spectrum in pure cultures of two GSB— Cbi. limicola , a green-colored representative and Cbi. phaeovibrioides , a brown-colored representative. The absorption spectra of intact cells are similar for both species, with a maximum in the red and a second peak in the blue region. Cbi. phaeovibrioides is better adapted to blue and green light, confirmed by the results of measuring H 2 S consumption and the absorption spectrum. On the other hand, Cbi. limicola was favored in white and red light. Both species performed better in red light than in white light, as expected from the absorption spectra of bacteriochlorophylls. The results are consistent with the data on the occurrence of bacteria in the water column that the green-colored GSB thrive better at shallower depths than the brown GSB, and they better tolerate the presence of PSB in the higher layers, since their absorption spectra are largely complementary ( Vila and Abella, 1994 ).

The only thermophilic representative of the GSB is Cba. tepidum , which grows fastest of all GSB in the presence of thiosulfate ( Jagannathan and Golbeck, 2009 ). In addition to basic minerals, sodium thiosulfate, EDTA, vitamin B 12 and a mixture of minerals were added to the medium. Cultivation took place at a temperature of 47°C and a light intensity of 40 μmol m −2 s −1 . The ratio of thiosulfate to H 2 S concentrations was further optimized. At high concentrations of H 2 S, when the redox potential fell below −330 mV, growth was inhibited. At a redox potential of −300 ± 20 mV, the optimal thiosulfate concentration was 12 mmol L −1 . Although thiosulfate is the preferred electron donor, cultivation was also successful only on H 2 S, but it was necessary to maintain its concentration below 0.1 mmol L −1 , redox potential around −300 mV and pH around 6.8. During the oxidation of H 2 S to elemental sulfur, the pH increased, while further oxidation of sulfur, on the contrary, decreased. Last but not least, bubbling and mixing, important elements of cultivation in a larger volume (scale-up), were optimized. More intense mixing ensures equal access of individual cells to resources, especially to light, but too intensive mixing is accompanied by large shear forces that can damage the cells. The best growth was achieved at a frequency of 300 rpm, which corresponds to a Reynolds number of 55.125 and a peripheral speed of 165 cm s −1 for the given stirrer. The presence of inclusions that change the flow direction and increase turbulence improved the growth rate. Bubbling the culture with gases was not optimal for growth, better growth and without form formation was obtained by supplying N 2 and CO 2 through surface gassing ( Mukhopadhyay et al., 1999 ).

4.3 Isolation from the environment and selection

When isolating GSB from the environment, the Winogradsky column can be used ( De Leon et al., 2023 ). This simulates the natural environment in the anaerobic zone at the bottom of water bodies and streams. Mud taken from the environment is enriched with a source of sulfur (CaSO 4 ) and organic carbon (for example, the roots of aquatic plants from the locality) and incubated in light in a suitable bottle. Elements circulate between heterotrophic and autotrophic bacteria, so the whole system can last for a long time without the supply of any chemicals, only requiring light. Gradually, zones inhabited by different microorganisms are created according to the lighting gradient and redox potential. GSB are most abundant in the lower (anaerobic) and illuminated part of the column, where they become visible as green spots. These green spots can be isolated and GSB can be further enriched. Molisch’s column is similar Winogradsky column, to which a piece of animal tissue is added. It decomposes in an anaerobic environment much faster than plant tissue, which will also accelerate the growth of microorganisms ( van Niel, 1971 ).

A method for isolation and purification of microorganisms is dilution to extinction ( Mauerhofer et al., 2019 ; Hanišáková et al., 2022 ). The method can be improved by the incorporation of micromanipulation techniques and removing a single cell from the culture. As GSB are sensitive to O 2 , they cannot grow on the surface of solidified agar when cultured exposed to air. The deficiency can be overcome by mixing the culture into a warm, still liquid medium at a temperature of around 45°C. Cultivation can be carried out in test tubes filled with a mixture of paraffin and paraffin oil to prevent access to O 2 . This mixture is preferable to paraffin alone, which shrinks during solidification and does not cover the entire surface of the medium well. Another problem is that GSB and PSB may thrive better in a consortium than in a pure culture, and their cells often stick close together, making pure culture isolation even more difficult ( van Niel, 1932 ).

An alternative option for purifying cultures of anoxygenic phototrophs involves the use of antibiotics. Two strains of Cbi. limicola and the PSB Allochromatium vinosum , Thiocapsa roseopersicina , and Thiocapsa sp. sensitivity to different antibiotics was determined. The bacteria were first cultured in liquid medium, then mixed into agarized medium, spread on Petri dishes, and cultured in an anaerobic N 2 atmosphere. Since H 2 S evaporates from the agar thioacetamide was added instead. Susceptibility to antibiotics was determined by the disk diffusion method. Cbi. limicola was particularly sensitive to amoxicillin, which caused complete lysis, but also to erythromycin, nalidixic acid, and nitrofurantoin. Cbi. limicola was also sensitive to gentamicin and netilmicin to some extent, but PSB were much more sensitive to these antibiotics, while they were not very sensitive to amoxicillin. Neither oxacillin nor trimethoprim and sulfamethoxazole significantly inhibited any of the cultures used. For selected antibiotics, critical concentrations of antibiotics, which should correspond to the limit of the inhibition zone, were also determined for A. vinosum and both strains of Cbi. limicola . Of these, both strains of Cbi. limicola had the lowest critical concentrations of mitomycin C (0.41 and 0.44 μg mL −1 ) and penicillin G (1.72 and 0.98 μg mL −1 ). Furthermore, penicillin G did not inhibit A. vinosum . If, on the other hand, the inhibition of A. vinosum and selection of Cbi. limicola , streptomycin would be the most suitable. The authors point out that the critical concentrations determined in this way may not fully correspond to the minimum inhibitory concentrations during cultivation in a liquid medium ( Nogales et al., 1994 ).

The use of antibiotics for the selection of phototrophs from consortia would also have other disadvantages: in addition to the need for a detailed study of the sensitivity of different strains to different antibiotics, it would involve the consumption of antibiotics and the risk of developing resistance. However, the method could be more practical than purification of cultures on solid soils, especially if we aimed to isolate a particular ecological group rather than a pure culture ( Wahlund and Madigan, 1995 ). Resistance to antibiotics can also be used as a selection marker to determine the success of the transfer of genetic information. During the selection of Cba. tepidum cells, in which conjugation was successful, solid medium with thioacetamide was used. Before selection, however, it was necessary to choose a suitable antibiotic to which Cba. tepidum is naturally sensitive. The antibiotic in the studied concentration was mixed directly into a culture medium solidified with agar. A small proportion of cells were resistant to kanamycin and streptomycin, especially when cultured at 37°C. This is not the temperature optimum, but it was chosen for conjugation as the optimum of E. coli donor cells. On the contrary, high sensitivity was observed to ampicillin, chloramphenicol and tetracycline ( Wahlund and Madigan, 1995 ).

5 Perspective of biotechnological application

5.1 desulfurization of gases and wastewater.

H 2 S in wastewater might be problematic because it is relatively toxic to aquatic animals, causes corrosion, inhibits methanogenesis, smells strongly, and has a high O 2 consumption requirement for oxidation. Kobayashi et al. were, to our knowledge, the first to investigate the possible use of phototrophic bacteria for wastewater desulfurization ( Kobayashi et al., 1983 ). In addition to desulfurization, phototrophic bacteria can also degrade mercaptans and lipids ( Imhoff, 2006 ). Rhodospirillaceae spp., representatives of the purple non-sulfur bacteria, are common in the environment and can consume H 2 S, but they can only tolerate it in low concentrations and are therefore not very suitable for wastewater desulfurization. In an experiment by Kobayashi et al., a column was used consisting of two concentric acrylic tubes. Into the inner tube, a 40 W tungsten light bulb was inserted. A consortium of anaerobic phototrophic bacteria isolated from the environment was inoculated into the column into the space between the outer and the inner tube and formed a biofilm. Brown and green colored zones with different bacteria developed in the column, the brown ones were in the more illuminated parts of the column. The most common morphological type were immobile rods with extracellular elemental sulfur. At the inlet, GSB dominated, further down the column, purple non-sulfur bacteria gradually became predominant ( Kobayashi et al., 1983 ). Purple non-sulfur bacteria were identified as Rhodopseudomonas acidophila , GSB as Cbi. limicola . Desulfurization was quite successful: efficiency was 81–95%. Desulfurization did not occur when the lights were turned off, confirming that it was provided by phototrophic bacteria ( Hurse et al., 2008 ). A commercial method employing chemotrophic sulfide-oxidizing bacteria within a fixed-film bioreactor under regulated O 2 conditions was published. Fixed-film or suspended-growth photobioreactors using anoxygenic phototrophic bacteria may be viable alternatives for economical H 2 S removal for example from biogas. These systems can operate for extended periods without needing a biomass separation step and can handle high and fluctuating sulfide loads ( Frigaard, 2016 ).

The tubular arrangement of the photobioreactor was more efficient than the column, which may have contributed to more uniform illumination and better flow, which ensured uniform mixing of the suspension and good contact of the cells with the H 2 S. In contrast to the column, elemental sulfur was not detected at the outlet from the tube; it was converted to sulfate at a retention time of 24 h. For further optimization, the authors of the study suggest focusing on the geometric arrangement of the photobioreactor. When choosing the lighting, the absorption spectrum of the pigments of the respective phototrophs was not taken into account ( Kobayashi et al., 1983 ).

An important source of wastewater with an increased content of H 2 S are oil refineries. Oil is commonly desulfurized by e.g., the Holmes-Stretford process, or stripping followed by the Claus process ( Kohl and Nielsen, 1997 ). Both are chemical methods. H 2 S is oxidized using O 2 to elemental sulfur and as elemental sulfur is solid, it is easier to separate than H 2 S it also loses its corrosive properties and can be used as a raw material for the production of sulfuric acid ( Saeid and Chojnacka, 2014 ). The disadvantages of the chemical methods are corrosion, the necessity of regular replacement of catalysts, consumption of additives, and work at high temperatures and pressures. Henshaw and Zhu grew GSB in a film in Tygon tubes, which are transparent and impermeable to O 2 . In their study, Cbi. limicola bacteria were grown on Pfennig’s medium under an infrared lamp ( Henshaw and Zhu, 2001 ). The well-grown culture was allowed to circulate through the tube for 3 days to allow the cells to adhere to its wall. Wastewater was then allowed to flow through the tube for 72–288 h. At an influent concentration of H 2 S up to 286 mg L −1 h −1 , its 100% removal was achieved, with 92–95% converted to elemental sulfur. The content of sulfates and bacteriochlorophyll was also monitored. On the contrary, the possibilities of the geometric arrangement of the reactor and methods of lighting were not studied ( Henshaw and Zhu, 2001 ). Hurse and Keller proposed another type of photobioreactor for wastewater desulfurization using GSB ( Hurse and Keller, 2004 ). Again, they used biofilm, but scale-up of a photobioreactor with biofilm in the tubes would be too difficult: simply magnifying the reactor would affect the ratio of irradiated surface (where the biofilm grows) to volume. A biofilm growing on optical fibers or transparent panels could overcome this problem. The variant with panels was also tested experimentally using artificial wastewater, with composition based on Pfennig’s medium ( Pfennig and Trüper, 1981 ; Overmann et al., 1992b ). The wavelength of the light was chosen as an interval between 720 and 780 nm. Compared to previous studies with tubes, desulfurization efficiency has decreased, but energy efficiency has increased ( Hurse and Keller, 2004 ; Cui et al., 2021 ).

Other raw materials in which H 2 S occurs as an impurity are oil and natural gas. They are also usually desulfurized chemically. Hydrodesulfurization is a chemical method that uses H 2 for reduction of all forms of sulfur to H 2 S, which is afterwards removed by absorption into a suitable solution ( Shafiq et al., 2022 ). Another option is to absorb up to sulfur dioxide from the flue gas. A consortium of GSB was isolated from the hot spring and tested for natural gas desulfurization. Since gas eliminates the problem of separation, the planktonic form of bacteria could be used. The bioreactor had a volume of 4.5 L, was bubbled with natural gas for 15 min and then saturated with CO 2 . Desulfurization took place for a total of 3 weeks, after each week samples were taken to determine the production of sulfur and bacteriochlorophyll. Although desulfurization was successful, it was less effective than in other studies using chemolithotrophic microorganisms ( Sharifian-Koupaiee et al., 2022 ).

In addition to natural gas, biogas can also be a gaseous fuel, which is produced from various wastes by their gasification with the help of microorganisms, the main components of which are methane and CO 2 ( Černý et al., 2018 ; Kushkevych et al., 2018b ; Struk et al., 2019 ). Even here, a problematic amount of H 2 S can occur (up to 3%, depending on the raw material). Struk et al. studied the desulfurization of synthetic biogas containing 70% methane, 29.5% CO 2 , and 0.5% H 2 S. The PSB A. vinosum and the GSB Cbi. limicola were compared. The bacteria were cultured photoautotrophically without the addition of organic substances. In a stirred batch reactor, the efficiency of desulfurization of synthetic biogas and the effect of H 2 S concentration and light intensity on it were monitored. After 7 days of incubation of both cultures with biogas, complete desulfurization occurred. Then H 2 S was added to a concentration of 1%. This added H 2 S was removed up to 100% in 2 days in the reactor with A. vinosum and up to 90% in 5 days in the case of Cbi. limicola ( Struk et al., 2023 ). For the abiotic control, approximately 33% H 2 S was removed by absorption into the water. About a third of the CO 2 in the biogas was consumed for photosynthesis, so both the proportion of methane in the biogas and the quality of the biogas increased. Desulfurization was also successful at increasing the H 2 S concentration. Both cultures grew best at a H 2 S concentration of 1%, but the process was also effective at 2%. An increased concentration provided enough electron donor for photosynthesis, thereby limiting light damage to the photosynthetic apparatus in the later stages of cultivation. Compared to the consortium of chemolithotrophs and cyanobacteria, which tolerate a H 2 S concentration not higher than 16 mg L −1 , anoxygenic phototrophs allow working with a concentration of up to 100–150 mg L −1 . At a light intensity of 10 kLx, desulfurization was more effective than at 25 kLx, when Cbi. limicola grew more slowly and A. vinosum stopped growing completely. An experiment with continuous desulfurization using Cbi. limicola was carried out as part of this study. The lag phase lasted 6 days, while the optical density practically did not increase and the consumption of H 2 S was also minimal. After that, H 2 S consumption began to increase, from the 10 th day 100% desulfurization was observed. In the later stages of cultivation, there was a decrease in efficiency, which could be due to the accumulation of biomass, which reduced light transmission. For practical use, a semi-continuous arrangement with regular biomass sampling could be more suitable. All experiments were performed sterile and inoculated with pure cultures, which could also be limiting in practice ( Struk et al., 2023 ).

5.2 Microbial electrochemical cells

A microbial electrochemical cell enables connection of the microbial metabolism with an electrical circuit and generation of electric current ( Logan et al., 2006 ; Hassan et al., 2021 ). At its anode, organic substances from the solution are oxidized by respiring bacteria, and the released electrons are transferred to the circuit ( Lovley, 2011 , 2012 ). Defined microbial consortia may be suitable for this purpose, for example, a consortium of fermentative Clostridium cellulolyticum and respiring Geobacter sulfurreducens successfully generated electricity by breaking down cellulose ( Desvaux, 2005 ; Poddar and Khurana, 2011 ). Other consortia also include phototrophic microorganisms. A microbial electrochemical cell can be inspired by biogeochemical sulfur cycling, for example using a consortium of sulfur-reducing bacteria and GSB ( Badalamenti et al., 2014 ). Badalamenti et al. examined a consortium of the GSB Cbi. limicola in planktonic form and the GSB Geobacter sulfurreducens forming a biofilm on the anode. Cbi. limicola assimilated CO 2 in the light and accumulated glycogen. Cultivation took place with alternating darkness (16 h) and light (8 h) ( Badalamenti et al., 2014 ). In the dark, Cbi. limicola consumed glycogen, which was fermented to acetate. Acetate could thus be oxidized by the bacteria G. sulfurreducens and the obtained electrons travel to the anode and further into the electrical circuit. When the consortium was cultivated in the dark, a current of 118 ± 16 μA was generated, but in the light it dropped to 61 ± 11 μA within 10 min. Dependence of electric current on light was not observed in monocultures of G. sulfurreducens or Cbi. limicola , which supports the model above. The glycogen content was also determined at each transition between dark and light. During the first and second light periods, the glycogen content of the consortium increased to a similar level, yet the current decreased in the following dark phase. In the case of Cbi. limicola monoculture, glycogen regeneration was much less efficient in the light phases. A possible explanation is that G. sulfurreducens cells transferred electrons not only to the anode, but also to elemental sulfur produced during photosynthesis, reducing it back to H 2 S. This allowed Cbi. limicola to be photosynthetically active for longer periods and to accumulate glycogen, while diverting some of the electrons away from the anode, so that a decrease in electrical current was observed. Overall, this study is groundbreaking in that a microbial electrochemical cell was assembled not requiring the supply of an organic substrate. As G. sulfurreducens is sensitive to O 2 , it was expected that better results would be obtained in the consortium with an anoxygenic phototroph than in previous experiments with an oxygenic phototroph ( Chlamydomonas ), which was confirmed ( Badalamenti et al., 2014 ). Possible applications of microbial electrochemical cells include bioremediation of wastewater, whether municipal or industrial. The advantage is that there is no need to work with an excess of activated sludge. Due to the high load of wastewater with heavy metals and other toxic inorganic substances, it is necessary to select such microorganisms that survive in this environment. The advantage of GSB is a high tolerance to H 2 S. Strains that have been isolated from a polluted environment might be particularly suitable. A consortium of the following bacteria for a microbial electrochemical cell was also used:

• Desulfuromonas acetoxidans , a sulfur-reducing bacterium capable of reducing Fe 3+ , a strain resistant to heavy metals ( Pfennig and Biebl, 1976 );

• Cbi. limicola , a GSB, consortium of GSB with Desulfuromonas acetoxidans also known from nature ( Kyndt et al., 2020 );

• Desulfuromusa , reduces sulfur, nitrate, nitrite, and various metal ions (Mn 4+ , Fe 3+ , Cu 2+ , and Cr 6+ ), resistant to the toxic effects of chromium ( Finster et al., 1997 );

• Geobacter sp., similar properties to the previous strain ( Jiang et al., 2022 ).

Heterotrophic representatives were cultivated as pure cultures on diluted landfill filtrate in a microbial electrochemical cell. All of them generated electric current; the highest specific power was achieved with D. acetoxidans . This bacterium was further cultured on diluted wastewater from yeast production. Many impurities were successfully removed, for example nitrates, nitrites, elemental sulfur, but also sulfates and sulfites, the use of which as electron acceptors was not yet known for this bacterium. On the contrary, the concentration of bicarbonate increased, probably due to the complete oxidation of organic substances. Further experiments were carried out with the participation of Cbi. limicola . Microbial electrochemical cells can also be used to remove hydrocarbons from water ( Fatehbasharzad et al., 2022 ). In a bioreactor using a naturally occurring microbial community from contaminated groundwater, not only hydrocarbons were removed, but also sulfates, which were reduced by sulfate-reducing bacteria. However, the effectiveness of removing sulfates was reduced by bacteria of the genus Chlorobium , which reoxidized H 2 S formed into sulfate and transferred electrons to the anode. The activity of these bacteria is considered undesirable by the authors, as they regenerate sulfate. The reduction of sulfate to H 2 S is referred to by the authors as sulfate removal, but it is not stated that H 2 S is a toxic compound and that it is also soluble in water, so it is not a more suitable form of sulfur than sulfate. On the other hand, modification of this bioreactor to produce elemental sulfur is being considered in the future ( Tucci et al., 2021 ).

Anoxygenic photosynthesis is a metabolic process utilized by some bacteria in which they generate energy from light without producing O 2 as a byproduct. In anoxic environments where O 2 is scarce or absent, various toxic compounds may accumulate, posing a threat to living organisms. Anoxygenic photosynthetic bacteria possess unique metabolic pathways that enable them to utilize alternative electron donors, such as H 2 S, H 2 , or ferrous iron, instead of water, to fuel their photosynthetic process. These bacteria play a crucial role in detoxification processes by converting harmful compounds into less toxic substances. By harnessing light energy, these bacteria can drive biochemical reactions that reduce the concentration of toxic compounds in their surroundings. Our review explores the ecological significance of anoxygenic photosynthetic bacteria in anaerobic environments. Additionally, the potential applications of these bacteria in bioremediation strategies are discussed with regard to mitigating environmental pollution that has been caused by toxic compounds. Overall, the role of anoxygenic photosynthesis is vital for maintaining ecosystem health and anoxygenic phototrophs may offer many still unexplored potential avenues in environmental management and biotechnology.

Author contributions

IK: Visualization, Writing – review & editing, Writing – original draft. VP: Writing – review & editing, Writing – original draft, Visualization. MV: Writing – review & editing, Writing – original draft. DD: Writing – review & editing, Writing – original draft. MA: Writing – review & editing. SK-MRR: Writing – review & editing, Writing – original draft.

The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This study was supported by Grant Agency of the Masaryk University (MUNI/A/1280/2022). Open access funding provided by the University of Vienna.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision.

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Abbreviations

Cbi. , Chlorobium ; Cba. , Chlorobaculum ; Chp. , Chloroherpethon ; Ptc. , Prosthecochloris ; Pld. , Pelodictyon .

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Keywords: bacterial photosynthesis, anoxygenic bacteria, bacterial physiology, microbiology, anaerobes, biotechnology

Citation: Kushkevych I, Procházka V, Vítězová M, Dordević D, Abd El-Salam M and Rittmann SK-MR (2024) Anoxygenic photosynthesis with emphasis on green sulfur bacteria and a perspective for hydrogen sulfide detoxification of anoxic environments. Front. Microbiol . 15:1417714. doi: 10.3389/fmicb.2024.1417714

Received: 15 April 2024; Accepted: 12 June 2024; Published: 11 July 2024.

Reviewed by:

Copyright © 2024 Kushkevych, Procházka, Vítězová, Dordević, Abd El-Salam and Rittmann. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Ivan Kushkevych, [email protected] ; Simon K.-M. R. Rittmann, [email protected]

Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

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Bubbling with curiosity: mizzou engineer investigates oceanic phenomenon.

July 09, 2024

Assistant Professor BinBin Wang and his research team are researching natural seeps to better understand these deep sea bubbles.

Missouri, a landlocked state, isn’t where one usually expects to find an expert on rare ocean phenomena. However, Mizzou Engineering faculty are tackling unexpected problems and researching innovative solutions to engineer a better world. 

Binbin Wang has spent years researching hydrocarbons in the Gulf of Mexico and discovering how natural seeps in the ocean floor affect the environment. He is now working on a long-term research project using a National Science Foundation research vessel. 

This summer, Wang, an assistant professor of civil and environmental engineering, and his research team traveled to the Gulf of Mexico to board the R/V Roger Revelle. They spent two weeks at sea documenting hydrocarbon bubbles released by natural seeps in the ocean floor, including the volume of the bubbles and how far they rose from their site of origin. 

“We’re solving a very challenging problem in an extreme environment,” Wang said. “We have observations and models; we’re now combining these methodologies to help us understand the movement of these bubbles.” 

Natural seeps are a key energy source for ocean floor ecosystems. They support bacterial growth through a process called chemosynthesis. Essentially, the bacteria survive on the released hydrocarbons, like how plants use photosynthesis to gain nutrients from the sun. These bacteria then support worms and other microbial species, which in turn feed shellfish and other ocean creatures. Like plants on dry land, they form the base of the food chain. 

Some researchers are concerned that the hydrocarbons, including methane, released by natural seeps will eventually make their way out of the ocean and into the atmosphere, contributing to global greenhouse gas emissions. An earlier study by Wang’s team showed that methane from deep ocean seeps fully dissolved in the water before reaching the surface. This project is investigating seeps in shallower conditions. 

“Our hypothesis is that bubbles from these natural seeps do not reach the ocean surface,” Wang said.  

Wang was joined on the research vessel by two graduate students, Mustahsin Reasad and Xuchen Ying, the latter of whom recently received a MO-AWWA Popalisky scholarship for his research in water resource and environmental science. The team used underwater imaging equipment, lasers and sonar to capture videos and photos of natural seeps 540 meters below the ocean’s surface. 

“We have a lot of data right now between videos to analyze and sonar images to process” Wang said. “We also plan to run models to predict the fate and transport of hydrocarbon bubbles. We’ll then compare this data with natural seeps at other sites.” 

In addition to the two weeks of data Wang and his team collected aboard the R/V Roger Revelle, the group will collect a periodical sample of video data over the next six months. Every 15 minutes the equipment turns on and records 5 minutes of high-resolution images to join the collection of observational data and broaden the information the team must work with. They will return to the Gulf in November 2024 to retrieve their equipment. 

“We’re contributing to a better understanding of natural phenomena on the ocean floor that has persisted for a really long time,” Wang said. “This research cruise will add another invaluable dataset to the natural seep community. Together, we can better understand natural seeps across the globe.” 

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