Dyes and Staining

 Dyes and Simple Staining

The dyes used to stain microorganisms have two features in common:

• ·     they have chromophore groups, (Chromophore is colored chemical substance that has ability to provide a color to dye)
• ·      they can bind with cells by ionic, covalent, or hydrophobic bonding.

Most dyes are used to directly stain the cell or object of interest. Some dyes (e.g., India ink and nigrosin) are used in negative staining, where the background but not the cell is stained; the unstained cells appear as bright objects against a dark background.

The most commonly used dyes bind cells by ionic interactions. These ionizable dyes may be divided into two general classes based on the nature of their charged group.

1. Basic dyes- methylene blue, basic fuchsin, crystal violet, safranin, malachite green—have positively charged groups (usually some form of pentavalent nitrogen) and are generally sold as chloride salts. Basic dyes bind to negatively charged molecules like nucleic acids, proteins.

Because the surfaces of bacterial cells also are negatively charged, basic dyes are most often used in bacteriology.

2. Acidic dyes— Nigrosine, Picric acid, India ink, eosin, rose bengal, and acid fuchsin—possess negatively charged groups such as carboxyls (—COOH) and phenolic hydroxyls (—OH). Acidic dyes, because of their negative charge, bind to positively charged cell structures.

The staining effectiveness of ionizable dyes may be altered by pH, since the nature and degree of the charge on cell components change with pH. Thus, acidic dyes stain best under acidic conditions when proteins and many other molecules carry a positive charge; basic dyes are most effective at higher pH.

Dyes that bind through covalent bonds or because of their solubility characteristics are also useful. For instance, DNA can be stained by the Feulgen procedure in which the staining compound (Schiff’s reagent) is covalently attached to its deoxyribose sugars. Sudan III (Sudan Black) selectively stains lipids because it is lipid soluble but will not dissolve in aqueous portions of the cell.

 

Staining Techniques

1.   Simple staining

2. Differential Staining 

-Gram stain

-Acid-fast staining

3. Special staining

-Capsule staining

-Negative staining

-Endospore staining

-Flagella staining

4. Fungal staining

  

 Simple staining/Monochrome staining

Microorganisms can be stained by simple staining, in which a single dye (monochrome) is used to determine the size, shape, and arrangement of procaryotic cells. The bacterial smear is stained with a single reagent, which produces a sharp contrast between the organism and its background. 

Basic stains with a positively charged chromogen are preferred because bacterial nucleic acids and certain cell wall components has a negative charge and will strongly attract and binds to the cationic chromogen. Basic dyes like crystal violet, methylene blue, and carbolfuchsin are frequently used in simple staining

Simple staining is characterised by its simplicity and ease of use. The fixed smear is immersed in one stain for a short period of time, followed by washing off the excess stain with water, and blotting the slide dry. 

The purpose of simple staining is to elucidate the morphology and arrangement of bacterial cells.

Bacilli and diplobacilli: Rod-shaped bacteria, purple
Cocci: spherical-shaped, bacteria, purple

 

STAINING OF SPECIMENS

 Living microorganisms can be directly examined with the light microscope, but they need fixing and staining to increase visibility, highlight specific morphological features, and preserve them for future study.

The first step in most bacterial staining procedures is the preparation of a smear.

Smear Preparation

Smear preparation is a process in which bacterial culture is spread uniformly or as a thin layer on glass slide.

Bacterial Smear Preparation

The crucial step in staining is the preparation of a good smear. A good smear is important to get a good staining result.

Requirements:

• Culture of Bacteria

• Glass slides

• Bunsen burner

• Inoculating loop

• Glassware marking pencil/ Permanent Marker

Step I: Preparation of the glass slide:

• Clean, grease free slides are needed for smear preparation.

• Grease or oil from the fingers on slides must be removed by washing the slides with soap and water

• Finally rinse the slide with 95% alcohol and dry it.

 • Hold the slide by their edge.

Step II: Labeling of slides:

• Proper labelling of the slide is essential- every slide should be labelled clearly.

• A lead pencil /permanent CD marker is used to write on the one end of the glass slide.

 Step III: Preparation of smear:

• An evenly spread smear should be prepared covering area of 15-20mm diameter.

• Avoid thick and dense smear because thick smear prevent light penetration to visualize the morphology of cell.

• A good smear is one that, when dried, appears as a thin whitish layer or film. The print of textbook should be legible through the smear.

Different techniques are used for smear preparation depending upon culture media

i. Broth cultures (liquid medium)

• Re-Suspend the culture by tapping the tube with your finger.

• Depending on the size of the loop, one or two loopfuls should be applied to the center of the slide with a sterile inoculating loop and spread evenly over an area.

• Allow the smear to air-dry

ii. Culture plates (Solid medium)

• Suspension is accomplished by spreading the cells in a circular motion in the drop of water with the loop. This helps to avoid cell clumping.

• The finished smear should occupy an area about the size of a nickel and should appear as a translucent, or semitransparent, whitish film

• Place a drop of water into the circle that has been created on the slide.

• Using a sterilized and cooled inoculation loop, obtain a very small sample of a bacterial colony from the culture palte.

• Gently mix the bacteria into the water drop.

Step IV: Air dry

• Smear should be allowed to dry completely at room temperature at safe place

  Step V: Fixation of smear:

• The purpose of fixation of smear is to preserve and prevent smear being washed away during staining.

• Heat fixation - After smear is air dried completely, rapidly pass the 3-4 times through flame of Bunsen burner or sprit lamp.  Avoid too much heating.

• After heat fix, allow the smear to cool before staining.

 

Fixation

The stained cells seen in a microscope should resemble living cells as closely as possible.

Fixation is the process by which the internal and external structures of cells and microorganisms are preserved and fixed in position. It inactivates enzymes that might disrupt cell morphology and toughens cell structures so that they do not change during staining and observation. A microorganism usually is killed and attached firmly to the microscope slide during fixation.

There are two fundamentally different types of fixation.

Heat fixation is routinely used to observe procaryotes. Typically, a film of cells (a smear) is gently heated as a slide is passed through a flame. Heat fixation preserves overall morphology but not structures within cells.

Chemical fixation is used to protect fine cellular substructure and the morphology of  delicate microorganisms. Chemical fixatives penetrate cells and react with cellular components, usually proteins and lipids, making them inactive, insoluble, and immobile. Common fixative mixtures contain components such as ethanol, acetic acid, mercuric chloride, formaldehyde, and glutaraldehyde.

Archaea Bacteria

 The word Archaea is derived from the Greek word Archaios which means ancient.

The Archaeabacteria, are quite diverse, both in morphology and physiology. They may be spherical, rod-shaped, spiral, lobed, cuboidal, triangular, plate-shaped, irregularly shaped, or pleomorphic. Some are single cells, whereas others form filaments or aggregates. They range in diameter from 0.1 to over 15 µm, and some filaments can grow up to 200 µm in length. Multiplication may be by binary fission, budding, fragmentation, or other mechanisms.

The Archaea are diverse physiologically- can be aerobic, facultatively anaerobic, or strictly anaerobic. They include psychrophiles, mesophiles, and hyperthermophiles that can grow above 100°C. Nutritionally they may be chemolithoautotrophs to organotrophs.

Ecology

Archaea are found in areas with either very high or low temperatures or pH, concentrated salts, or completely anoxic. These are generally referred to as “extreme environments.”

Extreme and hypersaline are situations where humans could not survive. Most of the Earth (the oceans) is an “extreme environment” where it is very cold (about 4°C), dark, and under high pressure. Many Archaea are well adapted to these environments, where they can grow to high numbers. Archaea constitute at least 34% of the prokaryotic biomass in some Antarctic coastal waters. In some hypersaline environments, the brine is red with archaeal pigments. Some archaea are symbionts in the digestive tracts of animals. Archaeal gene sequences have been found in soil and temperate and tropical ocean surface waters.

Thus, the Archaea are highly diverse with respect to morphology, reproduction, physiology, and ecology. Although best known for their growth in anoxic, hypersaline, and high-temperature habitats they also inhabit marine arctic, temperate, and tropical waters. Their RNA, ribosomes, elongation factors, RNA polymerases, and other components distinguish Archaea from Bacteria and eukaryotes. Much of archaeal metabolism appears similar to that of other organisms, but the Archaea differ with respect to glucose catabolism, pathways for CO₂ fixation, and the ability of some to synthesize methane.

Archaeal Cell Walls

The Archaeal cell wall, like the bacterial cell wall, is a semi-rigid structure which provide protection to the cell from the environment and from the internal cellular pressure. The cell walls of bacteria typically contain peptidoglycan, but it is absent in Archaea. 

The chemistry of Archaeal cell walls is different from that of Eubacteria. Archaea lack the muramic acid and D-amino acids that make up peptidoglycan. Thus they resist attack by lysozyme and β lactam antibiotics such as penicillin. 

Archaeal cell walls stain either Gram positive or Gram negative, depending on the thickness and mass of cell wall. 

Gram positive Archaea have a variety of complex polymers in their cell wall. Methanobacterium and some other methanogenic archaea have pseudomurein (a peptidoglycan-like polymer that is cross-linked with L-amino acids), N-acetyl talosaminuronic acid instead of N-acetyl muramic acid and β (1- 3) glycosidic bonds instead of β (1-4) glycosidic bonds. Methanosarcina and Halococcus lack pseudomurein and contain complex polysaccharides similar to the chondroitin sulfate of animal connective tissue. Other heteropolysaccharides are also found in Gram positive cellwalls. 

Gram negative Archaea have a layer of protein or glycoprotein (20-40 mm thick) outside their plasma membrane. Some methanogens (Methanolobus), Halobacterium, extreme thermophiles (Sulfolobus, Thermoproteus, Pyrodictium) have glycoproteins in their walls. Other methanogens (Methanococcus, Methanomicrobium, Methanogenium) and extreme thermophile Delsuphurococcus have protein walls.

Structure, function and chemical composition of archaeal cell membranes

One of the most distinctive archaeal features is their membrane lipids. 

Archaeal membrane lipids differ from those of other organisms in having glycerol connected to branched chain hydrocarbons by ether links. 

Bacterial and eukaryotic lipids have glycerol connected to fatty acids by ester bonds.

    Sometimes, two glycerol groups are linked to form long tetraethers. Usually, the diether hydrocarbon chains are 20 carbons in length, and the tetraether chains are 40 carbons. Cells adjust the  length of the tetraethers by forming pentacyclic rings. 

    Such pentacyclic rings are used by thermophilic archaea to help maintain the delicate balance of the membrane at high temperatures. Biphytanyl chains contain 1 to 4 cyclopentyl rings. 

        Phosphate, sulfur- and sugar-containing groups can be attached to the third carbons of the diethers and tetraethers, making them polar lipids -phospholipids, sulfolipids, and glycolipids.  These predominate in the membrane, making up 70 to 93% of the membrane lipids.  The remaining lipids (7-30%) are nonpolar and are usually derivatives of squalene.

Archaeal membranes may contain a mix of diethers, tetraethers and other lipids. These lipids are combined in different ways to yield membranes of various rigidity and thickness. A regular bilayer membrane is formed when C₂₀ diethers are used. A much more rigid monolayer membrane is formed when the membrane is constructed of C₄₀ tetraethers. 

The membranes of extreme thermophiles such as Thermoplasma and Sulfolobus contain  tetraether monolayers which provide stability. Archaea that live in moderately hot environments have a mixed membrane containing some regions with monolayers and some with bilayers.

Differences between Archaebacteria and Eubacteria

 Eu Bacteria and Archaea – The Major Differences  

	ArchaeaBacteria	EuBacteria
	-Ancient bacteria-	-True bacteria-
Complexity	Simple in their organization	Complex than archaebacteria
Habitat	Can sustain in extremely harsh environment such as oceans, hot springs, marshlands, hot springs and gut of animals	Found everywhere - soil, organic matter, earth’s crust, water, bodies of animals and plants, radioactive wastes, hot springs
Size	0.1-15 μm in diameter	0.5-5 μm in diameter
Shape	spheres, rods, plates, spiral, flat or square-shaped	cocci, bacilli, vibrio, rods, filaments or spiral in shape
Cell wall	Pseudopeptidoglycan	Lipopolysaccharide/ Peptidoglycan with muramic acid
Membrane lipids	Ether-linked, branched, aliphatic chains, containing D-glycerol phosphate	Ester-linked, straight chains of fatty acids, containing L-glycerol phosphates
RNA	Consists of single RNA	Three types of RNA
RNA polymerase	Complex subunit pattern	Simple subunit pattern
Introns (a long stretch of noncoding DNA found between exons (or coding regions) in a gene)	Present in archaebacteria	Absent in eubacteria
Metabolism	Methanogenesis- exhibit neither glycolysis nor Kreb’s cycle	Autotrophy, Aerobic and Anaerobic Respiration, Fermentation and Photosynthesis-exhibit both glycolysis and Kreb’s cycle
Reproduction and Growth	Asexual Reproduction, by fragmentation, budding and binary fission	Other than binary fission, budding and fragmentation, eubacteria can produce spores in order to remain dormant during unfavorable conditions
Types	Methanogens, halophiles and thermophiles	Gram positive and Gram negative
Examples	Halobacterium, Thermoproteus, Pyrobaculum, Thermoplasma and Ferroplasma	Mycobacteria, Bacillus, E. coli, Pseudomonas, Clostridium etc

 

Thus, in short, Archaebacteria are called ancient bacteria whereas the eubacteria are called true bacteria. Eubacteria are usually found in soil, water, living in and on of large organisms. Eubacteria are divided into two groups known as gram positive and gram negative bacteria. Archaebacteria are found in salt brines, ocean depths and hot springs.  Three types of archaebacteria are found: methanogens, halophiles and thermoacidophiles.

The Archaea (Archaebacteria)

• Archaea are ancient bacteria - believed to have evolved just after the evolution of first life on earth.

• Archaea are prokaryotic cells. The cell walls of Archaea contain no peptidoglycan, hence Archaea are not sensitive to some antibiotics that affect the Bacteria.

• Archaea have membranes composed of branched hydrocarbon chains (many also containing rings within the hydrocarbon chains) attached to glycerol by ether linkages. 

• The ether-containing linkages in the Archaea membranes is more stable than the ester-containing linkages in the Eubacteria  and are better able to withstand higher temperatures and stronger acid concentrations. 

• Archaea often live in extreme environments and include methanogens, extreme halophiles, and hyperthermophiles.

• Archaea contain rRNA that is unique to the Archaea, distinctly different from the rRNA of Bacteria and Eukarya.

  Archaea are found in Volcanic hot springs- Grand Prismatic Spring of Yellowstone National Park

                            The very cold and ultra-salty Deep Lake in East Antarctica is home to haloarchaea.

Hydrothermal vents on the ocean floor, where the surrounding water can reach over 300° Celsius, are home  for some archaeal species.

The Bacteria (Eubacteria)

Bacteria (also known as eubacteria or "true bacteria") are prokaryotic cells that are common in human daily life. Eubacteria can be found almost everywhere and serve as antibiotic producers and food digesters, pathogens etc. 

Bacteria are prokaryotic cells. They have membranes composed of unbranched fatty acid chains attached to glycerol by ester linkages. 

The cell walls of Bacteria contain peptidoglycan. Bacteria are sensitive to  antibacterial antibiotics.

Bacteria contain rRNA that is unique.

Bacteria include mycoplasmas, cyanobacteria, Gram-positive bacteria, and Gram-negative bacteria.

Interesting Read

https://www.ck12.org/c/biology/archaea/lesson/Introduction-to-Archaea-Advanced-BIO-ADV/

https://www.science.org.au/curious/earth-environment/what-are-archaea

Anoxygenic vs. oxygenic photosynthesis with reference to photosynthesis in cyanobacteria, green bacteria and purple bacteria

 

Anoxygenic and oxygenic photosynthesis differ in their electron donors, byproducts, and bacteriochlorophyll pigments, with anoxygenic types like green sulfur bacteria (GSB) using electron donors like hydrogen sulfide to produce elemental sulfur instead of oxygen. Oxygenic photosynthesis, performed by cyanobacteria and plants, uses water as an electron donor and releases oxygen as a byproduct. 

The Light Reaction in Oxygenic Photosynthesis 

Phototrophic eucaryotes and the cyanobacteria carry out oxygenic photosynthesis, so named because oxygen is generated when light energy is converted to chemical energy. Central to this process, and to all other phototrophic processes, are light-absorbing pigments. In oxygenic phototrophs, the most important pigments are the chlorophylls.

 Several chlorophylls are found in eucaryotes, the two most important are chlorophyll a- absorption peak at 665 nm and chlorophyll b -absorption peak at 645 nm. Chlorophylls absorb primarily in the red and blue ranges and green light is transmitted. Consequently many oxygenic phototrophs are green in color.

 Other photosynthetic pigments also trap light energy. The most widespread of these are the carotenoids, usually yellowish in color. Carotene is present in cyanobacteria belonging to the genus Prochloron and most photosynthetic protists; fucoxanthin is found in protists such as diatoms and dinoflagellates. Red algae and cyanobacteria have photosynthetic pigments called phycobiliproteins-phycoerythrin is a red pigment and phycocyanin is blue (maximum absorption at 620 to 640 nm). 

Carotenoids and phycobiliproteins are often called accessory pigments because of their role in photosynthesis. Accessory pigments are important because they absorb light in the range not absorbed by chlorophylls (the blue-green through yellow range; about 470–630 nm). This light is very efficiently transferred to chlorophyll. In this way accessory pigments make photosynthesis more efficient over a broader range of wavelengths. In addition, this allows organisms to use light not used by other phototrophs in their habitat. For instance, the microbes below a canopy of plants can use light that passes through the canopy. Accessory pigments also protect microorganisms from intense sunlight, which could oxidize and damage the photosynthetic apparatus. 

Chlorophylls and accessory pigments are assembled in highly organized arrays called antennas, which creates a large surface area to trap as many photons as possible. An antenna has about 300 chlorophyll molecules. Light energy is captured in an antenna and transferred from chlorophyll to chlorophyll until it reaches a special reaction-center chlorophyll pair directly involved in photosynthetic electron transport.

 In oxygenic phototrophs, there are two kinds of antennas associated with two different photosystems. Photosystem I absorbs longer wavelength light (680 nm) and funnels the energy to a special chlorophyll a pair called P700. The term P700 signifies that this molecule most effectively absorbs light at a wavelength of 700 nm. Photosystem II traps light at shorter wavelengths (680 nm) and transfers its energy to the special chlorophyll pair P680.

 When the photosystem I antenna transfers light energy to the reaction-center P700 chlorophyll pair, P700 absorbs the energy and is excited; it donates its excited, high-energy electron to a specific acceptor, probably a special chlorophyll a molecule or an iron-sulfur protein. The electron is eventually transferred to ferredoxin and can then travel in either of two directions.

 In the cyclic pathway, the electron moves in a cyclic route through a series of electron carriers and back to the oxidized P700. The pathway is termed cyclic because the electron from P700 returns to P700 after traveling through the photosynthetic electron transport chain. PMF is formed during cyclic electron transport in the region of cytochrome b6 and used to synthesize ATP. This process is called cyclic photophosphorylation because electrons travel in a cyclic pathway and ATP is formed. Only photosystem I participates.

Cyclic photophosphorylation

 

Electrons also can travel in a noncyclic pathway involving both photosystems. P700 is excited and donates electrons to ferredoxin as before. In the noncyclic route, however, reduced ferredoxin reduces NADP_ to NADPH. Because the electrons contributed to NADP cannot be used to reduce oxidized P700, photosystem II participation is required. It donates electrons to oxidized P700 and generates ATP in the process. The photosystem II antenna absorbs light energy and excites P680, which then reduces pheophytin a. Pheophytin a is chlorophyll a in which two hydrogen atoms have replaced the central magnesium. Electrons subsequently travel to the plastoquinone pool and down the electron transport chain to P700. Although P700 has been reduced, P680 must also be reduced if it is to accept more light energy. Thus, H₂O can be used to donate electrons to P680 resulting in the release of oxygen. ATP is synthesized by noncyclic photophosphorylation. One ATP and one NADPH are formed when two electrons travel through the noncyclic pathway.

 

 

In cyanobacteria, photosynthetic light reactions are located in thylakoid membranes within the cell.

The dark reactions require three ATPs and two NADPHs to reduce one CO₂ and use it to synthesize carbohydrate (CH₂O). 

CO₂ + 3ATP + 2NADPH +2H+ H2O ⎯⎯→ (CH₂O) + 3ADP + 3Pi +2NADP_ 

The noncyclic system generates one NADPH and one ATP per pair of electrons; therefore four electrons passing through the system will produce two NADPHs and two ATPs. A total of 8 quanta of light energy (4 quanta for each photosystem) is needed to propel the four electrons from water to NADP_. Cyclic photophosphorylation operates independently to generate the extra ATP. This requires absorption of another 2 to 4 quanta.

 

Thus, around 10 to 12 quanta of light energy are needed to reduce and incorporate one molecule of CO₂ during photosynthesis.

The Light Reaction in Anoxygenic Photosynthesis

 Certain bacteria carry out a second type of photosynthesis called anoxygenic photosynthesis. This phototrophic process derives its name from the fact that water is not used as an electron source and therefore O₂ is not produced. 

The process also differs in terms of the photosynthetic pigments used, the participation of just one photosystem, and the mechanisms used to generate reducing power. Three groups of bacteria carry out anoxygenic photosynthesis: phototrophic green bacteria, phototrophic purple bacteria, and heliobacteria. 

Sulfur bacteria use hydrogen sulfide which they oxidize to elemental sulfur, while non-sulfur bacteria can use a wider range of compounds, including some organic molecules (lactate, succinate etc)

 

 

Many differences found in anoxygenic phototrophs are due to their having a single photosystem. Because of this, they are restricted to cyclic electron flow and are unable to produce O₂ from H₂O. Indeed, almost all anoxygenic phototrophs are strict anaerobes.

 Anoxygenic phototrophs have photosynthetic pigments called bacteriochlorophylls. The absorption maxima of bacteriochlorophylls (Bchl) are at longer wavelengths than those of chlorophylls. Bacteriochlorophylls a and b have maxima in ether at 775 and 790 nm, respectively.

This shift of absorption maxima into the infrared region better adapts these bacteria to their ecological niches.

 

Purple bacteria

Purple bacteria has only one photosystem (similar to Photosystem II) with Pheophytin-Quinone/Type II Reaction Center.  Bacteriochlorophyll molecules absorb light energy, which is transferred to a reaction center called P₈₇₀. This process takes place in anoxic (oxygen-free) conditions, which are common in aquatic environments where these bacteria are found.

 

When bacteriochlorophyll P870 is excited, it donates an electron to bacteriopheophytin. Electrons then flow to quinones (Pheophytin-Quinone/Type II Reaction Center) and through an electron transport chain back to P870.  PMF created is used to drive ATP synthesis.

Purple bacteria

Both green and purple bacteria lack two photosystems, but the purple bacteria have a photosynthetic apparatus similar to photosystem II, whereas the green sulfur bacteria have a system similar to photosystem I.

 

Green bacteria

Photosynthesis in green sulfur bacteria (GSB) is similar to purple bacteria. Green sulfur bacteria has Fe-S Reaction Center (Type I Reaction Center). Excitation causes an electron to pass through a quinone (MK, Menaquinone) to the cytochrome bc1 complex and back to P 840.  PMF created is used for ATP synthesis.

Green Bacteria

GSB possess specialized light-harvesting chlorosomes containing bacteriochlorophyll, which are efficient at absorbing light energy, even in low-light conditions. Chlorosomes consist of bacteriochlorophyll (BChl) pigments, carotenoids, quinones, and proteins in a lipid-monolayer envelope. They efficiently capture light energy, even at very low light levels, and funnel it to the reaction center for photosynthesis.  They are essential for GSB to grow in extremely low-light environments, such as the deep parts of lakes and oceans. 

Anoxygenic phototrophs also require reducing power (NAD[P]H or reduced ferredoxin) for CO₂ fixation and other biosynthetic processes.

They generate reducing power in at least three ways, depending on the bacterium. Some have hydrogenases that are used to produce NAD(P)H directly from the oxidation of hydrogen gas.

Others, such as the photosynthetic purple bacteria, must use reverse electron flow to generate NAD(P)H. In this mechanism, electrons are drawn off the photosynthetic electron transport chain and “pushed” to NAD(P)_ using PMF or the hydrolysis of ATP. Electrons from electron donors such as hydrogen sulfide, elemental sulfur, and organic compounds replace the electrons removed from the electron transport chain in this way.

Phototrophic green bacteria and heliobacteria also must draw off electrons from their electron transport chains. Because the reduction potential of the component of the chain where this occurs is more negative than NAD_ and oxidized ferredoxin, the electrons flow spontaneously to these electron acceptors. Thus, these bacteria exhibit a simple form of noncyclic photosynthetic electron flow.

 

 

Carbon fixation

The thylakoids of cyanobacteria use the energy of sunlight to drive photosynthesis, a process where the energy of light is used to synthesize organic compounds from carbon dioxide..

The dark reaction, or Calvin cycle, is the second stage of photosynthesis where carbon dioxide is fixed into organic compounds like glucose. This process is independent of direct light but requires the ATP and NADPH produced during the light-dependent reactions. Microorganisms use these to convert atmospheric CO₂ into sugars for energy and growth with the involvement of the enzyme RuBisCO. Cyanobacteria have microcompartments known as carboxysomes, which store this CO₂-fixing enzyme, RuBisCO.

In eukaryotes like algae, the dark reaction occurs in the stroma of the chloroplast. In prokaryotes like cyanobacteria, it happens in the cytoplasm. It is "light-independent" because it doesn't use light energy directly, but it relies on the products (ATP and NADPH) of the light-dependent reactions, meaning it can only happen when light is available for the first stage to occur. 

Calvin cycle is driven by a series of enzyme-catalyzed reactions. It ultimately produces glucose from carbon dioxide, with ADP and NADP+ being recycled back to the light reactions

 

In short,

Phototrophs use light to generate a proton motive force (PMF), which is then used to synthesize ATP by a process called photophosphorylation (photo phos). The process requires light-absorbing pigments. When the pigments are chlorophyll or bacteriochlorophyll, the absorption of light triggers electron flow through an electron transport chain, accompanied by the pumping of protons across a membrane.

 The electron flow can be either cyclic (dashed line) or noncyclic (solid line), depending on the organism and its needs. Many phototrophs are autotrophs and must use much of the ATP and reducing power they make to fix CO2. 

In oxygenic photosynthesis, eucaryotes and cyanobacteria trap light energy with chlorophyll and accessory pigments and move electrons through photosystems I and II to make ATP and NADPH (the light reactions). 

Cyclic photophosphorylation involves the activity of photosystem I alone and generates ATP only. In noncyclic photophosphorylation photosystems I and II operate together to move electrons from water to NADP⁺ producing ATP, NADPH, and O₂

 Anoxygenic phototrophs differ from oxygenic phototrophs in possessing  bacteriochlorophyll and having only one photosystem. They use cyclic photophosphorylation to make ATP. They are anoxygenic because they do not use water as an electron donor for electron flow though the photosynthetic electron transport chain.

Purple sulfur bacteria contribute to nutrient cycling and play a significant role in primary production. These organisms contribute to the carbon cycle through carbon fixation and the phosphorus cycle & the iron cycle. Although purple sulfur bacteria are found in the anoxic layer of their habitat, they supply inorganic nutrients to the above oxic layer. Purple sulfur bacteria act as a source of food to other organisms.

Why are Cyanobacteria the most significant group of photosynthetic microorganisms?

Cyanobacteria are the largest group of photosynthetic prokaryotes, which harvest solar energy and perform photosynthesis through chlorophyll-a by fixing CO₂ and generating O₂. Cyanobacteria are important in global carbon fixation, and reduce atmospheric CO₂ levels. In addition to chlorophyll-a (green pigment), cyanobacteria produce accessory photosynthetic pigments carotenoids, which are protect against photooxidative damages & blue and red pigments known as phycobilin (phycocyanin (PC) and phycoerythrin (PE)), which enable them to grow under low-light conditions. Some cyanobacteria can also fix atmospheric nitrogen.