Phototrophy & Phototrophic Microorganisms

 Microorganisms derive energy not only from the oxidation of inorganic and organic compounds, but also from light energy, which they capture and use to synthesize ATP and reduce power (e.g., NADPH).

The process by which light energy is trapped and converted to chemical energy is called photosynthesis. Photosynthesis is one of the most significant metabolic processes on Earth because almost all our energy is ultimately derived from solar energy. It provides photosynthetic organisms with the ATP and reducing power necessary to synthesize the organic material required for growth. In turn these organisms serve as the base of most food chains in the biosphere.  

Photosynthesis carried out by a variety of organisms, both eucaryotic and bacterial, is also responsible for replenishing our supply of O₂.

 We mostly associate photosynthesis with the larger, more obvious plants, but over half the photosynthesis on Earth is carried out by microorganisms.

 Photosynthesis as a whole is divided into two parts. In the light reactions light energy is trapped and converted to chemical energy. This energy is then used to reduce or fix CO₂ and synthesize cell constituents in the dark reactions.

 Phototrophic Microorganisms 

Phototrophic organisms are a diverse group of organisms which  carry out photosynthesis. They synthesize their own food and provide energy and nutrients to other organisms. They are important in a variety of ecological and biogeochemical processes and play a vital role in our ecosystem.

 Phototrophic microorganisms are broadly classified as either oxygenic or anoxygenic, based on their ability to produce oxygen during photosynthesis. Major groups include Cyanobacteria (oxygenic) and various anoxygenic bacteria, such as purple bacteria (Rhodospirillineae), green bacteria (Chlorobiineae), and aerobic anoxygenic phototrophs (AAPs). 

 Beyond their photosynthetic pathway, microorganisms are grouped by how they obtain carbon: 

·        Photoautotrophs: 

Use sunlight to convert carbon dioxide into organic compounds, making their own food. This includes most oxygenic phototrophs and many anoxygenic ones.

·        Photoheterotrophs: 

Convert light into energy but require organic compounds from their environment to make their own food.

 Oxygenic Phototrophs

These organisms produce oxygen as a byproduct of photosynthesis. Eg, Cyanobacteria, eukaryotic microalgae

 1. Cyanobacteria: Often referred to as blue-green algae, these prokaryotes were crucial in forming Earth's oxygen-rich atmosphere. 

·        Cyanobacteria can perform oxygenic photosynthesis – producing oxygen from CO₂ and water. Due to their chlorophyll pigments, they are typically greenish blue in color and therefore also known as blue-green algae. They are found in a variety of aquatic and terrestrial habitats, including even extreme locations like hot springs and deserts.

·        Cyanobacteria play a crucial role in the global carbon cycle and have had a significant impact on the evolution of our planet's atmosphere. As one of the oldest organisms on Earth, they were responsible for releasing oxygen into the atmosphere, which initiated the transformation of the atmosphere and created the environment in which we live today.

·        Cyanobacteria are commonly used in research, both as model organisms for studying photosynthesis and as potential sources of biofuels and other useful compounds.

 2. Eukaryotic Microalgae: These are microscopic, single-celled eukaryotic organisms that perform oxygenic photosynthesis. eg. diatoms, dinoflagellates. green algae etc

 Eukaryotic microalgae perform photosynthesis to convert sunlight, carbon dioxide, and water into carbohydrates (like sugars) and oxygen. 

Photosynthesis takes place inside specialized organelles called chloroplasts, which are the site of both light and dark reactions. Chlorophyll, a green pigment, is crucial for absorbing sunlight. Other pigments may also be involved depending on the specific microalgae species.  Oxygen is a significant byproduct of the light-dependent reactions, released into the environment. The carbohydrates produced are the building blocks for vital organic molecules, including lipids and proteins, forming the microalgae's biomass. 

Microalgae are highly efficient photo-synthesizers and play a critical role in the global carbon cycle and oxygen production. 

Algal Blooms

Under conditions of high nutrient availability from sources like agricultural runoff or wastewater, these algae undergo rapid overgrowth, known as an algal bloom. These blooms can discolor water and, when the algae die, their decomposition consumes oxygen, creating "dead zones" harmful to other aquatic life, though some blooms are harmless

Excessive levels of nutrients, particularly nitrogen and phosphorus from human activities like farming and wastewater discharge, lead to proliferation of microalgae, resulting in an algal bloom. Besides nutrient enrichment, climate change can also cause blooms.

Blooms can be recognized by the visible change in the water's color due to the algae's pigments. As the microalgae in the bloom die and decompose, the process consumes large amounts of dissolved oxygen resulting in the death of fish and other aquatic life.  Some algal blooms, known as harmful algal blooms (HABs), produce potent toxins that can harm or kill other organisms and negatively impact human health and economies. 

 

Anoxygenic Phototrophs

These organisms perform photosynthesis without producing oxygen, as water is not their electron donor. eg, purple bacteria, green bacteria

 

1. Purple Bacteria:  Also known as Rhodospirillineae, purple bacteria uses bacteriochlorophyll and can be further divided into purple sulfur and nonsulfur bacteria. 

Purple bacteria, are a diverse group of phototrophic bacteria that perform anoxygenic photosynthesis, which means they do not produce oxygen. They are called purple bacteria because their main pigments (bacteriochlorophyll pigment a or b located on chromatophores and plasma membranes) give them a purple or red color. They are further divided into: the purple sulfur bacteria and the purple non-sulfur bacteria.

·       The main difference between sulfur and non-sulfur purple bacteria is the electron donor they use during photosynthesis. Purple sulfur bacteria use reduced sulfur compounds, such as hydrogen sulfide or thiosulfate, as electron donors for photosynthesis. In contrast,  purple non-sulfur bacteria use organic compounds, such as lactate or succinate, as electron donors.

·         Purple sulfur bacteria use sulfide or thiosulfate as their electron donor during photosynthetic pathways. They oxidize sulfide to elemental sulfur, which accumulates as internal globules or granules within the cell. The sulfur deposition occurs inside the bacterial cell.  The purple sulfur bacteria can be used to reduce the concentration of harmful compounds like methane and hydrogen sulfide.

·       Sulfur purple bacteria are usually found in environments where sulfur compounds are abundant, such as hot sulfur springs, swamps, and sediments, while non-sulfur purple bacteria are found in a wider range of environments, including freshwater ponds and lakes, soils, and microbial mats.

Eg., Allochromatium and Thiocapsa

 

2.     Green Bacteria: This group, including the Chlorobiineae, uses bacteriochlorophyll but has different pigments than purple bacteria.

·       Green sulfur bacteria are anoxygenic photosynthetic bacteria with a unique photosynthetic apparatus adapted to low light and anaerobic conditions. Most of them are nonmotile and obligate anaerobes. 

        Their name derives from their characteristic green color, which is due to the presence of chlorosomes – organelles that contain bacteriochlorophyll pigments. They have bacteriochlorophyll pigments c, d, a or e.        

·       They use sulphide as their ultimate electron donor for photosynthesis. Thus, they can thrive well in sulfur-rich environments with low light intensities. Most of these bacteria can reduce nitrogen to ammonia. This ammonia is later used to synthesise amino acids.

·       They are found in a variety of environments and can use different electron donors for photosynthesis, including hydrogen sulfide and elemental sulfur. 

·       These bacteria can synthesize large amounts of sulfur granules, which protect them from oxidative stress and give them a distinctive appearance, visible under a microscope. The sulfur granules are stored outside the cell as a byproduct of their anaerobic photosynthesis, distinguishing them from purple sulfur bacteria that store them intracellularly. 

·       They are also known for forming complex microbial communities in sulfide-rich environments, called mats or biofilms, playing crucial roles in biogeochemical cycling and ecosystem function.

Eg., Chlorobium tepidum and Chlorobium vibrioforme 

Purple Sulfur bacteria store sulfur inside their cells (intracellular storage)  allowing immediate access and faster regulation, when external availability becomes limited. Also, intracellular location protect the granules from being washed away in aquatic environments

Green Sulfur Bacteria store sulfur outside their cells (extracellular storage) thus avoiding cluttering and providing more cytoplasmic space for other metabolic activities. Extracellular storage may facilitate sharing or recycling of sulfur among nearby microbes in dense microbial mats or sediments.

    Both strategies offer adaptive advantages suited to their ecological niches, especially in anaerobic, sulfur-rich environments like lakes, sediments, and hot springs.

3.     Aerobic Anoxygenic Phototrophic Bacteria (AAPs): 

        These bacteria perform anoxygenic photosynthesis but require oxygen. They are mostly marine or freshwater genera. AAPB contain bacteriochlorophyll a as its main light harvesting pigment,. 

    Aerobic anoxygenic phototrophic bacteria are photoheterotrophic, meaning they obtain their carbon from organic compounds. They exist in a variety of aquatic environments and may constitute over 10% of the open ocean microbial community. AAPB are thought to play an important role in carbon cycling. 

1.      Acidobacteria and Heliobacteria 

 Heliobacteria perform anoxygenic photosynthesis and form endospore abilities, while Acidobacteria is a vast phylum with diverse metabolic capabilities, primarily found in soil.  

Acidobacteria are primarily chemoheterotrophs, though some such as Chloracidobacterium thermophilum are capable of photosynthesis. They are ubiquitous and abundant, especially in soil ecosystems, peatlands and mineral-rich environments. They are motile eg., Acidobacterium capsulatum and non-spore formers 

Heliobacteria use a unique form of chlorophyll, chlorophyll g as a light-harvesting pigment unlike other photosynthetic bacteria that use bacteriochlorophyll. They are obligate anaerobes and are typically found in anoxic environments such as freshwater sediments or soil. Heliobacteria are important members of the microbial community in these environments, where they play important roles in the cycling of nutrients and carbon. 

They are obligate anaerobes that are able to capture energy from light by photophosphorylation to produce ATP. Water is not used as an electron donor and, therefore, the production of oxygen is non-existent.

 Both phyla are important to soil ecology : 

·        Both are significant components of soil microbial communities. Acidobacteria are one of the most abundant phyla in soil, and heliobacteria are widespread in anaerobic soils.

·        Members of both groups are involved in biogeochemical cycles, including carbon and nitrogen cycling. Heliobacteria are known for their nitrogen-fixing capabilities.

·        The two phyla, show vast metabolic diversity found within bacteria. Acidobacteria possess a wide array of genes for degrading complex compounds, while heliobacteria use a unique photosynthetic process.  

Ecological Importance of Phototrophic Organisms 

Phototrophic organisms are essential for maintaining the balance of ecosystems and play a crucial role in the global carbon cycle. By converting light energy into chemical energy, phototrophic organisms produce organic matter that is then consumed by other organisms. This process forms the base of most food chains and supports the growth of all other living things in the ecosystem. 

Photosynthetic organisms are also responsible for producing the oxygen that we breathe. Oxygenic photosynthesis has played a critical role in the evolution of life on Earth, as oxygen is required by many organisms for respiration and other metabolic processes.

 In addition to producing oxygen and organic matter, phototrophic organisms also play a significant role in regulating the Earth's climate. By removing carbon dioxide from the atmosphere through photosynthesis, these organisms help to mitigate the effects of climate change.

 Phototrophic organisms have significant economic importance and are used in a wide range of applications in industry, agriculture, and medicine.

·       Plants, algae, and other phototrophs are the primary source of food for humans and many other animals. In addition, photosynthetic bacteria are used in the production of certain types of food, such as fermented dairy products and pickled vegetables. 

·       Photosynthetic organisms are also used in the production of biofuels. Biofuels, such as ethanol and biodiesel, are produced from organic matter, such as crops and algae, that have been grown using photosynthesis. These biofuels are considered to be more sustainable than traditional fossil fuels, as they produce fewer greenhouse gas emissions and are renewable.

·       In medicine, photosynthetic organisms are used in a variety of applications, such as the production of antibiotics and other pharmaceuticals. The ability of photosynthetic organisms to carry out complex biochemical reactions makes them valuable tools in biotechnology and bioengineering. They are an important area of research for the development of new and innovative applications.

 Modern photobioreactors will help to conduct meaningful research with these interesting organisms. Photobioreactors are closed systems designed to grow photosynthetic microorganisms, such as algae and cyanobacteria, under controlled conditions of light, temperature, and nutrient supply.  These systems are used for a variety of purposes, such as the production of biomass for food, feed, or biofuels, the removal of pollutants from wastewater, and the cultivation of microorganisms for research or biotechnology applications.

 

Effect of temperature on growth of microorganisms- TDT and TDP

 Aim

To determine the thermal death time and thermal death point of the given test organism

Principle

Temperature is one of the most important physical factors influencing the growth of microorganisms. Bacteria unlike eukaryotes lack homeostatic mechanism and they do not regulate the heat generated by metabolism thus are affected readily by changes in temperature. Enzymatic reactions have maximum efficiency at optimum temperature which varies with organisms. For every 10⁰C rise in temperature, there is 2 fold increase in the rate of enzyme catalyzed reactions for a limited range of temperature. At high temperatures, proteins are irreversibly denatured and there is a total enzyme destruction. At low temperature, the enzyme reactions are merely inactivated and are thus less harmful.

Bacteria are divided into three major groups with respect to their temperature requirements:

1)     Psychrophiles with optimum temperature between 0 and 20⁰C

2)     Mesophiles with optimum temperature between 20 and 40⁰C

3)     Thermophiles with optimum temperature between 40 and 60⁰C

Normally, the lethal range of temperature for bacteria is between 50 and 100⁰C. Time of exposure is a vital factor in assessing the lethal effect of high temperature on bacterial cells. Determination of thermal death time (TDT) and thermal death point (TDP) are done for this purpose.

1)     Thermal death point (TDP) – Temperature at which an organism is killed in 10 minutes of exposure. Lethal action of heat has a temperature-time relationship. Thermal death point is done to determine the degree of heat tolerance of the organism. Some factors such as pH, moisture, composition of media and age of cells influence TDP.

     2)     Thermal death time (TDT) – The time required to kill cells/spores at a given temperature. The length of time that the microbes are exposed to heat contributes to lethal effect. This is assessed by exposing cells to fixed temperature which is determined as thermal death time for increasing periods of time.

 

Materials required

Culture of  E. coli, nutrient tubes, nutrient agar plates, water bath, incubator etc

Methodology

1)     Thermal death point (TDP)

 Into each of the sterile test tubes, 5 ml of sterile nutrient broth was dispensed and tubes were marked 40, 50, 60, 70, 80, 90 and 100 ⁰C for different organisms. A loopful of culture was inoculated into the respective tubes and incubated for ten minutes at each temperature. They were then plated on nutrient agar plates and incubated at 37⁰C for 24 hours. The temperature above which the organism was completely killed and did not grow was noted and this was determined as the thermal death point of the organism.

2)     Thermal death time (TDT)

Each sterile tube containing 5 ml nutrient broth was inoculated with loopful of cultures and incubated at their thermal death point (TDP). A loopful of cultures from the tubes were taken at regular intervals of 3 minutes each, starting from 0 minutes till 15 minutes and plated on to nutrient agar followed by incubation at  37⁰C for 24 hours. The time period above which the organisms were completely killed and did not grow was determined as thermal death time (TDT).

Result

The thermal death time (TDT)  and thermal death point (TDP) for E. coli was 15 minutes at 80⁰C.

Hydrogen oxidation

Hydrogen-oxidizing bacteria are a group of facultative autotrophs that can use hydrogen as an electron donor. They oxidise H₂ (electron donor) and reduce O₂ (electron acceptor) via “knallgas” reaction.

"Knallgas” reaction is the reduction of O₂ with H₂. This reaction yields energy which is used in CO₂ fixation. Hydrogen oxidizing bacteria are also called Knallgas-bacteria. These include Hydrogenobacter thermophilus, Hydrogenovibrio marinus, and Helicobacter pylori. 

There are both Gram positive and Gram negative knallgas bacteria. They can be aerobes and anaerobes. Aerobic bacteria use hydrogen as an electron donor and oxygen as an acceptor while anaerobes use hydrogen as an electron donor and sulphate or nitrogen dioxide as electron acceptors.

Hydrogen is oxidized by a membrane-bound hydrogenase causing proton pumping along with electron transfer to various quinones and cytochromes. In many organisms, a second cytoplasmic hydrogenase is used to generate reducing power in the form of NADH, which is  used to fix carbon dioxide via the Calvin cycle.

Thus, many organisms are capable of using hydrogen (H₂) as a source of energy using hydrogenase enzyme which help in hydrogen oxidation. Most grow best under microaerophilic conditions because the hydrogenase enzyme used in hydrogen oxidation is inhibited by the presence of oxygen. Oxygen is still needed as a terminal electron acceptor. Typically, oxygen levels of about 5-10% support best growth of these bacteria.

Thus, the use of hydrogen as an electron donor and the ability to synthesize organic matter characterize the hydrogen-oxidizing bacteria.

Importance of Hydrogenase enzymes 

Hydrogenase enzyme is crucial for energy generation in hydrogen-oxidizing chemolithotrophs that use molecular hydrogen (H₂) as an electron donor. These enzymes catalyze the oxidation of H₂:

$$\; \; \; \; \; \; \; \; \; \; \; \; \; H₂\to 2{\text{H}}^{+ }+ 2e^{-}$$

The released electrons (e⁻) are then passed into the electron transport chain (ETC). 

As electrons flow through the ETC:

• A proton motive force (PMF) is generated across the membrane.

• This PMF drives ATP synthesis via ATP synthase (oxidative phosphorylation).

In addition, hydrogenase enzymes also contribute to the generation of reducing power (e.g., NADH or NADPH), which is essential for:

• Carbon fixation (e.g., via the Calvin cycle in autotrophs)

• Other biosynthetic reactions

Hydrogen oxidizing bacteria are both gram-positive and gram-negative. The best studied genera of this group of bacteria are Ralstonia, Pseudomonas, Paracoccus, and Alkaligenes; others are Acidovorax, Aquaspirillum, Hydrogenophaga, Hydrogenobacter, Bacillus, Aquifex, and Mycobacterium. Hydrogen-oxidizing bacteria have been isolated from a variety of environments, including fresh waters, sediments, soils, activated sludge, hot springs, hydrothermal vents etc. 

Almost all hydrogen-oxidising bacteria are facultative chemoautotrophs, i.e, they can also grow chemoheterotrophically. This means that the hydrogen-oxidising bacteria can switch between chemoautotrophic and chemoheterotrophic (chemoorganotrophic) modes of metabolism and generally do so whenever required. 

This is a major distinction between hydrogen oxidising bacteria and many sulphur-oxidising bacteria or nitrifying bacteria; most of the isolates from latter two groups are obligate chemoautotrophs.

Hydrogen-oxidizing organisms, such as Cupriavidus necator (formerly Ralstonia eutropha), often inhabit oxic-anoxic interfaces in nature to use hydrogen produced by anaerobic fermentative organisms while still maintaining a supply of oxygen.

Helicobacter pylori 

H. pylori,  is a Gram-negative, microaerophilic bacterium found in the stomach, identified in 1982 by Barry Marshall and Robin Warren. They found that it was present in patients with chronic gastritis and gastric ulcers, conditions that were not previously believed to have a microbial cause. It is also linked to the development of duodenal ulcers and stomach cancer. Over 80 percent of individuals infected with the bacterium are asymptomatic.  

More than 50% of the world’s population harbor H. pylori in their upper gastrointestinal tract.

Helicobacter pylori is a hydrogen oxidizing (H₂-oxidizing) bacterium, also known as a Knall-gas bacterium. It utilizes hydrogenase to oxidize molecular hydrogen, produced by other intestinal bacteria. This oxidation process generates energy for the bacterium, allowing it to respire and meet its metabolic needs. 

 This allows H. pylori to colonize the stomach and contributes to its ability to cause chronic inflammation and gastritis and stomach cancer.

While most H2-oxidizing bacteria can use carbon dioxide to fix carbon, H. pylori does not use the Calvin cycle. Instead, it uses organic carbon from its environment, making it a mixotroph. 

Hydrothermal vents

H₂ is an important electron donor in hydrothermal vents. Hydrogen oxidation represents a significant origin of energy in this environment  sufficient to perform ATP synthesis and autotrophic CO₂ fixation. So hydrogen-oxidizing bacteria form an important part of the ecosystem in deep sea habitats. The oxidation of sulphide and hydrogen is important among the main chemosynthetic reactions that take place in hydrothermal vents.

Uses

Given enough nutrients, H₂, O₂ and CO₂, many Knallgas bacteria can be grown quickly in vats using only a small amount of land area. 

For example, 

the polyhydroxybutyrate the bacteria produce can be used as a feedstock to produce biodegradable plastics in various eco-sustainable applications. 

Solar Foods is a startup that has sought to commercialize knallgas bacteria for food production, to grow a neutral-tasting, protein-rich food source for use in products such as artificial meat. 

Research studies have suggested that knallgas cultivation is more environmentally friendly than traditional agriculture.

 

Effect of aeration on growth of bacteria

 Aim

To study the effect of aeration on growth of bacteria

Principle

Microorganisms are classified into categories such as aerobic, facultative anaerobic, and anaerobic bacteria depending on their metabolic strategies influenced by the availability of oxygen. Aerobic bacteria or obligate aerobes, require oxygen for their growth and survival as they utilize oxygen as the final electron acceptor in their electron transport chain that yields a large amount of ATP. In contrast, anaerobic bacteria or obligate anaerobes do not require oxygen for growth and they rely on anaerobic respiration or fermentation, metabolic pathways that generate energy without oxygen by using alternative electron acceptors such as nitrate, sulfate, carbon dioxide or by relying solely on substrate-level phosphorylation. Facultative anaerobic bacteria possess metabolic flexibility to grow both in the presence or absence of oxygen.

Aeration is a critical factor in microbial growth, particularly for aerobic and facultative anaerobic microorganisms, as it directly impacts the availability of dissolved oxygen (DO) in the culture medium. Oxygen is essential for respiration and other metabolic processes in aerobic organisms, serving as the final electron acceptor in the electron transport chain.  For facultative anaerobic bacteria, even though they can survive and grow both with or without oxygen, aeration plays a significant role in their growth.  When oxygen is available, they preferentially switch to aerobic respiration since it is metabolically much more efficient. As a result, in well-aerated conditions, aerobes and facultative anaerobes will exhibit faster growth rates, reach higher cell densities, and utilize their carbon sources more completely than in unaerated conditions.

Materials required

1. Culture of E coli and Bacillus spp.

2. Sterile Nutrient broth flasks, Shaker incubator, other routine microbiological facilities

 

Procedure

1. Nutrient broth flasks were labelled with the organism to be inoculated, E coli and Bacillus spp.

2. One set of inoculated flasks was placed on an orbital shaker incubator set to 37°C with a shaking speed of 150-250 rpm. A second set of inoculated flasks was placed in a static incubator at 37°C. Both were incubated for 24-48 hours.

3. The flasks were observed, after incubation and the optical density at 600 nm was recorded

 

Observation and result

The bacterial species were found to have different growth patterns under aerated and static conditions.  For both organisms, maximum growth was observed under aerated conditions.

 

  

Effect of aeration on bacterial growth (left hand side)

Microorganism	OD at 600 nm
	Under static conditions	At shaker incubator 150 rpm
E coli	0.24	0.90
Bacillus sp	0.11	0.58

 

 

 

Aerobic and Anaerobic Chemolithotrophy

Chemolithotrophy is the oxidation of inorganic chemicals for the generation of energy. An inorganic compound is oxidized with the electrons being passed off to carriers in the electron transport chain.  A proton motive force is generated and is used to generate ATP with the help of ATP synthase. Reducing power NADPH also is produced in the process.

Electrons donors

Chemolithotrophs use a variety of inorganic compounds as electron donors, with the most common substances being hydrogen gas, sulfur compounds (such as sulfide and sulfur), nitrogen compounds (such as ammonium and nitrite), and ferrous iron.

• 
  Hydrogen oxidizers – these organisms oxidize hydrogen gas (H₂) with the use of a hydrogenase enzyme. Both aerobic and anaerobic hydrogen oxidizers exist, with the aerobic organisms eventually reducing oxygen to water. Several bacterial genera (eg. Alcaligenes, Hydrogenophaga & Pseudomonas spp.) can oxidize hydrogen gas to produce energy.                                                 H₂       2H⁺ + 2^e– 

• Sulfur oxidizers – as a group these organisms are capable of oxidizing a wide variety of reduced and partially reduced sulfur compounds such as hydrogen sulfide (H₂S), elemental sulfur (S⁰), thiosulfate (S₂O₃^2-), and sulfite (SO₃^2-). Sulfate (SO₄^2-) is frequently a by-product of the oxidation. Often the oxidation occurs in a stepwise fashion with the help of the sulfite oxidase enzyme. Thiobacillus can oxidize sulfur (S⁰), hydrogen sulfide (H₂S), thiosulfate (S₂O₃^2-), and other reduced sulfur compounds to sulfuric acid; therefore they have a significant ecological impact. Some of these are extraordinarily flexible metabolically. For example, Sulfolobus brierleyi and a few other species can grow aerobically as sulfur-oxidizing bacteria; in the absence of O₂, they carry out anaerobic respiration with molecular sulfur as an electron acceptor.

• Nitrogen oxidizers – the oxidation of ammonia (NH₃) is performed as a two-step process by nitrifying microbes such as Nitrosomonas and Nitrosospira, which oxidizes ammonia to nitrite (NO₂^-) and the second group Nitrobacter and Nitrococcus oxidizes the nitrite to nitrate (NO₃^-). The entire process is known as nitrification and is performed by small groups of aerobic bacteria and archaea, often found living together in soil or in water systems.

      

• Iron oxidizers – these organisms oxidize ferrous iron (Fe²⁺) to ferric iron (Fe³⁺). Since Fe²⁺ has such a positive standard reduction potential, the bioenergetics are not extremely favourable, even using oxygen as a final electron acceptor. Also, Fe²⁺ spontaneously oxidizes to Fe³⁺ in the presence of oxygen; so, the organisms must use it before that happens.   

    (Ferrous iron is a soluble form of iron that is stable at extremely low pH or under anaerobic conditions.  Under aerobic, moderate pH conditions ferrous iron is oxidized spontaneously to the ferric (Fe3+) form and is hydrolyzed abiotically to insoluble ferric hydroxide [Fe(OH)₃].)

There are three  types of ferrous iron-oxidizing microbes.

•  The first are acidophiles, such as the bacteria Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans, as well as the archaeon Ferroplasma.  These microbes oxidize iron in environments that have a very low pH and are important in acid mine drainage.
•  The second type of microbes oxidizes ferrous iron at near-neutral pH. These micro-organisms (Gallionella ferruginea or Leptothrix ochracea) live at the oxic-anoxic interfaces and are microaerophiles.
• The third type of iron-oxidizing microbes is anaerobic photosynthetic bacteria such as Rhodopseudomonas, which use ferrous iron to produce NADH for autotrophic carbon dioxide fixation.

 

Chemolithoautotrophs vs chemolithoheterotrophs

• Most chemolithotrophs are autotrophs (chemolithoautotrophs), where they fix atmospheric carbon dioxide to assemble the organic compounds that they need. These organisms require both ATP and reducing power (i.e. NADH/NADPH) in order to ultimately convert the oxidized molecule CO₂ into a greatly reduced organic compound, like glucose.
• Some microbes are chemolithoheterotrophs, using an inorganic chemical for their energy and electron needs, but relying on organic chemicals in the environment for their carbon needs. These organisms are also called mixotrophs, since they require both inorganic and chemical compounds for their growth and reproduction.

Thus the chemolithotrophs, are autotrophs and can use CO₂ as their carbon source.  Many will grow heterotrophically also, if they are supplied with reduced organic carbon sources like glucose or amino acids.

 Chemoautotrophs generally fall into several groups: methanogens, halophiles, sulfur oxidizers and reducers, nitrifiers, anammox bacteria, and thermoacidophiles.  Chemolithotrophic growth could be very fast, such as Thiomicrospira crunogena with a doubling time around one hour. 

Electron acceptors

    Chemolithotrophy can occur aerobically or anaerobically-the best electron acceptor is oxygen. Using a non-oxygen acceptor such as sulfate (SO₄²⁻), nitrate (NO₃⁻), elemental sulfur (S⁰), ferric iron (Fe³⁺) and CO₂ allows chemolithotrophs to have greater diversity and the ability to live in a wider variety of environments. 

Amount of ATP generated

Much less energy is available from the oxidation of inorganic molecules than from the complete oxidation of glucose to CO₂. As the electron donors and acceptors vary, the amount of ATP generated also vary widely for chemotrophs. An organism makes typically 32 molecules of ATP per glucose molecule using aerobic respiration, however, chemolithotrophs do not produce that much ATP - ATP yield is low to moderate; typically 1–3 ATP per molecule oxidized. 

Because the yield of ATP is so low, chemolithotrophs must oxidize a large quantity of inorganic material to grow and reproduce. Thus, they have a significant ecological impact. 

A lithotroph is thus an organism that uses an inorganic substrate (usually of mineral origin) for use in biosynthesis (e.g., carbon dioxide fixation) or energy conservation via aerobic or anaerobic respiration. 

Known chemolithotrophs are exclusively microbes; no known macrofauna possesses the ability to utilize inorganic compounds as energy sources. Macrofauna and lithotrophs can form symbiotic relationships, an example of this is chemolithotrophic bacteria in deep sea worms - Giant tube worms Riftia pachyptila have an organ containing chemosynthetic bacteria instead of a gut.

Chemotrophs thus, obtain energy through the oxidation of electron donor molecules in their environments.

• These molecules can be organic (chemoorganotrophs) or inorganic (chemolithotrophs).
• The chemotrophs are in contrast to phototrophs, which utilize solar energy.
• Chemotrophs can be either autotrophic or heterotrophic.
• Chemotrophs can be aerobic and anaerobic.

Ecological impact of chemolithotrophs

Chemolithotrophs play a crucial ecological role by driving essential biogeochemical cycles, involving nitrogen, sulfur, and iron. By oxidizing inorganic compounds such as ammonia, hydrogen sulfide, ferrous iron, and hydrogen, they act as the primary producers in environments where sunlight is unavailable, such as deep-sea vents and subsurface habitats. 

They have important roles in:

1. Nutrient Cycling:
• Chemolithotrophs convert reduced inorganic compounds into oxidized forms, facilitating the recycling of nutrients like nitrogen (through nitrification), sulfur (through sulfur oxidation), and iron.
• For example, nitrifying bacteria transform ammonia into nitrate, making nitrogen available in forms usable by plants and other organisms.
2. Supporting Ecosystems in Extreme Environments:
• In habitats lacking organic carbon or light (e.g., hydrothermal vents), chemolithotrophs form the base of the food web, supporting other communities by producing organic matter through chemosynthesis.
3. Influence on Soil and Water Chemistry:
• By oxidizing iron and sulfur compounds, chemolithotrophs influence soil pH and metal availability, affecting overall soil fertility and water quality.
• Their activities can lead to acid mine drainage, impacting aquatic ecosystems negatively, but also play roles in bioremediation.
4. Environmental and Industrial Applications:
• Chemolithotrophs can be used in waste treatment, bioleaching, and biogeochemical remediation processes.

Overall, chemolithotrophs are critical in maintaining ecosystem stability and productivity, especially in nutrient-poor or extreme environments. Their metabolic activities drive elemental cycles critical for the survival of diverse life forms.

 

Effect of temperature on growth of microorganisms- TDT and TDP

 Aim

To determine the thermal death time and thermal death point of the given test organism

Principle

Temperature is one of the most important physical factors influencing the growth of microorganisms. Bacteria unlike eukaryotes lack homeostatic mechanism and they do not regulate the heat generated by metabolism thus are affected readily by changes in temperature. Enzymatic reactions have maximum efficiency at optimum temperature which varies with organisms. For every 10⁰C rise in temperature, there is 2 fold increase in the rate of enzyme catalyzed reactions for a limited range of temperature. At high temperatures, proteins are irreversibly denatured and there is a total enzyme destruction. At low temperature, the enzyme reactions are merely inactivated and are thus less harmful.

Bacteria are divided into three major groups with respect to their temperature requirements:

1)     Psychrophiles with optimum temperature between 0 and 20⁰C

2)     Mesophiles with optimum temperature between 20 and 40⁰C

3)     Thermophiles with optimum temperature between 40 and 60⁰C

Normally, the lethal range of temperature for bacteria is between 50 and 100⁰C. Time of exposure is a vital factor in assessing the lethal effect of high temperature on bacterial cells. Determination of thermal death time (TDT) and thermal death point (TDP) are done for this purpose.

1)     Thermal death point (TDP) – Temperature at which an organism is killed in 10 minutes of exposure. Lethal action of heat has a temperature-time relationship. Thermal death point is done to determine the degree of heat tolerance of the organism. Some factors such as pH, moisture, composition of media and age of cells influence TDP.

     2)     Thermal death time (TDT) – The time required to kill cells/spores at a given temperature. The length of time that the microbes are exposed to heat contributes to lethal effect. This is assessed by exposing cells to fixed temperature which is determined as thermal death time for increasing periods of time.

 

Materials required

Cultures of S. aureus, E. coli, nutrient tubes, nutrient agar plates, water bath, incubator etc

Methodology

1)     Thermal death point (TDP)

 Into each of the sterile test tubes, 5 ml of sterile nutrient broth was dispensed and tubes were marked 40, 50, 60, 70, 80, 90 and 100 ⁰C for different organisms. A loopful of culture was inoculated into the respective tubes and incubated for ten minutes at each temperature. They were then plated on nutrient agar plates and incubated at 37⁰C for 24 hours. The temperature above which the organism was completely killed and did not grow was noted and this was determined as the thermal death point of the organism.

2)     Thermal death time (TDT)

Each sterile tube containing 5 ml nutrient broth was inoculated with loopful of cultures and incubated at their thermal death point (TDP). A loopful of cultures from the tubes were taken at regular intervals of 3 minutes each, starting from 0 minutes till 15 minutes and plated on to nutrient agar followed by incubation at  37⁰C for 24 hours. The time period above which the organisms were completely killed and did not grow was determined as thermal death time (TDT).

Result

The thermal death point (TDP) and thermal death time (TDT) for S. aureus was --------- and for E. coli was ----------.