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 & Methanogenesis

 Hydrogen-oxidizing bacteria are a group of facultative autotrophs that can use hydrogen as an electron donor. They oxidise H, (electron donor) and reduce O₂ (electron acceptor) via “knallgas” reaction, 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 subsequently used to fix carbon dioxide via the Calvin cycle.

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

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:

$$\text{H}_2 \rightarrow 2\text{H}^+ + 2e^-$$

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.  

Almost all hydrogen-oxidising bacteria are facultative chemoautotrophs, i.e, they can also grow chemoheterotrophically (chemoorganotrophically) with organic compounds as energy source. 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 bacteria have been isolated from a variety of environments, including fresh waters, sediments, soils, activated sludge, hot springs, hydrothermal vents and percolating water. 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, as an energy source for its respiration and survival. This allows H. pylori to colonize the stomach and contributes to its ability to cause chronic inflammation and gastritis and stomach cancer. 

• Source of Hydrogen: 

H. pylori obtains hydrogen from the fermentative metabolism of other intestinal bacteria. 

• Hydrogenase Enzyme: 

The bacterium contains a specific enzyme called hydrogenase, which is responsible for oxidizing the molecular hydrogen. 

• Energy Production: 

This oxidation process generates energy for the bacterium, allowing it to respire and meet its metabolic needs. 

• Role in Virulence: 

The hydrogen oxidation pathway, provides H. pylori with a high-energy non-carbon substrate. 

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. In this environment hydrogen oxidation represents a significant origin of energy, sufficient to conduct 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.

Strain MH-110

Ocean surface water is characterized by a high concentration of hydrogen. In 1989, an aerobic hydrogen-oxidizing bacterium was isolated from sea water - MH-110 strain of Hydrogenovibrio marinus is able to grow under normal temperature conditions and in an atmosphere with oxygen saturation of 40%. This differs from the usual behaviour of hydrogen-oxidizing bacteria, which in general thrive under microaerophilic conditions (<10% O₂ saturation). This strain is also capable of coupling the hydrogen oxidation with the reduction of sulfur compounds such as thiosulfate and tetrathionate.

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.

 

 Methanogenesis

 

Methanogenesis, or biomethanation, is a form of anaerobic respiration that uses carbon as the terminal electron acceptor, resulting in the production of methane by the reduction of CO₂ to CH₄. H₂ is commonly used as electron donor however, formate, CO₂ and even certain organic compounds such as alcohol may also be used as electron donors.

Methanogenesis (methane production) is characteristic to a group of obligate anaerobic archaea (archaebacteria) called the methanogens (e.g., Methanobacterium, Methanobrevibacter, Methanococcus, Methanogenium, Methanospirillum, Methanomicrobium, etc.).

Methanogenesis thus involves the anaerobic conversion of carbon compounds to methane and typically follows four steps: hydrolysis, acidogenesis, acetogenesis, and methanogenesis. During hydrolysis, complex organic matter is broken down, followed by acidogenesis to produce volatile fatty acids. Acetogenesis converts these fatty acids into acetate. Finally, methanogens (archaea) utilize acetate, CO₂, and hydrogen gas to produce methane. 

The reduction of CO₂ to methane can be summarised in the following way

1. Hydrolysis:

Complex organic matter (proteins, carbohydrates, lipids) are broken down into simpler, smaller molecules, such as sugars, fatty acids and amino acids, by hydrolytic bacteria e.g., Clostridium spp.

2. Acidogenesis:

Acidogenic bacteria further break down the simpler organic compounds into volatile fatty acids (VFAs), alcohols, H₂, and CO₂. e.g., Bacteroides, Lactobacillus. 

3. Acetogenesis:

Acetogenic bacteria convert the volatile fatty acids, alcohols and other products from the previous steps into acetate, carbon dioxide, and hydrogen gas. Acetogenic bacteria include Syntrophomonas, Syntrophobacter etc.

    Acetogenic bacteria occur in syntrophy with methanogens — hydrogen must be kept at low levels for the reaction to proceed efficiently.

4. Methanogenesis:

This is the final step, carried out by methanogenic archaea. They consume the acetate, CO₂, and H₂ produced in the earlier stages to generate methane. 

There are three primary types of methanogenesis, depending on the methanogens and the substrates they use:

• Hydrogenotrophic methanogenesis: Methanogens reduce CO₂ with hydrogen gas (H2) to produce methane. eg.,Methanobacterium 

        CO₂ + H₂ → CH₄ + H₂O (Hydrogenotrophic pathway)

• Aceticlastic methanogenesis: Methanogens cleave acetate directly into methane and CO₂. eg., Methanosarcina and Methanotricha

            CH₃COOH → CH₄ + CO₂ (Acetoclastic pathway)

• Methylotrophic methanogenesis: Methanogens convert methylated compounds, such as methanol or methylamines, into methane. eg., Methanomethylovorans 

            Methyl compounds → CH₄ (Methylotrophic pathway)

Ecological Role of Methanogens:

• • Key players in anaerobic ecosystems – they remove end products like H₂ and acetate, allowing upstream fermentative and syntrophic processes to continue.

  • Major contributors to global methane emissions, influencing climate change.

  • Used in biogas production for renewable energy (methane as fuel).

The process of methanogenesis is crucial for the degradation of organic matter in anaerobic environments, such as wetlands, animal digestive tracts, and anaerobic digesters used in waste treatment. Methanogenesis is the primary pathway that breaks down organic matter in landfills (can release large volumes of methane into the atmosphere if left uncontrolled). It can be used to treat organic waste and to produce useful compounds. Biogenic methane can be collected and used as a sustainable alternative to fossil fuels.

The production of methane is an important and widespread form of microbial metabolism, and in most environments, it is the final step in the decomposition of biomass. During the decay process, electron acceptors (such as oxygen, ferric iron, sulfate, and nitrate) become depleted, while hydrogen (H₂), carbon dioxide, and light organic compounds produced by fermentation accumulate. Without methanogenesis, a great deal of carbon (in the form of fermentation products) would accumulate in anaerobic environments.

Methanogenesis also occurs in the guts of humans and other animals, especially ruminants. In the rumen, anaerobic organisms, including methanogens, digest cellulose into forms usable by the animal. Without these microorganisms, animals such as cattle would not be able to consume grass. The useful products of methanogenesis are absorbed by the gut. Methane is released from the animal mainly by belching.The average cow emits around 250 liters of methane per day. 

Methane is one of the earth’s most important greenhouse gases, with a global warming potential 25 times greater than carbon dioxide. Therefore, the methane produced by methanogenesis in livestock contributes to global warming.