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

 

 

 

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.

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 ----------.