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 ----------.

Carbohydrate Fermentation by Different Microbes

 Aim

To determine the specific carbohydrate fermentation profiles of different bacteria.

Principle:

Microorganisms metabolize carbohydrates in various ways. Fermentation is an anaerobic process where microbes break down carbohydrates, typically producing acid and/or gas as byproducts. The carbohydrate fermentation test detects acid production as indicated by a change in the pH indicator (e.g., phenol red) in the medium from its initial red/orange colour to yellow. Gas production is indicated by the presence of gas bubbles collected in an inverted Durham tube placed inside the fermentation broth.

Materials Required

Bacterial cultures such as Staphylococcus aureus, Escherichia coli, Pseudomonas and Bacillus, Basal fermentation broth containing carbohydrates such as Glucose, Sucrose, Maltose and Lactose, other routine microbiological facilities.

Procedure

Procedure

1.  Basal fermentation broth containing carbohydrates such as glucose, sucrose, maltose and lactose were prepared

2.   The broth was dispensed into test tubes with Durham’s tube and sterilized.

3. Using a sterile inoculating loop, each labelled fermentation tube (glucose, sucrose, maltose and lactose) was aseptically inoculated with a small amount of the specific bacterial culture. A negative control was set up for each sugar without bacterial inoculation

4. All inoculated tubes were incubated at 37°C for 24 - 48 hours.

      5. The tubes were observed for colour change and gas production.

 Observation and result

Acid production is indicated if the medium turns yellow (from red/orange) and Gas production is indicated if bubbles are visible in the inverted Durham tube.

	Staphylococcus aureus	Escherichia coli	Pseudomonas	Bacillus
Glucose
Sucrose
Maltose
Lactose

         A = acid only (broth turned yellow)

       AG = acid and gas (broth turned yellow + bubble trapped in Durham tube)

Composition of Carbohydrate Broth

Peptone- 10.00 gm

Beef extract- 1.00 gm

Sodium Chloride- 5.00 gm

Glucose/Sucrose/Maltose/Lactose - 10 gm

Phenol Red- 0.018 gm

Distilled water – 1000 ml

Media formulation & Media components

 The word “fermentation” originates from the Latin word Fervere which means to boil. The boiling appearance is due to the appearance of bubbles on fruit/malted grain extracts which indicates the CO₂ production.

Fermentation is a process for the production of a product by the mass-culture of a microorganism under optimized conditions.

Based on the products formed, there are different types of Fermentation which are:

o   Production of Microbial cells/biomass

o   Production of Microbial enzymes

o   Production of Microbial metabolites

o   Production of Recombinant products

o   Production of Modified compound than which is added at the beginning- Transformation

Major Steps in Fermentation

• Media formulation- for inoculum development and for production fermenter
• Sterilization of medium, fermenters, ancillary equipments 
• Production of an active, pure culture in sufficient quantity to inoculate production vessel (Inoculum development) (Upstream processing- USP)
• Growth of the organism in the production fermenter under optimum conditions for product formation (Fermentation)
• Extraction of the product and its processing (Downstream processing- DSP)
• Disposal of effluents produced in the process

Media Formulation

Fermentation media must satisfy all the nutritional requirements of the microorganism and promote the synthesis of the target product, which is either the cell biomass or a specific metabolite. Most fermentations require liquid media/ broth (Submerged Fermentation-SmF/surface fermentation), although some fermentations employ solid substrates (solid state fermentation-SSF).

In an industrial fermentation process, media are required in inoculum (starter culture) build up and the main production fermentation. The media formulation is different for inoculum propagation and the actual fermentation process since they have different objectives. Inoculum media should allow rapid/uniform growth of the inoculum whereas the production medium should facilitate the production of desired product in good amounts. Also, if biomass or primary metabolites are the target product, the production medium should allow optimal growth of the microorganism. For secondary metabolite production, such as antibiotics, their biosynthesis is not growth related. Hence, the media should be designed to provide an initial period of cell growth, followed by starvation conditions. Here, the supply of one or more nutrients (carbon, phosphorus or nitrogen source) may be limited and rapid growth does not occur. This induces secondary metabolite production.

 The media components usually are,

■       Water

■       Carbon source

■       Nitrogen source

■       Oxygen

■       Sources of P, S, minor and trace elements

■       Vitamins – Biotin, riboflavin etc

■       Buffers

■       Antifoam

■       Precursor

■       Inducer

■       Inhibitor

Most fermentations, except those involving solid substrates, require large quantities of water in which the medium is formulated. General media requirements include a carbon source, which provides both energy and carbon units for biosynthesis, and sources of nitrogen, phosphorus and sulphur. Other minor and trace elements must also be supplied, and some microorganisms require added vitamins, such as biotin and riboflavin. Aerobic fermentations require oxygen, and even some anaerobic fermentations require initial aeration of media, e.g. beer fermentations. As the fermentation proceeds different metabolic products are formed which may change the pH of the media and it can interfere with the growth or product formation, So, to adjust the pH, media should contain buffers or the pH is controlled by addition of acid/ alkali. Antifoam agents are also required to control foam production. Some processes require compounds like precursor, inducer or inhibitor which are added certain stages of the fermentation.

The composition of a fermentation medium may be simple to complex, depending on the particular microorganism and its fermentation.  

Simple and complex media are further subdivided into two categories: synthetic and crude. In a synthetic medium, all the components are specifically defined and known compounds.  Each component is relatively pure and the exact concentrations are known.  Synthetic medium has defined components and concentrations and are expensive due to the relatively pure ingredients used. The concentration of one or several can be varied in order to determine the effect on cell growth and product yield. Individual components may be added or deleted as well. This has advantages in certain types of studies. However, yields derived from these media are relatively low.  Crude media contain crude factors or ill-defined sources of nutrients and growth.  They usually allow much higher yields.

The media adopted also depend on the scale of the fermentation. For small-scale laboratory fermentations pure chemicals are often used in well-defined media. However, this is not always possible due to cost, as media components may account for up to 60-80% of process expenditure. Industrial-scale fermentations primarily use cost-effective complex crude substrates, where many carbon and nitrogen sources are almost indefinable.  Most are derived from natural plant and animal materials, often by-products of other industries, with varied and variable composition.  The effects of such batch-to-batch variations must be determined.  Small scale trials are usually performed with each new batch of substrate, to examine the impact on product yield and product recovery.

            The main factors that affect the final choice of individual raw materials are as follows:

1 Cost and availability: ideally, materials should be inexpensive and of consistent quality and year round availability.

2 Ease of handling in solid or liquid forms, along with associated transport and storage costs, e.g., requirements for temperature control.

3 Sterilization requirements and any potential denaturation problems.

4 Formulation, mixing, complexing and viscosity characteristics that may influence agitation, aeration and foaming during fermentation and downstream processing stages.

5 The concentration of target product to be attained, its rate of formation and yield per gram of substrate utilized.

6 The levels and range of impurities and the potential for generating further undesired products during the process.

7 Overall health and safety implications.

Composition of a typical fermentation media

The composition of fermentation media is dependent on a number of factors characteristic of the particular fermentation.  The major ingredients of a typical fermentation media are:

■       Water

■       Carbon source

■       Nitrogen source

■       Sources of P, S, minor and trace elements

■       Vitamins – Biotic, riboflavin etc

■       Buffers

■       Antifoam

■       Precursor

■       Inducer

■       Inhibitor 

Water

Water is a major component of all fermentation media except solid-substrate fermentation. It also provides trace mineral elements. Water is also important for ancillary equipment and cleaning. Supply of large quantities of clean water, of consistent composition, is therefore essential. The quality of water in terms of pH, dissolved salts and effluent contamination is important.  The mineral content is important in brewing (mashing step) and has influenced the location of breweries and types of beer produced.  Before use, suspended solids, colloids and microorganisms should be removed. If the water is ‘hard’, it should be treated to remove salts such as calcium carbonate. Iron and chlorine should also be removed. For some fermentations, especially, plant and animal cell culture, the water must be highly purified. Since, water is becoming increasingly expensive, its recycle/reusage wherever possible should be encouraged. This minimizes water costs and reduces the volume requiring waste-water treatment.

Microbial Nutrition -requirements and modes of nutrition

    Microbial cells are structurally complex and carry out numerous functions. Nutrients are required for microbial growth to support biosynthesis and energy release. Microorganisms use nutrients and a source of energy to construct new cellular components and do cellular work.

Common Nutrient Requirements

Macroelements/Macronutrients : Macro elements or macronutrients are required by microorganisms in relatively large amounts. Over 95% of microbial cell dry weight is made up of a few major elements: carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus, potassium, calcium, magnesium, and iron, these are the Macroelements/Macronutrients. Carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus- C, O, H, N, S, and P- are components of carbohydrates, lipids, proteins, and nucleic acids.

Potassium, calcium, magnesium, and iron exist in the cell as cations and play a variety of roles. Potassium (K⁺) is required for activity by a number of enzymes, including some of those involved in protein synthesis. Calcium (Ca2⁺), contributes to the heat resistance of bacterial endospores among other functions. Magnesium (Mg2⁺) serves as a cofactor for many enzymes, complexes with ATP, and stabilizes ribosomes and cell membranes. Iron (Fe2⁺and Fe3⁺) is a part of cytochromes and a cofactor for enzymes and electron-carrying proteins.

Micronutrients or trace elements : Micronutrients or trace elements are required in small amounts for all microorganisms, in addition to macroelements. These include manganese, zinc, cobalt, molybdenum, nickel, and copper and are needed by most cells. These are required in such small amounts that they are available as contaminants from water, glassware, and regular media components. Often these are adequate for growth.

In nature, micronutrients are ubiquitous and probably do not usually limit growth. 

Micronutrients are normally a part of enzymes and cofactors, and they aid in the catalysis of reactions and maintenance of protein structure. For example, zinc (Zn2⁺) is present at the active site of some enzymes but can also be involved in the association of regulatory and catalytic subunits.  (e.g., E. coli aspartate carbamoyl transferase). Manganese (Mn2⁺) aids many enzymes that catalyze the transfer of phosphate groups. Molybdenum (Mo2⁺) is required for nitrogen fixation, and cobalt (Co2⁺) is a component of vitamin B12.

            Microorganisms may have particular requirements according to the environment or as part of their morphology. For example, bacteria/ archaea growing in saline lakes and oceans require high concentrations of sodium ion (Na⁺). Diatoms need silicic acid (H₄SiO₄) to construct their beautiful cell walls of silica [(SiO₂)n].

In general, microorganisms require a balanced mixture of nutrients. If an essential nutrient is in short supply, microbial growth will be limited regardless of the concentrations of other nutrients.

  

Requirements For Carbon, Hydrogen, Oxygen, And Electrons

All organisms need carbon, hydrogen, oxygen, and a source of electrons. Carbon is needed for the skeletons or backbones of all the organic molecules which make the organisms. Hydrogen and oxygen are also important elements found in organic molecules. Electrons are needed for electron transport chain and for other oxidation-reduction reactions which can provide energy for use in cellular work. Electrons are also needed to reduce molecules during biosynthesis (e.g., the reduction of CO₂ to form organic molecules).

Molecules serving as carbon sources also provide hydrogen and oxygen. Thus, the requirements for carbon, hydrogen, and oxygen are satisfied together. However, one carbon source, carbon dioxide (CO₂), supplies only carbon and oxygen because CO₂ is the most oxidized form of carbon, lacks hydrogen, and is unable to donate electrons during oxidation-reduction reactions. So it cannot be used as a source of hydrogen, electrons, or energy.

Organisms that use CO₂ as their sole or principal source of carbon are called autotrophs. Because CO₂ cannot supply their energy needs, they must obtain energy from other sources, such as light or reduced inorganic molecules.

Heterotrophs are organisms that use reduced, preformed organic molecules as their carbon source. They can also obtain hydrogen, oxygen, and electrons from the same molecules. Thus, organic carbon sources also provide electrons to be used in electron transport as well as in other oxidation-reduction reactions, so heterotrophs use their carbon source as an energy source also.

The more reduced the organic carbon source (i.e., the more electrons it carries), the higher its energy content. Thus, lipids have a higher energy content than carbohydrates.

Heterotrophic microorganisms can use many carbon sources. There is no naturally occurring organic molecule that cannot be used by some microorganism. Actinomycetes, common soil bacteria, will degrade amyl alcohol, paraffin, and even rubber. Some bacteria can use almost anything as a carbon source; for example, Burkholderia cepacia can use over 100 different carbon compounds. Microbes can degrade even relatively indigestible human-made substances such as pesticides. This is usually carried out by complex microbial communities (microbial consortia). These molecules sometimes are degraded in the presence of a growth-promoting nutrient that is metabolized at the same time—a process called cometabolism. Other microorganisms can use the products of this breakdown process as nutrients.

But some microbes are extremely fastidious and catabolize only a few carbon compounds. Cultures of methylotrophic bacteria metabolize methane, methanol, carbon monoxide, formic acid, and related one-carbon molecules. Parasitic members of the genus Leptospira use only long-chain fatty acids as their major source of carbon and energy.

Requirements For Nitrogen, Phosphorus, And Sulfur

Microorganisms require large quantities of nitrogen, phosphorus, and sulfur for growth. These elements may be acquired from the same nutrients/organic sources that supply carbon, or from other inorganic sources.

Nitrogen is needed for the synthesis of amino acids, purines, pyrimidines, some carbohydrates and lipids, enzyme cofactors, and other substances. Many microorganisms can use the nitrogen obtained in amino acids (organic source). Others incorporate ammonia directly through the action of enzymes such as glutamate dehydrogenase or glutamine synthetase and glutamate synthase. Most phototrophs and many chemotrophic microorganisms reduce nitrate to ammonia and incorporate the ammonia in a process known as assimilatory nitrate reduction (inorganic sources) A variety of bacteria (e.g., many cyanobacteria and the symbiotic bacterium Rhizobium) can assimilate atmospheric nitrogen (N₂) by reducing it to ammonium (NH₄). This is called nitrogen fixation.

Phosphorus is present in nucleic acids, phospholipids, nucleotides like ATP, several cofactors, some proteins, and other cell components. Almost all microorganisms use inorganic phosphate as their phosphorus source and incorporate it directly. Low phosphate levels can limit microbial growth in many aquatic environments. Some microbes, such as Escherichia coli, can use both organic and inorganic phosphate. Some organophosphates such as hexose 6-phosphates can be taken up directly by the cell. Other organophosphates are hydrolyzed in the periplasm by the enzyme alkaline phosphatase to produce inorganic phosphate, which then is transported across the plasma membrane.

Sulfur is needed for the synthesis of substances like the amino acids cysteine and methionine, and some carbohydrates, biotin, and thiamine. Most microorganisms use sulfate (inorganic source) as a source of sulfur and reduce it by assimilatory sulfate reduction. A few microorganisms require a reduced form of sulfur such as cysteine (organic source).

Growth Factors

Some microorganisms have the enzymes and biochemical pathways needed to synthesize all cell components using minerals and sources of energy, carbon, nitrogen, phosphorus, and sulfur. Some lack one or more of the enzymes needed to manufacture some indispensable constituents. So, they must obtain these constituents or their precursors from the environment. Organic compounds that are essential cell components or precursors of such components but cannot be synthesized by the organism are called growth factors. There are three major classes of growth factors:

(1) amino acids, (2) purines and pyrimidines, and (3) vitamins.

Amino acids are needed for protein synthesis; purines and pyrimidines for nucleic acid synthesis. Vitamins are small organic molecules that usually make up all or part of enzyme cofactors and are needed in only very small amounts to sustain growth.

 

Functions of Some Common Vitamins in Microorganisms
Vitamin	Functions	Microorganisms Requiring Vitamin
Biotin	Carboxylation (CO2 fixation) One-carbon metabolism	Leuconostoc mesenteroides Saccharomyces cerevisiae
Cyanocobalamin (B12)	Molecular rearrangements	Lactobacillus spp.
Folic acid	One-carbon metabolism	Enterococcus faecalis
Pyridoxine (B6)	Amino acid metabolism (e.g., transamination)	Lactobacillus spp.
Niacin (nicotinic acid)	Precursor of NAD and NADP	Haemophilus influenzae
Riboflavin (B2)	Precursor of FAD and FMN	Dictyostelium spp

 

Some microorganisms require many vitamins; for example, Enterococcus faecalis needs eight different vitamins for growth.

Other growth factors required are; heme (from hemoglobin or cytochromes) is required by Haemophilus influenzae, and some mycoplasmas need cholesterol.

Understanding the growth factor requirements of microbes has significant practical applications. Microbes with known, specific requirements and those that produce large quantities of a substance (e.g., vitamins) are useful.

Microbes with a specific growth factor requirement can be used in bioassays for the factor they need. A typical assay is a growth-response assay, which allows the amount of growth factor in a solution to be determined. The amount of growth in a culture is related to the amount of growth factor present. Ideally, the amount of growth is directly proportional to the amount of growth factor; if the growth factor concentration doubles the amount of microbial growth doubles. 

For example, Lactobacillus and Streptococcus can be used in microbiological assays of most vitamins and amino acids. Microbiological assays are specific, sensitive, and simple. They still are used in the assay of substances like vitamin B₁₂ and biotin, despite advances in chemical assay techniques.

Microorganisms which are able to synthesize large quantities of vitamins can be used to manufacture these compounds for human use. Several water-soluble and fat-soluble vitamins are produced partly or completely using industrial fermentations. Good examples of such vitamins and the microorganisms that synthesize them are

·       riboflavin (Clostridium, Candida, Ashbya, Eremothecium)

·       coenzyme A (Brevibacterium)

·       vitamin B12 (Streptomyces, Propionibacterium, Pseudomonas)

·       vitamin C (Gluconobacter, Erwinia, Corynebacterium)

·       carotene (Dunaliella)

·       vitamin D (Saccharomyces).

Research focuses on improving yields and finding microorganisms that can produce large quantities of other vitamins.

Nutritional Types of Microorganisms

Depending on how the need for carbon, energy, and electrons is fulfilled microorganisms can be classified. 

With respect to their preferred source of carbon, there are heterotrophs or autotrophs. There are only two sources of energy available to organisms: Phototrophs use light as their energy source; chemotrophs obtain energy from the oxidation of chemical compounds (either organic or inorganic). Microorganisms also have only two sources of electrons. There are Lithotrophs (i.e., “rock-eaters”) which use reduced inorganic substances as their electron source, and organotrophs which obtain electrons from reduced organic compounds.

Sources of Carbon, Energy, and Electrons
Carbon Sources
Autotrophs	CO2 sole or principal biosynthetic carbon source
Heterotrophs	Reduced, preformed, organic molecules from other organisms
Energy Sources
Phototrophs	Light
Chemotrophs	Oxidation of organic or inorganic compounds
Electron Sources
Lithotrophs		Reduced inorganic molecules
Organotrophs		Organic molecules

 

In spite of the great metabolic diversity seen in microorganisms, most can be placed in one of five nutritional classes based on their primary sources of carbon, energy, and electrons.

 

Major Nutritional Types of Microorganisms
Nutritional Type	Carbon Source	Energy Source	Electron Source	Representative Microorganisms
Photolithoautotrophy (photolithotrophic autotrophy)	CO2	Light	Inorganic e- donor	Purple and green sulfur bacteria, cyanobacteria
Photoorganoheterotrophy (photoorganotrophic heterotrophy)	Organic carbon but CO2 may also be used	Light	Organic e- donor	Purple nonsulfur bacteria, green nonsulfur bacteria
Chemolithoautotrophy (chemolithotrophic autotrophy)	CO2	Inorganic chemicals	Inorganic e- donor	Sulfur-oxidizing bacteria, hydrogen-oxidizing bacteria, methanogens, nitrifying bacteria, iron-oxidizing bacteria
Chemolithoheterotrophy/ mixotrophy (chemolithotrophic heterotrophy)	Organic carbon, but CO2 also used	Inorganic chemicals	Inorganic e-donor	Some sulfur-oxidizing bacteria (e.g., Beggiatoa)
Chemoorganoheterotrophy (chemoorganotrophic heterotrophy)	Organic carbon	Organic chemicals often same as C source	Organic e-donor often same as C source	Most non photosynthetic microbes, including most pathogens, fungi, many protists, and many archaea

 

The majority of microorganisms are either photolithotrophic autotrophs or chemoorganotrophic heterotrophs.

Photolithotrophic autotrophs (often called photoautotrophs or photolithoautotrophs) use light energy and have CO₂ as their carbon source. Photosynthetic protists and cyanobacteria use water as the electron donor and release oxygen. Other photolithoautotrophs, such as the purple and green sulfur bacteria, cannot oxidize water but get electrons from inorganic donors like hydrogen, hydrogen sulfide, and elemental sulphur.

Chemoorganotrophic heterotrophs (often called heterotrophs or chemoheterotrophs, or chemoorganoheterotrophs) use organic compounds as sources of energy, hydrogen, electrons, and carbon. Usually, the same organic nutrient will satisfy all these requirements. All pathogenic microorganisms are chemoheterotrophs.

The other nutritional classes are very important ecologically though they have only fewer known microorganisms. Some photosynthetic bacteria (purple and green bacteria) use organic matter as their electron donor and carbon source. These photoorganotrophic heterotrophs (photoorganoheterotrophs) are common in polluted lakes and streams. Some of these bacteria also can grow as photoautotrophs with molecular hydrogen as an electron donor.

Chemolithotrophic autotrophs (chemolithoautotrophs), oxidize reduced inorganic compounds such as iron, nitrogen, or sulfur molecules to derive both energy and electrons for biosynthesis. Carbon dioxide is the carbon source.

Chemolithoheterotrophs, also known as mixotrophs, use reduced inorganic molecules as their energy and electron source, but derive their carbon from organic sources. Chemolithotrophs contribute greatly to the chemical transformations of elements (e.g., the conversion of ammonia to nitrate or sulfur to sulfate) that continually occur in ecosystems.

 A particular species usually belongs in only one of the nutritional classes, but some show great metabolic flexibility and alter their metabolic patterns in response to environmental changes.

For example, many purple nonsulfur bacteria act as photoorganotrophic heterotrophs in the absence of oxygen but oxidize organic molecules and function chemoorganotrophically at normal oxygen levels. When oxygen is low, photosynthesis and chemoorganotrophic metabolism may function simultaneously. This sort of flexibility seems complex and confusing, but it gives these microbes an advantage if environmental conditions frequently change.

 Conclusion

     Microorganisms require about 10 elements in large quantities for the synthesis of macromolecules. Several other elements are needed in very small amounts and are parts of enzymes and cofactors.

     All microorganisms can be placed in one of a few nutritional categories on the basis of their requirements for carbon, energy, and electrons.