Effect of Oxygen: Microorganisms in their natural habitats require varying amounts of gases such as oxygen, methane, carbon dioxide, nitrogen etc. In microbial growth oxygen plays a major role. The importance of oxygen to the growth of an organism correlates with the processes it uses to conserve energy. Almost all energy-conserving metabolic processes involve the movement of electrons through a series of membrane-associated electron carriers called an electron transport chain (ETC). For chemotrophs, an externally supplied terminal electron acceptor is critical to the functioning of the ETC. In many cases, the terminal electron acceptor is oxygen. On the basis of their growth response to oxygen, microorganisms are divided into five physiological groups: Aerobic Microorganisms: Microbes that normally require oxygen for growth and can grow in a standard air atmosphere of 21% oxygen are classified as aerobes. These organisms are completely dependent on atmospheric oxygen for growth; that’s why; they are termed as obligate aerobes. Filamentous moulds and bacteria like Mycobacterium, Legionella, Microccusluteus are example of aerobic microbes. Microaerophilic Microorganisms: Microaerophiles are organisms which, like aerobes, can use oxygen for energy-yielding chemical reactions. However, unlike aerobes they cannot withstand the level of oxygen (21%) present in an air atmosphere and usually grow best at oxygen levels between 2 to 10%, This limited tolerance to oxygen is due to a high susceptibility to superoxide radicals and hydrogen peroxide, which are formed in aerobic condition, Examples of microaerophiles are: Campylobacter jejuni, Helicobacter pylori etc, Facultative Anaerobes: Microbes that does not require oxygen for growth but grow better in its presence is termed as facultative anaerobes, In the Presence of oxygen, they use oxygen. as the terminal electron acceptor during aerobic respiration, Under anaerobic condition they obtain energy by anaerobic respiration. Members of the bacterial family Enterobacteriaceae,such as Escherichia coli are facultative. Many types of yeast such as Saccharomyces cerevisiae are facultative anaerobe. Aerotolerant anaerobes: Aerotolerant anaerobes are anaerobic bacteria that are not killed by exposure to oxygen. These organisms grow equally well whether oxygen is present or not; they can tolerate oxygen, but they do not make use of it. Many have strictly fermentative metabolism and thus do not use oxygen in their energy-conserving processes. Such as Enterococcus faecalis which is an opportunistic pathogen that causes urinary tract infections. Anaerobic Microorganisms: Anaerobic microorganisms are those which may be poisoned by oxygen, cannot grow in an air atmosphere, and do not use oxygen for energy- yielding chemical reactions. Anaerobic (0,-free) microbial habitats are common in nature and include muds and other sediments, bogs, marshes, water-logged soils, intestinal tracts of animals, sewage sludge, the deep sub-surface of Earth, and many other environments, As far as is known, obligate anaerobiosis is characteristic of a wide variety of bacteria and archaea, a few fungi, and a few protozoa. Some of the best-known prokaryotic anaerobes are Clostridium, a genus of gram-positive endospore-forming Bacteria, and the methanogens, a group of methane-producing archaea. Mechanism of Oxygen toxicity in Anaerobes: In case of obligate anaerobic microbes, oxygen is toxic, and they are usually killed by the presence of oxygen. Obligate anaerobes cannot generate energy through aerobic respiration and employ other metabolic strategies such as fermentation or anaerobic respiration, neither of which requires oxygen. Toxic oxygen derivatives are formed when cellular proteins such as flavoproteins transfer electrons to 0,*, These toxic oxygen derivatives are called reactive Bn ann ee .02 + = —+ 05 (superoxide radical) Superoxi i i Peroxide radicals may cause damage to cells, but they also give rise to hydrogen peroxide, 4202 and hydroxyl radicals; both of which can destroy vital cell components. Hydroxyl radicals are produced from superoxide radicals in two steps. In the first step, two superoxide radicals react with each other to produce hydrogen peroxide. 20; + 2H*—> 0, + H20, In the second step, superoxide radicals react with the hydrogen peroxide in the presence of iron complexes to form hydroxyl radicals Oz + H,0, > 0,+ OH~ + OH" Superoxide anion and hydroxyl radicals are strong oxidizing agents that can oxidize macromolecules and any other organic compounds in the cell. Peroxides such as H202can also damage cell components but are not as toxic as 0; or OH’.Hydroxyl radicals are very short- lived, lasting less than 1/1000s. This is because they are among the most reactive chemical substance known. They can damage almost every kind of molecule found in living cells, including genetic material DNA. Aerobic microorganisms, facultative microorganisms can grow aerobically. Actually they have developed various protective mechanisms against these toxic forms of oxygen, One mechanism is the production of the enzyme superoxide dismutase (SOD), which eliminates superoxide radicals by rapidly converting them to hydrogen peroxide, cS Superoxide dismutase 20; + 2H+ ———"+, 0, + H,0, The hydrogen peroxide produced by this reaction can in turn be dissipated by two other enzymes. One is catalase and other enzyme is peroxidase. Catalase converts hydrogen peroxide to molecular oxygen and water. Peroxidase also converts hydrogen peroxide to water.Catalase 2H,0, > 2H,0 + 02 Peroxidase 2H,0, ++ NADH +H* ———> 2H,0 + NAD* Obligate anaerobes lack of these enzymes or have them in very low concentrations and therefore cannot tolerate oxygen. Effect of pH PH is a measure of the relative acidity of a solution and is defined as the negative logarithm of the hydrogen ion concentration pH = —log [H+] = | : is Soe 1g ial Thus, each pH unit represents a tenfold change in hydrogen ion concentration. pH values less than 7 are acidic and those greater than 7 are alkaline. Different species are adapted to grow at various pH values. But to grow well in an acidic or a basic environment, a microorganism must be able to maintain its intracellular pH at about 7.5, regardless of the external pH. A living cell has the ability, within limits, to keep constant internal pH by expelling hydrogen ions or by taking hydrogen ions into the cell. Thus the pH of its external environment usually has to change drastically before the inside of the cell is affected. Different species of microbes have different pH tolerances. Also, each organism shows a well-defined pH optimum, where growth occurs best. Most natural environments have a pH between 3 and 9, and organisms with pH growth optimum in this range are most common, On the basis of growth in depend upon the pH values the microorganisms are categorized as follows: Acidophiles: Organisms that grow best below pH 5.5 are called acidophiles. There are different classes of acidophiles, some growing best at moderately acidic pH and others at very low pH. Many fungi and bacteria grow best at pH 5 or even below, while a more restricted number grow best below pH 3. Most acidophiles cannot grow at pH 7 and many cannot grow at pH values more than two units above their optimum. A critical factor governing acidophilyi! 1s the stability of the cytoplasmic membrane. When the pH is raised to neutrality, the cytoplasmic membranes of strongly acidophilic bacteria are destroyed and the cells lyse. This Indicates that these organisms are not just acid tolerant but that high concentrations of protons are actually required for cytoplasmic membrane stability, Thiobacllus acidophilus, Acetobacter aceti, Helicobacter pylori are the example of acidophilic bacteria. Halarchaeum acidiphitum, Metallosphnera sedula are the acidophilic archaea. Alkaliphiles: Microorganisms growing pH optimum of 8 or higher are called alkaliphiles. Alkaliphilic microorganisms are typically found in highly alkaline habitats, such as soda lakes and high-carbonate soils. The most well-studied alkaliphilic bacteria are certain Bacillus species, such as Bacillus firmus. This organism is alkaliphilic but has an unusually broad range for growth, from pH 7.5 to 11. Some extremely alkaliphilic microbes are also halophilic (salt- loving), and most of these are Archaea. Some phototrophic purple bacteria are also strongly alkaliphilic. Certain alkaliphiles have commercial uses because they excrete hydrolytic enzymes such as proteases and lipases that maintain their activities at alkaline pH. These enzymes are added to laundry detergents to remove protein and fat stains, respectively, from clothing. Managing membrane bioenergetics is an obvious problem for alkaliphiles. B. firmus uses sodium (Na+) rather than H+ to drive transport reactions and rotate its flagellum; that is, it forms a sodium motive force instead of a proton motive force. Remarkably, however, B. firmus uses a proton motive force to drive ATP synthesis even though the external membrane surface is highly alkaline. Exactly how this happens is unclear, although it is thought that hydrogen ions are in some way kept very near the outer surface of the cytoplasmic membrane such that they cannot spontaneously combine with the abundant hydroxy! ions to form water. Organisms that grow optimally at a pH value in the range 5.5 to 7.9 are called neutrophiles. For example, the bacterium Escherichia coli is a neutrophile, Microorganisms respond to external pH changes using mechanisms that maintain a neutral cytoplasmic pH. Several mechanisms for adjusting to small changes in external PH have been Proposed. Neutrophiles appear to exchange potassium for Protons using an antiport transporter, if the external pH . a1 is. Howeve system, Internal buffering also may contribute to pH homeostasis a s becomes too acidic, other mechanisms come into play. When the PH drop: dE, coli synthesize an array of new prote conse, An ATPase enzyme contributes to this at the expense of ATP. If the external ock proteins are synthesized. ins as part Salmonella enterica serovar Typhimurium ani of what has been called their acidic tolerance resp‘ protective response by pumping protons out of the cell, PH decreases to 4.5 or lower, acid shock proteins and heat sh These prevent the denaturation of proteins and aid in refolding denatured proteins in acidic conditions. Metabolism: Energy Production Mechanisms In the living world, energy passes from one organism to another in the potential energy contained in the bonds of chemical compounds. Organisms obtain the energy from oxidation reactions, To obtain energy in a usable form, a cell must have an electron (or hydrogen) donor, which serves as an initial energy source within the cell. Electron donors are diverse and can include photosynthetic pigments, glucose or other organic compounds, elemental sulfur, ammonia, or hydrogen gas. Next, electrons removed from the chemical energy sources are transferred to electron carriers, such as the coenzymes NAD+, NADP+, and FAD. This transfer is an oxidation-reduction reaction; the initial energy source is oxidized as this first electron carrier is reduced. During this phase, some ATP is produced. In the third stage, electrons are transferred from electron carriers to their final electron acceptors in further oxidation reduction reactions, producing more ATP. In aerobic respiration, oxygen (Oz) serves as the final electron acceptor. In anaerobic respiration, substances from the environment other than oxygen, such as nitrate ions (N03) or sulfate ions ($0;), serve as the final electron acceptors. In fermentation, compounds in the cytoplasm serve as the final electron acceptors, In aerobic and anaerobic respiration, a series of electron carriers called an electron transport chain releases energy that is used by the mechanism of chemiosmosis to synthesize ATP. Regardless of their energy sources, allorganisms use similar oxidation-reduction reactions to transfer electrons and similar mechanisms to use the energy released to produce ATP. Nutritional types of microorganisms: Microorganisms have developed several strategies of metabolism for meeting their nutritional requirements. On the basis of their difference in procurement of organic metabolites i.e. the source of carbon the microorganisms are broadly categorized as ~ + Autotrophs: Greek ‘Auto’ means self and ‘tophe’ means nourshing. Autotrophs are those that synthesize|their organic metabolites (food) from|CO, supplied from external environment as raw material and an organic complex already existing in their cell with the help of their enzymatic equipment, That is, the autotrophs use CO, as their sole Source of carbon. This mode of food procurement is called autotrophy or autotrophic nutrition. Heterotrophs: Greek ‘Hetero’ means others and ‘tophe’ means nourshing. Heterotrophs however, cannot synthesize organic metabolites (food) on their own like autotrophs rather they must obtain organic metabolites (food) in prefabricated form from their (@temal environment: That is, the carbon already prepared in organic form by other living organisms. This mode of nutrition is| called heterotrophy or heterotrophic nutrition, To produce the food either by synthesis (autotrophy) or by absorption in pre-fabricated from (heterotrophy), all microorganisms need energy. This energy co mes either from sunlight (light energy) or from break-down of chemicals chemical energy). In this wi } / taking source of energy into account, the microorganisms can be categorized as *Phototrophs: that usc light as their energy source,ice of id isms also require sou In addition to the source of carbon and energy, the microorganisms ah electrons for growth to take place. Microorganisms can be categorized «Lithotroph: they us€ Ridced INOrgan BubSanCesUas their electron source. F Grganotroph: they uselGMON DRIED cil Ame. Nutritional Types of Microorganisms Energy Source Electron Source Carbon Source ‘Nutritional Type ee Photoorganoheterotroph Organic -heterotroph 120 -organo- Carbon dioxide e Light aiulatroph (ag known organisms) Photo- ‘Organic és Inorganic -heterotraph Go known organisms) -litho- Carbon dioxide : Sek Photolithoautotroph ‘Organic Orginic -heterotroph Chemoorgancheterotronh, -organo- Carbon dioxide = Steer (ag known organisms) ‘Organic - Inorganic -heterotroph Chemolithoheterotroph -litho~ Carbon dioxide -autotroph Chemolithoautotroph Photolithoautotroph: ‘Energy Source slight Gets electrons from Semen This group ne een ea bacteria (purple sulphur bacteria, such as Chromium, Thiospiriliom bacteria such aeraChemolithoautotroph: * Getcarbon from C0, © Among the best known chemolithoautotrophic microorganisms are the sulphur- oxidising bacteria iroivoxidising bacteria, fitrifying bacteria. Sulphur-oxidising bacteria: Sulphur-oxidising bacteria are those chemoautotrophs that oxidize Sulphur compounds as electron donors and energy-zleasing compounds. The sulphur compounds used by ieni/are HS, elemental! sulphui(S)/sg037/and SOs. "The best studied Sulphur-oxidising chemoautotroph is the genus Thidbacillis that contains several gram negative and rod-shaped bacteria such asl thioparlls, Ts neopolitani)T. thioxidans, - intermedius. Other Sulphur-oxidising chemoautotrophs are | Thiothriz, Thioploca, Thiomicrospira, Thiothrix etc. Iron-oxidising bacteria: Iron-oxidising bacteria cfiis@/6xidation of iron/from ferrous (Fe?*) to ferric (Fe) state that yields energy. Thiobacillis ferrvoxidans, Leptospirillum, Gallionella, Ferrobacilliis, Leptothrix, Cladothrix are the representatives of iron-oxidising bacteria. Chemolithoheterotroph: «Get energy from oxidation of inorganic compounds # Electron source is reduced inorganic moleciles + Lacks the enzymes of the Calvin-Benson cycle, so requires organic compounds as carbon sources. * Some sulphur oxidising bacteria like Beggiatoa Photoorganoheterotroph:an carbon from reduced organié molecules, oF from bi organic ‘ Rhodopseudomonas, © Purple ea bacteria Uke RUBIBMIeRbiim, Riodobacter, - Rhodospirillum ete. Chemoorganoheterotroph chemoheterotrophs, + Chemoorganotrophic rotrophs (often _called cnet) dae eeeine compounds ab sources of energy, hydrogen, «Frequently the sme fae SRE lah a EGER > ea ED osteie Chemolithoautotrophs + Chemolithotrophic autotrophs (chemolithoautotrophs), oxidize reduced inorganic compounds such as and lectrons for biosynthesis. eki i Pell Measurement of Microbial Growth: Growth as commonly applied in microbiology refers to the magnitude of the total population. Microbial growth can be measured by two different methods: ¥ Direct Measurement of Cell Numbers: Direct or Breed Method: A known volume of microbial cell suspension is spread uniformly over a glass slide covering a specific area. This smear is then fixed by heating, stained, and examined under oil immersion and the total cells are counted. Customarily, cells in a few microscopic fields are counted because it is not possible to scan the entire area of the smear. The counting of total number of cell is determined by calculating the total number of microscopic fields per one square cm, area of the smear. The total number of cells can be counted ~ Area of the microscopic field under the oil immersion lens = nr? = 3.14 x (0.08 mm)? = 0.02 sq.mm. whe! , ¢ (Gil immersion lens) = 0.08 mm Area of the smear one sq.cm = 100 sq. mm. Thus, the number of microscopic fields = “°° = 5000 No. of cells 1 sq. cm. (or per 0.01ml microbial cell suspension) = Average no. of microbes per microscopic field x 5000 Direct Microscopic Count: The number of cells in a population can be measured by taking direct microscopic count using Petroff-Hausser Counting chamber (for prokaryotic microorganisms) or hemocytometer (for larger eukaryotic microorganisms). Prokaryotic microorganisms are more easily counted by specially designed slides that have chambers of known depth with an etched grid on the chamber bottom, Each square on the grid has definite depth and volume. Total number of microorganisms in a sample can be calculated taking the count of number of bacteria per unit area of grid and multiplying it by conversion factortively quick method and isms. These specially ymber bottom. i ive and relat ‘The Petroff-Hausser counting chamber is easy, inexpensive phology of the microorgant also gives information about the size and mor] ‘ i with an etched grid on the chai designed slides have chamber of known depth Bacteria/mm® = (bacteria/square) (25 squares)/ (50) \ mple dilution. Bacteria can be counted by taking into account the chamber's volume and any samp! i i i d also it The disadvantage encountered in this method is that fairly large volume is required an is difficult to distinguish between living and dead cells. Microorganisms of larger sizes can. be unied by using electronic counters such as coulter counter; where in the number of cells 1 measured volume of liquid is counted. This method gives accurate results with larger cells and is extensively used in hospital laboratories to count red and white blood cells. ‘To calculate number per milliliter of sample: 12 cells x 25 large squares x 50x 103 = 1.5 x 10 Ridges that support coverslip Coverslip, NS + Number /mm2 Sample added here; care must 1_Number /mm3 betaken not to allow overflow; cells are counted in large square: 5 space between coverslip and 12 cells (in practice, several Number /em3 (ml) slide is 0.02 mm(, mm). Whole squares are counted and grid has 25 large squares,atotal the numbers averaged.) area of 1 mm? and a total volume of 0.02 mm’. Viable Count: A bacterial culture need not contain all living cells; there might be some dead cell as well. The culture when grown in proper medium and under standard set of growth conditions, only living cells grow and from colony. This fact is used to estimate number of living bacterial cells. ‘The estimations the number of living bacterial cells is called viable count. Several plating methods can be used to determine the number of viable microbes in a sample. These are referred to as either viable counting methods or plate counts because they count only those cells that are able to reproduce when cultured. Two commonly used Procedures are the spread-plate and the pour-plate techniques. Once the number of coloknown, the original number of viable microorganisms in the sample can be calculated from that number and the sample dilution, For example, if 1.0 milliliter of a solution diluted by @ factor of 1 10° yielded 150 colonies (ie,, 1.5 * 10? colonies), then the original sample contained around 1.5 x 10° cells per milliliter, However, because it is not possible to be certain that each colony arose from an individual cell; the results are often expressed in terms of colony forming units (CFU), rather than the number of microorganisms. Coulter Counter: Coulter counter is an electronic device used to count number the number of microorganisms. This device is provided with a tiny orifice 10 - 30 pm in diameter. This orifice connects the two counterparts of the counter which contain an electrically conductive solution (electrodes). In this method, the sample of bacterial cell is forced through the small orifice (small hole). On the both side of the orifice, electrodes are present to measure the electric resistance or conductivity when electric current is passed through the orifice. Every time a bacterial cell passes through the orifice, electrical resistance between the two compartments (electrodes) increases momentarily or the conductivity drops. This generates an electrical signal which is automatically counted. Each electrical signal represents the counting of one bacterial cell. Membrane -filter Technique: Microbial cell numbers are frequently determined using special membrane filters possessing millipores small enough to trap bacteria. In this technique, a water sample containing microbial cells is passed through the filter. The filter is then placed on solid agar ‘medium or on a pad soaked with nutrient broth (liquid medium) and incubated, Counting the colony gives the number of microorganisms in the filtered sample. This technique is especially useful in analyzing aquatic samples.MPN: Most probable number (MPN) determination is @ method where, numerous replicates i ; e E acul e al : ar ; in itable liquid a 5 ining a sui of several dilutions of a culture are made and added to tubes containing ibe is examined to determine if growth occurred. It is growth medium. After incubation, each tul th was assumed that the last tube in the dilution series that demonstrates growl lls, between one and 10 cells, while the next tube had between 11 and 100 cel ive In thi growth is observed, then the tube is assumed not to have received any cells. In inoculated with and so on. If no is way, the only used number of cells in the original sample is estimated. MPN values are most common'y when a selective medium can be employed that supports the growth of a specific type of microbe. It is often used in assessing microbial density in water samples. v Measurement of cell mass: Dry Weight Technique: The cell mass of a very dense cell suspension can be determined by this technique. In this technique, the microorganisms are removed from the medium by filtration and the microorganisms on filters are washed to remove all extraneous matter, and dried in dessicator by putting in weighing bottle (previously weighed). The dried microbial content is then weighed accurately. This technique is especially useful for measuring the growth of microfungi. Turbidometric Estimation: Rapid cell mass determination is possible using turbidometry method. Turbidometry is based on the fact that microbial cells scatter light striking them. Since the microbial cells in a population are roughly constant size, the amount of scattering is directly proportional to the biomass of cells present and indirectly related to cell number. One visible characteristic of growing bacterial culture is the increase in cloudiness of the medium (turbidity). When the concentration of bacteria reaches 10° cells per ml, the medium appears slightly cloudy or turbid. Further increase in concentration results in greater turbidity. When a beam of light is passed through a turbid culture, the amount of light transmitted is measured. Greater the turbidity, lesser would be the transmission of light through medium. Thus lightwillbe transmitted in inverse proportion to the number of bacteria. Turbidity can be measured by using instruments like spectrophotometer, colorimeter etc. Total Count: ‘The total count of bacteria means to say the total number of cell present in the sample irrespective of whether it is living or not. Total count = Viable cell + Dead cell Growth Yield: ‘The total growth of culture represent the difference between the initial cell mass or cell number and the final cell mass or cell number formed when population enter the stationary phase, When growth is limited by the concentration of particular nutrient there is fixed relationship between total growth and initial concentration of nutrient in the medium, The mass of cell material produce per unit mass of nutrient used is a constant which is known as growth yield. The amount of microbial mass produce from a nutrient can be expressed quantitatively as the growth yield. Thus, sold cy) = Mass of microorganism form Growth Wield (Y) = -rrass of substrate consumed The Y value often express in terms of gram of cell form per gram of substrate used. It is the index of the efficiency of conversion of nutrient into cell material.