Diversity of microorganisms

1. describe the general characteristics of representatives of the following groups of microorganisms, illustrated by the examples named in each group. FEATURE Cell wall Flagella Chlorophyll Membrane bound organelles Nucleus (envelope) Ribosomes Nucleic acid type Capsid BACTERIA Murein/ peptidoglycan Lack 9+2 microtubules Rare (archaeobacteria) Absent Absent (prokaryon) 70S (smaller) DNA (circular) & RNA is present Absent FUNGI Usually chitin Absent Absent Present Present (eukaryon) 80S DNA (linear) and RNA is present Absent VIRUSES Absent Absent Absent Absent Absent (akaryon) Absent DNA or RNA
(Never both)

Present

Bacteria
2. recall the structure of a bacterial cell and its inclusions, as illustrated by Escherichia coli;

Escherichia coli (Refer to unit one notes specification number 36.)

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3. understand that cell structure as a means of classifying bacteria; describe and understand the use of gram staining in identifying bacteria; Bacteria is classified as a prokaryote because of the absence of an envelope bound nucleus and membrane bound organelles. The DNA is circular (looped). The cell shape is also an important aid to classification. Some examples are given

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4. practical work to include use of Gram staining in the identification of bacteria. Another way of classifying and identifying bacteria was devised by Hans Christian Gram, a Danish scientist. According to the Gram staining technique, bacteria can either be Gram positive or Gram negative. The details of the Gram staining technique and its theoretical background are described below.

Gram positive bacteria have a thick murein cell wall (which retains crystal violet colour). In Gram negative bacteria the Murein cell wall is very thin so stain is removed easily. Gram negative bacteria have a lipopolysaccharide membrane outside the cell wall, which is washed away by ethanol.

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The diagram above represents the structure of the cell walls of Gram positive and Gram negative bacteria. The difference in the cell wall structure accounts for the difference in colour of the bacteria after Grams staining is carried out. The Gram+ bacteria appear purple and the Gram- bacteria appear pink. The Gram staining technique is described in the flow chart below. 5. understand that bacteria are agents of infection, invading and destroying host tissues, producing toxins; describe the production of exotoxins (Staphylococcus) and endotoxins (salmonella), and the invasion of host tissue (Mycobacterium tuberculosis). Pathogenic bacteria gain entry into body tissues by secreting enzymes like hyaluronidase which breaks down (hydrolyse) hyaluronic acid (substance binding cells together). This leads to tissue damage, once inside the host tissue, the bacteria secretes toxins which affects host cell metabolism. Staphylococcus aureus is a Gram positive bacteria which causes food poisoning by secreting exotoxins. These exotoxins are effective in very small quantities and the symptoms of food poisoning (Diarrhoea, Vomiting, abdominal pain, fever) become visible, 1 to 6 hours after ingestion of contaminated food. These toxins are enterotoxins (intestinal toxins). Salmonella enteritidis is a bacterium which causes food poisoning by producing Endotoxins. The bacteria is Gram negative and gains entry into intestinal tissue. When the bacterial cell is destroyed by lysis, the lipopolysaccharides of the outer membrane are released, which causes food poisoning. The symptoms of food poisoning are visible after 1 or 2 days of consumption of contaminated food, because the toxins are effective only in large quantities. Mycobacterium tuberculosis a Gram positive, rod shaped bacterium which causes tuberculosis. Spread from person to person through infected droplets in air. Inhaled bacteria are engulfed by macrophages (WBCs) in the lungs. The bacteria multiply inside the macrophages and are carried to the lymph nodes, where T cells (lymphocytes) activate macrophages to engulf more bacteria. The macrophages then surround the bacteria to form tubercles (macrophages + bacteria + calcium). This is the primary infection. The tubercles rupture later on and release the bacteria, which destroy tissues like the lungs, brain, bones, etc. This is the secondary infection. The secondary infection is the infectious stage, during which period the bacteria can be transferred to other people. Symptoms: loss of weight, loss of appetite, shortness of breath, coughing, etc.

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Fungi (yeasts and moulds)

6. describe the general characteristics of fungi; Fungi are made up of eukaryotic cells, containing an envelope bound nucleus, membrane bound organelles and 80S ribosomes. Fungi are non-chlorophyllous. Fungi have non-cellulose cell walls. Fungi never have flagella. Fungi usually reproduce by spores. Fungi are heterotrophic some are parasitic and others are saprobiontic. They usually exhibit extracellular digestion. 7. identify the differences in structure between yeasts and moulds as illustrated by Saccaromyces and penicillium.

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Differences between yeast and moulds.

Yeast Single celled Mononucleate Cell wall contains glucose and mannose Yeast does not form hypha. (may form chains pseudomycelium) Moulds Multicellular Multinucleated (coenocytic) Cell wall made of chitin Forms filamentous mycelium (hyphae).

8. understand that the classification is based on virus structure and nucleic acid types as illustrated by lambda phage (DNA), tobacco mosaic virus (RNA) and human immunodeficiency virus (RNA retrovirus). Viruses contain either DNA or RNA (never both), usually with associated enzymes. A protein coat (capsid) is present round the nucleic acid. This is made up of individual proteins called capsomeres held together by chemical bounds. (Sometimes) an envelope is present outside the protein coat. The envelope is made up of a phospholipid bilayer with embedded proteins / glycoprotein of the virus. The envelope is usually obtained from the host cell membrane, but host protein are replaced by viral proteins. The type of nucleic acid present and the shape / structure of capsid is a means of classifying viruses. All viruses are parasites, affecting specific host cells. Retroviruses are those viruses which contain RNA and reverse transcriptase. Virus name Nucleic acid Shape of capsid Host cell type Tobacco mosaic virus RNA Helical (non enveloped) Tobacco leaf cells (TMV) Lambda Phage virus DNA Complex - polyhedral head and Bacteria (E.coli) helical tail. (non enveloped) Human immune RNA Polyhedral (enveloped) T4 lymphocytes of deficiency virus (HIV) (Retroviruses) humans

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9. understand that viruses are agents of infection; the nature of host cell specificity; describe the cell infection cycle and latency as illustrated by human immunodeficiency virus (HIV)

Life cycle of HIV

Virus comes into contact with a T4 lymphocyte cell (WBC). Virus glycoprotein (gp120 on envelope) attaches to CD4 (receptor protein) receptors on T4 cell surface membrane. Virus (capsid) enters cell by endocytosis. Viral RNA and reverse transcriptase enzyme is released into T4 lymphocyte cytoplasm. Double stranded DNA copy of the single stranded viral RNA is made using reverse transcriptase Viral DNA inserts itself into host DNA. This is passed onto other cells during cell division. Number of infected cells increase. Viral DNA remains inactive. This is called latency period, which lasts for above 5 years At the end of latency, viral DNA becomes active and produces viral RNA (transcription), which synthesises viral proteins (translation). New viral particles assemble (capsid around viral RNA). Viral particles bud off from the cell membrane of the host by exocytosis. Lysis of cell occurs.

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10. describe the essential nutrients (carbon sources, nitrogen sources, mineral and growth factors) appropriate to the growth of heterotrophic microorganisms; Requirements for growth of microorganisms MACRONUTRIENTS (required in large quantities) ELEMENTS USES TO THE ORGANISM HOW SUPPLIED C,H,O For formation of lipids, proteins, C- organic acids, carbohydrates, amino acids. carbohydrates, nucleic acids, etc. H- H2O and organic compounds. For forming structural components O2 - Air, organic compounds and for energy N (Nitrogen) To make proteins, nucleic acids, ATP Ammonium ions, amino acids. N2 gas for Rhizobium. P (Phosphorous) For ATP, nucleic acids, phospholipids Phosphate ions (NH4) 3 PO4 S (sulphur) For proteins Sulphates, S containing amino acids. Fe (Iron) Act as enzyme activators As FeSO4 (salts) Na, K, Ca, Mg To maintain osmotic potential of the cells and for transport / absorption of As salts (NaCl, KCl, CaCl2 , MgSO4) substances across membranes Micronutrients (required in smaller amounts) V, Co, Mn, Zn act as enzyme activators. They are added to the culture medium in any soluble form. Vitamins, purines (Adenine, Guanine), pyrimidines (cytosine, Uracil, Thiamine) are needed as growth factors for formation of new DNA and RNA. The composition of the culture medium can be altered to suit the type of microorganisms being cultured (Refer to type of culture media). 11. understand the environmental influences of temperature, oxygen level and pH on growth.

Environmental factors affecting growth of microorganisms.

1- Temperature: Temperature affects enzyme activities. Low temperature decrease kinetic energy of enzyme and substrate molecules, high temperatures, above optimum, denatures enzymes so activity decreases. Enzymes regulate growth and metabolism Optimum temperature is determined by the type of microorganism being cultured. Psychrophiles (low optima) optimum temperature =13 0C. eg: Flavobacterium Mesophiles optimum temperature = 39 0C eg: E.coli Thermophiles optimum temperature = 60 0C eg: Bacillus stearothermophilus Hyperthermophiles optimum temperature = 88 0C eg: Desulphovibrio

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2- Oxygen: Different bacteria have different oxygen preferences. Obligate anaerobes grow only in the absence of oxygen. Oxygen is toxic to it. eg:Desulphovibrio. Obligate aerobes grow only in the presence of oxygen. eg: Micrococcus luteus. Facultative aerobes can grow without oxygen, but grow better if oxygen is present. Eg: E. Coli Microaerophillic aerobes require O2 at a lower concentration than air eg: Spirillum volutans
Diagram showing the distribution of bacteria grown in a test tube filled with nutrient broth.

In a large industrial fermenter the culture is supplied with O2 by bubbling sterile air through the medium. Anaerobic bacteria are grown in anaerobic jars which contain H2 gas and CO2 mixture along with a catalyst to remove O2. 3- pH microorganism have an optimum pH between 5 and 9. Fungi are more tolerant to acids, than bacteria. The pH of culture medium is kept constant by Buffers. The pH must be constantly monitored and regulated during the culture process, as microorganisms tend to release / absorb acids during metabolism. 12. culture techniques

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Aseptic techniques the systematic precautions taken to keep cultures pure and free from contamination. All apparatus (Petri dishes, pipettes, flasks, etc) must be sterilised by autoclaving at 1210C for 15 minutes. Or by irradiating with ultraviolet rays (Wavelength - 254nm). Using sterile culture medium Wiping bench / work surfaces with 70% ethanol before and after work. Flaming the necks of bottles. Flaming wire loops before and after use. Lids of containers not left on benches to prevent air borne contamination Petri dishes opened slightly during operation to avoid contamination Cultures after study and contaminated equipment must be autoclaved / sterilised before disposal or reuse.
Types of culture media

1- Defined media: The exact chemical composition of the medium is known. It is prepared by dissolving a known amount of specific compounds in distilled water. This is useful to investigate the effect of different nutrients on the growth of bacteria. Eg: KH2PO4 , K2HPO4 , (NH4 )2 SO4 , FeSO4 , KCl, C6H12O6 , NaCl, FeSO4 , H2O (contains all essential nutrients for growth). 2- Undefined media: The exact chemical composition of the medium is not known. Eg: yeast extract, malt extract, beef extract, peptone. 3- Selective media: It contains substances to inhibit the growth of some microorganism but promotes the growth of other microorganisms. Eg: Macconkey agar It contains lactose and bile salts which promotes the growth of E.coli, but inhibits the growth of other enteric (intestinal) bacteria. 4- Indicator media: It contains substances which indicate changes in pH of the media. The change in pH of the media occurs due to production of acid (more commonly), or alkalis during metabolism or growth of cultures. Changes in pH are good indicators of growth. Eg: Phenol red changes from red to yellow when the microorganisms produce acids by metabolism 5- Solid media: The nutrients are dissolved in water and a solidifying agent is added to it (eg: agar). The microorganisms grow in colonies on the surface of the medium. It is useful to isolate colonies of a particular species and to prepare slope / slant cultures to obtain pure or stock cultures by subculturing. Nutrients are neither added nor removed during the culturing process. 6- Liquid media: Nutrients are dissolved in distilled water and microorganisms grow while submerged / suspended in the medium. Nutrients and microorganisms can be added or removed during the culturing (fermentation) process. The medium must be aerated to supply O2 for aerobic respiration, agitation of medium is necessary to prevent microorganisms and nutrients from settling down. Agitation can be done by pumping compressed air through the broth (air - lift fermenters) or by using impellers with motor. Liquid media is commonly used in fermenters for large-scale production of microorganisms.

Selective as well as indicator medium

Eosin Methylene Blue (EMB) agar is an indicator as well as a selective medium. Methylene Blue inhibits Gram positive intestinal (enteric) bacterial growth (Staphylococcus). Gram Negative bacteria like E.coli and Salmonella (which secretes lactase) grow well as they can utilise glucose and lactose from the medium. Eosin changes colour to black when acids are produced by metabolism.

13. Describe the use of fermenters (bioreactors) for the production of mycoprotein and antibiotics; describe and understand the differences between batch and continues fermentation;
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Types of fermentation / cultures.

Batch culture: growing of microorganisms in a fixed volume of nutrient broth. Nothing is added or removed during the fermentation process, except oxygen / CO2. Lag phase, Exponential phase and stationary phase occurs. The culture / products are harvested in the stationary phase. Eg: penicillin production. During penicillin production, nutrients are added in calculated quantities during the stationary phase. This helps to sustain the culture in the stationary phase for a longer period of time and increase the yield of penicillin. This type of culture is called as a fed-batch culture or semi-continuous culture. Continuous culture: Spent nutrients and microorganisms are removed at regular intervals during the culture / fermentation process. Equal volumes of fresh nutrients are then added to maintain the culture in a continuous stage of exponential growth. This ensures a continuous supply of culture products. Eg: production of mycoproteins (Fungal proteins) 14. Understand that industrial processes involve the need for aseptic entry of material, culture inoculants, media, aeration, temperature, pH, agitation, product recovery; describe how culture conditions may be controlled.

The nutrient medium is sterilized by autoclaving at 121oC for 15 minutes. Ultraviolet radiation can also be used to sterilize nutrient medium or apparatus. Filter-sterilized air is pumped through the nutrient broth to provide oxygen for aerobic respiration. The inoculum syringe ensures that other microorganisms from the surrounding cannot enter the flask during inoculation. The harvest syringe ensures that microorganisms and used nutrient medium can be removed / added without allowing other microorganisms to enter the flask. This arrangement allows the culture to be free from unwanted microbial contamination. This reduces competition and

allows us to obtain pure cultures or culture products. The temperature of this fermenter can be regulated by using a water bath or an incubator in the laboratory. The pH can be maintained by the use of buffer solutions added to the nutrient medium. The culture can be agitated by using a stirrer. This type of small scale fermenter is useful to grow stocks of pure cultures for use in large industrial fermenters. The conditions for growth and nutrient requirements for microorganisms to be used in industrial fermentation can be studied before large scale production is attempted.

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PH c ontroller (acid base pum p) Inoc ulum (Corn steep liqour) Nutrients (uv treated) PH/temperature probe Motor Exhaust

Water out Im peller

S parger

Cooling jac ket Nutrient broth

Water in

S team Harvest line

S terile air Filter (for aerobic respiration)

The diagram represents a large industrial fermenter, which can be as large as 200,000 dm3. The nutrient medium can be sterilized by Ultraviolet radiation or by heating at 1210C for 15 minutes at a pressure of 103 kPa. The fermenter is sterilized by passing super-heated steam through the empty fermenter. Sterile air is pumped into the fermenter to provide aeration for aerobic respiration. The air can be sterilized by filtration or ultraviolet radiation. The sparger splits the air into small bubbles, which increase the surface area and efficiency of gas exchange. The impeller is connected to a shaft which is driven by a motor. This agitates the culture medium so that the microorganisms and nutrients do not settle down at the bottom. The cooling jacket maintains the optimum temperature for the growth of the culture. It removes the heat which is released by the respiration of microorganisms, and prevents overheating of the culture. pH is controlled by the addition of buffers. The products of the culture process are harvested from the harvest line. The fermenters are designed in such a way that the aseptic entry of nutrient medium, inoculants, buffers, air, etc is ensured. This enables the growth of pure cultures. Fermenter design should also ensure aseptic harvest of products. The design of the fermenter must be such that it allows us to monitor and regulate culture conditions like pH, temperature, growth, etc. This is necessary for the effective growth of cultures.

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Practical work to include preparation and sterilization of media, agar plate pouring and inoculation using sterile wire loops. Pipettes and spreaders; the investigation of the use of different carbon and nitrogen sources for growth using culture on agar plates or in liquid culture

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Understand the stages of growth of microorganisms in culture; describe and understand diauxic growth; describe and understand the production of secondary metabolism;

Growth curves
A. LAG phase: the number of organisms remains constant. They microorganisms may grow in size (not in number). They are preparing to secrete enzymes for growth and adjusting to the new environment. The duration of the lag phase depends on the growth phase of the inoculum, composition of the medium, physical conditions like pH, temperature, etc. B. LOG / exponential phase: population increases rapidly. No competition for resources (which are available in plenty). Cells are metabolically very active utilizing nutrients and dividing rapidly. The population doubles with every generation. The substances secreted by the microorganisms during this stage are needed for growth and are referred to as primary metabolites. The growth rate constant or mean doubling time depends on the genetic makeup of the microorganisms and the environmental factors like nutrients, pH, temperature, etc. C. Stationary phase: the population remains constant. Number of cells formed equals number of cells dying. The overall growth rate equals zero. Changes in pH, depletion of nutrients, accumulation of metabolites, etc. act as limiting factors. The conditions are sufficient to maintain metabolic activity, but not sufficient to promote growth of the cultures. The substances produced by microorganisms during this stage are not needed for growth and are called secondary metabolites. D. Death phase: The population decreases. The rate of death is greater than the rate of cell division. Both total cell count and viable cell count decreases due to lysis of cells. Lysis of cells may provide nutrients for some viable cells, but there is still an overall decrease in the number of cells.

DIAUXIC GROWTH: this is the growth curve obtained when microorganisms are cultured in a medium containing two carbon or energy sources. The microorganisms first use the preferred carbon source and then begin to use the second, less preferable carbon source. There is an initial lag phase, log phase, followed by another lag phase, log phase and stationary phase, as shown in the diagram below. E. coli when grown in a medium containing two sugars (Glucose and lactose) shows diauxic growth. The 1st growth phase uses glucose. When glucose is used up it enters the second lag phase during which time it is synthesizing lactase to hydrolyze lactose to glucose and galactose. These sugars are then utilized for the 2nd log phase (growth).

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Describe methods of measuring culture growth as illustrated by cell counts, dilution plating, mass and optical methods (turbidity); construct growth curves and calculate growth rate constant. Practical work to include the use of these methods to investigate the growth of a suitable microorganism in liquid culture The number of generations produced per hour is called the exponential growth rate constant (K). It can be calculated from the formula given below.

K = (log10Nt - log10N0 )/ 0.301 x t (log10Nt is the population at time t hrs and log10N0 is the population at 0 hrs)
Generation time: It is the time taken for the population to double. It is also referred to as the mean doubling time. It can be calculated from the growth rate constant (K). Mean doubling time = 1/K hrs OR 60/K mins Measuring cell growth: 1- Dry Mass Measurement Measuring dry mass of cultures. The culture is stirred thoroughly and a sample is obtained. The sample is then filtered and centrifuged. The dried pellet is then weighed to calculate the mass of microorganisms per unit volume of the medium. This method is suitable to measure filamentous fungi, grown in liquid medium. Unsuitable for measuring bacterial growth as an inaccuracy of 1g accounts for 5 x 109 cells. 2- Haemocytometry This is a method which gives the total cell count. It is particularly useful for counting of yeast cells or algae. A haemocytometer is used to count the actual number of cells in a known volume of sample under the microscope. Hence it is also called the direct cell count. The haemocytometer has a counting chamber of area 1mm2 and depth of 0.1 mm , hence it has a volume of 0.1mm3. This largest square is referred to as a Type A square. There are 25 smaller squares, each of area 0.04 mm2. The volume of each smaller square is 0.004 mm3. This is referred to as a Type B square. Each Type B square is marked off by triple lines, as shown in the diagram. Within each of the type B squares there are 16 smaller squares. These squares are referred to as type C squares. These squares make it easy to count the cells under the microscope.

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Generally the number of cells in each Type B (0.004 mm3) square is counted and the number is used to calculate the cells per mm3 of the culture.

Advantages: The haemocytometer can give the direct cell count / total cell count. Disadvantages: The haemocytometer cannot give the viable cell count. Clumping of cells can give inaccurate results. It is a time consuming and laborious process. Eg. The number of cells in one type B square (0.004mm3), as shown in the diagram, is 9. Therefore, the number of cells per mm3 is 9 / 0.004 = 2250 cells / mm3. If the sample of cells placed in the counting chamber had been diluted, then multiply by the dilution factor to find the number of cells in the original sample. If the sample in the diagram has a dilution of 1/200. Then the dilution factor is 200. So the number of cells in the undiluted sample would be 2250 X 200 = 450000 cells / mm3 3- Turbidometry This method gives the total cell count. A photometer or a spectrophotometer passes light through a sample of cells and finds the amount of unscattered light passing through the sample. The unit for measurement of photometer is called Klett units and the unit of measurement in a spectrophotometer is optical density (OD). However, the photometer must be calibrated to find the optical density/ turbidity which coincides with the corresponding number of microorganisms in the culture solution / sample under study. This can be done by using a method of direct counting like, haemocytometry or dry weight measurement in combination with a photometer. A calibration curve is then plotted and can be used for further measurement of the particular species of microorganism. There are standard calibration curves available for different species of microorganisms, in specific culture solutions. The advantage of using turbidometry is that it is a very quick process and saves labour and time when taking multiple readings. It can also be performed without disturbing or destroying the sample.

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Disadvantages: This method give only the total cell count, it cannot give the viable cell count. It must be calibrated before use, by using a haemocytometer or weight method. The solid line in the graph shows the calibration curve for yeast cells in a culture solution obtained by weighing method or haemocytometry. The dotted line shows the corresponding readings obtained by turbidometry. Note that the readings do not coincide as the number of cells in the medium increases. Clumping / settling down of cells gives lower than actual values. At high concentrations of cells the method is less accurate due to rescattering of light onto the photocell. Advantage: It is a Quick process, saving time and labour when taking multiple readings.

4- Dilution plating: This method gives the viable cell count. A known volume of culture is placed in a Petri dish and allowed to grow. The number of colonies formed after incubation is then counted to find the number of viable cells in the culture. A viable cell is one that can divide and form offspring or colonies. For this reason it is also called plate count or colony count. The assumption made here is that each viable cell can yield only one colony. There are two ways of performing a plate count- the spread plate method and the pour plate method, as shown in the diagram below. Disadvantage: It takes a long time, as viable cells have to grow into separate colonies on agar plates. The number of colonies obtained in a viable count depends not only on the inoculum but also on the culture medium and the incubation conditions. Sometimes cells may clump together and form a single colony.

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With both methods it is important that the number of colonies growing on the plates should not be too large, which may lead to fusion of colonies and erroneous measurements. The number of colonies should not be too small either, as this would make the statistical significance of the count too low. The usual practice is to count colonies on plates that have between 30 to 300 colonies. To obtain the appropriate colony number, the sample to be counted must always be diluted. Dilution can be carried out by adding 1ml of the original sample to 9ml of broth. Serial dilutions can then be carried out as shown in the diagram. 1ml or 0.1 ml of each diluted sample is then transferred to a Petri dish and incubated. The number of viable cell can then be calculated as explained in the diagram below.

The dilution factor is the reciprocal of the dilution. Thus if the dilution is 1/100, then the dilution factor is 102.

The number of cells = number of cells per mm3 of diluted sample X dilution factor.

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A.3 use of microorganism in biotechnology Food and Drink

Describe and understand the process involved in lactic acid fermentation leading to the production of yoghurt;

The nutritional value of yoghurt is the same as that of milk. However the type of nutrients is different from milk. Milk contains lactose, but yoghurt does not. This makes it suitable for consumption by lactose intolerant people. Yoghurt proteins are easier to digest, as the bacteria partially digest milk proteins. The yoghurt bacteria can synthesise vitamins in the alimentary canal. The low ph of yoghurt is unfavourable for bacterial growth. This enables yoghurt to be preserved longer than milk can be preserved at room temperature.

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describe the process involved in fermentation by yeast in brewing and dough production; understand the metabolic process involved;

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 Describe the production of mycoprotein. Practical works to include a study of the optimal conditions (eg: temperature, pH, nutrients) necessary for yoghurt production or for fermentation by yeast in brewing or dough production.

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Mycoprotein is obtained from the filamentous fungus Fusarium graminaerum to produce QUORNTM. The fungus is grown as a continuous culture in large bioreactors / fermenters. The sterile nutrient broth contains: GLUCOSE: which is a carbon and energy source for the culture. BIOTIN / CHOLINE: Growth factors, increase length and diameter of fungal hyphae. COMPRESSED AIR: Provides oxygen for respiration. It also agitates the culture medium preventing nutrients and microbes from settling down. This type of fermenter is called an Air Lift fermenter. AMMONIA: It is a source of nitrogen for protein and nucleic acid synthesis. It also maintains the pH of the culture medium between 6 to 6.7, by neutralizing carbonic acid and other organic acids produced by metabolism. The RNA content is reduced by thermal treatment or by using enzymes. This is a continuous culture which could run continuously for 100 days and can produce 150 tonnes of protein per day. One the advantage of a continuous culture is to ensure a continuous supply of products. Advantages of single cell protein Disadvantages of mycoprotein (SCP) High yield of proteins in a short period of time and in SCP are deficient in some amino acids limited space, when compared to plant/animal like lysine and Methionine (these must be proteins. added). Production independent of climate condition. High purine and RNA content (10%) must be reduce to about 2%. High RNA content Can be grown using agricultural or industrial waste as results in formation of uric acid, causing a culture medium. Gout like symptoms. Eg: molasses from sugar cane; methanol, ethanol from petroleum industry, citrus peel from juice industry; pulp Cell walls of fungi are indigestible. from paper mills; etc Industrial wastes, when used as a medium may contain toxin. Hence SCP must pass high protein and fibre content, low fat content prevents heart diseases and constipation. all toxicity trails before being available for human consumption. Shelf life longer when stored as a dry powder.
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describe the production of antibiotics; illustrated by penicillin form penicillium.

PH c ontroller (acid base pum p) Inoc ulum (Corn steep liqour) Nutrients (uv treated) PH/temperature probe Motor Exhaust

Water out Im peller

S parger

Cooling jac ket Nutrient broth

Water in

S team Harvest line

S terile air Filter (for aerobic respiration)

PENICILLIN PRODUCTION: penicillin is produced by growing the fungus Penicillium chrysogenum in large scale fermenters with a capacity of up to 200000dm3. Steam is passed through the empty fermenters under high pressure (1210 C, 15min, 103 kPa), to sterilise the vessel. The nutrients medium is sterilised by ultraheat treatment or UV radiation. The commonly used nutrients are corn steep liquor as a nitrogen source and lactose or glucose as a carbohydrate source. The nutrient medium inoculated with a pure strain of P.chrysogenum following aseptic techniques. Sterile air (filtered / heated and cooled) is pumped through the medium through the sparger. The impeller stirs the medium increasing the rate at which oxygen dissolves. It also prevents clumping of mycelia and promotes efficient heat exchange between media and cooling surfaces. It also maintains a concentration gradient between the cells and media for more efficient exchange of materials. The acid base reservoir can be used to regulate the pH between a range of 6.8 to 7.7. The cooling jacket regulates temperature at 250 C to 270 C. Penicillin is produced as a secondary metabolite during stationary phrase. The process is usually completed in about 6 to 7 days. Secondary metabolites are substances which are produced by microorganisms during the end of exponential phase or in the stationary phase. These substances are not required for growth of the culture, but are usually secreted to prevent the growth of other microbes in the culture medium. So, it must be obtained by BATCH CULTURES.

Down stream processing (product recovery)

Purification and modification of the desired product from the culture medium. Eg: distillation recovery of fuel alcohol and spirits (volatile liquids) Flotation recovery of yeast from yeast cultures during beer production(froth flotation method) Filtration clarification of sweets Wort in beer production (Removes cell debris) Centrifugation separation of cell from viscous medium (Beer) Solvent extraction for extraction and purification of penicillin using Amyl acetate.
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The process of downstream processing for penicillin, by solvent extraction is summarized below.

understand the effect of antibiotics (penicillin) on bacterial growth; understand antibiotic resistance and the reasons for its spread. Antibiotics are substances which inhibit the growth of microbes (bacteriostatic / microbiostatic), or kill microbes (bactericidal / microbicidal). These substances are usually secreted by bacteria and fungi to kill other microbes, which compete for available resources. However, today antibiotics may also be synthetic or semisynthetic. Penicillin is bacteriostatic. It prevent cell wall synthesis, so that new cells cannot be formed(lysis of the cells will occur during cell division). Thus population cannot increase and WBCs can easily destroy bacteria. Penicillin affects Gram positive bacteria. Penicillin cannot penetrate the outer lipopolysaccharide and lipoprotein membrane of Gram negative bacteria, so it cannot act on the cell wall (murein), hence Gram Negative bacteria are less affected. Antibiotic resistance is the acquired ability of microorganisms to resist the effect of an antibiotic to which it is normally susceptible. This may occur due to genetic variation (usually caused by mutations) in bacterial populations. Nature selects for the resistant strains and selects against the non resistant strains. These mutations (resistant strain) are inherited by successive generations, spreading resistance in the populations.

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