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Bacteriology


Bacterial Morphology (cell shape)
4 common and characteristics shapes of bacteria

 

         Cocci( spheres); Bacillus (Rods); Vibrio (curve rods/comma shaped)) and spiral

         Cocci – spherical bodies,  sometimes slightly elongated,plane of division generates uniquely shaped clusters – diplococcus, streptococcus, staphylococcus,sarcina e.g. Staphylococcus aureus ( coccus in clusters), Micrococcus luteus ( coccus in tetrads due to regular alternation of to plane of division).

         Bacillus –rod shaped : short coccobacillus,e.g. Bacillus cereus common soil inhabitant also found on food products, can form endospores, spores are resistant to heat, can germinate improper storage of food. Toxin formed:heat-labile during exponential phase(diarrhoea) and heat-stable formed during sporulation(vomitting)

         Vibrio – comma shaped e.g. Vibrio cholerae causes cholera. Bacteria grow in gut and produce toxin causing severe diarrhoeal disease

         Spiral -  e.g Rhodospirillum (purple non-sulphur bacteria) cockscrew movement by its polar flagella, spirochaetes e.g. Treponema pallidum causes syphilis.

 

The Gram Reaction

 

         1884 Christian Gram developed the staining technique

         Air dry and heat fixed smear, stain with crystal violet.

         Diluted Iodine solution added forming iodine –crystal violet complex

         Decolourisation with organic solvent(alcohol/acetone). It was thought dissolved lipid in cell wall, make cw leaky, complex leaks out, more lipid in cw more stain loss. Gm+ve retain deep blue –black complex and Gm-ve rapidly and completely decoourised

         Counterstaining with safranin resulted in pink colour for Gm-ve cells (cytoplasm picks up colour of safranin); Gm+ve purple blue, colour just intensified since cw  less leaky more complex retained

 

Method separates bacteria into TWO distinct types:

 

         Gram positive cells – crystal-violet complex retain by cells and thus appear purple

         Gram negative cells – decolourisation of cells by alcohol but then counterstaining by safranin gives pink cells.

 

Differences in staining reflects a fundamental difference in the organisation of the bacterial cell wall.

Gm+ve bacteria have thick wall of many layers of polymers peptidoglycan (murein) constituting a barrier to loss of complex,other suggestion says dehydration of cw becomes dehydrated by alcohol, causes pores to close prevents complex loss

Gm-ve bacteria – outer layer of cw rich in lipid, solvent easily enters cell and thin layer of peptidoglycan cannot prevent loss of complexes

 

         Gram Reaction is of a diagnostic value ONLY when applied to prokaryotes which have CELL WALL, does not yield taxonomically useful information when applied to Mycoplasma group or eukaryotic cells.

 

Gram Positive Bacteria

 

         Nearly all are chemoheterotrophs obtain energy from aerobic respiration or on fermentation.

         E.g. methanogenic bacteria are specialized anaerobic chemoautothrops, energy obtained from coupling oxidation of molecular hydrogen with reduction of carbon dioxide to methane.

         Divided into 4 major groups

 

Major constituent groups of Gm +ve bacteria distinguishable by structural characters

 

I. Unicellular bacteria with rod-shaped or spherical cells, reproducing by binary transverse fission: some produce endospores

 

II. Unicellular bacteria with a tendency to structural irregularity  Cells spherical , rod-shaped, or branched coryneform bacteria.

 

III. Bacteria which developed vegetatively as a mycelium and reproduce by mycelial fragmentation: the Proactinomycetes.

 

IV. Bacteria which form a persistant vegetative mycelium and reproduce by the formation of spores, formed in various ways at the tips of the hyphae: the Euactinomycetes.

 

The Gram-negative Bacteria:


Diverse both in structural and functional respects. Include all photosynthetic prokaryotes, most of the chemoautrophic bacteria and many groups of chemoheterotrophs

 

Many Gm-ve are characterised by swimming motility mediated by flagellation.

         Flagellation can be polar (at one pole of cell): monotrichous, multitrichous ( 20-30 flagella)

         Perithrichous ( all around the cell) slower movement

 

Other cellular movement: –

          swimming movement due to axial fibrils e.g. in Spirochaetes

         Gliding movement much slower than swimming movement, forms spreading and highly irregular structure colonies on solid media due to outward migration of cells

 

 

 

Structures found on bacterial cell:


1. Glycolyx layer
(polysaccharide, some protein, polyalcohols and amino sugars)

Capsule (extrapolymeric substance) –found outside the cell, composition varies from organisms to organisms

 

Function:

         Attachment to host’s cell

         Prevents ingestion by phagocytes (WBC) allowing

            bacteria to evade destruction

          virulence – associated with pathogen ability to cause disease

         Increased virulence may occur in pathogen if they have structures that allow them to overcome host immune defenses such as having a capsule

         Prevents drying

Examples: Streptococcus pneumoniae virulent pathogen in capsular form (can be found in non- capsulated form)

 

Slime layer- not as chemically organised as capsular material, more easily deformed layer, will not exclude particles

         Allows dental caries to attach to teeth forming dental plaques

 

2. Flagella – a structure for movement with base attached to cell membrane

         Almost all spiral bacteria and half of bacillus are motile

         Cocci generally non-motile

 

Types of flagellation:

Monotrichous- one flagellum originating from the cell membrane

Lophotrichous – a tuff of several flagella originating from cell membrane

Peritrichous-flagella located around entire cell structure (peri=around)

Some movement that appears to be due to flagella may be Brownian movement –not true movement ; occurs due to water /other liquid molecules striking the bacterium

 

Axial filament- found in spirochaetes fibres that spiral around the bacterium cell wall = their rotation cause a cockscrew-like movement, examples: Trepnomena pallidum (causes syphilis)

 

Pili – hair like appendages normally found in Gm-ve bacteria, smaller than flagella and not use for movement

Types of pili:

Sex pili- joining bacteria to begin transfer of DNA in conjugation

Common pili (fimbriae) – allows adherence of bacteria to mucous membrane to cause disease (indicate virulence of pathogen)

 

3. Cell Wall – affected by drugs – target, made up of peptidoglycan a polysaccharide of two monomer units (N-acetylglucosamine and N-acetylmuramic acid)

Function:

         Maintain shape of cell

         Prevents cell from rupturing during changes in osmotic state

         Chemical components differ in bacterial cell wall

 

4. Plasma Membrane- lies just outside the cell wall and determines what enters and leaves the cell

Function:

         Determines what enters and leaves the cell by being selectively permeable/semi-permeable

         Produces chemical energy(ATP) because there is no mitochondria in bacteria (prokaryotic cells)

 

ENDOSPORE

 

         Structure formed in adverse environmental conditions – drying, toxic chemicals, pH and temperature change, radiation

         Has thick specialised wall with low water content of enclosed resting cell

         Endospore can remain dormant but viable for many years

         6 genera of spore formers, only two of medically and economically important

         Genus Clostridium- Clostridium tetanus (tetanus), Clostridium perfringens (gas gangrene), Cl. botulinum (botulism)

         Genus Bacillus – Bacillus anthracis (anthrax)

 

Sporogenesis (formation of endospore)

 

         Vegetative cell –before endospore formation/development

         Sporulation – process of forming an endospore within a vegetative cell

         Dormant – a resting period, slowed metabolism may remain dormant for years

         Germination- a process which reverse the endospore state back to the vegetative cell state

 

5. Ribosomes – smallest cell structure , made up of two sub-units known as 30s & 50s for prokaryotic cells

 

Two kinds of genetic materials

a. Bacterial chromosomes (genome)

          single circular molecule of double stranded DNA

          contain most of the genetic instructions

         Not associated with histones (protein) found in eukaryotes

         Not found in nucleus but in the nucleoid region

Escherichia coli’s chromosome has been well studied and mapped for the gene location on the chromosome-use in genetic engineering

 

b. Plasmid

         Smaller circular macromolecule of genetic information

         Will not be found in all bacterial cells

         Contain limited amount of genetic information

 

Types of plasmid:

Mating plasmid- assist in establishing bacterial mating types, important  for conjugation

Resistance plasmid- confer resistance to antibiotics

 

MICROBIAL GROWTH

 

         Define as an orderly increase of all chemical components

         Generation time/doubling time -the time taken for the one cell to become two(differ from species to species)

         Increase in mass may not reflect growth-cell simply increasing their content of storage products such as glycogen or poly-- hydroxybutyrate

         Balanced growth- adequate medium allows doubling of biomass plus doubling of all measurable properties of the population, eg. DNA, RNA, protein and intracellular water resulting in constant chemical composition

         Since rate of increase of ALL components of the population is the same, rate of growth can be determine – measurement of any components

 

Genralised Growth Curve of A Bacterial Culture –
e.g. Escherichia coli

 

The curve can be divided into phases:

Lag/Adaptation phase

 

         cells transferred from the stationary phase to a fresh medium will undergo a change of chemical composition.

         Period of adjustment where new enzymes are produce, increase in cell mass no cell division.

         Extremely variable in duration where its length directly related to the length of the preceeding stationary phase

         Influence by age of the culture and type of medium –if young culture and transfer to same type of medium, lag phase will not be encountered; same type of medium will not have much influence but if from rich medium to poor medium longer lag phase

 

Exponential Phase

 

         One cell becomes two/doubling time

         Plenty of nutrient

         Young healthy cells

         Rate of growth influence by: species, environmental conditions (temperature & composition of medium)

 

 

 

Stationary Phase

 

         exponential growth - not indefinitely in batch culture

         Growth limited :essential nutrient used up, build up of waste product, inhibit growth, exponential growth ceases

         No nett increase or decrease in cell numbers

         Cell functions still continues

         Some cells grow some die

         Cells smaller

         Sur genes lead to rapid cell deaths as cell enter stationary phase

 

Death Phase

 

         Cells remain alive continue to metabolize

         Increase amount of toxic products

         Nutrients depleted

         reverse of exponential phase

 

STERILIZATION

 

         Definition:- A treatment that frees object of all living organisms by exposure to lethal physical or chemical agents or , in the special case of solutions, by filtration.

         Pactical goal of sterrilization – the probability that the object contains even one survivor should be infinitesimally small (Stanier, et el. 1984).

         Criterion of death in microorganism - irreversible loss of the ability to reproduce.

         Kinetics of death in a microbial population- nearly always exponential I.e.number of survivors decreases geometrically with time, if the number of survivor plotted against time of exposure- get straight line, negative slope defines death rate, initial population size must be known

         In practice – sterilization involved mixed population

         Spore suspension of resistance used as an assessment to rreliability of the sterilization methods

 

Sterilization by Heat

 

  1. Dry heat-using oven in atmosphere of air-

Disadvantages -requires high intensity and greater duration due to less conduction of heat in air compared to in steam. Bacteria can survive in completely desiccated state, intrinsic heat resistance of vegetative bacterial cells is increased almost to that of spores, lower death rate for dry cells compared to moist ones.

Application – to sterilize glassware, other heat stable solid materials. Objects wrapped in paper/procted from contamination, exposed to 170C or 118 C, 90 minutes

 

  1. Moist heat provided by wet steam - achieved by exposure to steam under pressure in an autoclave at 121 C /15 psi./15 minutes. Killed even bacterial which can survive several hours of boiling.

Disadvantage- not suitable for heat labile materials

Application – to sterilize aqueous solution

 

  1. Sterilization by chemical treatment – chemical used must be toxic and volatile (easily elminated after treatment), e.g.Ethylene oxide a liquid, bpt. 10.7 C, concentration used 0.5-1%, at 0 C to 4 C . But not commonly used (explosive and toxic to human), used too sterilize petri dishes. Sulphur dioxide, anti-fungal used to fumigate and preserve dry fruits/room fumigation in food industries.
  2. Filtration – use for very heat sensitive materials, use of filter with pore size able to retain the bacterial cells. Major therotical limitation – viruses smaller, may pass through filter, therefore never possible to be certain procedure gives  bacterium-free solution also free of viruses
  3. Irradiation – use of radiation to sterilize surgical instruments, some food, other heat sensitive materials.

 

What is food irradiation?

 

         use of ionizing radiation or ionizing energy to treat foods, either packaged or in bulk form.

         Ionizing radiation may come from one of three sources - radioactive isotopes of cobalt or caesium, electron accelorators or X-rays - produce similar effects, all fall in the short-wave, high energy region of the spectrum

         process cannot increase-

  1. the normal radioactivity level of the food, regardless of how long the food is exposed to the radiation, or
  2.  how much of an energy "dose" is absorbed. It can prevent the division of living cells, such as bacteria and cells of higher organisms, by changing their molecular structure.

          It can slow down ripening or maturation of certain fruits and vegetables by causing biochemical reactions in physiological processes of plant tissues.

 

Dosage

 

         quantity of radiation energy absorbed by food as it passes through radiation field during processing( gamma radiation, electron beam and x-ray irradiation).

         measured in Grays (G) or kiloGrays (kGy)  where 1 Gray = 0.001 kGy = 1 joule of energy absorbed per kilogram of food irradiated. Also measured in Rads (100 Rads = 1 Gray).

         Different dosages used to produce different effects in foods. extension of shelf-life of fruits (0.5-1.5 kGy)

         High energy rays of irradiation directly damage DNA of living organisms, inducing cross-linkages and other changes that make an organism unable to grow or reproduce.

         Rays interact with water molecules in an organism, generating transient free radicals causing additional indirect damage to DNA

 

Dosage Use to:

 

  1. Control of harmful bacteria in fresh meat and poultry (1.5-4.5 kGy)
  2. Control of insects, parasites or micro-organisms (0.15 to <1kGy)
  3. Delay of ripening (0.5-2 kGy)
  4. Inhibition of sprouting (0.05-0.15 kGy)

 

 

Why are more and more governments allowing a wider and wider range of food products to be irradiated?

 

         Presently over 30 countries approved applications to irradiate ~ 40 different foods-  include fruits, vegetables, spices, grains, seafood, meat and poultry.

         More than one half of a million tonnes of food now irradiated throughout the world on a yearly basis, amount represents only a fraction of the food consumed annually, it is constantly growing.

         Trend is due to three main factors -

  1. High losses of food due to insect infestation and spoilage: Economic losses between $5 and $17 billion yearly in the US alone. Food irradiation help reduce losses and also reduce dependence on chemical pesticides, some of which are extremely harmful to the environment (eg. methyl bromide).
  2. Increasing concern over food-borne illness: Deaths attributed to food poisoning in the US around 7000 annually.. Food irradiation can help to alleviate human suffering.
  3. Growing international trade in food products: As our economies become more global, food products must meet high standards of quality and quarantine to cross borders.  Irradiation  an important tool in the fight to prevent spread of deleterious insects and micro-organisms.

 

Other commodities irradiated

 

         include medical disposables and hospital supplies, cosmetics ingredients, joint implants, wine and bottle corks and food packaging materials

 

Safety of irradiated food

 

         World-wide standard for food irradiation was accepted in 1983, adopted by Codex Alimentarius Commission, a joint body of the FAO of the UN and WHO. Standard based on e results of a Joint Expert Committee on Food Irradiation study that stated that irradiation of any food commodity up to 10 kGy presents no toxicological or nutritional hazards in foods.

 

 

What about packaging?

 

         Foods can be irradiated either in bulk or in final packaging. Extensive studies have shown that most packaging materials can be irradiated effectively and safely. Approved lists are available from each country.

         Low doses of radiation needed to destroy certain bacteria in food can be useful in controlling foodborne disease. Considerable amounts of frozen seafoods, as well as dry food ingredients, are irradiated for this purpose in Belgium, France and the Netherlands. Electron beam irradiation of blocks of mechanically deboned, frozen poultry products is carried out industrially in France. Spices are being irradiated in many countries including Argentina, Brazil, Denmark, Finland, France, Hungary, India, Indonesia, Israel, Norway, United States, and Yugoslavia.

         The inability of countries to satisfy each other's quarantine and public health regulations is a major barrier to trade. For example, not all countries allow importation of chemically treated fruit. USA and Japan, have banned use of certain fumigants identified as health hazards.

         Problem - acute for developing countries whose economies are still largely based on food and agricultural production. Radiation processing offers an alternative to fumigation and some other treatments.

         US Center for Disease Control early 1990's showed even United States, foodborne diseases caused by pathogenic bacteria, Salmonella, Campylobacter , Trichinae and other parasites, claim estimated 9000 lives annually, causing 24-81 million cases of diarrhoeal disease. Economic losses associated with such foodborne diseases

 

How much food is being commercially irradiated?

 

         Each year about half a million tonnes of food products, ingredients irradiated worldwide, amount is small in comparison to total volumes of processed foods, not many of these irradiated food products enter international commerce.

         One factor influencing pace of development of food irradiation is public understanding and acceptance of  process. So far, this has been difficult to achieve, in view of the misconceptions and fears often surrounding nuclear-related technologies and the use of radiation.

 

  1. Chemical growth control

 

         Antimicrobial agentchemical that kills/inhibit growth of microorganisms, maybe synthetic chemical or natural products.A bacteriocidal kills bacteria, bacteriostatic only inhibit bacterial growth

         Antiseptics- chemical that kills/inhibit growth of microorganisms, sufficiently non-toxic to be applied to animal tissue

         Disinfectants - chemical that kills microorganisms and are used on inanimate objects – sterilants are actually disinfectant which kill microorganisms.

         Antibiotics- chemicals produced by prokaryotes, inhibit or kill other microorganisms. Gm+ more sensitive to antibiotics compared to Gm-ve. Broad spectrum antibiotics acts on both Gm+ and Gm-ve bacteria.

         One of most Important groups of antibiotics:

            - -lactam (penicillins, cephalosporins and cephamycins)

            Inhibits bacterial cell wall synthesis

 

  1. Viral control –antiviral chemotherpeutic agents, most successful is the nucleoside analog (zidovudine of azidothymidine AZT inhibit retrovirus HIV, interferons produced by many animal cells in response to viral infection

 

  1. Fungal control – antifungal agents, ergosterol inhibitors.

 

Pure Culture Technique:

 

         Bacteria and fungi can be grown on artificial media.

         Soild media and liquid media

         Pure Culturing technique- dilution of sample made, proceed with either streaking ( use of inoculating loop) onto surface of well-dried medium or use of spreading technique where inoculum/sample is spread using a glass spreader over surface of well dried medium. Single colonies obtain/visible growth of microorganisms/shows colonial morphology

         Dilution can also inoculated into broth/liquid medium. Growth observed as turbidity in medium.

         Viruses cannot be grown on artificial medium, requires live cells/tissue – use of cell/tissue –culturing technique.

 

Measurement of Microbial growth

 

         Total cell count by direct microscope  count – counting smple under the microscope, quick method.

         Limitation: dead cells also included, small cells difficult to see under microscope , some cells probably missed, precision difficult to achieve, phase contrast is required when cells not stained, not suitable for  cell suspension of low density

         Viable count – live cells counted, plate count or colony count – each individual cell yield one colony (spread plate and pour plate method)

         Disadvantages:

         Clumping of cells, not all cells deposited will give rise to colonies, dilution error but still very useful and commonly used method.

         Turbidimetric Measurement of cell numbers – cell suspension looks turbid, more cells more turbid (cloudy) more light scatter. Turbidity measured usinf photometer or spectrophotometer (pass light through suspension and detect amount of unscattered light emerged).

 

 

 

Metabolic Diversity:

 

         Prokaryotes, as a group, conduct all the same types of basic metabolism as other (higher) organisms,

         in addition- several types of energy-generating metabolism among prokaryotes non existent in eukaryotic cells or organisms.

         The diversity of prokaryotes - expressed by their great variation in modes of energy production and metabolism.

         within a prokaryotic species - may be great versatility in metabolism

 

         For example Escherichia coli :

  1. can produce energy for growth by fermentation or respiration.
  2. respire aerobically using O2 as a final electron acceptor,
  3. can respire under anaerobic conditions, using NO3 or fumarate as a terminal electron acceptor.
  4. use glucose or lactose as a sole carbon source for growth, with the metabolic ability to transform sugar into all necessary amino acids, vitamins and nucleotides that make up cells.

         A relative of E. coli, Rhodospirillum rubrum, has heterotrophic capabilities as E. coli, plus ability to grow by photoautotrophic, photoheterotrophic or lithotrophic means but require biotin as growth factor.

 

         most eukaryotes produce energy (ATP) through ethanol fermentation (e.g. yeast), lactic fermentation (e.g. muscle cells, neutrophils), aerobic respiration (e.g. molds, protozoa, animals) or oxygenic photosynthesis (e.g. algae, plants).

         These modes of energy-generating metabolism exist among prokaryotes, in addition to all following types of energy production which are virtually non existent in eukaryotes.

         Unique fermentations proceeding through the Embden-Meyerhof pathway

         Other fermentation pathways such as the phosphoketolase and Entner-Doudoroff pathways

         Anaerobic respiration: respiration that uses substance other than 02 as a final electron acceptor

         Lithotrophy: use of inorganic substances as sources of energy

         Photoheterotrophy: use inorganic compounds as a carbon source during bacterial photosynthesis

         Anoxygenic photosynthesis: photophosphorylation in the absence of O2

         Methanogenesis: an ancient type of archaeon metabolism that uses H2 as an energy source and produces methane

         Light-driven nonphotosynthetic photophosphorylation: unique archaeon metabolism that converts light energy into chemical energy

 

 

 

 

 

Metabolism

 

         sum of biochemical reactions required for energy generation and use of energy to synthesize cell material from small molecules in the environment.

         has an energy-generating component - catabolism,

          an energy-consuming, biosynthetic component, called anabolism.

         Catabolic reactions or sequences produce energy as ATP, which can be utilized in anabolic reactions to build cell material.

 

         During catabolism, useful energy is temporarily conserved in the "high energy bond" of ATP - adenosine triphosphate.

         Regardless of the form of energy used as its primary source -  energy is ultimately transformed and conserved as ATP (universal currency of energy exchange in biological systems).

         When energy is required during anabolism, spent as high energy bond of ATP (value of about 8 kcal per mole).

         conversion of ADP to ATP requires 8 kcal of energy, and hydrolysis of ATP to ADP releases 8 kcal.

 

Heterotrophic Types of Metabolism

 

Heterotrophy (i.e. chemoheterotrophy)

 

         use of an organic compound as a source of carbon and energy, complete metabolism package.

         cell oxidizes organic molecules to produce energy (catabolism) , then uses energy to synthesize cellular material from these the organic molecules (anabolism).

         Many Bacteria (but just a few Archaea) are heterotrophs, particularly those that live in associations with animals.

         Heterotrophic bacteria major decomposer and biodegraders in the environment.

         Heterotrophic metabolism is driven mainly by two metabolic processes: fermentations and respirations.

 

Fermentation

 

         metabolism in which energy is derived from partial oxidation of an organic compound using organic intermediates as electron donors and electron acceptors.

         No outside electron acceptors are involved; no membrane or electron transport system is required; all ATP is produced by substrate level phosphorylation.

         Fermentation may be as simple as two steps illustrated in the following model. some amino acid fermentations by the clostridia are this simple.

         But pathways of fermentation are a bit more complex, usually involving several preliminary steps to prime energy source for oxidation and substrate level phosphorylation.

 

         In biochemistry - fermentation pathways begin with glucose.

         glucose is the simplest molecule, requiring minimal catalytic steps, to enter pathway of glycolysis and central metabolism.

         In prokaryotes exist three major pathways of glycolysis (the dissimilation of sugars):

  1.  Embden-Meyerhof pathway, which is also used by most eukaryotes, including yeast (Saccharomyces)
  2.  phosphoketolase or heterolactic pathway related to the hexose-pentose shunt;
  3. Entner-Doudoroff pathway.

         Whether or not a bacterium is a fermenter, it will likely dissimilate sugars through one or more of these pathways

 

The Embden-Meyerhof Pathway

 

         Pathway of glycolysis most familiar to biochemists and eukaryotic biologists, as well as to brewers, breadmakers and cheeseheads.

  1. Pathway is used by Saccharomyces to produce ethanol and CO2:
  2. By (homo)lactic acid bacteria to produce lactic acid;
  3. Other bacteria to produce a variety of fatty acids, alcohols and gases.
  4. Some end products of Embden-Meyerhof fermentations are essential components of foods and beverages, and some are useful fuels and industrial solvents.
  5.  Diagnostic microbiologists use bacterial fermentation profiles (e.g. testing an organism's ability to ferment certain sugars, or examining an organisms's end products) for identification to genus level

 

         First three steps of the pathway prime (phosphorylate) and rearrange the hexose for cleavage into 2 trioses by the enzyme Fructose 1,6-diphosphate aldolase, the key (cleavage) enzyme in the E-M pathway.

         Each triose molecule is oxidized and phosphorylated followed by two substrate level phosphorylations that yield 4 ATP during the drive to pyruvate.

         Yeast reduce pyruvate to ethanol and CO2; lactic acid bacteria reduce pyruvate to lactic acid.

         Overall reaction is Glucose ----------> 2 lactate (or 2 ethanol +2 CO2 in yeast) with a net gain of 2 ATP.

         Oxidation of glucose to lactate yields a total of 56kcal per mole of glucose. Since cells harvest 2 ATP (16 kcal) as useful energy, efficiency of lactate fermentation is about 29 percent.

 

         Embden-Meyerhof fermentations in bacteria can lead to a wide array of end products depending on the pathways taken in the reductive steps after the formation of pyruvate. shows some of the pathways proceeding from pyruvate in certain bacteria. Usually, bacterial fermentations are distinguished by their end products into the following groups.

 

1. Homolactic Fermentation. Lactic acid is the sole end product. Pathway of the homolactic acid bacteria (Lactobacillus and most streptococci).

Bacteria are used to ferment milk and milk products:

yogurt, buttermilk, sour cream, cottage cheese, cheddar cheese, and most fermented dairy products.

 

2. Mixed Acid Fermentations. Mainly the pathway of the Enterobacteriaceae. End products are a mixture of lactate, acetate, formate, succinate and ethanol, with the possibility of gas formation (CO2 and H2) if the bacterium possesses the enzyme formate dehydrogenase, which cleaves formate to the gases.

 2a. Butanediol Fermentation. Forms mixed acids and gases as above, but, in addition, 2,3 butanediol from the condensation of 2 pyruvate. The use of the pathway decreases acid formation (butanediol is neutral) and causes the formation of a distinctive intermediate, acetoin. Water microbiologists have specific tests to detect low acid and acetoin in order to distinguish non fecal enteric bacteria (butanediol formers, such as Klebsiella and Enterobacter) from fecal enterics (mixed acid fermenters, such as E. coli, Salmonella and Shigella).

 

3. Butyric acid fermentations, as well as the butanol-acetone fermentation (below), are run by the clostridia, the masters of fermentation. In addition to butyric acid, these clostridia form acetate, CO2 and H2 from the fermentation of sugars. Small amounts of ethanol and isopropanol may also be formed.

 

3a. Butanol-acetone fermentation. Butanol and acetone were discovered as the main end products of fermentation by Clostridium acetobutylicum during the World War I. This discovery solved a critical problem of explosives manufacture (acetone is required in the manufacture gunpowder) and is said to have affected the outcome of the war. Acetone was distilled from the fermentation liquor of Clostridium acetobutylicum, which worked out pretty good if you were on our side, because organic chemists hadn't figured out how to synthesize it chemically. You can't run a war without gunpowder.

 

4. Propionic acid fermentation. This is a bizarre fermentation carried out by the propionic acid bacteria which include corynebacteria, Propionibacterium and Bifidobacterium. Although sugars can be fermented straight through to propionate, propionic acid bacteria will ferment lactate (the end product of lactic acid fermentation) to acetate, CO2 and propionate. The formation of propionate is a complex and indirect process involving 5 or 6 reactions. Overall, 3 moles of lactate are converted to 2 moles of propionate + 1 mole of acetate + 1mole of CO2, and 1 mole of ATP is squeezed out in the process. The propionic acid bacteria are used in the manufacture of Swiss cheese, which is distinguished by the distinct flavor of propionate and acetate, and holes caused by entrapment of CO2.

 

The Heterolactic (Phosphoketolase) Pathway:

 

         Distinguished by key cleavage enzyme, phosphoketolase, which cleaves pentose phosphate into glyceraldehyde-3-phosphate and acetyl phosphate.

         As a fermentation pathway- employed mainly by heterolactic acid bacteria, include some species of Lactobacillus and Leuconostoc.

         Glucose-phosphate is oxidized 6-phosphogluconate, which becomes oxidized and decarboxylated to form pentose phosphate.

         Unlike Embden-Meyerhof pathway, NAD-mediated oxidations take place before cleavage of substrate being utilized.

         Pentose phosphate subsequently cleaved to glyceraldehyde-3-phosphate (GAP) and acetyl phosphate - then converted to lactic acid by same enzymes as the E-M pathway.

         This branch of pathway contains an oxidation coupled to a reduction as illustrated in diagram. Two ATP produced by substrate level phosphorylation.

         Acetyl phosphate reduced in two steps to ethanol, which balances the two oxidations before cleavage but does not yield ATP.

         Overall reaction = Glucose ---------->1 lactate + 1ethanol +1 CO2 with net gain 1 ATP. Efficiency is about half that of the E-M pathway.

         Heterolactic species of bacteria occasionally used in fermentation industry. For example, fermented milk called kefir, analogous to yogurt which is produced by homolactic acid bacteria, is produced using a heterolactic Lactobacillus species. Sauerkraut fermentations use Leuconostoc species of bacteria to complete the fermentation.

 

The Entner-Doudoroff Pathway

 

         A few bacteria, most notably Zymomonas, employ Entner-Doudoroff pathway as a fermentation path.

         Many bacteria, especially those grouped around pseudomonads, use pathway as way to degrade carbohydrates for respiratory metabolism.

         E-D pathway yields 2 pyruvate from glucose (same as E-M pathway) but like phosphoketolase pathway, oxidation occurs before cleavage, and net energy yield per mole of glucose utilized is one mole of ATP

 

         In E-D pathway, glucose phosphate is oxidized to 2-keto-3-deoxy-6-phosphogluconate (KDPG) which is cleaved by KDPG aldolase to pyruvate and GAP.

         The latter is oxidized to pyruvate by E-M enzymes where  2 ATP are produced by substrate level phosphorylations.

         Pyruvate from either branch of pathway is reduced to ethanol and CO2, in the same manner as yeast, by the "yeast-like bacterium", Zymomonas).

         Overall reaction is Glucose ---------->2 ethanol +2 CO2, and a net gain of 1 ATP.

 

         Zymomonas  - a bacterium that lives on surfaces of plants, including succulent Maguey cactus indigenous to Mexico.

         Grapes are crushed and fermented by resident yeast to wine, so Maguey flesh may be crushed and allowed to ferment with Zymomonas, to produce "cactus beer" or "pulque”.

         Distilled pulque yields tequila in the state of Jalisco or mescal in the state of Oaxaca.

         Many cultures around the world prepare their native fermented brews with Zymomonas in deference to the Saccharomyces.

 

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