INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY

I. Microbial strain selection

In most cases, one can't use wild type microorganisms in industrial processes - they either don't grow well enough, make enough product, support enough plasmids or bacteriophages, or they may be environmentaly or medically unsafe. Therefore, new strains incorporating the best features possible must be developed.

A. Selection for given properties

(biochemical markers)

1. Plate out (or other technique) and pick those organisms with the desired characteristics; as observed by tests and/or descriminatory media

2. Select for desired characteristics (those without them will not grow or will die under selection conditions) using selective media and/or the presence or absence of specific chemicals

B. Mutation followed by selection

This allows for new forms to be developed, which might be better than the wild type organisms

1. Ultraviolet radiation

2. X-rays

3. Gama radiation, etc.

4. Chemical mutagens

C. Storage

1. Drying (on a filter?)

2. Lyophilization

3. Storage under liquid nitrogen

4. Use of low salt media and/or water agar

II. Recombinant DNA techniques

A. Gene identification

1. Classical method: protein --> mRNA --> cDNA --> hybridization to the chromosome at the site of the desired gene after digestion with restriction enzymes

B. Gene deletion

(if this is a deleterious gene) - after identifying and obtaining a "restriction map" of the gene and its neighborhood, cut out the gene out with restriction enzymes and religate the chromosome

C. Gene cloning

1. Identify useful gene

2. Cut out useful gene

3. amplify DNA - so that there will be enough to work with

a. Classical method - clone into bacteriophage

b. PCR (polymerase chain reaction)

4. Insert gene into host microorganism

a. Plasmid - utilizing restriction enzymes, ligation, etc.

i. E. coli (best known)

ii. B. subtilis (leaky)

iii. Yeast or other eukaryote

b. Genome (gene therapy?)

5. Production of gene product

III. Growth processes

Ideal is to maintain the optimal growth and production conditions for a large culture over an indefinate period so that the most product can be made. The growth process is called fermentation; this term is not related to the biochemical processes undergone by the organism when it is used in this manner. First, the process is developed in small, laboratory conditions, then 'scaled up' to large scale, production conditions. There may be unexpected physical and/or biological problems encountered during scaleup

A. Fermenters


1. Stirred

2. Lift tube (filtered air - or other gas mixture - provides stirring)

3. Solid state

4. Fixed bed reactor - microbes attached to particles in a fixed bed

5. Fluidized bed reactor - microbes attached to particles which become suspended in the medium

6. Dialysis culture unit - diffuse away wastes, diffuse in nutrients (substrates)

7. Continuous culture unit - (like chemostat) medium drips in, medium with cells drips out

B. Medium


Use as much low cost, redily availible constituents as possible. However, this can make it difficult to keep a consistant formula for the growth medium as these constituents can vary in composition from batch to batch, and they must be checked before use.

1. Formulia optimized for growth

2. Formulia may become limiting for a specific nutrient which might stimulate production and/or cause specific genes to become active.

a. Some factors (carbon source, etc.) must still be added constantly 'continuous feed' or in batches over time so that unwanted metabolites don't build up (as they can with excess substrate)

C. Conditions

1. Temperature

2. pH - buffering woth phosphates (P source), chalks. carbonates

3. Oxygenation

4. Other gases (CO2 etc.)

D. Problems

1. Contamination

2. Maintainance of conditions and/or reliable cycling of conditions

3. Clumping and formation of filamentous mats by the micrroorganisms (especially fungi) and/or their products

a. Non-Neutonian broth - viscous, resists stirrring and/or aeration

b. Blocks pipes etc.

IV. Microbial processes and products


pharmaceuticals (antibiotics etc.); bio/medical (hormones, steroids,etc.); solvents, organic acids (lactate, pyruvate, etc.); amino acids; enzymes; animal (and human?) feeds

A. Primary metabolites

Made during growth phase and related to cell growth and cell division products and/or products engineered to be activated during growth phase (trophophase) - such as amino acids, nucleotides, fermentation products (ethanol, acids), some enzymes, and varieties of bioengineered products

B. Secondary metabolites

Accumulate during stationary phase (idiophase), not related to cell growth, can be triggered by medium conditions - such as antibiotics, mycotoxind and some bioengineered compounds which have not been grafted to growth - related operator/promoter regions

C. Production

In most cases, tricks involving limiting or adding substances to the medium, using cell mutants and/or bioengineering, are used to get the microorganisms to overproduce the desired product(s), as most cells will only make enough of any product as is necessary for maximal growth and no more

1. Penicillin (Penicillium chrysogenum) - Primary metabolite under less than maximal growth; low glucose and/or lactose with limited nitrogen to slow growth plus specific precursors --> removal of molds --> isolation of antibiotics --> chemical modifications (if any)

2. Streptomycin (Streptomyces griseus) - Secondary metabolite --> growth --> idiophase - -> rise of antibiotic concentration with nitrogen limitation, increasing pH --> removal of supernatent --> isolation of antibiotic

3. Bioconversion - Microbial transformations or biotransformations - sort of an inside - out fermentor. Since enzymes and living systems are used, the energy input is minimized

a. Biocatalysts

i. purposeful utilization of microorganisms to perform specific tasks
Bacteria + 4 Fe+2(aq) + O2(g) + 4 H+ --> 4 Fe3+(aq) + 2 H2O. e.g. Thiobacillus thiooxidans,Thiobacillus ferrooxidans (Hardanger?) cause ferric iron reduction (in iron pyrites) when they oxidize sulfur, and are useful alone or, at best, together in purifying copper in mine tailings (which are usually acid). They work at acid pH (pH 1.6) and at 20 t0 35oC, and may attach themselvs to the pyrite crystals. , sometimes used together in culture to get maximum yields.
Leptospirillum ferrooxidans attaches directly to pyrite crystals and causes similar reactions. It is used in purifying Gold, Copper and Zinc from mine tailings.

b. Biodeterioration

i. problem causing, can be regulated using microbial inhibitors

c. Insecticides

d. Biosensors - used with microelectronics and microorganisms working together e.g. to monitor level of contaminants etc.

FOOD MICROBIOLOGY

I. Food production

(mostly fermentation) - aids in food preservation

A. Milk

Buttermilk, sour cream, yogurt, cottage cheeses, cheese. Fermentation by Lactobacillus, Streptococcus sp., Leuconostoc sp., Proprionibacterium, Brevibacterium, Penicillium sp. etc. Use starter culture, stop fermentation by cooling or by acid production. Rennin (a digestive enzyme from calf stomachs or recombinant bacteria) aids in curd formation

B. Meat and fish


1. Sausage - salami, lebanon etc.

2. Ham - cured

3. Fish sauces - mainly Japanese Bacillus sp., Lactobacillus sp., Aspergillis sp.

C. Wine, Beer etc.

Can use natural fermentation or treat by Pasteurization or Sulfur dioxide to kill native organisms and then add a defined culture. Grains must be crushed and mixed with water to form a mash before they can be fermented, and the supernatant wort then used for further treatment, unlike fruits which have readily available nutrients

1. Wines - Sacchromyces cerevisiae or Sacchromyces ellipsoides - ferment 3 - 5 days at 20 - 28oC ---> 10 - 18% EtOH. Microbes die and/or produce byproducts which are removed (most wines by centrifugation and/or filtration - some by pasteurization, champagnes by collecting the sediment in the bottle neck and then freezing the neck, removing the sediment and recorking). Vinegar is made by treating the wine or fruit juice with Acetobacter or Gluconobacter.

2. Beer and ale - Germination of barley --> enzyme action --> malt, add Sacchromyces carlsbergensis var. (bottom yeast) --> 7 - 12 days --> beer (pH 4.1- 4.2). If use Sacchromyces cerevisiae var. (top yeast) --> ale (pH 3.8). Age (lager) and add CO2, pasteurize or filter.

3. Distilled products - take any product made as in 1. or 2. above, heat in a still, collect the distillate produced at a given temperature range. Can add flavoring

4. Bread - Kneading moist dough releases amylases which digest the complex carbohydrates to limit dextrans and maltose. Add yeast (Sacchromyces cerevisiae var.) which ferments the maltose and gives off CO2 and ethanol (the CO2 makes the bread rise - about 2 hr at room temperature - the ethanol evaporates) and produces enzymes - maltase, zymase, invertase - which change the state of the dough, then bake. Some specialty breads use a starter mix with both microorganisms and/or special ingredients (caraway seeds etc.) that is made in advance and then added to the dough

5. Sauerkraut - first Leuconostoc sp. ferments until lactic acid is about 0.7 to 1% ; then Lactobacillus sp. ferments until acid concentration is 1.6 to 1.8%. pH kills off the bacteria

6. Pickles - done more or less as above, but with the addition of brine and dill seeds. In addition to the above bacteria, Streptococcus faecalis, Pediococcus cerevisiae and especially Lactobacillus are the major fermenting organisms

II. Spoilage

Proteolysis (putrefaction --> smelly amines; pectinolysis degrades pectins; fermentations and hydrolysis act on carbohydrates and fatty acids are broken down with fermentations and fatty acid degradation)

A. Extrinsic (environmental) factors

These can change with time during the shelflife of the food

1. Temperature - high temperatures 30 - 42oC best for growth of most microorganisms

2. Relative humidity - if it is high, microorganisms may grow even at low temperatures. Moisture can be absorbed by the food and promote growth

3. Gas(es) present - some plastic films such as those used in shrink wraps allow gases to diffuse across them. Excess CO2 lowers the pH and kills most gram (-) organisms, Lactobacillus sp can still grow. Oxygen allows aerobic growth, which is usually faster than anaerobic

4. Microorganisms present - obvious: can they utilize the food under the storage conditions

B. Intrinsic (to the food)

1. pH - if low, fungi grow best, if neutral or alkaline, bacteria grow best.

2. Aw - if too low, no growth, fungi grow until to 60%, bacteria only at 90 - 95%.

3. Redox potential - cooking lowers redox potential (reduces) of most foods so that anaerobes such as Clostridium can grow on them

4. Nutrients - need the proper nutrients for the bacteria present in the environment

5. Structure - larger surface areas - faster growth, whipping or grinding of food (ground meat, whipped cream) distribute microorganisms throughout food, let air in --> rapid spoilage

6. Antimicrobial agents - natural compounds in some foods - aldehydes, phenolics found in some spices, coumarins in fruit, lysozyme in eggs etc. can retard bacterial growth. Cooking can affect some of them.

III. Disease

most caused by poor hygiene, fecal - oral route

A. Food infection

Eat contaminated food, microorganisms grow and infect and/or produce toxins, cause "gastroenteritis"

1. Campilobacter jejuni - Campilobacteriosis appears 16 - 48 hr after ingestion, need 1 - 10 organisms to infect, produces toxins; sources - milk, pork, and especially poultry.
Prevention - cooking, good hygiene, washing utensils etc.

2. E. coli - E. coli enteritis appears in 6 - 36 hr, with or without toxins; sources - cheese, raw vegetables.
Prevention - washing, good hygiene, cooking

3. Listeria monocytogenes - Listeriosis - meningitis, abortion; source - dairy products.
Prevention -proper processing, good hygiene.

4. Salmonella typhimurium, enteridis - Salmonellosis - appears in 12 - 24 hrs, enterotoxins and cytotoxins; sources - meats, poultry, fish, eggs, dairy products.
Prevention - cleaning, cooking, good hygiene

5. Shigella sonnei, flexneri - Shigellosis - appears in 1 - 7 days, shigella toxins; sources - egg products, poultry.
Prevention - good hygiene, cooking

6. Vibrio parahemolyticus - gastroenteritis - appears in 16 - 48 hrs; sources - seafood, shellfish.
Prevention - cooking, good hygiene

7. Yersinia enterocolitica - Yersiniosis - toxins; sources - milk, meat products.
Prevention - proper treatment, cooking, good hygiene.

B. Food intoxication

Don't need live organisms, toxins left behind cause disease.

1. Bacillus cereus - Bacillus cereus food poisoning - appears in 1 - 6 hr; severe nausea and vomiting, 6 -17 hr; diarrhea; sources - meats, rice products and cereals, potatoes, puddings.
Prevention - proper treatment of food.

2. Clostridium botulinum - Botulism - appears in 12 - 36 hr, botulinum toxin (deadly) released after cell death; sources - fish, meats, canned foods that are low in acid.
Prevention - proper treatment of food, proper canning procedures.

3. Clostridium perfringins - Perfringins food poisoning - appears in 8 - 24 hr, heat labile enterotoxin produced during sporulation either in food or in intestine of ingester. Need 106 organisms/gm food. Heat food --> low O2, --> slow cooling, bacterium grows --> spore formation --> toxin production.
Prevention - quick cooling of food after cooking, proper procedures.

4. Staphylococcus aureus - Staphylococcal food poisoning - appears in 2 - 6 hr, enterotoxins; sources - meat, dairy products, poultry, custards, starchy foods. S. aureus in human nose, --> hands --> food --> grows at room temperature.
Prevention - good hygiene.

IV. Food preservation

A. Removal of microorganisms

Filtration (wine, beer, juices, soda, etc.), centrifugation (wine)

B. Low temperature

Lowers growth rate (fungi and other psychrophillic and psychrotrophic organisms will still grow)

C. High temperature

Pasteurization (can affect flavor), sterilization (canning approximates this), dehydration (heating or freeze-drying - lyophilization-)

D. Lower Aw

As in C. above - removes water and increases solute (mainly ionic) concentration, both reduce or stop growth of most microorganisms, but xerophillic or osmophilic organisms can still grow. Lower availability of water by adding sugar or salt, reduces growth of most organisms except for those mentioned above.

E. Chemical preservation

Sulfite (stops generation of Clostridium spores), but is allergenic to some people
ethylene oxide (can be highly toxic)
sodium nitrite (heat --> nitrosamines --> carcinogens)
ethyl formate
nisin (antibacterial compound made by S.lactis) is used, but not in the USA, to inhibit the growth of Clostridium sp.

F. Radiation

Uv light kills on surface only, won't penetrate, Cobalt 60 gamma radiation penetrates, kills by ionization of water inside microorganisms to peroxides, good for the preservation of seafoods, fruits, vegetables