Originally isolated from nature, but increasingly
"improved" by genetic manipulation via mutagenesis and selection
or recombinant DNA technology or protoplast fusion (fungi)
To be useful in industrial microbiology, an organism
must:
produce usable substance(s) or effect(s)
be available in pure culture
be genetically stable, but amenable to genetic
manipulation
produce spores or other reproductive structures to allow
easy inoculation
grow rapidly and produce product quickly in large-scale
culture
grow in such a way that the cells are easily separated from
the product
not be harmful to humans or agricultural plants and
animals, etc.
Growth Conditions
Composition of growth medium - generally use cheapest
sources of carbon (molasses, whey, grains), nitrogen (ammonia and
ammonium salts; distillers solubles), phosphorous, trace minerals
and other growth factors (either of which may be used to regulate
product generation)
Other considerations - use aseptic conditions and
maintain temperature, pH, oxygen concentration at optimal levels
in the microenvironment in which each individual cell is growing
and metabolizing
Fermentors - many types (lift-tube, solid state, fixed
bed, fluidized bed, dialysis, continuous) and sizes
aerobic fermentor - stainless steel cylinder with
temperature control (cooling jacket vs. internal coils) and
aeration system (sparger and impeller plus baffles) with
process control and monitoring devices (real-time acquisition
of data provides for "on-line" control of temperature, pH,
pO2, pCO2, cell concentration, foaming,
product concentration)
anaerobic fermentor - essentially the same as
aerobic, but does not need aeration
Scale-up of fermentation process: carried out by
biochemical engineers in conjunction with microbiologists
practical considerations - surface/volume ratio,
uniformity of mixing (maintain appropriate conditions,
especially oxygen transfer rate, at level of individual cell -
consider microenvironment)
Primary vs.
Secondary Metabolites
Primary metabolite
metabolite formed in parallel with growth, during
trophophase
example - alcoholic fermentation
Secondary metabolite
metabolite formed after growth has occurred, during
idiophase
characteristics:
formed by only a few organisms
not essential for growth and reproduction
dependent upon growth conditions - may be inducible
frequently produced from several intermediate products
formed during trophophase; may require an inducer produced
during trophophase
often produced as a group of related structures
possible to induce overproduction (not possible with
primary metabolites)
example - antibiotic production
Biotransformation - microbial
bioconversion
Microbes can act as biocatalysts to carry out complex
sequences of reactions very specifically
Cost-effective because of high cost of chemical
transformation alternatives
Major uses include:
production of organic acids (chemicals), steroid
hormones and antibiotics (pharmaceuticals)
precious metals recovery (Chlorella vulgaris)
bioleaching of metals - Thiobacillus ferrooxidans
provides recovery of up to 70% of copper from low-grade
ores
Microbial Products
Foods
spoilage (any condition that alters color, texture,
odor, and/or taste) and putrefaction (anaerobic breakdown of
proteins)
soft - cottage (Streptococcus lactis;
Leuconostoc cremoris), cream (S. cremoris, S.
diacetylactis, S. thermophilus, Lactobacillus bulgaricus,
Brie (S. lactis, S. cremoris; Penicillium camemberti, P.
candidum, Brevibacterium linens), mozzarella (S.
thermophilus, L. bulgaricus)
semi-soft - monterey jack (S. lactis, S.
cremoris), Muenster (S. lactis, S. cremoris;
Brevibacterium linens), bleu (Roquefort - S.
lactis, S. cremoris; Penicillium
roqueforti)
hard - cheddar (Streptococcus lactis, S.
cremoris, S. durans; Lactobacillus casei, L.
plantarum), edam (S. lactis, S. cremoris),
gouda (Streptococcus lactis, S. cremoris, S.
diacetylactis), swiss (S. lactis, L. helveticus,
S. thermophilus; Propionibacterium shermanii, P.
freudenreichii)
very hard - parmesan (goat milk fermented and
flavored by Streptococcus lactis, S. cremoris, S.
thermophilus; Lactobacillus bulgaricus), romano (cow
milk fermented and flavored by Streptococcus lactis,
S. cremoris, S. thermophilus; L. bulgaricus)
meat products (salami, summer sausage) -
Pediococcus cerevisiae and Lactobacillus
plantarum
baked goods - Baker's yeast (Saccharomyces
cerevisiae) aerobically generates carbon dioxide for breads
and pastries
"regular" bread - S. cerevisiae provides
flavor and carbon dioxide for "holes" to give light
texture
sourdough bread - S. exiguus (provides
flavor and carbon dioxide ) and Lactobacillus
(provides flavor)
vinegar (apple juice, wine) - Acetobacter
or Gluconobacter
e) microbes as a direct food source
single-cell protein - Candida grown
on sulfite waste "liquors" from paper manufacturing
approximately 50% protein, but also 16% nucleic
acid
taste is objectionable to many
may cause kidney stones or gout (high nucleic acid
content) when consumed in large quantities over long
periods of time
Spirulina (cyanobacterium -
photosynthetic)
mushrooms - Agaricus campestris
bisporus
food additives and supplements
amino acids - used as food additives or starting
materials in the chemical industry
microbes must be modified to overproduce their
products by eliminating feedback inhibition and
repression mechanisms as well as inducing secretion of
product
blood clotting factors (VII, VIII, IX) - restore
clotting mechanisms in hemophiliacs without chance of
transmitting AIDS or hepatitis
erythropoietin - treatment of certain forms of
anemia
cytokines - interferons (IFN), interleukins (IL),
and other cytokines that act as anticancer agents or immune
modulators
IFN-gamma stimulates cancer cells to produce
tumor-associated antigens so they can be detected and
eliminated by the immune system
IL-2 stimulates T cells to promote immune
responses
tumor necrosis factor alpha (TNFa) and
granulocyte-macrophage colony stimulating factor
(GM-CSF) work together with IL-2 in cancer therapy
vaccine antigens - prevention of bacterial,
fungal, metazoan, viral diseases (e.g., recombinant
Hepatitis B vaccine now in use)
monoclonal antibodies (mAb)
diagnostic applications - determine ovulation,
pregnancy; identification of infectious agents
therapeutic applications - specific drug
delivery in cancer therapy; destruction of
platelet-catalyzed blood clots in heart disease
therapy
chemotherapeutic agents -
antibiotics are secondary metabolites produced by
bacteria (Bacillus, Nocardia,
Streptomyces) or fungi (Aspergillus,
Cephalosporium, Penicillium)
more than 8000 known, several hundred discovered
per year (but most are unusable)
more than 100 tons produced annually, worth more
than $5 billion
cheaper to produce by fermentation than by chemical
synthesis, but their structures (and thus their activities)
may be modified by subsequent chemical steps (semisynthetic
antibiotics)
steps toward commercial production include:
isolation - usually by screening (cross-streak
method)
testing for toxicity and efficacy
optimization and purity of yield - gene
amplification, other genetic engineering of microbes
developing extraction and purification steps -
organic chemistry applications
examples:
ß-lactams
because they contain the ß-lactam group,
they inhibit cell wall synthesis by blocking the
transpeptidase that catalyzes peptide
cross-linking
examples - penicillin, ampicillin,
cephalosporins
aminoglycosides
these molecules containing amino sugars bonded by
glycosidic linkage inhibit protein synthesis in Gram
(-) bacteria by binding to 30S ribosomal subunits
examples - streptomycin, kanamycin,
gentamicin
macrolides
these lactone rings connected to sugar moieties
inhibit protein synthesis in Gram (+) bacteria by
binding to 50S ribosomal subunits
secondary metabolites produced by bacteria or fungi
during idiophase
useful (due to specificity of reaction) in food
processing (especially dairy products), pharmaceutical, and
textile industries
examples: proteases (detergent additives); amylase,
glucoamylase and glucose isomerase (polysaccharide digestion to
help start fermentations)
Biopolymers
exopolysaccharides can be used as stabilizers, etc.
microbial plastics:
poly-ß-hydroxybutyrate (PHB), which is commonly
used by some bacteria as a lipid storage material, can be
used as a raw material for plastic based packaging
materials
the nature of the raw material (and thus the plastic
that can be synthesized from it) can be selected by varying
the carbon source used to grow the bacteria - using acetate
(C2) and butyrate (C4) yields PHB; caproate (C6) yields
poly-ß-hydroxycaproate PHC; valerate (C5) yields
poly-ß-hydroxyvalerate PHV; mixtures yield
co-polymers
Biosensors - bioelectronics utilize the abilities of
microbes to measure pollutants and contaminants
Agricultural Uses of
Microbes
Rhizobium - facilitates nitrogen fixation in
symbiotic legumes
Agrobacterium tumefaciens - Ti plasmid is used
as a gene vector for transferring genes coding for important
functions to plants
Bacillus thuringiensis spores contain protein
crystals toxic for tomato worms
polyhedrosis virus is used to control pine caterpillars and
cotton bollworms
Biodeterioration
Management
Jet fuel - growth of Cladosporium resinal at water/fuel
interface is controlled with fungicides
Paper production - microbes and slime produced by them
controlled with biocides (environmental problems)
Electronics - microbes damage computer chips by growing
on trace contaminants at junctions (must use ultrapure water - no
microbes, no organics - to prevent this)
Paints - growth of fungi, etc. in paints (esp.
latex-based) is controlled by use of quaternary ammonium salts,
barium salts and chlorinated phenolics (mercury compounds no
longer used due to toxicity for humans)
Textiles and leather goods - fungal growth is
controlled with phenolics in textiles and copper compounds in
leathers
Metals and concrete - microbial metabolites (e.g.,
sulfuric acid produced by Thiobacilli) are frequently responsible
for corrosion of concrete sidewalks, highways, bridges and
building as well as sewer pipes
Biodegradation and Bioremediation
Bioremediation is also called Biodegradation Enhancement and
includes any purposful use of microbes to degrade unwanted substances
in the environment
natural products
petroleum - certain bacteria (some cyanobacteria,
pseudomonads, corynebacteria, mycobacteria), green algae and
fungi (several molds and yeasts) oxidize hydrocarbons at
aerobic water/oil interfaces (with optimal conditions, up to
80% removal within 1 year after a spill)
biodegradable plastics
photobiodegradable - structure of polymer altered
by UV light in sunlight so that it is now amenable to
biodegradation
biochemically biodegradable starch-linked
polymers - starch-digesting bacteria in soil attack the
starch, releasing polymer fragments which are degraded by
other microbes
xenobiotics - chemically
synthesized compounds not found in nature (pesticides, synthetic
polymers, etc.) and thus would seem unlikely to be degradable by
naturally existing microorganisms; these
products tend to be persistent in nature, and many nations are
working to ban the use of many of them; microbes that can
degrade xenobiotics are rather diverse and typically include both
bacteria and fungi
PCBs - certain Pseudomonas species have been
engineered to accelerate breakdown of polychlorinated biphenyls
(formerly used by electric industry as transformer
insulation)
PAHs (poly aromatic hydrocarbons) can be difficult
to degrade, but there are microbes in the environment that can
accomplish this task, especially when working together
pesticides - herbicides, insecticides and fungicides
these are typically rather complex molecules
some xenobiotics are good carbon sources and electron
donors for soil microbes, so they are more readily degraded
than others
other xenobiotics, such as chlorinated insecticides, are
recalcitrant to degradation; thus they have rather long
persistence times in the environment
lindane - 3 years for 75-100% disappearance
DDT - 4 years for 75-100% disappearance
chlordane - 5 years for 75-100% disappearance
to degrade these xenobiotics, microbes may employ
co-metabolism, in which an organic material other than the
xenobiotic is used as the primary energy source and the
xenobiotic is degraded as a secondary process
Genetically
engineered microbes in bioremediation
Microbes can be "engineered" to carry out the
biochemical processes needed for bioremediation
concerns about long-term the effects of
genetically engineered microbes on the environment are
manifold
fate of genetically engineered microbes is
similar to that of other allochthonous organisms, but even
more extreme because they are typically engineered to
require nutrients, etc. not naturally present in the
environments into which they may be introduced ... so they
will die out when the material they were engineered to
degrade has been removed from the area
Naturally-occurring microbes bioremediate just as well
as engineered microbes in many cases ... it is important to
adjust environmental conditions to favor their growth,
however
