41 Industrial Microbiology CHAPTER OVERVIEW Industrial ...

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Feb 12, 2013 (4 years and 7 months ago)

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41

Industrial Microbiology



CHAPTER OVERVIEW


Industrial microbiology exploits the range of genetic resources and natural products generated by
microorganisms to produce compounds with industrial or medical application.

Genetically engineered
microorga
nisms can be used to increase the efficiency of the industrial processes and to produce new or
modified products.

Major products of industrial microbiology is discussed, including agricultural
biotechnology and microbial energy conversion.


CHAPTER OBJECTI
VES


After reading this chapter you should be able to:




discuss the sources of microorganisms for use in industrial microbiology and biotechnology



discuss the genetic manipulation of microorganisms to construct strains that better meet the needs of an
indu
strial or biotechnological process



describe the design or manipulation of environments in which desired processes will be carried out



discuss the management of growth characteristics to produce the desired product



list the major products or uses of industr
ial microbiology and biotechnology



discuss the processes and value of microbial energy conversions



discuss the use of microorganisms in manufacturing nanotechnology, biosensors, and biopesticides


CHAPTER OUTLINE


I.

Microorganisms for Industrial Microbiolog
y

A.

Finding microorganisms in nature

major sources of microorganisms for use in industrial processes
are soil, water, and spoiled bread and fruits; only a minor portion of microbial species in most
environments have been identified and cultured

B.

Genetic manip
ulation of microorganisms

1.

Mutagenesis

cultures can be improved by mutagenesis with chemical agents and UV light

2.

Protoplast fusion

involves removal of cell walls (to create protoplasts), mixing two different
solutions of protoplasts, and growth in selective

media

3.

Transfer of genetic information between different organisms

functional genes are cloned and
inserted into new hosts where heterologous gene expression enables the production of specific
proteins and products

4.

Modification of gene expression

modificat
ion of regulatory molecules can increase gene
expression

5.

Directed evolution

a.

Construction of production strains by specifically targeting genes of interest for
mutagenesis

b.

Site
-
directed mutagenesis changes nucleotide sequences using a PCR
-
based approach
tha
t uses methylation patterns to protect mutated gene copies; only a few nucleotides are
changed at a time

c.

Combinatorial biosynthesis uses knowledge of pathways to create altered nonprotein
products (e.g., polyketide antibiotics)

d.

Systematic evolution of liga
nds by exponential enrichment (SELEX) creates large
populations of engineered RNA molecules (aptamers) for use in therapies




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e.

Selecting the best variants is more rapid using high
-
throughput screening (HTS) methods
often using robotic 96
-
well plate assays

6.

Met
agenomics

since most environmental microbes do not grow in the laboratory,
bioprospecting is often done by examining metagenomic libraries to identify new versions of
known genes and new genes through functional screening after expression in common
laborat
ory microbes

II.

Growing Microbes in Industrial Settings

A.

Industrial microbiologists use t
he term “fermentation” primarily to refer to the mass culture of
microorganisms; the term has many other meanings to other microbiologists

B.

Small
-
scale laboratory operation
s need to be scaled up to industrial
-
sized operations by maintaining
culture conditions during the transition

C.

In stirred fermenters, all steps in growth and harvesting must be carried out aseptically and
computers often are used to monitor microbial biomas
s, levels of critical metabolic products, pH,
input and exhaust gas composition, and other parameters

D.

Continuous feed of a critical nutrient may be necessary to prevent excess utilization, which could lead to
production and accumulation of undesirable meta
bolic waste products

E.

Microbial products often are classified as primary or secondary metabolites

1.

Primary metabolites are related to the synthesis of microbial cells in the growth phase; they
include amino acids, nucleotides, fermentation end products, and
exoenzymes

2.

Secondary metabolites usually accumulate in the period of nutrient limitation or waste product
accumulation that follows active growth; they include antibiotics and mycotoxins

III.

Major Products of Industrial Microbiology

A.

Antibiotics

1.

Predominantly p
roduced by actinomycetes in the genus Streptomyces and by filamentous fungi

2.

Penicillin

careful adjustment of medium composition is used to slow growth and to stimulate
penicillin production; side chain precursors can be added to stimulate production of par
ticular
penicillin derivatives; harvested product can then be modified chemically to produce a variety
of semisynthetic penicillins

B.

Amino acids

1.

Amino acids such as lysine and glutamic acid are used as nutritional supplements and as flavor
enhancers

2.

Amino a
cid production is usually increased through the use of regulatory mutants or through
the use of mutants that alter pathway architecture

C.

Organic acids

1.

These include citric, acetic, lactic, fumaric, and gluconic acids

2.

Citric acid is used in large quantities

by the food and beverage industry; produced largely by
Aspergillus niger

fermentation in which trace metals are limited to regulate glycolysis and the
TCA cycle, thereby producing excess citric acid

D.

Biopolymers

microbially produced polymers

1.

Used as stabi
lizers, agents for dispersing particulates, and as film
-
forming agents; they also
can be used to maintain texture in ice cream, as blood expanders and absorbents, to make
plastics, and as food thickeners; also used to enhance oil recovery from drilling mud

2.

Includes dextrans, polyesters, cellulose, and xantham gum

E.

Biosurfactants

1.

Biosurfactants are biodegradable agents used for emulsification, increasing detergency,
wetting and phase dispersion, as well as for solubilization

2.

The most widely used biosurfactant
s are glycolipids, which are excellent dispersing agents;
many have antimicrobial properties due to their amphipathic nature (disrupt membranes)

F.

Bioconversion processes

microbial transformations or biotransformations

1.

Microorganisms are used as biocatalysts
; bioconversions are frequently used to produce the
appropriate stereoisomer; are very specific, and can be carried out under mild conditions

G.

Microbial energy conservation

1.

Microbial conversion of biomass into ethanol a
n
d hydrogen; also microbial fuel cells

that
generate electricity by releasing electrons from organic chemicals




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2.

Ethanol fermentation starts with degradation of plant starches with amylase to release sugars;

these are then fermented to ethanol

3.

Hydrogen is produced anaerobically by a number of ph
ototrophs using nitrogenase or
hydrogenase

4.

Methane is commonly produced by archaea in anaerobic digesters in a mixture with carbon
dioxide called biogas

5.

Microbial fuel cells include microbes that can use an external electrode as an electron sink;
consists
of two
:

an aerobic and an anaerobic chamber; biomass in the anaerobic chamber is
digested to release electrons and protons; the protons move to the aerobic chamber and
produce water with a catalyst; the electrons are donated to an electrode using protein
n
anowires or chemical shuttles to generate a current

IV.

Agricultural Biotechnology

A.

The Ti plasmid from Agrobacterium tumefaciens is used to introduce genetic constructs into plant
cells; T
-
DNA within the plasmid transfers genes to plant cells in a process simi
lar to that of
transposons

B.

Biopesticides and bioinsecticides

include uses bacteria, viruses, and fungi, and their genes

1.

Bacillus thuringiensis

being used to control insects; accomplished by inserting toxin
-
encoding gene into the plant or by production of a

wettable powder that can be applied to
agricultural crops; the toxin gene also has been introduced into crop plants

V.

Microbes as Products

A.

Nanotechnology

1.

Diatom shells have precise structures at the micrometer scale; these can be grown and the
silicon oxide
s replaced by magnesium oxides

2.

Magnetosomes formed by bacteria are minute, perfectly formed magnetic beads with a
membrane envelope that can be used for drug delivery or diagnostic techniques

B.

Biosensors

1.

Biosensors make use of microorganisms or microbial en
zymes that are linked to electrodes in
order to detect specific substances by converting biological reactions to electric currents

2.

Biosensors have been or are being developed to measure specific components in beer, to
monitor pollutants, to detect flavor
compounds in foods, and to study environmental processes
such as changes in biofilm concentration gradients; they also are being used to detect glucose
and other metabolites in medical situations and to combat bioterrorism

3.

New immunochemical
-
based biosenso
rs are being developed; these are used to detect
pathogens, herbicides, toxins, proteins, and DNA


TERMS AND DEFINITIONS


Place the letter of each term in the space next to the definition or description that best matches it.


____ 1.

A process whereby a
critical nutrient is added periodically so that the organism will not have excess
substrate available to it at any time

____ 2.

Molecules produced by a microorganism that are directly related to synthesis of cell material during
the growth phase

____ 3.

Molecules produced by a cell that are not directly related to the synthesis of cell material and that
are usually produced after the growth phase has ended

____ 4.

Microbial polymers used to modify the flow characteristics of liquids and to serve as gelli
ng agents

____ 5.

Minor modifications in molecules that are carried out by nongrowing microbes

____ 6.

Microorganisms used to carry out biotransformations

____ 7.

Living microorganisms that are linked with electrodes to convert biological reactions to e
lectrical
currents

____ 8.

Bacteria, viruses, and fungi, or their products, that can be used to control insect pests

____ 9.

A system in which biomass is converted almost entirely to methane and carbon dioxide

____ 10.

A large vessel used to grow microbe
s in an industrial setting

____ 11.

A robotic system used to rapidly screen many molecules for useful activities

____ 12.

An engineered RNA molecule that exhibits a useful activity




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____ 13.

The transcription and translation of a foreign gene introduced int
o a host organism that does not
normally have that gene

____ 14.

Compounds generated by microbes that can be used to provide energy to vehicles or machines

____ 15.

Manipulation of genes to alter or create industrially important products

____ 16.

Increasin
g the volume of a culture to develop large
-
volume industrial systems

____ 17.

A compound produced by an organism that has industrial or medical application


a.

anaerobic digester

b.

aptamer

c.

biocatalysts

d.

biofuels

e.

bioinsecticides (biopesticides)

f.

biopolymers

g.

biosensors

h.

biotransformations

i.

continuous feed

j.

directed evolution

k.

fermenter

l.

heterologous gene expression

m.

high
-
throughput screening

n.

natural product

o.

primary metabolites

p.

scale
-
up

q.

secondary metabolites


FILL IN THE B
LANK


1.

To assure that physical factors are not limiting microbial activity, physical conditions used in industrial
processes must be considered at the level of the individual microbe. This is most critical in
____________, where a process developed in a
small shake flask, if successful, must be carried out in a
large fermenter.


2.

Compounds related to the synthesis of microbial cells and often involved in the growth phase are called
____________ ____________; those that have no direct relationship to the

synthesis of cell materials
and that are usually produced after the growth phase are referred to as __________ __________.


3.

Commercial production of amino acids is typically carried out using ____________ mutants, which over
produce the desired amino a
cid.


4.

A rapidly developing area of biotechnology concerns the linking of microorganisms to electronic
components to create ____________ that convert biochemical reactions into electrical current.


5.

The newest approach for creating new metabolic capabi
lities in a given microorganism is __________
__________ __________. This approach uses specific environmental stresses to force microorganisms to
mutate and adapt, processes described as __________ __________ and __________ __________.


6.

Microbiology is

a critical part of the area of __________ __________, which is concerned with tracking
the flo
w of elements and compounds through

the natural world (__________) and the social world
(__________).


7.

Increasingly microorganisms used in industrial processe
s are being improved by molecular methods. For
instance, __________ __________ involves the transfer of genes from one organism to another. Another
approach is __________ __________, in which small synthetic DNA molecules are inserted into a
microorganism
at a specific site.


8.

Microbial energy conversions include the transformation of organic materials into



.

Corn
is often fermented into



, while phototrophs can produce




using nitrogenase.

Anaerobic digesters can be used to generate gaseous



.







383

MULTIPLE CHOICE


For each of the questions below select the
one best

answer.


1.

Which of the following is NOT an accepted meaning for the term fermentation?

a.

any process involving the mass culture of microorganisms, either anaerobic or aerobic

b.

the

production of alcoholic beverages

c.

food spoilage

d.

All of the above are accepted meanings for the term fermentation.

2.

Agitation is used to maintain proper oxygen availability. However, this can be difficult when trying to
culture which of the followi
ng organisms?

a.

bacteria

b.

filamentous fungi

c.

yeasts

d.

It is a major problem with all of the above organisms.

3.

Which of the following methods is used to improve strains used to carry out microbial fermentations?

a.

mutation and selection

b.

recombin
ant DNA modification of gene expression

c.

protoplast fusion

d.

All of the above are used to improve strains used for microbial fermentations.

4.

Which of the following would NOT be considered a secondary metabolite?

a.

ethanol or some other fermentation e
nd product

b.

mycotoxins

c.

antibiotics

d.

All of the above are considered secondary metabolites.

5.

Which term refers to the use of microbes to make minor modifications to chemical compounds?

a.

bioconversions

b.

microbial transformations

c.

biotransforma
tions

d.

All of the above are correct.

6.

Which of the following best describes the production of semisynthetic penicillins?

a.

addition of a side
-
chain precursor molecule to a
Penicillium

culture to produce a desired
modification

b.

chemical modification
of a penicillin product after it has been released from the
Penicillium

culture

c.

Both (a) and (b) are correct.

d.

Neither (a) nor (b) is correct.

7.

Why are bioconversions increasingly replacing chemical synthesis of certain compounds?

a.

Bioconversions
yield only the desired stereoisomer.

b.

Bioconversions can be done under mild reaction conditions.

c.

Both (a) and (b) are correct.

d.

Neither (a) nor (b) is correct.


TRUE/FALSE


____ 1.

Industrial processes involving microorganisms require that the cult
ure be maintained in an active
growth phase in order to maximize product formation.

____ 2.

As with laboratory scale operations, industrial fermentations rely primarily on purified media
components in order to maintain better control of the process and of

the final product.

____ 3.

Continuous feed processes involve periodic additions of a critical nutrient so that the organism will
not have excess substrate available at any time.

____ 4.

Protoplast fusion can be used only between members of the same spec
ies.

____ 5.

Environmental conditions often have to be adjusted to switch from those conducive to microbial
growth to those conducive to product formation.




384

____ 6.

Biocatalysts are often immobilized by attaching them to ion exchange resins or by entrappi
ng them
in a polymerized matrix to enable their recovery and repeated use.

____ 7

Engineered proteins can be used to degrade previously recalcitrant molecules that were not
considered to be amenable to biological processing.

____ 8.

Plants have been made

pest
-
resistant by introducing a bacterial gene for an insect toxin into the
plant's DNA.

____ 9.

Although the terms can be used interchangeably, many differentiate industrial microbiology from
biotechnology because the former uses natural isolates modifi
ed by mutation and selection, whereas
the latter used molecular techniques to modify and improve microorganisms.

____ 10.

Most microorganisms of potential use in industrial microbiology have already been identified.

____ 11.

Because growth of microorganism
s in controlled environments is inexpensive, it is preferable to
carry out all microbial processes under such conditions.

____ 12.

Microorganisms can be used in controlled environments to make a variety of products.

They can
also be used in natural complex

environments to carry out environmental management processes.


CRITICAL THINKING


1.

Discuss how regulatory mutants are used (and why they are necessary) in the production of amino acids.
Cite a specific example and describe the nature of the regulatory c
hanges. Why is it desirable to increase
membrane permeability in these organisms as well?













2.

Genetically engineered microorganisms have been developed for use as pesticides. What steps should be
taken to test the safety of these organisms befor
e releasing them for use in a natural environment? Justify
your choices. If you do not think they should ever be used, state your reasons.













3.

Search the cabinets and shelves in your home and identify products containing compounds made by
microo
rganisms.

List the product, the microbial contribution, and describe the function of the compound
in the product.











385

ANSWER KEY


Terms and Definitions


1. i, 2. o, 3. q, 4. f, 5. h, 6. c, 7. g, 8. e, 9. a, 10. k, 11. m, 12. b, 13. l, 14. d, 15. h, 16.
p, 17. n


Fill in the Blank


1. scaleup 2. primary metabolites; secondary metabolites 3. regulatory 4. biosensors 5. natural genetic
engineering; forced evolution; adaptive mutations 6. industrial ecology; biosphere; anthrosphere 7.
combinatorial biology;
site
-
directed mutagenesis 8. biofuels; ethanol; hydrogen; methane


Multiple Choice


1. d, 2. b, 3. d, 4. a, 5. d, 6. b, 7. c


True/False


1. F, 2. F, 3. T, 4. F, 5. T, 6. T, 7. T, 8. T, 9. T, 10. F, 11. F, 12. T