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Chapter 41
Applied and Industrial
Microbiology
1
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Water Purification
and Sanitary Analysis
• water purification
– critical link in controlling waterborne
disease
2
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Water Purification
• water with high levels of suspended
material  sedimentation basin
– large particles settle out
• partially clarified water mixed with
chemicals such as alum and lime and
moved to a settling basin
– more material precipitates out in coagulation
or flocculation process
• removes microbes, organic matter, toxic
contaminants and suspended fine particles
3
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Water Purification…
• water  rapid sand filters
– physically traps fine particles and flocs
• water treated with disinfectant
– chlorine
• concern about the creation of disinfection
by-products such as trihalomethanes
which may be carcinogens
– ozone
4
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Water purification
sedimentation basin
settling basins
5
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Problem microbes
• not consistently removed by coagulation, rapid
sand filtration, and disinfection processes
– Giardia intestinalis
• “backpackers disease” (diarrhea)
• slow sand filters effectively remove Giardia cysts
– Cryptosporidium
• small protozoan with oocysts that escape usual
purification schemes
– Cyclospora
• protozoan that causes diarrhea
– viruses
• up to 99.9% are removed by usual purification
schemes, but this not considered sufficient protection
6
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Sanitary Analysis of Waters
• based on detecting indicator organisms
– indicate fecal contamination of water
supplies
– indicate possible contamination by human
pathogens
7
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“Ideal” indicator organism
•
•
•
•
•
suitable for analysis of all types of water
present whenever enteric pathogens are present
survives longer than hardiest enteric pathogen
does not reproduce in contaminated water
detected by highly specific test
– test easy to do and sensitive
• harmless to humans
• its level in water reflects degree of fecal
pollution
8
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Two commonly used indicators
• coliforms
• fecal streptococci
– increasingly used to test brackish and
marine water
9
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Coliforms
• facultative anaerobic, gram-negative,
nonspore-forming, rod-shaped bacteria that
ferment lactose with gas formation within 48
hours at 35°C
• e.g. Escherichia coli, Enterobacter aerogenes,
Klebsiella pneuminiae
• traditional method of detection is multiple-tube
fermentation test for presumptive test
10
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Multiple-tube
fermentation
test
confirmed test
completed test
11
presumptive test
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Other tests for indicator
organisms
• membrane filtration technique
• presence-absence (P-A) test
• defined substrate tests
12
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Membrane filtration technique
water passed through
filter

filter placed on surface
of growth medium

incubate

count colonies
13
Used to detect total coliforms,
fecal streptococci and fecal
coliforms
- from intestines of warm
blooded animals
- detected by incubation at
44.5°C
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14
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Presence-absence test
• modification of MPN
• uses larger water sample (100 ml)
• sample added to lactose containing medium
– contains pH indicator to detect acid
production
• based on assumption that no indicator
organisms should be present in 100 ml of
water
• detects total coliforms and fecal coliforms
15
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Defined substrate tests
• e.g., Colilert
• detects total coliforms and
fecal coliforms
• 100 ml sample added to
medium containing ONPG
and MUG
* ONPG: o-nitrophenyl-β-D-galactopyranoside
* MUG: 4-methylumbelliferyl-β-D-glucuronide
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(a)
Uninoculate control
(b)
Yellow color (o-nitrophenyl) due to the presence
of coliforms
(c)
Fluorescent reaction due to the presence of fecal
coliforms (E. coli modifies MUG to a fluorescent
product)
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Potable water
– suitable for human consumption
17
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Wastewater Treatment
• decreases organic matter and
number of microorganisms in
human waste-impacted water
• has lead to major reduction in
spread of pathogens
18
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Measuring Water Quality
• three major approaches
– total organic carbon (TOC)
– chemical oxygen demand (COD)
– biochemical oxygen demand (BOD)
19
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Total organic carbon
• quantifies all carbon
• organic matter oxidized at high
temperature
• amount of CO2 produced is measured
• fastest, but less informative
20
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Chemical oxygen demand
• quantifies organic matter (except lignin)
• organic matter reacted with strong acid
(permanganate)
• slower test
• high cost for chemical waste disposal
21
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Biochemical oxygen demand
• measures amount of dissolved oxygen
needed for microbial degradation of
organic matter
– indirect measure
– 5 days at 20oC
• can be affected by presence of ammonia
– nitrogen oxygen demand
• use of oxygen during nitrification process
• inhibited by addition of chemicals to sample
22
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23
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Wastewater Treatment Processes
• similar to self-purification processes
observed in rivers and streams
except are controlled and intensified
24
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An aerial view of a
modern conventional
sewage treatment plant
in New Jersey
25
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26
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Activated sludge system
27
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normal floc
– settles well
bulking sludge
- does not settle
properly
28
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Trickling filter system
29
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Extended aeration process
Extended aeration
period
30
Reduces amount of
sludge produced
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Anaerobic digestion
• often sludges from aerobic sewage
treatment, together with materials settled
out in primary treatment are further
treated by anaerobic digestion
• reduces the amount of sludge for disposal
• produces methane
31
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A sequential process during
anaerobic digestion
32
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Constructed wetlands
Employed in treatment
of liquid wastes and for
bioremediation
33
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Home Treatment Systems
• groundwater
– water in gravel beds and fractured rocks
below surface soil
• microbiological processes in groundwater
are not well understood
34
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Home septic tank system
Frequently fail to work properly, contributing to groundwater contamination.
35
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Choosing Microorganisms for
Industrial Microbiology
• must first find suitable microbes
– genetically stable
– easy to maintain and grow
– well suited for extraction or separation of
desire product
36
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Finding Microorganisms
in Nature
• bioprospecting
– hunting for new microorganisms
• most major sources of microbes for use in
industrial microbiology have been
natural materials
• hunting for industrially useful microbes
in nature continues because < 1% of
microbes have been cultured from nature
37
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38
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Genetic Manipulation
of Microorganisms
• Used to produce microorganisms with new
and desirable characteristics
– mutation
– protoplast fusion
• widely used with fungi
• can fuse protoplasts of different species
– insertion of short DNA sequences
– transfer of genetic information between
different organisms
– modification of gene expression
– natural genetic engineering
39
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Mutation of Penicillium chrysogenum
to increase penicillin yield
initial yield
X-ray (X)
UV (UV)
mutations used
to increase yield
of penicillin
Mustard gas (N)
40
Final yield
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Insertion of short DNA sequences
• site-directed mutagenesis
– insertion of chemically synthesized
DNA to create small genetic changes
– used in protein engineering
• deliberately making alterations in amino
acid sequence of a protein in order to
generate enzymes with new or improved
properties
41
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Transfer of Genetic Information
Between Different Organisms
• the transfer and expression of genes between
different organisms can give rise to novel
metabolic processes and products
• part of field of combinatorial biology
• heterologous gene expression
– the insertion of functional genes from one organism
into another
• e.g., creation of patented “superbug” with
enhanced ability to degrade hydrocarbons
42
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43
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e.g., recombinant
vaccine production
44
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Modification of Gene Expression
• allows for overproduction of a product,
production of more than one product by the
same organism, or synthesis of modified
products
• pathway architecture
– analysis, design, and modification of biochemical
pathways to increase process efficiency
– metabolic pathway engineering
• intentional alteration of metabolic pathway by
inactivation of specific genes
– metabolic control engineering
• alteration of control mechanisms of specific genes
45
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46
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Metabolic engineering to create modified antibiotics
6-deoxyerythronilide
47
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Protein Evolution
• one of the newest approaches for creating new
metabolic capabilities in a given microorganism
• employs forced evolution and adaptive mutations
– involve use of specific environmental stresses to “force”
microbes to mutate and adapt, thus creating microbes with
new biological capabilities
• in vitro evolution
– starts with purified nucleic acids rather than whole
organisms
• high-throughput screening enables the rapid selection
of a single desirable mutant or molecule from tens of
thousands of new constructed stains, molecules, or
compounds
48
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49
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Preservation of Microorganisms
• once microbe or virus has been selected
or created, it must be preserved in its
original form for further use and study
• periodic transfers can lead to mutations
and phenotypic changes
– to prevent this problem, a variety of culture
preservation techniques can be used
50
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Preservation of Microorganisms
51
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Microbial Growth in Controlled Environments
- Fermentation
52
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Medium Development
• to maintain economic competitiveness,
low-cost crude materials are frequently
used
• levels of minerals (especially iron) and
growth factors may be critical
53
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54
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Growth of Microorganisms in
an Industrial Setting
• requires precise control of agitation,
temperature, pH changes, and oxygenation
• non-Newtonian broth
– viscous, plastic medium resulting from growth of
filamentous fungi and actinomycetes
– resists stirring and aeration, making maintenance of
appropriate levels of O2 difficult
• scaleup
– modifications of growth process so that it works
efficiently in large fermenters
55
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Filamentous growth during fermentation
Initial culture
56
After 18 h growth
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A typical industrial
stirred fermentation
unit
57
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Supplying nutrients
• can supply a critical nutrient in limiting
amounts
– can cause shift from growth to production of
desired metabolites
• continuous feed
– continual addition of a critical nutrient so
that microbes will not have excess substrate
available at any given time
– prevents production and accumulation of
undesirable metabolic waste products
58
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Alternate methods for
mass culture
59
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Types of microbial products
• primary metabolites
– related to synthesis of microbial cells in the
growth phase
– include amino acids, nucleotides,
fermentation end products, and enzymes
• secondary metabolites
– accumulate following active growth
– have no direct relationship to synthesis of
cell material and natural growth
– include antibiotics and mycotoxins
60
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61
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Major Products of
Industrial Microbiology
• industrial and agricultural products
• food additives
• products for human and animal
health
• biofuels
62
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63
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Penicillin production
requires
precise control of
nutrients
Final product can be
modified to yield a variety
of semisynthetic penicillins
64
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Streptomycin production
65
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Amino Acids
• typically produced using regulatory
mutants, which have reduced ability
to limit synthesis of an end product
66
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Glutamic acid production by Corynebacterium glutamicum
67
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Organic Acids
68
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Specialty Products for Use in
Medicine and Health
69
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Biopolymers
• used to modify flow characteristics
of liquids and to serve as gelling
agents
• include:
– dextrans and other polysaccharides
– cyclodextrins
70
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(from Thermoanaerobacter sp.)
71
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Biosurfactants
• used for emulsification, increasing
detergency, wetting and phase dispersion,
and solubilization
• important in bioremediation, oil spill
dispersion, and enhancing oil recovery
• many have antibacterial and antifungal
activity; some inactivate enveloped
viruses
72
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Bioconversion Processes
• microbial transformations or
biotransformations
– minor changes in molecules carried out by
nongrowing microbes
– microbes act directly or indirectly as biocatalysts
• can be carried out by free enzymes or cells or
by immobilized enzymes or cells
• advantages
– produce appropriate stereoisomer
– very specific
– done using mild conditions
73
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e.g., Biotransformation to modify a steroid
progesterone
74
11α-hydroxyprogesterone
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Biodegradation and Bioremediation
by Natural Communities
• metabolic activities of microbes can be
exploited in natural environments
– where physical and nutritional conditions for
growth cannot be controlled
– a largely unknown microbial community is
present
75
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Biodegradation and Bioremediation
by Natural Communities
• examples
– use of microbial communities to carry out
biodegradation, bioremediation and
environmental maintenance processes
– addition of microbes to soils or plants for the
improvement of crop production
76
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Biodegradation Using Natural
Microbial Communities
• biodegradation has at least three
definitions
– minor changes
– fragmentation
– mineralization
77
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78
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Bioremediation
• the use of microbes to transform toxic
molecules to nontoxic degradation
products
• the degradation of toxic molecules
requires several stages, usually
performed by different microbes
• reductive dehalogenation
– removal of a halogen substituent while at the
same time adding electrons to the molecule
– usually occurs under anaerobic conditions
79
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Fate of a chemical in Nature
• structure and stereochemistry play critical
role in predicting the fate of specific chemical
– meta effect occurs when constituent is in meta, as
opposed to ortho position, the compound will be
degraded at a slower rate
• many compounds added to environments are
chiral
– possess asymmetry and handedness
• microbes often can degrade only one isomer of a
substance; the other isomer will remain in the
environment
80
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Meta effect
81
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Fate of a chemical in Nature
• microbial communities change their
characteristics in response to addition of
inorganic or organic chemicals
• acclimation
– occurs if chemical is repeatedly added, the
community adapts and faster rates of
degradation occur
82
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Repeated exposure and degradation rate
83
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Downside of biodegradation
• can lead to widespread damages and
financial losses if occurs in inappropriate
situation or in an uncontrolled manner
• e.g., corrosion of metals, especially iron
pipes
84
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Microbial-mediated metal corrosion (e.g. Desulfovibrio)
85
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Stimulating Biodegradation
• bioremediation usually involves stimulating
degradative activities of microbes already
present at contaminated sites
• it is necessary to determine the limiting factors
at the site (e.g., nitrogen or phosphorus or other
nutrients) and supply them or modify the
environment
86
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Changing Environmental Conditions
to Stimulate Biodegradation
• engineered bioremediation
– addition of O2 or nutrients to soils or water
in order to stimulate biodegradative
activities of microbes
– addition of easily metabolized organic
matter increases degradation of recalcitrant
compounds that are not usually used as
carbon or energy sources
• an example of cometabolism
87
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Some examples
• stimulation of hydrocarbon degradation
• phytoremediation
– use of plants
• stimulation of bioleaching of metals from
minerals
88
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Subsurface engineered bioremediation system
M: monitoring wells
89
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Phytoremediation
Hexachlorobenzene is cometabolized by microbes in the presence of
organic matters relased from plant roots.
90
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Types of phytoremediation
91
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Bioleaching
Leptospirillum ferrooxidans
Thiobacillus thiooxidans
92
(Fe2+)
(Fe3+)
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Bioaugmentation
• addition of microorganisms to complex microbial
communities
• generally has resulted in only short-term increases
in desired degradative activity
• outcome can be improved by providing protective
microhabitats
– living microhabitats (e.g., surface of a seed, root, or leaf)
– inert microhabitats (e.g., microporous glass)
• natural attenuation
– use of natural microbial communities to carry out
biodegradative process
93
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Microbes as Products
• nanotechnology
– e.g., use of diatom 3-D structures as templates
– e.g., use magnetosomes from magnetotactic
bacteria in magnetic resonance tomography
(MRI) applications and as probes to detect
cancer
94
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Marine diatom surface features
95
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Biosensors
• living microbes, enzymes or organelles
are linked to electrodes to detect specific
substances
– detection is done by converting biological
reaction products into electrical currents
• have a broad range of applications
96
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Biosensor design
97
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98
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Streptavidin-Biotin binding and biotechnology
99
Streptavidin: from Streptomyces avidini
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100
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Biopesticides
• biological agents, such as bacteria,
fungi, viruses, or their components,
which can be used to kill a
susceptible insect
101
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102
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Bacteria as biopesticides
• Bacillus thuringiensis
– parasporal body
• produced during sporulation as an intracellular
protein toxin crystal
• acts as microbial insecticide for specific groups of
insects
– insecticide (Bt)
• used for over 40 years
• unlike chemical insecticides, does not accumulate
in environment
103
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Mode of action of Bt toxin
Release of prototoxin from
the parasporal body
Modification of the
prototoxin by proteases
104
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68 kDa active toxins
105
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Impacts of Microbial Biotechnology
• industrial ecology
– concerned with tracking flow of elements and
compounds through natural world (biosphere) and
social world (anthrosphere)
• microbiologists must:
– understand potential impacts of new products and
processes on the broader society as well as on
microbiology
– communicate effectively with the various “societal
stakeholders” about the immediate and longer-term
potential impacts of biotechnologies
106