Download Biological processes in natural systems

How Do Engineered Systems Prevent and Manage
Pollution in Water and Soil? … and what is the
relevance to biotechnologists?
The demand for employees with the combined
expertise of environmental engineering and
microbiology is growing … .
Areas of emphasis include & not limited to:
· Biological methods for characterization and remediation of
contaminated sites
· Biological sensors or sensor technology development and
application
· Biological processes in natural systems
· Biological treatment of water and wastewater
· Biological aspects of the built environment
· Application of genetic techniques to characterizing
natural and engineered environmental systems
Some examples…
Water Treatment Systems
remove pollutants from lake or river or groundwater to
produce potable drinking water
Water Treatment Concerns:
Microbial Pathogens
 What kind are in the water to be treated?
 What is their source?
 Have they been removed by treatment?
Sewage Treatment Systems
remove pollutants from sewage to return water to a lake,
river or groundwater.
What constituents of sewage would require treatment?
A series of physical and
biological processes
The conventional biological
process employs activated
sludge.
These systems typically
consist of an aeration basin
and a clarifier
Aerobic
Up until recently, activated sludge was treated
as a “black box,” with little attention given to
the key microbial “players.”
The times that microbes get attention is when they:
a) Cause foaming or sludge bulking
Or
b) when they are specialized for a needed function, e.g.,
PAOs (Phosphorus Accumulating Organisms)
Typically biological sewage
treatment includes an
aeration basin and a clarifier
…and sewage treatment plants also
includes other microbiological
treatment to digest the solids
(sludge) that are collected.
Anaerobic
There are also many exciting new
innovations in wastewater treatment
that attempt to better mimic nature:
natural attenuation
The Living
Machine
…and constructed wetlands
Industrial Wastewater Treatment Systems
remove pollutants from industrial wastewater
to return water to a lake, river or groundwater
Water Reuse Systems
remove pollutants from treated wastewater so that
water can be reused for nonpotable & even potable use (?)
Groundwater Treatment Systems
Remove Pollutants from Groundwater
Pollution occurs due to leaks from septic tanks,
underground storage tanks, hazardous waste
dumping, landfills, lagoons, fuel spills, military
storage of chemical weapons, and agriculture
sources contributing fertilizers, herbicides, and
pesticides.
And each source creates a plume
Engineered Methods
Microbial treatment
Soil Treatment Systems
remove pollutants from soil
Soil becomes contaminated by
the same sources as
groundwater, but it can’t be
cleaned up in the same way
Biological methods have employed composting …
And “enhanced” bioremediation … .
Finally, there are biological systems
to treat gases … biofilters
Recent Topics: Risk Management and Biofuels
 Use of molecular techniques to protect the
environment, including Risk assessments of GMOs
 Renewable energy and resources: engineering plants
for the production of clean energy, biofuel, biomass,
and animals for food production, etc.
Environmental Biotechnology is the multidisciplinary
integration of sciences and engineering in order to utilise
the huge biochemical potential of microorganisms, plants
and parts thereof for the restoration and preservation of
the environment and for the sustainable use of resources
OUTLINE:
1. Molecular Ecology
2. Bioremediation (site restoration) and
Biotechnology for waste treatments
3. Biosensor (monitoring of pollution)
4. Environmental applications of genetically
modified organisms and Genetic Exchange in
Environment
5. Biofuel
1.
Molecular Ecology
Understanding nature by molecular techniques of:
 DNA fingerprinting for population genetic
studies; become more important for biodiversity
research to study kinship relationship
 Authentication; inspect endangered species
with minimal samples using non-invasive
technique
 Forensic analysis, to properly identify the
“evidence” for species identification
WHAT FOR?
 Phylogenetic study: e.g., horse family; compare
between species or strains
 Population study: compare within species
collected from different locations, e.g., compare
between Asian and African populations
 Molecular Ecology
 Authentication study: external morphology
cannot give positive identification of a species, e.g.,
specimen of meat samples or dried plants ground
in powder form
EcoRI digestions of Tilapia genomic DNA
MSL AFD T W F T
M U 1 2 3 1 2 1 2 3 1 2 M (50 bp)
mossam/horn
galilaeus
redalli
zillii
placidus
niloticus
horn
aureus
250 bp
2.
Bioremediation (site restoration) and
Biotechnology for Waste Treatments
 Ocean ranching for stock restoration (e.g., cultured salmon,
grouper and abalone released to the sea or artificial reef)
 Recovering of damaged sites to a “clean” or less harmful
site after dredging
 Remove chemicals using biological treatments on site (in
situ) or ex situ
 Chemicals: heavy metals, trace organics or mixtures
 Bacterial or fungal degradation of chemicals
 Engineered microbes for better and more efficient removal
of chemicals on-site
Redox Clean-Up Reactions
 Anaerobic or aerobic metabolism involve oxidation and
reduction reactions or Redox reactions for detoxification
 Oxygen could be reduced to water and oxidise organic
compounds. Anaerobic reaction can use nitrate
 In return, biomass is gained for bacterial or fungal growth
 In many cases, combined efforts are needed, indigenous
microbes found naturally in polluted sites are useful
Problems with bioremediation
 Work in vitro, may not work in large scale. Work
well in the laboratory with simulation, may not work
in the field. Engineering approach is needed
 Alternatively, select adapted species on site
(indigenous species) to remediate similar damage
 Most sites are historically contaminated, as a result
of production/transport/storage/dumping of waste.
They have different characteristics & requirements
 Those chemicals are persistent or recalcitrant to
microbial breakdown
Use of bacteria in bioremediation
 Greatly affected by unstable climatic and environmental
factors from moisture to temperature
 e.g., soil pH is slightly acidic; petroleum hydrocarbon
degrading bacteria do not work well at <10C
 These microbes are usually thermophilic anaerobes
 Fertilisers are needed. Seeding or bioaugmentation could
be useful too
 They contain monooxygenases and dehydrogenases to
break down organic matters including toxic substances
Pseudomonas
 Genetically engineered bacteria (Pseudomonas) with
plasmid producing enzymes to degrade octane and many
different organic compounds from crude oil
 However, crude oil contains thousands of chemicals
which could not have one microbe to degrade them all
 Controversial as GE materials involved
Use of fungi in bioremediation
 Lipomyces can degrade paraquat (a herbicide)
 Rhodotorula can convert benzaldehyde to benzyl alcohol
 Candida can degrade formaldehyde
 Gibeberella can degrade cyanide
 Slurry-phase bioremediation is useful too but only for
small amounts of contaminated soil
 Composting can be used to degrade household wastes
White rot fungi
 White rot fungi can degrade organic pollutants in soil and
effluent and decolorise kraft black liquor, e.g.,
Phanerochaete chrysosporium can produce aromatic
mixtures with its lignolytic system
 Pentachlorophenol, dichlorodiphenyltrichloroethane (e.g.,
DDT), even TNT (trinitrotoluene) can be degraded by
white rot fungi
Phyto-remediation
 Effective and low cost
 Soil clean up of heavy metals and organic compounds
 Pollutants are absorbed in roots, thus plants removed
could be disposed or burned
 Sunflower plants were used to remove cesium and
strontium from ponds at the Chernobyl nuclear power
plant
 Transgenic plants with exogenous metallothionein (a
metal binding protein) used to remove metals
Waste water treatments
 Bioremediation of water or groundwater or materials
recovered from polluted sites
 Ex situ: As many bacteria work better in controlled
conditions, e.g., anaerobic, higher temperature, effluent
(sewage treatment) or solid materials (composting) can be
treated with bacteria to decompose organic matters
 Primary treatment: screening and emulsification
 Secondary treatments: Nutrient removal and chemical
removal
Nutrient removal
 Phosphate removal by polyphosphate accumulating
organisms and glycogen accumulating organisms
 Nitrogen removal by Nitrosomonas which denitrify nitrite
to nitrogen gas. Anaerobic ammonium oxidation is also
important
 Algae could absorb many nutrients and pollutants.
Dunaliella. Chlorella and Spirulina are valuable species
Dye removal and chemical removal
 Azo-dye (N=N) removal
 Sensitive to redox and anaerobic treatments can
decolorise azo dyes
 Specific reductase enzymes are needed to detoxify the
dye after discoloration
 Chemical treatment or biological treatment, e.g.,
Candidatus Brocadia Anammoxidans for ammonia
removal
3.
Biosensor
(monitor pollution)
 Measurement of mutagenic activity (microtox and
mutatox tests with lux gene from Vibrio)
 Biomarkers of exposures to pollutants (stress proteins)
 Detection of pathogens by multiplex-PCR
 Detection of toxins (Ciguatoxin)
Ames 1973 developed a
rapid screening method
based on mutation of
Salmonella typhimurium. The
mutant strains used in the
Ames Tests are histidine
defective (unable to
synthesise histidine). Back
mutation make them able to
survive on plates without
histidine
BioDetection Systems
 CALUXR Bioassay
 A sensitive bioassay for
exposure to dioxins and related
compounds
 Synthetic gene promoter
created and linked to a reporter
gene which gives colour when
the gene promoter is turned on
 The synthetic gene promoter
contains multiple cis-acting
elements responsible for dioxin
(DRE) and dioxin receptor (Ah
receptor) binding.
 The reporter gene is tranfected
into a cell-line for the bioassay.
Stress Proteins
 Metallothionein for exposure to heavy metals
 Cytochrome P450 (CYP) IA1 for exposures to trace
organics
 Vitellogenin (an egg yolk protein) for exposure to
environmental estrogens
 Heat shock protein for general stress conditions
 These biomarkers are NOT biomarkers of toxic effects.
They are biomarkers of exposures. Thus, controversial
 Biomarkers have biological relevance and usually less
expensive than chemical analyses. Data could be
diagnostic and indicative
Pathogen detection
 Bacteria: coli form bacteria, salmonella, Legionella, Vibrio,
etc.
 Virus: Influenza, SARS, hepatitus, polio, etc.
 Algae: dinoflagellates, diatoms, toxic algae, ciguatoxin,
etc.
 Multiplex technology is being developed: one run for
many pathogens
 Collection with minimal amount of samples: water, soil, or
air
 Use PCR or real-time PCR techniques
Microarray technique
for environmental screening and detection
 NOT really quantitative, it’s qualitative
 A rapid screening procedure for
pathogens or multiple biomarkers to
monitor or identify the problem. Require
later verification and real-time PCR
detection with antibody confirmations
 Array of probes (biomarkers/pathogens)
placed on a piece of glass or other solid
surface. DNA or RNA from a test
environmental sample, is then applied
to the solid surface and wherever there
is a match with a probe sequence,
specific and sensitive hybridisation
occurs, resulting in the generation of a
signal
 Methods are still under development
4. Environmental applications of genetically
modified organisms
 Insect Bt resistance,
producing a bacterial
toxin from Bacillus
thuringiencis (Bt);
insects (dipterans) die
when eating the plants
New Traits
 Extensively used in the
past 20 years
Major GM crops
 Green groups
complained that this is
“gene pollution”
 74% Herbicide resistant
 19% Insect resistant
 7% Both
 58% Soybean
 23% corn
 12% cotton
 6% Canola
Genetic Exchange in the Environment
 Risk Assessments and Biotechnology Regulations (e.g.,
environmental use permits)
 To detect the 35s CaMV (Cauliflower mosaic virus)
promoter sequence or NOS (nopaline synthase gene
terminator) DNA sequence by Quantitative PCR for GMO
detection
 GMOs: Bacteria is associated with disease and hence is
always held up by fears. e.g., antibiotic –resistance
 GEM: The concern is antibiotic resistant plasmid
horizontally transferred to other microorganisms
5.
Bio-fuels
 Plant-derivedfuels: plant species
for hydrocarbon (oil) production,
e.g., rape-seed, sunflower, olive,
peanut oils. Or ethanol production
of sugars (or cellulose) derived from
plants
 Conversion of used cooking oil to
bio-fuel (called bio-diesel)
 Biogas: gases from composts or
landfill, but methane is a green
house gas
Bioethanol and biofuel cell
 Sugar cane, sugar beet wastes, high starch material
(cassava, potatoes, millet) to be hydrolysed by starch
hydrolysing enzyme to convert sucrose or glucose to
ethanol. Mainly used in Brazil
 Corn ethanol: 22% less carbon emission, used in the US.
 Bio-diesel: 68% less carbon emission; oils from soybean
(US) or canola oil (Germany)
 Cellulosic ethanol: 91% less carbon emission, but difficult
to change cellulose to ethanol
 Hydrogen energy however is the trend of future renewable
energy without carbon emission: a journey to forever … … .
 Problem is how to generate the hydrogen; too costly with
conventional chemical methods or reverse osmosis
A Pathway for our Future Energy?
Microbial biofuel cells:
A microbial bioreactor
providing fuel separated
from the anodic
compartment of the
electrochemical cell
A microbial bioreactor
providing fuel directly in
the anodic compartment
of the electrochemical cell
The Working Principle of An Enzyme Fuel Cell
The enzyme and mediator are
immobilised on the anode
Rough layout of the anode
structure
Other options
 Various bacteria and algae, for
example Escherichia coli,
Enterobacter aerogenes,
Clostridium butyricum,
Clostridium acetobutylicum, and
Clostridium perfringens have
been found to be active in
hydrogen production under
anaerobic conditions
 The most effective H2 production
is observed upon fermentation of
glucose in the presence of
Clostridium butyricum (strain IFO
3847, 35 mmol/h H2 evolution by
1 g of the microorganism at 37°C)
Summary of applied environmental Science/Biotech
 Potable water, Sewage, Industrial waste, Groundwater and Soil treatments
 Gas treatment - Treatment of gaseous waste. Biofilters – e.g., dechlorination of air.
 Detection, Monitoring, and effecting Change in Environmental pollution
 Effects on health and ecosystem
 Microorganisms in the prevention, elimination and evaluation of chemical pollution
 Environmental monitoring. Chemical and physical analyses. Determining populations & activities
 Biosensors. Screening for microbial toxicity. Regulations
 Microbial processes involved in the elimination of waste and pollutants
 Bioremediation of organically polluted soil, underground waters. Factors affecting biodegradation
 Bioavailability. Acclimatisation. Bioremediation technologies. Biosupplementation
 Bioremediation of soil and underground waters polluted with metals
 Phytoremediation of metals. Elimination of heavy metals from aqueous effluent.
 Precipitation, bioabsorption and transformation
 Measuring pollution in wastewater. Composition of effluent. Aerobic treatment of sludge.
Anaerobic digestion. Elimination of nitrogen, phosphorus and sulphur
 Biotechnologies to minimise the generation of waste and other products. Clean technologies.
 Microorganisms and fuels. Biofuels: bioethanol, biodiesel, biogas, hydrogen. Microbial extraction
of oil. Desulphurisation and denitrogenisation of oil. Solubilisation and desulphurisation of carbon
 Biomining. Bacterial leachate of metals by class. Microbial recovery of metals and minerals
 Microorganisms and agriculture. Use of symbionts and pathogens. Nitrogen fixers. Mycorrhiza.
Microbial biopesticides: Bt, fungal insecticides and baculovirus