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 (?)

Remove Pollutants from Groundwater Po llution 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. Groundwater Treatment Systems

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 aureus hornniloticus placidus zillii redalli galilaeus mossam/horn M 250 bp MSLAFD T W F T M (50 bp) U

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  Extensively used in the past 20 years  Green groups complained that this is “gene pollution” New Traits  74% Herbicide resistant  19% Insect resistant  7% Both Major GM crops  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 - derived fuels : 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?

A microbial bioreactor providing fuel directly in the anodic compartment of the electrochemical cell Microbial biofuel cells: A microbial bioreactor providing fuel separated from 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 H 2 production is observed upon fermentation of glucose in the presence of Clostridium butyricum (strain IFO 3847, 35 mmol/h H 2 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: B t, fungal insecticides and baculovirus