BIOREMEDIATION.

BIOREMEDIATION

The need for Bioremediation
The quality of life on Earth is linked inextricably to the overall quality of the environment. The problems associated with contaminated sites now assume increasing prominence in many countries. Contaminated lands generally result from past industrial activities when awareness of the health and environmental effects connected with the production, use, and disposal of hazardous substances were less well recognized than today.

Bioremediation Bioremediation is the use of biological systems (mainly microoganisms) for the removal of pollutants from aquatic or terrestrial systems. It is based on the extremely diverse metabolic potential of natural microbial communities. A challenge in this area is the existence of xenobiotics, i.e. compounds produced by chemical synthesis for industrial or agricultural purposes and having no counterparts in the natural world. Bioremediation is a key area of ‘white’ biotechnology, because the elimination of a wide range of pollutants from water and soils is an absolute requirement for sustainable development. The U.S. Environmental Protection Agency’s list of “priority pollutants” includes: industrial solvents building blocks of plastics polychlorobiphenyls pesticides (halogenated and nitroaromatics, polycyclic aromatic compounds)

Principles of Bioremediation
Bioremediation is based on the idea that organisms are capable to take in things from the environment and use it to enhance their growth and metabolism. With this unique characteristic lay the fundamental principle of Bioremediation, to use microorganism to take in contaminated substances from the environment or convert it to a non toxic form. Bacteria, Protista, and fungi are well known for degrading complex molecules and transform the product into part of their metabolism.

% Remaining Year 10 20 Bioremediation
There are three classifications of bioremediation: Biotransformation - the alteration of contaminant molecules into less or non-hazardous molecules Biodegradation - the breakdown of organic substances in smaller organic or inorganic molecules Mineralization - is the complete biodegradation of organic materials into inorganic constituents such as CO2 or H2O. % Remaining Year 10 20 Slow decrease in contaminant removal over time

Bioremediation as part of all remediation technologies
Source:

Compare the Costs Landfill disposal - $150 ~ $400 per m3 depending on hydrocarbon concentration. Time- 6 to 24 months . Thermal incineration- $250 to over $700 per m3. Time- fast (days to weeks). Bioremediation - $40 to $110 per m3. Time- 30 to 120 days Cost is site specific, but in most cases, bioremediation is very cost effective. Bioremediation can deal with lower concentrations of contaminants more effectively.

Limitations of Bioremediation
Contaminant type and concentration Environment Soil type Condition and proximity of groundwater Nature of organism Cost/benefit ratios: cost versus overall environmental impact Does not apply to all surface Length of bioremediation process Capabilities of bioremediation Reason for Failure presence of co-toxin physical constraints on electron acceptor delivery slow reaction rates (ex. low Temperature) conversion of contaminant to toxic metabolites heterogeneous distribution of contaminant lack of microorganisms with necessary biochemistry to degrade contaminant.

Bioremediation ADVANTAGES DISADVANTAGES
Minimal exposure of on site workers to the contaminant Long term protection of public health Minimal site disruption (buildings) Public acceptance The cheapest of all methods of pollutant removal The process can be done on site with a minimum amount of space and equipment Eliminates the need to transport of hazardous material Uses natural process Transform pollutants instead of simply moving them from one media to another Destroys dissolved and sorbed contaminants (vs P/T) Perform the degradation in an acceptable time frame Cost overrun Failure to meet targets Poor management Insufficient coordination and integration of project personnel Regulatory barriers Climate Issue Regulatory compliance concern Release of contaminants to environment Incomplete degradation can lead to odor and taste Scale up from bench/pilot difficult Unable to estimate the length of time it’s going to take, it may vary from site. It can takes a few month to as long as a few years. Not all organic compounds are biodegradable There are some concerns that the products of biodegradation may be more toxic then it’s parental form

CASE: Crude oil spill, Bemidji, Minnesota
First modern uses of Bioremediation CASE: Crude oil spill, Bemidji, Minnesota In the year 1979, in Bemidji, Minnesota a pipeline carrying crude oil suddenly exploded and releasing an enormous amount of oil. As a result of the oil spill, toxic chemicals were released which rapidly degraded the microbial population. The plume of contaminated ground water stopped enlarging after a few years as rates of the microbial degradation came in to balance with rates of the contaminant leaching.

First modern uses of Bioremediation
CASE: Exxon Valdez oil spill On March 24th, the Exxon Valdez suffered an accident and spilled 11 million gallons of oil on a reef in Prince William Sound, Alaska. Conventional cleaning proved unsuccessful, so the government turned to bioremediation to salvage the highly populated ecosystem. Phosphorus and nitrogen fertilizers were added to increase the efficiency of the microorganisms in 750 damaged areas. Over the next few years, scientists found that the level of contaminant fell five times faster in the bioremediated areas. On March 24th, 1989, the Exxon Valdez suffered an accident and spilled 11 million gallons of oil on a reef in Prince William Sound, Alaska. Conventional cleaning proved unsuccessful, so the government turned to bioremediation to salvage the highly populated ecosystem. Phosphorus and nitrogen fertilizers were added to increase the efficiency of the microorganisms in 750 damaged areas. Over the next few years, scientists found that the level of contaminant fell five times faster in the bioremediated areas.

Site characterization:
Bioremediation strategies Site characterization: Characterization of contaminants chemical composition concentration toxicity availability solubility volatility rates of biodegradation: hydrocarbon > substituted hydrocarbon > pesticides > highly chlorinated > monster molecules Relevant questions Are the contaminants biodegradable? Is it possible a biotreatment? Is the process a cometabolic one? Can the biodegradation be improved?

Bioremediation strategies Site characterization: Hydrogeochemical characterization geological properties hydraulic conductivity presence of nitrates and phosphates etc. presence of electron acceptors pH temperature Relevant questions Are the hydrogeological and environmental conditions favourable? Is the nutrient addition needed?

Bioremediation strategies Site characterization: Microbiological characterization dimension of microbial population catabolic diversity specific catabolic activities Relevant questions Is there the microbiological potential for biodegradation? Can the indigenous population be stimulated?

Site characterization - Soil Heterogeneity
Bioremediation strategies Site characterization - Soil Heterogeneity - dimension of microbial population → Community diversity - specific catabolic activities → Community function pore fungal hypha root bacteria mite clay soil particle nematode

Bioremediation Actors involved in bioremediation processes include:
• Bacteria - these generally use organic compounds (e.g. hydrocarbons) as an energy source and in the process convert them to innocuous by-products and ultimately into CO2 and H2O. • Fungi – these generally produce enzymes that can breakdown larger organic compounds (eg. lignin in woody material) into smaller by products that can be processed by bacteria. •Actinomycetes – a type of bacteria characterised by the presence of fine filaments. They are typically found in soil and give it its ‘earthy’ smell. Plants work with soil organisms to transform contaminants, such as heavy metals and toxic organic compounds, into harmless or valuable forms. The microorganisms may be indigenous to a contaminated area and stimulated in activity (biostimulation) or they may be isolated from elsewhere and brought to the contaminated site (bioaugmentation).

Bioremediation Bacteria
Beneficial characteristic of bacteria for bioremediation must include the following: Consume organic waste Grow and reproduce rapidly in selected environment Digest the waste quickly and completely Work without causing odours or poisonous compounds Non-pathogenic

Bacteria are characterized by their external sources of energy and carbon. Aerobic (with oxygen)- use available atmospheric oxygen to function. Food sources are converted to energy by the transfer of electrons to oxygen, which is an electron acceptor. Anaerobic (without oxygen)- break down chemical compounds to release the energy required to function. As electron acceptors, they utilize: nitrates sulphates carbon dioxide ferrous metals (such as iron)

Typical bacteria species include (in descending order of occurrence): Pseudomonas Arthobacter Alcaligenes Corynbacterium Flavobacterium Achrombacter Acinetobacter Micrococcus Nocardia Mycobacterium

Bioremediation Plant - Phytoremediation
In natural ecosystems, plants act as filters and metabolize substances generated by nature (Phytoremediation). Numerous plant types have been assessed and been found to be successful including various wetland plants (cattails, rushes, etc.), metal accumulating plants (astragalis), hybrid poplars (for TCE). Limited to surface sediments 1-2 m deep. Furthermore, contaminants that are strongly sorbed onto the physical soil matrix may be resistant to phytoremediation. Phytoremediation is used under satisfy environmental regulation and costs less then other alternatives. This process is very affective in cleaning polluted soil. How does it work Plants that are grown in polluted soil are specialized for the process of Phytoremediation. The plants roots can extract the contaminant, heavy metals, by one of the two ways, either break the contaminant down in the soil or to suck the contaminant up, and store it in the stem and leaves of the plant. Usually the plant will be harvested and removed from the site and burned. Phytoremediation is the use of plants to clean up potentially damaging spills. The plants work with soil organisms to transform contaminants, such as heavy metals and toxic organic compounds, into harmless or valuable forms.

Processes involved in Phytoremediation Phytovolatilization - removal of contaminants from the soil and subsequent release to the atmosphere Phytoextraction - extraction of contaminants by the plant Phytodegradation - plant metabolism of contaminants Rhizodegradation - microbial metabolism of contaminants in the rhizosphere

Soil Remediation (Schnoor, 2002)
Bioremediation Plant - Phytoremediation Soil Remediation (Schnoor, 2002) Application Description Contaminants Types of Plants Phytotransformation Sorption, uptake, and transformation of contaminants Organics, including nitroaromatics and chlorinated aliphatics Trees and grasses Rhizosphere Biodegradation Microbial biodegradation in the rhizosphere stimulated by plants Organics; e.g., PAHs, petroleum hydrocarbons, TNT, pesticides Grasses, alfalfa, many other species including trees Phytostabilization Stabilization of contaminants by binding, holding soils, and/or decreased leaching Metals, organics Various plants with deep or fibrous root systems Phytoextraction Uptake of contaminants from soil into roots or harvestable shoots Metals, inorganics, radionuclides Variety of natural and selected hyperaccumulators, e.g., Thalaspi,

Water/Groundwater Remediation (Schnoor, 2002) Application Description Contaminants Types of Plants Rhizofiltration Sorption of contaminants from aqueous solutions onto or into roots Metals, radionuclides, hydrophobic organics Aquatic plants, (e.g., duckweed, pennywort) Brassica, sunflower Hydraulic Control Removal of large volumes of water from aquifers by trees Inorganics, nutrients, chlorinated solvents Poplar, willow trees Phytovolatilization Uptake and volatilization from soil water and groundwater; conversion of Se and Hg to volatile chemical species Volatile organic compounds, Se, Hg Trees for VOCs in groundwater; Brassica, grasses, wetlands plants for Se, Hg in soil/sediments Vegetative Caps Use of plants to retard leaching of hazardous compounds from landfills Organics, inorganics, wastewater, landfill leachate Trees such as poplar, plants (e.g., alfalfa) and grasses

Bioremediation Plant - Phytoremediation BEFORE……………………….
……………………….AFTER

Bioremediation White-Rot Fungi
White Rot Fungi Bioremediation is very capable of degrading high to low levels of contaminants, as WRF are very effective in cleaning up a wide range of soil pollutants: Wood Preservation Polycyclic Aromatic Hydrocarbons Organoachlorines Polychlorinated Biphyenyls Dyes Pesticides Fungicides Herbicides

Bioremediation White-Rot Fungi The Mechanism
Fungi have ligninase, cellulase and oxidative enzymes that break down woody materials. This allows fungi to get the needed carbon and energy they require for growth. These enzymes are non-specific, meaning they can act on substrates like environmental pollutants. Growth of Bjerkandera adusta on a trunk LiP and MnP (ligninase manganese peroxidase) produce free radicals which have a high redox potential, with optimal function at pH =3 Laccase has the ability to oxidize phenolic cmpds extracellularly. Hyphae allow fungi to increase reaction surface area and get to the pollutant Physical Mechanism Hyphae allow fungi to expand their surface area, making it easier to contact the pollutant. Extracellular enzymes can then go to work.

Bioremediation White-Rot Fungi Phanerochaete chrysosporium
Phanerochaete chrysosporium acts to break down Organopollutants by adding an -OH group. Hydroxilating makes the compound more polar. Detoxification by hydroxilation (add -OH group) and excretion is standard for xenobiotic detoxification. Fungi can do this to pollutants too.

Bioremediation White-Rot Fungi Pleurotus ostreatus
Pleurotus ostreatus laccases are able to degrade OP pesticides and nerve agents. Organophosphorus pesticides and nerve agents

Bioremediation White-Rot Fungi Hebeloma crustuliniforme
Fungi are also used in conjunction with plant to remediate atrazine. Introduce fungi w/ host plant gives it better potential for successful survival. Atrazine is a widely used herbicide.

Bioremediation White-Rot Fungi PCB degradation by white rot fungi
PCB volatility, sorption on biological matrices, and xenobiotic extraction method were factors affecting remediation success. Drying the sample led to concentration different results.

Bioremediation White-Rot Fungi
Anthracene degradation by white rot fungi One mechanism to measure the degradation is by dye decolourisation. There was a high correlation between elimination of anthracene and Poly R-478 (dye) decolourisation. Interestingly, four strains were able to oxidize anthracene to anthraquinone even after HgCl2 poisoning. Biodegradation of Nitro-substituted explosives Wood and litter decaying fungi have been shown to metabolize TNT, but only a few of the species tested to date have been able to mineralize it. Problems: fungi have to compete with indigenous microflora, mineralization rate lower in soil than liquid medium, unclear biotransformation consequences. DNT →CO2 Figure shows the degradation pathway of 2,4-DNT by Phanerochaete chrysosporium (Valli et al., 1992) Polycyclic aromatic hydrocarbon PAH Antracene used in dyes, wood preservatives, and insecticides Explanation: HgCl2 killed cells without completely inhibiting extracellular enzymes involved in the oxidation. Anthraquinone is a dead-end metabolite which can be easily degraded by bacteria.

Bioremediation strategies
In situ bioremediation is when the spill is cleaned up exactly where it occurred. It is the most commonly used type of bioremediation because it is the cheapest and most efficient, so it’s generally better to use. Ex situ bioremediation, is when spills are taken out of the area to be cleaned up by the organisms. This type of bioremediation is generally only used when the site is threatened for some reason, usually by the spill that needs to be cleaned up. Ex situ bioremediation is only used when necessary because it’s expensive and damaging to the area, since the contaminated land is physically removed.

Intrinsic biodegradation (by native organisms) In situ: Does not accelerate cleanup, but prevents spread of further contaminants. Natural supply of organisms coupled with presence of additional organisms fast enough to prevent spreading. Engineered bioremediation (by applying nutrients or aeration) In situ and Ex situ: Increasing biotransformation rates by using engineering measures and adding materials. Cost-effective when concerned with deadlines, minimum exposure, or property sale. Through addition of micro-organism (bioaugmentation)

In situ technologies Criteria 1) microbes must exist that can bio-transform chemicals, 2) organisms must be able to convert chemicals at reasonable rates and meet regulatory standards, 3) toxic breakdown products must not be formed, 4) site must not contain specific microbial inhibitors, 5) favorable conditions must be present (achievable), 6) cost must be less or equivalent to other technologies.

In situ Bioremediation strategies
Bioaugmentation Bioaugmentation Bioaugmentation (seeding) has been used some in situ in soil and groundwater decontamination. A problem is that the non-indigenous microorganisms may not well enough with an indigenous population to develop and sustain long-term useful population levels. Biostimulation Bioventing Bioventing is the most common in situ treatment and involves supplying air and nutrients through wells to contaminated soil to stimulate the indigenous bacteria. Bioventing employs low air flow rates and provides only the amount of oxygen necessary for the biodegradation while minimizing volatilization and release of contaminants to the atmosphere. Biosparging Biosparging involves the injection of air under pressure below the water table to increase groundwater oxygen concentrations and enhance the rate of biological degradation of contaminants by naturally occurring bacteria. Biosparging increases the mixing in the saturated zone and thereby increases the contact between soil and groundwater.

In situ Bioremediation strategies
Amendment introduction via injection wells

Ex situ Bioremediation strategies
Landfarming (land treatment). Frequently used by oil industry to destroy oily wastes, also used for sludges from sewage plants, power plants, industrial facilities. Add wastes, contaminated water or contaminated soils to fertile soils, containing microbial populations. Important considerations: fertilization to achieve optimum C:N:P, supplementation to provide available oxygen (tilling), moisture control, inoculation (optional), pH control (optional). For these treatments, soil typically rests on liner system with engineered drainage systems in place. Biopiles The contaminated soil is piled in large heaps and air is pulled through with vacuum pumps Composting Contaminated material is mixed with readily degradable organic material (straw, bark, wood chips, etc.). Mixture is fertilized as appropriate, and adequate moisture and oxygen are maintained. In composting, microbial activity produces heat, which builds up in the compost (50-60 C° and higher). Composting has been successfully used for chlorophenols such as trinitrotoluene (TNT).

Landfarming (land treatment).

Biopiles

Bioreactors A bioreactor is a contained system where contaminated materials are mixed in a matrix and environmental variables can be controlled. Systems can be either static or stirred. Influent and effluent measurements determine relative success and provide for monitoring of final material. There are two main Bioreactor designs: batch and chemostat (continuous flow). Batch reactors have the advantage of simplicity, ease of disposal, control over effluent release. Chemostats offer more flexibility of controlling growth rates and degradation rates and avoid toxicity problems, associated with contaminants such as TCE. Another type of contained reactor system is the fixed film or immobilized cell bioreactor. Microbial cells become attached to an appropriate matrix (glass beads, fibers, polyurethane foam, alginate beads, activated carbon or polyacrylamide beads, etc.). The cells form a thin film, a biofilm, that becomes tolerant to greater concentrations of chemicals than free living cells are. The chemical solution is then passed over the biofilm, which results in rapid biodegradation due to high cell density and activity. The biofilter is a bioreactor designed to destroy volatile contaminants. Microorganisms are grown on solid matrices, and a contaminant gas stream is passed through the matrix. Microbial action destroys the contaminants.

How to circumvent bottlenecks? -By selecting soil microorganisms which have developed new properties in response to the introduction of xenobiotics By combining different metabolic abilities in the same microorganism for the degradation of a particular target compound By engineering microorganisms Engineering microorganisms Increase substrates detoxified - More individual compounds detoxified by one strain - Simultaneous detoxification Increase rate of detoxification - Increase expression Increase access to hydrophobic contaminants - If contaminant can be accessed, it will not persist - Hydrophobic contaminants persist

Engineering bacteria - Access to Contaminants Add genes to synthesize surfactants No surfactants With surfactants Problems with Genetically Engineered Microorganisms (GEMs) Don’t survive in the environment - Can’t compete with existing bacteria Cloning in survival or persistence genes raises regulatory issues Few field trials of GEMs, identified strains that can persist Greatest potential of GEMs for bioremediation is contained waste

Metagenomics and Bioremediation
Culture-independent DNA extracted from environmental sample Likely represents multiple species Gives information about populations individuals within populations Uses Samples taken from contaminated sites Can identify useful biodegradation activities Implementation in bioremediation strategies Diversity Metagenomics has revealed that microbial diversity is far greater than previously recognized Low-abundance organisms previously undetectable, even by microscopy 1L of seawater likely contains between 25,000 and 100,000 different microorganisms!

Genome-enabled techniques contribute to the development of models of how micro-organisms function in contaminated environment. Cells isolated from the environment provide the opportunity for in-depth physiological analysis as well as information on gene composition that can be used for the analysis of mRNA and proteins that are extracted directly from the environment. Genomic DNA extracted from the environment furnishes data on the genetic potential of as-yet-uncultured organisms. mRNA and proteins extracted from the environment provide information on gene expression under different environmental conditions. Analysis and comparison of pure cultures and mixed communities yields data for the development of models of microbial function in the environment.

Environmental transcriptomics / Meta-transcriptomics High throughput processing and sequence analysis Annotation and Data analysis Microbial community analysis broad PCR-DGGE (Bact. Fungi, Archea PCR-DGGE of specific bacterial groups, AOB, pseudomonads, etc.) Functional gene analyses (Q-PCR) PLFA / NLFA or FAME Phylogenetic microarray cDNA community profile cDNA synthesis (RT-PCR w/ short primers) DNA extraction Total RNA extraction rRNA subtraction (degradation) polyA-tail addition ligation of RNA oligo cDNA synthesis (polyT-based RACE ) cDNA synthesis with FAME label Cloning into E. coli Sample

Genomics and bioremediators!
Metagenomics and Bioremediation Genomics and bioremediators! See: Lovely, D.R. Cleaning up with Genomics: applying molecular biology to bioremediation. Nature Reviews Microbiology October 2003.

Bioremediation “Microbes can and will do anything: microbes are smarter, wiser and more energetic than microbiologist, chemists, engineers and others.” Perlmon, D. (1980): Developments in Industrial Microbiology, 31, XV

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