General overview of Bioremediation

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1 General overview of Bioremediation Presented during the Soil Remediation Workshop (With special presentation on Nextgen sequencing), Pretoria, SA, May 2014 By Damase Khasa Centre for Forest Research and Institute for Integrative and Systems Biology, Université Laval, Québec Canada G1V OA6

2 Outline of presentation Part I: Bioremediation Biostimulation Bioaugmentation Phytoremediation Part II: Canadian case study in the oil sands industry

3 Part I: Bioremediation, An overview Biostimulation Bioaugmentation Phytoremediation

4 Pictures depicting worldwide problems of pollution

5 Environmental contaminants Pollutants naturally-occurring compounds in the environment that are present in unnaturally high concentrations Examples: crude oil refined oil phosphates heavy metals Xenobiotics chemically synthesized compounds that have never occurred in nature. Examples: pesticides herbicides plastics S: Pearson education

6 Sources of contamination Modified from Capiro 2003 Industrial spills and leaks Surface impoundments Storage tanks and pipes Landfills and dumps Injection wells

7 Sources of contamination The major contributors to volatile organic compounds (air pollution) are from Paint industry Pharmaceutical industry bakeries printers dry cleaners auto body shops

8 Types of treatment technologies in use to remove contaminants from the environment Soil vapor extraction Air and hydrogen sparging soil washing, in situ soil flushing chemical oxidation/reduction soil excavation pyrometallurgical processes (thermal desorption, electrokinetical treatment) bioremediation

9 What is bioremediation? The use of microbes (bacteria and fungi) and plants to break down or degrade toxic chemical compounds that have accumulated in the environment into less toxic or non toxic substances

10 History of Bioremediation ~1900 Advent of biological processes to treat organics derived from human or animal wastes ~1950 Approaches to extend wastewater treatment to industrial wastes ~1960 Investigations into the bioremediation of synthetic chemicals in wastewaters ~1970 Application in hydrocarbon contamination such as oil spills and petroleum in groundwater (more pollution than the natural microbial processes could degrade the pollutants) ~1980 Investigations of bioremediation applications for substituted organics ~1990 Natural Attenuation of 70 and 90 ~2000 Development of in situ bioremediation; source zone reduction; bioaugmentation ~2003 Genomics era of Bioremediation (Cleaning up with genomics by Derek R.Lovley Nature Reviews 1: 35-44) S: modified from Capiro 2003

11 Types or techniques of bioremediation 1) Ex situ bioremediation: contaminants are treated off site 2) In situ bioremediation: contaminants are treated on site Natural Attenuation (slow process, not complete enough, not frequently occurring enough to be broadly used for some compounds, especially very difficult or recalcitrant substances) Enhanced Bioremediation or Biostimulation is to stimulate/enhance a site s indigenous subsurface microorganisms by the addition of nutrients (amendments) and electron acceptors such as P, N, O 2, C (e.g., in the form of molasses, biochar) Bioaugmentation is necessary when metabolic capabilities of microorganisms are not naturally present. Commercially prepared bacterial strains with specific catabolic activities are added (Novozymes Biologicals is a leader in the isolation and selection of novel microbial consortia, +25,000 characterized strains) Phytoremediation: extraction of soil pollutants by roots and accumulation or transformation by plants, e.g., hyperaccumulators

12 The advantages of bioremediation over other technologies permanence contaminant is degraded potentially low cost 60-90% less than other technologies (No additional disposal costs) Low maintenance Does not create an eyesore Capable of impacting source zones and thus, decreasing site clean-up time S: Pearson education

13 Economics of in-situ vs. ex-situ remediation of contaminated soils Cost of treating contaminated soil in place $80-$100 per ton Cost of excavating and trucking contaminated soil off for incineration is $400 per ton. Over 90% of the chemical substances classified as hazardous today can be biodegraded. S: Pearson education

14 Contaminants Potentially Amenable to Bioremediation Readily degradable fuel oils, gasoline Somewhat degradable creosote, coal tars Difficult to degrade chlorinated solvents (TCE) Generally recalcitrant dioxins ketones and alcohols pentachlorophenol (PCP) some pesticides and herbicides polychlorinated biphenyls (PCB) monocyclic aromatics bicyclic aromatics (naphthalene)

15 Some challenges for bioremediation of pollutants and xenobiotics Pollutants may exist at high, toxic concentrations degradation may depend on another nutrient that is in limiting supply Xenobiotics microbes may not yet have evolved biochemical pathways to degrade compounds may require a consortium of microbial populations

16 Fundamentals of biodegradation reactions Aerobic bioremediation Microbes use O 2 in their metabolism to degrade contaminants Anaerobic bioremediation Microbes substitute another chemical for O 2 to degrade contaminants Nitrate, iron, sulfate, carbon dioxide, uranium, technicium, perchlorate Cometabolic bioremediation microbes do not gain energy or carbon from degrading a contaminant. Instead, the contaminant is degraded via a side reaction

17 Bioremediation involves the production of energy in a redox reaction within microbial cells: an energy source (electron donor), an electron acceptor, and nutrients.

18 Electron Donors Alcohols and acids Almost any common fermentable compound Hydrogen apparently universal electron donor, but no universal substrate Hydrocarbon contaminants Surfactants Etc.

19

20 Metabolism of a Pollutant-degrading Bacterium ACETATE *Benzoate *Toluene *Phenol *p-cresol *Benzene ATP Fe(III) *U(VI) *Co(III) *Cr(VI) *Se(VI) *Pb(II) *Tc(VII) CO 2 *CCl 4 *Cl-ethenes *Cl-aromatics *Nitro-aromatics Fe(II)

21 How Microbes Use the Contaminant Contaminants may serve as: Primary substrate enough available to be the sole energy source Secondary substrate provides energy, not available in high enough concentration Cometabolic substrate fortuitous transformation of a compound by a microbe relying on some other primary substrate (Cometabolism is generally a slow process). Bacterium uses some other carbon and energy source to partially degrade contaminant (organic aromatic ring compound)

22 Genetic engineering of bacteria to remove toxic metals from the environment New gene/transport proteins E. coli bacterium Hg 2+ Hg 2+ -metallothein Hg 2+ Hg o New gene/enzyme Hg o (less toxic form of metal)

23 Phytoremediation 350 plant species naturally take up toxic materials Sunflowers used to remove radioactive cesium and strontium from Chernobyl site Water hyacinths used to remove arsenic from water supplies in Bangladesh, India S: Pearson education

24

25 Phytoremediation Drawbacks Only surface soil (root zone) can be treated Cleanup takes several years

26 7. Give products with economical value (woods, NTFP) Comparison between phytoremediation and soil excavation to restore mine site Technology Advantages Disadvantages 1. Expensive 1. Quick restoration 2. Inefficient on large areas Soil excavation (immediate) 3. Less aesthetic 2. Effective on limited areas 4. Very destructive 1. Returns site to its aesthetical value 2. Less expensive Phytoremediation 3. Less destructive 4. Ecological and sustainable method 5. Supports biodiversity 6. Allows carbon sequestration 1. Slow restoration 2. Additional cost needed for biomass storing for sites contaminated by dangerous products

27 Conclusions Many factors control biodegradability of a contaminant in the environment Before attempting to employ bioremediation technology, one needs to conduct a thorough characterization of the environment where the contaminant exists, including the microbiology, geochemistry, mineralogy, geophysics, and hydrology of the system Most organics are biodegradable, but biodegradation requires specific conditions: important to understand the physical and chemical characteristics of the contaminants of interest There is no Superbug: understand the possible catabolic pathways of metabolism and the organisms that possess that capability (functional genomics and specifically metabolomics)

28 Conclusions Contaminants must be bioavailable and in optimal concentrations Biodegradation rate and extent is controlled by limiting factors : ph, temperature, water content, nutrient availability, Redox Potential and oxygen content Understand the environmental conditions required to: Promote growth of desirable organisms Provide for the expression of needed organisms Engineer the environmental conditions needed to establish favorable conditions and contact organisms and contaminants

29 Part II: Canadian case study of phytobial remediation in the oil sands industry

30 Map of Canada

31

32 Canadian Natural Syncrude Albian Sands Syncrude Suncor Energy

33 Profile of oil sands deposit Overburden Oil sands

34

35

36 Oil extraction

37 Extraction Process Tar Sands Bitumen Tailings

38 Bitumen

39 Tailings Discharge to Storage Pond

40 Materials Requiring Reclamation Overburden storage Tailings sand Soft tailings Coke storage

41 Tailings sands and tailings water High ion content (eg. Na + ) Alkaline ph Nutrient depleted Residual hydrocarbons, NAs

42 Athabasca River at Fort McMurray erosion and run-off of bitumen continuous supply of hydrocarbons to river good source of natural hydrocarbon degraders potential source of obligate hydrocarbon degraders

43 Alders: Pioneer species Frankia sp.: N-fixing actinomycete

44 Definitions Actinorhizal plants (200 species, 25 genera) alder shrub and tree species (Alnus) are pioneer species colonizing very poor substrates Aulne rugueux (A. rugosa) Parc Forillon, 2002

45 Host Plants The symbiosis root nodulation similar to leguminous plants fixation rate ( kg / ha*year) Alnus crispa Alnus crispa, AVCi1

46 Growth and Inoculation of Alders in Greenhouse

47 Frankia inoculated (right) and non-inoculated control (left) green alder seedlings planted in oil sands areas in Spring The photos were taken in Fall 2007 after three growing season.

48 Frankia-inoculated alder (A. crispa)

49

50 Effects of Frankia inoculation on plant height (Height), root collar diameter (RCD), and stem volume of green alder outplanted on Syncrude W2 site (relatively better reclaimed site) after three growing seasons. Each value is the average of 24 seedlings (6 seedlings/replication).

51 Effects of Frankia inoculation on plant height (Height), root collar diameter (RCD), and seedling volume rate of green alder outplanted on Syncrude saline-alkaline harsh (Cell 46) after three growing seasons. Each value is the average of 24 seedlings (6 seedlings/replication).

52 Effects of Frankia inoculation on stem volume of green alder outplanted on Syncrude saline-alkaline harsh site (cell 46) and W2 site after three growing seasons. The data shows percent (%) increase in mean stem volume over control treatment on both sites

53 Hexadecane Hydrocarbon mineralization in bulk field soil planted with alders Naphthalene Phenanthrene

54 Hexadecane Hydrocarbon mineralization in the rhizosphere of alder planted soil Naphthalene Phenanthrene

55 alkb PCR in field plants

56 Detection of Frankia in endophytic community

57 DGGE Analysis of Rhizospheric and Endophytic Microbial Communities From Frankia-inoculated (F) and Non-Inoculated (C) Alders Arrow: Frankia

58 Conclusions and future prospects 1. The study of root symbioses in phytoremediation will help understand their sensitivity, tolerance and coadaptation capacity under stress 2. The biotechnology of root symbioses on contaminated soils will help understand and measure their impact on: a) the microbial density and diversity in the rhizosphere, b) the microbial degradation of contaminants, c) the global soil quality (phytotoxicity, nutrients).

59 Conclusions and future prospects 3. Appropriate experimental approaches are needed to assess the potential of these microorganisms in the management of disturbed soils following industrial activity. 4. Methagenomics, functional genomics (proteomics, transcriptomics and metabololomics) approaches will revolutionize the traditional studies of soil microbial ecology and de novo bioremediation

60 Acknowledgements Members of the Biomonitoring and Remediation Groups, EME, NRC-Montreal

61 THANK YOU VERY MUCH FOR YOUR ATTENTION 61

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