General overview of Bioremediation

Damase Khasa

Centre for Forest Research and Institute for

Integrative and Systems Biology, Université

Laval, Québec Canada G1V OA6

Presented during the

Soil Remediation Workshop (With special presentation on

Nextgen sequencing), Pretoria, SA, 27 - 28 May 2014

By

Part I: Bioremediation

Biostimulation

Bioaugmentation

Phytoremediation

Part II: Canadian case study in the

oil sands industry

Outline of presentation

Biostimulation

Bioaugmentation

Phytoremediation

Part I: Bioremediation, An

overview

Pictures depicting worldwide problems

of pollution

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

Sources of contamination

• Industrial spills and

leaks

•Surface

impoundments

•Storage tanks and

pipes

•Landfills and dumps

• Injection wellsModified from Capiro 2003

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

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

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

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 2003. Nature Reviews 1: 35-44)

S: modified from Capiro 2003

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, O2, 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

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

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

Contaminants Potentially Amenable to Bioremediation

____________________________________________Readilydegradable_____________

Somewhatdegradable_____________

Difficult todegrade_____________

Generallyrecalcitrant_____________

fuel oils, gasoline creosote, coaltars

chlorinatedsolvents (TCE)

dioxins

ketones andalcohols

pentachloro-phenol (PCP)

some pesticidesand herbicides

polychlorinatedbiphenyls (PCB)

monocyclicaromatics

bicyclic aromatics(naphthalene)

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

Fundamentals of biodegradation reactions

• Aerobic bioremediation

• Microbes use O2 in their metabolism to degrade

contaminants

• Anaerobic bioremediation

• Microbes substitute another chemical for O2 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

Bioremediation involves the production of energy in a

redox reaction within microbial cells: an energy source

(electron donor), an electron acceptor, and nutrients.

Electron Donors

• Alcohols and acids

• Almost any common fermentable compound

• Hydrogen apparently universal electron donor,

but no universal substrate

• Hydrocarbon contaminants

• Surfactants

• Etc.

ATP

ACETATE

CO2

Fe(III)

Fe(II)

Metabolism of a Pollutant-degrading Bacterium

*Benzoate

*Toluene

*Phenol

*p-Cresol

*Benzene

*U(VI)

*Co(III)

*Cr(VI)

*Se(VI)

*Pb(II)

*Tc(VII)

*CCl4

*Cl-ethenes

*Cl-aromatics

*Nitro-aromatics

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)

Genetic engineering of bacteria to remove

toxic metals from the environment

Hg2+-metallothein

Hg2+→HgoHg2+

New gene/enzyme

New gene/transport proteinsE. coli bacterium

Hgo (less toxic form of

metal)

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

Phytoremediation

• Drawbacks

• Only surface soil (root zone) can be treated

• Cleanup takes several years

Technology Advantages Disadvantages

Soil excavation

1. Quick restoration

(immediate)

2. Effective on limited areas

1. Expensive

2. Inefficient on large areas

3. Less aesthetic

4. Very destructive

Phytoremediation

1. Returns site to its

aesthetical value

2. Less expensive

3. Less destructive

4. Ecological and sustainable

method

5. Supports biodiversity

6. Allows carbon sequestration

7. Give products with

economical value (woods,

NTFP)

1. Slow restoration

2. Additional cost needed for

biomass storing for sites

contaminated by dangerous

products

Comparison between phytoremediation and soil

excavation to restore mine site

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)

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

Part II: Canadian case study of phytobial

remediation in the oil sands industry

Map of Canada

Syncrude

Albian Sands

Suncor Energy

Canadian Natural

Syncrude

Overburden

Oil sands

Profile of oil sands deposit

Oil extraction

Extraction Process

Bitumen Tailings

Tar Sands

Bitumen

Tailings Discharge to Storage Pond

Materials Requiring Reclamation

Overburden storage Tailings sand

Soft tailings Coke storage

• High ion content (eg. Na+)

• Alkaline pH

• Nutrient depleted

• Residual hydrocarbons, NAs

Tailings sands and tailings water

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

Frankia sp.:

N-fixing actinomycete

Alders:

Pioneer species

Definitions

• Actinorhizal plants (200 species, 25 genera)

– alder shrub and tree species (Alnus) are pioneer

species colonizing very poor substrates

Parc Forillon, 2002

Aulne rugueux (A. rugosa)

Host Plants

Alnus crispaAlnus crispa,

AVCi1

• The symbiosis

− root nodulation similar to leguminous plants

− fixation rate (40-300 kg / ha*year)

Growth and Inoculation

of Alders in Greenhouse

Frankia inoculated (right) and non-inoculated control (left) green

alder seedlings planted in oil sands areas in Spring 2005. The

photos were taken in Fall 2007 after three growing season.

Frankia-inoculated alder (A. crispa)

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).

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).

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

Hexadecane

Naphthalene Phenanthrene

Hydrocarbon mineralization in

bulk field soil planted with alders

Hexadecane

Naphthalene Phenanthrene

Hydrocarbon mineralization in the

rhizosphere of alder planted soil

alkB PCR in

field plants

Detection of Frankia

in endophytic community

DGGE Analysis of

Rhizospheric and

Endophytic Microbial

Communities From

Frankia-inoculated (F)

and Non-Inoculated (C)

Alders

Arrow: Frankia

Conclusions and future prospects1. 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).

Conclusions and future prospects3. 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

Members of the

Biomonitoring and

Remediation Groups, EME,

NRC-Montreal

Acknowledgements

61

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