Exploiting microbes to degrade wastes and xenobiotics

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• Bioremediation

• Exploiting microbes to degrade wastes and
  xenobiotics
• Dr. William Stafford
• wstafford_at_uwc.ac.za

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• Bioremediation is defined as the process whereby
  organic wastes are biologically degraded under
  controlled conditions to an innocuous state, or
  to levels below concentration limits established
  by regulatory authorities.
• Wastewater, Pesticides, herbicides,
  petrochemicals and products (eg. Oil, Plastics,,
  trichloroethene, polychlorobiphenyls (PCBs),
  dioxins dibenzofurans, and heavy metals can all
  persist in the environment.

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Agent orange 2,4 Dichlorophenoxyacetic acid and
2,4,5-Trichlorophenoxyacetic acid
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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 clean up contaminated site

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• In many cases the clean-up of contaminated sites
  has been carried out using physical and chemical
  methods such as immobilization, removal (dig and
  dump), thermal, and solvent treatments.
• Bioremediation is cheaper than the chemical and
  physical options, and can deal with lower
  concentrations of contaminants more effectively,
  although the process may take longer.
• Bioremediation typically uses naturally occurring
  bacteria and fungi or plants to degrade or
  detoxify substances hazardous to human health
  and/or the environment.

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Bioremediation methods

• Use the indigenous microbial population.
• Encourage the indigenous population.
• Bioaugmentation the addition of adapted or
  designed inoculants.
• Use of genetically modified micro-organisms.
• Phytoremediation.

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Bioremediation strategies
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In situ technologies Bioremediation Technologies

• Criteria for in situ bioremediation
• 1) microbes must exist that can biotransform
  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.

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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 1) fertilization to
  achieve optimum CNP, 2) supplementation to
  provide available oxygen (tilling) 3) moisture
  control, 4) inoculation (optional) 5) pH control
  (optional).
• For these treatments, soil typically rests on
  liner system with engineered drainage systems in
  place.

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Phytoremediation

• In natural ecosystems, plants act as filters and
  metabolize substances generated by nature.
• Phytoremediation uses higher plants to remove
  pollutants.
• Processes includes uptake into the plant and
  biodegradation by microorganisms colonizing root
  surfaces and rooting zone (rhizosphere).
• 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.

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Bioventing/Biosparging

• 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 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..

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Biostimulation

• Bioaugmentation
• Bioaugmentation (seeding) has been used some in
  situ in soil and groundwater decontamination. A
  problem is that the nonindigenous microorganisms
  may not well enough with an indigenous population
  to develop and sustain long-term useful
  population levels
• Composting technology
• 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
  (50-60C and higher). Composting has been
  successfully used for chlorophenols such as
  trinitrotoluene (TNT).

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30 VSS destructionAerobic, mostlyPasteurization
(50 to 70 C)Static pile 28 days then 30 day
cureWindrow 30 days, 55 CIn vesselFeed
sludge solids 40
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Ex situ technologies Bioremediation Technologies

• 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.

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• 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.

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• Bioremediation- transformations in detail
• Oxic Degradation of Hydrocarbons
• Microbes that utilize hydrocarbons
• Pseudomonas spp. ,Mycobacterium, Nocardia
• Some yeasts and molds
• Long chain aliphatic hydrocarbons are converted
  into fatty acids by oxidation of a terminal
  carbon (to an alcohol, then an aldehyde, then a
  carboxylic acid). The fatty acids thus generated
  are activated with CoA and simply fed into the
  normal pathway for fatty acid degradation - beta
  oxidation.
• Which of the following (a, b, c, d, e) would be
  recalcitrant to oxic degradation?
• Generally, hydrocarbons that have additional
  groups (e.g. -CH3 , -Cl atoms) at the position
  destined to become the beta-carbon in the beta
  oxidation pathway will be recalcitrant to oxic
  degradation. However, a hydroxyl group is
  favorable at this position since the pathway
  normally generates a beta alcohol anyway. A
  double bond between the alpha and the beta
  carbons is also favorable.

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• Oxic degradation of aromatic hydrocarbons
• Complex aromatic compounds are first converted to
  a "starting substrate" such as catechol. Probably
  using a monooxygenase
• A dioxygenase breaks open the aromatic ring of
  catechol, producing cis,cis-muconate, an
  unsaturated dicarboxylic acid
• This product is then oxidized to acetyl-CoAs by
  the aforementioned beta oxidation path.
  Substitution of aromatic rings (e.g. with -CH3 or
  -Cl) interferes with the oxic degradation of the
  aromatic ring.
• For instance, the herbicides 2,4-D and 2,4,5- are
  aromatic rings, respectively containing two and
  three chlorine substitutions. The additional
  chlorine atom makes 2,4,5-T five-fold less
  degradable compared to 2,4 D.

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Anoxic Degradation of Xenobiotics

• In anoxic environments, since oxygen is not
  available as an electron acceptor, microorganisms
  utilize alternates such as nitrate, sulfate and
  ferric iron. or xenobiotics as another electron
  acceptor.
• Two broad strategies for anoxic degradation
• Reductive dechlorination e.g. the substrate
  3-chlorobenzoate can be dechlorinated by
  reduction of the chlorine atom, producing
  benzoate and chloride anion
• e.g. denitrifiers and sulfate reducing bacteria
  (Desulfomonas).

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Reductive ring cleavage

• E.g. benzoate can be converted to a saturated
  dicarboxylic acid. There are three principal
  steps in the conversion
• (i) Activation - ATP energy is usred to attach
  CoA to the carboxylic acid group of benzoate,
  producing a reactive mixed anhydride.
• (ii) Reduction - NADPH is used to reduce the
  carbons within the aromatic ring to produce
  hexanyl-CoA
• (iii) Ring cleavage - carbon 2 of the hexanyl
  ring is oxidized (to an alcohol, then a
  ketone, then a carboxylic acid), leading to
  the cleavage of the ring system.
• (iv) The saturated dicarboxylic acid product
  is oxidized to acetyl-CoAs by the
  beta- oxidation pathway.

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• New possibilities

• Bioremediation Rocks JAMAIS CASCIO DEC12 2003
• Geobacter sulfurreducens -- get used to seeing
  that name. It may well be the key to cleaning up
  some of the most dangerous radioactive wastes
  sites around. Best of all, it's completely
  natural.
• G. sulfurreducens is a microbe that is able to
  turn the soluble form of uranium contaminating
  groundwater around nuclear weapons production
  sites (such as Rifle Mill in Colorado) into an
  easily-collected precipitate. Researchers with
  the Department of Energy have managed to use the
  bacteria to reduce uranium in the groundwater
  around Rifle Mill by 90. The microbe occurs
  naturally in the ground its growth is stimulated
  by adding vinegar to the soil.
• But now, biologists at The Institute for Genomic
  Research (TIGR) in Rockville, Maryland, have
  sequenced the microbe's DNA, figuring out how it
  manages to detect and "eat" uranium, producing
  minute amounts of electricity. Their report is in
  today's edition of Science the illustration at
  right is from their online supporting material.
  TIGR and University of Massachusetts in Amherst
  researchers believe that they will be able to
  manipulate the microbe's genome to make its
  uranium-electricity conversion faster and more
  efficient.

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