BIOREMEDIATION

BCH 404- BIOREMEDIATION

ASSIGNMENTS  #1, #2 and #3.

        Solutions

1.     Phytoextraction and phytoaccumulation is explained as the uptake of contaminants by plant roots and accumulation and movement  of the contaminants from the roots to above ground parts of the plant. Contaminants are generally removed from the sites by harvesting the plants.

2.     Phytostabilization occurs through contaminant accumulation in plant tissue and in the soil around the roots because of changes in the chemistry of the contaminants, which become insoluble and/or immobilized on soil components.

          Phytostabilization also refers to establishing a plant cover on the surface of the contaminated soil or sediment ,which greatly reduces exposure of the soil or sediment to wind, water and direct contact with humans or animal.

3.     Phytotransformation  is the uptake of organic contaminants from soil, sediments, or  water and  subsequently, their  transformation to more stable, less toxic or less mobile form.

4.     Rhizofiltration is a water remediation technique that involves the uptake of contaminants by plant roots.Rhizofiltration is used to reduce contamination in natural wetlands and estuary areas.

5.     Phytovolatilization is a technique whereby plants evaportranspirate selenium, mercury volatile hydrocarbons.

Question  2

2. Certain plants are able to extract hazardous substance such as arsenic, lead and uranium from soil and water. Explain in detail what is  meant by hyperaccumulation of Alpine pennycress and bracken fern. Name the specific metals in question

Solution

Hyperaccumulation  in  plants  involves  the  extraction  and  accumulation  of  larger  than  normal  metals  or  other  hazardous  substances from  the  soil  and water  into the  plant’s  roots  or other  parts.The  plants absorb  contaminants  through the  root  system  and  store  them  in the  root  biomass and/or  transport them  up  into  the  stems  and/or leaves..Alpine pennycress, a plant which naturally accumulates high levels of cadmium and zinc from the environment. Alpine pennycress is therefore known as a hyperaccumulator of these metals, which in unnaturally high levels would be poisonous to many plants.

Bracken fern extracts arsenic from the soil at a much greater rate than other plants. This arsenic is stored in the fern's leaves at as much as 200 times that present in the soil.

3) Outline a bioremediation process by which you would completely degrade tetrachloroethylene.  Justify your choice.

Solution

This complex compounds cannot be broken down to basic molecules easily.Addition of functional groups such as hydroxyl groups-(OH) to make it more polar. To degrade tetrachloroethlene completely, both anaerobic and aerobic processes are involved, first, an anaerobic step that is reductive dechlorination to remove some of the chlorines.

Tetrachloroethylene would act as the electron acceptor; an electron donor such as organic compound, H₂ is needed to be supplied.

This also reduces CO₂ to CH₄, which can be used in the aerobic step.

Secondly an aerobic step involves

Methanotrophs would use methane produced in the first step as their carbon and energy source, and would produce the enzyme methane monooxygenase.

Methane monooxygenase is non-specific and will degrade lightly chlorinated compounds like trichloroethylene produced in the first step.

This process might need to be supplied with methane and  oxygen.

4) (a) Starting with a soil contaminated with pentachlorophenol (PCP) and polycyclic aromatic hydrocarbon(PAH)-containing creosote, describe how you would obtain a microbial consortium which can rapidly degrade PCP and PAHs.  This consortium is to be used in a bioreactor in a bioaugmentation treatment of this soil.

Solution

Bioreator  method  involve removal of contaminants from the soil .There is  a continuous supply of nutrients, oxygen and microorganisms to the bioreator.

Landfarming is an aerobic process.  Therefore you would not expect reductive dechlorination.  Instead, oxygen would be added to the ring to  break open the ring, and PCP would be degraded to CO₂, H₂O, biomass, and Cl^- ions.

Mineralization experiment is done to verify that PCP and PAH is broken or minerilzed. Radio label  PCP  and put it in your microcosm along with required nutrients etc.  You would have a base trap (with KOH etc), which would trap the CO₂.  You would then analyze your base for radioactivity to determine whether mineralization was occurring.  You would also need an abiotic control to verify that the radioactivity in the traps is not just from volatilization.

(b) Design an experiment to determine whether (i) bioaugmentation is better than biostimulation and (ii) whether PCP and PAH disappearance is due to microbial activity.

Solution

(i)    Set up a series of microcosms, each with a soil sample in water:

Bioaugmentation test bottle; inoculate with culture obtained from enrichment process

Biostimulation test bottle; add  nutrients

Control; add nothing but Note: there will still be microbes present naturally in the soil, and whatever nutrients naturally occur in the soil as well

To determine whether bioaugmentation or biostimulation improves degradation, monitor concentration of contaminants with time.

(ii) To determine whether PCP and PAH disappearance is due to microbial activity:

Set up a mineralization experiment with radiolabelled PCP and PAH provided.  If radioactivity ends up in the base, you have mineralized the contaminant

Need an abiotic control to ensure that radioactivity in the base is not a result of volatilization.

In order to ensure that both PCP and PAH are being mineralized (and not just one or the other) could set up two test bottles, providing both PCP and PAH to each, but only radiolabelling one of the two. This would allow you to differentiate between CO₂ produced from PCP and CO₂ produced from PAH.

5) The following are the unbalanced equations that describe the energy and cell formation processes that occur during biological denitrification.

I C₆H₁₂O₆ +  H₂O  à  CO₂   +  H⁺  +  e^-

II NO₃ + H⁺  +  e^-  à  N₂  +  H₂O

III NO₃  +  CO₂  +  H⁺  +  e^-  à  C₅H₇O₂N  +  H₂O

I balance the equations

II pseudomonas bacteria are involved in the above dentrification  process. Explain in detail all the major  processes the bacteria undertake during the dentrification process.

Solution

I  C₆H₁₂O₆  + 6 H₂O  à 6 CO₂   +24H⁺  + 24e^-

II 2NO₃⁻ + 10e⁻ + 12H⁺ → N₂ + 6H₂O

III NO₃  +  5CO₂  +  H⁺ + e^- à  C₅H₇O₂N  +  7H₂O

Solution

II Pseudomonas bacteria transform nitrate into nitrogen gas as part of their metabolism. These bacteria use a carbon source (e.g. sugar) and nitrate both for energy and to build new cells. This means that some of the carbon in sugar and nitrogen in nitrate is incorporated into bacterial cells and some of the carbon and nitrogen is used for the bacteria to obtain energy. Bacteria obtain energy by breaking chemical bonds and forming new ones by “moving around” electrons. During denitrification, pseudomonas use sugar as an electron donor and nitrate as an electron acceptor.

1.     Microbial exudates (other than enzymes) can create a micro-environment in which certain polymers become chemically unstable. For example, sulfur bacteria produce sulfuric acid from sulfide or sulfur. Many fungi secrete organic acids while decomposing plant materials, while plant roots secrete both H+ and HCO3 - during the uptake of nutrients. Explain briefly how the above processes can influence polymer degradation.

Answer

             If these processes occur in the vicinity of acid - or base - susceptible polymers, they may increase the degradation rates of the polymers.

2.     Degradation of all polymers follows a sequence in which the polymer is first converted to its monomers, after which the monomers are mineralized. Why is it necessary for the conversion of the polymer to monomers interms of the polymer degradation within microbial cells­­.

Answer

 Most polymers are too large to pass through cellular membranes, so they must first be depolymerized to small monomers before they can be absorbed and biodegraded within microbial cells.

3.     Explain briefly the following i) Abiotic hydrolysis ii) Abiotic oxidation of polymer degradation, Specifying the type of polymer (give at least two examples) natural or synthetic been degraded under each of the processes.

Answer

i)                    Abiotic hydrolysis is the most important reaction for initiating the environmental degradation of synthetic polymers, hydrolysis acts as the initial step of splitting the polymer into its monomers, after which the monomers can be biodegraded.

             Examples include polycarboxylates, polylactic acids and silicones.

ii)                Abiotic oxidation can also initiate the degradation of some polymers. For example, polyethylene    undergoes an auto-oxidation, which gradually reduces its molecular weight to the point where biodegradation can proceed. Air pollutants such as ozone, nitric oxides and sulfuric oxides may also promote abiotic oxidation of polymers. Finally, sunlight which strikes the surface of many polymers will be absorbed, oxidizing the materials and thus beginning their degradation abiotically.

4.     State the conditions under which i) soil microorganisms to rapidly degrade cellulose and starch  ii) fungi  degrade wood

Answer

i)                   A ready supply of nutrients, oxygen, and water are needed for soil microorganisms to rapidly degrade cellulose and starch.

ii)                Fungi  which degrade wood are actually more active under poor nutrient conditions.

5.     Explain in detail the natural degradation of silicon.

Answer

Degradation of silicon follows a sequence in which the polymer is first converted to its monomers, after which the monomers are mineralized, in that it begins with an abiotic hydrolysis of the large polymer to small, water soluble monomers.

The monomer is then either biodegraded by soil microorganisms, the speed of degradation depends on the specific environmental conditions. For example, a ready supply of nutrients, oxygen, and water are needed for soil microorganisms to rapidly degrade, or it evaporates from the soil and should oxidize in the presence of sunlight. The cycle is thus completed: silicone, which is made from pure quartz sand, is eventually returned to the

6.When hydrogen peroxide is used as a source of oxygen, it discriminates in favor of peroxide-tolerant bacteria and against some efficient co metabolizers like methanotrophs. Why?

SOLUTION

Methanotrophs are bacteria that are able to metabolize methane as their only source of carbon and energy. They can grow aerobically or anaerobically and require single-carbon compounds to survive.However, hydrogen peroxide used as a source of oxygen increases the oxygen and assist in cleaning greatly. It releases too much oxygen too quickly for aerobic microorganism for biodegradation that is in situ.

When hydrogen peroxide is used as a source of oxygen, peroxide-tolerant bacteria are favoured, these are aerobic bacteria that use oxygen to become active, hence able to degrade the contaminants. Cometabolizers like methanotrophs do not use oxygen but require methane for degradation, so when hydrogen peroxide is used as a source of oxygen it affects methanotrophs degradation.

7.Explain in details what is meant by

i.                   Direct metabolism

ii.                 Aerobic co metabolism under aerobic mechanism of degradation of some chlorinated compounds.

SOLUTION

Direct metabolism;

Microorganisms use a wide range of metabolic pathways to harvest energy from their environment. In some cases, pollutants serve as the carbon and energy source for microbial growth, while in other cases; pollutants serve as the terminal electron acceptor. This manifests itself in the diverse ability of microbes to transform and degrade toxic molecules.

Direct metabolism is a mechanism of biodegradation in which chlorinated compounds are metabolized directly by microorganisms. They oxidize the carbon and excrete the chlorine as inorganic chloride. This is fairly uncommon, because there is little or no energy to be gained by oxidizing a carbon-chlorine bond.

Moreover direct metabolism also described as anaerobic reductive dechlorination is a biodegradation reaction in which bacteria gain energy and grow as one or more chlorine atoms on a chlorinated hydrocarbon are replaced with hydrogen. In that reaction the chlorinated compound serves as the electron acceptor and hydrogen serve as the direct electron donor.   Hydrogen used in the reaction typically is supplied indirectly through the fermentation of organic substrates.

i.                   Aerobic co metabolism under aerobic mechanism of degradation of some chlorinated compounds

A microorganism that lives on a nonchlorinated organic compound produces an enzyme that happens to break down the chlorinated compound. The microbe gains nothing by the dechlorination; the process works because the microbe is inefficient: Aerobic Co metabolism is a biodegradation reaction in which chlorinated hydrocarbon is fortuitously degraded by an enzyme or cofactor produced during microbial metabolism of another compound. In such case, biodegradation of the chlorinated compound does not appear to yield any energy or growth benefit for the microorganism mediating the reaction.

Under Aerobic conditions several different types of bacteira including methane oxidizing bacteria, ammonia oxidizing bacteria and some phenol utilizing bacteria can cometabolized  or cooxydized trichloethene, dichloethene, and vinyl chlroride. In general cometabolism of chlorinated ethenes is mediated by monoxygenase enzymes with relaxed specificity that oxidized a primary substrate and cooxidized the chlorinated compound. In the presence of methane for example, methanotrophs produce methane monoxygenase which oxydized methane for methanol and  can also cooxydized trichloroethene. For Aerobic cometabolism three key factors must be present; a primary substratek, oxygen, bacteria capable of producing nonspecific monoxygenase. These are biological processses responsible for biodegradation of some chlorinated compounds mentioned above.