Biotransformation

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Biotransformation

Biotransformation is the chemical alteration of compounds by living things; in particular, the microbial alteration of anthropogenic xenobiotics. Xenobiotics are chemicals made by humans that are not formed by 'natural' processes. It may also include 'natural' compounds produced primarily by human beings. The word means 'foreign to life'. Xenobiotics include such infamous things as:

Xenobiotics are often polycyclic, halogenated, highly reduced and lipophilic. As a result of the latter property, some can bioaccumulate up food chains. Because the liver (in mammals) cannot break many xenobiotics down fully, they accumulate in fat. Fats ingested by carnivores contain xenobiotics, which they then accumulate themselves. And so on up the foodchain. Top predators can receive very damaging doses of xenobiotics from their food, despite the environmental concentrations being quite low. Many xenobiotics are highly toxic. All are 'unusual' so far as ordinary metabolic processes are concerned, because they are generally not things that Life has been exposed to in its 3500 million year history. However, they are not that unusual: often, xenobiotics are degraded by enzymes that act on something else that occurs naturally. This is usually something chemically similar (for some definition of similar). e.g. polyaromatic hydrocarbons can be degraded by Phanerochaete, a white rot fungus, which usually degrades lignin, which is chemically somewhat similar.

Lignin is a highly reduced polymer of substituted benzene rings.
Lignin.

Polyaromatic compounds also contain benzene rings
Benzo[a]pyrene.

Xenobiotic compounds often sequester in (adsorb to) soil. Although they disappear from the aquatic phase, they have not been degraded, they have merely concentrated into a phase (such as sediment) in which they are not very available for analysis or degradation. PCP, DDT, etc., have very long half lives because they are not bioavailable.

Xenobiotics are frequently degraded by oxidases and dehalogenases. This probably reflects the nature of the compounds humans tend to synthesise, rather than anything more profound.

PCP is polychlorinated.
Chlorinated (dehalogenase required).

Benz[a]anthracene is highly reduced.
Highly reduced (oxidase required).

Polyaromatic hydrocarbons (PAH) are a well understood group of xenobiotics. They are formed naturally by burning, and humans make even more of them by burning fossil fuels. Creosote (coal-tar) is 90% or more PAH. Low RMM ones are acutely toxic, and high RMM ones are carcinogenic.

Pyrene is named after its chief source- burning.

Phanerochaete chrysosporium is a fungus that causes white rot. The wood goes white as it rots because lignin (which is brown-ish) is destroyed. It is a basidiomycete, and degrades lignin to access the cellulose in the wood as a food source. White rots are the main organisms involved in the recycling of lignin in the environment. Note the chemical similarity between lignin and PAH shown above. Like lignin, PAH can be partially degraded by ligninase. Ligninase contains a cytochrome-P450 that has extremely high oxidising power. It removes electrons from veratryl alcohol (VA) and dumps them onto peroxide. VA+ radical ions then pull electrons out of aromatic rings.

Ligninase degrades lignin by generating highly oxidising free-radicals.

VA pulls electrons out of PAH rings, generating quinones by nucleophilic attack.

Veratryl alcohol oxidises lignin-like compounds.

Bacteria can also actively metabolise PAH < 4 rings, by dioxygenation followed by ring cleavage. Aliphatic bits can be metabolised by Krebs cycle.

Dioxygenation leads to ring cleavage and production of organic acids that can be metabolised easily.

Monooxygenation of BAP by mammalian liver enzymes produce an extremely carcinogenic epoxide. This epoxide can form an adduct with DNA, causing mutation.

BAP monooxygenation yields a carcinogenic epoxide.

Aldicarb (an insecticidal carbamate) is also transformed to even more toxic compounds by oxidation.

Aldicarb sulfone is more toxic than the parent compound.

Clearly, ligninase was not 'designed' to degrade PAH. Likewise, the degradation of other xenobiotics is a happy accident that may provide a selective advantage. Bacterial degradative enzymes are often plasmid-borne, and can transfect into other bacteria.

An interesting example of evolution being caught in the act is that of pentachlorophenol dechlorination. Pentachlorophenol is a very toxic chlorinated phenol. It is degraded by successive dechlorination to form hydroquinones. Seemingly unrelatedly, the synthesis of tyrosine requires a glutathione (GSH)-mediated cis/trans isomerase. This enzyme also catalyses the GSH- reductive dechlorination of tetrachlorohydroquinone.

PCP is degraded by successive dehalogenation.

Some bacteria can even use chlorinated aromatics as terminal electron acceptors. Tetrachloroethene (TCE) is used as TES by Dehalococcoides ethenongenes.

R-Cl + 2e− + 2H+ → R-H + HCl.

Another important group of xenobiotics are the quaternary ammonium compounds (QACs), used in detergents. Molecularly, these are like aberrant membrane lipids, with a long fatty acid chain and a polar head group. When degraded, the head group is cleaved, and the tail is degraded by β-oxidation, just like fats.

Benzalkonium chloride - click for Jmol version
Benzalkonium chloride is a quaternary ammonium compound found in many antiseptic creams.

Evolution loves a preadaptation.

Bacteria in biotransformation

Bacterial biotransformation can be useful for:

Bacteria that degrade xenobiotics can be isolated using enrichment culture. This provides the xenobiotic as the sole source of an element (usually carbon). This selects for bacteria able to metabolise the chemical, over those that just modify or resist it.

Bacterial degradation can (somewhat arbitrarily) be divided into metabolic vs. cometabolic activity. Metabolic degradation actually uses the xenobiotic as a source of carbon, nitrogen, energy, or some combination of the above. Cometabolic degradation is 'fortuitous' transformation that does not lead to energetic/nutritional gain. Cometabolic degradation can still be adaptive, as it may reduce local toxicity.

IPBC

An example of cometabolic degradation is the degradation of the wood preservative IPBC.

IPBC is a wood preservative containing a terminally iodinated propynyl group.

IPBC appears to be degraded cometabolically by bacteria: the bacteria cannot survive on IPBC as a sole source of carbon, and degradation appears to be limited to reductive dehalogenation.

Certain bacteria turn up time and time again in these degradation studies.

Fecund gene transfer, enzymatic competence and thick cell walls are probably important in some or all of these bacteria. They are also (sometimes facultatively) aerobic, and easy to culture on Luria, bringing up the unfortunate question: Is what we see in term of bacterial biotransformation more to do with the conditions in a microbiology lab than reality?

Sometimes, several bacteria will acts together to degrade a compound. This is a form of cross-feeding. Selection is for a consortia of genes across several organisms (Richard Dawkins would like this: the individual organisms are irrelevant, so long as between them, the correct genes are present).

Atrazine is degraded by a consortium of bacteria.









Pseudomonas ATZ1 does this bit.









And this bit.








Pseudomonas CN1 does this bit, and mineralises the cyanurate produced.



Clavibacter degrades the amines.

Bioremediation

Bioremediation is the use of microorganisms to remove chemical pollutants from an area, or (equivalently), the human manipulation of environmental biotransformation.

It is easier to remediate point sources of pollutants, such as contaminated soil near a chemical plant or oil-spills and other accidental releases. It is more difficult to bioremediate distributed sources, which must be collected for remediation, such as pallet boards and sewage waste.

There are many targets for bioremediation. these include soil contaminated with pesticides, biocides, radioisotopes, heavy metals, or hydrocarbon (oil) pollution; water contaminated with any of the above; and sewage.

There are many reasons we might want to clear up a contaminated area: cleaning up contaminated industrial areas so they can be used for other purposes; removing toxins from the groundwater meeting human needs, and the recovery of fibres from preserved wood for paper making. These are all essentially forms of recycling: recycling wood, land or water.

There are essentially to approaches to bioremediation. Ex situ bioremediation is said to occur when we excavate contaminated soil (or pump out contaminated water), then clean it up in a bioreactor. In situ bioremediation is said to occur when we encourage microbial biotransformation in soil or water without moving it elsewhere. The intrinsic rate of degradation is that achieved without human help. The augmented rate of degradation is that achieved with human intervention to encourage the bioremediation.

In situ techniques

Ex situ techniques

This generally involves a bioreactor, which is a vessel in which biotransformation occurs. This does not necessarily need to be very high tech.

Note that these two approaches are not exclusive: in situ may be used to treat contaminated soil, whilst ex situ is used to treat associated groundwater contamination.

Combined in situ soil remediation and ex situ water remediation.

An important consideration of how we encourage bioremediation is whether the degradation of the xenobiotics in question proceeds most quickly under aerobic or anaerobic conditions. PCP is degraded by reductive dehalogenation and this proceeds better under anaerobic conditions. Anaerobic conditions may be encouraged by the addition of electron acceptors. Degradation of DCP (dichlorophenol) proceeds better under aerobic condition, which may be encouraged by sparging with air.

Case studies

Intrinsic - remediation of heavy metals

Bacteria can be used to remove metal contaminants from the acidic effluent streams of metal mines in situ. Sulfate-reducing bacteria reduce sulfate to sulfide, and metal ions are precipitated as sulfide salts. The pH increases as sulfuric acid is removed.

Bioaugmentation - oil pollution

When Exxon Valdez spilt in Prince William Sound, bioaugmentation by Inipol EAP-22 was used. This consisted of a mixture of urea, oleic acid and tris-(laureth-4-)phosphate. This encouraged the natural in situ flora on beaches, and increased degradation rates 5-fold over the intrinsic.

Bioaugmentation - oil slick surfactants

Surfactants can be used to increase the solubility of oil in situ, but this has had mixed results. The increased surface area may lead to increased degradation rate, but the decreased attachment to particles any decrease the degradation rate.

Inoculation - Rhodococcus and PCP

In vitro studies indicated Rhodococcus chlorophenolicus could degrade PCP very rapidly. This bacterium can be inoculated into PCP-contaminated soil, and mineralisation of PCP (conversion to inorganic CO2, H2O and Cl−) can be demonstrated to occur in situ.

Inoculation - Pseudomonas and DCP

However, other studies have been abject failures. Pseudomonas can degrade dichlorophenol readily in culture, but it cannot do this in soil (in situ). It appears that there are many factors besides enzymatic competence that determine whether inoculation will succeed. These include:

Phytoremediation

Some plants sequester heavy metals in their vacuoles using malate. You can grow the plants to remove the metals in situ, and burn the plants to recover the metals. You can even use this to 'mine' the soil for metals.

Lead is sequestered in the vacuole by malate.

Composting - explosives waste

This is an example of high temperature bioremediation: soil is excavated and treated ex situ in a big compost heap. Chlorophenols and nitroarene explosives degrade well in composting systems with Pseudomonas. The system requires aeration: turning or pumping.

Composting - Phanerochaete and PCP

This fungus degrades PCP in ex situ composting situations, but not very quickly. Is it possible to use it in situ? It can be encouraged by adding wood substrate to the soil, and seems promising with PAH, but this is still in early stages.

Activated sludge - sewerage

Contaminated water is collected ex situ. Sludge is strongly aerated to encourage growth of aerobic microbes, which degrade faecal waste. This may be adapted for PAH remediation by the inoculation of the sludge with PAH-degradative bacteria.

Biofilters - sewerage

Trickle bed filters in sewerage are a form of these. They have a solid substrate with a biofilm of degradative organisms growing on it. Contaminated water (ex situ) is trickled through. Organic pollutants such as acetone and toluene are degraded. This soil bed can be used to degrade pollutants in groundwater.

Trickle bed filter.

Biofilters - contaminated air and water

Contaminated water may be treated by sparging if it contains volatile toxins. Gas can then be collected and subjected to ex situ treatment by forcing them through a biofilter to remove the pollutants.

Soil bed air filter.

Biofilters - immobilised bacteria

Bacteria may also be immobilised in microparticles and packed into columns. Polluted water can then be trickled through the bioreactor to effect ex situ degradation.

Bacteria may be immobilised in beads and packed into a column.

Fermenters - methanotrophs and TCE

Methanotrophic bacteria can be used to remediate TCE (tetrachloroethene, an industrial solvent) in an ex situ bioreactor. TCE is oxidised cometabolically by methane monooxygenase. Cells are grown in a fermenter, then drip fed into a reactor supplied with TCE-contaminated water and methane. You can also use them immobilised. In situ remediation may be achieved by bioaugmentation with methane.

In conclusion, when planning a bioremediation strategy, you must consider many factors:

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