Molecular detection of microbial pollutant-biodegrading genes in environmental samples is useful in assessing the potential bioremediation activity of a particular soil. Beller et al. (2002) developed a real-time PCR method to quantify hydrocarbon-degrading bacteria in sediment samples. A gene coding for the alpha-subunit of benzylsuccinate synthase, bssA, a catabolic gene involved in toluene and xylene degradation, was used as a target. The real-time PCR method was sensitive over seven orders of magnitude, and was used to study the changes in a toluene-degrading bacterial community. Microcosms were incubated anaerobically with BTEX (benzene, toluene, ethylbenzene, and xylenes) and NO- in thepresenceandabsenceofethanol. Microcosms with the most rapid toluene degradation also had the highest numbers of bssA copies. In microcosms with rapid toluene degradation, numbers of bssA copies increased 100- to 1000-fold over the first 4 days of incubation.
Catechol 2,3-dioxygenase genes have also been used as indicators for biodegradation ability of compounds such as benzene, toluene, xylenes, phenol, naphthalene, and biphenyl (Mesarch et al. 2000). This technique can be used to accurately and reproducibly quantify catechol 2,3-dioxygenase genes in complex environments such as petroleum-contaminated soil.
The distribution of genes responsible for the degradation of sulphur-containing hydrocarbons was studied in soil samples contaminated with sulphurous oil (Duarte et al. 2001). Genes that encode enzymes involved in the desulphurisation of hydrocarbons, i.e. dszA, dszB, and dszC, were present in all contaminated soil samples, whereas a range of unpolluted soils gave negative results.
Novel hydrocarbon-degrading genes belonging to the class II aromatic ring-hydroxylating dioxygenase (RHD) gene family were detected in soils using PCR (Taylor et al. 2002). This suggested that distinct groups of novel aromatic hydrocarbon-degrading bacteria exist in soils.
Phenol-degrading genes have been detected in TC-DNA (Watanabe et al. 1998). A gene encoding the largest subunit of multi-component phenol hydroxylase (LmPH) was amplified from phenol-digesting activated sludge. Two major LmPH gene bands became apparent after the activated sludge became acclimated to phenol.
Herbicides such as atrazine are common pollutants; the degradation potential of two wetland sites was assessed by PCR and Southern hybridisation to detect a gene coding for atrazine chlorohydrolase (Anderson et al. 2002). Sediments from constructed wetlands receiving agricultural run-off tested positive for atzA correlating with observed atrazine mineralisation. Sediment from a natural fen that did not show a high level of mineralisation was negative for atzA using PCR and Southern hybridisation. This study indicates that the detection of specific degradation genes in TC-DNA is a good predictor of degradation activity
In addition to bioremediation of organic compounds, microorganisms have also been implicated in the bioremediation of heavy metals such as selenium, uranium, cadmium and mercury. Metal remediation can be achieved by precipitation and immobilisation of contaminants (microbes can reduce metal which can result in detoxification and precipitation; e.g. mercury is taken up into the cell and delivered to the NADPH-dependent flavoenzyme mercuric reductase, which catalyses the reduction of Hg2+ to volatile, low-toxicity HgO; Nascimento and Chartone-Souza 2003), by biosorption (passive sequestration by interaction with live or dead biological material; bacteria can be genetically engineered to incorporate metal-binding polysaccharides in their cell surface, or to enhance metal transporters with metal binding metallothioneins in the cytoplasm), or by biomineralisation (formation ofinsoluble metal precipitates by interactions with microbial metabolites thus achieving concentration and reduction in volume of contaminants). A review of this topic was published by Barkay and Schaefer (2001). It is necessary to keep in mind that not all microbial transformation must necessarily result in reduction of toxicity. For example, environmental production of methylmercury is highly undesirable as this represents the most toxic and readily accumulated form of mercury (National Research Council 2000). Success of any microbial transformation also depends on various factors including catabolic capabilities and physiology of the organism, on environmental conditions, nature of contamination, and on societal and economical considerations. The majority of studies on genes involved in remediation have been on bacterial isolates, with very few attempts at in situ detection in complex environments such as soil. One study on mercury resistance in freshwater illustrated a 29-fold increase in resistance genes in water enriched with mercury compared to control samples (Barkay et al. 1989).
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