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Abstract
Chlorinated benzenes (CBs) are known carcinogens that have been used as pesticides and as industrial intermediates. Improper disposal practices have turned this group of carcinogens into a major concern for the environment and for human health all over the world. Incineration is the most commonly used technology to treat sites contaminated with these compounds, however, this method implies high operation costs and may also lead to the formation of more toxic compounds like dioxins or furans from hexachlorobenzene (HCB). Therefore biodegradation has become a more suitable, lower cost, environmental friendly technology to address contamination with these compounds.
To date, only two genera of strict anaerobes able to utilize CBs as electron acceptors in an energy yielding process known as organohalide respiration, have been successfully isolated, Dehalococcoides mccartyi CBDB1 and Dehalobacter sp. 12, 13, 14DCB1. The aim of this study was to obtain either mixed or pure native Australian cultures able to reductively dechlorinate CBs, in particular HCB, since Australia holds the largest HCB stockpile in the world. Groundwater from an aquifer contaminated with aliphatic organohalides below the stockpile site was used as a starting inoculum. Under strict anoxic conditions, two different CB degrading cultures were developed.
AusHCB, a lactate-fed mixed culture, was able to reductively dechlorinate HCB extracted from samples of the previously mentioned Stockpile, without exogenous addition of H2. The sole end product of HCB reduction was 1,3,5-TCB in cultures without mechanical agitation. Molecular techniques (DGGE, qPCR and Pyrosequencing) revealed that reduction of HCB to 1,3,5-TCB was driven by an uncultured Dehalococcoides since this was the only organohalide respiring bacterium (OHRB) found in AusHCB. Reducing equivalents necessary for this process came from the fermentation of lactate and citrate. The influence of vitamin B12 (VB12) was tested in AusHCB revealing it was essential for activity. The effect of mechanical agitation on AusHCB to promote mixing in order to increase HCB reductive dechlorination was studied in a Stirred Tank Bioreactor, where the specific dechlorination rate was 9.16x10-11 nmolHCB cell-1s-1 and the resulting concentration of 1,3,5-TCB within the same incubation period as cultures without agitation was 2-fold higher.
A novel Dehalobacter sp. strain TeCB1 was isolated using the same inoculum, when cultures were incubated with direct addition of acetate and H2 and replacing HCB with 1,2,4,5-TeCB, after dilution-to-extinction. Phenotypic characterization of strain TeCB1 showed that this bacterium is a strict hydrogenotroph, of the substrates tested, and could only use 1,2,4,5-TeCB and 1,2,4-TCB as final electron acceptors. Genomic annotation and analysis demonstrated that strain TeCB1 possesses genes that encode for 24 putative reductive dehalogenases. This strain also harbors a full set of genes for uptake hydrogenases, ATP-ase synthase, menaquinone biosynthesis, all of which relate to electron transport. Combining genomic annotation with Blue Native PAGE and LC-MS/MS a novel reductive dehalogenase (RDase) from strain TeCB1 was identified, TcbA. This RDase was able to catalyze the reduction of 1,2,4,5-TeCB to 1,2,4-TCB, 1,2-, 1,3- and 1,4-DCB. Carbon isotopic fractionation during the reductive dechlorination of 1,2,4,5-TeCB by Dehalobacter sp. TeCB1 in cell suspensions appears to be negligible.
The development of AusHCB would have significant relevance to the treatment of HCB stockpile and to other sites contaminated with chlorinated benzenes. On the other hand, the isolation and characterization of Dehalobacter sp. Strain TeCB1 and the identification of TcbA will broaden the knowledge of organohalide respiration.