Dorsamy Pillay

Aerobic biodegradation of dichloroethenes by indigenous bacteria isolated from contaminated sites in Africa.

The widespread use of tetrachloroethene (PCE) and trichloroethene (TCE) as dry cleaning solvents and degreasing agents for military and industrial applications has resulted in significant environmental contamination worldwide. Anaerobic biotransformation of PCE and TCE through reductive dechlorination frequently lead to the accumulation of dichloroethenes (DCEs), thus limiting the use of reductive dechlorination for the biotransformation of the compounds. In this study, seven bacteria indigenous to contaminated sites in Africa were characterized for DCE degradation under aerobic conditions. The specific growth rate constants of the bacterial isolates ranged between 0.346-0.552 d(-1) and 0.461-0.667 d(-1) in cis-DCE and trans-DCE, respectively. Gas chromatographic analysis revealed that up to 75% of the compounds were degraded within seven days with the degradation rate constants ranging between 0.167 and 0.198 d(-1). The two compounds were also observed to be significantly degraded, simultaneously, rather than sequentially, when present as a mixture. Phylogenetic analysis of the 16S rRNA gene sequences of the bacterial isolates revealed their identity as well as their relation to other environmentally-important bacteria. The observed biodegradation of DCEs may contribute to PCE and TCE removal at the aerobic fringe of groundwater plumes undergoing reductive dechlorination in contaminated sites.

In vitro screening of sugarcane to evaluate smut susceptibility

Tissue-cultured plantlets of three sugarcane (Saccharum spp.) cultivars having a known field smut reaction were screened for susceptibility to Ustilago scitaminea H&P Sydow. Plantlets were inoculated with 0.5 μ l of a suspension of equally mixed quantities of plus and minus mating type sporidia of U. scitaminea at concentrations ranging from 1 × 101 to 1 × 106 cells. Fungal sori (whips) were produced in cultivar N12 (intermediate) 6 weeks following inoculation with 1 × 105 mixed sporidia and thereafter in cultivar NCo310 (susceptible) but not in cultivar N19 (resistant). Sori bearing teliospores were produced up to 3 months following inoculation and incubation at 26 °C. No sori were produced at mixed sporidial concentrations lower than 1 × 105cells. The in vitro soral production in cultivars N19, N12 and NCo310 was 0, 27.5 and 47.5% respectively. Plantlets inoculated with 1 × 105sporidia of only one mating-type did not produce sori in any of the three cultivars tested. Blind scoring of an unknown sugarcane cultivar by this method corresponded exactly with its field smut rating.

Synergistic utilization of dichloroethylene as sole carbon source by bacterial consortia isolated from contaminated sites in Africa

The widespread use and distribution of chloroethylene organic compounds is of serious concern owing to their carcinogenicity and toxicity to humans and wildlife. In an effort to develop active bacterial consortia that could be useful for bioremediation of chloroethylenecontaminated sites in Africa, 16 combinations of 5 dichloroethylene (DCE)-utilizing bacteria, isolated from South Africa and Nigeria, were assessed for their ability to degradecis- andtrans-DCEs as the sole carbon source. Three combinations of these isolates were able to remove up to 72% of the compounds within 7 days. Specific growth rate constants of the bacterial consortia ranged between 0.465 and 0.716 d−1 while the degradation rate constants ranged between 0.184 and 0.205 d−1, with 86.36–93.53 and 87.47–97.12% of the stoichiometric-expected chloride released during growth of the bacterial consortia, incis- andtrans-DCE, respectively. Succession studies of the individual isolates present in the consortium revealed that the biodegradation process was initially dominated byAchromobacter xylosoxidans and subsequently byAcinetobacter sp. andBacillus sp., respectively. The results of this study suggest that consortia of bacteria are more efficient than monocultures in the aerobic biodegradation of DCEs, degrading the compounds to levels that are up to 60% below the maximum allowable limits in drinking water.

Amplified fragment length polymorphisms reveal genetic differentiation among strains of Xanthomonas albilineans.

Xanthomonas albilineans, the causative agent of leaf scald disease (LSD), colonizes the vascular system of sugarcane (Saccharum spp. hybrids). In this study X. albilineans strains from 28 countries were differentiated by using two methods of amplified fragment length polymorphism (AFLP). In the manual procedure, AFLP reactions were performed on 57 X. albilineans strains and after selective amplification using radiolabelled primers, the resulting products were separated using polyacrylamide gel electrophoresis. The autoradiographs were analyzed using GelCompar version 4.1 software (Applied Maths) to construct dendograms from similarity matrices. Fluorescent AFLP (FAFLP) was also employed on 52 X. albilineans strains using three fluorescently labelled primer combinations (automated AFLP). The FAFLP data was converted to a binary format using the Genemapper Software 3.7 (Applied Biosystems). Thereafter, dendograms were generated using the NTSYSpc. Software (USA). Distinct AFLP profiles were produced for the majority of the strains and were found to be useful in differentiating X. albilineans strains from various geographical locations. Fingerprints unique to each strain were reproducibly obtained and may be used to create a database for use in the identification of the various X. albilineans strains. It can be also concluded from the results obtained that the FAFLP has considerable technical advantages compared with the manual AFLP and also that the FAFLP is more sensitive than AFLP using radiolabelled primers in differentiating X. albilineans.

Characterization of two bacteria isolated from a wastewater treatment plant in South Africa for aerobic dehalogenation of some aliphatic chlorinated compounds

Two bacteria with the ability to utilize some short‐chain aliphatic chlorinated compounds were isolated from a wastewater treatment plant in South Africa and identified as belonging to the genera Pseudomonas and Acinetobacter. Their specific growth rate constants in the compounds ranged between 0.414 and 1.048 d− 1, while the maximum specific substrate utilization rate ranged variously between 25.668 and 57.429 nmol/h/mg of protein. Dehalogenases produced by these organisms demonstrated wide substrate specificities with optimum activities of 285.0 and 345.7 mU/mg protein at a pH levels of 7.5 and 9.0, respectively. This treatment plant could therefore be a good source of organisms that may be applicable for possible bioremediation of sites polluted with this group of compounds.

Genetic sequences derived from suppression subtractive hybridization analysis provides insight into their possible roles in Xanthomonas albilineans

Leaf scald disease (LSD) is caused by the Gram-negative bacterium, Xanthomonas albilineans. Genomic DNA from X. albilineans and Xanthomonas hyacinthi were analyzed by suppression subtractive hybridization (SSH) using X. albilineans as the tester from which unique sequences were sought and X. hyacinthi as the driver. Following the SSH procedure, amplification products within the size range of 100 - 600 bp were generated, purified, directly cloned with the Promega pGEM-T vector cloning kit, and transformed into ultracompetent Escherichia coli X L2-blue MRF’ cells (Stratagene, La Jolla, CA). Clones selected were sequenced (using a Perkin Elmer ABI PRISM Dye terminator cycle sequencing kit and ABI Model 377 DNA sequencer) in one direction with SP6 and T7 primers (Promega). Clone Xa 6 revealed very close homology with a probable bacterioferritin from Pseudomonas aeruginosa. Clone X. albilineans 12 showed 92% homology to the acetate repressor proteins and clone X. albilineans 18 displayed 85% homology to the plasmid pTOM9 from Alcaligenes xylosoxidans. Sequencing data also revealed homology to various hypothetical proteins.