Identification of metal-tolerant organisms isolated from the Plankenburg River, Western Cape, South Africa

Identification of metal-tolerant organisms isolated from the Plankenburg River, Western Cape, South Africa VA Jackson1,3, AN Paulse1,2, JP Odendaal2, S Khan1and W Khan3* Department of Biomedical Sciences, Faculty of Health and Wellness Sciences, Cape Peninsula University of Technology, Bellville 7535, South Africa Department of Environmental and Occupational Studies, Faculty of Applied Sciences, Cape Peninsula University of Technology, Cape Town 8000, South Africa 3 Department of Agricultural and Food Sciences, Faculty of Applied Science, Cape Peninsula University of Technology, Cape Town 8000, South Africa 1 2 Abstract The ability of biofilms to resist pollutants makes them advantageous for use in bioremediation. The objective of this investigation was to isolate metal-tolerant micro-organisms from a site along the Plankenburg River. Microbial biofilms cultivated in multi-channelled flow cells were exposed to varying concentrations of aluminium (Al), iron (Fe), copper (Cu), manganese (Mn), nickel (Ni) and zinc (Zn), stained with the BacLightTM viability probe, visualised using epifluorescence microscopy and analysed using ScionImage. Exposure to the highest Al, Fe, Cu and Mn concentrations increased the percentages of dead cells. A difference in live and dead cells after exposure to varying Zn and Ni concentrations was not evident. When exposed to the lowest concentrations, no notable difference could be detected in comparison with the untreated control. Possible metal-tolerant micro-organisms were identified from the exposed flow cells using polymerase chain reaction (PCR) and deoxyribonucleic acid (DNA) sequencing, followed by ClustalX alignment and phylogenetic analysis. Phylogenetic analysis identified a variety of organisms, including Bacillus sp., Pseudomonas sp., Delftia tsuruhatensis strain A90, Kocuria kristinae strain 6J-5b, Comamonas testosteroni WDL7, Stenotrophomonas maltophilia strain 776, Staphylococcus sp. MOLA:313, Micrococcus sp. TPR14, Sphingomonas sp. 8b-1 and Microbacterium sp. PAO-12. Two major clusters could be distinguished based on their Gram-reactions. Keywords: BacLightTM viability probe, biofilms, phylogenetic analysis, river water, ScionImage Introduction Population growth and urbanisation results in increased water resource utilisation. Continued deposition of point- and nonpoint source pollutants, including those originating from industrial effluents, agricultural runoff, leaking sewers, on-site sanitation at informal housing and waste irrigation (DEAT, 1996), amongst others, adversely affects the surrounding environment. Metal contamination in the environment can also be attributed to the natural occurrence of metals in soil, atmospheric deposits and the corrosion of building materials (Maanan et al., 2004). The metals most commonly associated with most river water systems are lead (Pb), copper (Cu), iron (Fe), cadmium (Cd), aluminium (Al), mercury (Hg), arsenic (As) and manganese (Mn) (Wright and Welbourne, 2002). Zinc (Zn), nickel (Ni), As, Hg, cobalt (Co) and Mn concentrations were studied in water and fish samples isolated from the Aba River, Nigeria, into which waste from various industries is discharged. Atomic absorption spectrometry (AAS) analysis revealed elevated concentrations of Zn, Mn and As in fresh fish and elevated Ni and Hg concentrations in frozen fish found in a nearby market (Allinnor, 2005). In water and the environment, micro-organisms exist mostly as biofilm communities attached to surfaces; microbial biofilms exhibit high affinities for contaminants due to the ability of the exopolymers to bind and sequester antimicrobial * To whom all correspondence should be addressed.  +27 21 460-9095; fax: +27 21 460-3193; e-mail: Received 28 January 2011; accepted in revised form 14 December 2011. http://dx.doi.org/10.4314/wsa.v38i1.5 Available on website http://www.wrc.org.za ISSN 0378-4738 (Print) = Water SA Vol. 38 No. 1 January 2012 ISSN 1816-7950 (On-line) = Water SA Vol. 38 No. 1 January 2012 agents from the surrounding environment (Teitzel and Parsek, 2003). Biofilms have been shown by Roane and Pepper (2000) to be one of the most effective treatments for the removal of metals from metal-contaminated water. Flow cell systems have been used to cultivate microbial biofilms in vivo (Caldwell et al., 2002). They are multichannelled to allow for experimental replication and simplified handling. Teitzel and Parsek (2003) used a flow cell system to visualise the behaviour of biofilm-bound micro-organisms in response to Cu and Zn. Confocal laser scanning microscopy (CLSM) analysis revealed that the majority of cells in the outer layers were dead, in comparison to the untreated control, where the majority of cells were alive. Microbial composition can be determined genetically, through the amplification of the 16S or 23S rRNA region of the genomic DNA, using specific primers (Amann, 1995). The diversity of tolerant micro-organisms depends on nucleotide sequence variations (Martin, 2002), ranging from 20% to 80% G+C (Ochman et al., 2005) among individual species sharing common ancestry. This genetic variation can then be visualised with phylogenetic trees (Martin, 2002). Chien et al. (2007) studied the microbial diversity in soil contaminated with effluent from a chemical industrial factory, using 16S rDNA. The organisms isolated were Polyangium spp., Sphingomonas spp., Variovorax spp., Hafina spp., Clos tridia, Acidobacteria, the enterics and some uncultured strains. Acinetobacter, Enterobacter and Stenotrophomonas spp. also exhibited the ability to tolerate high concentrations of Cd. The objective of this investigation was to isolate metaltolerant micro-organisms from a metal-contaminated site along the Plankenburg River. The micro-organisms were cultured 29 included Site A (Agricultural farming and residential areas) and Site B (Industrial area at Adam Tas Bridge). Results from this study showed that the highest concentrations of metals were recorded at Site C (Substation in the industrial area), which is why this particular site was selected to investigate the efficiency of the bioreactor systems to reduce metal concentrations in the river water. Ten litres of river water was collected from Site C (Fig. 1) in a 10 ℓ plastic container and transported at 4°C. SITE A Agricultural Farming and Residential Areas Informal Settlement SITE B Closest point to Informal Settlement SITE C Substation in Industrial Area Metal concentrations in river water Stellenbosch SITE D Industrial Area at Adam Tas Bridge The Boord Residential Area To determine the concentrations of Al, Cu, Fe, Mn, Ni and Zn in water, 5 mℓ samples were digested with 10 mℓ 55% nitric acid at 40°C for 60 min and then at 120°C for 180 min, using a Grant dry-block heater. A blank (control) of 10 mℓ 55% nitric acid was analysed along with the collected samples to check for possible contamination. The samples were cooled to room temperature, filtered wi (...truncated)