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Aims: To compare Thiobacillus thioparus population dynamics in a control and a test activated sludge (AS) bioreactor, used for hydrogen sulfide (H2S) degradation. Methods and Results: Denaturing gradient gel electrophoresis (DGGE) was used to confirm the presence of T. thioparus, and real-time PCR was used to quantify the level of this bacterium in the AS samples. The DGGE analysis showed a band for T. thioparus in all samples, with the band being more prominent in the test sample with H2S diffusion. It also showed that although a change occurred in the diversity of the microbial population in the test sludge after 6 weeks of H2S diffusion, the microbial community structure of the test and control was still similar. Thiobacillus thioparus-specific PCR primers confirmed that 50% of the isolates from both the test and control bioreactors were T. thioparus. The thiobacilli population became more efficient at degrading the diffused H2S. This increase in efficiency was confirmed by a significant increase in the number of isolates from the test sludge compared with those from the control sludge, when they were grown in a thiosulfate-rich liquid medium. Conclusions: It was concluded that the use of AS process for H2S removal encourages the population of T. thioparus to increase even at times when the total biomass concentration shows a decrease. Significance and Impact of the Study: The research results give an insight into the dynamics of the microbial population in an AS pilot plant used in a dual role, to treat the wastewater and H2S.