The utilisation of bacterial species diversity as a bioindicator for biocide efficacy in water systems
Thesis (PhD)--Stellenbosch University, 2019.
ENGLISH ABSTRACT: The poor quality of cooling water, utilised by South African power plants, necessitates the use of biocides and bio dispersants to manage microbiological fouling. The two most common biocides used are isothiazolone and dibromonitrilopropionamide (DBNPA). Management of the dosing of these chemicals is historically not controlled or the efficacy thereof monitored. Cooling water microbiology is mostly performed on the planktonic (free-living) bacterial count in the water, which requires complex analyses in a microbiological laboratory. In order to manage the efficacy of biocide dosing, a simple, effective test is required, which does not need complex technical analysis. The Darwinian concept of survival of the fittest can be applied to bacterial communities too. This implies that when a biocide is dosed into a cooling water system, there should be a change in the bacterial species diversity of the community (Koonin and Wolf, 2012, Spencer, 2018). The monitoring of metabolic changes in the water offers insight to the bacterial species diversity present. In order to evaluate this Biolog Ecoplates ® were used to compare the carbon substrate utilisation (bacterial species diversity) of samples pre and post biocide dosing. In order to verify these results total bacterial plate counts were conducted as were denaturing gradient gel electrophoresis (DGGE) finger prints. The culturable analyses (total bacteria and Biolog Ecoplates®) showed good correlation when an appropriate concentration was dosed but there was little correlation when biocides were under dosed. The effect of biocidal resistance was also evaluated to determine whether the metabolism of the resident bacterial community changes when impacted by sequentially increasing, sub lethal concentrations of biocide. These results, for both carbon substrate utilisation and DGGE, indicate a small change in bacterial species diversity. However the addition of a secondary biocide (DBNPA) post resistance development caused a decrease in the size of the bacterial community, to levels below detection limits for both the Biolog Ecoplate and the total plate count. In an attempt to evaluate the concomitant development of biocidal and antibiotic resistance, Biolog Phenotypic Microarrays (PM) ® 1 to 20 were used. These plates offer an indication of variations in the metabolisms of bacterial communities in different samples. Biocidal resistance was again induced by sequential dosages of sub lethal concentrations of isothiazolone. Samples were collected at three specific times: 1) pre dosing, day 1, time 0 hrs (1-0), 2) when the because the non-resilient bacteria were all killed off and only the resilient bacteria survived, (lowest bacterial plate count) (day 9, 24 h post dosing, 9 -24) and after the biocide residual was eliminated from the system (day 9, 48 h post dosing, 9-48). Each of the three samples was loaded onto a set of the Biolog PM® plates. The plates were incubated in an Omnilog® plate reader and the Omnilog® measurement units compared for the three samples. This technique, based on a colour change, indicated that microorganisms in the community were resistant to the antibiotics Rifamycin, Aztreonam and Ethionamide. The study indicates that Biolog Ecoplates can be utilised to evaluate the efficacy of biocide dosing by monitoring changes in carbon substrate utilisation. The study highlights the need for optimal dosing of the various biocides. The possibility of antibiotic resistance development with biocidal resistance should be considered with the optimisation of dosing. However, the use of biocides with differing modes of action does minimise this risk.
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