Identification and characterisation of chloramine decaying proteins and control of impact in chloraminated systems

Abstract

Chloramine is the second most popular disinfectant behind chlorine used in water distribution systems. The main advantages of using chloramine over chlorine are; it provides a longer lasting disinfectant residual and forms a less amount of the regulated halogenated disinfection by-products. However, at times, microbial chloramine decay can overwhelm stability and is identified as one of the serious problems that needs addressing. One of the mechanisms of microbial chloramine decay is by production of soluble microbial products (SMP), which substantially affects the chloramine decay. The SMP are usually composed of proteins, polysaccharides, humic acids, fulvic acids, nucleic acids, enzymes and structural compounds, but it was suspected that the chloramine decaying SMP could be protein(s) due to the catalytic effect that was noted in the samples. It was noted in soluble form in water after the onset of nitrification in a chloraminated system. When the SMP was filtered out of the water and was run on the SDS page to identify the compound 25 different proteins were seen with weaker signals. Therefore, the identity of chloramine decaying soluble microbial products (SMP), which microbes produce them and how to control them are not known. If SMP was produced by nitrifiers and since nitrifiers could be inhibited by higher organic carbon levels when changing dissolved organic carbon (DOC) level in the water, it could alter the production of chloramine decaying SMP. Therefore, batch rechloramination tests were conducted for reactor sets with variable DOC levels to understand their effect on the impact of SMP. The results revealed as the highest production of SMP had been stimulated with low DOC level (0-1mg-C.L-1) compared to other DOC levels (2-3, 4-5 and 7-8 mg-C.L-1). To recognize microbial community variations with the impression of identifying CDP producing micro-organisms, microbial community analysis was also carried out in the same reactor sets. Significant differences in bacterial types against DOC variations could not be detected. However, some of the bacterial types such as Micobacterium, AOB, Bradyrhizobium sp., Methylobacterium and family Sphingomonadaceae recognized in this study are known to produce extracellular polymeric substances (EPS). My early work has identified that SMP are proteins; hence, named as chloramine decaying proteins (CDP). In Relation to the context of these experiments, the proteins consisting within EPS can be considered as CDP. Questioning if nitrifiers always produce CDP, two nitrified reactors - one with chloramine (chloraminated reactor) and the other with ammonia (ammoniated reactor) - were operated using nutrient added Milli-Q water as the feed water in a way nitrification occurs within the reactor. MilliQ was selected since it produced the highest concentration of CDP. It was expected that CDP could be easily separated amongst 25 previously found proteins. Therefore, nitrified bulk water and biofilm samples from both reactors were subjected to protein separation (2-dimensional gel electrophoresis and SDS-polyacrylamide gel electrophoresis) and protein identification (mass spectrometry-MS). Furthermore, bacterial community variations on ammoniated and chloraminated reactors were characterised by sequencing of 16S RNA. The batch rechloramination results obtained from the reactors for the first time established the production of CDP as a microbial response to chloramine stress. The bacterial community characterisations on each of the reactors did not show major differences in identified bacterial strains. However, the EPS producing bacterial strains (AOB, Bradyrhizobium sp. and family Sphingomonadaceae) identified in chloraminated reactors were suspected to be responsible for CDP production. Chloraminated and ammoniated bulk water samples were not resulting in enough concentrations, therefore, for comparison of protein spots and MS analysis, the biofilm samples (which are believed to have more CDP) were analysed. The major proteins detected were ammonia monooxygenase subunit A and putative porin related to Nitrosomonas sp. and Bradyrhizobium sp., respectively. However, their relation to CDP has to be further investigated. Conclusively, every aspect of this study is directing towards discovering a better control mechanism for the microbial/ CDP induced accelerated chloramine decay. Silver is a known inhibitor for several micro-organisms. Therefore, experiments were conducted to reveal the optimum dose of silver on inactivating nitrifying microbes and CDP for controlling the fast decay of chloramine. Interestingly, 2 I¼g-Ag.L-1 silver (which is far lower than the recommended level- 0.1 mg-Ag.L-1) was found to be effective for improving chloramine residuals in tested bulk waters. This study concludes by further emphasising the need for extensive study/research in further identification of CDP and bacteria communities responsible for chloramine decay in chloraminated drinking water distribution systems.

Date of Award	2019
Original language	English