Desalination technology based on Reverse Osmosis (RO) membrane filtration has been resorted to provide high-quality drinking water. RO produced drinking water is characterized by a low bacterial cell concentration. Monitoring microbial quality and ensuring membrane-treated water safety has taken advantage of the rapid development of DNA-based techniques. However, the DNA extraction process from RO-based drinking water samples needs to be evaluated regarding the biomass amount (filtration volume) and residual disinfectant such as chlorine, as it can affect the DNA yield. We assessed the DNA recovery applied in drinking water microbiome studies as a function of (i) different filtration volumes, (ii) presence and absence of residual chlorine, and (iii) the addition of a known Escherichia coli concentration into the (sterile and non-sterile, chlorinated and dechlorinated) tap water prior filtration, and directly onto the (0.2 μm pore size, 47 mm diameter) mixed ester cellulose membrane filters without and after tap water filtration. Our findings demonstrated that the co-occurrence of residual chlorine and low biomass/cell density water samples (RO-treated water with a total cell concentration ranging between 2.47 × 102–1.5 × 103 cells/mL) failed to provide sufficient DNA quantity (below the threshold concentration required for sequencing-based procedures) irrespective of filtration volumes used (4, 20, 40, 60 L) and even after performing dechlorination. After exposure to tap water containing residual chlorine (0.2 mg/L), we observed a significant reduction of E. coli cell concentration and the degradation of its DNA (DNA yield was below detection limit) at a lower disinfectant level compared to what was previously reported, indicating that free-living bacteria and their DNA present in the drinking water are subject to the same conditions. The membrane spiking experiment confirmed no significant impact from any potential inhibitors (e.g. organic/inorganic components) present in the drinking water matrix on DNA extraction yield. We found that very low DNA content is likely to be the norm in chlorinated drinking water that gives hindsight to its limitation in providing robust results for any downstream molecular analyses for microbiome surveys. We advise that measurement of DNA yield is a necessary first step in chlorinated drinking water distribution systems (DWDSs) before conducting any downstream omics analyses such as amplicon sequencing to avoid inaccurate interpretations of results based on very low DNA content. This study expands a substantial source of bias in using DNA-based methods for low biomass samples typical in chlorinated DWDSs. Suggestions are provided for DNA-based research in drinking water with residual disinfectant.

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Cocoa-bean fermentation has been associated with the involvement of diverse microbial assemblages which consist of a wide array of bacteria and yeast. We attempted to screen and to identify the potential antifungal yeast from this assemblage against phytopathogenic fungi. We employed in-vitro antagonism assay using agar plug methods to performpreliminary screening from 35 yeast isolates followed by total protein production and measurement with Bradford methods. We found three yeast strains that were effective against Trichoderma sp. T009, and two moulds associated with cocoa (Penicilliumsp. Cocoa2 and Fusariumsp.Cocoa 1). The three bioactive yeast strains were identified as Saccharomyces cerevisiae IDI-002, Hanseniasporauvarum IDE-056 and Hanseniasporauvarum IDE-271 based on molecular identification and phylogenetic analysis. The production of antifungal protein from pure cultures on YEPG media resulted intotal protein concentration between 6.20 - 8.17 mg/L. Cell suspension showed higherinhibitory activity compared to thecell-free supernatant gave indication that antifungal proteins in the bulk fermentation was below the minimal inhibitory concentration to cause the inhibitory effect. Further characterization, purification, and optimization are still needed before the up-scale production of antifungal metabolites and its biological control application.