Enhancing microbial detection in engineered anaerobic systems: improved efficiency of hybridization chain reaction fluorescence in situ hybridization (HCR-FISH) with cell wall treatment

Microbial monitoring plays a pivotal role in ensuring water quality and managing the key microorganisms in various water systems and environments. Traditional methods of microbial detection often involve time-consuming and labor-intensive procedures, which can hinder timely decision-making and response 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[1, 2]. Fluorescence in situ hybridization (FISH) is a powerful tool for detecting of specific targeted microorganisms in environment or engineered water systems [3]. However, the low ribosomal RNA content and the complexity of the cell wall structure of anaerobic microorganisms decreased the efficiency of FISH [4]. In this research, cell wall treatment and the application of hybridization chain reaction (HCR) FISH were used to detect the spatial microbial distribution in samples of anaerobic sludge from anaerobic hybrid reactor (AHR), compared to standard FISH detection. The objective of this research was to investigate the effect of cell wall treatment on improving the efficiency of standard FISH and HCR FISH for detecting targeted anaerobic microorganisms.

     The results show that lysozyme treatment applied in HCR FISH resulted in a significant increase in the detection rates of Archaea and Bacteria groups compared to standard FISH. With cell wall treatment, the detection rates of Archaea and Bacteria groups by HCR FISH were 55.5% and 49.9%, which were significantly higher than those of standard FISH (47.2% and 41.4%), respectively, as shown in Table 1. The amplification of fluorescence-labelled probes during the HCR FISH step contributed to the significant improvement of detection rate and brighter signal [4]. In addition, cell wall treatment with lysozyme allowed more probes to penetrate microbial cells, resulting in higher detection rates compared to non-treatment cells. Cell wall treatment with lysozyme for both FISH techniques was then applied to detect spatial microbial distribution in anaerobic granule from a full-scale UASB reactor treating cassava starch wastewater.

     Remarkable results were achieved with improved HCR FISH, which was able to detect the spatial microbial distributions of Archaea and Bacteria groups at the outer, middle, and inner layers of the anaerobic granule, as shown in Fig. 1. In contrast, standard FISH only detected the spatial microbial distributions of these microbial groups at the outer and middle layers of granule. Additionally, the fluorescence intensity of HCR FISH was stronger compared to standard FISH with relative units of 72.6 RU and 52.8 RU, respectively.

The capability of HCR FISH combined with cell wall treatment is crucial for accurate and timely microbial monitoring in water management systems which lead to better water quality management, improved treatment processes, and more effective quality control in wastewater treatment plant. The adoption of HCR FISH and cell wall treatment protocols in routine water monitoring can thus play a pivotal role in maintaining safe and high-quality water in both environmental and engineered systems.