Evaluation and application of molecular denitrification monitoring methods in the northern Lake Tai, China

Abstract

Worldwide, excessive reactive nitrogen in groundwater and surface waters is a growing problem, especially in areas that face rapid urbanization and industrialization. One example for environmental nitrogen pollution is the Lake Tai, China's third largest freshwater Lake, located in the Yangtze River basin. Due to the rapid development of the surrounding area, nitrogen compounds like nitrate are discharged into the Lake. Consequently, eutrophication and harmful algae blooms increased and led to the production of toxins directly affecting water consumers through the water supply chain. Denitrification is the main process that attenuates nitrate by converting it into atmospheric nitrogen and represents an intrinsic natural process to compensate the excess reactive nitrogen. In this study, the methodology to detect nitrate reducing bacteria on a functional gene and transcriptional level was optimized and verified in laboratory experiments with a pure culture of Pseudomonas veronii, isolated from Lake Tai. We demonstrated that transcripts analysis (mRNA) did correspond with nitrate reduction activity. Subsequently, the abundance and the activity of nitrate reducing bacteria in Lake Tai were assessed using the developed methods. We demonstrated that nitrate reducing bacteria can be found throughout all sediment and water samples taken from the northern Lake Tai in September 2017. Measurements of narG transcripts also indicated the activity of the membrane-bound nitrate reductase in the water samples. However, the bioinformatic analysis of narG sequences showed varying binding efficiency of primer and gene sites in dependence of phylogenetic groups, which may lead to an underestimation in the qPCR method. Thus, it is important to point out the precautions and limitations of primer systems to monitor nitrogen transformation by qPCR in the environment. Based on this study, mRNA detection methods are suitable for improved microbiological monitoring of denitrification, as an intrinsic process in Lake Tai to mitigate the inflowing reactive nitrogen compounds.

Keywords: Denitrification; Functional genes; Lake Tai; Nitrate pollution; Nitrate reduction; mRNA analysis.