Abstract
In this study, the specific amplifications of six denitrification-associated genes using PCR(Polymerase Chain Reaction) primer sets were compared. Thereafter, the PCR primer sets that were determined to be suitable for each denitrification-associated gene were used to test samples from sixteen aqueous environments (three from groundwater, three from stream water, and ten from hyporheic zone water). The specific amplification was determined using PCR primer sets for denitrification-associated genes and nucleic acids from eleven types of strains. NosZ was the most frequently amplified gene from the nucleic acid of type, with a specific band seen in all eleven strains. The specific band amplification and PCR time of the strains were analyzed to select one PCR primer set for each gene. The selected PCR primer sets were used to analyze sixteen samples from the aqueous environments in which denitrifying microorganisms were expected to be present. Specific bands of narG, nirS, and nosZ were most frequently observed in the hyporheic water samples. The results showed that microorganisms containing nirG (involved in the reduction of nitrate to nitrite), nirS (involved in the reduction of nitrite to nitric oxide), and nosZ (involved in the reduction of nitrous oxide to nitrogen gas) were the most abundant in the hyporheic zone samples used in this study.
Highlights
The nitrogen cycle is one of the most important nutrient cycles in terrestrial ecosystems and is carried out via nitrogen fixation, mineralization, nitrification, and denitrification [1]
Thereafter, the PCR primer sets that were determined to be suitable were selected for each denitrification-associated gene and tested using sixteen environmental samples
The specific band amplification and PCR time of the type strains were analyzed to select one PCR primer set for each gene
Summary
The nitrogen cycle is one of the most important nutrient cycles in terrestrial ecosystems and is carried out via nitrogen fixation, mineralization, nitrification, and denitrification [1]. The nitrogen cycle involves biological and non-biological processes, where the biological processes usually involve microorganisms [2]. Denitrification is the last step of the nitrogen cycle, in which the nitrogen in fixed forms, such as nitrates, returns to the atmosphere in the form of nitrogen gas. This process can prevent nitrate contamination of groundwater and surface water that arises from the overuse of nitrogen fertilizers [3]. Microorganisms involved in the nitrogen cycle are labeled as either nitrogen-fixing, nitrifying, or denitrifying microorganisms according to their characteristics [2]. Of these, denitrifying microorganisms are typically found in the soil, mud, sewage, sea, and wetland, and are phylogenetically diverse, belonging to Firmicutes [4], Actinobacteria [5], Proteobacteria [6], Archaebacteria [7], and fungi [8,9]
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