Nitrite Reductase Genes As Functional Markers To Investigate Diversity Of Denitrifying Bacteria During Agricultural Waste Composting

Composting is the widely used method to stabilize agriculturalorganic wastes through the degradation of biodegradable components by microbialcommunities under controlled condition. The lossing of nitrogen during the process ofcomposting will result odor and reduce the quality of compost products. Studying thetransfer and transformation of nitrogen during composting has important practicalsignificance. Denitrification is a crucial microbial process in the Ncycle in which nitrogen oxides (NO3--N and NO2-N) are reducedstepwise to gaseous end products (NO, N2O, N2). So denitrification playsan important role in the nitrogen cycle. We choose the denitrifying as the object of theresearch, using the polymerase chain reaction-denaturing gradient gel electrophoresistechnology to study the diversity and community composition of denitrifiercommunities in the composting， and explore the relationship between denitrifiercommunities and composting parameters. The aim of this study was to providetheoretical guidance for optimizing the composting process.Denitrification is an anaerobic process, occuring only in low-oxygen or anoxicenvironments and consists of four reaction steps by which nitrate is reduced todinitrogen gas sequentially by the four metalloenzyme classes of nitrate reductases,nitrite reductases, nitric oxide reductases and nitrous oxide reductases. The nitritereductase is the key enzyme of this respiratory process since it catalyses the reductionof soluble nitrite into gas. Two types of nitrite reductases, that differ in terms of theirstructures and prosthetic metal, have been characterized: a copper nitrite reductaseencoded by the nirK gene and a cytochrome cd1-nitite reductase encoded by the nirSgene that is the key enzyme. The dynamic succession of denitrifying bacteriacommunity during the agricultural waste composting was investigated using thepolymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). ThenirK and nirS genes were abundant throughout the composting process. The clones ofthe two genes obtained in this study spread throughout the trees and affiliated toProteobacteria. The phylogenetic tree of the nirK gene was divided into six majorclusters: Alcaligenaceae、Phyllobacteriaceae、Bradyrhizobiaceae、Rhizobiaceae、Rhodobacteraceae、Brucellaceae. The nirS tree was divided into two major clusters:Pseudomonadaceae、Rhodocyclaceae. DGGE showed the diversity indices of the twogenes were various in the different composting sample time. Higher diversity indices were appeared during the thermophilic stage, and the diversity index reached thelowest value on day15.The changes of pile temperature, pH, moisture content, soluble NH4+-N andNO3--N were measured during different composting periods. The results showed thatthe pile was insulated and temperatures above50℃were maintained for8days. Thehighest temperature in the compost was62℃,which can reach the requirements tokill pathos. The pH ranged from7.15~9.45, which was beneficial for microbes. Thecontent of NH4+-N accumulated rapidly and reached peak values on day5. Afterwards,its content decreased to low level by the NH3volatilization and the immobilization bymicroorganisms. The content of NO3--N showed a significant increase in the first3days. Then the content decreased sharply to974.60mg·kg-1(dry weight) on the12thday. Afterwards, it gradually increased to1401mg·kg-1(dry weight) during the laterthermophilic stage and cooling stage.The goal of this study was to identify and prioritize some of thephysico-chemical parameters that contributed to denitrifier community compositionsduring agricultural waste composting. Relationships between those parameters anddenitrifier community compositions determined by PCR-DGGE were simultaneouslyevaluated by redundancy analysis (RDA). The results showed that the temporalvariation of nirK and nirS genes community composition were both significantlyrelated to pile temperature and pH (P <0.05). Significant amounts of the variation(49.2%and38.3%for nirK and nirS genes species data, respectively) were explainedby those parameters, suggesting that those parameters were the most likely ones toinfluence, or be influenced by the nirK and nirS genes species. Pile temperature solelyexplained14.4%(P=0.002) of the variation in the nirK gene DGGE profiles,whereas pH explained19.6%(P=0.004). For the nirS gene, pile temperatureexplained16.4%(P=0.002) of the variation, and pH explained7.9%(P=0.04).