Molecular analysis of nitrogen cycling related microorganisms in forest soils and engineering systems

Abstract

Chemical transformation processes of the nitrogen cycle are catalyzed by a wide variety of microorganisms, including some recently discovered ones. Extremely acidic forest soils of Nanling Nature Reserve in Southern China were analyzed for microbial community and composition responsible for nitrogen fixation, denitrification, anammox and nitrite-dependent methane oxidation (n-damo). In this forest ecosystem, nifH gene as a biomarker was used to detect the community and diversity of nitrogen-fixing microorganisms in both natural and re-vegetated forest soils using MiSeq sequencing platform, and to quantify the abundance of diazotrophs. Both diazotrophic archaea and bacteria were detected in all soil samples, but diazotrophic bacteria especially Proteobacteria were the dominant ones contributing to nitrogen fixation. Nitrogen-fixing bacteria of Cyanobacteria, Firmicutes, Proteobacteria, Spirochates, Verrucomicrobia were observed in both summer and winter. Bradyrhizobium, Metholybacterium from α-Proteobacteia, and Desulfovibrio from δ-Proteobacteia were the core genera in these soils. 16S rRNA gene for anammox and pmoA gene for n-damo bacteria were used to construct clone libraries to investigate the occurrence of them in these forest soils. Candidatus Brocadia and Ca. Kuenenia of anammox bacteria were detected while Ca. Methylomirabilis oxyfera and a newly enriched Ca. Methylomirabilis species were found in these forest soil samples. Additionally, clone libraries constructed by nirK and nosZ genes were used to estimate denitrifying bacteria. The community was closely related to Proteobacteria revealed by both nirK and nosZ genes. The abundance of diazotrophic microorganisms, anammox, n-damo bacteria, and denitrifiers was ~107, 103-106, 103-106 and 106-107 copies/g dry soil, respectively. Basically, water content, pH, NO3-‒N, NH4+‒N and some other physiochemical parameters were significantly effects on different nitrogen cycle microorganisms in acidic forest soils. Anammox process has a great importance in developing a new generation of wastewater treatment technology. An inoculation study showed anammox process was successfully re-established in conventional wastewater treatment plants (WWTPs) without re-design of the treatment system to save construction costs. Meanwhile, a sewage treatment plant and four landfill leachate treatment plants with different time periods after inoculation of anammox sludge, were selected for an investigation of simultaneous removal of ammonium and methane by responsive microorganisms. Specifically, Ca. Brocadia, Ca. Kuenenia and Ca. Jettenia of anammox bacteria were detected by hzsB gene, with n-damo bacteria investigated by pmoA gene co-occurring in all WWTPs. The abundance of anammox bacteria was apparently higher in four high N landfill-leachate WWTPs than the sewage treatment plant, especially the inoculated ones up to 1012-1013 copies per gram of dry solids from the immobilizing materials. Different from anammox bacteria, copy numbers of co-occurred n-damo bacteria in these plants were stable at 107-108 per gram of dry solids and showed no direct relationship to the inoculation. Tropical climate with high temperature and humidity provides a suitable condition for microorganisms to colonize and grow on the stone surfaces. Microbial contribution to biodeterioration of cultural heritage is one of the most serious issues in protection of sandstone monuments in Cambodia. The nitrogen cycle-related microorganisms, in particular ammonia-oxidizing archaea, were responsible for acidity production and attack of the stone than ammonia-oxidizing bacteria.