Cultivation of nitrite-dependent anaerobic methane-oxidizing bacteria: impact of reactor configuration

Nitrite-dependent anaerobic methane oxidation (n-damo) is mediated by bacteria that anaerobically oxidize methane coupled with nitrite reduction and is a potential bioprocess for wastewater treatment. In this work, the effect of reactor configuration on n-damo bacterial cultivation was investigated. A magnetically stirred gas lift reactor (MSGLR), a sequencing batch reactor (SBR), and a continuously stirred tank reactor (CSTR) were selected to cultivate the bacteria. Microbial community was monitored by using quantitative PCR, 16S rRNA gene sequencing, pmoA gene sequencing, and fluorescence in situ hybridization (FISH). The effects of substrate inhibition, methane mass transfer, and biomass washout in the three reactors were focused on. The results indicated that the MSGLR had the best performance among the three reactor systems, with the highest total and specific n-damo activities. Its maximum volumetric nitrogen removal rate was up to 76.9 mg N L^?1 day^?1, which was higher than previously reported values (5.1–37.8 mg N L^?1 d^?1).     

稻田土壤亚硝酸盐型甲烷厌氧氧化菌群落结构的时空特征

稻田是温室气体甲烷的重要排放源之一,对全球气候变化具有重要影响.由隶属于NC10门的Candidatus Methylomirabilis oxyfera(M.oxyfera)-like细菌介导的亚硝酸盐型甲烷厌氧氧化是控制稻田甲烷排放的新途径.目前,有关此类微生物群落在稻田土壤中的时空分布特征及其环境影响因素尚不明确...     

Diversity and enrichment of nitrite-dependent anaerobic methane oxidizing bacteria from wastewater sludge

Recently discovered microorganisms affiliated to the bacterial phylum NC10, named "Candidatus Methylomirabilis oxyfera", perform nitrite-dependent anaerobic methane oxidation. These microorganisms could be important players in a novel way of anaerobic wastewater treatment where ammonium and residual dissolved methane might be removed at the expense of nitrate or nitrite. To find suitable inocula for reactor startup, ten selected wastewater treatment plants (WWTPs) located in The Netherlands were screened for the endogenous presence of M. oxyfera using molecular diagnostic methods. We could identify NC10 bacteria with 98% similarity to M. oxyfera in nine out of ten WWTPs tested. Sludge from one selected WWTP was used to start a new enrichment culture of NC10 bacteria. This enrichment was monitored using specific pmoA primers and M. oxyfera cells were visualized with fluorescence oligonucleotide probes. After 112 days, the enrichment consumed up to 0.4 mM NO(2)(-) per day. The results of this study show that appropriate sources of biomass, enrichment strategies, and diagnostic tools existed to start and monitor pilot scale tests for the implementation of nitrite-dependent methane oxidation in wastewater treatment at ambient temperature.     

Spatial and temporal distribution of nitrite-dependent anaerobic methane-oxidizing bacteria in an intertidal zone of the East China Sea

Nitrite-dependent anaerobic methane oxidation (N-DAMO), which couples anaerobic methane oxidation and nitrite reduction, is a recently discovered bioprocess coupling microbial nitrogen and carbon cycles. The discovery of this microbial process challenges the traditional knowledge of global methane sinks and nitrogen losses. In this study, the abundance and activity of N-DAMO bacteria were investigated and their contributions to methane sink and nitrogen loss were estimated in different seasons and different partitions of an intertidal zone of the East China Sea. The results showed that N-DAMO bacteria were extensively and continuously present in the intertidal zone, with the number of cells ranging from 5.5 × 10⁴ to 2.8 × 10⁵ copy g⁻¹ soil and the potential activity ranging from 0.52 to 5.7 nmol CO₂ g⁻¹ soil day⁻¹, contributing 5.0–36.6% of nitrite- and sulfate-dependent anaerobic methane oxidation in the intertidal zone. The N-DAMO activity and its contribution to the methane consumption were highest in the spring and in the low intertidal zone. These findings showed that the N-DAMO process is an important methane and nitrogen sink in the intertidal zone and varies with the seasons and the partitions of the intertidal zone.

     

A new intra-aerobic metabolism in the nitrite-dependent anaerobic methane-oxidizing bacterium Candidatus 'Methylomirabilis oxyfera'

Biological methane oxidation proceeds either through aerobic or anaerobic pathways. The newly discovered bacterium Candidatus 'Methylomirabilis oxyfera' challenges this dichotomy. This bacterium performs anaerobic methane oxidation coupled to denitrification, but does so in a peculiar way. Instead of scavenging oxygen from the environment, like the aerobic methanotrophs, or driving methane oxidation by reverse methanogenesis, like the methanogenic archaea in sulfate-reducing systems, it produces its own supply of oxygen by metabolizing nitrite via nitric oxide into oxygen and dinitrogen gas. The intracellularly produced oxygen is then used for the oxidation of methane by the classical aerobic methane oxidation pathway involving methane mono-oxygenase. The present mini-review summarizes the current knowledge about this process and the micro-organism responsible for it.     

氮依赖型甲烷厌氧氧化菌的整合

氮依赖型甲烷厌氧氧化菌(nitrite-dependent anaerobic methane oxidation bacteria,n-damo细菌,属于NC10门)是最近10年来微生物生态学领域的研究热点。然而,对该类群基于现有数据的生态分布、群落结构和系统进化的整合分析还未见报道。【目的】为了更好地将近年来针对该类群的研究做一次全面梳理,本文通过整合前人已有发表数据和结合自身实验数据两方面进行。【方法】一方面,利用NCBI数据库(数据搜集到2016年11月)中所有n-damo细菌序列对其进行生物信息学分析;另一方面,对大九湖泥炭地表层泥炭利用16S rRNA二代测序技术对该类群进行检测,并同前人数据进行对比。【结果】n-damo细菌主要在沉积物、湿地和水稻土检出;基于pmo A基因的n-damo细菌的平均检出率是基于16S rRNA基因检出率的7倍,但是这两类基因分子标记物所得到的多样性指数保持相对稳定(1.4-3.4);贫氮的大九湖泥炭其NC10的丰度仅为0.067%。【结论】n-damo类群种群相对稳定,暗示其行使的生态功能相对单一;贫氮的大九湖泥炭其极低的NC10丰度暗示氮对NC10是限制因子;具有真正氮依赖型甲烷厌氧氧化细菌的Group A可能只占很少的一部分(小于20%),暗示出该类群真正的生态潜能需要进一步评估。本次整合分析为更好的理解n-damo细菌的生活环境、评估不同基因分子标记物下n-damo细菌的检出率、不同亚类群比如Group A和Group B等的丰度和真正的潜在生态功能提供参考。     

A newly designed degenerate PCR primer based on pmoA gene for detection of nitrite-dependent anaerobic methane-oxidizing bacteria from different ecological niches

A new pmoA gene-based PCR primer set was designed for detection of nitrite-dependent anaerobic oxidation of methane (n-damo) bacteria from four different ecosystems, namely rice paddy soil, freshwater reservoir, reed bed, and sludge from wastewater treatment plant. This primer set showed high specificity and efficiency in recovering n-damo bacteria from these diverse samples. The obtained sequences showed 88–94 and 90–96 % similarity to nucleotide and amino acid sequences, respectively, with the known NC10 phylum bacterium. According to the UniFrac principal coordinates analysis (PCoA), DNA sequences retrieved by the new PCR primer set in this study formed a separate group from the reported sequences, indicating higher diversity of n-damo in the environment. This newly designed PCR primer is capable of amplifying not only the currently known n-damo bacteria but also those that have not been reported, providing new information on the ecological diversity and distribution of this group of microorganisms in the ecosystem.     

Molecular and stable isotopic evidence for the occurrence of nitrite-dependent anaerobic methane-oxidizing bacteria in the mangrove sediment of Zhangjiang Estuary,China

Applied Microbiology and Biotechnology - Nitrite-dependent anaerobic methane oxidation (n-damo), which is mediated by “Candidatus Methylomirabilis oxyfera-like” bacteria, is unique in...     

Spatial change of reservoir nitrite-dependent methane-oxidizing microorganisms

Nitrite-dependent anaerobic methane oxidation (n-damo), catalyzed by microorganisms affiliated with bacterial phylum NC10, can have an important contribution to the reduction of the methane emission from anoxic freshwater sediment to the atmosphere. However, information on the variation of sediment n-damo organisms in reservoirs is still lacking. The present study monitored the spatial change of sediment n-damo organisms in the oligotrophic freshwater Xinfengjiang Reservoir (South China). Sediment samples were obtained from six different sampling locations and two sediment depths (0–5 cm, 5–10 cm). Sediment n-damo bacterial abundance was found to vary with sampling location and layer depth, which was likely influenced by pH and nitrogen level. The presence of the n-damo pmoA gene was found in all these samples. A remarkable shift occurred in the diversity and composition of sediment n-damo pmoA gene sequences. A variety of distinctively different n-damo pmoA clusters existed in reservoir sediments. The pmoA sequences affiliated with Candidatus Methylomirabilis oxyfera formed the largest group, while a significant proportion of the obtained n-damo pmoA gene sequences showed no close relationship to those from any known NC10 species. In addition, the present n-damo process was found in reservoir sediment, which could be enhanced by nitrite nitrogen amendment.     

自然生境中亚硝酸盐型厌氧甲烷氧化细菌研究进展

随着功能微生物介导的亚硝酸盐型厌氧甲烷氧化(nitrite-dependent anaerobic methane oxidation,N-DAMO)过程被发现,人们对自然界的碳氮循环有了全新的认识,该过程成为自然生态系统中温室气体甲烷的汇,同时还是氮污染的消减途径。本文系统介绍了N-DAMO过程反应机理以及参与该过程的亚硝酸盐型厌氧甲烷氧化细菌(Candidatus Methylomirabilis oxyfera)的生理生化特征,并对研究该功能菌的分子微生物方法进行了汇总。通过对不同自然生境中该细菌的研究报道进行总结分析,揭示各生境中年均降水量、年均温度、所处不同自然区等大尺度宏观环境因子及碳源、氮源、pH和氧气含量等生存因子对其群落结构的潜在影响,最后在展望中提出此功能菌在未来可深入研究的方向,期望能厘清厌氧甲烷氧化过程及其功能菌在碳、氮循环中的生态学功能。     

Dry-thermophilic anaerobic digestion of organic fraction of the municipal solid waste: focusing on the inoculum sources

The effect of inoculum source on anaerobic thermophilic digestion of separately collected organic fraction of municipal solid wastes (SC_OFMSW) has been studied. Performance of laboratory scale reactors (V: 1.1 L) were evaluated using six different inoculums sources: (1) corn silage (CS); (2) restaurant waste digested mixed with rice hulls (RH_OFMSW); (3) cattle excrement (CATTLE); (4) swine excrement (SWINE); (5) digested sludge (SLUDGE); and (6) SWINE mixed with SLUDGE (1:1) (SWINE/SLUDGE). The SC_OFMSW was separately and collected from university restaurant. The selected conditions were: 25% of inoculum, 30% of total solid and 55 degrees C of temperature, optimum in the thermophilic range. The six inoculum sources showed an initial start-up phase in the range between 2 and 4 days and the initial methane generation began over 10 days operational process. Results indicated that SLUDGE is the best inoculum source for anaerobic thermophilic digestion of the treatment of organic fraction of municipal solid waste at dry conditions (30%TS). Over 60 days operating period, it was confirmed that SLUDGE reactor can achieve 44.0%COD removal efficiency and 43.0%VS removal. In stabilization phase, SLUDGE reactor showed higher volumetric biogas generated of 78.9 mL/day (or 35.6 mLCH(4)/day) reaching a methane yield of 0.53 LCH(4)/gVS. Also, SWINE/SLUDGE and SWINE were good inoculums at these experimental conditions.     

Growth and population dynamics of anaerobic methane-oxidizing archaea and sulfate-reducing bacteria in a continuous-flow bioreactor

The consumption of methane in anoxic marine sediments is a biogeochemical phenomenon mediated by two archaeal groups (ANME-1 and ANME-2) that exist syntrophically with sulfate-reducing bacteria. These anaerobic methanotrophs have yet to be recovered in pure culture, and key aspects of their ecology and physiology remain poorly understood. To characterize the growth and physiology of these anaerobic methanotrophs and the syntrophic sulfate-reducing bacteria, we incubated marine sediments using an anoxic, continuous-flow bioreactor during two experiments at different advective porewater flow rates. We examined the growth kinetics of anaerobic methanotrophs and Desulfosarcina-like sulfate-reducing bacteria using quantitative PCR as a proxy for cell counts, and measured methane oxidation rates using membrane-inlet mass spectrometry. Our data show that the specific growth rates of ANME-1 and ANME-2 archaea differed in response to porewater flow rates. ANME-2 methanotrophs had the highest rates in lower-flow regimes (mu(ANME-2) = 0.167 . week(-1)), whereas ANME-1 methanotrophs had the highest rates in higher-flow regimes (mu(ANME-1) = 0.218 . week(-1)). In both incubations, Desulfosarcina-like sulfate-reducing bacterial growth rates were approximately 0.3 . week(-1), and their growth dynamics suggested that sulfate-reducing bacterial growth might be facilitated by, but not dependent upon, an established anaerobic methanotrophic population. ANME-1 growth rates corroborate field observations that ANME-1 archaea flourish in higher-flow regimes. Our growth and methane oxidation rates jointly demonstrate that anaerobic methanotrophs are capable of attaining substantial growth over a range of environmental conditions used in these experiments, including relatively low methane partial pressures.     

Current advances in molecular methods for detection of nitrite-dependent anaerobic methane oxidizing bacteria in natural environments

Nitrite-dependent anaerobic methane oxidation (n-damo) process uniquely links microbial nitrogen and carbon cycles. Research on n-damo bacteria progresses quickly with experimental evidences through enrichment cultures. Polymerase chain reaction (PCR)-based methods for detecting them in various natural ecosystems and engineered systems play a very important role in the discovery of their distribution, abundance, and biodiversity in the ecosystems. Important characteristics of n-damo enrichments were obtained and their key significance in microbial nitrogen and carbon cycles was investigated. The molecular methods currently used in detecting n-damo bacteria were comprehensively reviewed and discussed for their strengths and limitations in applications with a wide range of samples. The pmoA gene-based PCR primers for n-damo bacterial detection were evaluated and, in particular, several incorrectly stated PCR primer nucleotide sequences in the published papers were also pointed out to allow correct applications of the PCR primers in current and future investigations. Furthermore, this review also offers the future perspectives of n-damo bacteria based on current information and methods available for a better acquisition of new knowledge about this group of bacteria.     

Nitrogen source effects on the denitrifying anaerobic methane oxidation culture and anaerobic ammonium oxidation bacteria enrichment process

The co-culture system of denitrifying anaerobic methane oxidation (DAMO) and anaerobic ammonium oxidation (Anammox) has a potential application in wastewater treatment plant. This study explored the effects of permutation and combination of nitrate, nitrite, and ammonium on the culture enrichment from freshwater sediments. The co-existence of NO₃ ⁻, NO₂ ⁻, and NH₄ ⁺ shortened the enrichment time from 75 to 30 days and achieved a total nitrogen removal rate of 106.5 mg/L/day on day 132. Even though ammonium addition led to Anammox bacteria increase and a higher nitrogen removal rate, DAMO bacteria still dominated in different reactors with the highest proportion of 64.7% and the maximum abundance was 3.07 ± 0.25 × 10⁸ copies/L (increased by five orders of magnitude) in the nitrite reactor. DAMO bacteria showed greater diversity in the nitrate reactor, and one was similar to M. oxyfera; DAMO bacteria in the nitrite reactor were relatively unified and similar to M. sinica. Interestingly, no DAMO archaea were found in the nitrate reactor. This study will improve the understanding of the impact of nitrogen source on DAMO and Anammox co-culture enrichment.