滨海湿地生态系统微生物驱动的氮循环研究进展

滨海湿地生态系统介于陆地生态系统和海洋生态系统之间,其类型多种多样,环境差异极大,微生物种类丰富。近年来,随着人为氮源的大量输入,造成滨海湿地生态系统富营养化污染问题日趋严重。本文主要总结了滨海湿地生态系统微生物驱动的固氮、硝化、反硝化、厌氧氨氧化、NO_3~-还原成铵等主要氮循环过程,并综述了通过功能基因(如nifH、amoA、hzo、nirS、nirK、nrfA)检测微生物群落多样性及其环境影响因素的相关研究,旨在更好理解微生物驱动氮循环过程以去除氮,以期为减轻富营养化和危害性藻类爆发提供科学依据。     

Molecular analysis of halophilic bacterial community for high-rate denitrification of saline industrial wastewater

A denitrification system for saline wastewater utilizing halophilic denitrifying bacteria has not been developed so far. In this study, denitrification performance and microbial community under various saline conditions were investigated using denitrifying sludge acclimated under low-salinity condition for a few years as seed sludge. A continuous denitrification experiment showed that denitrification performance and microbial community at 10% salinity was higher than that at 1% salinity. The microbial community in the denitrification sludge that was acclimated under low salinity was monitored by terminal-restriction fragment length polymorphism (T-RFLP) analysis during acclimation to high-salinity condition. T-RFLP profiles and clone analysis based on 16S rRNA-encoding genes in the sludge of the denitrification system with 10% salinity indicated that the γ-Proteobacteria, particularly Halomonas spp., were predominant species, suggesting that these bacterial members were possibly responsible for a high denitrification activity under high-salinity conditions. Furthermore, the investigation of denitrification performance under various saline conditions revealed that 4–10% salinity results in the highest denitrification rate, indicating that this salinity was optimal for predominant bacterial species to exhibit denitrification activity. These results indicate the possibility that an appropriate denitrification system for saline wastewater can be designed using acclimated sludge with a halophilic community.     

Urban stream denitrifier communities are linked to lower functional resistance to multiple stressors associated with urbanization

The microbial communities in urban stream ecosystems are subject to complex combinations of stressors. These same microbial communities perform the critical ecosystem service of removing excess reactive nitrogen. We asked whether the denitrifying microbial communities in urban streams differ in their functional resistance to common urban stressors from communities from nonurban streams. We exposed inocula from a highly polluted urban stream and a nearby nonurban stream to three different stressors, added alone and in combination. Stressors represent the common urban impacts of thermal pollution (10°C), trace metal exposure (ionic silver (Ag⁺)), and salinization (addition of NaCl). We used reduction in nitrite (NO₂ ^?) concentrations under anaerobic conditions as a proxy for denitrification potential. Nonurban stream denitrifying microbial communities were more diverse than their urban counterparts. Denitrification potential for both communities was unaffected by exposure to any individual stressor. However, denitrification rates by the less diverse urban microbial inoculum decreased in response to combined heavy metal and salt stress, while nonurban communities were unaffected. These findings support the hypothesis that higher diversity may confer greater functional resistance in response to multiple stressors and do not support the idea that stressful conditions select for communities that are functionally resilient to multiple stressors.     

海洋最小含氧带氮流失过程与机制

对全球大洋氮循环的研究发现,大洋输入和输出的氮存在严重的不平衡,所固定的氮中有相当一部分被还原为N₂或N₂O从大洋中流失,而海洋最小含氧带(OMZ)被认为是发生氮流失的最主要区域,通过反硝化作用和厌氧氨氧化作用,固定氮在OMZ海区内损失量可达40～450 Tg·a^-1.对不同海区OMZ内固定氮损失的两种主要作用总结发现,异养反硝化作用在热带太平洋东部、阿拉伯海的OMZ内以及海洋沉积物内占有显著优势,在智利、秘鲁沿岸海域及阿拉伯海域也已发现自养反硝化作用的存在;而在黑海、非洲西南部的本格拉上升流、智利北部沿岸等地,厌氧氨氧化作用强烈,且其在陆架区的作用强度和面积要大于大洋区.OMZ氮的流失除受氮流失过程自身影响外,固氮作用、硝化作用、硝酸盐异化还原作用等都可能对OMZ海区内氮收支不平衡造成影响.其中固氮作用的影响最不能忽视,其在全球OMZ内固定的氮的总量可达15～40 Tg·a^-1,是对OMZ氮流失量的重要补充.区分反硝化作用和厌氧氨氧化作用对OMZ氮流失的相对贡献,明确氮流失的另一产物N₂O的形成机制和定量评估方法是当前OMZ氮流失研究中存在的最主要问题.本文针对存在问题提出了相应的研究设想,以期为海洋最小含氧带的研究提供参考.     

反硝化菌功能基因及其分子生态学研究进展

由微生物推动的反硝化作用是地球氮素循环的重要分支,尽管已被发现广泛存在于细菌、真菌和古生菌中,其功能基因的研究仍仅限于很少几个物种。现代分子生物学的发展为研究环境微生物提供了行之有效的方法,以反硝化功能基因作为分子标记的分子生态学研究迅猛发展。综述近年来国内外微生物反硝化功能基因研究及以其为标记的分子生态学研究进展。     

不同季节艾比湖湿地盐角草根际nirS-型与nirK-型反硝化细菌群落组成分析

旨在了解艾比湖湿地盐生植物盐角草根际与非根际中不同类型反硝化细菌的分布及其随季节变化情况,为温带干旱地区荒漠盐化生态系统的代表-艾比湖湿地在生态植被恢复过程中,由微生物推动的土壤氮素循环过程提供数据支撑。采集了艾比湖湿地夏、秋、春三个季节的盐角草根际和非根际土壤样本,通过高通量测序技术,比较分析了nirS-型和nirK-型两种类型的反硝化细菌的多样性和群落结构特点;利用RDA (redundancy analysis)探究了土壤理化因素对反硝化细菌多样性及群落结构的影响。艾比湖湿地盐角草根际与非根际中,nirS-型和nirK-型反硝化细菌多样性最高的为秋季根际土壤样本;各土壤样本中的反硝化细菌多样性均呈现根际>非根际。盐角草各土壤样本中的nirS-型反硝化细菌在门分类水平上隶属于变形菌门(Proteobacteria),厚壁菌门(Firmicutes)和放线菌门(Actinobacteria),而nirK-型反硝化细菌在门水平上分类仅包括了Proteobacteria和Firmicutes。Proteobacteria在各土壤样本中的占比均较高;其中Gamma-Proteobacteria的盐单胞菌属(Halomonas)和假单胞菌属(Pseudomonas)是各土壤样本所共有的nirS-型反硝化菌的优势菌属,但它们在每个土壤样本中的相对丰度各有差异。Alpha-Proteobacteria的根瘤菌属(Rhizobium)是盐角草各土壤样本中较为广泛存在的nirK-型反硝化细菌。艾比湖湿地盐角草各土壤样本中的反硝化细菌群落结构存在着一定的差异。RDA结果显示含水量、有机质、全氮和铵态氮等对各土壤样本中的nirS-型反硝化细菌的多样性影响较大,含水量、有机质、全氮、碱解氮等是nirK-型反硝化细菌多样性的主要影响因素。土壤电导率、全磷、全钾、全氮和碱解氮协同影响nirS-型反硝化细菌的群落结构,有机质、速效钾、速效磷、pH和硝态氮是nirK-型反硝化细菌群落结构组成的主要影响因素。艾比湖湿地反硝化细菌呈现季节性变化,nirS-型和nirK-型反硝化细菌以不同的主要菌属,共同推进湿地反硝化作用。而对于湿地生态系统的保护,则需要进行长期而广泛的土壤状态评估和土壤反硝化微生物菌群的动态监测。     

Denitrification likely catalyzed by endobionts in an allogromiid foraminifer

Nitrogen can be a limiting macronutrient for carbon uptake by the marine biosphere. The process of denitrification (conversion of nitrate to gaseous compounds, including N₂ (nitrogen gas)) removes bioavailable nitrogen, particularly in marine sediments, making it a key factor in the marine nitrogen budget. Benthic foraminifera reportedly perform complete denitrification, a process previously considered nearly exclusively performed by bacteria and archaea. If the ability to denitrify is widespread among these diverse and abundant protists, a paradigm shift is required for biogeochemistry and marine microbial ecology. However, to date, the mechanisms of foraminiferal denitrification are unclear, and it is possible that the ability to perform complete denitrification is because of the symbiont metabolism in some foraminiferal species. Using sequence analysis and GeneFISH, we show that for a symbiont-bearing foraminifer, the potential for denitrification resides in the endobionts. Results also identify the endobionts as denitrifying pseudomonads and show that the allogromiid accumulates nitrate intracellularly, presumably for use in denitrification. Endobionts have been observed within many foraminiferal species, and in the case of associations with denitrifying bacteria, may provide fitness for survival in anoxic conditions. These associations may have been a driving force for early foraminiferal diversification, which is thought to have occurred in the Neoproterozoic era when anoxia was widespread.     

Effects of pentachlorophenol on the bacterial denitrification process

The use of pentachlorophenol (PCP) was banned or restricted in many countries worldwide because of its adverse influences on the ecological environment and humans. However, the potential disrupting effects of PCP on denitrifying microorganisms have warranted more analysis. In this study, the impacts of PCP on denitrification were investigated by using Paracoccus denitrificans as a model denitrifying bacterium. Compared with the control, the presences of 10 and 50 μM of PCP were found to significantly decrease the denitrification efficiencies from 98.5 to 87.2% and 68.7%, respectively. The mechanism studies showed that PCP induced the generation of reactive oxygen species, which decreased the vital enzymes activities related to glycolysis process, causing the disturbance of the metabolism of P. denitrificans utilizing carbon source (glucose) and the growth of the cell, and subsequently the generation of electron donor (NADH) for denitrification via NAD⁺ reduction was severely depressed. Further studies indicated that PCP also decreased the genes expression of several key enzymes responsible for denitrification, such as napA of nitrate reductase (NAR), nirS of nitrite reductase, norB of nitric oxide reductase, and nosZ of nitrous oxide reductase; however, there was only the enzyme activity of NAR was remarkably inhibited.     

Identifying causes for N2O accumulation in a lab-scale sequencing batch reactor performing simultaneous nitrification, denitrification and phosphorus removal

The recently described process of simultaneous nitrification, denitrification and phosphorus removal (SNDPR) has a great potential to save capital and operating costs for wastewater treatment plants. However, the presence of glycogen-accumulating organisms (GAOs) and the accumulation of nitrous oxide (N(2)O) can severely compromise the advantages of this process. In this study, these two issues were investigated using a lab-scale sequencing batch reactor performing SNDPR over a 5-month period. The reactor was highly enriched in polyphosphate-accumulating organisms (PAOs) and GAOs representing around 70% of the total microbial community. PAOs were the dominant population at all times and their abundance increased, while GAOs population decreased over the study period. Anoxic batch tests demonstrated that GAOs rather than denitrifying PAOs were responsible for denitrification. N(2)O accumulated from denitrification and more than half of the nitrogen supplied in a reactor cycle was released into the atmosphere as N(2)O. After mixing SNDPR sludge with other denitrifying sludge, N(2)O present in the bulk liquid was reduced immediately if external carbon was added. We therefore suggest that the N(2)O accumulation observed in the SNDPR reactor is an artefact of the low microbial diversity facilitated by the use of synthetic wastewater with only a single carbon source.     

Nitrogen-Cycling Genes in Epilithic Biofilms of Oligotrophic High-Altitude Lakes (Central Pyrenees,Spain)

Microbial biofilms in oligotrophic environments are the most reactive component of the ecosystem. In high-altitude lakes, exposed bedrock, boulders, gravel, and sand in contact with highly oxygenated water and where a very thin epilithic biofilm develops usually dominate the littoral zone. Traditionally, these surfaces have been considered unsuitable for denitrification, but recent investigations have shown higher biological diversity than expected, including diverse anaerobic microorganisms. In this study, we explored the presence of microbial N-cycling nirS and nirK (denitrification through the conversion of NO₂ ^? to NO), nifH (N₂ fixation), anammox (anaerobic ammonium oxidation), and amoA (aerobic ammonia oxidation, both bacterial and archaeal) genes in epilithic biofilms of a set of high-altitude oligotrophic lakes in the Pyrenees. The concentrations of denitrifying genes determined by quantitative PCR were two orders of magnitude higher than those of ammonia-oxidizing genes. Both types of genes were significantly correlated, suggesting a potential tight coupling nitrification-denitrification in these biofilms that deserves further confirmation. The nifH gene was detected after nested PCR, and no signal was detected for the anammox-specific genes used. The taxonomic composition of denitrifying and nitrogen-fixing genes was further explored by cloning and sequencing. Interestingly, both microbial functional groups were richer and more genetically diverse than expected. The nirK gene, mostly related to Alphaproteobacteria (Bradyrhizobiaceae), dominated the denitrifying gene pool as expected for oxygen-exposed habitats, whereas Deltaproteobacteria (Geobacter like) and Cyanobacteria were the most abundant among nitrogen fixers. Overall, these results suggest an epilithic community more metabolically diverse than previously thought and with the potential to carry out an active role in the biogeochemical nitrogen cycling of high-altitude ecosystems. Measurements of activity rates should be however carried out to substantiate and further explore these findings.     

Antarctic microbial diversity: the basis of polar ecosystem processes

Microorganisms are fundamental to the functioning of Antarctic ecosystems. Although microbial biomass can be immense in Southern Ocean blooms and freshwater cyanobacterial mats, species richness is generally more restricted than it is in temperate regions. However, there are representatives of a broad variety of taxa providing a diverse gene pool. Species diversity may be low while metabolic flexibility is high so that a few strains can provide most necessary functions. In this context, biodiversity is the sum of biological potential. This Special Issue highlights aspects of microbial ecology that can be studied only in Antarctica or which are defined most clearly in Antarctic habitats. Relatively simple microbial communities, or conspicuous species within them, can be used as indicators of microbial processes and responses to environmental change. These include the palaeological record of benthic diatoms and response of soil cyanobacterial communities to regional warming and UV-B stress. The climatic conditions and relict babitats of the Antarctic dry valleys are a valuable analogue for detecting microbial life and diversity on Mars. The global microbial biodiversity initiative Diversitas and international Antarctic networks such as BIOTAS (Biological Investigations of Terrestrial Antarctic Systems) harness taxonomic and ecophysiological expertize to understand better these unique polar ecosystems.     

Soil microbes of an urban remnant riparian zone have greater potential for N removal than a degraded riparian zone

Soils in the riparian zone, the interface between terrestrial and aquatic ecosystems, may decrease anthropogenic nitrogen (N) loads to streams through microbial transformations (e.g., denitrification). However, the ecological functioning of riparian zones is often compromised due to degraded conditions (e.g., vegetation clearing). Here we compare the efficacy of an urban remnant and a cleared riparian zone for supporting a putative denitrifying microbial community using 16S rRNA sequencing and quantitative polymerase chain reaction of archaeal and bacterial nitrogen cycling genes. Although we had no direct measure of denitrification rates, we found clear patterns in the microbial communities between the sites. Greater abundance of N-cycling genes was predicted by greater soil ammonium (N-NH₄), organic phosphorus, and C:N. At the remnant site, we found positive correlations between microbial community composition, which was dominated by putative N oxidisers (Nitrosomonadaceae, Nitrospiraceae and Nitrosotaleaceae), and abundance of ammonia-oxidizing archaea (AOA), nirS, nirK and nosZ, whereas the cleared site had lower abundance of N-oxidisers and N cycling genes. These results were especially profound for the remnant riparian fringe, which suggests that this region maintains suitable soil conditions (via diverse vegetation structure and periodic saturation) to support putative N cyclers, which could amount to higher potential for N removal.     

Characterization of microbial community in an aerobic moving bed biofilm reactor applied for simultaneous nitrification and denitrification

A continuous-flow moving bed biofilm reactor (MBBR) under aerobic conditions was established for simultaneous nitrification and denitrification (SND), and microbial communities were investigated by a combination of denaturing gel gradient electrophoresis (DGGE) and fluorescence in situ hybridization (FISH). DGGE analysis has revealed more similar microbial community structures formed in the biofilms with more similar carbon nitrogen (C/N) ratios. FISH analysis shows that the dominance of both Betaproteobacteria ammonia-oxidizing bacteria and Nitrospira-like nitrite-oxidizing bacteria were negatively correlated to C/N ratios. Sequence analysis of DGGE bands has indicated the presence of anoxic denitrifying bacteria Agrobacterium tumefaciens and Rhizobium sp., suggesting that the oxygen gradient inside the biofilm may be responsible for the mechanism of SND in aerobic MBBRs. The study confirms that appropriate control of microbial community structure resulting from optimal C/N ratio is beneficial in improving SND, thus optimizing nitrogen removal in aerobic MBBR. The established SND-based MBBR can save operation space and time in comparison to the traditional nitrogen removal process, and might be very attractive for future practical applications.     

Targeted metagenomics reveals extensive diversity of the denitrifying community in partial nitritation anammox and activated sludge systems

The substantial presence of denitrifiers has already been reported in partial nitritation anammox (PNA) systems using the 16S ribosomal RNA (rRNA) gene, but little is known about the phylogenetic diversity based on denitrification pathway functional genes. Therefore, we performed a metagenomic analysis to determine the distribution of denitrification genes and the associated phylogeny in PNA systems and whether a niche separation between PNA and conventional activated sludge (AS) systems exists. The results revealed a distinct abundance pattern of denitrification pathway genes and their association to the microbial species between PNA and AS systems. In contrast, the taxonomic analysis, based on the 16S rRNA gene, did not detect notable variability in denitrifying community composition across samples. In general, narG and nosZa2 genes were dominant in all samples. While the potential for different stages of denitrification was redundant, variation in species composition and lack of the complete denitrification gene pool in each species appears to confer niche separation between PNA and AS systems. This study suggests that targeted metagenomics can help to determine the denitrifying microbial composition at a fine‐scale resolution while overcoming current biases in quantitative polymerase chain reaction approaches due to a lack of appropriate primers.     

不同恢复阶段热带森林土壤nirS型反硝化微生物群落结构及多样性特征

探明热带森林土壤反硝化微生物群落结构及多样性,对于理解反硝化引起的N₂O排放及缓解全球变暖具有重要意义。本研究以西双版纳3个不同恢复阶段热带森林类型[即白背桐（Mallotus paniculatus,MP）、崖豆藤（Millttia leptobotrya,ML）群落、群落及高檐蒲桃（Syzygium oblatum,SO）群落]为研究对象,揭示土壤nirS型反硝化微生物群落组成及多样性的干湿季变化,分析热带森林恢复过程中土壤理化环境变化对nirS型反硝化细菌群落的影响。结果表明,变形菌门（Proteobacteria）和酸杆菌门（Acidobacteria）相对丰度表现为恢复前期高于恢复后期,而脱氯单胞菌属（Dechloromonas）、嗜盐单胞菌属（Halomonas）和罗思河小杆菌属（Rhodanobacter）表现为恢复后期高于恢复前期;绿弯菌门（Chloroflexi）和放线菌门（Actinobacteria）均随恢复年限增加而增加,而贪铜菌属（Cupriavidus）和假单胞菌属（Pseudomonas）的相对丰度表现为随恢复年限增加而降低。9月份各样地新检测出的属数量表现为：SO （19种） > MP （13种） > ML （7种）。土壤nirS型反硝化微生物群落的Shannon多样性指数表现为：高檐蒲桃群落 > 崖豆藤群落 > 白背桐群落,且9月（湿季） > 3月（干季）。相关分析表明,热带森林恢复引起土壤N库（全氮、NH₄⁺、NO₃^-）、C有效性（微生物量碳、易氧化碳）及微气候（土壤含水率与温度）的改变,能够显著影响nirS型反硝化细菌群落的结构及多样性。主成分分析结果表明,土壤硝态氮、微生物量碳、全氮及易氧化碳是调控不同恢复阶段热带森林土壤nirS型反硝化细菌群落结构及多样性变化的主控因子,其次为土壤水分、温度、水解氮、pH、铵态氮、有机碳、容重及C/N。     

Nitrogen Turnover in Drying Sediments of an Amazon Floodplain Lake

In the Amazon floodplain large areas are subject to annual cycles of drying and rewetting. The turnover of nitrogen in the periodically drying sediments is an important regulator of floodplain fertility. In the present study the transition of a lake sediment from flooded to dry conditions was studied with respect to microbial nitrogen turnover. Soil nitrogen pools, as well as the activity and abundance of denitrifying and nitrifying bacteria, were investigated during one dry season. During the first weeks after drying, most of the inorganic nitrogen vanished from the sediment. The process was inhibited by a nitrification inhibitor, showing that coupled nitrification–denitrification was responsible for the nitrogen loss. Assimilation by plants or microbes, as well as leaching, were not important mechanisms of nitrogen loss. During a period of only 10 days, 59% of the total denitrification and 94% of the total N₂O emission during the dry period occurred. Cell numbers of denitrifiers were not correlated with activities. Denitrification was not correlated with other sediment variables but was regulated by the patchy distribution of reduced and oxidized zones in the uppermost centimeters of the sediment. This heterogeneity was probably introduced by the bioturbation of small insects, which was restricted to a rather short time period shortly after drying.     

Prokaryotic community dynamics and nitrogen-cycling genes in an oxygen-deficient upwelling system during La Niña and El Niño conditions

Dissolved oxygen regulates microbial distribution and nitrogen cycling and, therefore, ocean productivity and Earth's climate. To date, the assembly of microbial communities in relation to oceanographic changes due to El Niño Southern Oscillation (ENSO) remains poorly understood in oxygen minimum zones (OMZ). The Mexican Pacific upwelling system supports high productivity and a permanent OMZ. Here, the spatiotemporal distribution of the prokaryotic community and nitrogen-cycling genes was investigated along a repeated transect subjected to varying oceanographic conditions associated with La Niña in 2018 and El Niño in 2019. The community was more diverse during La Niña and in the aphotic OMZ, dominated by the Subtropical Subsurface water mass, where the highest abundances of nitrogen-cycling genes were found. The largest proportion of the Gulf of California water mass during El Niño provided warmer, more oxygenated, and nutrient-poor waters towards the coast, leading to a significant increase of Synechococcus in the euphotic layer compared with the opposite conditions during La Niña. These findings suggest that prokaryotic assemblages and nitrogen genes are linked to local physicochemical conditions (e.g. light, oxygen, nutrients), but also to oceanographic fluctuations associated with ENSO phases, indicating the crucial role of climate variability in microbial community dynamics in this OMZ.     

Denitrification performance and microbial diversity in a packed-bed bioreactor using PCL as carbon source and biofilm carrier

Polycaprolactone (PCL) was used as both carbon source and biofilm support for denitrifying bacteria in a packed-bed bioreactor. The denitrification performance and microbial diversity were investigated. The microbial community of biofilm developed on the surface of PCL in the reactor was analyzed by pyrosequencing method. The experimental results showed the average nitrate removal efficiency reached 93 % at stable operation. ESEM observation and FTIR analysis were conducted to characterize the PCL structure before and after microbial utilization. For the microbial community, Betaproteobacteria predominated, and most of the PCL-degrading denitrifying bacteria assigned to the family of Comamonadacea. Denitrifying bacteria accounted for more than 20 % in the total population, indicating that PCL is a good carrier and carbon source for biological denitrification.