莫克兰冷泉区沉积物原核微生物群落组成及其环境响应

【目的】冷泉系统广泛存在于大陆边缘地区,其典型特征是在海底渗漏出大量富含以甲烷为主的碳氢化合物和硫化氢等成分的低温流体。冷泉也因其独特的地球化学条件孕育着独特的原核微生物群落结构,然而,原核微生物组成与冷泉环境之间的响应关系却并不清楚。【方法】本文以莫克兰大陆边缘活跃冷泉区沉积物柱状样为研究对象,沿深度剖面分析了沉积物中的CH4以及孔隙水SO42–、H2S浓度等关键地球化学参数,并基于16S rRNA基因高通量测序对冷泉沉积物原核微生物的群落结构及其空间变化进行了系统分析。【结果】根据其硫酸盐-甲烷浓度剖面特征,从上向下,将沉积物垂向剖面划分为硫酸盐还原区(SZ)、硫酸盐-甲烷转换区(SMTZ)和产甲烷区(MZ)。通过原核微生物α多样性与基因定量研究发现,随着深度增加微生物多样性与丰度呈逐渐降低的趋势。16SrRNA基因高通量测序结果表明,SZ中以硫氧化细菌γ-变形菌纲、α-变形菌纲和埃普西隆杆菌门为主,且以硫酸盐为电子受体的与有机质降解相关的原核微生物JS1、绿弯菌门、洛基古菌纲、深古菌纲及底栖古菌纲的相对含量也较高;SMTZ存在较高含量的ANME-1a、ANME-1b与SEEP-S...     

Combined removal of sulfur compounds and nitrate by autotrophic denitrification in bioaugmented activated sludge system

An autotrophic denitrification process using reduced sulfur compounds (thiosulfate and sulfide) as electron donor in an activated sludge system is proposed as an efficient and cost effective alternative to conventional heterotrophic denitrification for inorganic (or with low C/N ratio) wastewaters and for simultaneous removal of sulfide or thiosulfate and nitrate. A suspended culture of sulfur-utilizing denitrifying bacteria was fast and efficiently established by bio-augmentation of activated sludge with Thiobacillus denitrificans. The stoichiometry of the process and the key factors, i.e. N/S ratio, that enable combined sulfide and nitrogen removal, were determined. An optimum N/S ratio of 1 (100% nitrate removal without nitrite formation and low thiosulfate concentrations in the effluent) has been obtained during reactor operation with thiosulfate at a nitrate loading rate (NLR) of 17.18 mmol N L(-1) d(-1). Complete nitrate and sulfide removal was achieved during reactor operation with sulfide at a NLR of 7.96 mmol N L(-1) d(-1) and at N/S ratio between 0.8 and 0.9, with oxidation of sulfide to sulfate. Complete nitrate removal while working at nitrate limiting conditions could be achieved by sulfide oxidation with low amounts of oxygen present in the influent, which kept the sulfide concentration below inhibitory levels.     

南海Formosa冷泉区沉积物微生物多样性与分布规律研究

【目的】当前对全球冷泉生态系统微生物生态学研究显示，冷泉生态系统中主要微生物类群为参与甲烷代谢的微生物，它们的分布差异与所处冷泉区生物地球化学环境密切相关。但在冷泉区内也存在环境因子截然不同的生境，尚缺乏比较冷泉区内小尺度生境间微生物多样性和分布规律的研究。本研究旨在分析南海Formosa冷泉区内不同生境间微生物多样性差异，完善和理解不同环境因子对冷泉内微生物群落结构的影响。【方法】对采集自南海Formosa冷泉区不同生境(黑色菌席区、白色菌席区和碳酸盐岩区)沉积物样本中古菌和细菌16S rRNA基因进行测序，结合环境因子，比较微生物多样性差异，分析环境因子对微生物分布的影响。【结果】发现在Formosa冷泉内的不同生境中，甲烷厌氧氧化古菌(anaerobic methanotrophic archaea,ANME)是主要古菌类群，占古菌总体相对丰度超过70%；在菌席区ANME-1b和ANME-2a/b是主要ANME亚群，碳酸盐岩区则是ANME-1b。硫酸盐还原菌(sulfate-reducing bacteria,SRB)和硫氧化菌(sulfur-oxidizing bacteria...     

Benthic organic carbon influences denitrification in streams with high nitrate concentration

1. Anthropogenic activities have increased reactive nitrogen availability, and now many streams carry large nitrate loads to coastal ecosystems. Denitrification is potentially an important nitrogen sink, but few studies have investigated the influence of benthic organic carbon on denitrification in nitrate‐rich streams. 2. Using the acetylene‐block assay, we measured denitrification rates associated with benthic substrata having different proportions of organic matter in agricultural streams in two states in the mid‐west of the U.S.A., Illinois and Michigan. 3. In Illinois, benthic organic matter varied little between seasons (5.9–7.0% of stream sediment), but nitrate concentrations were high in summer (>10 mg N L⁻¹) and low (<0.5 mg N L⁻¹) in autumn. Across all seasons and streams, the rate of denitrification ranged from 0.01 to 4.77 μg N g⁻¹ DM h⁻¹ and was positively related to stream‐water nitrate concentration. Within each stream, denitrification was positively related to benthic organic matter only when nitrate concentration exceeded published half‐saturation constants. 4. In Michigan, streams had high nitrate concentrations and diverse benthic substrata which varied from 0.7 to 72.7% organic matter. Denitrification rate ranged from 0.12 to 11.06 μg N g⁻¹ DM h⁻¹ and was positively related to the proportion of organic matter in each substratum. 5. Taken together, these results indicate that benthic organic carbon may play an important role in stream nitrogen cycling by stimulating denitrification when nitrate concentrations are high.     

Nitrification, denitrification, and nitrate ammonification in sediments of two coastal lagoons in Southern France

Summary Seasonal and diurnal variations in sediment-water fluxes of O₂, NO ₃ ^– , and NH ₄ ⁺ as well as rates of nitrification, denitrification, and nitrate ammonification were determined in two different coastal lagoons of southern France: The seagrass (Zostera noltii) dominated tidal Bassin d'Arcachon and the dystrophic Etang du Prévost. Overall, denitrification rates in both Bassin d'Arcachon (<0.4 mmol m^–2 d^–1) and Etang du Prévost (<1 mmol m^–2 d^–1) were low. This was mainly caused by a combination of low NO ₃ ^– concentrations in the water column and a low nitrification activity within the sediment. In both Bassin d'Arcachon and Etang du Prévost, rates of nitrate ammonification were quantitatively as important as denitrification.Denitrification played a minor role as a nitrogen sink in both systems. In the tidal influenced Bassin d'Arcachon, Z. noltii was quantitatively more important than denitrification as a nitrogen sink due to the high assimilation rates of the plants. Throughout the year, Z. noltii stabilized the mudflats of the bay by its well- developed root matrix and controlled the nitrogen cycle due to its high uptake rates. In contrast, the lack of rooted macrophytes, and dominance of floating macroalgae, made nitrogen cycling in Etang du Prévost more unstable and unpredictable. Inhibition of nitrification and denitrification during the dystrophic crisis in the summer time increased the inorganic nitrogen flux from the sediment to the water column and thus increased the degree of benthic-pelagic coupling within this bay. During winter, however, benthic microalgae colonizing the sediment surface changed the sediment in the lagoon from being a nitrogen source to the over lying water to being a sink due to their high assimilation rates. It is likely, however, that this assimilated nitrogen is liberated to the water column at the onset of summer thereby fueling the extensive growth of the floating macroalgae, Ulva sp. The combination of a high nitrogen coupling between sediment and water column, little water exchange and low denitrification rates resulted in an unstable system with fast growing algal species such as phytoplankton and floating algae.     

Diverse sulfate-reducing bacteria of the Desulfosarcina/Desulfococcus clade are the key alkane degraders at marine seeps

Biogeochemical and microbiological data indicate that the anaerobic oxidation of non-methane hydrocarbons by sulfate-reducing bacteria (SRB) has an important role in carbon and sulfur cycling at marine seeps. Yet, little is known about the bacterial hydrocarbon degraders active in situ. Here, we provide the link between previous biogeochemical measurements and the cultivation of degraders by direct identification of SRB responsible for butane and dodecane degradation in complex on-site microbiota. Two contrasting seep sediments from Mediterranean Amon mud volcano and Guaymas Basin (Gulf of California) were incubated with ¹³C-labeled butane or dodecane under sulfate-reducing conditions and analyzed via complementary stable isotope probing (SIP) techniques. Using DNA- and rRNA-SIP, we identified four specialized clades of alkane oxidizers within Desulfobacteraceae to be distinctively active in oxidation of short- and long-chain alkanes. All clades belong to the Desulfosarcina/Desulfococcus (DSS) clade, substantiating the crucial role of these bacteria in anaerobic hydrocarbon degradation at marine seeps. The identification of key enzymes of anaerobic alkane degradation, subsequent β-oxidation and the reverse Wood–Ljungdahl pathway for complete substrate oxidation by protein-SIP further corroborated the importance of the DSS clade and indicated that biochemical pathways, analog to those discovered in the laboratory, are of great relevance for natural settings. The high diversity within identified subclades together with their capability to initiate alkane degradation and growth within days to weeks after substrate amendment suggest an overlooked potential of marine benthic microbiota to react to natural changes in seepage, as well as to massive hydrocarbon input, for example, as encountered during anthropogenic oil spills.     

Nitrogen mass balances and denitrification rates in central Ontario Lakes

Nitrogen mass balances for seven unproductive lakes and 20 forested catchments in central Ontario were measured between 1977 and 1989. Average annual lake denitrification rates calculated with the N/P ratio method were strongly correlated with summer anoxic factor (extent of surficial sediment anoxia) whereas denitrification rates calculated with a²¹⁰Pb sediment N accumulation method were poorly correlated with the anoxic factor suggesting that the N/P method is superior. Substantial denitrification occurred in all lakes — an average of 36% of TN inputs or 75% of the net gain. On a regional area-weighted basis, 67% of bulk atmospheric TN deposition was stored or denitrified terrestrially, 12% was denitrified in lakes, 4% was stored in lake sediments, and 17% was exported from lakes. N/P ratios were generally less in streams than in precipitation suggesting preferential N retention in catchments, whereas the N/P ratios in lake outputs were slightly higher than lake input ratios, suggesting preferential P retention in lakes. This is consistent with the notion that P-limited lakes can exist adjacent to N-limited forests.     

Potential denitrification in submerged natural and impacted sediments of Lake Batata,an Amazonian lake

Lake Batata is one of many clear water lakes located on the floodplain of the Trombetas River in the northern Brazilian Amazon. Lake Batata is distinctly different from other lakes of this region because, for a period of 10 years, its waters received tremendous amounts of aluminum ore tailings from a bauxite mining operation. Approximately 30% of the sediments of the upper basin of the 2100 hectare lake were covered by tailings before dumping was curtailed. The goal of this research was to identify factors controlling denitrification in the natural and impacted sediments of Lake Batata. Rates of denitrification in sediments were estimated in the laboratory by the acetylene blockage method. Denitrification was measured under four conditions: without amendment; amended with glucose; amended with nitrate; and amended with glucose and nitrate. Denitrification was observed only in assays amended with nitrate suggesting that availability of nitrate is a principle factor for controlling denitrification in the sediments of Lake Batata. Effects of nitrate amendments are most pronounced when the water level is low, i.e. during the hydroperiods of draw-down and low-water.     

Simultaneous occurrence of denitrification and nitrate ammonification in sediments of the French Mediterranean Coast

Dissimilatory nitrate reductions in coastal marine sediment of Carteau Cove (French Mediterranean Coast) were studied between April 1993 and July 1994. Simultaneous determination of denitrification and dissimilatory nitrate reduction to ammonium was achieved by using a combination of acetylene blockage and 15N techniques. After short incubations (maximum 5 h), a part of 15N labelled nitrate added to the sediment was recovered as ammonium without incorporation in organic matter. The result indicate that a fraction of nitrate was reduced to ammonium by a dissimilatory mechanism instead of denitrifying. Denitrifying and nitrate ammonifying activities ranged from 0 to 19.8 μmol l-1 d-1 and from 2.3 to 83.2 μmol l-1 d-1, respectively. Denitrification rates were highest in early spring whereas nitrate ammonification were highest in fall. The recovery of nitrate reduced as N2O-N plus ammonium was between 40 and 100%, the highest nitrogen losses were recorded in July. Depending on the station and time of year denitrification accounted for between 0 and 43% of the total nitrate reduction whereas dissimilatory nitrate reduction to ammonium (DNRA) accounted for between 18 and 100%. The reduction rate data suggest that the pathway of nitrate reduction to ammonium may be important in coastal sediments. This revised version was published online in July 2006 with corrections to the Cover Date.     

Quantification of mcrA by quantitative fluorescent PCR in sediments from methane seep of the Nankai Trough

A quantitative fluorogenic PCR method for group-specific methyl coenzyme M reductase subunit A genes (mcrA) from methanotrophic archaea was established and applied to the characterization of microbial communities in anoxic methane seep sediments at the accretionary prism of the Nankai Trough. All of the previously identified subgroups of anaerobic methanotroph (ANME) mcrA genes were detected in the cores up to 25 cm below the seafloor, but distributional patterns of mcrA genes were found to differ according to depth. These findings suggest a distinct distribution of phylogenetically and physiologically diverse methanotrophic archaea that mediate methane oxidation in the anoxic sediments. This quantification method will contribute to future investigations of methanotrophic microbial ecosystems in anoxic marine sediments.     

Identity of epibiotic bacteria on symbiontid euglenozoans in O2-depleted marine sediments: evidence for symbiont and host co-evolution

A distinct subgroup of euglenozoans, referred to as the ‘Symbiontida,'' has been described from oxygen-depleted and sulfidic marine environments. By definition, all members of this group carry epibionts that are intimately associated with underlying mitochondrion-derived organelles beneath the surface of the hosts. We have used molecular phylogenetic and ultrastructural evidence to identify the rod-shaped epibionts of the two members of this group, Calkinsia aureus and B.bacati, hand-picked from the sediments of two separate oxygen-depleted, sulfidic environments. We identify their epibionts as closely related sulfur or sulfide-oxidizing members of the epsilon proteobacteria. The epsilon proteobacteria generally have a significant role in deep-sea habitats as primary colonizers, primary producers and/or in symbiotic associations. The epibionts likely fulfill a role in detoxifying the immediate surrounding environment for these two different hosts. The nearly identical rod-shaped epibionts on these two symbiontid hosts provides evidence for a co-evolutionary history between these two sets of partners. This hypothesis is supported by congruent tree topologies inferred from 18S and 16S rDNA from the hosts and bacterial epibionts, respectively. The eukaryotic hosts likely serve as a motile substrate that delivers the epibionts to the ideal locations with respect to the oxic/anoxic interface, whereby their growth rates can be maximized, perhaps also allowing the host to cultivate a food source. Because symbiontid isolates and additional small subunit rDNA gene sequences from this clade have now been recovered from many locations worldwide, the Symbiontida are likely more widespread and diverse than presently known.     

脱氮微生物及脱氮工艺研究进展

脱氮是大部分污水处理系统中不可缺少的一环。由于具有经济高效、工艺简单和无二次污染等显著优势,生物脱氮工艺在最近数十年中备受关注。根据脱氮微生物的生理特性和脱氮机制不同,文中分类综述了近年来生物脱氮工艺的研究进展,重点对比分析了硝化菌、反硝化菌和厌氧氨氧化菌以及以这些菌为基础的不同生物脱氮工艺的优缺点,为复杂污水环境的脱氮工艺选择提供参考。基于微生物脱氮机制,通过合成生物学技术开发高效脱氮菌株,结合不同工艺优点并应用自动化模拟最佳条件,从而建立经济高效的脱氮工艺将是未来发展的重要方向。     

Potential rates and pathways of microbial nitrate reduction in coastal sediments

Nitrate reduction plays a key role in the biogeochemical dynamics and microbial ecology of coastal sediments. Potential rates of nitrate reduction were measured on undisturbed sediment slices from two eutrophic coastal environments using flow-through reactors (FTR). Maximum potential nitrate reduction rates ranged over an order of magnitude, with values of up to 933 nmol cm(-3) h(-1), whereas affinity constants for NO(3) (-) fell mostly between 200 and 600 microM. Homogenized sediment slurries systematically yielded higher rates of nitrate reduction than the FTR experiments. Dentrification was the major nitrate removal pathway in the sediments, although excess ammonium production indicated a contribution of dissimilatory nitrate reduction to ammonium under nitrate-limiting conditions.     

Elevated nitrate enriches microbial functional genes for potential bioremediation of complexly contaminated sediments

Nitrate is an important nutrient and electron acceptor for microorganisms, having a key role in nitrogen (N) cycling and electron transfer in anoxic sediments. High-nitrate inputs into sediments could have a significant effect on N cycling and its associated microbial processes. However, few studies have been focused on the effect of nitrate addition on the functional diversity, composition, structure and dynamics of sediment microbial communities in contaminated aquatic ecosystems with persistent organic pollutants (POPs). Here we analyzed sediment microbial communities from a field-scale in situ bioremediation site, a creek in Pearl River Delta containing a variety of contaminants including polybrominated diphenyl ethers (PBDEs) and polycyclic aromatic hydrocarbons (PAHs), before and after nitrate injection using a comprehensive functional gene array (GeoChip 4.0). Our results showed that the sediment microbial community functional composition and structure were markedly altered, and that functional genes involved in N-, carbon (C)-, sulfur (S)-and phosphorus (P)- cycling processes were highly enriched after nitrate injection, especially those microorganisms with diverse metabolic capabilities, leading to potential in situ bioremediation of the contaminated sediment, such as PBDE and PAH reduction/degradation. This study provides new insights into our understanding of sediment microbial community responses to nitrate addition, suggesting that indigenous microorganisms could be successfully stimulated for in situ bioremediation of POPs in contaminated sediments with nitrate addition.     

Molecular phylogenetic and chemical analyses of the microbial mats in deep-sea cold seep sediments at the northeastern Japan Sea

Microbial communities inhabiting deep-sea cold seep sediments at the northeastern Japan Sea were characterized by molecular phylogenetic and chemical analyses. White patchy microbial mats were observed along the fault offshore the Hokkaido Island and sediment samples were collected from two stations at the southern foot of the Shiribeshi seamount (M1 site at a depth of 2,961 m on the active fault) and off the Motta Cape site (M2 site at a depth of 3,064 m off the active fault). The phylogenetic and terminal-restriction fragment polymorphism analyses of PCR-amplified 16S rRNA genes revealed that microbial community structures were different between two sampling stations. The members of ANME-2 archaea and diverse bacterial components including sulfate reducers within Deltaproteobacteria were detected from M1 site, indicating the occurrence of biologically mediated anaerobic oxidation of methane, while microbial community at M2 site was predominantly composed of members of Marine Crenarchaeota group I, sulfate reducers of Deltaproteobacteria, and sulfur oxidizers of Epsilonproteobacteria. Chemical analyses of seawater above microbial mats suggested that concentrations of sulfate and methane at M1 site were largely decreased relative to those at M2 site and carbon isotopic composition of methane at M1 site shifted heavier (¹³C-enriched), the results of which are consistent with molecular analyses. These results suggest that the mat microbial communities in deep-sea cold seep sediments at the northeastern Japan Sea are significantly responsible for sulfur and carbon circulations and the geological activity associated with plate movements serves unique microbial habitats in deep-sea environments.     

Estimating denitrification rates in estuarine sediments: A comparison of stoichiometric and acetylene based methods

We compared denitrification rates obtained using an adaptation of the acetylene block technique to rates estimated from benthic flux nutrient stoichiometry in the subtidal sediments of Tomales Bay, California (USA). By amending whole cores with acetylene and saturating nitrate concentrations, we obtained potential denitrification rates, which ranged between 4 and 30 mmol N m^–2 d^–1. We determined the apparent Michaelis constant (K_app) and the maximum potential rate (V_mp) of the denitrifying community and used these constants in a rectangular hyperbola to estimatein situ denitrification rates. Both the K_app and V_mp of the denitrifying community exhibited significant variation over both depth in the sediment column and time of sampling.Estimates ofin situ denitrification obtained using our kinetic-fix adaptation of the acetylene block ranged between 1.8 (March) and 9 (Sept.) mmol N m^–1 d^–1. Denitrification rates obtained using benthic flux stoichiometry ranged between 0.7 and 4.1 mmol N m^–2 d^–1. Average denitrification rates obtained using the kinetic-fix acetylene block approach exceeded those obtained from net benthic flux stoichiometry; however, these differences were not significant. We conclude that our kinetic-fix adaptation of the acetylene block technique provides realistic estimates of denitrification in sediments, even when pore water nitrate concentrations are low and nitrification and denitrification are closely coupled.     

High rates of denitrification and nitrous oxide emission in arid biological soil crusts from the Sultanate of Oman

Using a combination of process rate determination, microsensor profiling and molecular techniques, we demonstrated that denitrification, and not anaerobic ammonium oxidation (anammox), is the major nitrogen loss process in biological soil crusts from Oman. Potential denitrification rates were 584±101 and 58±20 μmol N m⁻² h⁻¹ for cyanobacterial and lichen crust, respectively. Complete denitrification to N₂ was further confirmed by an ¹⁵NO₃⁻ tracer experiment with intact crust pieces that proceeded at rates of 103±19 and 27±8 μmol N m⁻² h⁻¹ for cyanobacterial and lichen crust, respectively. Strikingly, N₂O gas was emitted at very high potential rates of 387±143 and 31±6 μmol N m⁻² h⁻¹ from the cyanobacterial and lichen crust, respectively, with N₂O accounting for 53–66% of the total emission of nitrogenous gases. Microsensor measurements revealed that N₂O was produced in the anoxic layer and thus apparently originated from incomplete denitrification. Using quantitative PCR, denitrification genes were detected in both the crusts and were expressed either in comparable (nirS) or slightly higher (narG) numbers in the cyanobacterial crusts. Although 99% of the nirS sequences in the cyanobacterial crust were affiliated to an uncultured denitrifying bacterium, 94% of these sequences were most closely affiliated to Paracoccus denitrificans in the lichen crust. Sequences of nosZ gene formed a distinct cluster that did not branch with known denitrifying bacteria. Our results demonstrate that nitrogen loss via denitrification is a dominant process in crusts from Oman, which leads to N₂O gas emission and potentially reduces desert soil fertility.     

Roseobacter clade bacteria are abundant in coastal sediments and encode a novel combination of sulfur oxidation genes

Roseobacter clade bacteria (RCB) are abundant in marine bacterioplankton worldwide and central to pelagic sulfur cycling. Very little is known about their abundance and function in marine sediments. We investigated the abundance, diversity and sulfur oxidation potential of RCB in surface sediments of two tidal flats. Here, RCB accounted for up to 9.6% of all cells and exceeded abundances commonly known for pelagic RCB by 1000-fold as revealed by fluorescence in situ hybridization (FISH). Phylogenetic analysis of 16S rRNA and sulfate thiohydrolase (SoxB) genes indicated diverse, possibly sulfur-oxidizing RCB related to sequences known from bacterioplankton and marine biofilms. To investigate the sulfur oxidation potential of RCB in sediments in more detail, we analyzed a metagenomic fragment from a RCB. This fragment encoded the reverse dissimilatory sulfite reductase (rDSR) pathway, which was not yet found in RCB, a novel type of sulfite dehydrogenase (SoeABC) and the Sox multi-enzyme complex including the SoxCD subunits. This was unexpected as soxCD and dsr genes were presumed to be mutually exclusive in sulfur-oxidizing prokaryotes. This unique gene arrangement would allow a metabolic flexibility beyond known sulfur-oxidizing pathways. We confirmed the presence of dsrA by geneFISH in closely related RCB from an enrichment culture. Our results show that RCB are an integral part of the microbial community in marine sediments, where they possibly oxidize inorganic and organic sulfur compounds in oxic and suboxic sediment layers.