Gas chromatography and gas chromatography/mass spectrometry applied to the determination of a new pathway of pristane degradation by a marine mixed bacterial population

Degradation of pristane is rapidly achieved with high efficiency by a mixed bacterial population isolated from a marine biotope highly polluted with hydrocarbons.Analysis of the degradation products by gas chromatography and gas chromatography/mass spectrometry reveals that pristane is not only attacked according to classical mechanisms of β- and ω-oxidation, but also according to a new mechanism beginning with attack at the third carbon atom.Thanks to that mechanism, the population must be particularly suited to degradation of substrates resistant to α- and β-oxidations; this has been confirmed with an α-disubstituted structure.     

潮间带沉积物厌氧烃降解细菌的多样性及Desulfovibrio subterraneus ND17的分离鉴定

潮间带作为海陆交界处,易受到来自海洋的石油污染,且各类石油烃进入沉积物后的降解过程尚不清楚。前人在各类生境中对好氧微生物烃降解方面已有较多研究,但对近海潮间带环境中的厌氧烃降解鲜有报道。本研究对青岛女岛湾潮间带沉积物深层样品以混合烃（中长链烷烃、多环芳烃）为碳源,硫酸盐作为电子受体进行厌氧富集培养。富集菌群的细菌多样性表明在混合烃作为碳源的作用下,优势菌群转变为脱硫叠球菌科（Desulfosarcinaceae）、脱硫杆菌科（Desulfobacteraceae）等具有石油烃降解潜力的硫酸盐还原菌。经分离纯化得到一株厌氧烃降解菌ND17,与地下脱硫弧菌属模式种Desulfovibrio subterraneus HN2T 16S rRNA基因序列的相似度为99.93%。进一步实验表明,菌株ND17在厌氧条件下对二十四烷和菲的降解率可分别达到53.9%和35.7%。这也是首次对脱硫弧菌属单菌在厌氧条件下进行石油烃降解的研究。脱硫弧菌作为一种广泛分布在厌氧环境的细菌,本研究为进一步认识其在海洋石油污染环境中的修复潜力提供了支撑。     

Bacterial production and carbon conversion based on saltmarsh plant debris

There is a well-defined succession of micro-organisms which colonize powdered leaf debris from Spartina alterniflora and Juncus roemerianus, and aged natural detrital material when these were incubated in estuarine water at temperatures near to those recorded in the habitat at the time of collection. The natural assemblage of free-living bacteria in estuarine water rapidly enters logarithmic growth, subsequently declining with the increase in numbers of bactivorous microflagellates. These are then replaced by a mixed population of ciliates, choanoflagellates, amoeboid forms and attached bacteria which form part of a complex microbial community associated with particulate debris. The rate of increase of bacterial cells (μ), in both spring and summer experiments ranged from 0·010–0·108 h^?1 whilst estimates of bacterial carbon production ranged from 1·5 to 10·1 μg C 1^?1 h^?1, values which conform well with estimates obtained from natural assemblages of marine bacteria in coastal and estuarine waters elsewhere. Although both the ease of degradation of the detrital substrate and incubation temperature are of importance, enrichment of both powdered Spartina leaf debris and aged natural detritus with inorganic nutrients evidently enhances bacterial production under experimental conditions. In addition, the amount of carbon utilized to sustain bacterial carbon production shows a significant reduction following enrichment with NH₄, NO₃ or combinations of NO₃ + PO₄. The bacterial carbon conversion efficiency (μg C incorporated into bacterial production per μg C consumed) × 100, based on powdered Spartina leaves, and aged natural detritus, is thus increased from 9–14%, to as much as 38% in nutrient enriched media. Since NH₄, NO₃ and PO₄ values are generally low in the water column, it seems likely that bacteria achieve a carbon conversion of only 9–14% on natural suspended detrital material, with the possibility of an enhanced conversion of up to 38% occurring at the sediment-water interface where ammonia regeration occurs. This suggests that suspended bacteria which characterize estuarine waters of salt marsh areas may be responsible for the oxidation of 86–91% of the carbon which enters water column microheterotroph food chains, and are probably implicated in the large CO₂ fluxes recently recorded from coastal wetland habitats.     

海洋附着细菌对冠瘤海鞘幼体附着和变态的影响

从冠瘤海鞘、皱瘤海鞘及乳突皮海鞘的被囊表面及其附着基和附近的海水中分离到290株细菌,根据其形态和生理生化特征,将其鉴定至弧菌属、肠杆菌属和无色杆菌属等10个属.分析海鞘被囊表面及其附着基和海水中的细菌组成,并从各属中挑出的29株细菌中分离筛选出附着能力较强的9株.将这9株细菌分别制成不同的单种细菌黏膜及9种细菌混合黏膜,检测细菌黏膜对冠瘤海鞘幼体附着和变态的影响.结果表明不同细菌黏膜对冠瘤海鞘幼体的附着和变态起不同的作用.无色杆菌属H-13显著地促进海鞘的附着和变态;弧菌属的H-4对海鞘的附着和变态均起抑制作用;弧菌属H-9明显地促进附着却强烈抑制海鞘幼体变态;其余细菌仅仅对海鞘幼体附着或变态的一个过程起作用或是对两者都没有明显效果.     

胶州湾沉积物中氨基酸对有机质降解及细菌源贡献的指示作用解析

基于对胶州湾表层沉积物中总可水解氨基酸（THAA）的含量、组成、构型及分布特征的系统研究,通过氨基酸碳氮归一化产率（THAA-C%,THAA-N%）、降解因子DI、反应活性指数RI以及D型氨基酸占比（D-AA%,摩尔百分比）等指标结合碳氮比（TOC/TN）、碳稳定同位素（δ13C）探析了胶州湾沉积物中有机质的来源与降解状态,利用细菌源有机质及胞外肽酶活性（EEA）探讨了微生物在有机质迁移转化过程中的作用与贡献。结果表明,胶州湾表层沉积物中氨基酸平均含量为（7.60±3.64）μmol/g,在陆源与海源混合影响下,其水平分布呈现湾内高于湾外、湾内东部高于西部的特点,表明湾内东部陆源输入对沉积物THAA具有较高贡献。THAA-C%、THAA-N%、DI、RI以及D-AA%等指示因子均显示胶州湾表层沉积物中有机质的降解程度呈现湾外高于湾内、湾内东部高于西部的变化趋势,有机质来源、微生物活性与上覆水水深共同影响了有机质的降解程度。胶州湾表层沉积物中细菌源有机碳的贡献率为（29.35±18.73）%,其水平分布显示出湾内西部与湾外相近且高于湾内东部的特点。细菌胞外肽酶活性（EEA）平均为（0.81±1.31）nmol/（g·h）（以MCA计）,整体分布趋势与细菌贡献率相反,呈现湾内东部高于湾内西部和湾外的特性。沉积物中有机质的不同海源、陆源占比决定了有机质的可降解性,而有机质的降解程度进一步影响了细菌源有机质的贡献与胞外肽酶活性。     

Assessing the apparent imbalance between geochemical and biochemical indicators of meso- and bathypelagic biological activity: What the @$♯! is wrong with present calculations of carbon budgets?

Metabolic activity in the water column below the euphotic zone is ultimately fuelled by the vertical flux of organic material from the surface. Over time, the deep ocean is presumably at steady state, with sources and sinks balanced. But recently compiled global budgets and intensive local field studies suggest that estimates of metabolic activity in the dark ocean exceed the influx of organic substrates. This imbalance indicates either the existence of unaccounted sources of organic carbon or that metabolic activity in the dark ocean is being over-estimated. Budgets of organic carbon flux and metabolic activity in the dark ocean have uncertainties associated with environmental variability, measurement capabilities, conversion parameters, and processes that are not well sampled. We present these issues and quantify associated uncertainties where possible, using a Monte Carlo analysis of a published data set to determine the probability that the imbalance can be explained purely by uncertainties in measurements and conversion factors. A sensitivity analysis demonstrates that the bacterial growth efficiencies and assumed cell carbon contents have the greatest effects on the magnitude of the carbon imbalance. Two poorly quantified sources, lateral advection of particles and a population of slowly settling particles, are discussed as providing a means of closing regional carbon budgets. Finally, we make recommendations concerning future research directions to reduce important uncertainties and allow a better determination of the magnitude and causes of the unbalanced carbon budgets.     

太平洋深海沉积物来源的菌株Tenacibaculum sp.HMG1对孔雀石绿降解特性的研究

本文在太平洋深海沉积物中分离得到一株孔雀石绿降解菌株,鉴定命名为Tenacibaculum sp.HMG1。通过菌株生长实验和高效液相色谱的研究表明, HMG1菌株可以在20 mg/L的孔雀石绿中维持较快的生长速率,并且在12 h内可降解98.8%的孔雀石绿,这证明该菌株具有很高的孔雀石绿耐受能力和降解活性。通过基因组测序在HMG1菌株发现一条过氧化物酶基因可能参与了孔雀石绿的降解,随后利用原核表达获得了相应的重组蛋白。实验表明,该重组过氧化物酶具有极强的活性,可在1000 mg/L的孔雀石绿中发挥降解功能。本文利用液相色谱-质谱联用（LC-MS）技术对孔雀石绿的菌株降解产物和重组酶降解产物进行鉴定,并基于鉴定结果推测了两种降解途径。结果发现两种降解方式存在共同的降解途径。此外,孔雀石绿降解条件的实验结果证明重组过氧化物酶可以在低温（20℃）、复杂的pH值（6.0–9.0）、高盐度（100 mmol/L）、金属离子和EDTA等反应条件下依旧维持很高的孔雀石绿降解活性。以上实验结果表明,HMG1菌株和重组过氧化物酶均在孔雀石绿污染生物修复方面具有很大潜力。     

New and important roles for DMSP in marine microbial communities

The algal osmolyte dimethylsulfoniopropionate (DMSP) is recognised as the major precursor of marine dimethylsulfide (DMS), a volatile sulfur compound that affects atmospheric chemistry and global climate. Recent studies, using ³⁵S-DMSP tracer techniques, suggest that DMSP may play additional very important roles in the microbial ecology and biogeochemistry of the surface ocean. DMSP may serve as an intracellular osmolyte in bacteria that take up phytoplankton-derived DMSP from seawater. In addition, DMSP appears to support from 1 to 13% of the bacterial carbon demand in surface waters, making it one of the most significant single substrates for bacterioplankton so far identified. Furthermore, the sulfur from DMSP is efficiently incorporated into bacterial proteins (mostly into methionine) and DMSP appears to be a major source of sulfur for marine bacterioplankton. Assimilatory metabolism of DMSP is via methanethiol (MeSH) that is produced by a demethylation/demethiolation pathway which dominates DMSP degradation in situ. Based on the linkage between assimilatory metabolism of DMSP and bacterial growth, we offer a hypothesis whereby DMSP availability to bacteria controls the production of DMS by the competing DMSP lyase pathway. Also linked with the assimilatory metabolism of DMSP is the production of excess MeSH which, if not assimilated into protein, reacts to form dissolved non-volatile compounds. These include sulfate and DOM–metal–MeSH complexes, both of which represent major short-term end-products of DMSP degradation. Because production rates of MeSH in seawater are high (3–90 nM d⁻¹), reaction of MeSH with trace metals could affect metal availability and chemistry in seawater. Overall, results of recent studies provide evidence that DMSP plays important roles in the carbon, sulfur and perhaps metal and DOM cycles in marine microbial communities. These findings, coupled with the fact that the small fraction of DMSP converted to DMS may influence atmospheric chemistry and climate dynamics, draws attention to DMSP as a molecule of central importance to marine biogeochemical and ecological processes.     

海洋环境微生物腐蚀机理研究进展

微生物腐蚀(microbiologically influenced corrosion, MIC)是海洋环境中一种非常重要的腐蚀形式,其是微生物、环境、材料共同作用的结果,本文自MIC对海洋环境、菌株、材料性质的依赖性三个方面概述其机理的研究进展。在MIC对海洋环境的依赖性方面,突出溶解氧浓度和营养水平的影响规律与机制。在MIC对菌株的依赖性方面,首先介绍典型单菌株的腐蚀影响机制,然后分析混合菌株间不同菌株的腐蚀协同与拮抗作用,最后阐述复杂微生物群落的影响。在MIC对材料性质的依赖性方面,在总结基体化学成分与结构影响的基础上,介绍对MIC有抑制作用的典型表面的特性及其作用机制。     

Evolution des hydrocarbures dans des sédiments côtiers méditerranéens prélevés à proximité d'un emissaire d'une raffinerie de pétrole

Coastal Mediterranean sediments highly polluted by refinery effluents have been studied for their hydrocarbon content and bacterial activity. The study has taken into account both the distance from the refinery and depth of sampling. Very high hydrocarbon concentrations have been found (1–250 g/kg dry sediment). Micro-organisms in the sediments have a high bacterial activity and a good correlation exists between hydrocarbon concentrations and bacteria able to use these substrates as a carbon and energy source.     

南海北部海水中氨基酸的分布及其对溶解有机物降解行为的指示研究

于2015年6月对南海北部海区5个断面共26个站位海水中溶解态氨基酸（THAA）、溶解有机碳（DOC）和叶绿素a（Chl a）的浓度进行了科学调查。结果表明:夏季南海北部海水中THAA的浓度范围为0.40~1.95 μmol/L,平均值为（0.80±0.40） μmol/L,THAA的水平分布总体上体现出近岸高、远海低的特点,表明陆源输入对南海北部海域表层THAA分布有重要影响。THAA在断面上的垂直分布呈现出由近岸至远岸、由表层至底层逐渐降低的趋势。THAA浓度与两种D型氨基酸（D-谷氨酸:D-Glu和D-丙氨酸:D-Ala）含量之间存在显著负相关性,与天门冬氨酸/β-丙氨酸（Asp/β-Ala）和谷氨酸/γ-氨基丁酸（Glu/γ-Aba）比值之间存在显著正相关性,表明细菌的消耗是影响南海海水中THAA浓度的重要因素。D-Ala作为细菌肽聚糖中相对稳定的氨基酸,根据其占DOC的含量估算南海海水中的细菌源有机碳对DOC的贡献率为（29.32±14.32）%,其水平分布显示出近岸低、远岸高的特点;而其垂直分布则呈现出从表层至底层逐渐增加的趋势。THAA占DOC百分比（THAA-C%）的变化范围为1.02%~5.49%,平均值为（2.97±1.38）%。THAA-C%、活性因子和降解因子的高值均出现在珠江口外围区域。随着海水深度增加3种降解因子的数值均显著降低,这表明底层海水中有机物比表层海水中的有机物降解程度更大。     

On the sensitized photo-oxidation of alkylbenzenes in seawater

Several homologous monoalkylated benzenes were photo-oxidized as surface films on and dissolved in pure water and purified natural seawater by solar and equivalent artificial UV irradiation in the presence of anthraquinone as photo-sensitizer. The main reaction products were the 1-phenylalkanones, the corresponding secondary alcohols, benzaldehyde and, when the side chain contained four or more carbon atoms, the products of what appears to be a Norrish type II photo-degradation. The detection among the reaction products of an aldehyde with one carbon atom less than the olefin produced by the Norrish type II photo-degradation suggests a photolytic decomposition of 1-alkenes, resulting in the formation of highly reactive compounds capable of abiotic reactions in the electronic ground state with a variety of organic compounds occurring in seawater.     

石油降解菌对石油烃的降解能力及影响因素研究

采取油浓度梯度升高的方法,从胜利油田石油污染土壤中富集分离出以柴油为碳源和能源的优势除油混合菌B4。混合菌中分离出7株可培养菌。混合菌在含油0.5%的无机盐液体培养基中培养7天后柴油的去除率达到90.4%。该菌有较好的耐受性,当盐度达3.5时,油浓度达1.0%,亦有较好长势。混合菌对油污染土壤的修复有较好的效果,特别是加入营养盐,降解率达85.6%。混合菌脱氢酶动态变化的初步研究结果表明微生物活性变化与降解率有很好的相关性。     

珠江口外近海沉积有机质化学及稳定同位素组成的早期成岩改变

理解早期成岩过程中有机质的化学和同位素分馏对于研究海洋和湖泊环境中的生物地球化学过程是很重要的。将珠江口外近海生物成因有机质分为可水解氨基酸、碳水化合物、脂类和酸不溶四个部分,分析了有机质的化学和同位素组成(δ13C,δ15N),借以讨论沉积有机质在埋藏的早期成岩过程中所发生的化学和同位素改变,结果表明,从浮游生物→悬浮颗粒物→表层沉积物→沉积柱内部,易降解组分可水解氨基酸、碳水化合物、脂类占样品总有机碳的份额依次降低。沉积物及四个有机部分的稳定碳同位素组成在纵向上随深度保持相对恒定,而在不同有机部分之间差异明显。不同类型有机物的分解速率差异在改变有机质化学成分的同时,导致其δ13C发生小幅度负向漂移;细菌有机质的形成和分解对有机质化学成分和同位素组成演化也有重要贡献,并且在一定程度上抵消了上述δ13C的负向漂移,其结果导致沉积有机质的δ13C略低于浮游生物;另一方面,由于异养菌生长过程中的氮同位素分馏系数与可利用氮源的特征和培养基的性质等多种因素有关,导致沉积物的δ15N变化范围增大。在这里δ13C可以可靠地指示该海域沉积有机质的来源,而δ15N变化范围较大且规律不明显,难以用作沉积有机质来源的指示。     

Benthic mineralization rates at two locations in the southern North Sea

Benthic oxygen uptake, sulphate reduction and benthic bacterial production were measured at two contrasting locations in the southern North Sea: the shallow and turbulent Broad Fourteens area in the Southern Bight, and the deeper Oyster Grounds, a deposition area, where thermohaline stratification occurs during summer. Oxygen uptake and sulphate reduction showed a clear seasonal pattern in the Broad Fourteens area, indicating a supply of carbon to the benthic system that is closely related to the standing stock of carbon in the water column. This close benthic-pelagic coupling is probably due to the influence of the tide in this part of the North Sea, which keeps the water column permanently mixed. At the Oyster Grounds, no seasonal pattern was observed. Peaks in oxygen uptake and sulphate reduction were found in winter. Irregularly occurring events, such as storms and fishery-related activities, are likely to affect the benthic mineralization patterns in this area. Annual benthic carbon mineralization rates estimated from oxygen uptake rates were 44 gC·m⁻² at the Broad Fourteens, and 131 gC·m⁻² at the Oyster Grounds, of which 26 and 28%, respectively, could be attributed to sulphate reduction (assuming an annual sulphide reoxidation rate of 100%). Although sulphate reduction rates in the southern North Sea are higher than previously suggested, aerobic respiration is the most important pathway for benthic carbon mineralization at the stations visited. Production rates of benthic bacterial carbon measured with labelled leucine were much higher than carbon mineralization rates based on oxygen uptake or sulphate reduction. This may either imply a very high bacterial carbon conversion efficiency, or point to shortcomings in the accuracy of the techniques. A critical evaluation of the techniques is recommended.     

海洋石油降解菌群的固定化及石油降解特性

为克服岸滩溢油生物修复过程中海浪冲刷等不利环境对石油降解菌(群)岸滩定植的影响,本文利用聚乙烯醇(PVA)和海藻酸钠作为载体对石油降解菌群DC10进行固定化,通过研究细菌固定化微球的机械性能、传质性能及石油降解特性等参数,确定石油降解菌群的最优固定化条件。实验结果表明:6%PVA,2%海藻酸钠及0.5%活性炭制备的凝胶可以通过蠕动泵方便快捷制备细菌固定化微球,其粘度小、易成型、机械强度高。海洋石油降解分析表明,与游离菌体(FB)相比,固定化菌群12d石油降解率提高了近7%;GC-MS分析显示,石油烷烃和芳烃降解效果显著。实验证明,石油降解菌群DC10经过固定化处理,其石油降解活性提高,连续降解能力增强,该研究为溢油岸滩的生物修复提供新的技术方法。     

Particulate carbohydrates and uronic acids in the northern East China Sea

Carbohydrate species, such as uronic acids, play an important role in oceanic carbon cycling, coagulation and adsorption processes. Concentrations of particulate carbohydrates (PCHO) and uronic acids (PURA) were measured in the northern East China Sea (ECS) during June and November, 2006. In June, maximum concentrations of PCHO and PURA were observed in the surface layer of coastal waters. Their concentrations rapidly decreased with depth, suggesting that they are both bio-reactive. Moreover, phytoplankton abundance and bacterial biomass seem to be associated with observed PCHO and PURA concentrations in the ECS during June, suggesting that production of carbohydrate species in the ECS is regulated by phytoplankton assemblages, bacterial assimilation or degradation. In November, however, PCHO and PURA concentrations were homogenous within the water column due to strong vertical mixing. No strong correlations were observed between carbohydrate species (PCHO and PURA) and phytoplankton or bacterial biomass, suggesting that production of these compounds in November might be caused by the physiological difference between nutrient limited and non-nutrient limited phytoplankton. Furthermore, strong negative correlations between nutrients and PCHO species suggest that nutrient levels may be one of the driving forces behind the production of these compounds in the ECS.     

菲降解菌的分离鉴定及其降解性研究

采用微生物富集培养分离法对南洋油田不同油样中的菲降解菌进行了分离鉴定。分离得到2株以菲作为唯一碳源和能源的细菌菌株S17和S28,经革兰氏染色及显微镜形态观察,发现两者都是具有极生单鞭毛的革兰氏阴性、无芽孢杆菌。根据生理生化特性分析,以及16S rDNA序列同源性分析,两者属于假单胞菌属的不同种。菌株S17与食树脂假单孢菌(Pseudomonas resinovorans)序列同源性为97%;菌株S28则为高温假单胞菌(Pseudomonas thermaerum),其序列同源性达到100%。在以菲为唯一底物的条件下,菌株S28的生长速度是S17的2倍多,第4天即达到最大生长量,但是对于菲的降解而言,两者都在第10天达到最大,降解率分别为88.86%和82.02%,但是二者在起始的两天内对菲的降解效率最高,分别达到70.21%和72.74%,因此可用于菲污染的快速治理领域。     

Seasonal DOC accumulation in the Black Sea: a regional explanation for a general mechanism

During three cruises in the Black Sea, organised in July 1995 and April–May 1997, biological and chemical parameters that can influence the carbon budget were measured in the water column on the NW shelf, particularly in the mixing zone with Danube River waters. We observed in early spring (end of April–May) conditions an important input of freshwater organisms that enhanced the microbial activity in the low salinity range. High bacterial activity regenerates nitrogen in the form of nitrates, but is also responsible for an important consumption of ammonium and phosphate, leading to a high N/P ratio and a strong deficit in phosphorus. The consequence is a limitation of phytoplankton development but also a production of carbohydrates that accumulate all along the salinity gradient. These mechanisms are responsible for a seasonal accumulation of dissolved organic carbon (DOC) that increases from 210 μM in winter to about 280 μM in summer. All this excess DOC disappears during winter, probably degraded by bacterial activity. The degradation of carbon-rich organic matter increases the phosphorus demand by bacteria bringing limitation to phytoplankton primary production.     

Determination of the distribution of dissolved organic carbon in the Indian sector of the Southern Ocean

During France JGOFS campaign ANTARES 2 (R.V. Marion Dufresne), samples were taken along a section of the 62°E meridian from 49° to 66°S. The high temperature catalytic oxidation (HTCO) method was used to determine the concentration of dissolved organic carbon (DOC). The analyses were conducted both on-board ship and after the cruise in the laboratory. Collecting and storing acidified samples for post-cruise analysis induced no significant differences. The use of two separate but identical channels on the carbon analyzer increased the number of samples analysed per day and allowed independent monitoring of the instrument blank and the calibration of the detector response. The mixed layer concentrations of organic carbon varied from about 52 μM C in the Antarctic Divergence (64°S) to about 63 μM C in the Polar Frontal Zone (49°S). Vertical profiles showed a slight, but significant, decrease in organic carbon below the mixed layer, to about 42 μM C below 2000 m across the transect. The homogeneity and low concentration of organic carbon in deep water is consistent with values recently reported for the equatorial Atlantic and Pacific Ocean and supports the evidence for a constant deep water DOC concentration. In addition, this provides a verification of the instrument performance, thus validating observed DOC data trends and allowing a comparison with the ‘modern' DOC literature. In general, the organic carbon concentration in the mixed layer was lower than previously published data of the main ocean basins, which might -reflect the low chlorophyll a concentration (<0.5 μg/l) encountered in this region. Along the 62°E meridian section, organic carbon showed a trend with corresponding measurements of phytoplankton biomass and bacterial production, underlining the dependence of bacterial growth on a pool of ‘freshly' produced DOC. Organic carbon was found to exhibit a weak inverse trend versus apparent oxygen utilization (AOU). This suggests that only a small part of the oxygen consumption is due to the mineralisation of DOC.