黄土高原石油污染土壤微生物群落结构及其代谢特征

针对污染胁迫下土壤微生物群落变化和代谢变异等问题,基于平板稀释法和Biolog微平板分析方法,研究了陕北黄土高原石油污染土壤微生物群落结构、代谢特征及其功能多样性。结果表明,不同类群的土壤微生物对石油污染胁迫的响应不同,污染土壤细菌和真菌数量高出清洁土壤1个数量级,而污染土壤的放线菌数量极显著减少(P0.01);污染土壤和清洁土壤微生物对糖类和多聚物类碳源较易利用,污染土壤微生物总体上代谢碳源的种类和活性均低于清洁土壤。微生物群落主成分分析(PCA)表明,石油污染土壤和清洁土壤的微生物群落存在显著差异(P0.01),起分异作用的碳源主要为糖类,其次是羧酸类和氨基酸类;随着土壤石油含量增加,典型变量值变异(离散)增大,土壤微生物群落结构稳定性降低。微生物群落多样性分析表明,Shannon丰富度指数(H)、McIntosh均一度指数(U)和Simpson优势度指数(1/D)均达到极显著差异(P0.01),污染土壤微生物群落H和U低于清洁土壤,但是一定浓度的石油污染可以刺激土壤微生物群落中优势种群的生长,1/D增高。研究结果为陕北黄土高原石油污染区土壤微生物修复提供理论基础。     

Microbial Community Composition in Crude Oils and Asphalts from the Kurdistan Region of Iraq

Abstract

To identify hydrocarbon-degrading microorganisms contributing to the formation of heavy oil we investigated the microbial community composition in different types of crude oils from oil-production facilities and in crude oil and asphalt from different natural seeps from the Kurdistan Region of Iraq (KRI). Crude oils from five out of six production facilities did not contain microorganisms detectable by 16S rRNA gene PCR amplicon sequencing likely reflecting a low microbial abundance in these samples. Crude oil and asphalt from the natural seeps hosted diverse microbial communities. The same phylotypes of uncultivated Deferribacteres and Thermodesulfobacteraceae were predominant community members across crude oils and asphalts from separate geographical locations. Soils surrounding seeps did not contain these phylotypes suggesting that they originate from the subsurface and although they seem commonly detected in hydrocarbon-rich environments their role in hydrocarbon-degradation is unknown. GC-MS analyses showed that mainly aromatic hydrocarbons were present in the crude oil and asphalt and that they were undergoing biodegradation - likely with sulfate and nitrate as terminal oxidants. In agreement, only bssA gene, but not assA gene-carrying microorganisms were detectable in the analyzed sampled. Overall our study identified several abundant uncultivated taxa with likely roles in transformation of nitrate, sulfate and hydrocarbons.     

Comparison of biodegradability of crude and fuel oils.

Crude and fuel oils were compared for ability to support growth of a mixed population of estuarine bacteria. A total of four oils, two crude and two fuel oils, were examined. It was found that each of the oils supported a unique population of bacteria and yeasts, with respect to generic composition. Low-sulfur, high-saturate, South Louisiana crude oil was found to be highly susceptible to degradation. In contrast, the dense, high-sulfur, high-aromatic, Bunker C fuel oil was strongly refractory to microbial degradation.     

芦苇克隆整合对石油污染湿地土壤微生物群落结构和生物量的影响

克隆植物形体相连的无性个体(分株)之间可以进行水分、养分和光合产物的传递和共享,并且这种克隆整合可以显著提高分株对环境胁迫的耐受能力,从而可能进一步影响分株周围的土壤微生物群落结构和生物量。尽管国内外已经开展了大量有关克隆整合对分株耐受胁迫能力影响的研究,但克隆整合对土壤微生物群落结构和生物量影响的研究却十分缺乏。以黄河三角洲芦苇(Phragmites australis)湿地生态系统为研究对象,将直径60 cm的圆形样方进行三个水平的石油添加处理(不添加石油或每年添加5 mm或10 mm厚的石油),同时通过切断或不切断样方内外芦苇根状茎的连接来控制克隆整合的有无。实验连续开展了两年(2014—2015年),每年8月份在样方内进行土壤样品取样,在实验室内采用磷酸脂肪酸(PLFA)法测定土壤微生物总量及主要微生物类群含量,并测定土壤微生物生物量碳和氮含量。取样时间显著影响土壤微生物PLFA总量、微生物碳和氮含量,这3个变量在2015年均显著高于2014年。石油添加在2015年显著增加了土壤微生物PLFA总量,但在2014年却无显著效应;同时,石油添加在2014年降低了土壤微生物碳和氮含量,而在2015年却增加了其含量。然而,无论在2014年还是2015年,芦苇的克隆整合对土壤微生物PLFA含量、微生物碳和氮含量均没有显著影响。土壤微生物PLFA总量与土壤微生物碳和氮含量呈正相关关系。这些结果表明,石油污染可以显著影响湿地土壤微生物动态,但克隆整合却无显著效应。     

Efficiency of the EPS emulsifier produced by <Emphasis Type="Italic">Ochrobactrum anthropi</Emphasis> in different hydrocarbon bioremediation assays

Ochrobactrum anthropi strain AD2 was isolated from the waste water treatment plant of an oil refinery and was identified by analysis of the sequence of the gene encoding 16S rDNA. This bacterium produced exopolysaccharides in glucose nutrient broth media supplemented with various hydrocarbons (n-octane, mineral light and heavy oils and crude oils). The exopolysaccharide AD2 (EPS emulsifier) synthesized showed a wide range of emulsifying activity but none of them had surfactant activity. Yield production varied from 0.47 to 0.94 g of EPS l⁻¹ depending on the hydrocarbon added. In the same way, chemical composition and emulsification activity of EPS emulsifier varied with the culture conditions. Efficiency of the EPS emulsifier as biostimulating agent was assayed in soil microcosms and experimental biopiles. The AD2 biopolymer was added alone or combined with commercial products frequently used in oil bioremediation such as inorganic NPK fertilizer and oleophilic fertilizer (S200 C). Also, its efficiency was tested in mixture with activated sludge from an oil refinery. In soil microcosms supplemented with S200 C + EPS emulsifier as combined treatment, indigenous microbial populations as well as hydrocarbon degradation was enhanced when compared with microcosms treated with NPK fertilizer or EPS emulsifier alone. In the same way EPS emulsifier stimulated the bioremediation effect of S200 C product, increasing the number of bacteria and decreasing the amount of hydrocarbon remained. Finally, similar effects were obtained in biopile assays amended with EPS emulsifier plus activated sludge. Our results suggest that the bioemulsifier EPS emulsifier has interesting properties for its application in environment polluted with oil hydrocarbon compounds and may be useful for bioremediation purposes.     

独山子区优势草本植物根际与非根际土壤微生物功能多样性

以独山子区3种优势草本植物的根际与非根际土壤为研究对象,采用Biolog-ECO微平板法对土壤微生物功能多样性进行了研究。结果表明:3种不同植物根际与非根际土壤微生物代谢活性(AWCD)、丰富度指数Shannon(H)和均匀度指数Mc Intosh(U)均存在不同差异,且博乐蒿根际土壤微生物功能多样性均优于非根际土壤及其他两种植物;根际土壤微生物对糖类、脂类、酸类和胺类碳源物质比较敏感,非根际土壤微生物敏感于酸类、氨基酸类以及糖类碳源,根际土壤微生物对碳源的利用能力更强,且不同植物根际环境微生物碳源利用特征不同;微生物活性、丰富度指数和微生物均匀度指数与土壤pH值、SOM、AP和NO-3-N存在显著正相关(P0.05);博乐蒿根际土壤养分含量与微生物活性均较高,对环境的适应性更强,在独山子区生态环境管理与建设中可对其进行关注。     

高效石油降解菌群的构建及对芳香烃化合物萘的协同降解性能

【背景】石油作为一类混杂有机化合物,一旦产生污染就会对人类和环境造成严重的危害。【目的】从新疆石油污染土壤中分离筛选石油降解菌,为石油污染土壤的生物修复提供数据支持及技术参考。【方法】以石油为唯一碳源,通过富集培养、筛选分离得到123株单菌,根据菌落形态挑选出30个不同形态菌株,通过16S rRNA基因序列确定其种属,构建系统发育树;通过原油降解实验筛选出高效石油降解菌,以芳香烃的标志化合物萘为唯一碳源筛选出高效降解菌株,并分别筛选可降解水杨酸、邻苯二酚的菌株。【结果】分离筛选出5株高效石油降解菌,降解率高于85%;萘、水杨酸和邻苯二酚降解菌株各获得一株,将3种菌株按照1:1:1的接种比例对萘进行降解,萘的降解率从单菌60.74%提升到89.40%,菌株间的分工协作可以提高有机物的降解效率。【结论】筛选得到的菌株丰富了石油降解微生物菌种库,不同微生物菌株之间的分工协作为石油污染物的降解提供了新思路,为进一步研究石油污染治理提供参考。     

高羊茅草对盐碱地原油污染土壤微生物的影响

采用常规土壤理化分析和Biolog-Eco微孔板鉴定系统,研究了松嫩平原盐碱土地区高羊茅草的生长对原油污染土壤pH、总盐含量和微生物群落的影响.结果表明: 石油污染导致表征土壤微生物代谢活性的平均颜色变化率(AWCD)、物种多样性指数和碳源利用数增加, 微生物碳源利用模式发生改变.高羊茅草对石油污染土壤有较好的修复作用,降低了土壤pH和石油烃(TPH)含量,提高了土壤含水率.高羊茅草根际土壤的AWCD和碳源利用丰富度指数明显高于裸地土壤,为其根际微生物的生长发育提供了适宜的环境.     

Anaerobic microbial communities and their potential for bioenergy production in heavily biodegraded petroleum reservoirs

Most of the oil in low temperature, non-uplifted reservoirs is biodegraded due to millions of years of microbial activity, including via methanogenesis from crude oil. To evaluate stimulating additional methanogenesis in already heavily biodegraded oil reservoirs, oil sands samples were amended with nutrients and electron acceptors, but oil sands bitumen was the only organic substrate. Methane production was monitored for over 3000 days. Methanogenesis was observed in duplicate microcosms that were unamended, amended with sulfate or that were initially oxic, however methanogenesis was not observed in nitrate-amended controls. The highest rate of methane production was 0.15 μmol CH₄ g⁻¹ oil d⁻¹, orders of magnitude lower than other reports of methanogenesis from lighter crude oils. Methanogenic Archaea and several potential syntrophic bacterial partners were detected following the incubations. GC–MS and FTICR–MS revealed no significant bitumen alteration for any specific compound or compound class, suggesting that the very slow methanogenesis observed was coupled to bitumen biodegradation in an unspecific manner. After 3000 days, methanogenic communities were amended with benzoate resulting in methanogenesis rates that were 110-fold greater. This suggests that oil-to-methane conversion is limited by the recalcitrant nature of oil sands bitumen, not the microbial communities resident in heavy oil reservoirs.     

Long-chain n-alkanes occurring during microbial degradation of petroleum.

Five axenic cultures and a mixed culture were examined for ability to degrade South Louisiana, Brass River Nigerian, Anaco Venezuelan, and Altamont crude oils. A wax was observed during microbial degradation of Altamont crude oil, but not during weathering of the oil. The high-boiling n-alkanes in the wax were associated with microbial degradation of the oil and appeared to be similar to components of tarballs found in the open ocean.     

Effects of crude oil on marine microbial communities in short term outdoor microcosms

To assess the effects of crude oil spills on marine microbial communities, 10 L outdoor microcosms were manipulated over an exposure period of 8 days. The responses of microbial organisms exposed to five crude oil concentrations in 10 to 10,000 ppm (v/v) were monitored in the microcosms. The abundance of microalgae and copepods decreased rapidly upon the addition of crude oil at concentrations over 1,000 ppm, whereas the total density of heterotrophic bacteria increased dramatically at the higher concentrations. Bacterial diversity, determined by denaturing gradient gel electrophoresis, was increased at higher concentrations. In particular, the intensity of the bands representing Jannaschia sp. and Sulfitobacter brevis increased with the addition of oil. These results indicate that crude oil spills with concentrations over 1,000 ppm seriously affected the structure of the microbial communities.     

Effects of Bacillus subtilis O9 biosurfactant on the bioremediation of crude oil-polluted soils

The application of a surfactant from Bacillus subtilis O9 (Bs) on the bioremediation of soils polluted with crude oil was assayed in soil microcosms under laboratory conditions. Three concentrations of biosurfactant were assayed (1.9, 19.5, and 39 mg kg(-1) soil). Microcosms without biosurfactant were prepared as controls. During the experiment, the crude oil-degrading bacterial population, the aliphatic and aromatic hydrocarbons were monitored in each microcosm. The results indicated that applying Bs did not negatively affect the hydrocarbon-degrading microbial population Concentrations of 19 and 19.5mg (Bs) per kilogram of soil stimulated the growth of the population involved in the crude oil degradation, and accelerated the biodegradation of the aliphatic hydrocarbons. However, none of the assayed Bs concentrations stimulated aromatic hydrocarbon degradation.     

凋落物对土壤有机碳与微生物功能多样性的影响

森林凋落物是影响土壤微生物群落和有机碳含量的重要因素,但其作用的程度和机制尚不清楚,研究该问题对于分析森林生态系统碳循环和资源管理具有重要意义。研究凋落物去除与添加处理下土壤有机碳含量与土壤微生物对碳源利用的差异,明确凋落物去除与添加对土壤微生物群落代谢功能及其多样性的影响,探究不同处理下SOC含量变化的土壤微生物群落代谢机理。选取承德市雾灵山1405-1435 m海拔范围内核桃楸-蒙古栎混交林的表层土壤,采用室内培养结合Biolog-ECO方法,测定了培养第21天的土壤有机碳（soil organic carbon,SOC）含量及微生物群落的AWCD值、Shannon-Wiener多样性指数、Simpson优势度指数、McIntosh均匀度指数、Pielou丰富度指数,分析培养期内凋落物的不同处理下SOC含量与微生物功能多样性的变化特征。结果表明：1）不同凋落物处理对SOC含量与土壤微生物群落多样性具有显著影响（P<0.05）,DL > HL > NL > CK;2）不同凋落物处理下土壤微生物群落代谢活性和土壤微生物对碳源的利用程度具有显著差异（P<0.05）,碳水化合物类和氨基酸类是土壤微生物的主要碳源;3）不同处理的SOC含量与土壤微生物多样性具有正相关关系。双倍凋落物添加在短期内对土壤微生物多样性影响难以达到显著水平且在一定程度上对土壤微生物的代谢活性具有抑制作用,土壤微生物群落功能多样性对SOC含量具有重要影响。     

Biogeochemical Importance of the Bacterial Community in Uranium Waste Deposited at Key Lake,Northern Saskatchewan

The long-term stability of immobilized elements of concern in uranium tailings deposited in the Deilmann Tailings Management Facility (DTMF), northern Saskatchewan, is dependent upon maintenance of highly oxic conditions within the tailings mass. The main objective of this study was to investigate the effect of stimulating microbial activity on the redox potential and state of ferrihydrite, which are considered to be the primary controlling condition and mineral phase, respectively, within the tailings. To determine the potential for biologically mediated decreases in redox potential and ferrihydrite reduction, a series of microcosm assays were performed. Non-sterile material from the tailings–water interface of the DTMF site was inoculated with indigenous flora previously isolated from the tailings material and enriched with a carbon source (50 ppm trypticase soy broth) and incubated under continuous-flow or intermittent-flow conditions, and compared with an uninoculated, no-carbon control that received continuous flow. Highly reducing conditions with redox potentials of less than ?300 mV were detected after 2 days of incubation within the carbon-enriched tailings of microcosms receiving continuous flow, and less than ?280 mV after 11 days of incubation within carbon-enriched tailings in microcosms receiving intermittent flow. The lowest recorded Eh value (?545 mV) was recorded after 14 days in a carbon-enriched microcosm receiving intermittent flow. In contrast, the redox conditions in the control microcosm never dropped below ?93 mV; thus, it was clear that microbial activity and available carbon drove the Eh conditions to become highly reducing. The occurrence of low redox conditions was concomitant with the bulk chemical detection of Fe (II) in the effluent of treated microcosms. Sites of microbial ferrihydrite reduction were also detected using scanning transmission X-ray microscopy where Fe (II) species were observed in close proximity with bacterial cells. Analysis of the microbial diversity present within the microcosms confirmed that microbes indigenous to the DTMF system have the potential to generate conditions suitable for the proliferation of sulfate and iron reducing bacteria, such as Desulfosporosinus, which was detected by high-throughput 16S rRNA gene sequencing.     

Ratio [13C]/[12C] as an index for express estimation of hydrocarbon-oxidizing potential of microbiota in soil polluted with crude oil

The hydrocarbon-oxidizing potential of soil microbiota and hydrocarbon-oxidizing microorganisms introduced into soil was studied based on the quantitative and isotopic characteristics of carbon in products formed in microbial degradation of oil hydrocarbons. Comparison of CO₂ production rates in native soil and that polluted with crude oil showed the intensity of microbial mineralization of soil organic matter (SOM) in the presence of oil hydrocarbons to be higher as compared with non-polluted soil, that is, revealed a priming effect of oil. The amount of carbon of newly synthesized organic products (cell biomass and exometabolites) due to consumed petroleum was shown to significantly exceed that of SOM consumed for production of CO₂. The result of microbial processes in oil-polluted soil was found to be a potent release of carbon dioxide to the atmosphere.     

高寒草甸不同植被土壤微生物数量及微生物生物量的特征

用稀释平板法和氯仿熏蒸法研究了藏嵩草草甸、小嵩草草甸、矮嵩草草甸、禾草草甸、杂类草草甸及金露梅灌丛土壤的微生物数量、生物量及有机质的变化特征.结果表明:0～40 cm土层细菌和放线菌数量、微生物生物量碳和土壤有机质含量均以藏嵩草草甸最高,其微生物生物量及土壤有机质显著高于其它5种植被;真菌数量以金露梅灌丛最高;由表层到深层,随着土壤深度的增加以上各项指标均呈下降趋势.通径分析表明:土壤各生物环境因子之间存在着不同程度的相关性;土壤微生物数量、生物量及土壤有机质含量均与土壤水分含量存在显著的相关关系,说明土壤水分是调节高寒草甸生态系统土壤微生物代谢及物质转化的关键因子.     

Movement of Viruses between Biomes

Viruses are abundant in all known ecosystems. In the present study, we tested the possibility that viruses from one biome can successfully propagate in another. Viral concentrates were prepared from different near-shore marine sites, lake water, marine sediments, and soil. The concentrates were added to microcosms containing dissolved organic matter as a food source (after filtration to allow 100-kDa particles to pass through) and a 3% (vol/vol) microbial inoculum from a marine water sample (after filtration through a 0.45-μm-pore-size filter). Virus-like particle abundances were then monitored using direct counting. Viral populations from lake water, marine sediments, and soil were able to replicate when they were incubated with the marine microbes, showing that viruses can move between different ecosystems and propagate. These results imply that viruses can laterally transfer DNA between microbes in different biomes.     

Evaluating microbial contaminations of alternative heating oils

Since 2008, European and German legislative initiatives for climate protection and reduced dependency on fossil resources led to the introduction of biofuels as CO₂-reduced alternatives in the heating oil sector. In the case of biodiesel, customers were confronted with accelerated microbial contaminations during storage. Since then, other fuel alternatives, like hydrogenated vegetable oils (HVOs), gas-to-liquid (GtL) products, or oxymethylene ether (OME) have been developed. In this study, we use online monitoring of microbial CO₂ production and the simulation of onset of microbial contamination to investigate the contamination potential of fuel alternatives during storage. As references, fossil heating oil of German refineries are used. Biodiesel blends with fossil heating oils confirmed the promotion of microbial activity. In stark contrast, OMEs have an antimicrobial effect. The paraffinic Fischer–Tropsch products and biogenic hydrogenation products demonstrate to be at least as resistant to microbial contamination as fossil heating oils despite allowing a diversity of representative microbes. Through mass spectrometry, elemental analysis, and microbial sequencing, we can discuss fuel properties that affect microbial contaminations. In summary, novel, non-fossil heating oils show clear differences in microbial resistance during long-term storage. Designing blends with an intrinsic resistance against microbial contamination and hence reduced activity might be an option.     

Inhibition of light-induced pH increase and O2 evolution of marine microalgae by water-soluble components of crude and refined oils.

Light-induced alkalinization of the extracellular medium was found to be a common feature of the primary photosynthetic process of several marine microalgae. The light-induce PH increase of suspensions of whole cells was immediately and severely inhibited by a single dose of water-soluble components from crude and fuel oils. Differential effects on the rates of microalgal photosynthetic O2 evolution and cell suspension pH increase suggest different toxicity mechanisms of the water-soluble components of no. 2 fuel oil as compared with Southern Louisiana and Jay Crude oils. These short-term studies on the nature of sublethal petroleum toxicity to microalgae indicate that the primary effect may be through direct action on the energy-yielding electron transport systems.     

Inhibition of light-induced pH increase and O2 evolution of marine microalgae by water-soluble components of crude and refined oils.

Light-induced alkalinization of the extracellular medium was found to be a common feature of the primary photosynthetic process of several marine microalgae. The light-induce PH increase of suspensions of whole cells was immediately and severely inhibited by a single dose of water-soluble components from crude and fuel oils. Differential effects on the rates of microalgal photosynthetic O2 evolution and cell suspension pH increase suggest different toxicity mechanisms of the water-soluble components of no. 2 fuel oil as compared with Southern Louisiana and Jay Crude oils. These short-term studies on the nature of sublethal petroleum toxicity to microalgae indicate that the primary effect may be through direct action on the energy-yielding electron transport systems.