不同石油污染程度土壤细菌群落多样性及优势菌群分析

  目的  探究辽河油田不同石油污染程度土壤中理化性质及细菌群落多样性和组成的变化规律,并对石油污染土壤中的石油降解菌进行分离培养和鉴定。  方法  采集了辽河油田不同石油污染程度土壤,采用高通量测序技术和化学分析法对土壤细菌群落组成和土壤理化性质进行测定,并进一步筛选出石油降解菌株。  结果  在出油口（A）、距离出油口50 m（B）和距离出油口150 m（C）采集的三个土壤样品,其土壤总石油烃含量分别为2467.44 mg kg?1、884.99 mg kg?1和141.63 mg kg?1,三个土壤样品具有不同的石油污染程度。石油污染显著提高了土壤总有机碳含量,土壤总石油烃含量与总有机碳含量呈现正相关（P < 0.001）。土壤细菌群落多样性和丰富度指数与土壤石油烃的浓度呈显著负相关（P < 0.01）。不同石油污染程度土壤具有不同的细菌群落组成和结构,土壤石油烃含量是影响细菌群落变化的主要因素。出油口石油污染土壤样品（A）中,变形菌门（Proteobacteria）为优势菌门,假单胞菌属（Pseudomonas）、假黄单胞菌属（Pseudoxanthomonas）、博代氏杆菌属（Bordetella）和伯克氏菌属（Burkholderia）为优势菌属。从出油口石油污染土壤（A）中分离出3株石油降解菌株,通过16S rRNA基因测序分别被鉴定为Pseudomonas baetica、黄褐假单胞菌（Pseudomonas fulva）和施氏假单胞菌（Pseudomonas stutzeri）,其石油降解率分别为37.2%、46.9%和57.8%。此结果与A样品高通量测序属水平组成分析相吻合,表明石油污染能够选择性富集土壤中具有石油降解能力的假单胞菌属。  结论  石油污染提高了土壤总有机碳含量,降低了土壤细菌群落多样性,富集了具有烃类降解能力的优势菌属,是造成土壤细菌群落组成和结构改变的主要因素,并筛选出具有潜在开发应用价值的石油降解假单胞菌株。     

石油降解菌的筛选、鉴定及菌群构建

从胜利油田石油污染土壤中富集、分离得到236株能以石油作为唯一碳源和能源的石油降解菌株；采用选择性培养基进行复筛得到直链烷烃降解菌31株、环烷烃降解菌28株、芳烃降解菌3株以及表面活性剂产生菌24株；从3种不同烃类降解菌和表面活性剂产生菌中选择菌株，构建石油降解微生物菌群，结果表明，由菌株SL-51、SL-84、SL-133和SL-163组成的菌群c9降解石油能力最强，菌群C9在含原油浓度为0．5％的无机盐培养液中，5d内原油的降解率达到了55．5％；气相色谱分析结果证明，菌群C9能有效降解原油中的饱和烃和芳烃组分；通过16SrDNA序列分析，初步鉴定SL-51和SL-163属于红球菌属（Rhodococcus spp．），SL-84、SL-133两株菌分别属于苍白杆菌属（Ochrobactrum sp．）、铜绿假单胞菌属（Pseudomonas sp．）。     

石油污染土壤中芳烃降解菌及邻苯二酚2,3双加氧酶的克隆

石油污染土壤中的芳烃降解菌是进行土壤修复的主要生物资源,本研究对某炼油厂附近土壤中的芳烃降解菌及邻苯二酚2,3双加氧酶基因进行了研究。结果表明,部分石油烃污染土壤中存在着大量的芳烃降解菌;对其中一个土壤样本中的邻苯二酚2,3双加氧酶基因进行克隆,获得了7个不同的邻苯二酚双加氧酶基因序列,序列分析表明这些基因可能来源于土壤中的假单胞菌,且该基因在土壤中的丰度与污染水平及芳烃降解菌的数量相关。可见,土壤中芳烃降解菌数量及降解基因的丰度和多样性,可以对石油污染土壤的生物修复进行监控并为生物修复提供丰富的微生物资源。     

盐渍化土壤中土著菌的石油烃降解潜力研究

自盐渍化地区（黄河三角洲）采集4种不同石油污染程度的土壤样品,从中筛选出高效降解石油烃的4个菌系和8个单菌株。分别以原油、柴油、烷烃和多环芳烃（PAHs）为底物进行培养,测定降解菌的生物量和降解率,研究其对不同底物的耐受浓度和降解潜力。结果表明,获得的石油烃降解菌为轻度嗜盐菌;不同菌株对不同底物的耐受浓度不同,混合菌系对不同底物的降解能力强于单菌株,对单一组分底物的降解优于复杂组分的底物;单菌株I-2、3、5、7能较好地降解PAHs并且对原油的降解能力高于柴油,单菌株I-1、4、6、8能够利用烷烃且对柴油的降解能力要比原油高;降解菌对柴油和原油的最高降解率分别可达78．4％和70．7％,对正十六烷和菲的生物降解率分别高达87．7％和88．1％,表现出较强的降解能力。研究结果表明黄河三角洲盐渍化土壤中土著菌对石油烃污染土壤具有较强的牛物修复潜力。     

两株真菌的分离及其在石油污染土壤修复中的作用

从石油污染的盐碱土壤中分离获得2株真菌,并对其生理生化性质进行初步研究。将2株菌扩大培养,制成混合菌剂,通过盆栽试验,以石油烃降解率、脱氢酶活性和土壤的微生物多样性等为指标,研究了不同剂量的混合菌剂对石油污染土壤修复的作用。结果表明,添加菌剂各处理的石油烃降解率、脱氢酶活性和微生物多样性明显高于对照;石油烃降解率随菌剂加入剂量的增大而提高,加入8%的菌剂,70 d石油烃降解率可达63%,是对照组的1.44倍。     

12C6+重离子诱变海迪茨氏菌及其修复柴油污染土壤的研究

应用重离子束辐射改良海迪茨氏菌(Dietzia maris)菌株在降解方面的能力,将离子辐射学与环境修复学相结合,论证该菌在以柴油为代表的污染土壤中的修复能力.利用12C6+重离子能量为80MeV、LET为35.5keV·μm-1、吸收剂量率1Gy· min-1、辐射剂量为5～120Gy辐射具有降解石油烃的代表菌株Dietzia maris,经初选、复选筛选得到了突变菌株DMYR9,并对其在构建污染土壤样品中分解柴油率及修复前后土壤系统中酶活性相关参数与化学改良修复前后污染土壤样品相关数据进行对比分析.结果表明,土壤受柴油污染不久后过氧化氢酶、脱氢酶和脂酶的活性呈现上升趋势,由突变菌体DMYR9修复后的污染土壤系统中脂酶活性呈现最低下降趋势,随着土壤中柴油被DMYR9降解,脂酶活性又不断降低,突变菌体数量及柴油降解率都具有很好的正相关性.突变菌株DMYR9进行生物修复的效果优于化学法修复,这对今后在土壤污染治理中开发利用此突变菌体具有指导意义,并具有较高的后续应用利用价值.     

石油污染土壤的生物修复研究

通过田间试验研究了玉米和向日葵两种植物对石油污染土壤的修复作用,考察了外源菌（OX-9）对植物修复的强化和协同效应,对“外源菌一植物”修复效果进行了初步评价。结果表明,在10000mg·k-1污染浓度下,150d玉米、向13葵试验区土壤中石油降解率分别为42．5％和46．4％,较对照区提高了100．5％和118．9％。外源节细菌的施加可使生物修复速度显著加快,150d“DX-9-玉米”和“DX-9-向日葵”试验区石油烃降解率分别达到72．8％和76．4％,较同期单独植物修复的降解率提高了71.3％和64．7％。500d各试验区土壤中石油烃降解率分别为95．5％、96．1％、97．6％和98．9％,土壤中石油烃含量均低于国家标准规定限量（〈500mg·kg-1）;土壤主要理化性质、生物群落分布、呼吸强度及植物不同部位中石油烃的残留量与对照无显著差异。结果表明：玉米、向日葵与节细菌对石油污染土壤的联合生物修复效果显著;经过两年修复,污染土壤恢复健康状态。     

外源菌群及与植物联合修复华北中度石油污染土壤效果研究

为探讨不同微生物菌群对华北地区中度石油污染土壤的修复效果,在实验室模拟条件下分别进行优势外源石油降解菌群的筛选和优势菌群与植物联合修复试验。结果显示:4种外源石油降解菌群中,PDC-3菌群对中度石油污染土壤总石油烃(TPH,total petroleum hydrocarbons)去除率为84.07%,在各菌群中效果最优;该优势菌群与植物联合修复中度石油污染土壤比单独使用优势菌群修复可获得更好的效果,优势菌群与黑麦草联用及优势菌群与紫花苜蓿联用120 d TPH的去除率分别为91.58%和89.30%,修复后土壤中TPH含量均小于500 mg/kg;同时优势菌群与黑麦草联合修复在90 d即可去除89.32%的土壤TPH,相比选用紫花苜蓿可有效缩短修复周期;优势菌群对土壤TPH的去除起主要作用,其贡献率远高于土著微生物菌群或植物的贡献率;植物对土壤TPH去除的贡献率为4.09%～6.48%,且其作用主要发生在修复过程的中后期;优势菌群单独使用或与植物联合修复中度石油污染土壤120 d均可有效去除C10～C12及C22～C40石油烃组分,去除率为85.14%～100.00%;然而,C13～C21石油烃组分含量表现出阶段性的积累效应;除对土壤石油烃的去除作用外,使用优势菌群进行生物修复存在一定的调节土壤pH、增加土壤肥力,且有利于恢复修复后的土壤功能。     

一株蒽降解菌的分离筛选及降解特性初析

多环芳烃(PAHs)是土壤中最常见的典型持久性有机污染物,蒽作为其典型代表,常被用作降解PAHs的模型化合物和检测PAHs污染的指示物。采集北京郊区受污染土样,通过驯化和富集,分离出185株具有蒽降解潜力的菌株,采用高效液相色谱法定量测定菌株的蒽降解效率,筛选获得一株可高效降解蒽的菌株R4004-2。经菌落形态观察、16SrRNA和gyrB基因系统发育分析、基因组框架图ANI、DDH和dDDH值分析,确定R4004-2为施氏假单胞菌(Pseudomonas stutzeri)。菌株R4004-2以蒽为唯一碳源,对蒽的降解率可达82.3%,在肉汤培养基中的生长速度最快。在蒽含量为0.25mg/kg的污染土壤中,施氏假单胞菌R4004-2可以有效定殖,具有修复污染土壤的潜力。基于转录组以及代谢组结果,施氏假单胞菌R4004-2通过双加氧酶进行对蒽的第一步氧化,后续分别通过蒽醌的氧化以及1-羟基-2-萘甲酸酯的分解两个途径完成对蒽的降解,初步解析了其降解机制。     

石油污染土壤的生物修复技术研究

从炼油厂污水池底泥中驯化、分离、筛选,得到4种优势石油降解菌。采用摇床培养,研究了各优势菌和混合菌对石油烃的降解性能;采用黄豆、苜蓿和混合菌对石油污染土壤进行了植物-微生物联合修复试验。结果表明,4种菌和混合菌25天可将初始质量浓度为10000mg/L的石油烃依次降解74.36%、54.36%、78.19%、62.17%和83.73%;运行120天,苜蓿、黄豆试验田污染土壤中的石油烃减少46.83%和41.27%;外源混合菌的施加使两种植物的降解率分别提高到67.14%和56.92%。苜蓿或黄豆-土著微生物-外源混合菌联合修复石油污染土壤效果显著。     

Bioremediation of petroleum-contaminated soils using Streptomyces sp. Hlh1

Purpose

Bioremediation using microorganisms is a promising strategy to remediate soil with petroleum hydrocarbons. Streptomyces sp. Hlh1, an endophytic strain, has previously demonstrated the ability to degrade crude petroleum in liquid culture. To apply this strain at field scale, it is necessary to test its ability to colonize the soil, compete with native microbiota, and remove the petroleum hydrocarbons under unfavorable conditions. Herein, a study was conducted to evaluate the performance of Streptomyces sp. Hlh1 to remove crude petroleum from contaminated sterilized and non-sterilized soils.

Materials and methods

Soils samples, contaminated with 2%, 5%, and 10% of petroleum, were inoculated with Streptomyces sp. Hlh1, and incubated at 30 °C for 4 weeks. At the end of bioremediation assays, the pollutant concentrations were determined by Gas chromatography flame ionization detector and the degradation rates were also calculated. The survival of the strain in the soil was estimated and the toxicity of metabolites was evaluated on Lactuca sativa.

Results and discussion

Streptomyces sp. Hlh1 was able to grow and remove total petroleum hydrocarbons (TPH), n-alkanes, and aromatic hydrocarbons found in soil samples. In sterilized soil samples, Streptomyces sp. Hlh1 removed up to 40% of TPH at an initial concentration of 10%. Whereas, the maximum TPH removal reached was 55% in non-sterilized soil at an initial concentration of 2%. In addition, it was observed that the degradation of aromatic hydrocarbons was more active than n-alkanes. The strain grew well and produced high biomass in contaminated soil. Lettuce seedling was found to be the adequate bioindicator to assess the toxicity of petroleum end products. Streptomyces sp. Hlh1 performed a successful bioremediation, which was confirmed through the phytotoxicity test.

Conclusions

The study shows the first insight of the contribution of free endophytic actinobacterial strain in the bioremediation of petroleum-contaminated soil; therefore, it suggests that Streptomyces sp. Hl1 can be usefully exploited at field scale.

     

复合菌群的构建及其对石油污染土壤修复的研究

从石油污染土壤中富集分离、筛选出3株高效降解石油的微生物菌株,通过生理生化特性研究及16SrRNA基因序列分析,确定3株菌均属于红球菌属（Rhodococcus sp）,研究和比较了它们与实验室保存的4株菌（分别属于Gordonia sp,Comamonas sp,Pesudomonas sp）降解石油的能力。这7株菌株对石油的不同组分具有不同的降解能力,对7株菌进行不同的组合用以研究复合菌群对石油的降解。结果表明,由两株Rhodococcus sp,一株Gordonia sp和一株Pesudomonas sp组成的复合菌群D,降解石油的能力超过任何单一菌株和其他组合菌群。混合菌群D在5d的培养中能降解70.3%的石油总量和71.4%的芳香化合物。混合菌群D能降解99.8%的C13-19烷烃,92.6%的C20-26烷烃,82.2%的C27-32烷烃以及90.2%的植烷。在实验室模拟条件下,对土壤中石油的降解率达到50%以上。降解土壤中石油的最适温度为10～30℃、pH值为6.5～9.5,接种量需要在106CFU·g-1以上。     

Changes in Hydrocarbon Composition and Autochthonous Microorganism Growth of Contaminated Mining Soil During Bioremediation

The goal of this study was to evaluate the effect of high Tween 80 concentrations on hydrocarbon contaminants and hydrocarbonoclastic microorganisms present in contaminated mining soil during a bioremediation process. The independent variables included Tween 80 concentration and process time. The elected response variables included concentration of total petroleum hydrocarbons (TPH), the chemical composition of contaminants, viable microbial count, and CO₂ production. These were measured at various time points during the bioremediation process, which was conducted at room temperature. Higher removal of pollutants occurred at low Tween 80 concentrations. An analysis of the normalized variables shows that the rate of TPH degradation depended on microbial growth rate, which depended on Tween 80 concentration. The addition of Tween 80 changed the concentration and type of the residual hydrocarbon contaminants present, as well as the count and genus of the hydrocarbonoclastic microorganisms that grew during the bioremediation process. Higher concentrations of Tween 80 increased the levels of the chemical compounds present in the bioremediated soil while reducing the viable count and microbial diversity during the bioremediation process. Conversely, low Tween 80 concentrations produced only monoethylhexyl phthalate, which is not a petroleum hydrocarbon derivative. Overall, these results prove that the removal of TPH is dependent on the count and genus of active indigenous hydrocarbonoclastic microorganisms in soil, and these conditions may be controlled by modulating the concentration of Tween 80 that is applied.     

微生物与有机肥混合剂修复石油污染土壤的研究

采用含有4种菌的菌剂与多种有机肥联合修复石油污染土壤,通过盆栽实验对不同浓度菌剂处理土壤中的石油烃降解率、16种多环芳烃（PAHs）浓度、脱氢酶活性、pH、阳离子交换量和微生物多样性等变化进行了研究。结果表明,腐植酸、诺沃肥和生物有机钙等有机肥和菌剂（4%处理）的加入使土壤盐碱环境得到明显改善,土壤pH稳定于6.9,阳离子交换量为201.94cmol·kg-1;对比4个不同浓度菌剂处理的效果,4%菌剂处理与有机肥联合作用修复效果最显著,石油烃降解率可达到73%,大部分所测PAHs浓度显著降低,其中萘、蒽、苯并（a）芘和苯并（g,h,i）芘降解率分别达到了65.5%、57.7%、74.7%和55.5%,土壤微生物数量增加,多样性更为丰富。     

A Comparison of Five Bioassays to Monitor Toxicity During Bioremediation of Pentachlorophenol-Contaminated Soil

Five bioassays were used to measure toxicity during bioremediation of a soil contaminated with pentachlorophenol (PCP; 335 ppm), polycyclic aromatic hydrocarbons (PAHs; 1225 ppm) and petroleum hydrocarbons (19 125 ppm). Different bioremediation treatments were tested in soil microcosms including amendment with phosphorus and/or PCP-degrading Pseudomonas sp. UG30, either as free cells or encapsulated in κ-carrageenan. Soil toxicity was monitored using the solid-phase Microtox test, SOS-chromotest, lettuce seed germination, earthworm survival and sheep red blood cell (RBC) haemolysis assays. PCP levels were reduced in all treatments after 210 days. The RBC lysis assay, Microtox test and SOS-chromotest indicated reduced toxicity in most of the microcosms by day 210. Trends depicted by lettuce seed germination and earthworm survival LC50 values varied with each treatment. For example, in soil amended with phosphorus, both the seed germination and earthworm survival LC50 data suggested increased soil toxicity. However, for soil treated with encapsulated Pseudomonas sp. UG30 cells, the earthworm survival LC50 data indicated reduced toxicity while seed germination LC50 values showed little change from values obtained prior to bioremediation. Our results show that toxicity trends in a contaminated soil during bioremediation differ according to the assay used.     

石油污染盐碱土壤的淋洗施肥修复

采用淋洗施肥修复方法处理石油污染盐碱土壤,评价该修复方法对石油污染盐碱土壤的修复效果,并且采用最大或然数法和Biolog方法对土壤微生物数量和微生物群落水平生理特性进行研究。结果表明,经过182d的培养,淋洗施肥处理中油和脂的降解率分别比对照处理和施肥处理高（19.7±4.3）%和（13.8±3.4）%,土壤盐分去除率分别比对照处理和施肥处理高（66.5±2.9）%和（41.3±6.2）%,说明该处理是一种修复石油污染盐碱土壤的有效方式。淋洗施肥处理明显提高异养细菌、石油烃降解菌、烷烃降解菌和多环芳烃降解菌数量和土壤微生物活性,促进了微生物对土壤中油和脂的降解。此外,淋洗施肥处理提高了土壤微生物Shannon多样性指数和Simpson指数,促进了微生物种群的稳定,这暗示着土壤微生物种群正在逐渐恢复。     

采用通气堆沤对石油烃污染土壤进行生物修复

Laboratory simulation studies and a composting pilot study were conducted to evaluate the capacity of three strains of fungi, indigenous fungus Fusarium sp. and Phanerochaete chrysosporium and Coriolus Versicolor, to remediate petroleum-contaminated soils. In laboratory, the fungi were inoculated into a liquidculture medium and the petroleum-contaminated soil samples for incubation of 40 and 50 days 5 respectively. In the 200-day pilot study, nutrient contents and moisture were adjusted and maintained under aerobiccondition in composting units using concrete container (118.5 cm × 65.5 cm × 12.5 cm) designed specially for this study. The laboratory simulation results showed that all the three fungi were effective in degrading petroleum in the liquid culture medium and in the soil. At the end of both the laboratory incubations, the degradation rates by Phanerochaete chrysosporium were the highest, reaching 66% after incubation in liquid culture for 50 days. This was further demonstrated in the composting pilot study where the degradation rate by P. chrysosporium reached 79% within 200 days, higher than those of the other two fungi (53.1% and 46.1%), indicating that P. chrysosporium was the best fungus for bioremediation of soil contaminated with petroleum. Further research is required to increase degradation rate.