泽普油田石油降解功能菌株的分离与鉴定

目的:在研究泽普油田产区菌株多态性的基础上,对石油降解功能菌株进行探讨。方法:通过稀释平板涂布法分离微生物;采用TLC和HPLC方法对分离菌株进行多糖成分鉴定,并进行16S rRNA和18S rRNA序列分析;采用溶血圈实验、排油圈法、石油黏度测定等方法,测定石油降解菌株降解能力。结果:分离菌株中有5株细菌和4株真菌,且菌株多糖的TLC分析结果与形态学鉴定结果基本相同;5株分离细菌中,2株细菌隶属于Bacillus属,3株细菌隶属于Enterobacter。另外4株真菌被鉴定为隶属于Penicillium属;分离菌株B2能降解石油。结论:泽普油田石油产区微生物的群落中存在一定丰度的细菌和真菌,含有B2的原油发酵液黏度为0.67±0.20 mPa.s.和原油在0.05水平上有显著性差异。推断B2对石油具有一定的降解能力。     

中国东海和南海海域可培养烃类降解细菌的筛选及功能

【背景】海洋环境中蕴藏着丰富的微生物资源,其种类繁多而且功能多样,在驱动物质循环及能量流动等方面起着重要的作用。目前,海洋中烷烃化合物降解菌的分离筛选和降解功能研究已有文献报道;但是对海洋中尤其是我国东海和南海海域,具有降解芳香烃类化合物功能的菌株分离筛选及其多样性研究鲜有报道。【目的】分离筛选我国东海和南海海域具有烃类降解能力的可培养菌株,并对其降解功能和多样性进行初步研究。【方法】分别从东海和南海海底沉积物样品中筛选菌株,选择不同的烃类化合物为菌株筛选的唯一碳源,采用梯度稀释和平板划线法分离纯化得到单菌落,并利用相应烃类为唯一碳源进行生长验证获得该化合物降解菌。【结果】以肉桂酸、碱木素、十六烷等12种烃类化合物为唯一碳源,从样品中共分离到63株具有烃类化合物降解能力的菌株,分别属于3个门4个纲8个目10个属,主要为红球菌属(Rhodococcus)、不动杆菌属(Acinetobacter)、弧菌属(Vibrio)、盐单胞菌属(Halomonas)、假单胞菌属(Pseudomonas)。两大海域优势降解菌差别较大,其中东海沉积物降解菌株主要为不动杆菌属(Acinetobacter),而南海沉积物降解菌株主要为红球菌属(Rhodococcus)。【结论】我国东海和南海海域蕴藏着丰富的烃类化合物降解菌株资源,两大海域优势降解菌种类存在明显差异,这将为我国未来可能的海洋环境石油污染的微生物治理储备菌种资源。     

高浓度氯苯优势降解菌的筛选及其降解酶的纯化

[目的]分离纯化出一株高浓度氯苯优势降解菌株,对其所产氯苯降解酶进行分离与纯化,为该菌株及其氯苯降解酶的研究提供理论参考.[方法]利用梯度富集培养技术和无菌滤纸片平板法分离菌株,通过形态特征及16S rRNA基因序列分析初步鉴定菌株,用气相色谱法测定培养液中氯苯浓度,以单位细胞氯苯降解率评价菌株对氯苯的降解能力,以氯苯降解率表示氯苯降解酶的活性.取纯化菌株的发酵酶液制备粗酶液,经硫酸铵梯度盐析、透析脱盐、DE-52离子交换层析、G-100凝胶层析和透析浓缩后,进行SDS-PAGE凝胶电泳检验酶的纯度并测定酶的分子量.[结果]从氯苯长期驯化的成熟期活性污泥中筛选到一株以氯苯为唯一碳源和能源的氯苯优势降解细菌LW13,该菌株在以2000 mg/L氯苯为唯一碳源的无机盐培养基中仍能正常生长,其单位细胞氯苯降解率可达1.37 ×10-10.扫描电镜观察到该菌株细胞大小约为2.3 ×0.8μm,长有数根端生鞭毛.16S rRNA基因序列相似性比较表明该菌株与Lysinibacillus fusiformis(溶藻菌)的相似性达95.5％.所纯化的氯苯降解酶为胞外酶,带正电荷,其分子大小约为57 kDa.整个纯化过程中酶纯化倍数化达8.0倍,酶活回收率达52.51％,酶量回收率达6.57％.纯化后的氯苯降解酶在30℃-55℃和pH在6.0-8.0之间都保持较高的酶活性,其最适反应温度和pH分别在40℃和pH8.0左右.[结论]所分离的氯苯优势降解菌属于Lysinibacillus属菌株,该菌株能有效降解高浓度(500-2000 mg/L)氯苯废水,通过逐级分离纯化,可获得氯苯降解酶纯酶,纯化指标符合分离纯化基本规律,纯化效果较为理想.     

原油微生物群落构成及降解菌降解特性的研究

为分离得到高效原油降解菌,直接向原油中加入营养物刺激,培养一段时间后原油乳化降解。气相色谱法、红外光谱法和紫外吸收均表明降解菌体系具有较强的降解原油烃的能力。采用细菌16S rDNA通用引物和PCR扩增等方法,构建原油降解菌体系16S rDNA克隆文库,并对所构建的文库进行分析。同时,从降解菌体系中分离得到一株降解菌,鉴定结果表明所分离的细菌为短小芽孢杆菌(Bacillus pumilus)。采用气相色谱技术对降解过程中的原油烃的变化进行分析,结果显示原油烃类组分会随着降解时间的变化而发生变化,降解菌表现出其对烷烃类物质的降解能力。     

大蜡螟和黄粉虫肠道菌中聚乙烯地膜降解细菌的筛选及其降解性能

【背景】在我国农业生产中大量使用的聚乙烯(polyethylene,PE)地膜难以降解,在土壤中长期累积影响农作物生长并破坏生态环境,发掘微生物资源,寻求聚乙烯生物降解途径对治理“白色污染”具有重要意义。【目的】以不同来源的啮食塑料昆虫大蜡螟、黄粉虫为材料,从肠道菌群中分离筛选出对PE具有降解能力的细菌菌株,研究其降解农用地膜的效能。【方法】饲喂PE膜片驯化大蜡螟、黄粉虫幼虫,采集肠道液富集培养、共代谢驯化、选择培养基筛选等方法从肠道细菌中分离出以PE为唯一碳源的细菌菌株。将菌株接种到以PE膜片为唯一碳源的培养基中共培养,通过测定菌体生长量,定期检测膜片失重率,结合高分辨场发射扫描电子显微镜观察、红外扫描分析和膜片力学性能测定,评价菌株对聚乙烯地膜的降解效果。对筛选出的降解性能良好的菌株通过16S rRNA基因扩增和序列分析进行菌株鉴定。【结果】从新疆蜜蜂蜂巢中的土著大蜡螟肠道分离获得的聚乙烯降解菌菌株最多,其聚乙烯的降解效率高于其他来源的分离菌株。从中筛选出具有较高降解能力的3个菌株XJDLM-3、XJDLM-8和XJDLM-12,它们能利用PE膜片生长,扫描电镜观察经过30 d降解的PE膜片表面出现明显的侵蚀孔洞和裂痕,红外扫描图谱发生改变,拉伸强度、断裂伸长率和弹性模量等力学性能显著下降,膜片失重率分别达到了8.06%、5.66%和5.39%。从新疆蜜蜂蜂巢中的土著大蜡螟肠道分离出降解效果较好的细菌菌株,经鉴定XJDLM-8和XJDLM-12为Bacillus cereus,XJDLM-3为Enterobacter bugandensis。【结论】证明了新疆蜜蜂蜂巢中的土著大蜡螟肠道存在对PE具有较高降解能力的菌株,丰富了PE降解菌的菌种资源,在PE地膜降解中具有开发应用的潜力。     

高效降解机油微生物的筛选及除油效果初探

从多处受石油及其制品污染的土壤中筛选到六株对高浓度机油等相关石油制品具有降解能力的微生物菌种。对该六株菌种的单独和混合降解机油的效果进行了研究,并考察了营养成分对机油降解的影响。实验表明:Px01菌种具有最强的除油能力,在合适条件下,Px01培养液的机油降解速率可达0.502g/L.d。混合菌的除油效果明显好于单菌,最高降解速率可到0.64g/L.d。混合菌的营养要求较低,添加有机营养元素对混合微生物的生长和机油降解没有显著的促进作用。由于所筛选驯化得到的微生物菌种针对高浓度含机油废水的降油效果显著,因此具有良好的应用前景。     

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。     

高效降解石油细菌的分离鉴定及降解能力的研究

目的:为获得高效降解原油的菌株,从石油污染严重的土壤中采样,富集分离得到原油降解菌,并初步考察它们降解原油的能力。方法:通过富集培养、多次筛选分离得到三株优势菌,编号为SWH-1、SWH-2和SWH-3。通过16S rDNA序列分析和NCBI数据库的Blast比对分析,对其鉴定到种。通过差量法测定它们在室内摇瓶中对原油的降解率。结果:经鉴定,这三株菌分别为枯草芽孢杆菌(Bacillus subtillus)、多食鞘氨醇杆菌(Sphingobacterium multivorum)和嗜温鞘氨醇杆菌(Sphingobacterium thalpophi-lum)。在0.5g/L的原油培养基内培养1w,SWH-1和SWH-2的降解率较高,分别为33.89%和46.31%。将这两株菌进行混合培养降解原油,降解率高达51.73%。结论:所筛选到的枯草芽孢杆菌和多食鞘氨醇杆菌在生物修复方面具有很好的应用潜力,而且多食鞘氨醇杆菌在石油降解方面的报道尚属首次。     

从麦草浆污泥中分离产木聚糖酶的木质纤维降解真菌

采用涂布平板法在马丁氏培养基平板上对麦草浆污泥作真菌分离研究,共分离纯化获得27株真菌,其中青霉属(Penicillium)、曲霉属(Aspergillus)、麦轴梗霉属(Tritirachium)根霉属(Rhizopus)和毛霉属(Mucor)为其优势菌群;在麦芽提取物-OPP培养基平板上分离得到10株真菌,其中拟指突孢曲霉属(Emercellopsis)为其优势菌群。通过对麦草浆污泥连续富集培养得到真菌10株,经初步鉴定为曲霉属(Aspergillus)和青霉属(Penicillium)。对富集培养分离的菌种进行木聚糖酶产酶特性研究,结果表明:最优菌株为Aspergillussp.MC-JAⅡ,其木聚糖酶酶活性最高值达到2060 U/mL,发生在产酶培养的第84 h,相应纤维素酶活为99.5 U/mL。为国内首次报道麦草浆污泥中的降解木质纤维真菌的分离、鉴定和产酶研究。     

一株可乳化降解原油的Compostibacillus的分离及特性

【背景】通过实施多轮次微生物采油,华北油藏产出液菌浓达到了106个/mL以上,油藏内部已经形成了较稳定的微生物发酵场,从其中筛选出能够乳化降解原油的微生物,并在地面对其进行扩大培养,然后再应用到微驱油藏,以进一步提高微生物采油实施效果。【目的】筛选乳化降解原油性能良好的菌株,对其进行多相分类学鉴定和性能评价。【方法】利用原油为底物筛选乳化降解性能良好的菌株,通过形态特征观察、生理生化测定、16S rRNA基因序列分析等确定菌株的分类地位。通过乳化能力、降解率等方法确定菌株的原油乳化降解特性。【结果】从华北油田采集的地层水样品中分离得到一株乳化原油的菌株BLG74,经多相分类鉴定表明其是土壤堆肥芽孢杆菌(Compostibacillus humi)的新菌株,亲源性99.6%。该菌株的生长温度为30-60℃ (最适温度45℃),pH6.5-9.5(最适pH7.0),NaCl浓度0%-7%(质量体积比)。菌株BLG74在玉米浆培养基中培养,其发酵液的表面张力为56.3 mN/m,乳化力约95%,在初始原油质量浓度0.5%、温度45℃的条件下培养20d,对原油的降解率可达40.8%。【结论】菌株BLG74是可乳化降解原油的新成员,其在热盐条件下乳化降解原油的特性在石油开采中有一定的潜力。     

Obligate oil-degrading marine bacteria

Over the past few years, a new and ecophysiologically unusual group of marine hydrocarbon-degrading bacteria - the obligate hydrocarbonoclastic bacteria (OHCB) - has been recognized and shown to play a significant role in the biological removal of petroleum hydrocarbons from polluted marine waters. The introduction of oil or oil constituents into seawater leads to successive blooms of a relatively limited number of indigenous marine bacterial genera--Alcanivorax, Marinobacter, Thallassolituus, Cycloclasticus, Oleispira and a few others (the OHCB)--which are present at low or undetectable levels before the polluting event. The types of OHCB that bloom depend on the latitude/temperature, salinity, redox and other prevailing physical-chemical factors. These blooms result in the rapid degradation of many oil constituents, a process that can be accelerated further by supplementation with limiting nutrients. Genome sequencing and functional genomic analysis of Alcanivorax borkumensis, the paradigm of OHCB, has provided significant insights into the genomic basis of the efficiency and versatility of its hydrocarbon utilization, the metabolic routes underlying its special hydrocarbon diet, and its ecological success. These and other studies have revealed the potential of OHCB for multiple biotechnological applications that include not only oil pollution mitigation, but also biopolymer production and biocatalysis.     

Selection and characterization of active psychrotrophic microbial oil-degrading microorganisms

The ability of 96 microbial strains degrading oil and 32 strains degrading polycyclic aromatic hydrocarbons (PAHs) to consume diesel fuel and oil at 4–6 and 24°C and at elevated NaCl concentrations was studied. The temperature range, salt tolerance, ability to produce biosurfactants, range of substrates, and antibiotic resistance were determined. The eleven most active oil-degrading and PAH-degrading strains were genotyped by a polymerase chain reaction with BoxA1R primers and a restriction analysis of ribosomal DNA amplicons. For six strains, the degree of oil degradation at 4–6°C was higher than at 24°C. For the most active strains, the degree of oil degradation in liquid mineral medium ranged from 15 to 26% at 24°C and from 28 to 47% at 4–6°C. An association of six of the strains degraded the oil by 46% at 24°C.     

耐高温石油烃降解菌的筛选及性能研究

目的筛选耐高温石油烃降解菌并对降解条件进行优化。方法以大庆地区石油污染土壤的堆肥样品为研究材料,通过富集培养后分离得到耐高温石油降解菌株,选取降解率最高的菌株,对其降解条件进行了探讨。结果得到6株耐高温石油烃降解菌,其中WY 2最适温度52~58℃,pH值7,石油浓度0.5%,接种量2mL,最佳氮源为硫酸铵,通过16SrDNA序列分析,确定该菌株为地衣芽孢杆菌。结论确定了耐高温石油烃降解菌的最佳降解条件。     

Establishing oil-degrading biofilms on gravel particles and glass plates

A method is described for “artificially” establishing biofilms rich in hydrocarbon degrading bacteria on gravel particles and glass plates. The microbial consortia in the biofilms included in additions, filamentous cyanobacteria, picoplankton and diatoms. Phototrophic microorganisms were pioneer colonizers. Hydrocarbon utilizing bacteria, namely Acinetobacter calcoaceticus and nocardioforms were in part attached to filaments of cyanobacteria. In batch cultures, it was shown that those artificial biofilms had an attenuation effect on crude-oil in contaminated sea water samples. The potential use of these biofilms for preparing trickling filters (gravel particles), and in bioreactors (glass plates) for attenuating hydrocarbons in oily liquid wastes before their disposal in the open environment is suggested and discussed.     

Change of oil-degrading activity in microorganisms stored under laboratory conditions

Change of the oil-degrading activity was studied in psychrophilic microbial strains Rhodococcus spp. DS-07, DS-21 and Pseudomonas spp. DS-09, DS-22 maintained on various media: rich and synthetic with a selective agent. After 2.5 years of storage on rich medium, the oil-degrading activity decreased by 50-60%, whereas this decrease was insignificant in the medium with oil. Passages to selective medium with oil after the storage partly restored the activity. It was found that storage of oil-degrading microorganisms caused loss of biodegradation plasmids. Their recovery and long-term preservation demand the presence of the selective agent in the medium.     

Change of oil-degrading activity in microorganisms stored under laboratory conditions

Change of the oil-degrading activity was studied in psychrophilic microbial strains Rhodococcus spp. DS-07, DS-21 and Pseudomonas spp. DS-09, DS-22 maintained on various media: rich and synthetic with a selective agent. After 2.5 years of storage on rich medium, the oil-degrading activity decreased by 50–60%, whereas this decrease was insignificant in the medium with oil. Passages to selective medium with oil after the storage partly restored the activity. It was found that storage of oil-degrading microorganisms caused loss of biodegradation plasmids. Their recovery and long-term preservation demand the presence of the selective agent in the medium.     

大连湾原油降解菌的分离和多样性分析

[目的]研究大连湾原油污染海域可培养原油降解菌的多样性,并获得新的原油降解菌.[方法]通过大连湾海水、海泥和海绵样品采集,以原油作为唯一碳源,培养、富集、分离筛选原油降解菌,根据16S rRNA基因序列确定其系统进化地位.[结果]通过形态观察和16S rRNA基因分析,共获得22个属的50株菌.其中,有6株菌的16S rRNA序列与最相近的菌株序列一致性仅为95％-97％,可能是潜在的新菌.单菌实验表明,45株菌具有石油降解能力.[结论]揭示了大连湾可培养原油降解菌的多样性,并获得了新的原油降解菌,为海洋石油污染的生物治理提供新资源.     

Predominant culturable crude oil-degrading bacteria in the coast of Kuwait

Total of 272 crude oil-degrading bacteria were isolated from seven locations along the coast of Kuwait. The analysis of the 16S rDNA sequences of isolated bacteria revealed the predominance of six bacterial genera: Pseudomonas, Bacillus, Staphylococcus, Acinetobacter, Kocuria and Micrococcus. Investigation of the factors associated with bacterial predominance revealed that, dominant culturable crude oil-degrading bacteria were better crude oil utilizers than the less frequently occurring isolates. Bacterial predominance was also influenced by the ability of bacteria to adapt to the level of organic content available. Predominant culturable bacteria constituted 89.7–54.2% of the total crude oil-degrading bacterial communities. Using 16S-RFLP analyses to assess the diversity of the dominant crude oil-degrading bacterial genera, four phylotypes of Pseudomonas sp. and seven phylotypes of Bacillus sp. were determined. This suggested high degree of diversity of crude oil-degrading bacterial population at the strain level, but low diversity at the genus level.     

Development of an oil-degrading biopreparation by activation of aboriginal hydrocarbon-oxidizing microflora

A method of activation of aboriginal hydrocarbon-oxidizing microorganisms for remediation of soil and water basins polluted with oil products was developed. The optimum composition of activating additives was found (g/l): mineral components, 10.0; oil, 5.0; and a synthetic detergent, 0.2. The resulting biopreparations increased the degree of purification by factors of 4-8 in soil and 18-24 in water when applied at a concentration of 10(7) cells/g(ml).