耐高温沼渣降解菌的筛选及复合菌剂降解效果研究

分离筛选耐高温纤维素降解细菌，构建可快速降解沼渣的复合菌剂，缩短堆肥腐熟时间。采用高温富集筛选法，从宁夏中卫市秸秆堆和畜禽粪便中分离筛选耐高温细菌，经羧甲基纤维素钠水解试验和滤纸条崩解试验复筛获得具有纤维素降解能力的菌株。通过纤维素酶和木质素酶活性比较，确定目标菌株并进行分类鉴定。开展拮抗性试验和菌种复配，构建复合型菌剂；经沼渣堆肥场地试验验证复合菌剂的促腐熟效果。本研究筛选获得2株耐高温纤维降解细菌——蜡样芽胞杆菌(Bacillus cereus)W44菌株和解蛋白芽胞杆菌(Bacillus proteolyticus)X51菌株，纤维素酶活性分别为34 812.69 U/L和34 159.10 U/L。比较菌株W44/X51配比分别为1:1、1:2和2:1的复合菌剂对沼渣堆肥的促腐熟效果，添加不同的复合菌剂均缩短了沼渣堆肥腐熟时间，其中1:2处理腐熟时间最短，较CK提前18 d。蜡样芽胞杆菌W44和解蛋白芽胞杆菌X51按1:2配比的复合菌剂可用于沼渣快速堆肥发酵。     

石油烃降解菌的筛选与鉴定

从胜利油田的石油污染土壤中经富集培养分离出50株细菌,其中33株菌在以石油为惟一碳源和能源的选择性培养基中生长良好.采用紫外分光光度法对菌株的降解能力进行测定,结果有16株菌在石油初始浓度为2 500 mg·L-1的培养液中振荡培养4 d降解率超过30%,其中PU-34、PU-15、PU-2、PU-4、PU-1降解能力较高,4 d能够使石油烃类含量分别减少58.38%、55.55%、55.17%、53.09%、52.36%,在生物修复石油污染技术中具有应用前景.结合形态特征观察、生理生化特性和16S rDNA序列分析的方法对这5株菌进行菌种鉴定,确定PU-34为假黄单胞菌(Pseudoxanthomonas sp.),PU-15和PU-2为戈登氏菌(Gordonia sp.),PU-4为红球菌(Rhodococcus sp.),PU-1为假单胞菌(Pseudomonas sp.).     

高效石油降解菌的筛选及其降解性能研究

从长期被石油污染的土壤中驯化筛选、分离出2株高效石油降解菌Y-7和Y-9,通过形态学特征的观察和生理生化试验对其进行初步鉴定,鉴定结果分别为假单胞菌属(Pseudomonas sp.)和芽孢杆菌属(Bacillus sp.)。同时,研究并分析了不同pH、温度、初始石油浓度、接种量、吐温80等条件对菌体生长和石油降解率的影响。结果表明,在试验条件下,2株优势菌在初始pH为7左右,对石油的降解率可分别高达68.7%,74.5%,偏酸或偏碱的环境均不利于菌体的生长;培养温度对2株菌体生长和石油降解率的影响较大,最佳温度为35℃,降解率达到最大,分别为73.1%和69.6%;石油初始浓度大于0.4g/L时,Y-7降油率从69%降到49%,Y-9基本变化不大,控制石油物质浓度在0.4g/L,有利于对石油的生物处理;最佳接种量为2mL/L;吐温80对石油的降解促进作用有待进一步分析与研究。     

耐高温木质素降解菌筛选及降解效果研究

为提高育苗基质中废弃物木质素降解速率,在废弃物堆腐生产育苗基质高温阶段取样,筛选耐高温木质素降解菌,并对菌种进行鉴定,同时测定其对秸秆木质素和菌糠木质素的降解效果。获得了1株较好的木质素高温降解菌HZ11,鉴定为解淀粉芽胞杆菌（Bacillus amyloliquefaciens）,结果显示,该菌株对秸秆木质素和菌糠木质素降解效果较好,50 ℃条件下,20 d木质素降解率分别为46.7%和42.4%。菌株HZ11在降解秸秆和菌糠方面具有很好的应用潜力,为利用农业废弃物生产育苗基质提供更加丰富的菌种资源,具有重要的参考价值。     

耐盐碱石油烃降解菌的筛选、鉴定及其耐盐碱性研究

从大港油田区石油污染盐碱化土壤和油泥中筛选得到10株耐盐碱石油烃降解菌,通过形态特征、生理生化特征和16S r RNA序列分析确定这些菌株为苍白杆菌属、葡萄球菌属、迪茨菌属、棒状杆菌属、无色杆菌属、微杆菌属、芽孢杆菌属。通过液体培养试验,研究了10株菌的耐盐碱能力。结果表明,除B07仅能耐受3%盐度外,其他菌株均能耐受5%或者更高的盐度环境,其中B02和B05在盐度高达11%时仍具有较高的生长活性;10株菌均能耐受p H为9的碱度环境,B01、B03、B04、B06、B09能耐受p H为10的环境,其中,B03和B04在p H为11时仍具有较高的生长活性。研究表明石油烃降解菌在不同微生物种属中广泛存在,并具有较好的耐盐碱特性,有望在石油污染盐碱化土壤修复中广泛应用。     

一株石油烃降解菌分子鉴定及特性分析

从受污海水中分离筛选了具有石油烃降解能力的降解菌Bac1020,经PCR扩增得到1 497 bp长16S rDNA序列,通过Blast比对,与主要石油烃降解菌属16S rDNA序列构建系统发生树,鉴定其为不动杆菌(Acinetobactersp.)。降解菌(Acinetobactersp.)在72 h内生长稳定,对石油烃的降解率随时间的延长而不断增加。建立了快速筛选及鉴定石油烃降解菌的方法,应用于海洋石油烃污染的生物降解。     

采用酶液活性测定法快速筛选及构建石油烃高效降解菌系

快速筛选复杂有机物降解微生物混合菌系,在污染物治理过程中具有重要的实践意义.本研究首次尝试利用MicroResp^TM技术分析微生物酶液活性的方法,快速标定高效降解菌及混合菌系的石油烃降解能力,并采用传统的摇瓶培养检测法予以验证.通过微生物胞内、胞外及混合酶液的活性分析,考察了不同酶系(胞外、胞内及混合酶液)、菌系对石油烃分子的降解情况.结果表明: 结合MicroResp^TM技术的酶液活性测定法能够快速检测石油烃代谢酶系的降解能力,其灵敏度好、通量高,与传统的菌株摇瓶培养方法的检测结果基本一致.其中,7株菌株的120种全组合菌系活性测定试验在12 h周期内1次完成.筛选周期由传统摇瓶培养所需的7 d缩短10倍以上.以酶活性测定结果为指导设计的复配菌系具有较高的降解效率,最高石油烃降解率达(56.1±1.6)%.表明本筛选方法精度高、通量高,可用于石油烃降解功能菌系的构建.     

海洋石油降解菌AH07的分离鉴定及其石油降解性能分析

以原油为唯一碳源,从大连新港石油污染区域海底沉积物中分离获得1株石油高效降解菌AH07。通过形态学观察、生理生化特征检验及16S rDNA序列分析,确定菌株AH07为人苍白杆菌(Ochrobactrum anthropi),GenBank序列登录号为KT831449。通过单因素试验确定了菌株AH07的最优生长条件,即培养温度为30℃,培养基pH 7.0。为了进一步了解并提高菌株AH07的降解性能,选用5种氮源考察不同氮源对菌株石油降解性能的影响。结果表明,玉米粉为最佳有机氮源,NH_4NO_3为最佳无机氮源;28℃、150 r/min振荡培养10 d,菌株AH07对原油的降解率分别为58.25%和31.98%。     

丢糟和磷矿粉高温堆肥中耐高温解磷菌的筛选及性能分析

为了解决高温极端环境下的磷素溶出问题,从高温堆肥中分离出具有耐高温能力的解磷微生物。该研究利用无机磷选择培养基,从添加磷矿粉和丢糟的高温堆肥样品中,分离筛选出5株耐高温解磷菌(细菌为GDB1-2;真菌为GDF1-3),并对这5株菌株进行形态学和分子生物学鉴定及解磷能力分析。研究结果表明,筛选获得的解磷菌株分别为枯草芽孢杆菌(Bacillus subtilis)、地衣芽孢杆菌(Bacillus licheniformis)、多枝横梗霉(Lichtheimia ramosa)、烟曲霉(Aspergillus fumigatus)及构巢曲霉(Aspergillus nidulans)。该5株菌具有较好的耐高温和耐pH性能,各菌株耐高温范围为40-60℃,在50℃条件下其解磷能力均达到最大值,当初始pH 在5-9范围时各菌株均能保持一定的解磷能力。此外,通过解磷曲线发现各菌株的最高解磷量范围在136.85-174.33 μg/mL。该研究结果为后续开发高温环境微生物资源提供了素材,具有良好的应用推广前景。     

低温石油降解菌LHB16的筛选及降解特性研究

目的:筛选、鉴定低温石油降解菌并对其降解特性进行研究.方法:富集分离低温石油降解菌;采用形态学、生理生化实验和分子生物学方法进行菌种鉴定;紫外分光光度法和GC-MS检测石油降解特性.结果:自盘锦油田低温环境土样中分离到1株低温菌,命名为LHB16,该菌能以石油烃为惟一碳源和能源.经鉴定为嗜麦芽窄食单胞菌(Stenotrophomonas maltophilia).该菌生长温度范围0℃～35℃,最适生长温度15℃.在接种量为2%(V/V),原油浓度为0.5%(W/V),振荡培养10 d时,降解率可达80.16%.石油中长链烷烃C15～C32被完全降解.传代培养数代,降解率为81.06%,降解性能稳定.结论:菌株LHB16在低温地区石油污染的生物治理中有良好的应用前景.     

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.     

大连新港原油降解微生物的分离纯化及降解效率评价

目的 在大连新港原油污染海域分离纯化出可降解原油的“土著”微生物,评价其原油降解能力,并研究提高降解效率的方法.方法 取海水样品进行富集培养,分离纯化出“土著”原油降解微生物,以16S rDNA测序法鉴定微生物种类,并采用MEGA 5.0进行多序列比对分析,选用最大相似法构建系统发育树.在实验室纯培养的条件下以气相色谱法对微生物的原油降解能力进行分析,选出优势菌种,再将优势菌种混配分析最佳原油降解条件.结果 分离纯化得到的“土著”原油降解微生物分属枯草芽孢杆菌属、动性球菌属、嗜冷菌属等多个菌属,“土著”原油降解微生物资源丰富,优势菌种的混配有助于加快和提高原油降解效率,是有效且对生态环境友好的生物处理法.     

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.     

Establishment of oil-degrading bacteria associated with cyanobacteria in oil-polluted soil

N.A. SORKHOH, R.H. AL-HASAN, M. KHANAFER AND S.S. RADWAN. 1995. A unique natural microbial cocktail with promising potential for remediating oil-polluted desert in the Gulf region is reported. Oil-degrading micro-organisms immobilized within dense cyanobacterial mats on oily coasts of the Arabian Gulf were successfully established in oil-contaminated sand. Those micro-organisms biodegraded 50% of the oil within 10–20 weeks. Nocardioforms belonging to the genus Rhodcoccus predominated in the first few weeks, but after 22 weeks Pseudomonas spp. increased, sharing Rhodococcus in the predominance. Other oil-utilizing bacterial genera included Bacillus and Arthrobacter. Filamentous actinomycetes belonging to the genera Streptomyces and probably Thermoactinomyces , as well as fungi belonging mainly to Aspergillus and Penicillium increased in the contaminated sand during the experiment but declined later. Representative strains grew on spectra of the tested n -alkanes with chain lengths between C₁₀ and C₄₀, as sole sources of carbon and energy.     

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.     

Thermotolerant oil-degrading bacteria isolated from soil and water of geographically distant regions

Oil-degrading bacteria were isolated from soil and water samples taken in Russia, Kazakhstan, and the Antarctic; 13 of 86 strains proved to be thermotolerant. These bacteria utilized crude oil at 45–50°C; their growth optimum (35–37°C) and range (20–53°C) differ from those of mesophilic bacteria. Thermotolerant strains were identified as representatives of the genera Rhodococcus and Gordonia. It was shown that their ability to degrade petroleum products does not differ at 24 and 45°C. The strains Rhodococcus sp. Par7 and Gordonia sp. 1D utilized 14 and 20% of the oil, respectively, in 14 days at 45°C. All of the isolated thermotolerant bacteria grew in a medium containing 3% NaCl; the medium for the strains Gordonia amicalis 1B and Gordonia sp. 1D contained up to 10% NaCl. The bacteria G. amicalis and Rhodococcus erythropolis were able to utilize crude oil and individual hydrocarbons at higher (up to 50°C) temperatures.     

Ultrastructure of two oil-degrading bacteria isolated from the tropical soil environment

Two oil-degrading bacteria identified as Pseudomonas aeruginosa and Micrococcus luteus were isolated from crude-oil-polluted soils in Nigeria. The organisms were grown on n-hexadecane and sodium succinate and then examined for the presence of hydrocarbon inclusions. Inclusion bodies were found in n-hexadecane-grown cells and were absent in succinate-grown cells. Formation of hydrocarbon inclusion bodies appears to be a general phenomenon among hydrocarbon utilizers.     

印度洋表层海水石油降解菌的多样性分析

【目的】为了研究印度洋石油降解菌多样性,并获得新的石油降解菌。【方法】本研究通过印度洋表层海水样品采集、以柴油与原油1:1混合物作为碳源,从中富集、分离筛选石油降解菌,并通过PCR-DGGE对13个站点富集菌群的菌群结构进行分析。【结果】通过形态观察、生理生化反应和16SrRNA分析,共得到共29个属的51株不同的细菌,它们主要是属于α亚群和γ亚群。其中,Alcanivorax属(占18%),Novosphingobium属(占10%)和Marinobacter(占6%)Thalassospira(占6%)为主要的优势菌。通过生态多样性分析表明,Shannon-Winner指数(H)为4.57968,说明其具有较高的多样性;均匀度指数(E)为0.8664771,表示其分布比较均匀。单菌实验表明,49株具有石油降解能力其中,Sinomonas,Knoellia,Mesoflavibacter等属的细菌为首次发现有降解能力。DGGE分析表明Alcanivorax属的细菌是印度洋表层海水中的重要石油降解菌。【结论】本研究首次揭示了印度洋表层海水中石油降解菌的多样性,并获取了若干在海洋石油污染生物修复中具有应用前景的降解菌。