石油降解菌的筛选优化及其对油污土壤的修复特性

分别以牛肉膏蛋白胨-布氏哈斯培养基、蓝色凝胶培养基作为初筛和复筛培养基,从石油污染土壤中筛选出2株可产生微生物表面活性剂的石油烃降解菌。并将菌株投加到油污土壤中进行修复研究,考查了不同影响因素对修复效果的影响。研究结果表明,(1)2株菌对中度石油污染土壤有较好的修复效果,向油污土壤中直接投加菌株修复70 d时对石油烃的去除率为52%;(2)向油污土壤中投加降解菌并同时补充氮营养液,修复70 d时对土壤中总石油烃的去除率可达到75%;对土壤中正构烷烃的去除率为66%;(3)与土壤的含水率及土著菌的降解效果相比,向油污土壤中投加降解菌以及补充氮磷营养液是影响石油污染土壤修复效果的关键因素。     

用于石油污染土壤降解的高效降解菌的筛选及其降解条件优化

经过富集、分离优选出高效石油降解菌L-1,根据形态观察和生理生化特征初步鉴定为琼氏不动杆菌;采用单因素花盆实验模拟微生物原位修复并对其降解条件进行优化。结果表明,将高效石油降解菌应用于修复石油污染土壤,适宜接种量、表面活性剂浓度、CNP比、翻耕频率分别为15%、0.1%、100∶10∶1和1 d 1次;在该降解条件下修复28 d,可达到16.80%的石油降解率,远远高于土著微生物6.92%的降解率。     

代谢表面活性剂菌处理含油污泥的研究

试验采用异位生物修复技术堆肥法,对某炼厂油泥进行生物修复处理研究.用微生物代谢的表面活性剂对油泥进行预处理,洗脱油泥中部分油分后进行堆肥试验,投加从油田含油土壤中获得的以石油为唯一碳源、代谢高效生物表面活性剂的微生物C-2菌、F-2菌以及无机营养物和疏松剂(锯末),降解油泥中的石油污染物.经过外源微生物和内源微生物共同作用120 d,油泥中的石油烃总量由22 910 mg/kg下降到3 000 mg/kg以下.试验利用色谱-质谱联用方法分析了降解前后石油组分的变化.菌株经传统方法鉴定为蜡状芽孢杆菌、枯草芽孢杆菌.     

石油污染土壤生物修复菌Z1a-B的分离鉴定与调控效应研究

从山东东营胜利油田附近的石油污染土壤中分离筛选得到一株高效石油降解菌Z1a-B,通过菌落形态及显微镜个体形态观察对其初步鉴定到属,并采用气相色谱/质谱(GC/MS)法分析了Z1a-B的石油降解性能,采用投加石油降解菌、调节土壤N、P含量和优化环境因素等措施,进行了为期60d的石油污染土壤室外自然堆制生物修复实验。结果表明,Z1a-B为链霉菌属白孢类群,其摇瓶培养的石油降解率为66.4%;Z1a-B有着很宽的烷烃降解谱;N、P最佳的添加量组合为KNO32.50g/kg、K2HPO40.35g/kg,即N/P(质量比)为5.57:1.00,此时的石油降解率达63.5%,土壤脱氢酶活性达最高值,为2.99μL/g;石油降解的最佳环境条件为:将石油质量分数为3.3%的100g土样调节pH至8.5后,装入容积为300mL的锥形瓶中灭菌,再接种孢子密度为2.7×108个/mL的菌剂5.5mL,于28℃下进行生物降解,在此条件下的石油降解率可达76.5%;土壤脱氢酶活性的测定结果可以作为检验石油污染土壤生物修复效果的重要指示指标之一;室外自然堆制生物修复实验中,添加菌剂、锯末、秸秆以及N、P后,石油降解率可达69.9%,总体来说,室外自然堆制生物修复是一种投资少、见效快、治理效果较好的石油污染土壤治理方法。     

CaO2及H2O2类Fenton降解土壤石油烃污染

采用H_2O_2/Fe(Ⅲ)/柠檬酸类Fenton体系和CaO_2/Fe(Ⅲ)/柠檬酸类Fenton体系修复土壤石油污染,考察了氧化剂种类、氧化剂投加量、 Fe(Ⅲ)浓度和柠檬酸浓度对柴油降解效果的影响,并进一步研究比较了CaO_2/Fe(Ⅲ)/柠檬酸和H_2O_2/Fe(Ⅲ)/柠檬酸2种修复方式对土壤原著微生物群落变化及豌豆植株生长所带来的生态毒性效应。单因素实验结果表明:在其他条件相同的情况下,CaO_2类Fenton降解柴油效果优于H_2O_2类Fenton降解效果;柴油降解率随着氧化剂投加量、Fe(Ⅲ)和柠檬酸浓度的增大呈现先增后降的趋势。当CaO_2浓度为166.67 mmol·L~(-1)、Fe(Ⅲ)浓度为27.78 mmol·L~(-1)、柠檬酸浓度为27.78 mmol·L~(-1)时,反应24 h后,土壤中柴油降解率达到44.14%。生态毒性实验表明:CaO_2类Fenton处理后土壤微生物群落的丰富度和多样性指数均有所提高,H_2O_2类Fenton处理后均有所降低,2种处理方式均在不同程度上改变了土壤微生物群落的优势菌门构成;CaO_2及H_2O_2类Fenton处理均抑制了豌豆植株的生长,发芽率、植株干重、株高、叶绿素含量等测试指标均下降,其中H_2O_2类Fenton处理的抑制效果更为明显。进一步分析可知,CaO_2类Fenton处理技术比H_2O_2类Fenton处理技术更适用于石油污染土壤修复。     

石油污染土壤的生物修复室内模拟实验研究

在实验室模拟的条件下,利用从克拉玛依的石油污染土壤中筛选出的4株高效降解菌,以石油烃降解率、脱氢酶活性、呼吸强度、微生物量碳氮和土壤毒性作为评价指标,研究不加生物菌剂不翻耕、不加生物菌剂翻耕、加生物菌剂不翻耕、加生物菌剂翻耕、加固定化菌剂不翻耕和加固定化菌剂翻耕6种不同实验条件对石油污染土壤修复的效果。结果表明,在63 d的修复过程中,加固定化菌剂翻耕实验F组的石油去除率达到了78.7%,比不加生物菌剂不翻耕实验A组的石油去除率提高了49.5%。随着土壤毒性逐渐降低,玉米(Zea mays L.)和赤子爱胜蚓(Eisenia fetida)可以在F组土壤中良好的生长,达到了修复的效果。     

植物-微生物联合修复石油污染土壤的实验研究

筛选高效石油降解菌并考察菌株的石油降解能力,通过植物-微生物联合修复石油污染土壤室内实验,在修复过程中测定了土壤中细菌和固氮菌,碱解氮、速效磷和速效钾的含量变化,同时采用傅立叶变换离子回旋共振质谱(ESI FT-ICR MS)考察了植物-微生物联合修复效果。结果表明,菌株3#、4#的生长适应性较强,其混合菌的降解效果最好,将其混合菌液与植物进行植物-微生物联合修复不同浓度的石油污染土壤,经过150 d的温室降解,最高降解率达到73.47%。ESI FT-ICR MS分析结果表明,与空白组相比,植物组的O1、O2和N1类等化合物相对丰度都发生了显著变化,石油污染物得到一定程度的生物降解。     

生物泥浆体系修复氯酚污染的土壤

利用生物泥浆体系研究了受氯酚污染土壤的修复特性。结果表明，对4-氯酚污染浓度为300mg／kg的土壤，土著微生物基本没有降解活性；投加高效菌可大大加快4-氯酚的降解进程。生物泥浆体系修复氯酚污染土壤的最佳工艺参数为：温度35℃，水土比3：1，投菌量5％。外加碳源和氮源可明显促进4-氯酚的降解，但外加磷源对降解的影响并不显著。     

固定化耐低温混合菌对焦化土壤多环芳烃的降解研究

研究了固定化耐低温真菌-细菌混合菌在低温环境下,对焦化厂污染土壤中的菲(Phe)和苯并[b]荧蒽(BbF)降解的动态变化,利用高通量测序技术分析了降解过程中微生物群落多样性变化。结果表明:在低温条件下固定化混合菌对土壤中Phe、BbF的去除率远高于游离混合菌与固定化单菌,在60d的降解周期下,固定化混合菌对土壤中Phe和BbF的降解率分别可达59.61%和45.24%。处理前,土壤中细菌与真菌初始Shannon多样性指数分别为2.79和0.33,细菌远高于真菌,土壤中土著微生物以细菌为主,高丰度的细菌抑制了真菌的生长代谢。处理后,细菌的Shannon多样性指数下降至1.33,真菌的Shannon多样性指数增加至1.01,Phe和BbF的降解与细菌多样性呈负相关,且细菌多样性的降低减少了其对真菌的抑制作用。对比分析了处理前后土壤中微生物群落组成的变化,结果表明:投加固定化混合菌后,固定化混合菌中的假单胞菌(Pseudomonas sp.)SDR4和高山被孢霉(Mortierella alpina)JDR7在低温下生长代谢良好,并成为降解过程中的优势菌,其物种相对丰度分别提高至79.84%与58.63%。固定化混合菌对低温环境有良好的耐性,固定化混合菌的投加提高了菌株对多环芳烃(PAHs)的生物利用有效性,改变了土壤中微生物群落的结构和丰度,可应用于低温土壤PAHs的原位修复。     

不同处理条件对石油污染土壤植物修复的影响

针对石油烃植物修复过程中的主要影响因素,研究了不同植物种类、不同土壤调理剂和菌剂使用等不同条件对土壤中石油烃植物修复效果的影响.结果表明,不同种类的植物修复可使总石油烃的年降解率达到37.8％ ～ 73.98％,其中大豆和碱蓬具有较好的修复效果;3种不同土壤调理剂对石油烃污染土壤修复的效果为商业添加剂＞牛粪＞蛭石;先微生物修复后种植植物的处理要优于单独的微生物修复及微生物、植物修复同步进行的处理.     

Plant species affect colonization patterns and metabolic activity of associated endophytes during phytoremediation of crude oil-contaminated soil

Plants coupled with endophytic bacteria hold great potential for the remediation of polluted environment. The colonization patterns and activity of inoculated endophytes in rhizosphere and endosphere of host plant are among the primary factors that may influence the phytoremediation process. However, these colonization patterns and metabolic activity of the inoculated endophytes are in turn controlled by none other than the host plant itself. The present study aims to determine such an interaction specifically for plant-endophyte systems remediating crude oil-contaminated soil. A consortium (AP) of two oil-degrading endophytic bacteria (Acinetobacter sp. strain BRSI56 and Pseudomonas aeruginosa strain BRRI54) was inoculated to two grasses, Brachiaria mutica and Leptochloa fusca, vegetated in crude oil-contaminated soil. Colonization patterns and metabolic activity of the endophytes were monitored in the rhizosphere and endosphere of the plants. Bacterial augmentation enhanced plant growth and crude oil degradation. Maximum crude oil degradation (78 %) was achieved with B. mutica plants inoculated with AP consortium. This degradation was significantly higher than those treatments, where plants and bacteria were used individually or L. fusca and endophytes were used in combination. Moreover, colonization and metabolic activity of the endophytes were higher in the rhizosphere and endosphere of B. mutica than L. fusca. The plant species affected not only colonization pattern and biofilm formation of the inoculated bacteria in the rhizosphere and endosphere of the host plant but also affected the expression of alkane hydroxylase gene, alkB. Hence, the investigation revealed that plant species can affect colonization patterns and metabolic activity of inoculated endophytic bacteria and ultimately the phytoremediation process.     

Enhancement of biodegradation of oil adsorbed on fine soils in a bioslurry reactor

Techniques for enhancing the biodegradation of oil-contaminated fine soils in a slurry-phase bioreactor were investigated. Using a model system consisting of kaolin particles containing adsorbed n-dodecane as a diesel fuel surrogate, we investigated how increasing the temperature and adding a surfactant and various hydrophobic support media affected the biodegradation rate of n-dodecane. Increasing the temperature from 25 to 35 degrees C decreased the time required for complete degradation of n-dodecane by 30%, from 110h to 80h. Addition of the surfactant polyethylene glycol p-1,1,3,3-tetramethylbutylphenyl ether decreased the degradation time to less than 48h at 35 degrees C, although a high concentration of the surfactant (3000mgl(-1)) was required. We suspect that the surfactant increased the degradation rate by solubilizing the n-dodecane into the solution phase in which the microorganisms were suspended. We tested five types of organic polymers as support media for the microorganisms and found that the biodegradation time could be reduced by approximately 50% with a support medium made from polyurethane; in the presence of this medium, only 36h was required for complete decomposition at 35 degrees C. The reduction in the degradation time was probably due to transfer of the n-dodecane from the soil to the support medium, which improved contact between the n-dodecane and the microorganisms. The polyurethane support medium bearing the microorganisms was stable and could be reused.     

老化石油污染土壤的清洗处理

以华北油田老化长达1年以上的石油污染土壤为研究对象,采用自行选配的清洗剂对该污染土壤进行了一次清洗和二次清洗处理.实验结果表明,一次清洗后,污染土壤样品的含油率从26.34%～29.90%降到6.34%～7.84%,洗油率达80.06%～81.06%;经二次清洗处理后,污染土壤样品的含油率从26.34%～29.90%降到4.05%～4.85%,洗油率达88.06%～88.19%.在一次清洗和二次清洗的基础上,通过模拟实验确定了洗油污水回用的最佳回用率为80%,最佳加药质量浓度为0.4 g/L,该条件下污水的最终产生量也较少.按照该参数对华北油田的石油污染土壤进行了清洗实验,洗油率达79.20%～80.51%.     

固定化降解菌Q5去除喹啉的性能研究

为了研究固定化微生物在土壤生物修复中的应用,以实验室筛选出来的高效降解菌 Q5 为生物活性物质,利用生物大分子仿生合成出的纳米多孔氧化硅为载体,通过表面吸附同定化方法将其固定,制备出固定化微生物.考察固定化微牛物初始 pH 值、温度、摇床转速和菌种的接种量对喹啉去除的影响,得到适宜的去除条件,在相同条件下比较固定化微生物与游离菌种对底物的去除情况,研究单一固定化菌种对不同浓度的喹啉的去除情况,考察固定化微生物的稳定性.实验结果表明,菌株 Q5 经固定化后,对喹啉的去除能力大大增强,在 500 mg/L 浓度下,40 h 固定化 Q5 对底物去除率达96.6%,远高于未固定化 Q5 的去除率 56.1%;对于高底物浓度,固定化微生物的去除效果明显,初始底物浓度为1 500 mg/L,反应 70 h 后去除率为 91.6%,且这种固定化微生物的重复使用性能良好.     

Bioremediation of oil-contaminated soil using Candida catenulata and food waste

Even though petroleum-degrading microorganisms are widely distributed in soil and water, they may not be present in sufficient numbers to achieve contaminant remediation. In such cases, it may be useful to inoculate the polluted area with highly effective petroleum-degrading microbial strains to augment the exiting ones. In order to identify a microbial strain for bioaugmentation of oil-contaminated soil, we isolated a microbial strain with high emulsification and petroleum hydrocarbon degradation efficiency of diesel fuel in culture. The efficacy of the isolated microbial strain, identified as Candida catenulata CM1, was further evaluated during composting of a mixture containing 23% food waste and 77% diesel-contaminated soil including 2% (w/w) diesel. After 13 days of composting, 84% of the initial petroleum hydrocarbon was degraded in composting mixes containing a powdered form of CM1 (CM1-solid), compared with 48% of removal ratio in control reactor without inoculum. This finding suggests that CM1 is a viable microbial strain for bioremediation of oil-contaminated soil with food waste through composting processes.     

Bioremediation of petroleum hydrocarbon-contaminated soil by composting in biopiles

Composting of contaminated soil in biopiles is an ex situ technology, where organic matter such as bark chips are added to contaminated soil as a bulking agent. Composting of lubricating oil-contaminated soil was performed in field scale ( [Formula: see text] m(3)) using bark chips as the bulking agent, and two commercially available mixed microbial inocula as well as the effect of the level of added nutrients (N,P,K) were tested. Composting of diesel oil-contaminated soil was also performed at one level of nutrient addition and with no inoculum. The mineral oil degradation rate was most rapid during the first months, and it followed a typical first order degradation curve. During 5 months, composting of the mineral oil decreased in all piles with lubrication oil from approximately 2400 to 700 mg (kg dry w)(-1), which was about 70% of the mineral oil content. Correspondingly, the mineral oil content in the pile with diesel oil-contaminated soil decreased with 71% from 700 to 200 mg (kg dry w)(-1). In this type of treatment with addition of a large amount of organic matter, the general microbial activity as measured by soil respiration was enhanced and no particular effect of added inocula was observed.     

Fenton氧化技术处理稠油污染土壤

利用Fenton氧化技术对稠油污染土壤进行氧化处理,分析对后续微生物修复的促进作用。向1 000 g石油类含量为8%的稠油污染土壤中加入10.0 mL 18 mmol/L Fe2+溶液与10.0 mL 30%H2O2,反应时间为2 h。氧化处理后土壤中石油烃的总去除率可达到31.38%,胶质去除率为45.22%,沥青质去除率为51.26%,胶质的分子量由1 841下降到1 472,沥青质的分子量由5 831下降到5 073。Fenton氧化可使土壤酶活、各类微生物的数量及呼吸强度有不同程度的下降,但在氧化后30 d内,土壤各类微生物数量都超过了原有水平,其中细菌数量最高达到9.84×105CFU/g,是氧化前的数量的1.57倍。以上实验结果表明,Fenton氧化可以有效去除土壤中胶质和沥青质,并且使土壤中微生物的生长速率加快。因此,Fenton氧化能够促进后续的微生物修复。     

秸秆固定化石油降解菌降解原油的初步研究

用秸秆做载体固定嗜碱芽孢杆菌(Bacillus alcalophilus SG)降解原油,其原油去除率为73.88%,高于单纯投加菌液或者菌液与秸秆的混合物的原油去除率.秸秆的最佳投加量(干重)为25.0 g/L,最佳固定化时间为30 h.用预处理过的秸秆固定SG,降低了固定化SG的原油去除率.在固定化培养基中添加无机盐离子,促进了固定化SG对原油的降解.不同初始pH的原油培养基在固定化SG降解原油的过程中逐渐呈中性或偏碱性.固定化SG在pH 6.0～10.0时对原油均有不错的降粘能力.