固定化微生物菌种的筛选与鉴定

以菲、芘作为降解对象，对多种菌株进行筛选，从中挑选出降解率最高的两株菌，细菌和真菌各一株，并对其生长曲线进行测定，为菌种的固定化提供了一定的依据。同时对菌种进一步鉴定，细菌为动胶杆菌(Zoogloea sp．)，在72h内对菲、芘的降解率为87．82％和49．22％；真菌为镰刀菌(Fuarium sp．)，在216h内对菲、芘的降解率为93．32％和96．29％。     

菌源对多环芳烃降解菌的筛选及降解性能的影响

高效降解菌的筛选对利用生物修复技术有效去除环境中的多环芳烃具有重要意义。分别以石油污染土壤和焦化废水活性污泥为菌源,分离出芘降解菌和混合PAHs(菲、荧蒽和芘)降解菌共14株并对其降解性能进行对比研究。结果表明,筛选得到的菌株分别属于9个菌属,其中2种菌源共有的菌属为Mycobacterium sp.、Ralstonia sp.和Shinella sp.。芘和PAHs的高效降解菌(CP16和CM32)均属于分支杆菌属(Mycobacterium),来源于焦化废水活性污泥;菌株CP16对芘(50mg/L)的7 d降解率为74.99%,CM32对PAHs(菲50 mg/L、荧蒽和芘各10 mg/L)的7 d降解率为100%。因此,以焦化废水活性污泥为菌源更有利于获得高效的多环芳烃降解菌。     

铜绿假单胞菌NY3所产表面活性剂对原油降解的影响

铜绿假单胞菌NY3是从石油污染土壤中分离出的一株能快速代谢疏水性化合物的菌种。研究了该菌产表面活性剂及对原油降解的作用。实验表明,在敞开体系中,投加82 mg/L NY3菌产的鼠李糖脂,240 h能使NY3对原油的降解率提高50%。投加甘油使NY3产鼠李糖脂与降解原油同步进行,与投加鼠李糖脂对原油降解的促进作用相近。投加9‰甘油使NY3在168 h对原油的降解率提高43%。NY3菌能同时降解原油中的直链烷烃及菲(Pr)和芘(Ph)等多环芳烃。在敞开体系中用少量甘油使产鼠李糖脂和降解原油同步进行,节约处理费用。研究结果为NY3菌株在露天石油污染环境修复中的应用奠定了基础。     

石油污染土壤中芘高效降解菌群的筛选及降解特性研究

从长期受石油污染土壤中驯化筛选到能以芘为惟一碳源生长的混合菌群GP3,其主要由假单胞菌株GP3A(Pseudomonas sp.)和菌株GP3B(Pandoraea pnomenusa)组成.采用摇瓶振荡培养方法,研究了不同环境条件对混合菌GP3降解芘效能的影响.结果表明,在30℃,150 r/min振荡培养下,混合菌GP3对15 mg/L芘的7 d降解率为90.6%.混合菌GP3降解芘的最适宜温度为35℃,最佳pH值为6.2.加入低浓度葡萄糖(100 mg/L)或菲(10 mg/L)作为共代谢底物,均可提高GP3对芘的降解率.混合菌对芘的降解速率(PDR)与芘的初始浓度呈正相关.研究重金属离子胁迫下GP3对芘的降解时发现,10 ms/L Zn2 的存在对芘降解效能影响较小,Cu2 对芘的降解有抑制作用,Cd2 对混合菌GP3有很强的毒性.     

固定化混合菌修复油污染地表水的研究

从辽河油田受石油污染的河床底泥中筛选出一株芽孢杆菌(Bacillus sp.)和一株黄杆菌(Flavobacterium sp.)，采用二次交联化学方法对2株细菌单独及混合固定，分别进行了不同接种量的固定化细菌对油的降解，以及固定化混合菌对环境的耐受性和在自然地表水中对油的降解进行研究，结果表明，在相同时间内固定化混合菌对油的降解效果明显优于固定化单株菌，而且都优于游离菌。固定化混合菌pH在6～10、温度在20～40℃范围内能保持较好的活性。在120 h时，固定化混合菌对自然地表水中油的降解率达94.5%,对地表水中COD的去除率达89.6%。扫描电子显微镜分析显示，固定化载体的微观结构适合细菌的生长，固定化混合菌在载体内部形成高密度的菌群。以上的研究为固定化微生物应用于油污染地表水的生物修复提供了一定的理论基础。     

三株优势菌对多环芳烃的降解性能研究

多环芳烃具有毒性、生物蓄积性和半挥发性，并能在环境中持久存在。生物修复处理具有费用低、效果好、污染物残留量低、不产生二次污染、能够保持或改善植物生长的土壤结构等优点。实验分别在对单一菌株、两两混合菌株及三株菌混合等三种情形下，对蒽、菲、芘的降解作用进行了研究。研究结果表明：单一菌株对蒽、菲、芘有一定的降解能力，菌株混合时，对蒽、菲的降解率有所提高；三株菌种混合时，黄杆菌对放线菌、红球菌的生长过程有抑制作用；蒽、菲、芘的降解过程会不断交替进行着产酸和脱羧过程，使降解液pH值出现波动；同时在降解过程中，菌体能产生表面活性物质，这有利于蒽、菲、芘的生物降解转化。     

固废拆解场污染土壤中多环芳烃降解菌的筛选鉴定与降解特性

以腐植酸(HA)溶液为吸附剂、从受多环芳烃污染的土壤中分离出来的降解菌制成为生物修复剂,以多环芳烃(PAHs)萘、菲、芘、荧蒽、苯并蒽、苯并芘为土壤污染物,对PAHs污染土壤进行修复实验。目的是筛选与分离吸附于HA的PAHs降解菌,研究HA与降解菌的协同效应对PAHs的降解效率的影响。用经过HA吸附的PAHs富集分离培养出1株高效降解菌株,命名为Tzyx3,鉴定其为解脂耶氏酵母菌(Yarrowia lipolytica)。15 d后,土壤中萘、菲、芘、荧蒽、苯并蒽、苯并芘的降解率分别为90.7%、91.0%、74.7%、86.9%、84.7%和74.7%,表明Tzyx3和HA在PAHs污染土壤中存在协作关系,Tzyx3能够直接利用HA对土壤中的多环芳烃进行降解。     

芘降解菌DY-1的条件优化及降解动力学特性分析

选用已筛选出以芘为唯一碳源的假单胞菌DY-1(Pseudomonas sp.)作为芘降解菌,采用摇瓶振荡培养方法,研究了不同环境条件对菌DY-1降解芘效率的影响以及降解动力学特性。结果表明,在含芘50 mg/L的条件下培养9 d,降解率达83.2%。最适宜温度为30℃,pH值为7.5,摇床转速为120 r/min,接种量为1.5 mL;在不同培养条件下芘的降解符合一级动力学模型;低浓度Zn2+,Cd2+,Cr6+的存在对芘降解效果影响较小,Cu2+,Pb2+对芘的降解有较强的毒性;加入低浓度有机物质,蔗糖可提高DY-1芘的降解;低浓度萘或蒽的存在可促进芘的降解。     

沉积物中2株多环芳烃降解菌的分离鉴定及其对菲、荧蒽的降解特性

从长江重庆主城段近岸表层沉积物中,分离出2株能以菲和荧蒽为碳源和能源生长的菌株(命名为CJ1、CJ2),经鉴定分别为黄杆菌属(Flavobacterium sp.)和克雷伯氏杆菌属(Klebsiella sp.)。进行了菲和荧蒽在初始浓度为20~200 mg/L条件下的生长代谢过程与降解动力学分析。结果表明,CJ1对菲和荧蒽的降解效能总体优于CJ2。15 d时CJ1和CJ2对菲的降解率最高分别为74.3%和70.3%;30 d时CJ1和CJ2对荧蒽的降解率最高分别为58.2%和49.9%。初始浓度为200 mg/L时,两菌株对菲、荧蒽的降解受到一定程度抑制。     

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。     

Degradation mechanisms of benzo[a]pyrene and its accumulated metabolites by biodegradation combined with chemical oxidation

A high degradation extent of benzo[a]pyrene (BaP) should not be considered as the sole desirable criterion for the bioremediation of BaP-contaminated soils because some of its accumulated metabolites still have severe health risks to human. Two main metabolites of BaP, benzo[a]pyrene-1,6-quinone (BP1,6-quinone) and 3-hydroxybenzo[a]pyrene (3-OHBP) were identified by high performance liquid chromatography (HPLC) with standards. This study was the first time that degradation of both BaP and the two metabolites was carried out by chemical oxidation and biodegradation. Three main phases during the whole degradation process were proposed. Hydrogen peroxide-zinc (H(2)O(2)-Zn), the fungus - Aspergillus niger and the bacteria - Zoogloea sp. played an important role in the different phases. The degradation parameters of the system were also optimized, and the results showed that the effect of degradation was the best when fungus-bacteria combined with H(2)O(2)-Zn, the concentration range of BaP in the cultures was 30-120mg/l, the initial pH of the cultures was 6.0. However, as co-metabolites, phenanthrene significant inhibited the degradation of BaP. This combined degradation system compared with the conventional method of degradation by domestic fungus only, enhanced the degradation extent of BaP by more than 20% on the 12d. The highest accumulation of BP1,6-quinone and 3-OHBP were reduced by nearly 10% in the degradation experiments, which further proved that the combined degradation system was more effective as far as joint toxicity of BaP and its metabolites are concerned.     

Sequential UV-biological degradation of polycyclic aromatic hydrocarbons in two-phases partitioning bioreactors

A method based on UV-irradiation in organic solvent followed by transfer of the remaining pollutants into silicone oil for subsequent biodegradation in a biphasic system inoculated with a phenanthrene degrading Pseudomonas sp. was tested for the treatment of various mixtures of PAHs. Acetone was first selected as the most suitable solvent compared to methanol, acetonitrile and silicone oil for the removal of pyrene and phenanthrene. The sequential treatment was then applied to the treatment of a mixture of fluorene, phenanthrene, anthracene, fluoranthrene, pyrene, benzo(a)anthracene and benzo(a)pyrene in acetone. These compounds were photodegraded in the following order of initial removal rates (mg l(-1) d(-1)): benzo(a)pyrene (7.8) > anthracene (5.0) > benzo(a)anthracene (2.5) > fluoranthrene (1.8) > pyrene (1.5) > phenanthrene (1.2) > fluorene (0.2). UV-treatment allowed complete removal of, anthracene, benzo(a)anthracene and benzo(a)pyrene and removals of 63% of pyrene and 37% of fluorene after 434 h or irradiation. The subsequent biological treatment removed the remaining phenanthrene and fluorene by 100% and 90%, respectively, after 790 h of cultivation. Although less efficient due to the presence of interfering compounds, the UV-biological treatment of a soil extract allowed a 63% removal of the seven PAHs named above. Microbial growth did not occur when the pollutants were directly supplied to the microorganism showing that biphasic systems reduced the toxicity effects cause by mixtures of PAHs at high concentrations. This study demonstrates the potential of selective UV treatment of high molecular weight PAHs followed by biological treatment of the low molecular weight species in biphasic systems.     

真菌和细菌对染料吸附脱色的高效共培养体系研究

在含有真菌G 1培养液中加入染料厂污水排放口的污泥样品 ,从发生快速脱色降解染料的混合培养液中分离出 2株染料脱色细菌L_1和L_2 ,经API鉴定系统鉴定 ,确定菌株L_1为Enterobactersp .,菌株L_2为Peudomonassp .。研究比较了单一和不同组合混合的真菌G_1菌株 (Penicilliumsp .)、细菌L_1菌株 (Enterobactersp .)和L_2菌株 (Pseu domonassp .)对偶氮染料红M - 3BE(C .I .ReactiveRed 2 41)和蒽醌染料艳蓝KN -R(C .1.ReactiveBlue 19)的去除情况 ,发现G - 1真菌和 2种细菌组合的共培养体系对 5 0mg/L红M - 3BE和艳蓝KN -R处理 5h去除率达 10 0 %和 97.9% ,并且是以脱色降解作用为主 ,建立了染料脱色降解菌的最佳组合 ;进一步测定了此最佳共培养体系对另外 13种不同结构染料的脱色降解 ,结果表明 ,除对蒽醌染料R - 478脱色降解较差外 ,对其他染料均可在lh— 3d被完全脱色降解 ,表现出脱色降解染料的广谱性 ;向培养 4d的共培养体系中依次加入 8种染料 ,菌体可对染料连续脱色 ,维持脱色能力达 8d左右     

Combined UV-biological degradation of PAHs

The UV-photolysis of PAHs was tested in silicone oil and tetradecane. In most cases, the degradation of a pollutant provided within a mixture was lower than when provided alone due to competitive effects. With the exception of anthracene, the larger pollutants (4- and 5-rings) were always degraded first, proving that UV-treatment preferentially acts on large PAHs and thereby provides a good complement to microbial degradation. UV-photolysis was also found to be suitable for treatment of soil extract from contaminated soils. The feasibility of UV-biological treatment was demonstrated for the removal of a mixture of phenanthrene and pyrene in silicone oil. UV-irradiation of the silicone oil led to 83% pyrene removal but no phenanthrene photodegradation. Subsequent treatment of the oil in a two-phases partitioning bioreactor (TPPB) system inoculated with Pseudomonas sp. was followed by complete phenanthrene biodegradation but no further pyrene removal. Totally, the combined process allowed 92% removal of the PAH mixture. Further work should focus on characterizing the photoproducts formed and studying the influence of the solvent on the photodegradation process.     

Metabolism of aromatic hydrocarbons by the filamentous fungus Cyclothyrium sp

The metabolism of biphenyl, naphthalene, anthracene, phenanthrene, pyrene and benzo[a]pyrene by Cyclothyrium sp. CBS 109850, a coelomycete isolated for the first time in Brazil from industrially polluted estuarine sediment, was studied. The metabolites were extracted and separated by high performance liquid chromatography (HPLC) and characterized by UV spectral analyses and mass, and proton nuclear magnetic resonance ((1)H NMR) spectrometry. Cyclothyrium sp. transformed biphenyl to 4-hydroxybiphenyl and anthracene to anthracene trans-1,2-dihydrodiol. This isolate metabolized 90% of [9-(14)C]phenanthrene, producing phenanthrene trans-9,10-dihydrodiol as a major metabolite, phenanthrene trans-3,4-dihydrodiol, 1-hydroxyphenanthrene, 3-hydroxyphenanthrene, 4-hydroxyphenanthrene, and a novel metabolite, 2-hydroxy-7-methoxyphenanthrene. Circular dichroism spectra analyses indicated that the major enantiomers of phenanthrene trans-9, 10-dihydrodiol, phenanthrene trans-3,4-dihydrodiol and pyrene trans-4,5-dihydrodiol, a pyrene metabolite produced previously by Cyclothyrium sp. CBS 109850, were predominantly in the (R,R) configuration, revealing a high stereoselectivity for initial monooxygenation and enzymatic hydration of phenanthrene and pyrene by Cyclothyrium sp. CBS109850. The results also show a high regioselectivity since the K-regions of phenanthrene and pyrene were the major sites of metabolism.     

Treatment of PAHs in contaminated soil by extraction with aqueous DNA followed by biodegradation with a pure culture of Sphingomonas sp

The biodegradation of polycyclic aromatic hydrocarbons (PAHs) in aqueous deoxyribonucleic acid (DNA) solution from contaminated soil washing was investigated. Initial data with a model effluent consisting of anthracene, phenanthrene, pyrene and benzo[a]pyrene that were individually dissolved in 1% aqueous DNA solution confirmed their positive degradation by Sphingomonas sp. at around 10(8)CFU mL(-1) initial cell loading. For anthracene and phenanthrene, complete removal was achieved within 1h treatment. Degradation of pyrene and benzo[a]pyrene took a relatively longer time of a few days and weeks, respectively. DNA-dissolved PAHs were also degraded relatively faster than PAH crystals in aqueous medium to suggest that the binding of the PAHs in the polymer does not pose serious constraint to bacterial uptake. The DNA was stable against the PAH-degrading bacteria. Parallel experiments with actual DNA solutions obtained during pyrene extraction from an artificially spiked soil also showed similar results. Close to 100% pyrene degradation was achieved after 1d treatment. With its chemical stability, the cell-treated DNA was re-used up to four cycles without a considerable decline in extraction performance.     

Remediation of PAH contaminated soils: application of a solid-liquid two-phase partitioning bioreactor

The feasibility of a two-step treatment process has been assessed at laboratory scale for the remediation of soil contaminated with a model mixture of polycyclic aromatic hydrocarbons (PAHs) (phenanthrene, pyrene, and fluoranthene). The initial step of the process involved contacting contaminated soil with thermoplastic, polymeric pellets (polyurethane). The ability of three different mobilizing agents (water, surfactant (Biosolve) and isopropyl alcohol) to enhance recovery of PAHs from soil was investigated and the results were compared to the recovery of PAHs from dry soil. The presence of isopropyl alcohol had the greatest impact on PAH recovery with approximately 80% of the original mass of PAHs in the soil being absorbed by the polymer pellets in 48 h. The second stage of the suggested treatment involved regeneration of the PAH loaded polymers via PAH biodegradation, which was carried out in a solid-liquid two-phase partitioning bioreactor. In addition to the PAH containing polymer pellets, the bioreactor contained a microbial consortium that was pre-selected for its ability to degrade the model PAHs and after a 14 d period approximately 78%, 62% and 36% of phenanthrene, pyrene, and fluoranthene, respectively, had been desorbed from the polymer and degraded. The rate of phenanthrene degradation was shown to be limited by mass transfer of phenanthrene from the polymer pellets. In case of pyrene and fluoranthene a combination of mass transfer and biodegradation rate might have been limiting.     

Effect of expandable clays and cometabolism on PAH biodegradability

Pyrene and phenanthrene degradation was examined in both single and binary slurry systems for three different natural soils. It was found that the amount of total expandable clays (smectite and vermiculite) was in a good agreement with the achieved rate and extent of biodegradation. For instance, the intrinsic phenanthrene biodegradation rate was 626 microg/L/day for the soil with the largest expandable clay and 3203 microg/L/day for the soil with the least. Similarly, the smallest total pyrene biodegradation (65%) was found for the soil rich in expandable clays, compared to an 82% pyrene reduction in the soil that had the lowest amount. Mass transfer limitation after compound sorption to the clays was more pronounced for the more hydrophobic pyrene. In the presence of phenanthrene, total pyrene biodegradation increased by 2 to 7% due to cometabolism, while the total phenanthrene biodegradation was only enhanced by 0.5 to 5% in the binary system. This research demonstrated that expandable clays might govern the substrate availability to microorganisms and microbial accessibility to substrates. Therefore, the contribution of organic matter and expandable clays to sorption, desorption and biodegradation should be taken equally into account in order to better understand complex bioremediation issues.     

Plant uptake, accumulation and translocation of phenanthrene and pyrene in soils

Uptake, accumulation and translocation of phenanthrene and pyrene by 12 plant species grown in various treated soils were comparatively investigated. Plant uptake and accumulation of phenanthrene and pyrene were correlated with their soil concentrations and plant compositions. Root or shoot accumulation of phenanthrene and pyrene in contaminated soils was elevated with the increase of their soil concentrations. Significantly positive correlations were shown between root concentrations or root concentration factors (RCFs) of phenanthrene and pyrene and root lipid contents. The RCFs of phenanthrene and pyrene for plants grown in contaminated soils with initial phenanthrene concentration of 133 mgkg(-1) and pyrene of 172 mgkg(-1) were 0.05-0.67 and 0.23-4.44, whereas the shoot concentration factors of these compounds were 0.006-0.12 and 0.004-0.12, respectively. For the same soil-plant treatment, shoot concentrations and concentration factors of phenanthrene and pyrene were generally much lower than root. Translocations of phenanthrene and pyrene from shoots to roots were undetectable. However, transport of these compounds from roots to shoots usually was the major pathway of shoot accumulation. Plant off-take of phenanthrene and pyrene only accounted for less than 0.01% of dissipation enhancement for phenanthrene and 0.24% for pyrene in planted versus unplanted control soils, whereas plant-promoted biodegradation was the predominant contribution of remediation enhancement of soil phenanthrene and pyrene in the presence of vegetation.