假单胞菌Nwu1-mu对陕北石油污染土壤的生物修复作用研究

选用假单胞菌Nwu1-mu对陕北地区石油污染土壤进行生物修复。通过检测修复过程中土壤样品的菌体生长数量、脱氢酶活性、表面张力和石油烃及其组分降解率综合考察了菌株对石油烃类物质的降解作用。结果表明,在不添加营养物质的前提下假单胞菌Nwu1-mu在60d内对石油污染土壤中的石油烃类物质降解率达到了86.5%,尤其对石油烃中的C24～C28和〉C28组分有突出的降解效果;菌株产脱氢酶和表面活性剂显示出了良好的性能,在石油烃类物质的降解过程中均起着关键的促进作用。假单胞菌Nwu1-mu显示出的强大的生物修复潜力及其不依赖于外来营养源的特性,为黄土高原的土壤生态环境修复提供了新的解决思路。     

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。     

石油污染土壤的生态风险评价和生物修复Ⅲ.石油污染土壤的植物—微生物联合修复

在对石油污染土壤理化性质和微生物数量研究的基础上,通过接种含石油降解菌的菌剂、添加营养、定期翻动以及栽种植物等方式对其进行了修复。试验表明,添加氮磷等营养后,土壤中的烃降解菌明显增加,石油降解速率显著加快,但接种菌剂以及定期翻动对降解率没有显著影响。在修复试验进行到120 d后,对部分处理进行了植物修复。试验表明栽种狼尾草后,土壤中石油降解速率显著加快,生物毒性明显降低。另外,试验结束后对土壤中的总DNA进行了提取和基于16S rDNA V3区的PCR-DGGE研究。结果表明,经过修复处理改变了土壤中优势细菌的群落结构,使土壤细菌多样性增强。     

多环芳烃污染土壤生物联合强化修复研究进展

多环芳烃(polycyclic aromatic hydrocarbons,PAHs)是广泛存在于环境中的一类有毒有机污染物。在PAHs污染土壤修复领域中,运用一些生物化学的方式来强化生物联合修复技术可以有效缩短生物修复的时间,大大提高修复效率,最具发展前景和应用价值。本文主要以植物-微生物、植物-微生物-土壤动物两种生物联合修复方式为对象,结合各自的特点、机理和实例,推断了其修复机制的内在原因,总结了影响土壤中PAHs降解效率的主要因素(包括:PAHs的浓度水平、根系分泌物的种类、外源添加降解菌和土壤动物的数量和种类、菌属或土壤动物之间的种间竞争和部分环境因素等);同时通过综述近年来国内外强化生物联合修复PAHs污染土壤的技术原理、应用成果和存在的一些问题,指出了不同情况下制约PAHs强化降解进程的潜在限制因子(包括:表面活性剂和固定化微生物的添加量、不同表面活性剂的适度混合、载体材料的性质、固定化方式的选取、土壤养分和水分含量等);并强调在进行强化修复的过程中,要注重现场应用和安全性评价,为多环芳烃污染土壤的生物联合强化修复研究提供了理论依据和技术参考。     

石油污染土壤的生态风险评价和生物修复 Ⅱ.石油污染土壤的理化性质和微生物生态变化研究

采用常规土壤理化分析、微生物培养法以及碳素利用法(Biolog)和发光细菌生物毒性测定等新兴微生态研究方法,研究了石油污染引起的土壤理化性质和微生物功能多样性等微生态的变化。结果表明,石油污染使土壤有机质含量增加,pH降低,而对全磷、全钾、速效钾、全氮和水解氮无显著影响。平板计数显示石油污染对土壤中的细菌总量没有显著影响,但由于石油污染物的刺激作用导致污染土壤中总烃降解菌数以及芳烃降解菌数增加了几个数量级。Biolog的研究也表明,石油污染导致土壤微生物Gini指数、McIn-tosh指数和McIntosh均匀度等多样性指数增加,说明石油污染刺激了土壤中微生物的生长,使土壤中微生物多样性增加。而生物毒性试验表明,石油污染土壤对发光细菌具有毒性,其EC50为1 950μg ml-1。     

影响土壤中PAHs降解的环境因素及促进降解的措施

土壤中的多环芳烃(PAHs)类有机污染物的生物有效性低,不易降解。本文综述了影响污染土壤中多环芳烃降解的环境因素和促进降解措施的研究进展。影响土壤中多环芳烃降解的因素,包括水分、养分、土壤物理条件等;促进土壤中多环芳烃降解的措施有:向污染土壤添加有机溶剂、利用冯顿反应、添加堆肥和有机物料等。从目前研究来看,应当通过促进多环芳烃从土壤上解吸和培育具有较高多环芳烃降解能力的微生物来促进多环芳烃污染土壤的修复。     

通气对石油污染土壤生物修复的影响

为了探讨石油污染土壤的生物修复的有效方法,本研究就通气对石油污染土壤生物降解的影响,在自制反应器中进行了为期50天的堆腐试验。结果表明,通气可为石油烃污染土壤中的微生物提供充足的电子受体,可保持土壤pH稳定,从而促进了微生物的生物活性,强化了它们对石油污染物的氧化降解作用。通过在反应器中,调控通气量使污染土壤中石油烃的降解率进一步提高,为石油污染土壤生物修复技术的应用奠定了科学基础。     

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。     

降解菌对堆肥中多环芳烃降解作用的初步研究

通过在堆肥中加入经过驯化的降解菌这种土壤有机污染生物修复技术,以超声波提取－高效液相色谱（ＨＰＬＣ）分离测定的方法,对堆肥材料中多环芳烃的浓度变化进行监测,从而了解降解解菌对堆肥中多环芳烃的降解作用。实验结果表明,降解菌对堆肥中的多环芳烃有明显的降解作用。     

油污土壤修复微生物的筛选及其影响因素

[目的]探讨微生物原位修复的主要影响因素及水平(正交设计)之间的关系,为石油污染场地生物修复工程的参数设计提供一定理论依据。[方法]选取5因素4水平的正交设计,考察污染强度、营养物、氧化剂、表活剂、接菌量等因素对土壤修复效果的影响。[结果]以原油为唯一碳源经过初步筛选,获得16株石油烃降解优势菌,经过菌群复筛,获得2株偏利共生协同真菌DPF2,DPF4,协同降解率最高,7d达87.77%。选择其进行室内油污土壤的微生物修复模拟试验,60d石油污染强度为10的油污土壤降解率最高,可达94.12%。污染强度为25的油污土壤降解率为90.17%,SPSS数据分析表明生物修复影响的最大影响因素是氧化剂、表活剂和营养物,其次是污染强度、接菌量。[结论]初期添加表活剂、氧化剂、营养剂能对石油生物修复具有重要意义。污染强度仅在35d前有一定影响,在修复后期影响最小。在整个修复过程,接菌量方差均值与其他因素比较都最小,因此其因素水平对石油降解能力的影响不显著。     

黄河三角洲土壤土著菌的石油烃降解潜力

The bioremediation potential of bacteria indigenous to soils of the Yellow River Delta in China was evaluated as a treatment option for soil remediation. Petroleum hydrocarbon degraders were isolated from contaminated soil samples from the Yellow River Delta. Four microbial communities and eight isolates were obtained. The optimal temperature, salinity, pH, and the ratios of C, N, and P （C：N：P） for the maximum biodegradation of diesel oil, crude oil, n-alkanes, and polyaromatic hydrocarbons by indigenous bacteria were determined, and the kinetics changes in microbial communities were monitored. In general, the mixed microbial consortia demonstrated wider catabolic versatility and faster overall rate of hydrocarbon degradation than individual isolates. Our experimental results demonstrated the feasibility of biodegradation of petroleum hydrocarbon by indigenous bacteria for soil remediation in the Yellow River Delta.     

食细菌线虫和葡萄糖对恶臭假单胞菌去除菲的影响

Bacterial-feeding nematodes can promote the bacterial activity through feeding.Bacterial abundance and their activity affect the degradation of polycyclic aromatic hydrocarbons (PAH) such as phenanthrene.The effects of bacterial-feeding nematodes,bacteria,and their interactions on the degradation of phenanthrene with or without glucose were studied through a microcosm experiment.The results showed that up to 57.0％ of phenanthrene in mineral medium contaminated with phenanthrene was degraded in the control with bacteria alone and bacteria with the presence of nematodes and/or glucose increased the degradation of phenanthrene by 25.6％ to 36.6％.Although both nematode and bacteria abundance decreased gradually,catechol 2,3-dioxygenase (C23O) activity increased during the incubation period.Compared with bacteria alone,the presence of nematodes significantly increased C23O activity as well as the abundance of bacteria;this effect was more pronounced when glucose was present.The results imply that nematodes might promote the removal of phenanthrene from medium by stimulating bacteria and C23O activities.     

Rhizosphere effects on soil bacterial abundance and diversity in the Yellow River Deltaic ecosystem as influenced by petroleum contamination and soil salinization

In this study, we compared the differences of bacterial abundance and diversity between rhizosphere and surrounding bulk soils under soil salinization and petroleum contamination in the Yellow River Delta on a 110-km-distance scale. In comparison with bulk soils, rhizosphere soils were mainly characterized by lower salinity and higher water content in saline soils. For bacterial abundance, the numbers of total bacteria and hydrocarbon degraders were significantly higher in rhizosphere soils than those in bulk soils. Although there was no significant difference in total petroleum hydrocarbon (TPH) concentration between the two types of soils, TPH had distinctly different effects on bacterial abundance in rhizosphere and bulk soils. TPH concentration was the major determinant of total bacterial abundance and had positive effects on abundances of hydrocarbon degraders. However, the abundances of total bacteria and hydrocarbon degraders in bulk soils were primarily determined by soil salinity and water content. Great abundance of rhizosphere bacteria suggested that plant roots could alleviate the stresses from soil salinization and provide more favorable microhabitats for bacterial growth. TPH had positive effects on bacterial diversity of both rhizosphere and bulk soils. Our results support the view that petroleum in the environments functions as both toxic chemicals and carbon sources to soil bacteria. Great abundance and diversity of total bacteria in plant rhizospheres would potentially improve the roles of bacteria in maintaining ecosystem functioning in the degraded ecosystems. Our results would improve our understanding of the relationships between rhizosphere effects and multiple environmental stresses that control the development of bacterial community in fragile anthropologically-affected ecosystems.     

Bioremediation of Mangroves Impacted by Petroleum

The majority of oil from oceanic oil spills (e.g. the recent accident in the Gulf of Mexico) converges on coastal ecosystems such as mangroves. Microorganisms are directly involved in biogeochemical cycles as key drivers of the degradation of many carbon sources, including petroleum hydrocarbons. When properly understood and managed, microorganisms provide a wide range of ecosystem services, such as bioremediation, and are a promising alternative for the recovery of impacted environments. Previous studies have been conducted with emphasis on developing and selecting strategies for bioremediation of mangroves, mostly in vitro, with few field applications described in the literature. Many factors can affect the success of bioremediation of oil in mangroves, including the presence and activity of the oil-degrading microorganisms in the sediment, availability and concentration of oil and nutrients, salinity, temperature and oil toxicity. More studies are needed to provide efficient bioremediation strategies to be applicable in large areas of mangroves impacted with oil. A major challenge to mangrove bioremediation is defining pollution levels and measuring recuperation of a mangrove. Typically, chemical parameters of pollution levels, such as polycyclic aromatic hydrocarbons (PAHs), are used but are extremely variable in field measurements. Therefore, meaningful mangrove monitoring strategies must be developed. This review will present the state of the art of bioremediation in oil-contaminated mangroves, new data about the use of different mangrove microcosms with and without tide simulation, the main factors that influence the success of bioremediation in mangroves and new prospects for the use of molecular tools to monitor the bioremediation process. We believe that in some environments, such as mangroves, bioremediation may be the most appropriate approach for cleanup. Because of the peculiarities and heterogeneity of these environments, which hinder the use of other physical and chemical analyses, we suggest that measuring plant recuperation should be considered with reduction in polycyclic aromatic hydrocarbons (PAHs). This is a crucial discussion because these key marine environments are threatened with worldwide disappearance. We highlight the need for and suggest new ways to conserve, protect and restore these environments.     

Effects of nutrient concentration on the biodegradation of crude oil and associated microbial populations in the soil

The influence of nutrient amendments on the biodegradation of a crude oil and on associated microbial populations in an agricultural soil was studied in microcosms during a 150-day experiment. Concentration and chemical composition of residual hydrocarbons (HC) were periodically monitored in series of polluted soils unfertilized and fertilized with increasing concentrations of nutrients. The decrease in HC concentration was effective in all soils over time. The maximal biodegradation extent was 62%, meanwhile the natural attenuation contributed to 47% of degradation. A permanent inhibition of hydrocarbons assimilation was recorded with a high input of nutrients. The biodegradation of saturates, aromatics and polars was respectively, permanently, temporally and not reduced by excessive fertilization in soil. Accumulation of polar metabolic by-products was demonstrated. Enumerations of total heterotrophic bacteria and hydrocarbon-adapted bacteria showed a strong stimulation in both populations. Maximum stimulation was observed with the highest input of nutrients. However, the extents of biodegradation were not concurrently improved, thus indicating that the microbial degraders were selected depending on the nutrient supply. The permanent and/or temporally inhibition of the saturated and unsaturated HC assimilation revealed that different nutrient supplies were optimum for the degradation of aliphatic and aromatic HC.     

Dissipation of polycyclic aromatic hydrocarbons (PAHs) in soil microcosms amended with mushroom cultivation substrate

The potential of mushroom cultivation substrate (MCS) in bioremediation was examined in polycyclic aromatic hydrocarbon (PAH)-contaminated soil. After a 60-day incubation, 32.9% dissipation of the 15 studied PAHs was observed in MCS-amended microcosms, with anthracene, benzo(a)pyrene and benzo(a)anthracene being the most degradable PAHs. MCS significantly increased the abundance and changed the community compositions of bacteria, fungi and aromatic hydrocarbon degraders. Two species belonging to the Sordariomycetes of the Ascomycota were enriched in all MCS-treated soil samples, and coupled with the unique changes in the PAH profile, this implies the involvement of laccase-like enzymes. Limited improvement was observed after adding Pleurotus ostreatus, possibly because of its poor colonization of the soil. In addition, alfalfa appeared to antagonize the bioremediation effects of MCS. The results of this study suggest that MCS can be a cost-effective and green biostimulation agent, thereby providing support for the development of MCS-based biostimulation of PAH-contaminated soil.     

Ecological evaluation of oil-contaminated soils (Sakhalin) using enchytraeidae

The ecological status of oil-contaminated soils of Sakhalin and their background analogues has been evaluated with the use of soil invertebrates. The survival rates of Enchytraeus albidus in soils with different textures and the contents of organic carbon and nutrients have been compared. The indicative role of soil mesofauna (Enchytraeus albidus) for the ecological evaluation of oil-contaminated soils with due account for their properties has been shown. The permissible residual concentration of oil hydrocarbons in some soils of Sakhalin—acid brown forest soils (Umbrisols), high-moor peat soils (Histosols), acid meadow alluvial soils (Fluvisols), cultivated meadow soddy soils (Anthrosols), and mucky-podzolic surface-gleyed soils (Gleysols)— has been determined from data on the response of Enchytraeus albidus to different levels of the soil contamination with oil hydrocarbons.     

Bioremediation of oil-contaminated soil by combination of soil conditioner and microorganism

Purpose

Oil hydrocarbons are widespread pollutants in soil which pose serious threats to ecological environment. Thus, this study carried out the bioremediation of oil-contaminated soil by using the efficient petroleum-degrading bacteria and soil conditioner, to investigate the changes of physicochemical properties of contaminated soil during bioremediation, reveal the relationship among the exogenous degradation strains and indigenous microbe, and finally illuminate the effects of soil conditioner and microbe on the bioremediation of oil-contaminated soil.

Materials and methods

A PAH-degrading strain named Stenotrophomonas maltophilia was used in this study, which was isolated from an e-waste dismantling area. The soil conditioner in this present study was developed previously by using agricultural wastes, which was in a powdered form and rich in N, P, and K. The simulated experiments were conducted under the control environmental conditions of greenhouse, to study the effects of inoculation and soil conditioner on bioremediation of oil-contaminated soil. Then, the physicochemical properties of soil and the degradation rates of oil were measured at different set times to evaluate the bioremediation effect.

Results and discussion

Adding 1% soil conditioner could significantly improve the soil conditions and offer microorganism enough N, P, and K, which would promote microbial growth and played a key role on bioremediation of oil-contaminated soil. Although in polluted soil, the microorganism could maintain metabolic activity and use the petroleum as carbon source. The soil indigenous microbe was more easily to adapt to the contaminated surrounding. However, when both of them co-existed in soil, they would restrain each other, and go against the oil decomposition. Thus, making two types of microorganisms work to achieve synergy was the key to gain much better remediation effect. Because the indigenous microbe was good at decomposing low molecular compounds and saturated hydrocarbons, while the oil-degrading strains can effectively decompose high molecular weight aromatics.

Conclusions

The soil nutrient and microorganism, including the exogenous oil-degrading strains and the soil indigenous microbe, had an important effect on degradation of petroleum. The addition of soil conditioner, presence of indigenous microbe, and inoculation of oil-degrading strains all were conducive to bioremediation of oil-contaminated site, but the key was to control the proportion and relationship of the three.

Graphical abstract

     

In‐situ remediation of contaminated farmland by horizontal transfer of degradative plasmids among rhizosphere bacteria

Horizontal transfer of catabolic genes for pollutant degradation among rhizobacteria plays an important role in environmental bioremediation, but lacks support from field trial data. To address this problem, Pseudomonas fluorescens strain TP13 was inoculated into the soils of a phenol‐contaminated farmland on which tomato seedlings were growing in April 2009, 2010, 2011 and 2012. Results consistently showed that introduction of TP13 strain significantly reduced phenol content and increased plant biomass after 20 days, compared with controls. Strain TP13 was able to colonize the plant rhizosphere and the number of rhizosphere bacteria which were grown on phenol and contained the plasmids containing the gene encoding for catechol 2, 3‐dioxygenase (C23O) increased gradually in the later stages of the experiment. The increase in magnitude of the plasmid‐containing rhizosphere bacteria correlated well with plant biomass, while the number of plasmid‐containing rhizosphere bacteria and phenol content was strongly negatively correlated. Furthermore, six strains (T1‐T6) of rhizosphere bacteria were isolated and found to possess large plasmids containing identical C23O genes and similar HindIII restriction patterns. Sequence alignment showed that the C23O genes from strains T1‐T6 contained almost identical sequences and the sequence of the C23O of strain T1 was the same as that of strain TP13. These data indicated that the plasmids were transferred from strain TP13 to these rhizosphere bacteria and that horizontal gene transfer stimulated phenol degradation and plant growth in the contaminated farmland. This is an important finding for in situ remediation of contaminated farmland.     

Effects of Lumbricus terrestris, Allolobophora chlorotica and Eisenia fetida on microbial community dynamics in oil-contaminated soil

Oil spills are one of the most common types of soil pollution. Bioremediation has become an attractive alternative to physicochemical methods of remediation, where feasible. Earthworms have been shown to stimulate the degradation of petroleum hydrocarbons in soil, and it was hypothesized that the role of earthworms in remediation lies in the enhancement of an oil degrading microbial community. The aim of this study was to characterize microbial activity and community dynamics in oil-contaminated soil incubated with or without earthworms. Three earthworm species (Eisenia fetida, Allolobophora chlorotica and Lumbricus terrestris) were incubated in crude oil polluted soil (ca. 10,000 mg/kg total petroleum hydrocarbons (TPH)) and a reference soil for 28 d. Control treatments with manual mixing and/or cattle dung amendment were also included. In the oil-contaminated soil, respiration and concentration of microbial biomass was significantly enhanced by earthworm amendment, and TPH concentrations decreased significantly. These effects were less evident in treatments with A. chlorotica, possibly due to a difference in behavior, since individuals of this endogeic species were found in a state of inactivity (aestivation). Microbial community dynamics were described by phospholipid fatty acid (PLFA) analyses. After 28 d, similar shifts in the soil PLFA composition were observed in the oil-contaminated soil irrespective of worm species. Fungal:bacterial ratios were increased in the presence of worms, but also by addition of dung as a food source, indicating a non-specific effect of metabolizable substrates. In contrast, the fatty acids 17:1ω8 (=Δ9-heptadecenoic acid) and 20:4ω6c (arachidonic acid) were specifically stimulated by the presence of earthworms in the oil-contaminated soil. The results showed that earthworms can contribute positively to bioremediation of oil-contaminated soil, but that the effect may be species-dependent.