Monitoring bioremediation in creosote-contaminated soils using chemical analysis and toxicity tests

Three soils with a history of creosote contamination (designated NB, TI and AC) were treated in bench-scale microcosms using conditions (nutrient amendment, moisture content and temperature) which had promoted mineralization of ¹⁴C-pyrene in a preliminary study. Bioremediation was monitored using the solid-phase Microtox test, seed germination and earthworm survival assays, SOS-chromotest, Toxi-chromotest and a red blood cell (RBC) haemolysis assay. Contaminant concentrations in the AC soil did not change after 150 days. Polycyclic aromatic hydrocarbon (PAH) concentrations decreased in the NB soil, and toxicity decreased overall according to the earthworm, seed germination and Microtox tests. Although total petroleum hydrocarbons (TPHs) in the TI soil were reduced following treatment, results of the earthworm, seed germination, RBC and Microtox tests suggested an initial increase in toxicity indicating that toxic intermediary metabolites may have formed during biodegradation. Toxicity testing results did not always correlate with contaminant concentrations, nor were the trends indicated by each test consistent for any one soil. Each test demonstrated a different capacity to detect reductions in soil contamination. Journal of Industrial Microbiology & Biotechnology (2000) 24, 132–139. Received 14 June 1999/ Accepted in revised form 12 November 1999     

Multi-Species Ecotoxicity Assessment of Petroleum-Contaminated Soil

In 1992, a study was begun to compare the effect of landfarming vs. natural attenuation on the restoration of soil that had been contaminated with crude oil. Each of three lysimeters was filled with a sandy loam topsoil, and crude oil was applied to two of the lysimeters. One of the contaminated lysimeters was tilled, watered, and received a one-time application of fertilizer (N, P, K). No amendments were added to the second contaminated lysimeter, and the third was left uncontaminated. The lysimeters were monitored for 6 months and then left unattended. In 1995 and again in 1997 we sampled these lysimeters to evaluate the long-term effects of contamination and bioremediation. In 1995 we found marked effects on soil chemistry, bacterial, fungal, nematode, and plant populations and a higher rate of bioremediation in the fertilized-contaminated lysimeter (Lawlor et al., 1997). Data from 1997 and previously unreported data from 1995 are the subject of the current report. In 1997, low densities of hydrocarbon-degrading bacteria were found in all the lysimeters and little loss of TPH from the two contaminated lysimeters, suggesting a decreased rate of bioremediation. Nevertheless, there were increases in diversity and number of functional groups of bacteria, nematodes, and native plant species. However, molecular analyses revealed marked differences remained in the composition of dominant eubacterial species, and tests of soybeans indicated field conditions remained unsuitable for these plants.     

Soil lipase activity – a useful indicator of oil biodegradation

The evaluation of soil lipase activity as a tool to monitor the decontamination of a freshly oil-polluted soil was tested in a laboratory study. An arable soil was experimentally contaminated with diesel oil at 5 mg hydrocarbons g^–1 soil dry weight and incubated with and without fertilization (N-P-K) for 116 days at 20°C. Lipase activity and counts of oil-degrading microorganisms were measured at regular time intervals, and the correlations with the levels of hydrocarbon concentrations in soil were investigated. The residual soil hydrocarbon concentration correlated significantly negatively with soil lipase activity and with the number of oil-degrading microorganisms, independent of fertilization. The induction of soil lipase activity is a valuable indicator of oil biodegradation in naturally attenuated (unfertilized) and bioremediated (fertilized) soils.     

Effects of Bacillus subtilis O9 biosurfactant on the bioremediation of crude oil-polluted soils

The application of a surfactant from Bacillus subtilis O9 (Bs) on the bioremediation of soils polluted with crude oil was assayed in soil microcosms under laboratory conditions. Three concentrations of biosurfactant were assayed (1.9, 19.5, and 39 mg kg(-1) soil). Microcosms without biosurfactant were prepared as controls. During the experiment, the crude oil-degrading bacterial population, the aliphatic and aromatic hydrocarbons were monitored in each microcosm. The results indicated that applying Bs did not negatively affect the hydrocarbon-degrading microbial population Concentrations of 19 and 19.5mg (Bs) per kilogram of soil stimulated the growth of the population involved in the crude oil degradation, and accelerated the biodegradation of the aliphatic hydrocarbons. However, none of the assayed Bs concentrations stimulated aromatic hydrocarbon degradation.     

Aerobic Cr(VI) Reduction by Bacteria in Culture and Soil Conditions

We compared the performance of aerobic Cr(VI)-reducing bacteria isolated from Cr(VI)-contaminated soil in pure and mixed cultures of five isolated strains. The mixed culture had increased reduction rates compared to individual cultures. Cr(VI) reduction was observed in sterile soil inoculated with Pseudomonas fluorescens and in non-sterile soil with and without inoculation with P. fluorescens at initial pore water concentrations up to 1,600 mg Cr(VI)/L, whereas in culture the maximum inhibitory concentration was 500 mg Cr(VI)/L. Linear rates of Cr(VI) reduction in non-sterile soil amended with peptone were ～5 to 8 times higher than those observed in the mixed culture. Inoculation of non-sterile soil with P. fluorescens did not further enhance Cr(VI) reduction rates. Our results indicate that evaluation of Cr(VI) reduction capacity in Cr(VI)-contaminated soil for in-situ bioremediation purposes should not be done solely in pure culture. Although the latter may be used initially to assess the effects of process parameters (e.g., pH, temperature), the rate and extent of Cr(VI) reduction should be determined in soil for bioremediation design purposes.     

国外环境生物技术的发展和展望

环境生物技术在社会的发展中起着越来越重要的积极作用,相关产业也随之迅速发展起来,环境治理要依赖对生物,尤其是微生物及其生理,生化特性的了解和认识,从而可以对其生理,生化和遗传方面的性能加以利用。在这方面,分离解毒微生物及阐明毒物降解过程和机理仍是现在研究的焦点,与此同时,对遗传基因方面的研究和利用还有许多研究工作待进行,监测和跟踪微生物已进入到了分子水平,而传感监测方面已有一些可喜的成果正在向生产转化,生物修复作为消除污染的手段正治理中发挥巨大的作用,但环境保护的根本应是在于进行无污染产生的生产,也称之为绿色生产,未来的社会中,环境生物技术仍对将社会产生巨大的影响,对环境保护起到重要的作用,特别是在新型生物能源的开发和探索方面,本文同时也对世界不同地区在环境生物技术的发展及其特点进行了综述。     

大庆油田石油开采对土壤线虫群落的影响

选择大庆油田环境一致的6口油井,研究石油开采干扰对土壤线虫的影响。共鉴定出土壤线虫18科15属。食细菌线虫最多,共15属占总数的49.2%,其次为植物寄生线虫共9属,占30.6%,食真菌线虫4属,占17.6%,杂食/捕食线虫2属,占2.6%。其中优势类群为丝尾垫刃属Filenchus和头叶属Cephalobus,占总数的42.5%。常见类群包括13个属,占总数的51.7%。稀有类群15个属占总数的6.34%。土壤的线虫生活史策略以c-p 2所占比例最大,其次是c-p3和c-p 1类群,受油井开采作业等影响,线虫通过大量繁殖来应对环境的干扰与压力。不同油井线虫总数差异不显著,但所有油井线虫总数比对照明显减少(p<0.001)。不同油井线虫群落结构差异不大,各油井线虫富集指数和结构指数表明所有油井线虫受干扰程度较高,土壤食物网趋于退化。随着采样距离增加,线虫总量差异不显著,但线虫群落的物种数量增加,优势度指数和多样性指数逐渐增加。结果表明油井开采对土壤线虫群落有一定的影响,开采距离对线虫群落结构影响较大。     

Enhanced biodegradation of transformer oil in soils with cyclodextrin – from the laboratory to the field

The use cyclodextrins for the intensification of bioremediation by improving the mobility and bioavailability of contaminants has recently been studied. In this work, the role of randomly methylated -cyclodextrin in the bioremediation of soils contaminated with transformer oil was studied both in bench scale bioreactors and through field experiments. The aims of this research were to (a) establish the scientific background of a cyclodextrin-based soil bioremediation technology, (b) demonstrate its feasibility and effectiveness in the field, and (c) develop an integrated methodology, consisting of a combination of physical, chemical, biological and ecotoxicological analytical methods, for efficiently monitoring the technology performances. The stepwise increasing scale of the experiments and the application of the integrated analytical methodology supported the development of a scientifically established new technology and the identification of the advantages and the limitations of its application in the field. At each phase of the study, randomly methylated -cyclodextrin was found to significantly enhance the bioremediation and detoxification of the transformer oil-contaminated soils employed by increasing the bioavailability of the pollutants and the activity of indigenous microorganisms.     

Physical and Biological Treatment of Oil-Contaminated Soil in a Baffled Roller Bioreactor

Physical and biological removal of diesel oil from contaminated soil was studied in a baffled roller bioreactor. Initially, the effects of four factors (soil loading, temperature, pH, and surfactant) on physical removal of diesel oil were investigated. Only the presence of a surfactant (sodium dodecyl sulfate [SDS]) demonstrated a significant effect on diesel oil removal. Diesel oil removal efficiency was increased from 32.0% to 63.9% in the presence of 100 mg/L SDS. Using a microbial culture enriched from contaminated soil, biological treatment of diesel oil polluted soil under different soil loadings (15% to 50%), different diesel oil concentrations (1 to 50 g/L), and different types of soil (sand, silt, and clay) was then investigated in the baffled roller bioreactor. Biodegradation consisted of both fast and slow stages for degradation of light and heavy compounds, respectively. All biodegradation experiments demonstrated significant decreases in diesel oil concentrations (88.3% in 14 days for initial diesel oil concentrations of 1000 mg/L and a wide range of soil loadings). The presence of silty or sandy soils enhanced the biodegradation rate compared to the control bioreactor (without soil). The sandy soil loading had no effect on the biodegradation results. Using the enriched culture, the baffled roller bioreactor was able to biodegrade high diesel concentrations (up to 50 g/L) with biodegradation rates of 112.2 and 39.3 mg/L· h during fast and slow stages, respectively.     

Geochemical, Microbiological, and Geophysical Assessments of Anaerobic Immobilization of Heavy Metals

Bioremediation methods that precipitate contaminants in situ as solid (mineral) phases can provide cost-effective options for removing dissolved metals in contaminated groundwater. The current field-scale experiments demonstrate that indigenous bacteria can be stimulated to remove metals by injection of electron-donating substrates and nutrients into a contaminated aquifer. Groundwater at the investigation site is aerobic and contains high levels of lead, cadmium, zinc, copper, and sulfuric acid (pH = 3.1) derived from a car-battery recycling plant. During the experiments, lead, cadmium, zinc, and copper were almost completely removed by precipitation of solid sulfide phases, as pH increased from 3 to ∼ 5 and Eh dropped from +400 mV to -150 mV. X-ray and transmission electron microscopy (TEM) analyses of filtered material from the treated groundwater indicated the presence of newly formed nanocrystalline metal sulfides. Genetic sequencing indicated that the principal species of sulfate-reducing bacteria involved in the bioremediation process was Desulfosporosinus orientis. Geochemical modeling shows that oxidation of added substrates and subsequent bacterial sulfate reduction produced desired geochemical conditions (i.e., decreasing Eh and increasing pH) for the precipitation and sorption of metal sulfides. Geophysical survey results suggest that bioremediation lowers electrical conductance of groundwater and possibly increases the magnetic susceptibility of porous media. This study demonstrates that integrated geochemical, geophysical, and microbiological analyses, combined with theoretical modeling, can successfully track and predict the progress of subsurface bioremediation.     

Petroleum bioremediation — a multiphase problem

Microbial degradation of hydrocarbons is a multiphase reaction, involving oxygen gas, water-insoluble hydrocarbons, water, dissolved salts and microorganisms. The fact that the first step in hydrocarbon catabolism involves a membrane-bound oxygenase makes it essential for microorganisms to come into direct contact with the hydrocarbon substrate. Growth then proceeds on the hydrocarbon/water interface. Bacteria have developed two general strategies for enhancing contact with water-insoluble hydrocarbons: specific adhesion mechanisms and production of extracellular emulsifying agents. Since petroleum is a complex mixture of many different classes of hydrocarbons, of which any particular microorganism has the potential to degrade only part, it follows that the microorganisms must also have a mechanism for desorbing from used' oil droplets.The major limitations in bioremediation of hydrocarbon-contaminated water and soil is available sources of nitrogen and phosphorus. The usual sources of these materials, e.g. ammonium sulfate and phosphate salts, have a high water solubility which reduces their effectiveness in open systems because of rapid dilution. We have attempted to overcome this problem by the use of a new controlled-release, hydrophobic fertilizer, F-1, which is a modified urea-formaldehyde polymer containing 18% N and 10% P as P₂O₅. Microorganisms were obtained by enrichment culture that could grow on crude oil as the carbon and energy source and F-1 as the nitrogen and phosphorus source. The microorganisms and the F-1 adhered to the oil/water interface, as observed microscopically and by the fact that degradation proceeded even when the water phase was removed and replaced seven times with unsupplemented water — a simulated open system. Strains which can use F-1 contain a cell-bound, inducible enzyme which depolymerizes F-1.After optimizing conditions in the laboratory for the use of F-1 and the selected bacteria for degrading crude oil, a field trial was performed on an oil contaminated sandy beach between Haifa and Acre, Israel, in the summer of 1992. The sand was treated with 5 g F-1 per kg sand and inoculated with the selected bacteria; the plot was watered with sea water and plowed daily. After 28 days the average hydrocarbon content of the sand decreased from 5.1 mg per g sand to 0.6 mg per g sand. Overall, there was an approx. 86% degradation of pentane extractables as demonstrated by dry weight, I.R. and GLC analyses. An untreated control plot showed only a 15% decrease in hydrocarbons. During the winter of 1992, the entire beach (approx. 200 tons of crude oil) was cleaned using the F-1 bacteria technology. The rate of degradation was 0.06 mg g^-1 sand day^-1 (10°C) compared to 0.13 mg g^-1 sand day^-1 during the summer (25°C).     

基于土壤线虫群落分析的油页岩废渣地不同植被类型恢复过程中的土壤食物网能流状况

土壤健康与生态系统功能是当前生态学研究的重要课题。土壤线虫是评价生态系统健康状况的指示生物,特别是用于评价土壤污染和恢复的过程。对广东茂名油页岩废渣场人工实施不同生态恢复改造状况下的土壤线虫属的重要值及土壤线虫反映的土壤食物网能量通道进行了分析,以通过污染地土壤食物网内的能量流动状况反映种植不同植物物种相同时间后的土壤养分与健康状况。结果表明在废渣场种植不同植物物种约8年后,乌墨林和大叶相思林下土壤线虫类群数(属)分别为54和45,重要值最高的是食细菌的棱咽属(Prismatolaimus)和拟丽突属(Acrobeloides);而红荷林和荒草地的线虫类群数分别为41和38,重要值最高的是杂食性的真矛线属(Eudorylaimus);废渣地线虫属的数量最少,仅为34个,重要值最高的是食真菌的丝尾垫刃属(Filenchus)和滑刃属(Aphelenchoides)。食物网能流分析表明:细菌能流通道比重最高的是大叶相思林和乌墨林,而真菌能流通道比重最高的是废渣地,红荷林和荒草地居中,各样地植物能流通道比重都比较小,仅为2%—10%之间。总体来讲,在有植被覆盖的生态系统,养分周转更快,特别是乌墨和大叶相思林,而无植被的废渣地,土壤养分周转速率倾向于更慢的真菌能流通道,这表明在养分条件比较差的情况下,真菌通道的食物网可能会起更大的作用。研究还说明了在油页岩废弃地恢复过程中,植物资源的输入不仅仅刺激植物能流通道,同时也会刺激细菌和真菌能流通道,植被是土壤线虫群落发展的主要驱动力。     

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...     

Enhanced activity of oligotrophic endogenous bacteria in clay‐rich sediments by nutrient injection

A controlled field experiment was performed in which the microbiology and geochemistry of clay‐rich fluvial‐deltaic sediments were characterized both before and after nutrient injection into a shallow well. Acetate addition (with nitrogen and phosphate) initially increased the heterotrophic bacteria population in the groundwater within 21 days after nutrient addition. Consumption of oxygen and injected nutrient resulted in an expected stimulation of copiotrophic bacterial growth (31–48 days), then a noticeable “trough phase”; reflecting minimal or no bacterial growth followed by a secondary peak reflecting another bacterial population growth surge (62–85 days). This secondary surge was apparently supported by the nutrients generated by the decomposing biomass (initial population peak), and by oxygen replenishment supplied by continual ground water flow. During the intervening trough phase, bacterial counts by most‐probable‐number analysis of soil samples indicated that the denitrifying population increased to a greater degree than the remainder of the heterotrophic population. An increase in methane gas was detected in the head space of water samples collected during the trough phase, suggesting an increase in bacterial methanogenesis. There was no evidence of a concomitant increase in bacterial sulfate‐reducing activity. This rapid progression from aerobic to microaerophilic and anaerobic growth probably resulted from the microbial biodiversity present in clay‐rich sediments, which in turn is related to the development of microhabitats. These microhabitats developed because of the hydrogeologic character of the clay‐rich sediments in which flow is controlled by macropores while fluid in the clay matrix (micropores) is relatively isolated from the groundwater in the macropores.     

Effect of Competing Electron Acceptors on the Reduction of U(VI) by Desulfotomaculum reducens

The biological reduction of soluble U(VI) to the less soluble U(IV) has been proposed as a strategy to remediate uranium-contaminated sites. However, the majority of the contaminated sites contain, in addition to U(VI), competing electron acceptors (CEAs) that can either enhance or inhibit U(VI) reduction. Desulfotomaculum reducens MI-1 is a sulfate-reducing bacterium able to reduce a variety of electron acceptors including U(VI). We characterized U(VI) reduction by D. reducens in the presence of pyruvate and three CEAs: sulfate, nitrate or soluble ferric iron. In the presence of sulfate or ferric iron and U(VI), cell growth was driven by respiration of the CEA. Nitrate was not used as an electron acceptor for growth and vegetative cells grew instead by fermenting pyruvate. Sulfate remaining after sulfate reduction has ceased or the presence of nitrate did not affect U(VI) reduction. However, in the case of sulfate, the addition of H₂ after the depletion of pyruvate greatly enhanced U(VI) reduction. Contrary to sulfate and nitrate, the presence of Fe(II), the product of Fe(III) reduction, abolished U(VI) reduction. The results from this investigation suggest that this microorganism and others with similar characteristics may play a role in U(VI) bioremediation efforts but only after the soluble Fe(II) produced by Fe(III) reduction has been advected away.     

中国水青冈分布,生长和更新特点

中国水青冈分布、生长和更新特点吴刚（中国科学院生态环境研究中心,北京１０００８５）Ｄｉｓｔｒｉｂｕｔｉｏｎ,ＧｒｏｗｔｈａｎｄＲｅｖｅｇｅｔａｔｉｏｎａｌＣｈａｒａｃｔｅｒｉｓｔｉｃｓｏｆＦａｇｕｓｉｎＣｈｉｎａ．ＷｕＧａｎｇ（ＲｅｓｅａｒｃｈＣｅｎ...     

Hydrocarbon bioremediation of a mineral-base contaminated waste from crude oil extraction by indigenous bacteria

The susceptibility to bioremediation of the hydrocarbons contained in a waste from crude oil extraction was examined. Laboratory scale batch reactors were inoculated with indigenous bacteria and biodegradation was followed for 45 days. The total hydrocarbon content was reduced to 70% of its initial value at the end of the experiments. Saturated and aromatic hydrocarbons were the most readily degraded fractions with, respectively, 70% and 60% of the fraction remaining at the end of the experiment. A minor degradation was observed in the resins fraction (20%), whereas the asphaltenes fraction remained almost constant.The substrate preferences of the natural population towards various fractions of the crude oil were determined by both the length of the lag phase and the slope of the exponential growth in a mineral salt-base medium containing either of the different hydrocarbon fraction as the sole source of carbon. The highest consumption rate for every fraction during the time course experiments was in agreement with the shortest lag phase and the greatest exponential growth slope in the corresponding selective media, indicating changes in the population composition.