Influence of oil contamination levels on hydrocarbon biodegradation in sandy sediment

The influence of oil concentration on hydrocarbon biodegradation in a sandy sediment was studied in polyvinyl chloride reactors (0.45 x 0.28 x 0.31 m) containing 76.8 kg of beach sand in natura, where the upper layer was artificially contaminated with petroleum. The oil-degrading microorganisms used consisted of a mixed culture named ND, obtained from landfarming and associated with indigenous microorganisms. On the 28th day of the process, the degradation in reactors containing sandy sediment contaminated with light Arabian oil and presenting an initial oil content of 14, 21 or 28 g kg-1 reached the following levels (%): 33.7, 32.9 and 28.9 for oil and grease; up to 88.3, 35.3 and 13.0 for C14-C26 n-alkanes; and 100, 61.3 and 59.4 for pristane, respectively. Phytane removal (37.1%) was only detected in the reactor contaminated with the lowest oil concentration studied. These results, together with the expressive bacterial growth observed (from 10(6) to 10(11) cfu g-1) give strong support to the argument that biodegradation was the dominant component of the remediation process. Susceptibility to biodegradation was inversely proportional to increasing oil contamination. The degradation of branched alkane: pristane was not repressed by the presence of n-alkanes.     

Degradation of pentachlorophenol by pure and mixed cultures in two different soils

GOAL, SCOPE AND BACKGROUND: Pentachlorophenol (PCP) is the second highest volume pesticide used in the United States. It is a mutagenic compound whose exposure poses significant health effects, One of the most desirable, environmentally friendly treatment methods is bioremediation. For soil-based contamination, the effectiveness of bioremediation will also be affected by the presence of an active indigenous population, sorption of the contaminant onto the soil, and environmental parameters. METHODS: Two pure strains and their mixed culture were used to evaluate PCP biodegradation in two different field soils, Columbia (CO) and New Mexico (NM). Biostimulation of the indigenous microbes was evaluated by adding nutrients. The efficiency of adding bacteria strains (bioaugmentation) for degrading PCP was determined with Arthrobacter sp., Flavobacterium sp. and a 50:50 mixture of the two bacteria strains. RESULTS: In CO soil, only 24%, 12% and 25% of the initial PCP concentration were degraded by Flavobacterium sp., Arthrobacter sp. and mixed culture, respectively. Arthrobacter sp. was used in NM soil with two initial concentrations and achieved degradation efficiencies of 57% and 61% for 361 and 95 mg kg- concentrations, respectively. Discussion. Analysis via statistical methods showed that the bacteria had different efficiencies on PCP degradation in each soil. 2 CONCLUSIONS: All bacteria catalyzed a higher PCP degradation when present in NM soil. Second, Flavobacterium sp. degraded more PCP than Arthrobacter sp. in CO soil. The mixed culture achieved the highest degradation efficiency regardless of the initial concentration or soil origin. RECOMMENDATIONS AND PERSPECTIVES: The effect of the soil properties, such as the soil organic matter (SOM) on PCP biodegradation should be investigated. Future work can also investigate the effect of aging time on biodegradation.     

嗜盐菌的筛选及原油降解性能

油气开发过程含油废液中过高的盐含量是影响其生物处理效果不佳的一个重要因素。针对含油废液的特点,实验从油田废弃泥浆中筛选分离出一株高效嗜盐降解菌,该菌呈杆状,经BIOLOG鉴定系统与分子序列鉴定分析,该菌为芽孢杆菌Bacillus subtilis strain;研究了嗜盐菌的耐盐碱性及原油降解性能,结果表明,该菌适宜于碱性环境,适盐浓度范围为5 000~200 000 mg/L,7 d内对高盐含油模拟废水中原油的降解率高达60%,最佳降解条件为：菌液/培养液体积比1：12.5,pH=9,NaCl浓度范围为10 000~50 000 mg/L,最佳N源和P源分别为（NH2）2CO和K2HPO4·3H2O。嗜盐菌的研究为高盐含油废液的生物处理拓展了一条新的技术途径。     

Influence of salinity on bioremediation of oil in soil

Spills from oil production and processing result in soils being contaminated with oil and salt. The effect of NaCl on degradation of oil in a sandy-clay loam and a clay loam soil was determined. Soils were treated with 50 g kg(-1) non-detergent motor oil (30 SAE). Salt treatments included NaCl amendments to adjust the soil solution electrical conductivities to 40, 120, and 200 dS m(-1). Soils were amended with nutrients and incubated at 25 degrees C. Oil degradation was estimated from the quantities of CO(2) evolved and from gravimetric determinations of remaining oil. Salt concentrations of 200 dS m(-1) in oil amended soils resulted in a decrease in oil mineralized by 44% for a clay loam and 20% for a sandy-clay loam soil. A salt concentration of 40 dS m(-1) reduced oil mineralization by about 10% in both soils. Oil mineralized in the oil amended clay-loam soil was 2-3 times greater than for comparable treatments of the sandy-clay loam soil. Amending the sandy-clay loam soil with 5% by weight of the clay-loam soil enhanced oil mineralization by 40%. Removal of salts from oil and salt contaminated soils before undertaking bioremediation may reduce the time required for bioremediation.     

Mineralization of phenanthrene and fluoranthene in yardwaste compost

The purpose of the study was to evaluate the potential of phenanthrene and fluoranthene biodegradation in yardwaste compost materials. These polynuclear aromatic hydrocarbons were chosen for this work because they are relatively readily biodegradable and ubiquitous in the environment. Compost samples were incubated in biometers with 14C-labeled phenanthrene and the evolution of "4CO2 was assessed as a measure of mineralization. The '4CO2 evolution varied widely among replicate biometers, possibly as the result of (1) uneven and patchy colonization of phenanthrene-degrading microorganisms on compost particles, and (2) non-uniform dispersion of the labeled substrate spike into the yardwaste microenvironment. Mineralization of phenanthrene reached about 40%extent of 14CO2 evolution at best before leveling off, but the maximum varied from sample to sample and could be as low as 1%after three months. Active mineralization occurred at mesophilic and thermophilic temperatures. Methanol extraction was used to recover "4C from biometer samples that were spiked with "4C-labeled phenanthrene. Extraction for 24-48 h yielded 1-14% recovery of 14C, depending on the length of the preceding incubation. The low extraction yield and relatively low maximum mineralization(<40%) indicated that residual phenanthrene was sorbed and bound within the compost matrix in the biometer. Amendment ofbiometers with 0.05% Tween 80 or addition of water did not consistently enhance the mineralization. Variability in mineralization was greatly reduced in liquid samples taken from pre-enriched compost samples. Mineralization of 14C-labeled fluoranthene was negligible in biometers but could be stimulated by pre-enrichment with salicylate or naphthalene. Pre-enrichment also accelerated the mineralization of phenanthrene.     

Chlorpyrifos degradation in Turkish soil

Degradation of chlorpyrifos was evaluated in laboratory studies. Surface (0-15 cm) and subsurface (40-60 cm) clay loam soils from a pesticide-untreated field were incubated in biometer flasks for 97 days at 25 degrees C. The treatment was 2 micrograms g-1 [2,6-pyridinyl-14C] chlorpyrifos, with 74 kBq radioactivity per 100 g soil flask. Evolved 14CO2 was monitored in KOH traps throughout the experiment. Periodically, soil subsamples were also methanol-extracted [ambient shaking, then supercritical fluid extraction (SFE)], then analyzed by thin-layer chromatography. Total 14C and unextractable soil-bound 14C residues were determined by combustion. From the surface and subsurface soils, 41 and 43% of the applied radiocarbon was evolved as 14CO2 during 3 months incubation. The time required for 50% loss of the parent insecticide in surface and subsurface soils was about 10 days. By 97 days, chlorpyrifos residues and their relative concentration (in surface/subsurface) as % of applied 14C were: 14CO2 (40.6/42.6), chlorpyrifos (13.1/12.4), soil-bound residues (11.7/11.4), and 3,5,6-trichloropyridinol (TCP) (3.8/4.8). Chlorpyrifos was largely extracted by simple shaking with methanol, whereas TCP was mainly removed only by SFE. The short persistence of chlorpyrifos probably relates to the high soil pH (7.9-8.1).     

嗜盐菌的筛选及原油降解性能简

油气开发过程含油废液中过高的盐含量是影响其生物处理效果不佳的一个重要因素。针对含油废液的特点,实验从油田废弃泥浆中筛选分离出一株高效嗜盐降解菌,该菌呈杆状,经BIOLOG鉴定系统与分子序列鉴定分析,该菌为芽孢杆菌Bacillus subtilis strain;研究了嗜盐菌的耐盐碱性及原油降解性能,结果表明,该菌适宜于碱性环境,适盐浓度范围为5 000~200 000 mg/L,7 d内对高盐含油模拟废水中原油的降解率高达60%,最佳降解条件为：菌液/培养液体积比1：12.5,pH=9,NaCl浓度范围为10 000~50 000 mg/L,最佳N源和P源分别为（NH₂）₂CO和K₂HPO₄·3H₂O。嗜盐菌的研究为高盐含油废液的生物处理拓展了一条新的技术途径。     

固定化微生物修复石油污染土壤影响因素研究

针对石油污染土壤修复,利用实验室已筛选的高效石油降解单菌SM-3,以天然有机材料为载体,吸附法制备固定化微生物。将游离与固定化微生物应用于室内花盆模拟修复石油污染土壤,对C/N/P、微生物投加量、石油含量、氧化剂和表面活性剂设计5因素4水平正交实验,探讨不同修复时期各影响因素的重要性顺序,最佳条件下各菌株的修复效果。结果表明,不同微生物在不同降解时期,各影响因素的重要性会发生变化;经过21 d的修复,固定化单菌SM-3石油降解率为22.77%,修复过程中,接种量是最重要的影响因素,营养元素N、P投加影响较大,表面活性剂和氧化剂影响次之。     

脂肽生物表面活性剂作用下多菌种石油生物降解影响因素优化

探索多种菌种降解石油过程中菌种和脂肽生物表面活性剂的作用,筛选石油降解的主要影响因素及最佳组合,并为石油污染物的降解机理研究和石油污染修复提供指导。基于正交实验筛选主要影响因素,采用Box-Behnken实验探讨各因素最佳水平。正交实验中脂肽生物表面活性剂是多菌种降解石油过程中最主要的影响因素,在Box-Behnken实验中,其能显著地影响石油降解率。菌种降解能力是石油饱和烃组分生物降解的最主要影响因素,但脂肽生物表面活性剂是芳烃、胶质和沥青质组分降解的最主要的影响因素。研究所用菌种中,解淀粉芽孢杆菌（Bacillus amyloliquefaciens）和假单胞菌（Pseudomonas aeruginosa）在石油降解过程最重要,是本实验的石油降解最优菌。菌种和脂肽生物表面活性剂的添加浓度配比对于石油降解具有重要的影响。解淀粉芽孢杆菌和假单胞菌添加量5%,脂肽生物表面活性剂粗品添加量200 mg·L-1的降解效果最优,理论上,最高降解率可达63.78%,验证降解率达到了53.89%,相对于多菌种正交实验最高降解率提高了5.54%。利用正交实验和Box-Behnken实验筛选最优降解菌和最优菌种组合的方法,具有分析因素多、实验量少等优点,具有较好的应用前景。     

Biodegradation of trifluralin in Harran soil

Degradation of trifluralin (alpha, alpha, alpha-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) was investigated in soils taken from three different locations at Harran region of Turkey under laboratory conditions. Surface (0-10 cm) soils, which were taken from a pesticide untreated field Gürgelen, Harran-1 and Ikizce regions in the Harran Plain. were incubated in biometer flasks for 350 days at 25 degrees C. Ring-UL-14C-trifluralin was applied at the rate of 2 microg g(-1) with 78.7 kBq radioactivity per 100 g soil flask. Evolved (14)CO2 was monitored in KOH traps throughout the experiment. Periodically, soil sub-samples were removed and extracted by supercritical fluid extraction (SFE). Unextractable soil-bound 14C residues were determined by combustion. During the 350 days incubation period 6.6, 5.4, and 3.3/' of the applied radiocarbon was evolved as (14)CO2 from the Harran-1, Gürgelen, and Ikizce soil, respectively. At the end of 350 days the SFE-extractable and bound 14C-trifluralin residues were 39.0 and 29.2% of the initially applied herbicide in Gürgelen soil. The corresponding values for Harran-1 and Ikizce soils were 36.2, 28.4% and 41.6, 18.5% respectively.     

聚乙烯醇生物降解菌群的结构分析及优势菌株降解特性

对堆肥中降解聚乙烯醇材料的微生物菌群结构进行了分析。结果表明：降解聚乙烯醇材料的优势菌群属于芽胞杆菌科(Bacillaceae)。从降解了3年的材料表面筛选出了1株聚乙烯醇降解菌DG01,鉴定为苏云金杆菌(Bacillus thuringiensis sp.)。分别以聚乙烯醇(poly(vinyl alcohol),PVA)浓度和二氧化碳排放量为指标,对PVA的降解动力学进行了研究。结果表明：PVA生物降解过程符合一级动力学模型,R²分别为0.984 0和0.983 5。对摇瓶培养条件进行了单因素优化实验。最佳降解温度,初始pH和酵母粉浓度分别为41 ℃、7和1.40 g·L⁻¹。优化后,48 h内PVA初始浓度为3 g·L⁻¹的降解率达到了45.21%,提高了2.10倍。     

热水洗辅助微生物修复高含油落地油泥

为解决油田落地油泥含油率高但不能直接进行生物无害化处置的问题,探索一种热水洗辅助微生物修复工艺。利用热水洗实验考察了清洗配方和水温对降低含油率的影响;从落地油泥中分离出石油烃降解菌,以用于含油残砂的微生物降解过程,同时探究填料和烃降解微生物对残砂中石油烃降解效果的影响。结果表明,鼠李糖脂清洗剂在总用量为0.5%、水洗时间30 min和50 ℃条件下,可将油泥含油率由107.1 mg·g⁻¹降至为39.3 mg·g⁻¹;水洗后残砂中的土著菌群数量出现了明显下降。分离获得了2株烃降解菌,并优选出具有增加油泥溶氧和保水的沼渣。将烃降解微生物和优选沼渣加入残砂后,石油烃生物降解效率得到显著提升,200 d后含油率降为4.62 mg·g⁻¹。在此过程中,微生物优先降解分子量小的饱和烃和芳香烃组分,对胶质和沥青质等组分降解程度偏低。该研究结果可为油田高含油落地油泥的环保处置提供参考。     

响应面法优化石油降解菌性能的研究

从延长油田石油污染土壤中筛选出4株能以原油为唯一碳源生长的细菌单菌株,鉴定其优势菌株YC-2为枯草芽孢杆菌;利用响应面法进行实验设计,选定培养温度、pH值、氮磷比以及盐度为影响因素,对原油含量为0.5%的培养基进行YC-2菌株降解工艺优化,得到了原油降解率与4种因素之间的非线性回归方程,确定了降解原油的最优工艺条件:培养温度28℃;pH值6.5,氮磷比5.2:1,盐度0.45%。在最优条件下,预测降解率为53.9%,实验验证值为52.5%,结果显示,建立的模型具有较高的精度。     

Biodegradation of methyl red by Bacillus sp. strain UN2: decolorization capacity,metabolites characterization,and enzyme analysis

Azo dyes are recalcitrant and refractory pollutants that constitute a significant menace to the environment. The present study is focused on exploring the capability of Bacillus sp. strain UN2 for application in methyl red (MR) degradation. Effects of physicochemical parameters (pH of medium, temperature, initial concentration of dye, and composition of the medium) were studied in detail. The suitable pH and temperature range for MR degradation by strain UN2 were respectively 7.0–9.0 and 30–40 °C, and the optimal pH value and temperature were respectively 8.0 and 35 °C. Mg²⁺ and Mn²⁺ (1 mM) were found to significantly accelerate the MR removal rate, while the enhancement by either Fe³⁺ or Fe²⁺ was slight. Under the optimal degradation conditions, strain UN2 exhibited greater than 98 % degradation of the toxic azo dye MR (100 ppm) within 30 min. Analysis of samples from decolorized culture flasks confirmed biodegradation of MR into two prime metabolites: N,N′dimethyl-p-phenyle-nediamine and 2-aminobenzoic acid. A study of the enzymes responsible for the biodegradation of MR, in the control and cells obtained during (10 min) and after (30 min) degradation, showed a significant increase in the activities of azoreductase, laccase, and NADH-DCIP reductase. Furthermore, a phytotoxicity analysis demonstrated that the germination inhibition was almost eliminated for both the plants Triticum aestivum and Sorghum bicolor by MR metabolites at 100 mg/L concentration, yet the germination inhibition of parent dye was significant. Consequently, the high efficiency of MR degradation enables this strain to be a potential candidate for bioremediation of wastewater containing MR.     

Potential of the microbial community present in an unimpacted beach sediment to remediate petroleum hydrocarbons

The potential of the microbial communities present in the intertidal zone of an unimpacted beach (a beach that did not suffer any significant oil spill) to degrade hydrocarbons was investigated. For that, laboratory-based microcosms (50-ml flasks) were set up with sandy beach sediment spiked with crude oil and incubated with local seawater for 15 days in the dark. Three bioremediation treatments were tested (biostimulation (BS), autochthonous bioaugmentation (AB), and combined treatment of biostimulation + bioaugmentation (BS + AB)) and the results were compared with natural attenuation (NA). Visual inspection showed clearly an oil solubility increase (confirmed by a higher hydrocarbons concentration in supernatant solutions) for all tested treatments when compared to NA. Significant degradation of the oil, shown by different profiles of petroleum hydrocarbons, was also observed for the different treatments particularly for BS + AB. Therefore, the microbial community of this unimpacted beach sediment could respond to an oil spill, degrading hydrocarbons. But to increase the natural attenuation pace, obtained results indicated that BS + AB is an appropriate approach for the bioremediation of beaches recently impacted by an oil spill. The autochthonous microbial cultures can be obtained “before” or “after” the contamination of the target site, being inoculated into the site right after it contamination.     

Isolation and characterization of 1,2,4-trichlorobenzene mineralizing Bordetella sp. and its bioremediation potential in soil

A soil which has been polluted with chlorinated benzenes for more than 25 years was used for isolation of adapted microorganisms able to mineralize 1,2,4-trichlorobenzene (1,2,4-TCB). A microbial community was enriched from this soil and acclimated in liquid culture under aerobic conditions using 1,2,4-TCB as a sole available carbon source. From this community, two strains were isolated and identified by comparative sequence analysis of their 16S-rRNA coding genes as members of the genus Bordetella with Bordetella sp. QJ2-5 as the highest homological strain and with Bordetella petrii as the closest related described species. The 16S-rDNA of the two isolated strains showed a similarity of 100%. These strains were able to mineralize 1,2,4-TCB within two weeks to approximately 50% in liquid culture experiments. One of these strains was reinoculated to an agricultural soil with low native 1,2,4-TCB degradation capacity to investigate its bioremediation potential. The reinoculated strain kept its biodegradation capability: (14)C-labeled 1,2,4-TCB applied to this inoculated soil was mineralized to about 40% within one month of incubation. This indicates a possible application of the isolated Bordetella sp. for bioremediation of 1,2,4-TCB contaminated sites.     

Studies on biodegradation of nicotine by Arthrobacter sp. strain HF-2

A nicotine-degrading bacterium, strain HF-2, was isolated from tobacco waste-contaminated soil and identified as a member of Arthrobacter sp. based on morphology, physiological tests, 16S rDNA sequence and phylogenetic characteristics. At thermal denaturation test indicated that the G + C mol% of strain HF-1 was 63.5. The relationship between the growth of the isolate and the nicotine degradation suggested that strain HF-2 could utilize nicotine as sole sources of carbon, nitrogen and energy. Blue pigment was observed during the nicotine degradation by strain HF-2. The isolate grew well at 20 to 33 degrees C, initial pH 6.5 to 8.0 and 0.5 to 2.0 g L-1 of nicotine concentration in the nicotine inorganic salt media. The maximum growth and nicotine degradation occurred at 30 degrees C, initial pH 7.0 and 0.7 g.L-1 of nicotine concentration in media under natural incubation condition. Strain HF-2 could degrade 100% of nicotine under the optimized incubation conditions for 43 h. The concentrations of nicotine were monitored by high performance liquid chromatography. This study demonstrates Arthrobacter sp. strain HF-2 had a great ability to degrade nicotine, and it may be available for the application to the bioremediation of environments contaminated by tobacco waste.     

一株嗜油不动杆菌(Acinetobacter oleivorans)的分离鉴定及石油降解特性

从石油污染土壤分离到30株石油降解菌,经复筛得到一株适应能力强、对石油降解效率高的优势菌株JC3-1。该菌株在温度为20~40℃、pH 5~9、盐度(NaCl)为0~3%(W/V)条件下生长良好。在以4 g/L原油为惟一碳源的培养基中生长15 d后对原油的降解率达42.15%。通过形态学观察、生理生化实验及16S rDNA序列分析,初步鉴定属于嗜油不动杆菌(Acinetobacter oleivorans)。菌株在以正戊烷、正己烷、十六烷、十八烷、苯、甲苯、二甲苯、邻苯二酚、萘、芘为惟一碳源培养基中都能很好生长。用特异性PCR检测发现,JC3-1具有酰基辅酶A脱氢酶、苹果酸合成酶、异柠檬酸裂合酶、TonB-依赖性铁载体受体、铁载体受体蛋白、多功能脂肪酸氧化酶复合体α亚单位等代谢功能基因,及烷烃单加氧酶、芳烃双加氧酶、联苯双加氧酶、邻苯二酚双加氧酶、萘双加氧酶、甲苯双加氧酶等降解功能基因。     

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.     

Ex situ bioremediation of a soil contaminated by mazut (heavy residual fuel oil)--a field experiment

Mazut (heavy residual fuel oil)-polluted soil was exposed to bioremediation in an ex situ field-scale (600 m(3)) study. Re-inoculation was performed periodically with biomasses of microbial consortia isolated from the mazut-contaminated soil. Biostimulation was conducted by adding nutritional elements (N, P and K). The biopile (depth 0.4m) was comprised of mechanically mixed polluted soil with softwood sawdust and crude river sand. Aeration was improved by systematic mixing. The biopile was protected from direct external influences by a polyethylene cover. Part (10 m(3)) of the material prepared for bioremediation was set aside uninoculated, and maintained as an untreated control pile (CP). Biostimulation and re-inoculation with zymogenous microorganisms increased the number of hydrocarbon degraders after 50 d by more than 20 times in the treated soil. During the 5 months, the total petroleum hydrocarbon (TPH) content of the contaminated soil was reduced to 6% of the initial value, from 5.2 to 0.3 g kg(-1) dry matter, while TPH reduced to only 90% of the initial value in the CP. After 150 d there were 96%, 97% and 83% reductions for the aliphatic, aromatic, and nitrogen-sulphur-oxygen and asphaltene fractions, respectively. The isoprenoids, pristane and phytane, were more than 55% biodegraded, which indicated that they are not suitable biomarkers for following bioremediation. According to the available data, this is the first field-scale study of the bioremediation of mazut and mazut sediment-polluted soil, and the efficiency achieved was far above that described in the literature to date for heavy fuel oil.