Bioprospection and selection of bacteria isolated from environments contaminated with petrochemical residues for application in bioremediation

The use of microorganisms with hydrocarbon degrading capability and biosurfactant producers have emerged as an alternative for sustainable treatment of environmental passives. In this study 45 bacteria were isolated from samples contaminated with petrochemical residues, from which 21 were obtained from Landfarming soil contaminated with oily sludge, 11 were obtained from petrochemical industry effluents and 13 were originated directly from oily sludge. The metabolization capability of different carbon sources, growth capacity and tolerance, biosurfactant production and enzymes detection were determined. A preliminary selection carried out through the analysis of capability for degrading hydrocarbons showed that 22% of the isolates were able to degrade all carbon sources employed. On the other hand, in 36% of the isolates, the degradation of the oily sludge started within 18–48 h. Those isolates were considered as the most efficient ones. Twenty isolates, identified based on partial sequencing of the 16S rRNA gene, were pre-selected. These isolates showed ability for growing in a medium containing 1% of oily sludge as the sole carbon source, tolerance in a medium containing up to 30% of oily sludge, ability for biosurfactant production, and expression of enzymes involved in degradation of aliphatic and aromatic compounds. Five bacteria, identified as Stenotrophomonas acidaminiphila BB5, Bacillus megaterium BB6, Bacillus cibi, Pseudomonas aeruginosa, and Bacillus cereus BS20 were shown to be promising for use as inoculum in bioremediation processes (bioaugmentation) of areas contaminated with petrochemical residues since they can use oily sludge as the sole carbon source and produce biosurfactants.     

Biotreatment of Total Petroleum Hydrocarbons from an oily Sludge Using Co-Composting Approach

Discharging of non-treated oily sludge from oil refineries has undesirable impacts on the environment. In this research, the biotreatment of total petroleum hydrocarbons (TPHs) from Abadan Petroleum Refinery, Iran was done using co-composting method. A 5 kg mixture of oily sludge and compost (oily sludge: compost ratios 1:0, 1:0.1, 1:0.3, 1:0.5, and 1:0.7 w/w) and a bulking agent of wheat straw were used as treatments. All treatments were placed in a wooden box inside a laboratory and aerated every 2–3 days by mixing during two months of the experiments. The variation of TPHs concentration, bacterial density, C/N ratio, pH, and temperature were assessed during the 63 days- experiments. Results showed that the majority of TPHs of samples was removed at first 30 days. The maximum TPHs removal (65%) was obtained in the sludge: compost = 1:0.5 at the operation time of 63 day. The significant different between removal efficiency of oily sludge: compost ratios and the control sample (p value < 0.05) indicated the appropriate density of TPHs degrader-bacteria in treated samples. The highest variation in C/N ratio was observed 15± 0.58 in the oily sludge: compost = 1:0.1. At the beginning of experiments, the pH values of all treatments was alkaline, but became neutral at the end of the reaction time. The TPHs degradation kinetic for all oily sludge: compost ratios followed pseudo second-order model. In general, co-composting has high efficiency in oily sludge remediation and can be potentially applied as a simple and cost-effective approach for remediation of oily sludge.     

Oily sludge degradation by bacteria from Ankleshwar, India

Three bacterial strains, Bacillus sp. SV9, Acinetobacter sp. SV4 and Pseudomonas sp., SV17 from contaminated soil in Ankleshwar, India were tested for their ability to degrade the complex mixture of petroleum hydrocarbons (such as alkanes, aromatics, resins and asphaltenes), sediments, heavy metals and water known as oily sludge. Gravimetric analysis showed that Bacillus sp. SV9 degraded approx. 59% of the oily sludge in 5 days at 30 °C whereas Acinetobacter sp. SV4 and Pseudomonas sp. SV17 degraded 37% and 35%. Capillary gas chromatographic analysis revealed that after 5 days the Bacillus strain was able to degrade oily sludge components of chain length C₁₂–C₃₀ and aromatics more effectively than the other two strains. Maximum drop in surface tension (from 70 to 28.4 mN/m) was accompanied by maximum biosurfactant production (6.7 g l⁻¹) in Bacillus sp. SV9 after 72 h, these results collectively indicating that this bacterial strain has considerable potential for bioremediation of oily sludge.     

Pollution Characteristics and Risk Assessment of Polycyclic Aromatic Hydrocarbons (PAHs) in Soils in Winter around Zhongyuan Oilfield,China

The degree of contamination with polycyclic aromatic hydrocarbons (PAHs) in soil samples in winter was determined. The contents of PAHs in samples were analyzed with HPLC. The PAHs contents of soil samples in winter around three different oily sludges from high to low represented the Third Wenming Plant of the oily sludge (3W), the Third Mazhai plant of the oily sludge (3M), and the Fourth Wener Plant of the oily sludge (4W), respectively. PAHs with 2–4 rings were major pollutants in oily sludge. Based on Nemero index P, the classification evaluation showed that soils around oily sludge were heavily polluted in winter. The health risk assessment and ecological risk assessment in soil in winter around oily sludge in Zhongyuan Oil Field was also analyzed.     

Evaluation of genetic diversity among Pseudomonas citronellolis strains isolated from oily sludge-contaminated sites

The diversity among a set of bacterial strains that have the capacity to degrade total petroleum hydrocarbons (TPH) in soil contaminated with oily sludge (hazardous hydrocarbon waste from oil refineries) was determined. TPH is composed of alkane, aromatics, nitrogen-, sulfur-, and oxygen-containing compound, and asphaltene fractions of crude oil. The 150 bacterial isolates which could degrade TPH were isolated from soil samples obtained from diverse geoclimatic regions of India. All the isolates were biochemically characterized and identified with a Biolog microbial identification system and by 16S rDNA sequencing. Pseudomonas citronellolis predominated among the 150 isolates obtained from six different geographically diverse samplings. Of the isolates, 29 strains of P. citronellolis were selected for evaluating their genetic diversity. This was performed by molecular typing with repetitive sequence (Rep)-based PCR with primer sets ERIC (enterobacterial repetitive intergenic consensus), REP (repetitive extragenic palindromes), and BOXAIR and PCR-based ribotyping. Strain-specific and unique genotypic fingerprints were distinguished by these molecular typing strategies. The 29 strains of P. citronellolis were separated into 12 distinguishable genotypic groups by Rep-PCR and into seven genomic patterns by PCR-based ribotyping. The genetic diversity of the strains was related to the different geoclimatic isolation sites, type of oily sludge, and age of contamination of the sites. These results indicate that a combination of Rep-PCR fingerprinting and PCR-based ribotyping can be used as a high-resolution genomic fingerprinting method for elucidating intraspecies diversity among strains of P. citronellolis.     

不同结构配比的鼠李糖脂乳化活性与油泥清洗效果

以大庆油田原油和含油污泥为对象,研究不同结构配比鼠李糖脂表面活性剂乳化活性及其对含油污泥清洗效果的影响,并优化清洗工艺参数。结果表明:单鼠李糖脂比例越高,其表面活性越好;双鼠李糖脂比例越高,其对原油的乳化能力越强;临界胶束浓度随着双鼠李糖脂比例的增加而逐渐加大;单、双鼠李糖脂配比不同的表活剂对油泥的清洗效果也不同,质量比为50∶50时清洗效率最高;鼠李糖脂浓度为1.0 g·L^-1、热洗时间为1.5h、热洗温度为65℃、洗脱强度为220 r·min^-1、固液质量比为1∶5条件下,油泥的清洗效率最高,可达81.3%;含油率为29.6%的落地油泥,经一级洗涤后油泥残油率降至5.5%,原油回收率达到87.3%,清洗出的原油无明显乳化,易于分离。由此可知,鼠李糖脂的单、双糖脂比例不同对其理化性质和清洗含油污泥的效果均有不同程度的影响。     

Evaluation of Genetic Diversity among Pseudomonas citronellolis Strains Isolated from Oily Sludge-Contaminated Sites

The diversity among a set of bacterial strains that have the capacity to degrade total petroleum hydrocarbons (TPH) in soil contaminated with oily sludge (hazardous hydrocarbon waste from oil refineries) was determined. TPH is composed of alkane, aromatics, nitrogen-, sulfur-, and oxygen-containing compound, and asphaltene fractions of crude oil. The 150 bacterial isolates which could degrade TPH were isolated from soil samples obtained from diverse geoclimatic regions of India. All the isolates were biochemically characterized and identified with a Biolog microbial identification system and by 16S rDNA sequencing. Pseudomonas citronellolis predominated among the 150 isolates obtained from six different geographically diverse samplings. Of the isolates, 29 strains of P. citronellolis were selected for evaluating their genetic diversity. This was performed by molecular typing with repetitive sequence (Rep)-based PCR with primer sets ERIC (enterobacterial repetitive intergenic consensus), REP (repetitive extragenic palindromes), and BOXAIR and PCR-based ribotyping. Strain-specific and unique genotypic fingerprints were distinguished by these molecular typing strategies. The 29 strains of P. citronellolis were separated into 12 distinguishable genotypic groups by Rep-PCR and into seven genomic patterns by PCR-based ribotyping. The genetic diversity of the strains was related to the different geoclimatic isolation sites, type of oily sludge, and age of contamination of the sites. These results indicate that a combination of Rep-PCR fingerprinting and PCR-based ribotyping can be used as a high-resolution genomic fingerprinting method for elucidating intraspecies diversity among strains of P. citronellolis.     

Methanogenic Microbial Community Composition of Oily Sludge and Its Enrichment Amended with Alkanes Incubated for Over 500 Days

Methanogenic microbial community is responsive to the availability of hydrocarbons and such information is critical for the assessment of hydrocarbon degradation in remediation and also in biologically enhanced recovery of energy from non-producing oil reserves. In this study, methanogenic enrichment cultures from oily sludge amended with n-alkanes (C₁₅-C₂₀) showed a development of active methanogenic alkanes-degrading consortium for over a total of 1000 days of incubation at 37°C. Total genomic DNAs were extracted from three types of samples, the original oily sludge (OS), the sludge after incubation for 500 days under methanogenic condition without any external carbon addition (EC), and the enrichment culture from the EC amended with n-alkanes (ET) incubated for another 500 days. The phylogenetic diversities of microbial communities of the three samples were analyzed by PCR amplification of partial 16S rRNA genes. The catabolic genes encoding benzylsuccinate synthase (bssA) and alkylsuccinate synthase (assA) were also examined by PCR amplification. These results provide important evidence in that microbial populations in an oily sludge shifted from methanogenic aromatic compounds degrading communities to potential methanogenic alkane-degrading communities when the enrichment was supplemented with n-alkanes and incubated under anaerobic conditions.     

Biodegradation of Total Petroleum Hydrocarbons from Oily Sludge

The present study concerns the biotreatment of oily sludge of petroleum refineries. Experiments were performed to assess the degradation potential of the microbial species present in the oily sludge through augmentation, and using the augmented microbial inoculums to treat oily sludge in a slurry form containing mineral medium and water. The optimum pH of the slurry was found to be 8 for the biodegradation of oily sludge. The effect of oily sludge concentration, stirring rate, and treatment time on degradation of total petroleum hydrocarbons (TPH) was studied. It was found that the degradation process consists of two consecutive stages and that each stage follows a first-order kinetics. The first stage lasted 4 days followed by a second stage up to 7 days. The faster first stage had a rate constant of 0.1 day^?1, whereas the slower second stage had a rate constant of 0.056 day^?1. The kinetics was found to be time-dependent. The study showed that the mineral medium provided essential nutrients to the microbial species and that the degradation efficiency of the whole microbial species present in the oily sludge was quite high (～90%).     

Biodegradation potential of oily sludge by pure and mixed bacterial cultures

The biodegradation capacity of aliphatic and aromatic hydrocarbons of petrochemical oily sludge in liquid medium by a bacterial consortium and five pure bacterial cultures was analyzed. Three bacteria isolated from petrochemical oily sludge, identified as Stenotrophomonas acidaminiphila, Bacillus megaterium and Bacillus cibi, and two bacteria isolated from a soil contaminated by petrochemical waste, identified as Pseudomonas aeruginosa and Bacillus cereus demonstrated efficiency in oily sludge degradation when cultivated during 40 days. The bacterial consortium demonstrated an excellent oily sludge degradation capacity, reducing 90.7% of the aliphatic fraction and 51.8% of the aromatic fraction, as well as biosurfactant production capacity, achieving 39.4% reduction of surface tension of the culture medium and an emulsifying activity of 55.1%. The results indicated that the bacterial consortium has potential to be applied in bioremediation of petrochemical oily sludge contaminated environments, favoring the reduction of environmental passives and increasing industrial productivity.     

预水解发酵碳源对铜绿假单胞菌NY3产鼠李糖脂特性及应用效能的影响研究

发酵碳源对铜绿假单胞菌NY3(Pseudomonas aeruginosa NY3)产鼠李糖脂(Rhamnolipids,Rha)的特性影响较大。研究了利用废弃动物油作为发酵碳源时,其碱预水解和酶预水解对NY3菌发酵产鼠李糖脂产量、产物结构和性能的影响,从碳源水解酸值与水解产物、鼠李糖脂组分结构和实际应用效果进行了研究。碱、酶预水解实验发现,碳源酸值由初始的19.81 mg/g分别提高到72.04 mg/g和73.75 mg/g,气质联用(GC-MS)分析检测结果表明,碱、酶预水解后,碳源均释放7种C14-C18碳链的脂肪酸,鼠李糖脂产量由未预水解的8.28 g/L分别提高到15.35 g/L和17.63 g/L。液质联用(LCMS-IT-TOF)分析结果表明,用未预水解及碱、酶预水解碳源发酵时,NY3菌所产鼠李糖脂中单糖脂含量分别为62.07%、65.67%、87.32%。利用NY3菌在中试条件下处理高浓度石化企业油污泥,发现鼠李糖脂能促进NY3菌去除油污泥中的石油烃,且促进作用强弱顺序为未预水解产Rha碱预水解产Rha酶预水解产Rha。     

Biodegradation of petroleum industry oily-sludge using Jordanian oil refinery contaminated soil

The main objective of this study was to evaluate the effect of oily sludge concentration on its biodegradability in soil. Oily sludge was collected and applied to microcosms at full-, half-, or quarter-strength concentrations equivalent to 44.2, 22.2, and 11.1 g kg^?1 soil, respectively, of total petroleum hydrocarbons (TPH) contained in oily sludge. The biodegradability of oily sludge was evaluated by measuring CO₂ evolution and by measuring removal of TPH as well as its main composing fractions; namely; alkanes, aromatics, NSO-compounds, and asphaltenes. The collected soil contained 3.63 × 10⁶ cfu g^?1 soil of hydrocarbon-degrading bacteria, which is satisfactory to drive successful biodegradation of hydrocarbons in soil. These numbers increased significantly with oily sludge addition at a rate proportional to the added TPH reaching 3.35 × 10⁷ cfu g^?1 soil in the half-strength treatment. TPH mineralization rate followed the same pattern. However, TPH-mineralization efficiency was the greatest in quarter-strength treatment at 18.3%. TPH-removal efficiency was also highest in quarter-strength treatment at 30.9%. Nutrients addition caused mineralization inhibition. Since nutrients were added as a ratio of the added carbon, inhibition was the greatest with the highest TPH treatment. While alkanes were degraded, aromatics and asphaltenes were not, and NSO-compounds were enriched. Although SDS was completely biodegradable in soil, its addition promoted mineralization and removal of TPH from soil.     

Biodegradation of chrysene by the bacterial strains isolated from oily sludge

The biodegradation studies were conducted to test the ability of the bacterial strains (Chry2 and Chry3) isolated from the oily sludge obtained from Gujarat refinery, India, for utilization of chrysene in the liquid medium. Biodegradation of the compound was confirmed using gas chromatography and the percent degradation was calculated to be 15.0 and 17% by Chry2 and Chry3, respectively. The biodegradation results were supported by increase in viable cell count and dry biomass, in the presence of chrysene as the sole carbon source. Both the cultures produced biosurfactant which was indicated by the reduction in surface tension of the growth medium. Presence of catechol 2, 3-dioxygenase gene in Chry3 indicated its potential for degradation of PAHs through meta cleavage degradation pathway. Both the strains were found to possess catechol 1,2-dioxygenase and catechol 2,3-dioxygenase enzyme activities. Based on morphological and biochemical tests, the cultures were tentatively identified as Bacillus sp. (Chry2) and Pseudomonas sp. (Chry3).     

Screening of herbaceous plants for peat-enhanced rehabilitation of contaminated soil with oily sludge

A batch pot experiment using nine herbaceous species were conducted for peat enhanced rehabilitation of contaminated soil with oily sludge in the initial contents of 0%, 1.3%, 7.4%, and 12.2%, respectively. The results showed that petroleum hydrocarbons removal, plant growth indices and enzyme activities varied depending on plant species and oil contents. Cotton, ryegrass and tall fescue were effective in the rehabilitation of oily sludge contaminated soils. The total petroleum hydrocarbon (TPH) removal ranged from 30.0% to 40.0% after 170 days of treatment. Plant biomass was shown to be the preferred indicator for screening phytoremediation plant because it was closely correlated with TPH removal and enzyme activities. Peat-enhanced plant rehabilitation could be a good strategy for the treatment of oily sludge contaminated saline soils.     

Distribution and physiology of microorganisms in petrochemical oily sludge of plant

The occurrence, vertical distribution, and the physiological state of microorganisms in a petrochemical oily sludge deposit were studied. The total number and the number of viable microbial cells at depths of 0.2 and 3 m were about 10 and 10(8) cells/g dry wt. sludge. Most microbial cells taken from the middle (1 m deep) and the bottom (3 m deep) sludge horizons showed a delayed colony-forming ability, which suggested that the cells occurred in a hypometabolic state. The relative number of microaerobic denitrifying microorganisms steeply increased with depth. The amount of microorganisms tolerant to 3, 5, and 10% NaCl and capable of growing at 7 and 40 degrees C varied from 10(2) to 10(8) CFU/g dry wt. sludge. Petrochemical oily sludge was found to maintain the growth of heterotrophs, among which the degraders of oily sludge and ten different individual polycyclic aromatic hydrocarbons were detected. The occurrence of highly adaptable microorganisms with an adequate metabolic potential in the petrochemical oily sludge deposit implies that its bioremediation is possible without introducing special microorganisms.     

Anaerobic mineralization of quaternary carbon atoms: isolation of denitrifying bacteria on dimethylmalonate.

The microbial capacity to degrade simple organic compounds with quaternary carbon atoms was demonstrated by enrichment and isolation of five denitrifying strains on dimethylmalonate as the sole electron donor and carbon source. Quantitative growth experiments showed a complete mineralization of dimethylmalonate. According to phylogenetic analysis of the complete 16S rRNA genes, two strains isolated from activated sewage sludge were related to the genus Paracoccus within the alpha-Proteobacteria (98.0 and 98.2% 16S rRNA gene similarity to Paracoccus denitrificans(T)), and three strains isolated from freshwater ditches were affiliated with the beta-Proteobacteria (97.4 and 98.3% 16S rRNA gene similarity to Herbaspirillum seropedicae(T) and Acidovorax facilis(T), respectively). Most-probable-number determinations for denitrifying populations in sewage sludge yielded 4.6 x 10(4) dimethylmalonate-utilizing cells ml(-1), representing up to 0.4% of the total culturable nitrate-reducing population.     

Comparison of bio-augmentation and composting for remediation of oily sludge: A field-scale study in China

Two bioremediation technologies were performed in order to explore a better treatment process for an oily sludge restoration in China during 2004. The bioremediation by augmentation of biopreparation was compared with a conventional composting. The oily sludge and oil-polluted soil were received from an oil production plant. The total hydrocarbon content (THC) varied from 327.7 to 371.2 g kg⁻¹ of dry sludge and the THC in contaminated soil was 151.0 g kg⁻¹. Before application of preparation, straw, sawdust, top sand and pure soil were added in different proportions to the sludge and soil and mixed thoroughly. Such sludge and soil composites were used for negative controls and for activation of indigenous oil degrading microorganisms with addition of fertilizer (positive controls). For composting, crude manure and straw were added to the oily sludge and the THC was 101.4 g kg⁻¹. The biopreparation was applied every 2 weeks and experiment lasted 56 days under the ambient temperature. The sludge was mixed and watered every 3 days. After three times of biopreparation application, the THC decreased by 46–53% in the oily sludge and soil, while in the positive controls (activation of indigenous microorganisms) the THC decreased by 13–23%, and there was no oil degradation in negative controls After composting, the THC decreased by 31% in the oily sludge. The planting of Tall Fescue (Festuca arundinace) revealed a decrease of sludge toxicity after application of both bioremediation technologies and additionally decreased the THC by 5–7%.     

Distribution and Physiological State of Microorganisms in Petrochemical Oily Sludge

The occurrence, vertical distribution, and physiological state of microorganisms in a petrochemical oily sludge deposit were studied. The total number and the number of viable microbial cells at depths of 0.2 and 3 m were about 10¹⁰ and 10⁸ cells/g dry wt sludge. Most microbial cells taken from the middle (1 m deep) and the bottom (3 m deep) sludge horizons showed a delayed colony-forming ability, which suggested that the cells occurred in a hypometabolic state. The relative number of microaerobic denitrifying microorganisms steeply increased with depth. The amount of microorganisms tolerant to 3, 5, and 10% NaCl and capable of growing at 7 and 40°C varied from 10² to 10⁸ CFU/g dry wt sludge. Petrochemical oily sludge was found to maintain the growth of heterotrophs, among which the degraders of oily sludge and ten different individual polycyclic aromatic hydrocarbons were detected. The occurrence of highly adaptable microorganisms with an adequate metabolic potential in the petrochemical oily sludge deposit implies that its bioremediation is possible without introducing special microorganisms.     

New halo- and thermotolerant fermenting bacteria producing surface-active compounds

Two new strains of fermenting bacteria were isolated from oily sludge under conditions of enhanced salt concentration (approx. 8% w/v) and temperature (50°C). They produced considerable amounts of surface-active compounds that were detected by a newly developed quick and easy half-quantitative test of emulsion stabilization, and were quantified by tensiometry. The chemical structure of the surfactant is unknown. The strains grew fast with inexpensive substrates such as sugars and might be of interest for application in microbially improved oil recovery. Morphological, cytological, and physiological characterization allowed affiliation of the two strains to the genus Bacteroides.     

The response of maize (Zea mays L.) plant assisted with bacterial consortium and fertilizer under oily sludge

The objective of this study was to evaluate the role of PGPR consortium and fertilizer alone and in combination on the physiology of maize grown under oily sludge stress environment as well on the soil nutrient status. Consortium was prepared from Bacillus cereus (Acc KR232400), Bacillus altitudinis (Acc KF859970), Comamonas (Delftia) belonging to family Comamonadacea (Acc KF859971) and Stenotrophomonasmaltophilia (Acc KF859973). The experiment was conducted in pots with complete randomized design with four replicates and kept in field. Oily sludge was mixed in ml and Ammonium nitrate and Diammonium phosphate (DAP) were added at 70 ug/g and 7ug/g at sowing. The plant was harvested at 21 d for estimation of protein, proline and antioxidant enzymes superoxide dismutase (SOD) and peroxidase (POD). To study the degradation, total petroleum hydrocarbon was extracted by soxhelt extraction and extract was analyzed by GC-FID at different period after incubation. Combined application of consortium and fertilizer enhanced the germination %, protein and, proline content by 90,130 and 99% higher than untreated maize plants. Bioavailability of macro and micro nutrient was also enhanced with consortium and fertilizer in oily sludge. The consortium and fertilizer in combined treatment decreased the superoxide dismutase (SOD), peroxidase dismutase (POD) of the maize leaves grown in oily sludge. Degradation of total petroleum hydrocarbon (TPHs) was 59% higher in combined application of consortium and fertilizer than untreated maize at 3 d. The bacterial consortium can enhanced the maize tolerance to oily sludge and enhanced degradation of total petroleum hydrocarbon (TPHs). The maize can be considered as tolerant plant species to remediate oily sludge contaminated soils.