Oil Palm Empty Fruit Bunch and Sugarcane Bagasse Enhance the Bioremediation of Soil Artificially Polluted by Crude Oil

Contamination of soil by petroleum hydrocarbons is becoming prevalent in Malaysia. Infiltration of soil contamination into groundwater poses a great threat to the ecosystem and human health. Bioremediation can occur naturally or can be enhanced with supplementation of microorganisms and fertilizers. However, fertilizers are expensive and therefore alternative nutrient-rich biomaterials are required. In this study, two organic wastes from agricultural industry (i.e., sugarcane bagasse and oil palm empty fruit bunch) were investigated for possible enhanced bioremediation of soil contaminated with Tapis crude oil. Two bacterial strains isolated and characterized previously (i.e., Pseudomonas aeruginosa UKMP-14T and Acinetobacter baumannii UKMP-12T) were used in this study. Sugarcane bagasse (5% and 15%, w/w) and oil palm empty fruit bunch (20%, w/w) were mixed with soil (500 g) spiked with Tapis crude oil (3%, v/w). The treated soils as well as controls were incubated for 20 days under controlled conditions. Sampling was carried out every four days to measure the number of bacterial colonies (CFU/g) and to determine the percentage of oil degradation by gas chromatography. The two biostimulating agents were able to maintain the soil moisture holding capacity, pH, and temperature at 38-40% volumetric moisture content (VMC), 7.0, and 29–30°C; respectively. The growth of bacteria consortium after 20 days in the treatment with sugarcane bagasse and oil palm empty fruit bunch had increased to 10.3 CFU/g and 9.5 CFU/g, respectively. The percentage of hydrocarbon degradation was higher in the soil amended with sugarcane bagasse (100%) when compared to that of oil palm empty fruit bunch (97%) after 20 days. Our results demonstrated the potential of sugarcane bagasse and oil palm empty fruit bunch as good substrates for enhanced bioremediation of soil contaminated with petroleum crude oil.     

高效石油降解菌群的构建及对芳香烃化合物萘的协同降解性能

【背景】石油作为一类混杂有机化合物,一旦产生污染就会对人类和环境造成严重的危害。【目的】从新疆石油污染土壤中分离筛选石油降解菌,为石油污染土壤的生物修复提供数据支持及技术参考。【方法】以石油为唯一碳源,通过富集培养、筛选分离得到123株单菌,根据菌落形态挑选出30个不同形态菌株,通过16S rRNA基因序列确定其种属,构建系统发育树;通过原油降解实验筛选出高效石油降解菌,以芳香烃的标志化合物萘为唯一碳源筛选出高效降解菌株,并分别筛选可降解水杨酸、邻苯二酚的菌株。【结果】分离筛选出5株高效石油降解菌,降解率高于85%;萘、水杨酸和邻苯二酚降解菌株各获得一株,将3种菌株按照1:1:1的接种比例对萘进行降解,萘的降解率从单菌60.74%提升到89.40%,菌株间的分工协作可以提高有机物的降解效率。【结论】筛选得到的菌株丰富了石油降解微生物菌种库,不同微生物菌株之间的分工协作为石油污染物的降解提供了新思路,为进一步研究石油污染治理提供参考。     

Isolation and Identification of hydrocarbon degrading bacteria from Ennore creek

The widespread problem caused due to petroleum products, is their discharge and accidental spillage in marine environment proving to be hazardous to the surroundings as well as life forms. Thus remediation of these hydrocarbons by natural decontamination process is of utmost importance. Bioremediation is a non-invasive and cost effective technique for the clean-up of these petroleum hydrocarbons. In this study we have investigated the ability of microorganisms present in the sediment sample to degrade these hydrocarbons, crude oil in particular, so that contaminated soils and water can be treated using microbes. Sediments samples were collected once in a month for a period of twelve months from area surrounding Ennore creek and screened for hydrocarbon degrading bacteria. Of the 113 crude oil degrading isolates 15 isolates were selected and cultivated in BH media with 1% crude oil as a sole carbon and energy source. 3 efficient crude oil bacterial isolates Bacillus subtilis I1, Pseudomonas aeruginosa I5 and Pseudomonas putida I8 were identified both biochemically and phylogenetically. The quantitative analysis of biodegradation is carried out gravimetrically and highest degradation rate, 55% was recorded by Pseudomonas aeruginosa I5 isolate.     

Removing of Crude Oil From Polluted Areas Using the Isolated Fungi From Tehran Oil Refinery

The pollution of soil and the subsurface environment by crude oil spill and petroleum products spill is a major concern around the world. The aim of this research was to investigate the ability of fungi isolated from Tehran oil refinery area in removing crude oil and to evaluate their enzymatic activities. Plant root samples were collected from the polluted and control areas, and rhizospheral fungi were isolated and determined using the laboratory methods and taxonomic keys. Seven fungal species were isolated and then cultured in potato dextrose agar (PDA) media containing 0–15% (v/v) crude oil. Oil removal was determined after a one-month growth of fungal colonies and then compared with the control media. The results showed that the studied fungi were able to remove crude oil from the media. The highest removal efficiency was observed in Aspergillus sp. Total protein content and enzymatic activity (of peroxidase and catalase) increased with increasing crude oil pollution. The highest enzymatic activity was evaluated in Aspergillus sp. growing in media containing 15% petroleum and the lowest activity was found in non-polluted groups. Results showed that there is a direct correlation between oil-removing potency and enzymatic activity. Aspergillus sp. showed the highest enzyme activity and also the highest petroleum removal efficiency.     

陕北地区石油污染土壤中不动杆菌属的筛选、鉴定及降解性能

从陕北地区石油污染土壤中分离鉴定得到两株不动杆菌属(Acinetobacter sp.)的高效石油降解菌A.sp 1和A.sp 2,分别从盐浓度、pH值、氮源、磷源和接种量等因素进行研究以确定其最佳石油降解条件,并进一步通过GC-MS(Gas ChromatographyMass Spectrometer)方法分析其在最佳条件下对原油组分的不同降解性能。结果显示:A.sp 1在盐浓度为1%、pH值为6—7、磷源为KH2PO4和K2HPO4、氮源为尿素和接种量为4%的条件下,最高降解率可达到60%。A.sp 2在盐浓度为1%、pH值为7—9、磷源为KH2PO4和K2HPO4、氮源为硝酸铵和接种量为8%的条件下,最高降解率可达到67%。GC-MS分析结果表明,菌株A.sp 1对石油烃类C21—C25有明显的降解效果,菌株A.sp 2对石油烃类C20—C30的降解效果较好。     

高效降解石油细菌的分离鉴定及降解能力的研究

目的:为获得高效降解原油的菌株,从石油污染严重的土壤中采样,富集分离得到原油降解菌,并初步考察它们降解原油的能力。方法:通过富集培养、多次筛选分离得到三株优势菌,编号为SWH-1、SWH-2和SWH-3。通过16S rDNA序列分析和NCBI数据库的Blast比对分析,对其鉴定到种。通过差量法测定它们在室内摇瓶中对原油的降解率。结果:经鉴定,这三株菌分别为枯草芽孢杆菌(Bacillus subtillus)、多食鞘氨醇杆菌(Sphingobacterium multivorum)和嗜温鞘氨醇杆菌(Sphingobacterium thalpophi-lum)。在0.5g/L的原油培养基内培养1w,SWH-1和SWH-2的降解率较高,分别为33.89%和46.31%。将这两株菌进行混合培养降解原油,降解率高达51.73%。结论:所筛选到的枯草芽孢杆菌和多食鞘氨醇杆菌在生物修复方面具有很好的应用潜力,而且多食鞘氨醇杆菌在石油降解方面的报道尚属首次。     

The properties of hydrocarbon-oxidizing bacteria isolated from the oilfields of Tatarstan, Western Siberia, and Vietnam

Eleven strains of hydrocarbon-oxidizing bacteria, isolated from oilfields, representing the genera Rhodococcus, Gordonia, Dietzia, and Pseudomonas, were characterized as mesophiles and neutrophiles. Rhodococci were halotolerant microorganisms growing in a media containing up to 15% NaCl. All the strains oxidized n-alkanes of crude oil. An influence of the cultivation temperatures (28 or 45 degrees C) and organic supplements on the degradation of C12-C30 n-alkanes in oxidized oil by two bacterial strains of the genus Pseudomonas was shown. The introduction of acetate, propionate, butyrate, ethanol, and sucrose led mainly to the decreased oxidation of petroleum paraffins. At certain cultivation temperatures, the addition of volatile fatty acid salts increased the content of individual n-alkanes in oxidized vs. crude oil.     

A Comparative Study on Biosurfactant Activity of Crude Oil–Degrading Bacteria and Its Correlation to Total Petroleum Hydrocarbon Degradation

In this study, 11 bacteria isolated from Tapis crude oil–contaminated sites were identified by using biochemical tests and 16S rDNA gene sequencing. Their abilities to biodegrade Tapis crude oil was determined by gas chromatography before they were further screened for biosurfactant activity by employing qualitative (blood agar hemolysis, microplate assay, drop-collapse test), semiquantitative (emulsification formation), and quantitative (surface tension measurement) methods. Four isolates, namely, Acinetobacter baumanii UKMP-12T, Pseudomonas aeruginosa UKMP-14T, Rhodococcus sp. UKMP-5T, and Rhodococcus sp. UKMP-7T, exhibited high percentages in total petroleum hydrocarbon (TPH) degradation. A strong correlation between the emulsification index (E ₂₄) and surface tension measurement (r _s = +.866) as shown by Spearman rank correlation analysis suggested that these two methods were more reliable to predict biosurfactant activity. The TPH removal was also positively correlated to the ability of bacterial isolates to reduce the surface tension of growth medium, as revealed by Pearson correlation test (r_p = +.886). In conclusion, not all the biosurfactant detection protocols employed were effective. Nevertheless, the measurement of surface tension and E ₂₄ determination provided a rather rapid, easy, reproducible, and accurate result in identifying bacteria with biosurfactant-producing ability.     

Isolation and characterization of long-chain alkane–degrading Acinetobacter sp. BT1A from oil-contaminated soil in Diyarbakır,in the Southeast of Turkey

A strain of long-chain alkane–degrading bacteria, BT1A, was isolated from oil-contaminated soil in Diyarbak?r, in the southeast of Turkey. Morphological, biochemical, and physiological characterization and 16S rRNA gene sequence analysis showed that the strain BT1A was a member of Acinetobacter genus, and it was found to be closely related to Acinetobacter baumannii. The strain BT1A was able to utilize crude petroleum as carbon and energy sources in order to grow. Among the aliphatic hydrocarbons, growth was observed only in the medium containing long-chain alkanes (tridecane, pentadecane, and hexadecane) and squalene. Hexadecane was the most preferred hydrocarbon among the long-chain alkanes. Gas chromatography–mass spectrometry (GC-MS) analysis showed that BT1A degraded 83% of n-alkanes of 1% crude oil in 7 days. The present study indicates that the isolated strain can well be used for biodegradation of hydrocarbons in oil-contaminated sites.     

Isolation of Bacteria Degrading Carbazole under Microaerobic Conditions,i.e. Nitrogen Gas Substituted Conditions

Microorganisms degrading carbazole (CA), a model substrate of heterocyclic nitrogen compounds in crude petroleum oil, were screened under microaerobic conditions, i.e. nitrogen gas substituted conditions. Eight bacteria were isolated and identified. For example, Bacillus sp. KUKK-4 degraded 31 % of CA when cultivated for 28 days in a medium initially containing CA at 1000 mg/l with shaking under the micro aerobic conditions.     

Bioremediation of oil by marine microbial mats

Cyanobacterial mats developing in oil-contaminated sabkhas along the African coasts of the Gulf of Suez and in the pristine Solar Lake, Sinai, were collected for laboratory studies. Samples of both mats showed efficient degradation of crude oil in the light, followed by development of an intense bloom of Phormidium spp. and Oscillatoria spp. Isolated cyanobacterial strains, however, did not degrade crude oil in axenic cultures. Strains of sulfate-reducing bacteria and aerobic heterotrophs were capable of degrading model compounds of aliphatic and aromatic hydrocarbons. Results indicate that degradation of oil was done primarily by aerobic heterotrophic bacteria. The oxygenic photosynthesis of oil-insensitive cyanobacteria supplied the molecular oxygen for the efficient aerobic metabolism of organisms, such as Marinobacter sp. The diurnal shifts in environmental conditions at the mat surface, from highly oxic conditions in the light to anaerobic sulfide-rich habitat in the dark, may allow the combined aerobic and anaerobic degradation of crude oil at the mat surface. Hence, coastal cyanobacterial mats may be used for the degradation of coastline oil spills. Oxygen microelectrodes detected a significant inhibition of photosynthetic activity subsequent to oil addition. This prevailed for a few hours and then rapidly recovered. In addition, shifts in bacterial community structure following exposure to oil were determined by denaturing gradient gel electrophoresis of PCR-amplified fractions of 16S rRNA from eubacteria, cyanobacteria and sulfate-reducing bacteria. Since the mats used for the present study were obtained from oil-contaminated environments, they were believed to be preequilibrated for petroleum remediation. The mesocosm system at Eilat provided a unique opportunity to study petroleum degradation by mats formed under different salinities (up to 21%). These mats, dominated by cyanobacteria, can serve as close analogues to the sabkhas contaminated during the Gulf War in Kuwait and Saudi Arabia. Electronic Publication     

Development of a Microbial Consortium from a Contaminated Soil That Degrades Pentachlorophenol and Wood-Preserving Oil

An indigenous microbial consortium capable of degrading pentachlorophenol (PCP) and petroleum hydrocarbons (C₁₀-C₅₀) was produced from a soil contaminated with wood-preserving oil. Two 10-L stainless steel soil slurry (10% w/v) bioreactors were operated in fed-batch mode. To verify the growth and efficiency of PCP degraders in the presence of other contaminants, one bioreactor was fed with a PCP-based wood-preserving mixture (WPM) and a second reactor was fed with technical-grade NaPCP. During the 90-day period of activation, PCP, C₁₀-C₅₀, Cl^-, pH, and dissolved oxygen levels were monitored. The microbial community was monitored using specific most probably number (MPN) bacterial counts and mineralization tests. PCP degradation rates increased similarly in both reactors, from 19 to 132 mg/L-d in the NaPCP reactor, and from 41 to 112 mg/L-d in the WPM reactor. Contaminant loss calculations showed that 99.5% of PCP and 92.5% of C₁₀-C₅₀ added to the WPM reactor were biodegraded. It also revealed that 83% of polychlorinated dioxins and furans were removed. PCP-degrading bacteria increased from 7×10² to 1.6×10⁶ bacteria/mL in both reactors, and petroleum hydrocarbon degraders increased from 1.7×102 to 3.4×108 bacteria/mL in the WPM reactor, indicating that the activity of PCP degraders was not inhibited by the presence of microorganisms growing on petroleum hydrocarbons.     

Bioremediation potential of hydrocarbon degrading bacteria: isolation,characterization, and assessment

Oil contamination is a worldwide concern now. However, oil contaminated environment is enriched with microorganisms that can utilize petroleum oil and use hydrocarbon for their growth, nutrition and metabolic activities. In the present study, bacteria present in the oil contaminated soil were isolated by enrichment culture technique using Minimal Salt (MS) media supplemented with diesel oil and burned engine oil as a sole carbon source. The isolated bacteria were characterized by morphological and biochemical tests and identified by molecular tool through cycle sequencing method. Three isolates were morphologically characterized as gram-negative, cocci shaped and 16S rRNA sequence analysis revealed that the isolates are closely related to Pseudomonas sp., Acinetobacter sp., and Enterobacter sp. respectively. Growth condition was optimized at pH 7.0 and temperature 37 °C. All the isolates were susceptible to several antibiotics and they have no antagonistic effect with soil beneficial bacteria. Three isolates were grown in two different concentrations of diesel oil and burned engine oil (4% v/v and 8%, v/v) respectively. Study revealed that with increasing the concentration of diesel oil in the media the growth rate of all the isolates were decreased. In contrast, the growth rates of all the three isolates were increased, with increasing concentration of burned engine oil. In our study, all the isolates showed their degradation efficacy in 4% v/v diesel oil and in 8% v/v burned engine oil. So, our research clearly shows that the isolates could be potentially used for bioremediation purposes for cleaning up petroleum polluted area.     

Biodegradation of petroleum hydrocarbons by filamentous fungi (Aspergillus ustus and Purpureocillium lilacinum) isolated from used engine oil contaminated soil

The use of microorganisms for remediation and restoration of hydrocarbons contaminated soils is an effective and economic solution. The current study aims to find out efficient telluric filamentous fungi to degrade petroleum hydrocarbons pollutants. Six fungal strains were isolated from used engine (UE) oil contaminated soil. Fungi were screened for their ability to degrade crude oil, diesel and UE oil using 2.6-dichlorophenol indophenol (DCPIP). Two isolates were selected, identified and registered at NCBI as Aspergillus ustus HM3.aaa and Purpureocillium lilacinum HM4.aaa. Fungi were tested for their tolerance to different concentration of petroleum oils using radial growth diameter assay. Hydrocarbons removal percentage was evaluated gravimetrically. The degradation kinetic of crude oil was studied at a time interval of 10 days. A.ustus was the most tolerant fungi to high concentration of petroleum oils in solid medium. Quantitative analysis showed that crude oil was the most degraded oil by both isolate; P. lilacinium and A. ustus removed 44.55% and 30.43% of crude oil, respectively. The two fungi were able to degrade, respectively, 27.66 and 21.27% of diesel and 14.39 and 16.00% of UE oil. As compared to the controls, these fungi accumulated high biomass in liquid medium with all petroleum oils. Likewise, crude oil removal rate constant (K) and half-lives (t_1/2) were 0.02 day⁻¹, 34.66 day and 0.015 day⁻¹, 46.21 day for P. lilacinium and A. ustus, respectively. The selected fungi appear interesting for petroleum oils biodegradation and their application for soil bioremediation require scale-up studies.     

The properties of hydrocarbon-oxidizing bacteria isolated from the oilfields of Tatarstan, western Siberia, and Vietnam

Eleven strains of hydrocarbon-oxidizing bacteria, isolated from oilfields and representing the genera Rhodococcus, Gordonia, Dietzia, and Pseudomonas, were characterized as mesophiles and neutrophiles. Rhodococci were halotolerant microorganisms growing in a media containing up to 15% NaCl. All the strains oxidized n-alkanes of crude oil. An influence of the cultivation temperatures (28 or 45°C) and organic supplements on the degradation of C₁₂-C₃₀ n-alkanes in oxidized oil by two bacterial strains of the genus Pseudomonas was shown. The introduction of acetate, propionate, butyrate, ethanol, and sucrose led mainly to decreased oxidation of petroleum paraffins. At certain cultivation temperatures, the addition of volatile fatty acid salts increased the content of certain n-alkanes in oxidized oil as compared to crude oil.     

石油降解细菌的表型特性和系统发育分析

从3种不同土壤中分离和纯化得到10个石油降解细菌菌株,分离菌株均为好氧菌、革兰氏阴性菌、不形成芽孢的杆菌,10个菌株均能利用中等链长的烷烃、柴油和原油作为碳源,而不能以单环芳烃和多环芳烃为碳源。根据其生理生化性状和16SrDNA序列分析结果表明,分离菌株EVA5,EVA6,EVA7,EVA8和EVA9为假单胞菌（Pseudomonas spp.）,EVA10、EVA11、EVA12、EVA13和EVA14为不动杆菌（Acinetobacter spp.）,均属于Proteobac-teria的γ亚群。     

Thermotolerant oil-degrading bacteria isolated from soil and water of geographically distant regions

Oil-degrading bacteria were isolated from soil and water samples taken in Russia, Kazakhstan, and the Antarctic; 13 of 86 strains proved to be thermotolerant. These bacteria utilized crude oil at 45–50°C; their growth optimum (35–37°C) and range (20–53°C) differ from those of mesophilic bacteria. Thermotolerant strains were identified as representatives of the genera Rhodococcus and Gordonia. It was shown that their ability to degrade petroleum products does not differ at 24 and 45°C. The strains Rhodococcus sp. Par7 and Gordonia sp. 1D utilized 14 and 20% of the oil, respectively, in 14 days at 45°C. All of the isolated thermotolerant bacteria grew in a medium containing 3% NaCl; the medium for the strains Gordonia amicalis 1B and Gordonia sp. 1D contained up to 10% NaCl. The bacteria G. amicalis and Rhodococcus erythropolis were able to utilize crude oil and individual hydrocarbons at higher (up to 50°C) temperatures.     

胜利油田滩涂区石油降解菌的筛选、鉴定及其多样性分析

基于传统的实验方法,对胜利油田滩涂区土壤中石油降解菌进行了筛选和鉴定,并利用PCR-DGGE (变性梯度凝胶电泳)技术分析了胜利油田滩涂区的菌群多样性.结果表明：由研究区土壤中筛选出13株石油降解菌,其中,6株石油降解菌的石油降解率>90%,能降解大部分C₁₂～C₂₆的石油烷烃,系统发育学鉴定为Alcanivorax、Halomonas和Marinobacter,均属于γ-proteobacteria分支;胜利油田滩涂区未培养优势菌有Sulfurovum、Gillisia和Arcobacter;该区优势菌群中γ-proteobacteria所占比重较大,其次为α-proteobactiria、ε-proteobactiria、Actinobacteria 和Flavobacteria.     

Enumeration of petroleum-degrading microorganisms.

A variety of factors, including concentration of oil, antibiotics, dyes, and inoculum washes, were examined to determine their effect on the total counts of microorganisms on oil-containing media. The media found to be best for enumerating petroleum-degrading microorganisms contained 0.5% (vol/vol) oil and 0.003% phenol red, with Fungizone added for isolating bacteria and streptomycin and tetracycline added for isolating yeasts and fungi. Washing the inoculum did not improve recovery of petroleum degraders. Specifically, silica gel-oil medium and a yeast medium are recommended for enumeration of petroleum-degrading bacteria and yeasts and fungi, respectively. It is suggested that counts of petroleum degraders be expressed as percentage of the total population rather than total numbers of petroleum degraders per se. Incubation temperature and presence of oil was found to influence the numbers of petroleum-degrading microorganisms at a given sampling site.     

Biotechnological Potential of Bacillus salmalaya 139SI: A Novel Strain for Remediating Water Polluted with Crude Oil Waste

Environmental contamination by petroleum hydrocarbons, mainly crude oil waste from refineries, is becoming prevalent worldwide. This study investigates the bioremediation of water contaminated with crude oil waste. Bacillus salamalaya 139SI, a bacterium isolated from a private farm soil in the Kuala Selangor in Malaysia, was found to be a potential degrader of crude oil waste. When a microbial population of 10⁸ CFU ml^-1 was used, the 139SI strain degraded 79% and 88% of the total petroleum hydrocarbons after 42 days of incubation in mineral salt media containing 2% and 1% of crude oil waste, respectively, under optimum conditions. In the uninoculated medium containing 1% crude oil waste, 6% was degraded. Relative to the control, the degradation was significantly greater when a bacteria count of 99 × 10⁸ CFU ml^-1 was added to the treatments polluted with 1% oil. Thus, this isolated strain is useful for enhancing the biotreatment of oil in wastewater.