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%).     

石油污染土壤的生物修复技术

1　前　言在石油生产、贮运、炼制加工及使用过程中 ,由于事故 ,不正常操作及检修等原因 ,都会有石油烃类的溢出和排放。例如 ,油田开发过程中的井喷事故 ;输油管线和贮油罐的泄漏事故 ;油槽车和油轮的泄漏事故 ;油井清蜡和油田地面设备检修 ;炼油和石油化工生产装置检修等。石油烃类大量溢出 ,应当尽可能予以回收 ,但有的情况下回收很困难 ,即使尽力回收 ,仍会残留一部分 ,对环境 (土壤、地面和地下水 )造成污染。其进入土壤后 ,会破坏土壤结构 ,分散土粒 ,使土壤的透水性降低。其富含的反应基能与无机氮、磷结合并限制硝化作用和脱磷酸作…     

Enhanced Biodegradation of Petroleum Hydrocarbons in Contaminated Soil

Soil samples taken from a contaminated site in Northern Quebec, Canada, exhibited a low capacity for biodegradation of total petroleum hydrocarbons (TPH), despite a high capacity for the mineralization of aromatic hydrocarbons and a low toxicity of soil leachates as measured by Microtox assay. Toxicity assays directly performed on surface soil, including earthworm mortality and barley seedling emergence, indicated moderate to high levels of toxicity. Soil biostimulation did not improve the removal of petroleum hydrocarbons, while bioaugmentation of soil with a developed enrichment culture increased the efficiency of hydrocarbon removal from 20.4% to 49.2%. A considerable increase in the removal of TPH was obtained in a bioslurry process, enhancing the mass transfer of hydrocarbons from soil to the aqueous phase and increasing the efficiency of hydrocarbon removal to over 70% after 45 days of incubation. The addition of ionic or nonionic surfactants did not have a significant impact on biodegradation of hydrocarbons. The extent of hydrocarbon mineralization during the bioslurry process after 45 days of incubation ranged from 41.3% to 58.9%, indicating that 62.7% to 83.1% of the eliminated TPH were transformed into CO₂ and water.     

Enhanced Biodegradation of Petroleum Hydrocarbons in Contaminated Soil

Soil samples taken from a contaminated site in Northern Quebec, Canada, exhibited a low capacity for biodegradation of total petroleum hydrocarbons (TPH), despite a high capacity for the mineralization of aromatic hydrocarbons and a low toxicity of soil leachates as measured by Microtox assay. Toxicity assays directly performed on surface soil, including earthworm mortality and barley seedling emergence, indicated moderate to high levels of toxicity. Soil biostimulation did not improve the removal of petroleum hydrocarbons, while bioaugmentation of soil with a developed enrichment culture increased the efficiency of hydrocarbon removal from 20.4% to 49.2%. A considerable increase in the removal of TPH was obtained in a bioslurry process, enhancing the mass transfer of hydrocarbons from soil to the aqueous phase and increasing the efficiency of hydrocarbon removal to over 70% after 45 days of incubation. The addition of ionic or nonionic surfactants did not have a significant impact on biodegradation of hydrocarbons. The extent of hydrocarbon mineralization during the bioslurry process after 45 days of incubation ranged from 41.3% to 58.9%, indicating that 62.7% to 83.1% of the eliminated TPH were transformed into CO₂ and water.     

Importance of Environmental Black Carbon to Dissolved Petroleum Hydrocarbons Sorption on Soil

Importance of environmental black carbon (BC) to sorption of dissolved petroleum hydrocarbons (DPH) on two soils with high BC:TOC ratios (33% and 11%, respectively) was evaluated at a relatively high concentrations (mg/L ～ μ g/L range). Sorption isotherms of DPH were determined for the two original soils and soils combusted at 375°C (only BC). The sorption isotherms of the original soils were linear, whereas the isotherms of the combusted soils were highly nonlinear (n _F = 0.45, 0.60). It is indicated that intrinsic BC-water sorption coefficient is not possible to be used to estimate total sorption to the original soil, even in our relatively high concentrations. From the sorption isotherms, Freundlich coefficient of environmental BC sorption, K _F,BC ^env of 10 ^{2.55 ± 0.21} was calculated and could be used as a generic starting point for environmental modeling purposes. From the data, it could be deduced that BC was responsible for 50% of the total sorption at concentrations of 45 and 4 μ g/L (μ g/L range), which were significantly higher than literature concentrations (ng/L range). These results demonstrate that in soil with high BC:TOC ratio BC is the most important geosorbent constituent with respect to sorption of DPH at relatively high concentrations ranged in μ g/L.     

Co-Composting of Residual Fuel Contamination in Soil

A remediation program was designed and implemented at a site in southeastern Australia that had become contaminated with nonvolatile, n-alkane total petroleum hydrocarbons (TPH). The remediation was conducted in two stages. The excavation, validation and reinstatement of two contaminated areas on the site was first conducted, followed by development of a composting treatment process. The total volume of contaminated soil (i. e., TPH concentration >1000?mg/kg C₁₀?C₃₆) was ～4300?m³ with a concentration of 3100±1270?mg/kg. The soil was stockpiled into four windrows, on a compacted, bunded clay base. Approximately 35% (v/v) of raw materials (green tree waste, cow manure, gypsum, and nutrients) were added to initiate composting. The piles were kept moist during the summer months, but no other maintenance was conducted. Once the composting process was initiated, the windrows were sampled at 2 and 6 months. After 6 months treatment, the average TPH concentration (C₁₀?C₃₆) was 730?mg/kg (with a 95% CI of 1020?mg/kg), which met the relevant clean fill criteria applicable to the site. There were no other contaminants of significance in the treated soil compost and it posed no unacceptable risk to human health or the environment, allowing it to be used as fill at the site.     

Biodegradation of Petroleum Hydrocarbons in a Soil Polluted Sample by Oil-Based Drilling Cuttings

Microbial degradation of hydrocarbons in soils polluted by oil-based drilling mud and cuttings has been investigated by static methods such as composting or biopiling. Bioremediation of polluted soils by oil-based drilling cuttings through a slurry bioreactor has not previously been reported. The main aim of this work is to monitor hydrocarbon biodegradation in slurry of drilling cuttings and unpolluted soils and the effects of nutrients on it. Indigenous, bacterial-mixed culture isolated from a polluted soil by drilling cuttings adapted to drilling mud concentrations up to 15% (v/v) was done during a 15-month program. The total petroleum hydrocarbons’ (TPHs) removal efficiency in C/N/P 100/5/1 ratio was 90.5 and 79.85% under experimental and control conditions, respectively. The microbial count on the first day, 15 × 10⁷ CFUg^?1, reached 20 × 10⁹ CFUg^?1on the twenty-first day at experimental conditions. The TPH removal efficiency in C/N/P 100/10/2 was 92.5 and 82.25% at experiment and control, respectively. Increasing nitrogen and phosphorous amount couldn't increase microbial count in comparison with C/N/P ratio 100/5/1. The measured biomass contents and microbial counts in experiments were significantly higher than the control and confirmed hydrocarbons’ biodegradation during the time. Results showed that slurry bioreactors could accelerate the biodegradation of TPHs and reduce remediation time in soil polluted by oil-based drilling cuttings.     

Potential for Aerobic and Anaerobic Biodegradation of Petroleum Hydrocarbons in Boreal Subsurface

We studied the role of aerobic and anaerobic petroleum hydrocarbon degradation at a boreal, light-weight fuel and lubrication oil contaminated site undergoing natural attenuation. At the site, anoxic conditions prevailed with high concentrations of CH4 (up to 25% v/v) and CO2 (up to 18% v/v) in the soil gas throughout the year. Subsurface samples were obtained mainly from the anoxic parts of the site and they represented both the unsaturated and saturated zone. The samples were incubated in microcosms at near in situ conditions (i.e. in situ temperature 8 degrees C, aerobic and anaerobic conditions, no nutrient amendments) resulting in the removal of mineral oil (as determined by gas chromatography) aerobically as well as anaerobically. In the aerobic microcosms on average 31% and 27% of the initial mineral oil was removed during a 3- and 4-month incubation, respectively. In the anaerobic microcosms, on average 44% and 15% of the initial mineral oil was removed during a 12- and 10-month anaerobic incubation, respectively, and e.g. n-alkanes from C11 to C15 were removed. A methane production rate of up to 2.5 microg CH4 h(-1) g(-1) dwt was recorded in these microcosms. In the aerobic as well as anaerobic microcosms, typically 90% of the mineral oil degraded belonged to the mineral oil fraction that eluted from the gas chromatograph after C10 and before C15, while 10% belonged to the fraction that eluted after C15 and before C40. Our results suggest that anaerobic petroleum hydrocarbon degradation, including n-alkane degradation, under methanogenic conditions plays a significant role in the natural attenuation in boreal conditions.     

Biotransformation and Dissolution of Petroleum Hydrocarbons in Natural Flowing Seawater at Low Temperature

The objective of this study was to establish methods for controlled studies of hydrocarbon depletion from thin oil films in cold natural seawater, and to determine biotransformation in relation to other essential depletion processes. Mineral oil was immobilized on the surface of hydrophobic Fluortex fabrics and used for studies of microbial biodegradation in an experimental seawater flow-through system at low temperatures (5.9-7.4 degrees C) during a test period of 42 days. The seawater was collected from a depth of 90 m, and microbial characterization by epifluorescence microscopy, fluorescence in situ hybridization, and most-probable number analysis showed relatively larger fractions of archaea and oil-degrading microbes than in the corresponding surface water. Chemical analysis of hydrocarbons attached to the fabrics during the test period showed that n-alkanes (C10-C36) were decreased by 98% after 21 days, while naphthalenes were depleted by 99-100% during the same period. At the end of the period 4-5 ring polyaromatic hydrocarbon (PAH) compounds were removed by 82% from the fabrics. Analysis of the recalcitrant pentacyclic triterpane C30 17alpha(H),21beta(H)-hopane showed that the oil remained adsorbed to the fabrics during the test period. Comparison of depletion analysis with calculation of hydrocarbon dissolution in a flow-through system indicated that naphthalenes and smaller PAH compounds (alkylated 2-ring and 3-ring compounds) were removed from the fabrics by dissolution. The data further implied that depletion of n-alkanes and 4-5 ring PAH hydrocarbons were the result of biotransformation processes. PCR amplification of bacterial 16S rRNA genes from microbes adhering on the immobilized oil surfaces showed the dominance of a few bands when analysed in denaturing gradient gel electrophoresis (DGGE). Sequence analysis of DGGE bands revealed phylogenetic affiliation to the alpha- and gamma-subdivisions of proteobacteria and to the Chloroflexus-Flavobacterium-Bacteroides group.     

Field Note: Phytoremediation of Petroleum Sludge Contaminated Field Using Sedge Species,Cyperus Rotundus (Linn.) and Cyperus Brevifolius (Rottb.) Hassk

The aim of this study was to degrade total petroleum hydrocarbon (TPH) in a petroleum sludge contaminated site (initial TPH concentration of 65,000–75,000 mg.kg^–1) with two native sedge species namely Cyperus rotundus (Linn.) and Cyperus brevifolius (Rottb.) Hassk. Fertilized and unfertilized treatments were maintained separately to record the influence of fertilizer in TPH degradation. The average biomass production (twenty plants from each treatment) of C. rotundus was 345.5 g and that of C. brevifolius was 250.6 g in fertilized soil during 360 days. Decrease in soil TPH concentration was higher in fertilized soil (75% for C. rotundus and 64% for C. brevifolius) than in unfertilized soil (36% for C. rotundus and 32% for C. brevifolius). In unvegetated treatments, decrease in soil TPH concentration in fertilized (12%) and unfertilized soil (8%) can be attributed to natural attenuation and microbial degradation. TPH accumulation in roots and shoots was significantly higher in fertilized soil in comparison to unfertilized soils (p < 0.05). Most probable number (MPN) in planted treatments was significantly higher than in unplanted treatments (p < 0.05).     

工业化废水处理反应器污泥总DNA提取方法

根据工业化废水处理反应器污泥特性,对常规的溶菌酶-SDS-酚/氯仿环境样品总DNA提取方法进行改进,增强样品预处理,强化细胞裂解,提高杂质去除效率,获得了一种工业化污泥总DNA提取的通用方法,并采用该方法对石家庄若干实际运行的工业化厌氧、好氧反应器的污泥样品进行了总DNA提取研究.结果表明,该方法对所选污泥样品均有效,具有普适性.提取的污泥总DNA杂质含量少,纯度高,A260/A280在1.8左右;提取效率较高.总DNA产率都在0.7 mg/g以上,最大产率可达0.85 mg/g.所提取的污泥总DNA可以直接作为模板进行PCR反应,PCR产物直接进行变性梯度凝胶电泳(DGGE),能够得到较好的DGGE谱图,表明该方法提取的污泥总DNA样品可满足后续分析研究的要求.     

Distribution of Petroleum and Aromatic Hydrocarbons at a Former Crude Oil and Natural Gas Production Facility

Site characterization and remediation activities were performed at a former crude oil and natural gas production facility prior to redevelopment of the site. Field activities included delineation, excavation and segregation of approximately 1,250,000 m³ of soil impacted by total petroleum hydrocarbons (TPH) and the aromatic volatile organic compounds (VOCs) benzene, toluene, ethylbenzene, and xylenes (hereafter, collectively referred to as BTEX). Petroleum hydrocarbon chain length information was used to determine whether remediation was required in impacted areas, because the site-specific cleanup values for TPH compounds, established by the California State Regional Water Quality Control Board (RWQCB), were based on hydrocarbon chain length. Site-specific cleanup levels were also established by the RWQCB for BTEX. Subsurface investigation activities performed at the site indicated that the mean percentage of condensate and TPH compounds in the gasoline range was significantly greater at depths ranging from 4.6 to 18 m than in shallower samples. There was no significant difference in the mean concentration of BTEX compounds and mean percentage of diesel range and heavier hydrocarbons with depth. The occurrence of BTEX, diesel range, and heavier hydrocarbons at depth may result from preferential pathways for downward migration of contaminants, including blown out wells, abandoned wellbores, and the presence of faults. Vapor phase diffusion may also be a major transport mechanism controlling movement of BTEX compounds beneath the site.     

Anaerobic Hydrogen Production from Sucrose Using an Acid‐Enriched Sewage Sludge Microflora

A novel and high‐rate anaerobic sequencing bath reactor (ASBR) process was used to evaluate the hydrogen productivity of an acid‐enriched sewage sludge microflora at a temperature of 35 °C. In this ASBR process a 4 h cycle, including feed, reaction, settle, and decant steps, was repeatedly performed in a 5 L reactor. The sucrose substrate concentration was 20 g COD/L; the hydraulic retention time (HRT) was maintained at 12–120 h at the initial period and thereafter at 4–12 h. The reaction/settle period ratio, which is the most important parameter for ASBR operation was 1.7. The experimental results indicated that the hydrogenic activity of the sludge microflora was HRT‐dependent and that proper pH control was necessary for a stable operation of the bioreactor. The peak hydrogenic activity value was attained at an HRT of 8 h and an organic loading rate of 80 kg COD/m³ × day. Each mole of sucrose in the reactor produced 2.8 mol of hydrogen and each gram of biomass produced 39 mmol of hydrogen per day. An overly‐short HRT might deteriorate the hydrogen productivity. The concentration ratios of butyric acid to’acetic acid, as well as volatile fatty acid and soluble microbial products to alkalinity can be used as monitoring indicators for the hydrogenic bioreactor.     

Human Risk Assessment of Contaminated Soils by Oil Products: Total TPH Content Versus Fraction Approach

Petroleum hydrocarbons may cause risks for humans and the environment that must be properly managed. Some methodologies cluster hundreds of hydrocarbon substances into one single parameter, total petroleum hydrocarbon (TPH) ranged from C10 to C40. Several national policies establish a maximum acceptable concentration in soil to directly consider if a site is seriously contaminated; this scope may be described as a total content approach. Another approach considers TPH division into fractions according to their physico-chemical and toxicological properties, performed in terms of the environmental behavior (aliphatic and aromatic compounds) and the equivalent carbon number (EC). This approach lets us determine the associated risk for human health through the Human Risk Index (HRI). The consequences of application of the total content and fraction approaches is discussed in this study, evaluating the differences in the approach for volatile and semi-volatile hydrocarbons and also in regard to the origin of the contamination. When focusing on volatile substances, the fraction approach is much more restrictive than the total content approach where all oil products are assessed in the same way. When assessing semi-volatile hydrocarbons, their behavior varies depending on the oil product. This work contributes to the implementation of risk-based assessment for petroleum hydrocarbons.     

An Investigation of the Ability of a 14C-Labeled Hydrocarbon Mineralization Test to Predict Bioremediation of Soils Contaminated with Petroleum Hydrocarbons

Bioremediation is a widely accepted technology for the remediation of hydrocarbon-contaminated soil. Treatability studies are usually carried out to assess the biodegradation potential of the contaminants and to design optimal treatments. Laboratory studies measuring soil respiration are often used. One method consists of monitoring the mineralization of a ¹⁴C-labeled hydrocarbon surrogate added to the contaminated soil. This study investigates the ability of this method to properly predict the removal of the hydrocarbon contaminants initially found in soils. Mineralization of ¹⁴C-labeled hexadecane was monitored in seven soils contaminated with various hydrocarbon mixtures, both fresh and weathered, in microcosm experiments. Reduction of total petroleum hydrocarbon (TPH) concentrations was measured simultaneously in separate microcosms. Both types of microcosms were subjected to the same amendment regimes. For all soils, poor correlation was observed between the mineralization and TPH reduction data sets. Mineralization data supported contaminants removal data in only one soil. Findings indicate that the radioactive surrogate method does not reliably predict the extent of, and the effect of amendments on, the removal of the hydrocarbons initially present in soil, and may therefore predict suboptimal treatment regimes. Recommendations for soil treatability protocols are provided.     

Development of Sulfidogenic Sludge from Marine Sediments and Trichloroethylene Reduction in an Upflow Anaerobic Sludge Blanket Reactor

The importance of microbial sulfate reduction relies on the various applications that it offers in environmental biotechnology. Engineered sulfate reduction is used in industrial wastewater treatment to remove large concentrations of sulfate along with the chemical oxygen demand (COD) and heavy metals. The most common approach to the process is with anaerobic bioreactors in which sulfidogenic sludge is obtained through adaptation of predominantly methanogenic granular sludge to sulfidogenesis. This process may take a long time and does not always eliminate the competition for substrate due to the presence of methanogens in the sludge. In this work, we propose a novel approach to obtain sulfidogenic sludge in which hydrothermal vents sediments are the original source of microorganisms. The microbial community developed in the presence of sulfate and volatile fatty acids is wide enough to sustain sulfate reduction over a long period of time without exhibiting inhibition due to sulfide. This protocol describes the procedure to generate the sludge from the sediments in an upflow anaerobic sludge blanket (UASB) type of reactor. Furthermore, the protocol presents the procedure to demonstrate the capability of the sludge to remove by reductive dechlorination a model of a highly toxic organic pollutant such as trichloroethylene (TCE). The protocol is divided in three stages: (1) the formation of the sludge and the determination of its sulfate reducing activity in the UASB, (2) the experiment to remove the TCE by the sludge, and (3) the identification of microorganisms in the sludge after the TCE reduction. Although in this case the sediments were taken from a site located in Mexico, the generation of a sulfidogenic sludge by using this procedure may work if a different source of sediments is taken since marine sediments are a natural pool of microorganisms that may be enriched in sulfate reducing bacteria.     

Optimization of PAHs Oxidation from a Concentrate of Soil Attrition Using Potassium Permanganate

The treatment of soils contaminated with organic compounds, such as polycyclic aromatic hydrocarbons (PAHs), by attrition produced large amounts of highly concentrated attrition sludge (PAH – attrition concentrate – PAC). This paper studied the performance of an oxidation process using potassium permanganate (KMnO₄) to degrade PAHs that were initially present in attrition concentrates. The influence of operating conditions (temperature, concentration of KMnO₄ and reaction time) was studied, and these parameters were optimized using a response surface methodology (RSM). The results showed that the temperature and the reaction time had a significant and positive effect on the degradation of PAHs for the experimental domain studied (temperature between 20 and 60°C and reaction time between 1 and 7 h). The interaction between the temperature and the concentration of KMnO₄ significantly influenced the degradation of the PAHs. The temperature and the concentration of KMnO₄ were the main parameters that influenced the degradation of both phenanthrene (Phe) and benzo [a] pyrene (BaP). For benzo [a] anthracene (BaA), the temperature was the most influential factor. According to our results, the optimal conditions were defined as [KMnO₄] = 0.4 M for 5.5 h at 60°C. These optimal conditions led to degradations of 42.9%, 40.8%, 41.0% and 46.0% of the total PAHs, Phe, BaA and BaP, respectively.     

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.     

Investigating the Effects of Probiotics on Pneumococcal Colonization Using an In Vitro Adherence Assay

Adherence of Streptococcus pneumoniae (the pneumococcus) to the epithelial lining of the nasopharynx can result in colonization and is considered a prerequisite for pneumococcal infections such as pneumonia and otitis media. In vitro adherence assays can be used to study the attachment of pneumococci to epithelial cell monolayers and to investigate potential interventions, such as the use of probiotics, to inhibit pneumococcal colonization. The protocol described here is used to investigate the effects of the probiotic Streptococcus salivarius on the adherence of pneumococci to the human epithelial cell line CCL-23 (sometimes referred to as HEp-2 cells). The assay involves three main steps: 1) preparation of epithelial and bacterial cells, 2) addition of bacteria to epithelial cell monolayers, and 3) detection of adherent pneumococci by viable counts (serial dilution and plating) or quantitative real-time PCR (qPCR). This technique is relatively straightforward and does not require specialized equipment other than a tissue culture setup. The assay can be used to test other probiotic species and/or potential inhibitors of pneumococcal colonization and can be easily modified to address other scientific questions regarding pneumococcal adherence and invasion.     

Genotoxic evaluation of an industrial effluent from an oil refinery using plant and animal bioassays

Polycyclic aromatic hydrocarbons (PAHs) are genotoxic chemicals commonly found in effluents from oil refineries. Bioassays using plants and cells cultures can be employed for assessing environmental safety and potential genotoxicity. In this study, the genotoxic potential of an oil refinery effluent was analyzed by means of micronucleus (MN) testing of Alium cepa, which revealed no effect after 24 h of treatment. On the other hand, primary lesions in the DNA of rat (Rattus norvegicus) hepatoma cells (HTC) were observed through comet assaying after only 2 h of exposure. On considering the capacity to detect DNA damage of a different nature and of these cells to metabolize xenobiotics, we suggest the association of the two bioassays with these cell types, plant (Allium cepa) and mammal (HTC) cells, for more accurately assessing genotoxicity in environmental samples.