Optimization of soil physical and chemical conditions for the bioremediation of creosote-contaminated soil

Mispah type soil (FAO : Lithosol) contaminated with >250 000 mg kg^-1 creosote was collected from the yard of a creosote treatment plant. The soils carbon, nitrogen and phosphorus contents were determined. Due to creosote contamination, thecarbon content of the soil was found to be 130,000 mg C kg^-1. This concentration was found to greatly affect the nitrogen content (0.08%). The phosphorus content was less affected (4.5%). It was estimated that a nutrient amendment to bring the soil to a C : N 10 : 1 would be adequate to stimulate microbial growth and creosote degradation. The soil was amended with a range of C : N ratios below and above the estimated ratio. In one of the treatments, the phosphorus content was amended. Sterile and natural controls were also set up. The soil was incubated at 30 °C on a rotaryshaker at 150 rpm in the dark for six weeks. Water content was maintained at 70% field capacity. The lowest nitrogen supplementation (C : N = 25 : 1) was more effective in enhancing microbial growth (3.12E + 05) and creosote removal (68.7%) from the soil. Additional phosphorus was not very effective in enhancing the growth of microorganisms and removal of creosote. The highest nitrogen supplementation(C : N = 5 : 1) did not enhance microbial growth and creosote removal.A relationship between mass loss and creosote removal was also observed. Phenolics and lower molecular mass polycyclic aromatic hydrocarbons (PAHs) were observed to be more susceptible to microbial degradation than higher molecular mass compounds. Nutrient concentration, moisture content and pH were thus observed to play very significant roles in the utilization of creosote in soil. These results are being used for the development of a bioremediation technology for the remediation of creosote contaminated soils in a treatment plant in South Africa.     

Evaluation of a creosote-based medium for the growth and preparation of a PAH-degrading bacterial community for bioaugmentation

Creosote was evaluated as an inexpensive carbon source for growing inocula of a polycyclic aromatic hydrocarbon (PAH)-degrading bacterial community (community five). Creosote was a poor growth substrate when provided as sole carbon source in a basal salts solution (BSM). Alternatively, peptone, yeast extract or glucose in BSM supported high growth rates, but community five could not subsequently degrade pyrene. A combination of creosote and yeast extract in BSM (CYEM) supported growth and maintained the pyrene-degrading capacity of community five. Optimum pyrene-degrading activity occurred when the inocula were grown in creosote and yeast extract concentrations of 2 ml L⁻¹ and 1 g L⁻¹ respectively: concentrations outside these values resulted in either low biomass yields or loss of PAH-degrading activity. CYEM-grown community five inocula degraded 250 mg L⁻¹ of pyrene in BSM at a rate comparable to cultures inoculated with community five grown in BSM-pyrene. However, the CYEM-grown community showed a 40% lower rate of PAH degradation in a synthetic PAH mixture compared with pyrene-grown cells and there was an increase in the lag period before the onset of PAH degradation. This appears to reflect a weaker induction of PAH catabolism by CYEM compared to BSM-pyrene. Journal of Industrial Microbiology & Biotechnology (2000) 24, 277–284. Received 24 August 1999/ Accepted in revised form 20 January 2000     

Microbial community structure changes during bioremediation of PAHs in an aged coal-tar contaminated soil by in-vessel composting

The microbial community structure changes of an aged-coal-tar soil contaminated with polycyclic aromatic hydrocarbons (PAHs) were investigated during simulated bioremediation at the laboratory-scale using an in-vessel composting approach. The composting reactors were operated using a logistic three-factor factorial design with three temperatures (T=38, 55 or 70 °C), four soil to green-waste amendment ratios (S:GW=0.6:1, 0.7:1, 0.8:1 or 0.9:1 on a dry weight basis) and three moisture contents (MC=40%, 60% or 80%). Relative changes in microbial populations were investigated by following the dynamics of phospholipid fatty acid (PLFA) signatures using a ¹³C-labeled palmitic acid internal standard and sensitive GC/MS analysis during in-vessel composting over 98 days. The results of this investigation indicated that fungal to bacterial PLFA ratios were significantly influenced by temperature (p<0.05), and Gram-positive to Gram-negative bacterial ratios were significantly influenced by temperature (p<0.001) and S:GW ratio (p<0.01) during in-vessel composting. Additionally, the Gram-positive to Gram-negative bacterial ratios were correlated to the extent of PAH losses (p<0.005) at 70 °C.     

分枝杆菌TZh51菌株的分离鉴定及其生物修复污染土壤的特性

[目的]为获得降解芘的微生物菌株,并用其生物修复被多环芳烃污染的土壤.[方法]芘降解菌的分离采用平板升华法.根据表型观察、生理生化特性和16S rDNA的序列同源性分析,对菌株进行分类学鉴定.通过活菌计数、HPLC测定多环芳烃的残留量,研究菌株在固体、液体无机盐培养基以及在污染土壤中降解多环芳烃(polycyclic aromatic hydrocarbons,PAHs)的能力.[结果]分离到4株能降解芘的菌株TZh51、TZh52、TG42和TG52.实验结果表明,TZh51降解PAHs的能力强于其余3株菌.TZh51被鉴定为分枝杆菌属(Mycobacterium sp.),但与已发表的分枝杆菌菌株M11为不同的种.TZh51接种在芘膜的固体无机盐培养基上,测定获得最大芘降解量的条件是培养温度为3512和芘膜厚度为130 ng/mm2.在芘浓度为50、100 mg/L的液体无机盐培养基中培养,6天时TZh51的芘降解率分别达到91.9%、71.8%,10天时菌体数量分别达到最大值为2.0、6.0×108cfu/mL;TZh51降解芘的效果强于M11.在种植作物的处理中,到第6周时TZh51的菌体数量达到每克干土含7.2×108个菌落数,到第8周时菲、荧蒽和芘的降解率分别达到91.4%、86.9%和85.8%;[结论]TZh51具有很强降解PAHs的能力;另外,TZh51与作物联合生物修复污染土壤的效果明显.     

Bioremediation of creosote-contaminated soil in South Africa by landfarming

AIMS: To determine the combined effects of biostimulation and bioaugmentation in the landfarming of a mispah form (lithosol; food and Agriculture Organisation (FAO)) soil contaminated with >310000 mg kg-1 creosote with a view to developing a bioremediation technology for soils heavily contaminated with creosote. METHODS AND RESULTS: The excavated soil was mixed with 2500 kg ha-1 dolomitic lime and 2000 kg ha-1 mono-ammonium phosphate (MAP) before spreading over a treatment bed of shale reinforced with clay. Sewage sludge (500 kg) was ploughed into 450 m3 of contaminated soil in the second and sixth months of treatment. A further 1000 kg ha-1 MAP was added to the soil at the end of the fifth month. Moisture was maintained at 70% field capacity. Total creosote was determined by the US Environmental Protection Agency (EPA) method 418.1 and concentrations of selected creosote components were determined by gas chromatography/flame ionisation detection (GC/FID). Total creosote was reduced by more than 90% by the 10th month of landfarming. The rate of reduction in creosote concentration was highest after the addition of sewage sludge. The three-ring PAHs were more slowly removed than naphthalene and the phenolic compounds. The four- and five-ring PAHs, although persist until the end of treatment, were reduced by 76-87% at the end of the experiment. CONCLUSIONS: A combination of biostimulation and bioaugmentation during landfarming could enhance the bioremediation of soils heavily contaminated with creosote. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides information on the management of a combination of biostimulation and bioaugmentation during landfarming, and contributes to the knowledge and database necessary for the development of a technology for bioremediating creosote-contaminated land.     

Fungal metabolism of polycyclic aromatic hydrocarbons: past, present and future applications in bioremediation

This article examines the importance of non-ligninolytic and ligninolytic fungi in the bioremediation of polycyclic aromatic hydrocarbon contaminated wastes. The research from the initial studies in Dave Gibson’s laboratory to the present are discussed. Received 10 August 1997/ Accepted in revised form 15 August 1997     

微生物降解多环芳烃的研究进展

多环芳烃(PAHs)是具有严重危害的环境污染物质。介绍PAHs的降解菌,降解机理和PAHs的生物修复方面的研究进展。土壤中PAHs的生物修复被认为是解决污染的有效方法,目前,菲的生物降解途径已经比较清楚,但对结构更为复杂的多环芳烃研究较少。文章还对消除环境中多环芳烃的相关生物技术提出展望。     

Development and assessment of a preliminary kinetic model for in situ pah bioremediation

Polycyclic aromatic hydrocarbons (PAHs) are a group of organic nonaqueous‐phase liquid (NAPL) contaminants of critical environmental concern. The treatment method used for a contaminated soil depends primarily on the nature and extent of the contamination as well as the cost effectiveness of the method. Current research has shown that bioremediation is perhaps the simplest and most economic process for the treatment of large contaminated areas. Although bioremediation feasibility and effectiveness has been well documented, additional information is required to fully understand subsurface kinetics. Specifically, the importance and effect of interactions between bacteria, supplemental nutrients, oxygen source, contaminant, and soil type must be understood. Preliminary respirometer experiments have been conducted to address these factors for the development of a kinetic model for both steady‐state and unsteady‐state conditions.     

Simazine treatment history determines a significant herbicide degradation potential in soils that is not improved by bioaugmentation with Pseudomonas sp. ADP

AIMS: To study biological removal of the herbicide simazine in soils with different history of herbicide treatment and to test bioaugmentation with a simazine-degrading bacterial strain. METHODS AND RESULTS: Simazine removal was studied in microcosms prepared with soils that had been differentially exposed to this herbicide. Simazine removal was much higher in previously exposed soils than in unexposed ones. Terminal restriction fragment length polymorphism analysis and multivariate analysis showed that soils previously exposed to simazine contained bacterial communities that were significantly impacted by simazine but also had an increased resilience. The biodegradation potential was also related to the presence of high levels of the atz-like gene sequences involved in simazine degradation. Bioaugmentation with Pseudomonas sp. ADP resulted in an increased initial rate of simazine removal, but this strain scarcely survived. After 28 days, residual simazine removals were the same in bioaugmented and not bioaugmented microcosms. CONCLUSIONS: In soils with a history of simazine treatment bacterial communities were able to overcome subsequent impacts with the herbicide. The success of bioaugmentation was limited by the low survival of the introduced strain. SIGNIFICANCE AND IMPACT OF THE STUDY: Conclusions from this work provided insights on simazine biodegradation potential of soils and the convenience of bioaugmentation.     

Effects of humic substances and soya lecithin on the aerobic bioremediation of a soil historically contaminated by polycyclic aromatic hydrocarbons (PAHs)

The high hydrophobicity of polycyclic aromatic hydrocarbons (PAHs) strongly reduces their bioavailability in aged contaminated soils, thus limiting their bioremediation. The biodegradation of PAHs in soils can be enhanced by employing surface-active agents. However, chemical surfactants are often recalcitrant and exert toxic effects in the amended soils. The effects of two biogenic materials as pollutant-mobilizing agents on the aerobic bioremediation of an aged-contaminated soil were investigated here. A soil historically contaminated by about 13 g kg(-1) of a large variety of PAHs, was amended with soya lecithin (SL) or humic substances (HS) at 1.5% w/w and incubated in aerobic solid-phase and slurry-phase reactors for 150 days. A slow and only partial biodegradation of low-molecular weight PAHs, along with a moderate depletion of the initial soil ecotoxicity, was observed in the control reactors. The overall removal of PAHs in the presence of SL or HS was faster and more extensive and accompanied by a larger soil detoxification, especially under slurry-phase conditions. The SL and HS could be metabolized by soil aerobic microorganisms and enhanced the occurrence of both soil PAHs and indigenous aerobic PAH-degrading bacteria in the reactor water phase. These results indicate that SL and HS are biodegradable and efficiently enhance PAH bioavailability in soil. These natural surfactants significantly intensified the aerobic bioremediation of a historically PAH-contaminated soil under treatment conditions similar to those commonly employed in large-scale soil bioremediation.     

Remediation of benzo[a]pyrene and chrysene-contaminated soil with industrial hemp (Cannabis sativa)

The phytoremediation, with industrial hemp (Cannabis sativa), of a Hawaiian silty clay soil contaminated with two polycyclic aromatic hydrocarbons (PAHs), chrysene and benzo[a]pyrene, was studied. Hemp showed a very high tolerance to the contaminants. The growth rates of hemp, compared with control, in soils fortified with chrysene and benzo[a]pyrene at concentrations of each varying from 25 to 200 micrograms/g were consistently above 100%. The plants grew from seed for 45 days in soil fortified with PAHs at concentrations of 25, 50, and 75 micrograms/g. Controls were pots with contaminated soil but no plant. PAHs levels were significantly reduced in all pots (control and seeded pots), expect for one set at a high concentration of chrysene, which may be due to uneven spiking. A time course study over 28 days was done to monitor changes of microbial count and levels of chrysene. Little changes were observed for the total microbial count in the soil, and the concentration of chrysene in the soil decreased slightly in the pots containing plants. However, the chrysene levels in those pots were consistently lower than those in the pots without plants.     

Degradation of Polycyclic Aromatic Hydrocarbons in the Rhizosphere of Festuca arundinacea and Associated Microbial Community Changes

A phytoremediation growth chamber study was conducted to evaluate the contribution of soil microbial diversity to the contaminant degradation. Target contaminant removal from soil was assessed by monitoring concentrations of polycyclic aromatic hydrocarbons (PAHs), along with changes in the bacterial community structure over a time period of 10 months in the presence of tall fescue (Festuca arundinacea). Enhanced degradation of PAHs was observed in rhizosphere soil, with a maximum reduction in pyrene at a rate 36% higher than that noted for the unvegetated control. The dissipation of < 4-ring PAHs, 4-ring PAHs, and > 4-ring PAHs in unvegetated soil was 70%, 54%, and 49% respectively, whereas a higher dissipation rate was observed in tall fescue treated soil of 78%, 68%, and 61% at the end of the study. Microbial enumeration results showed greater total bacterial numbers and PAH-degrading bacteria in rhizosphere soil when compared to unvegetated soil. The results from the terminal restriction fragment length polymorphism (T-RFLP) analysis indicated that there was a shift in the rhizosphere bacterial community during the phytoremediation process.     

新疆富蕴地震断裂带植被恢复对土壤古菌群落的影响

选取新疆富蕴地震断裂带8种次生植物根际土壤,以同土层裸地为对照,测量土壤化学性质,利用末端限制性片段长度多态性技术研究塌陷区次生植物对土壤古菌群落的影响.结果表明,多数植物根际土壤养分显著(P＜0.05)高于对照,其主要古菌类群为泉古菌门(Crenarchaeota)和广古菌门(Euryarchaeota),群落间多样性差异大,相似性低.不同植物根际土壤古菌的优势类群数量差异较大,鼠掌老鹳草高达18种,西北绢蒿少为2种.典范对应分析表明,土壤有机质含量对古菌优势类群分布影响最大(Hha Ⅰ酶切:r=0.94;Rsa l酶切:r=0.74),速效磷含量与古菌群落各多样性指数呈显著正相关(P＜0.05),总氮含量与均匀度指数E呈显著正相关(P＜0.05).新疆地震断裂带植被的恢复可影响根际土壤古菌群落的分布、组成和结构,其原因与植物根际土壤化学性质有关,同时在改善土壤肥力方面也有显著效果.     

新疆低阶煤本源生物甲烷转化中微生物群落组成及变化

【目的】探究新疆低阶煤生物甲烷转化过程微生物群落组成及多样性。【方法】采用厌氧培养方法和末端限制性片段长度多态性技术(Terminal restriction fragment length polymorphism,T-RFLP)分析新疆低阶煤本源微生物对甲烷转化及有机酸含量的影响,分析新疆哈密大南湖长焰煤生物甲烷转化过程中微生物群落动态变化。【结果】研究表明长焰煤和褐煤对本源微生物产甲烷影响较小,随着低阶煤生物甲烷转化时间的延长,甲烷产量呈上升趋势,转化60 d后长焰煤甲烷产量高达10.28 m L/g,挥发性有机酸(VFA)浓度则最低;微生物多样性指数变化不明显,不同转化时间微生物主要类群为放线菌门(Actinobacteria),拟杆菌门(Bacteroidetes),厚壁菌门(Firmicutes),变形菌门(Proteobacteria);甲烷菌的群落结构相对于细菌较简单,在整个低阶煤生物转化产甲烷过程中共有古菌类群为甲烷八叠球菌属(Methanosarcina)、甲烷盐菌属(Methanohalobium)、甲烷叶菌属(Methanolobus)、甲烷食甲基菌属(Methanomethylovorans),它们是构成群落结构的基本菌群。【结论】低阶煤生物甲烷转化过程微生物群落具有丰富的多样性,且不同时期多样性有较大差异。甲烷菌群落结构相对于细菌较简单,共有类群明显。     

Biodegradation of selected UV-irradiated and non-irradiated polycyclic aromatic hydrocarbons (PAHs)

Biodegradation of UV-irradiated anthracene, pyrene,benz[a]anthracene,and dibenz[a,h]anthracene was comparedto that of the non-irradiated samples, individuallyand in synthetic mixtures with enrichment cultures.Combined treatment was repeated for individual anthraceneand for the PAH mixture with Sphingomonas sp.strain EPA 505 and Sphingomonas yanoikuyae.Enrichment culture studies were performed on the PAHmixtures in the presence of the main photoproduct ofanthracene, pure 9,10-anthracenedione. Photochemicallypretreated creosote solutions were also subjected tobiodegradation and the results were compared tothose of the non-irradiated solutions. The primaryinterest was on 16 polycyclic aromatic hydrocarbons(PAHs) listed as priority pollutants by European Union(EU) and the United States Environmental ProtectionAgency (USEPA). Irradiation accelerated thebiodegradation onset for anthracene, pyrene, andbenz[a]anthracene when they were treatedindividually. The biodegradation of irradiatedpyrene started with no lag phase andwas complete by 122 h whereas biodegradation of thenon-irradiated sample had a lag of 280 h andresulted in complete degradation by 720 h. Biodegradation ofPAHs was accelerated in synthetic mixtures, especiallyin the presence of pure 9,10-anthracenedione.In general, irradiation had no effect on the biodegradation of PAHsincubated in synthetic mixtures or with pure cultures. Undercurrent experimental conditions, the UV-irradiation invariablyreduced the biodegradation of PAHs in creosote. Based onthe results of the present and previous photochemical-biologicalstudies of PAHs, the influence of the photochemical pretreatmenton the biodegradation is highly dependent on the compoundsbeing treated and other process parameters.     

三七根腐病株根际土壤真菌群落组成与碳源利用特征研究

根腐病是药用植物三七Panax notoginseng的多发性病害,而土壤真菌群落组成与根腐病的发生存在一定的联系。利用末端限制性酶切片段长度多态性分析和Biolog技术研究了云南文山三七种植园健康和发病三七植株根际土壤真菌群落结构和碳源利用特征,结果表明健康和发病植株的根际土壤真菌多样性和代谢活性差异不显著,但是真菌群落组成存在差异。健康和发病植株根际土壤都以小茎点霉Phoma exigua及镰孢菌属Fusarium真菌为主要种群,而健康植株根际土壤中火丝菌属Pyronema和被孢霉属Mortierella等真菌类群的相对丰度显著高于发病植株根际土壤,尖孢镰孢菌F.ox‐ysporum和刺座霉属的Volutella colletotrichoides的相对丰度则显著低于发病植株。     

Comparison of DNA‐ and RNA‐based bacterial community structures in soil exposed to 2,4‐dichlorophenol

Aims: To examine the effect of the pollutant 2,4‐dichlorophenol on DNA‐ and RNA‐based bacterial communities in soil. Methods and Results: Soil was exposed to 100 mg kg^?1 of 2,4‐dichlorophenol (2,4‐DCP), and degradation was monitored over 35 days. DNA and RNA were coextracted, and terminal restriction fragment length polymorphism (T‐RFLP) was used to report changes in bacterial communities in response to the presence of the chlorophenol. The phylogenetic composition of the soil during degradation was determined by creating a clone library of amplified 16S rRNA sequences from both DNA and reverse‐transcribed RNA from exposed soil. Resulting clones were sequenced, and putative identities were assigned. Conclusions: A significant difference between active (RNA‐based) and total (DNA‐based) bacterial community structure was observed for both T‐RFLP and phylogenetic analyses in response to 2,4‐DCP, with more pronounced changes seen in RNA‐based communities. Phylogenetic analysis indicated the dominance of Proteobacteria in both profiles. Significance and Impact of the Study: This study describes the response of soil bacterial communities to the addition of the xenobiotic compound 2,4‐DCP, and highlights the importance of including RNA‐based 16S rRNA analysis to complement any molecular study in a perturbed soil.     

Microbial populations related to PAH biodegradation in an aged biostimulated creosote-contaminated soil

A previous bioremediation survey on a creosote-contaminated soil showed that aeration and optimal humidity promoted depletion of three-ringed polycyclic aromatic hydrocarbons (PAHs), but residual concentrations of four-ringed benzo(a)anthracene (B(a)A) and chrysene (Chry) remained. In order to explain the lack of further degradation of heavier PAHs such as four-ringed PAHs and to analyze the microbial population responsible for PAH biodegradation, a chemical and microbial molecular approach was used. Using a slurry incubation strategy, soil in liquid mineral medium with and without additional B(a)A and Chry was found to contain a powerful PAH-degrading microbial community that eliminated 89% and 53% of the added B(a)A and Chry, respectively. It is hypothesized that the lack of PAH bioavailability hampered their further biodegradation in the unspiked soil. According to the results of the culture-dependent and independent techniques Mycobacterium parmense, Pseudomonas mexicana, and Sphingobacterials group could control B(a)A and Chry degradation in combination with several microorganisms with secondary metabolic activity.     

Changes in bacterial community during fermentative hydrogen and acid production from organic waste by thermophilic anaerobic microflora

AIMS: Changes in fermentation pattern during the treatment of organic wastes containing solid materials by thermophilic anaerobic microflora were investigated with respect to product formation and bacterial community structure during hydrogen production. METHODS AND RESULTS: Anaerobic microflora enriched from sludge compost was cultivated using artificial garbage slurry in a continuous flow-stirred tank reactor. Product formation varied depending on pH and hydraulic retention time (HRT) applied. Community analysis by terminal restriction fragment length polymorphism and clone library analysis of polymerase chain reaction-amplified bacterial 16S rDNA indicated that difference in the fermentative product distribution could be caused by different populations of micro-organisms in the microflora. CONCLUSION: Hydrogen fermentation with acetate/butyrate formation was optimized at <1.0 d HRT at pH 5.0 and 6.0. Thermoanaerobacterium thermosaccharolyticum was the dominant hydrogen-producing micro-organism. Conversely, unidentified organisms became dominant after 4.0 d HRT at pH 7.0 and 8.0, where relatively high-solubilization efficiency of solid materials was observed with no production of hydrogen. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing product formation in the fermentation of solid organic wastes by a mixed population of micro-organisms. Various fermentation patterns including hydrogen fermentation were characterized and evaluated from engineering and microbial aspects.