苯系物(BTEX)长期污染对土壤和地下水微生物群落及代谢潜能的影响

苯、甲苯、乙苯和二甲苯统称为苯系物(BTEX)，是化工污染场地检出率最高的芳香族有机污染物。为研究BTEX长期污染对土壤和地下水微生物群落结构和代谢潜能的影响，采集了江苏省某搬迁化工厂的浅层土、地下水和深层土样品，利用16S rRNA基因扩增子测序和宏基因组测序技术对BTEX长期污染场地展开分析。结果表明：相比较未受污染的土壤样品，长期BTEX污染显著改变了微生物群落结构和多样性，其中以变形菌门改变最为显著。共现性网络分析表明，污染场地中随着样品取样深度的增加，微生物网络复杂性和群落稳定性降低。BTEX代谢功能基因注释表明，地下水样品中污染物代谢基因丰度和多样性更高，并且在地下水和浅层土中同时存在完整的好氧降解途径，但在地下水中厌氧降解基因的丰度更高。BTEX降解途径中benABC和bcrCBAD基因簇在浅层土中更完整，但通过构建BTEX开环的关键基因bamA的系统发育树表明，地下水中可能存在新的BTEX开环基因。这些结果证明BTEX长期污染的不同生境中存在高度多样的微生物群落与降解途径，为相关污染场地的微生物修复提供了科学依据。     

Part 2: A Field Study of Enhanced Remediation of Toluene in the Vadose Zone Using a Nutrient Solution

The objective of this study was to test the effectiveness of a nitrate-rich nutrient solution and hydrogen peroxide (H₂O₂) to enhance in-situ microbial remediation of toluene in the unsaturated zone. Three sand-filled plots were tested in three phases (each phase lasting approximately 2 weeks). During the control phase, toluene was applied uniformly via sprinkler irrigation. Passive remediation was allowed to occur during this phase. A modified Hoagland nutrient solution, concentrated in 150 L of water, was tested during the second phase. The final phase involved addition of 230 moles of H₂O₂ in 150 L of water to increase the available oxygen needed for aerobic biodegradation. During the first phase, measured toluene concentrations in soil gas were reduced from 120 ppm to 25 ppm in 14 days. After the addition of nutrients during the second phase, concentrations were reduced from 90 ppm to about 8 ppm within 14 days, and for the third phase (H₂O₂), toluene concentrations were about 1 ppm after only 5 days. Initial results suggest that this method could be an effective means of remediating a contaminated site, directly after a BTEX spill, without the intrusiveness and high cost of other abatement technologies such as bioventing or soil-vapor extraction. However, further tests need to be completed to determine the effect of each of the BTEX components.     

联苯菊酯降解菌的筛选、鉴定及降解特性研究

联苯菊酯是一种广谱高效杀虫剂,大规模的应用使其广泛残留在环境中,因此筛选联苯菊酯的高效降解菌具有重要意义。从扬州农药厂附近的地表土壤取样,利用富集驯化培养分离得到一株编号为S8的降解细菌,经表形特征、生理生化特性和16S rDNA序列分析其为醋酸钙不动杆菌（Acinetobacter calcoaceticus）,该菌株在pH7.0和30 ℃的条件下,对100 mg·L-1联苯菊酯的3 d降解率达56.4%,半衰期为60.7 h。其最适生长条件为：pH6.0~8.0,温度30~35 ℃,接种量5%。研究结果可为今后治理联苯菊酯残留污染提供理论参考。     

Response of microbial communities to different doses of chromate in soil microcosms

The toxic effect of chromate on soil microbial communities is not well documented, although microorganisms control biogeochemical cycling, contribute to formation of soil structure, regulate the fate of organic matter applied to soil. In this study the effects of short- and middle-term chromate on the soil microbial community were investigated. The shifts in the size and in the diversity of culturable heterotrophic bacterial community, the resistance to Cr(VI) of heterotrophic bacteria, the presence of cyanobacteria, the activity of 19 enzymes, and the ATP content were monitored over time (120 days) in soil microcosms artificially contaminated with three concentrations of chromate (50, 250 and 1000 mg kg⁻¹ soil). The chromate contamination affected the structure and the diversity of the soil bacterial community. Bacterial strains isolated from the microcosm contaminated with the highest concentration of chromate were identified by 16S rDNA gene sequencing. All isolates belonged to the genus Pseudomonas, were able to reduce Cr(VI), and showed a high resistance to chromate. To our knowledge, this is the first report that shows Pseudomonas strains having the capability to resist up to 40 mM of Cr(VI) on minimal medium. The cyanobacterial group was more sensitive to chromate contamination than culturable heterotrophic bacteria. No cyanobacterial growth was detected in enrichment cultures from the soil polluted with the highest chromate concentration. Some enzymes were inhibited by high concentrations of chromate, whereas others were stimulated. The ATP content in microcosms was strongly affected by chromate. We conclude that the soil microbial community responds to chromate pollution through changes in community structure, in metabolic activity, and in selection for Cr(VI)-resistance.     

Ecological evaluation of polymetallic soil quality: the applicability of culture-dependent methods of bacterial communities studying

Purpose

This work aimed to study the effect of long-term polymetallic contamination on the state and parameters of soil bacterial communities, including the abundance of different groups of culturable bacteria and the activity of nitrification.

Materials and methods

Monitoring plots were located in the dry lake and surrounding area, which had been formerly used for the discharge of industrial waste. The soils in the 16 plots were characterized by extremely high levels of heavy metal pollution. This study evaluated the main soil physicochemical properties by various methods, total metal contents by X-ray analysis, mobile metal content by atomic absorption spectrophotometry, the abundance of chosen groups of culturable bacteria by inoculation on solid media, and nitrification activity from ammonium and nitrite oxidation rates.

Results and discussion

High adaptation capacity of microbial communities to long-term pollution was revealed through marked lack of decrease in the abundance of some of the bacterial groups in soils with high contamination levels. Among the bacteria determined by the colony count method, copiotrophic and spore-forming bacteria were the least sensitive to contamination, and actinomycetes were the most sensitive. The high levels of soil pollution with heavy metals had pronounced adverse effects on nitrification activity. The decrease in activity was strongly correlated with pollutant concentrations. The oxidation of nitrite was shown to be more affected by pollution that the oxidation of ammonium.

Conclusions

Some groups and parameters of culturable microorganisms can be used for soil status estimation under pollution conditions even though they are only a small fraction of the microbial community. The most sensitive parameter was the nitrification rate, while the number of actinomycetes was found to be most promising parameter among the groups of bacteria determined by plate counts. The use of sensitive groups of culturable microorganisms for bioindication purposes is a method, which may provide a cheap and sufficiently reliable tool for large-scale soil monitoring studies.

     

The contribution of biogas residues to soil organic matter formation and CO2 emissions in an arable soil

The biogas production process generates as side-products biogas residues containing microbial biomass which could contribute to soil organic matter formation or induce CO₂ emissions when applied to arable soil as fertilizer. Using an isotope labelling approach, we labelled the microbial biomass in biogas residues, mainly G⁺ bacteria and methanogenic archaea via KH¹³CO₃, and traced the fate of microbial biomass carbon in soil with an incubation experiment lasting 378 days. Within the first seven days, 40% of the carbon was rapidly mineralized and after that point mineralization continued, reaching 65% by the end of the experiment. Carbon mineralization data with 93% recovery could be fitted to a two-pool degradation model which estimated proportions and degradation rate constants of readily and slowly degrading pools. About 49% of the carbon was in the slowly degrading pool with a half-life of 1.9 years, suggesting mid-term contribution to living and non-living soil organic matter formation. Biogas residues caused a priming effect at the beginning, thus their intensive application should be avoided.     

多环芳烃污染土壤的植物-微生物联合修复初探

在温室盆栽条件下,通过种植紫花苜蓿单独或联合接种菌根真菌(Glomus caledonium L.)(AM)和多环芳烃专性降解菌(DB),研究了利用植物-微生物强化修复多环芳烃(PAHs)长期污染土壤的效果。试验结果表明,接种菌根真菌和PAHs专性降解菌能促进紫花苜蓿的生长和土壤中PAHs的降解。经过90天修复试验,种植紫花苜蓿接种AM、DB和DB+AM处理的PAHs的降解率分别为47.9%、49.6%、60.1%,均高于只种植紫花苜蓿的对照处理(CK)(21.7%)。另外,随着PAHs苯环数的增加,其平均降解率逐渐降低,但是接种PAHs专性降解菌能够提高4环和5环PAHs的降解率。同时也发现土壤中脱氢酶活性和PAHs降解菌数量越高的处理,土壤PAHs的降解率也越高,这也是种植紫花苜蓿接种微生物能够有效促进土壤PAHs降解的原因。     

Isotopic evidence and quantification assessment of in situ RDX biodegradation in the deep unsaturated zone

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is an explosive compound whose extensive use has resulted in significant contamination of soils and groundwater worldwide. We studied its in situ biodegradation along the unsaturated zone beneath an explosives wastewater lagoon using compound-specific isotope analysis (CSIA) of RDX in the unsaturated zone, together with biodegradation slurry experiments under anaerobic conditions. We found the highest degradation potential of RDX and its nitroso derivatives in the upper part of the soil profile while in the lower parts, RDX-degradation potential was lower and the nitroso derivatives tended to persist. This was also observed in the field, as reflected by the isotopic composition of RDX along the profile. We also found a correlation between biodegradation potential and clay content: biodegradation was further enhanced in layers characterized by high-clay content or in those influenced by the high-clay layers. In addition, in the presence of high organic matter content, further enhancement of biodegradation was observed. We obtained different isotopic enrichment factors (?) for RDX biodegradation in different sections of the unsaturated profile and suggest that different degradation pathways exist simultaneously in situ, in variable proportions. Using the range of enrichment factors, we were able to assess the biodegradation extent of RDX at different sampling points along the profile, which ranged between 30 and 99.4%. The novel application of CSIA together with slurry experiments provides better insight into degradation processes that are otherwise difficult to detect and assess.     

降解菌HD接种和非接种根围土壤中丁草胺的降解动力学研究

测定了小麦、棉花、水稻和玉米根围土壤和非根围土壤中丁草胺的降解特征和降解菌变化动态。结果表明,种植作物丰富了土壤微生物,根围土壤丰富的微生物对丁草胺的降解具有显著的促进作用。根围土壤中丁草胺的降解是非根围土壤的1.63～2.34倍,相应的半衰期缩短为非根围土壤的 42.2％～72.8％。根围土壤接种处理后这种促进作用得到进一步加强,其降解速率是非根围土壤的1.68～2.83倍,半衰期为非根围土壤的34.4％～59.4％。试验结果表明,作物根围是丁草胺残留快速降解的微环境,作物根围接种处理可以强化丁草胺残留的微生物降解。     

Cross-enhancement of accelerated biodegradation of organophosphorus compounds in soils: Dependence on structural similarity of compounds

Rates of degradation of seven organophosphate nematicides and insecticides were examined in two soils known to show accelerated biodegradation of fenamiphos and one soil known to show accelerated biodegradation of chlorpyrifos. The results indicated that several organophosphate insecticides and one nematicide were susceptible to cross-enhanced degradation in the soil showing accelerated biodegradation of chlorpyrifos. No cross-enhancement was observed in the two soils showing accelerated degradation of fenamiphos. Fumigation resulted in the complete inhibition of pesticide degradation in all soils. The data suggested that the cross-enhancement of selected pesticides in chlorpyrifos-degrading soil was dependent on the structural similarity of the compounds. Mechanisms of degradation of pesticide in soil support this hypothesis, where structurally similar compounds (diazinon, parathion, coumaphos and isazofos) were hydrolysed by microbial activity in chlorpyrifos-degrading soil but the degradation products were accumulated. Enhanced degradation of chlorpyrifos and fenamiphos was found to be stable in the laboratory condition for a period of one year.     

四环素对芘污染农田土壤微生物修复的影响及响应过程

针对城郊农田土壤中多环芳烃和抗生素复合污染的新特征,通过室内模拟土培实验,研究四环素(Tetracycline,TC)胁迫下,降解菌Sphingobium sp.PHE3对长三角典型农田土壤中芘的降解效果和影响机制。研究表明,接种降解菌处理(B)能明显促进土壤中芘的降解,TC的引入可显著抑制土壤中芘的深度降解过程(P0.05)。经过90天培养后,B处理与接菌+添加TC处理(BTC)的降解率分别为40.1%、25.7%,较对照分别提高了23.0倍、14.1倍。通过土壤微生物群落结构多样性分析发现,降解菌数量在经历90天的土壤环境适应期后逐渐快速增加,其数量变化与污染物芘在土壤中含量消减趋势呈负相关;引入芘和四环素对土壤细菌群落结构多样性和功能稳定性具有显著影响(P0.05),然而对土壤真菌群落影响不显著(P0.05)。此外,B和BTC处理条件下,土壤过氧化氢酶活性、荧光素二乙酸酯酶活性和土壤微生物生物量碳氮值显著高于单独添加芘处理(P)和单独添加TC处理(TC),但P处理与TC处理之间无显著差异(P0.05),说明外源污染物(芘或四环素)对于土壤酶活性和微生物生物量碳氮具有显著抑制作用(P0.05),致使降解菌功能作用受到抑制。综上研究结果表明TC可明显抑制土壤中典型四环多环芳烃的微生物降解过程,针对多环芳烃与抗生素复合有机污染农田土壤的微生物强化修复技术有待深入研究。     

Effects of nutrient concentration on the biodegradation of crude oil and associated microbial populations in the soil

The influence of nutrient amendments on the biodegradation of a crude oil and on associated microbial populations in an agricultural soil was studied in microcosms during a 150-day experiment. Concentration and chemical composition of residual hydrocarbons (HC) were periodically monitored in series of polluted soils unfertilized and fertilized with increasing concentrations of nutrients. The decrease in HC concentration was effective in all soils over time. The maximal biodegradation extent was 62%, meanwhile the natural attenuation contributed to 47% of degradation. A permanent inhibition of hydrocarbons assimilation was recorded with a high input of nutrients. The biodegradation of saturates, aromatics and polars was respectively, permanently, temporally and not reduced by excessive fertilization in soil. Accumulation of polar metabolic by-products was demonstrated. Enumerations of total heterotrophic bacteria and hydrocarbon-adapted bacteria showed a strong stimulation in both populations. Maximum stimulation was observed with the highest input of nutrients. However, the extents of biodegradation were not concurrently improved, thus indicating that the microbial degraders were selected depending on the nutrient supply. The permanent and/or temporally inhibition of the saturated and unsaturated HC assimilation revealed that different nutrient supplies were optimum for the degradation of aliphatic and aromatic HC.     

Cocomposting of Cattle Manure and Hydrocarbon Contaminated Flare Pit Soils

The potential of using composting technology to remediate clayey soils with high levels of crude oil contamination was evaluated. An open air windrow comprised of flare pit soil, manure and wood chips was constructed at Olds College, Composting Technology Centre. Aeration and mixing were carried out by a skid steer loader and composting parameters were monitored for ten months. Temperature profile of this windrow gave cyclic patterns of high and low temperature recordings corresponded to the turning events. Most of the microbial metabolic activity occurred within the mesophilic temperature range and the hydrocarbon degrading microorganism populations remained high throughout the trial. Complete removal of BTEX compounds was achieved within six months and extractable carbons from C₅ to C₁₀ were reduced by 98.7% compared to the initial contaminated soil. Vegetative growth on the composted soil was also evaluated. Barley and timothy plants grown in the composted soil were compared to the control off-lease soil, contaminated soil, and other treatments of varying salinity and organic matter levels. Plant germination, survival, and biomass production was significantly better in the composted soil than in the contaminated soil. Furthermore, barley plants grown in the composted soil were more resilient than those grown in the control off-lease soil.     

Continuous defoliation of perennial ryegrass (Lolium perenne) and white clover (Trifolium repens) and associated changes in the composition and activity of the microbial population of an upland grassland soil

A microcosm study was conducted to investigate the effect of continuons plant defoliation on the composition and activity of microbial populations in the rhizosphere of perennial ryegrass (Lolium perenne) and white clover (Trifolium repens). Continuons defoliation of ryegrass and clover resulted in sigmficant (P <0.01) increases in soil microbial biomass, although whilst increases were measured from day 2 in soil sown with clover significant increases were only seen from day 21 in soil sown with ryegrass. These increases were paralleled, from day 10 onwards, by increases in the numbers of culturable bacteria. Numbers ofPsendomonas spp. also increased in the later stages of the study. No influence on culturable fungal populations was detected. Whilst shifts in the composition of the microbial populations were measured in response to defoliation there was little effect on microbial activity. No changes in either dehydrogenase activity or microbial respiration in the rhizosphere of ryegrass or clover were measured in response to defoliation, but both dehydrogenase activity and microbial respiration were greater in ryegrass than clover when values over the whole study were combined. Continuous defoliation resulted in significant (P <0.001) reductions in the root dry weight of ryegrass and clover, of the order 19% and 16%, respectively.     

Soil Biological Activities in Monitoring the Bioremediation of Diesel Oil-Contaminated Soil

The effects of two different biological treatments on hydrocarbon degradation and on soil biological activities were determined during a 100-d incubation period. An evaluation of soil biological activities as a monitoring instrument for the decontamination process of diesel-oil contaminated soil was made using measurements of organic carbon content, soil microbial respiration, soil ATP and dehydrogenase, β-glucosidase, lipase enzyme activities. Five samples were used: S (control, uncontaminated soil), CS (contaminated soil), SCS (sterilized contaminated soil), CFS (contaminated soil plus N and P), CCS (contaminated soil plus compost). The relationships between soil parameters and the levels of total petroleum hydrocarbons (TPH) residues were investigated. Results showed that inorganic nutrients NP and compost stimulated hydrocarbon biodegradation but not all biological activities to a significant extent. The residual hydrocarbon trend was positively related with that of the organic C content, microbial respiration and with β-glucosydase activity, while both soil lipase and dehydrogenase activities were negatively related with the hydrocarbon trend. Lipase activity was found to be the most useful parameter for testing hydrocarbon degradation in soil.     

Continuous defoliation of perennial ryegrass (Lolium perenne) and white clover (Trifolium repens) and associated changes in the composition and activity of the microbial population of an upland grassland soil

A microcosm study was conducted to investigate the effect of continuons plant defoliation on the composition and activity of microbial populations in the rhizosphere of perennial ryegrass (Lolium perenne) and white clover (Trifolium repens). Continuons defoliation of ryegrass and clover resulted in sigmficant (P <0.01) increases in soil microbial biomass, although whilst increases were measured from day 2 in soil sown with clover significant increases were only seen from day 21 in soil sown with ryegrass. These increases were paralleled, from day 10 onwards, by increases in the numbers of culturable bacteria. Numbers ofPsendomonas spp. also increased in the later stages of the study. No influence on culturable fungal populations was detected. Whilst shifts in the composition of the microbial populations were measured in response to defoliation there was little effect on microbial activity. No changes in either dehydrogenase activity or microbial respiration in the rhizosphere of ryegrass or clover were measured in response to defoliation, but both dehydrogenase activity and microbial respiration were greater in ryegrass than clover when values over the whole study were combined. Continuous defoliation resulted in significant (P <0.001) reductions in the root dry weight of ryegrass and clover, of the order 19% and 16%, respectively.     

Lumbricid macrofauna alter atrazine mineralization and sorption in a silt loam soil

Atrazine is a widely used herbicide and is often a contaminant in terrestrial and freshwater ecosystems. It is uncertain, however, how the activity of soil macrofauna affects atrazine fate and transport. Therefore, we investigated whether earthworms enhance atrazine biodegradation by stimulating herbicide degrading soil microflora, or if they increase atrazine persistence by facilitating herbicide sorption. Short (43 d) and medium term (86 d) effects of the earthworms Lumbricus terrestris and Aporrectodea caliginosa on mineralization, distribution, and sorption of U-ring-¹⁴C atrazine and on soil C mineralization was quantified in packed-soil microcosms using silt loam soil. A priming effect (stimulation of soil C mineralization) caused by atrazine supply was shown that likely lowered the earthworm net effect on soil C mineralization in atrazine-treated soil microcosms. Although earthworms significantly increased soil microbial activity, they reduced atrazine mineralization to ¹⁴CO₂-C from15.2 to 11.7% at 86 d. Earthworms facilitated formation of non-extractable atrazine residues within C-rich soil microsites that they created by burrowing and ingesting soil and organic matter. Atrazine sorption was highest in their gut contents and higher in casts than in burrow linings. Also, gut contents exhibited the highest formation of bound atrazine residues (non-extractable atrazine). Earthworms also promoted a deeper and patchier distribution of atrazine in the soil. This contributed to greater leaching losses of atrazine in microcosms amended with earthworms (3%) than in earthworm-free microcosms (0.003%), although these differences were not significant due to high variability in transport from earthworm-amended microcosms. Our results indicated that earthworms, mainly by casting activity, facilitated atrazine sorption, which increased atrazine persistence. As a consequence, this effect overrode any increase in atrazine biodegradation due to stimulation of microbial activity by earthworms. It is concluded that the affect of earthworms of atrazine mineralization is time-dependent, mineralization being slightly enhanced in the short term and subsequently reduced in the medium term.     

拟除虫菊酯类农药微生物降解研究进展

拟除虫菊酯类农药是杀虫剂中的第三大类,这类农药残留已成为目前农产品中的主要农药残留类型之一。而微生物在降解农药残留中具有重要的作用,微生物降解技术已成为去除农药残留的绿色生产技术。拟除虫菊酯类农药的微生物降解国内外已有的研究主要集中在降解现象,菌株的分离、鉴定及生理生化特性,酶学,不同光学异构体的降解、降解途径等方面,本文对此进行了较详细的回顾,并对将来的研究方向进行了展望。     

Biodegradation of chlorinated phenols in subsurface soils

Microcosm studies were employed to determine the subsurface biodegradation rates of phenol, 2-chlorophenol (2-CP), 2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TCP), and pentachlorophenol (PCP). Soil samples were taken from sites in Pennsylvania and Virginia from depths up to 31 m, and all samples contained significant microbial populations. Soil from both sites readily biodegraded all five compounds. Biodegradation rates increased as initial concentrations increased, and all biodegradation rates appeared to follow first-order kinetics with regard to the initial compound concentrations. Biodegradation rates for the five compounds followed the order: phenol = 2-CP > 2,4,6-TCP > 2,4-DCP. PCP was degraded more slowly than phenol or 2-CP, but similarly to 2,4,6-TCP and 2,4-DCP. Different soils exhibited different degradation rates, and the soil characteristics that may influence the rates are discussed. The data suggest that biological degradation is a significant attenuation mechanism for phenol and its chlorinated derivatives in subsurfaces saturated and unsaturated zones.     

Cross-enhancement: enhanced biodegradation of isothiocyanates in soils previously treated with metham sodium

Rates of degradation of 2-propenyl isothiocyanate (PrITC), benzyl isothiocyanate (BeITC) and 2-phenylethyl isothiocyanate (2-PeITC) in a soil known to biodegrade methyl isothiocyanate (MITC) at an accelerated rate, but never previously exposed to the other ITCs, were higher (persistence in soil increased by 1150, 80 and 100%, respectively,) than in a similar non-degrading soil. The rate of degradation of the same three ITCs was significantly lower in sterilised (autoclaved) soils than in the degrading soil. These results indicate that the three ITCs are susceptible to enhanced cross-biodegradation in soils where enhanced biodegradation of MITC has been induced by use of metham sodium soil fumigant. When Brassica plant tissue containing sinigrin (2-propenyl glucosinolate) as the predominant glucosinolate (GSL) was added to the degrading soil, the amount of PrITC present after 24 h was significantly lower than in the non-degrading soil at the same amendment rates. The toxicity to an insect test organism of the PrITC produced from the biofumigant plant tissue was correlated with the concentration of PrITC measured in the two soils, with 67% more plant tissue required in the degrading soil to cause 100% mortality as in the non-degrading soil (3.0 vs 5.0 mg g⁻¹). The effectiveness of biofumigation using ITC-producing Brassica plants may be diminished in soil suffering from enhanced biodegradation of MITC.