产漆酶食用菌的粗酶液对多环芳烃降解研究

漆酶是一种含铜的多酚氧化酶,可以降解多种有机污染物,其中包括多环芳烃。但是由于纯漆酶的价格昂贵,限制了在环境修复中的应用。采用液态和固态2种方式培养了食用菌Pleurotus ostreatus和Coprinus comatus,并考察了固态培养的粗酶液对多环芳烃的降解和去毒能力,结果发现2种真菌均可产漆酶,但不产木质素过氧化物酶和锰过氧化物酶,其中固态培养的漆酶产量高于液态培养,且C.comatus的漆酶产量大于P.ostreatus。2种真菌固态培养的粗酶液对多环芳烃均有一定的降解和去毒作用,且P.ostreatus粗酶液的漆酶活性虽然较低,但是对多环芳烃降解率和去毒效果却优于C.comatus,这可能是P.ostreatus的粗酶液中含有较多的调节物质造成的。P.ostreatus的固态发酵粗酶液对蒽、苯并[a]芘、苯并[a]蒽、苊等均有较高的降解率(50%以上),但是对生物有效性高的萘的降解率却较低,这种降解特征可能与底物的电离势大小有关。     

Restoring biochemical activity and bacterial diversity in a trichloroethylene-contaminated soil: the reclamation effect of vermicomposted olive wastes

Background, aim, and scope  In this work, the potential for using olive-mill solid waste as an organic amendment for biochemical and biological restoration of a trichloroethylene-contaminated soil, which has previously been stabilized through vermicomposting processes, has been explored. Materials and methods  Trichloroethylene-contaminated water was pumped into soil columns with a layer of vermicompost at 10-cm depth (biobarrier system). The impacts of the trichloroethylene on the microbial community were evaluated by determining: (1) the overall microbial activity (estimated as dehydrogenase activity) and enzyme activities related to the main nutrient cycles (β-glucosidase, o-diphenoloxidase, phosphatase, urease, and arylsulphatase activities). In addition, isoelectric focusing of the soil extracellular humic-β-glucosidase complexes was performed to study the enzymatically active humic matter related to the soil carbon cycle. (2) The soil bacterial diversity and the molecular mechanisms for the bacterial resistance to organic solvents were also determined. For this, polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) was used to detect changes in bacterial community structure and PCR-single-strand conformational polymorphism (SSCP) was developed and optimised for detection and discrimination of the resistance-nodulation-division (RND) genes amplified from the contaminated soils. Results  Vermicompost reduced, with respect to the unamended soil, about 30% of the trichloroethylene leaching during the first month of the experiment. Trichloroethylene had a marked negative effect on soil dehydrogenase, β-glucosidase, urease, phosphatase, and arylsulphatase activities. Nevertheless, the vermicompost tended to avoid this toxic effect. Vermicompost also displays stable humic-β-glucosidase complexes that increased the extracellular activity related to C-cycle in the contaminated soils. The isoelectric focusing technique showed a more biochemically active humic matter in the soil sampled under the vermicompost. The behaviour of the three main phyla of bacteria isolated from the DGGE bands was quite different. Bands corresponding to Actinobacteria disappeared, whereas those affiliated with Proteobacteria remained after the trichloroethylene contamination. The disappeared Actinobacteria became visible in the soil amended with the vermicompost. Bands corresponding to Bacteriodetes appeared only in columns of contaminated soils. In this study, six types of RND proteins were detected by PCR-SSCP in the natural soil, three in the trichloroethylene-contaminated soil and 7/5 in trichloroethylene-contaminated soil above/below the vermicompost in the biobarrier columns. Trichloroethylene tended to reduce or eliminate all the clones detected in the uncontaminated soil, whereas new efflux pumps appeared in the biobarrier columns. Discussion  Although enzymes incorporated into the humic substances of vermicomposted olive wastes are quite stable, trichloroethylene also inhibited the background levels of the soil extracellular β-glucosidase activity in the amended soils. The decrease was less severe in the biobarrier system, but in any case, no relation was found between the levels of trichloroethylene in soil and extracellular β-glucosidase activity, or between the latter and the quantity of humic carbon in soils. The isoelectric focusing technique was carried out in the humic fraction to determine whether the loss of activity occurred in overall extracellular β-glucosidase or in that linked to stable humic substances (humic–enzyme complexes). The contaminated soils showed the lower enzyme activities, whereas contaminated and amended soils presented greater quantity of focalised (and therefore stable) humic carbon and spectra heterogeneity: very different bands with higher enzyme activities. No clear relationship between trichloroethylene concentration in soil and diversity of the bacterial population was noted. Similar patterns could be found when the community structures of bacteria and microbial activity were considered. Since the use of the dehydrogenase assay has been recognised as a useful indicator of the overall measure of the intensity of microbial metabolism, these results could be attributed to PCR-DGGE methodology, since the method reveals the presence of dominant populations regardless of their metabolic state. Trichloroethylene maintained or even increased the number of clones with the DNA encoding for RND proteins, except for the contaminated soil located above the vermicompost. However, the main effect of trichloroethylene was to modify the structure of the community in contaminated soils, considering the type of efflux pumps encoded by the DNA extracted from soil bacteria. Conclusions  Trichloroethylene inhibited specific functions in soil and had a clear influence on the structure of the autochthonous bacterial community. The organic matter released by the vermicomposted olive waste tended to avoid the toxic effect of the contaminant. Trichloroethylene also inhibited the background levels of the soil extracellular β-glucosidase activity, even when vermicompost was present. In this case, the effect of the vermicompost was to provide and/or to stimulate the humic-β-glucosidase complexes located in the soil humic fraction >10⁴, increasing the resistance of the enzyme to the inhibition. The bacterial community from the soil presented significantly different mechanisms to resistance to solvents (RND proteins) under trichloroethylene conditions. The effect of the vermicompost was to induce these mechanisms in the autochthonous bacterial community and/or incorporated new bacterial species, able to grow in a trichloroethylene-contaminated ambient. Coupled biochemical and molecular methodologies are therefore helpful approaches in assessing the effect of an organic amendment on the biochemical and biological restoration of a trichloroethylene-contaminated soil. Recommendations and perspectives  Since the main biochemical and biological effects of the organic amendment on the contaminated soil seem to be the incorporation of biochemically active humic matter, as well as new bacterial species able to grow in a trichloroethylene-contaminated ambient, isoelectric focusing and PCR-SSCP methodologies should be considered as parts of an integrated approach to determine the success of a restoration scheme.     

Metal-related oxidative stress in birds

Metals can cause oxidative stress by increasing the formation of reactive oxygen species (ROS), which render antioxidants incapable of defence against growing amounts of free radicals. Metal toxicity is related to their oxidative state and reactivity with other compounds. Our aim is to review the mechanisms on how metals cause oxidative stress and what is known about metal-induced oxidative stress in wildlife. Taking birds as model organisms, we summarize the mechanisms responsible for antioxidant depletion and give a view of how to detect metal-induced oxidative stress in birds by using different biomarkers. The mechanisms producing the harmful effects of oxidative stress are complex with different biomolecular mechanisms associated with ecotoxicological and ecological aspects. The majority of the studies concerning metals and ROS related to oxidative stress have focused on the biomolecular level, but little is known about the effects at the cellular level or at the level of individuals or populations.     

Assessment of the mechanisms of detoxification of chemical compounds and antioxidant enzymes in the digestive gland of mussels, Mytilus galloprovincialis, from Mediterranean coastal sites

In this study the effects of the main marine pollutants (metals, PAHs, PCBs and DDTs) were assessed in native mussels from the Mediterranean coast of Spain. For this purpose several biomarkers such as benzo[a]pyrene hydroxylase (BPH), DT-diaphorase (DTD), glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPs), glutathione reductase (GR), metallothionein (MT) and lipid peroxidation (LPO) were measured in the digestive gland. Results showed increased LPO levels in mussels which accumulated high loads of organic compounds and arsenic in their tissues. BPH levels correlated to the concentrations of organic compounds in mussel tissues, though the range of BPH response was low in relation to the high gradient of accumulation of organic pollutants. Increased BPH levels, concomitant to low DTD and GST activities, were detected in mussels which presented high levels of organic pollutants in their tissues. This suggests that signs of LPO present in these organisms are related to the imbalance between phase I and phase II biotransformation processes. Furthermore, the increased levels of MT and CAT detected in mussels which showed high levels of Cd in their tissues appear to reflect a coordinated response which protects against the toxicity of this metal. The application of these biomarkers in environmental assessment is discussed.     

Study of the effect of refinery waste water exposure on Allium cepa's enzymatic antioxidant defense: probing potential biomarkers

The effects of heavy metals, hydrocarbons, and various toxicants present in Mathura Refinery Waste Water (MRWW) on Allium cepa were examined as a model plant system. The study was based on exposure of A. cepa to different concentrations of MRWW and compared to untreated control (exposure of onion bulbs with aquaguard purified water) to determine the total protein content and activities of certain antioxidant enzymes. These enzymes were evaluated for their efficacy to serve as biomarkers of refinery waste water pollution. DNA damaging potential of MRWW was also investigated. Data demonstrated maximal enhancement in ascorbate peroxidase activity subsequent to MRWW insult, although a significant increase in activities was also noted for other enzymes in the following order: superoxide dismutase?>?glutathione-S-transferase?> catalase. This is suggestive of their potency as a biomarker of MRWW toxicity. Increase in activities of monodehydroascorbate reductase (MDHAR; 152%) and deoxyribonuclease (345%) were also found in the A. cepa system as a result of MRWW exposure. In conclusion, A. cepa system might thus serve as an appropriate tool for monitoring water pollution, especially produced by petroleum waste and heavy metals in term of induction of glutathione peroxidase, MDHAR, and deoxyribonuclease activity.     

Induction of glutathione reductase enzyme activity by β‐aminobutyric acid in plant leaves

Abstract

Treatment of pea and tobacco leaf discs with the resistance inducer DL‐β‐amino‐n‐butyric acid (BABA) led to a substantial induction of glutathione reductase (GR, E.C. 1.6.4.2.) enzyme activity. After exposure to 1 mM BABA for 96 hrs, the GR activities were 3.2‐fold and 2.9‐fold higher in pea and tobacco leaf discs, respectively, than GR activities in untreated controls. Elevated GR levels may contribute to the antioxidative protection of plants during pathogen attack.     

Tissue distribution, isozyme abundance and sensitivity to chlorpyrifos-oxon of carboxylesterases in the earthworm Lumbricus terrestris

A laboratory-based study was conducted to determine the basal carboxylesterase (CbE) activity in different tissues of the earthworm Lumbricus terrestris, and its sensitivity to the organophosphate (OP) pesticide chlorpyrifos-oxon (CPx). Carboxylesterase activity was found in the pharynx, crop, gizzard, anterior intestine, wall muscle and reproductive tissues of L. terrestris, and multiple tissue-specific isozymes were observed by native gel electrophoresis. Esterase activity and sensitivity to CPx inhibition varied on a tissue- and substrate-specific basis, suggesting isoforms-specific selectivity to OP-mediated inhibition. Three practical issues are recommended for the use of earthworm CbE activity as a biomarker of pesticide exposure: (i) CbE should be measured using several routine substrates, (ii) it should be determined in selected tissues instead of whole organism homogenate, and (iii) earthworm CbE activity should be used in conjuncture with other common biomarkers (e.g., ChE) within a multibiomarker approach to assess field exposure of OPs, and potentially other agrochemicals.     

基于综合生物标志物响应指数评价0~#柴油和平湖原油胁迫下的缢蛏(Sinonovacula constricta)毒性效应

选择0#柴油和平湖原油乳化液对缢蛏(Sinonovacula constricta)进行氧化胁迫实验,选取典型的抗氧化酶-超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷光甘肽硫转移酶(GST)及过氧化物酶(POD)用于衡量油类污染物对生物体造成的氧化压力大小.此外,结合综合生物标志物响应(Integrated Biomarker Responses,IBR)指标,对2种石油污染物对缢蛏的毒性响应进行定量化评价.结果表明,不同浓度的0#柴油和平湖原油对缢蛏消化腺中的4种酶表现出不同程度的诱导效应,各试验组在暴露前期均表现出诱导或抑制,但对4种酶的影响存在时间顺序性,SOD、CAT和GST的酶活性表现为升高-降低的过程,POD表现为降低-升高的过程,活性达到峰值的时间SOD和CAT要早于GST和POD.结合计算出的IBR数值来看,高浓度0#柴油能够引起最为显著的生物效应变化,显示该石油污染物高毒性的特征,0#柴油生物毒性大于平湖原油生物毒性.     

贵州省典型矿区土壤重金属污染对蚯蚓的毒性效应评估

选取我国贵州省某矿区典型的重金属复合污染土壤开展28 d慢性毒性暴露试验,以蚯蚓体内2种抗氧化酶活性——过氧化氢酶(CAT)和超氧化物歧化酶(SOD)、8-羟基脱氧鸟苷(8-ODHG)和金属硫蛋白(MT)含量为生物指标,评估了以上生物指标与土壤重金属含量的相关性,探讨该研究方法用于矿区土壤重金属生态风险评估的可行性。结果表明,CAT和SOD在暴露周期内均出现先诱导后抑制的动态响应过程,表明蚯蚓通过提高CAT和SOD的活性来清除机体因污染胁迫而产生的自由基,以适应环境变化,在毒性缓解后,蚯蚓体内代谢产物的积累抑制了抗氧化氢酶活性,CAT和SOD活性下降并趋于稳定。暴露于重金属污染亚致死浓度时,蚯蚓体内的8-OHDG含量随着暴露时间的延长显著降低,随着暴露浓度的增加,损伤存在由低到高的转变。在28 d暴露周期内,蚯蚓体内MT在重金属胁迫下发挥了解毒作用,MT含量在蚯蚓暴露后第7天增加,随后逐渐降低。本文基于生物毒性试验综合评估了矿区典型重金属污染土壤的生态风险,研究结果对指导重金属复合污染土壤的修复与再利用具有重要意义。     

丛枝菌根真菌(AMF)对铯胁迫宿根高粱生长及根际土壤酶的影响

通过盆栽试验研究了接种透光球囊霉(Glomus diaphanum)、摩西球囊霉(Glomus mosseae)、地表球囊霉(Glomus versiforme)及幼套球囊霉(Glomus etunicatum)等4种丛枝菌根真菌(AMF)对铯污染下宿根高粱生长及根际土壤酶活性的影响.结果表明,与未接种对照相比,接种AMF处理均显著增加了宿根高粱的株高和根长,同时增强了过氧化氢酶、蔗糖酶活性及根系活力(P0.05),且以接种G.etunicatum增强效果最好,上述酶活性和根系活力分别相当于对照的1.11、2.25和4.04倍,而接种Glomus diaphanum最显著增强了酸性磷酸酶活性,使其提高了24.53 mg·g-1·d-1·FW(P0.05),宿根高粱对铯的耐性指数均高于对照,说明AMF在一定程度上能缓解铯污染胁迫对根际土壤酶的抑制作用,进而减轻核素铯对植物的毒害;接种AMF处理显著抑制了脲酶活性,表明脲酶可作为反应土壤核素铯污染的一个有效指标.同时也表明AMF有效地增强了宿根高粱对核素铯的耐性能力.