黄孢原毛平革菌所产木素过氧化物酶系对染料降解的研究

白腐真菌黄孢原毛平革菌所产生的胞外过氧化物酶系由两类同功酶木素过氧化物酶和锰过氧化物酶组成，木素过氧化物酶对所研究的染料都有解作用，锰过氧化物酶只对部分染料起作用。加入H2O2，胞外酶液可以降解多种酶性染料如卡布龙红、酸性大兰、弱酸黄和弱酸大红，它们的脱色速率依次下降，以卡布龙红为例研究了各种条件因素对其降解脱色的影响，最佳H2O2浓度是100μmol/L，最佳PH为4．0，温度越高，脱色越快，但高于50℃酶将明显失活，木素过氧化物酶活性越高脱色速率越快，高浓度染料对其脱色有抑制作用，最佳条件下，50mg/L浓度卡布龙红第一分钟的脱色率达70％。随着稀释倍数的增加，酶液的降解速率呈抛物线加速下降，表明酶液中的某些组分如元素离子的浓度也是一个重要影响因素。     

不同培养条件下锰过氧化物酶(MnP)的合成及其对甲基橙的降解

高效、大规模、低成本合成木质素降解酶是直接采用其降解难降解有机污染物所必须解决的问题.对锰过氧化物酶(MnP)降解甲基橙和在非灭菌的反应器中连续合成MnP的可行性进行考察.结果表明,在采用2 mmol H2O2和1.5 mmol MnSO4的降解体系中,获最大脱色效果,且100、200和300 U/L的MnP可在8h内将甲基橙分别脱色18％、23％和35％;在非灭菌的反应器水平上实现了固定化培养的P.chrysosporium连续23 d合成MnP,但MnP酶活仅为2～ 23 U/L,难以酶解甲基橙;然而,在摇瓶培养条件下固定化的P.chrysosporium合成的MnP却能达1 152 U/L.因此,直接采用MnP对污染物进行降解以及在非灭菌的反应器中持续合成MnP是可行的,但就在非灭菌条件下如何提高MnP的合成量还有待开展深入的研究.     

降解菌对堆肥中多环芳烃降解作用的研究

通过在堆肥中加入经过驯化的降解菌这种土壤有机污染生物修复技术 ,对堆肥中多环芳烃的浓度变化进行监测 ,从而了解降解菌对堆肥中多环芳烃的降解作用。实验结果表明 ,降解菌的加入能明显地提高多环芳烃的降解率 ,本次实验中 ,菲、芴的去除率提高了 2 5 %左右 ,芘的去除率提高了约 4 5 %。     

白腐菌P.sordida YK-624及其锰过氧化物酶对2-氯酚的转化

利用白腐菌P.sordida YK-624和产自该菌株的锰过氧化物酶(MnP)处理2-氯酚(2-CP).结果表明,当2-CP质量浓度为50mg/L时,培养5d, P.sordida YK-624能降解50.42%的2-CP;采用300 U的MnP处理150mg/L的2-CP, 24h时2-CP降解率可达75.00%.利用紫外-可见分光光度计、红外光谱仪和高效液相色谱仪对MnP处理过程中的产物进行了分析,表明MnP氧化2-CP过程中有多种产物生成,可能的产物包括醌类和聚合产物.     

一株多环芳烃降解菌的筛选及其降解特性

微生物修复是治理土壤多环芳烃(polycyclic aromatic hydrocarbons, PAHs)污染的主要方法,而高效降解菌筛选是微生物修复技术的重要基础。从北京焦化厂土壤中筛选分离得到一株PAHs降解菌Q3,通过生理生化和16S rDNA等分析手段鉴定其为Rhodococcus rhodochrous。结果表明：该菌株对芘的耐受能力较强,可降解初始浓度为200 mg·L⁻¹的芘;该菌株具有降解广谱性,可利用苯并[a]芘、苯并[b]荧蒽、二苯并[a,h]蒽、苯并[g,h,i]苝等9种PAHs为唯一碳源进行代谢,特别是对苯并[a]芘等高环PAHs具有较好的降解效果;此外,该菌株可有效降解模拟液中的混合PAHs,并且对野外被PAHs长期污染的土壤具有较好的强化修复效果。投加菌株处理后的处理组与对照组相比,土壤PAHs总去除率提高了24%。以上结果表明该菌株对环境中被PAHs污染的土壤具有较好的强化修复潜力,可为PAHs污染土壤的微生物修复技术提供技术参考。     

三株优势菌对多环芳烃的降解性能研究

多环芳烃具有毒性、生物蓄积性和半挥发性,并能在环境中持久存在。生物修复处理具有费用低、效果好、污染物残留量低、不产生二次污染、能够保持或改善植物生长的土壤结构等优点。实验分别在对单一菌株、两两混合菌株及三株菌混合等三种情形下,对蒽、菲、芘的降解作用进行了研究。研究结果表明：单一菌株对蒽、菲、芘有一定的降解能力,菌株混合时,对蒽、菲的降解率有所提高;三株菌种混合时,黄杆菌对放线菌、红球菌的生长过程有抑制作用;蒽、菲、芘的降解过程会不断交替进行着产酸和脱羧过程,使降解液pH值出现波动;同时在降解过程中,菌体能产生表面活性物质,这有利于蒽、菲、芘的生物降解转化。     

菌源对多环芳烃降解菌的筛选及降解性能的影响

高效降解菌的筛选对利用生物修复技术有效去除环境中的多环芳烃具有重要意义。分别以石油污染土壤和焦化废水活性污泥为菌源,分离出芘降解菌和混合PAHs(菲、荧蒽和芘)降解菌共14株并对其降解性能进行对比研究。结果表明,筛选得到的菌株分别属于9个菌属,其中2种菌源共有的菌属为Mycobacterium sp.、Ralstonia sp.和Shinella sp.。芘和PAHs的高效降解菌(CP16和CM32)均属于分支杆菌属(Mycobacterium),来源于焦化废水活性污泥;菌株CP16对芘(50mg/L)的7 d降解率为74.99%,CM32对PAHs(菲50 mg/L、荧蒽和芘各10 mg/L)的7 d降解率为100%。因此,以焦化废水活性污泥为菌源更有利于获得高效的多环芳烃降解菌。     

黄孢原毛平革菌降解磺胺甲噁唑的影响因素

采用摇瓶实验考察固定化培养黄孢原毛平革菌(Phanerochaete chrysosporium)对磺胺甲噁唑(SMX)的降解效果并对抗生素降解条件进行优化。结果表明,黄孢原毛平革菌在固定化培养后对SMX的降解能力有所提高,10 d后培养液中抗生素去除率达到了100%,而游离培养液中去除率为74%。固定化黄孢原毛平革菌降解磺胺甲噁唑的较优碳源和氮源分别是葡萄糖和酒石酸铵,初始pH范围在3.0~5.0之间、培养温度为30~40℃时较适宜。黄孢原毛平革菌对磺胺甲噁唑的降解受碳源种类影响较大,而在限氮条件下氮源的差异对其影响较小。     

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。     

降解菌对堆肥中多环芳烃降解作用的研究

通过在堆肥中加人经过驯化的降解菌这种土壤有机污染生物修复技术，对堆肥中多环芳烃的浓度变化进行监测，从而了解降解菌对堆肥中多环芳烃的降解作用。实验结果表明，降解菌的加人能明显地提高多环芳烃的降解率，本次实验中，菲、芴的去除率提高了25％左右，芘的去除率提高了约45％。     

Study of the degradation of dyes by MnP of Phanerochaete chrysosporium produced in a fixed-bed bioreactor

The production of ligninolytic enzymes by the fungus Phanerochaete chrysosporium in a fixed-bed tubular bioreactor, filled with cubes of nylon sponge, operating in semi-solid-state conditions, was studied. Maximum individual manganese-dependent peroxidase (MnP) and lignin peroxidase (LiP) activities of 1293 and 225 U/l were detected.The in vitro decolourisation of two structurally different dyes (Poly R-478, crystal violet) by the extracellular liquid obtained in the above-mentioned bioreactor was monitored in order to determine its degrading capability. The concentration of some compounds (sodium malonate, manganese sulphate) from the reaction mixture was optimised in order to maximise the decolourisation levels. A percentage of Poly R-478 decolourisation of 24% after 15 min of dye incubation was achieved.On the other hand, a methodology for a long treatment of these dyes based on the continuous addition of MnP enzyme and H(2)O(2) was developed. Moreover, this enzymatic treatment was compared with a photochemical decolourisation process. The former allowed to maintain the degradation rate almost constant for a long time, resulting in a decolourisation percentage of 70% and 30% for crystal violet and Poly R-478, respectively, after 2 h of treatment. As for the latter, it was not able to degrade Poly R-478, whereas crystal violet reached a degradation of 40% in 2 h.     

紫外诱变黄孢原毛平革菌染料脱色研究

对黄孢原毛平革菌孢子悬浮液进行了紫外诱变，并将筛选出的优势菌ZWYB1与未经诱变的菌作对比实验，结果表明：诱变筛选得到的ZWYB1不仅可缩短对染料的脱色时间，提高对杂环类染料蕃红花红及高浓度染料400mg／L的孔雀石绿的脱色效果，而且诱变后的菌种具有一定的脱色能力稳定性。     

紫外诱变黄孢原毛平革菌染料脱色研究

对黄孢原毛平革菌孢子悬浮液进行了紫外诱变,并将筛选出的优势菌ZWYB1与未经诱变的菌作对比实验,结果表明:诱变筛选得到的ZWYB1不仅可缩短对染料的脱色时间,提高对杂环类染料蕃红花红及高浓度染料400 mg/L的孔雀石绿的脱色效果,而且诱变后的菌种具有一定的脱色能力稳定性.     

Cellobiose dehydrogenase-dependent biodegradation of polyacrylate polymers by Phanerochaete chrysosporium

When Phanerochaete chrysosporium was cultured using conditions which promote the expression of cellobiose dehydrogenase (CDH), but not the ligninolytic peroxidases, the fungus effectively solubilized and mineralized an insoluble, crosslinked polyacrylate and an insoluble polyacrylate/polyacrylamide copolymer. Addition of iron to the cultures increased CDH activity in the cultures and the rate and extent of solubilization and mineralization of both polymers. Solubilization of both polymers was observed when incubated with purified CDH, ferric iron and hydrogen peroxide.     

Phanerochaete chrysosporium吸附载体的选择及染料降解研究

研究了游离细胞与载体吸附培养、不同载体材料对Phanerochaete chrysosporium进行连续染料脱色及产酶能力的影响。结果表明,P.chrysosporium可在载体上良好生长,甚至生长到载体内部。木屑、玉米芯、花生壳3种载体材料中,以木屑载体吸附培养物的持续脱色和产酶效果最佳,该培养物经三轮连续脱色后对染料RB5仍能达到最高95%的脱色率,并产生596 U/L锰依赖过氧化物酶（MnP）和1 326 U/L木质素过氧化物酶（LiP）,对染料的持续脱色和产酶能力明显优于游离细胞培养物。     

载体吸附培养黄孢原毛平革菌(Phanerochaete chrysosporium)和云芝(Trametes versicolor)对染料的脱色

比较研究了不同载体吸附培养的黄孢原毛平革菌(Phanerochaete chrysosporium)和云芝(Trametes versicolor)对染料连续脱色的效果。结果表明:(1)白腐真菌黄孢原毛平革菌和云芝分别在含有球形的木屑、玉米芯和花生壳载体的液体环境中振荡培养,菌体以膜状或团状形式大量附着生长在载体表面。(2)连续4轮脱色过程中,不论黄孢原毛平革菌还是云芝,都是木屑为载体的培养液的持续脱色和产酶能力最好,宜选择木屑为载体。其中,木屑为载体的黄孢原毛平革菌培养液经过2轮连续12d脱色后对活性黑RB5的最高脱色率仍能达到97%,在第3轮脱色中对活性红M-3BE的最终脱色率接近96%,并且能产生最高611U/L的锰依赖过氧化物酶(MnP)和1 477U/L的木质素过氧化物酶(LiP)。(3)在实际应用中应该将木屑为载体的黄孢原毛平革菌和云芝培养液都投加到含染料废水处理系统中,强化生物处理效果。     

Cellular and molecular damage of Phanerochaete chrysosporium by the oxidation hair dyes

Introduction

The toxic effect of the oxidation hair dyes on Phanerochaete chrysosporium was investigated by exposure of this fungus in a nitrogen-limited culture medium to various concentrations of the oxidation hair dyes.

Results

The results showed that both the size and the dry weight of the mycelial pellets of P. chrysosporium could be reduced when the concentration of the oxidation hair dyes was higher than 300?mg/L. By using the AFLP analysis and the UPGMA dendrogram, the DNA damage of P. chrysosporium by the oxidation hair dyes was also detected. Comparing with that in the control, the percent polymorphism under different concentrations of the oxidation hair dyes increased. In the meantime, the DNA similarity was decreased, which meant that the DNA damage was aggravated with an increase in the concentrations of the oxidation hair dyes.

Conclusion

Thus, as an environmental pollutant, the oxidation hair dyes have a toxic effect on P. chrysosporium at both cellular and molecular levels.     

Growth of Phanerochaete chrysosporium on diesel fuel hydrocarbons at neutral pH

Generally, the white-rot fungus Phanerochaete chrysosporium performs its biodegradative activities in liquid culture while growing on easily utilized carbon sources such as malt- or potato-extract. However, less is known about the potential of this organism to grow directly on environmental pollutants without regard to special conditions. Growth of P. chrysosporium on a middle fraction (MF) of diesel fuel at neutral pH in mineral medium under non-ligninolytic conditions was explored. After 14 d, the GC-analyzable n-alkanes of 1000 mg l(-1)MF were reduced to background, with most biodegradation occurring by day 7 when quantified relative to the biodegradation of the internal fuel biodegradation marker, pristane. Investigations with n-hexadecane and unmodified diesel fuel further confirmed these biodegradation results. Biomass production was monitored and indicated that fungal biomass was more than 10 times less than positive controls (potato dextrose broth, PDB) but that biomass increased relative to negative controls. When P. chrysosporium was incubated with diesel fuel and PDB, fuel biodegradation was delayed for at least 4d and inhibited overall through 14 d. Experiments with P. chrysosporium growing on n-hexadecane in the presence of 1 mM 1-aminobenzotriazole (ABT), an inhibitor of the cytochrome P-450 enzyme system, resulted in inhibition of biomass production relative to positive controls implicating the utilization of this enzyme system in n-alkane metabolism. Finally, when P. chrysosporium was incubated in a non-aqueous phase liquid (NAPL) mixture of polycyclic aromatic hydrocarbons (PAHs) and MF, n-alkanes and phenanthrene were degraded in 2 weeks while anthracene, chrysene and benzo[a]pyrene were not.