Polycyclic aromatic hydrocarbon biodegradation and extracellular enzyme secretion in agitated and stationary cultures of Phanerochaete chrysosporium

The extracellular enzyme secretion and biodegradation of polycyclic aromatic hydrocarbons (PAHs) were studied in agitated and shallow stationary liquid cultures of Phanerochaete chrysosporium. Veratryl alcohol and Tween80 were added to cultures as lignin peroxidase (LIP) and manganese peroxidase (MnP) inducer, respectively. Shallow stationary cultures were suitable for the production of enzyme, whereas agitated cultures enhanced overall biodegradation by facilitating interphase mass transfer of PAHs into aqueous phases. The use of a LIP stimulator, veratryl alcohol, did not increase PAH degradation but significantly enhanced LiP activity. In contrast, Tween80 increased both MnP secretion and PAH degradation in shallow stationary cultures. On the other hand, high PAH degradation was observed in agitated cultures in the absence of detectable LIP and MnP activities. The results suggested that extracellular peroxidase activities are not directly related to the PAH degradation, and the increased solubility rather than enzyme activity may be more important in the promotion of PAH degradation.     

Cooperation between ligninolytic enzymes produced by superior mixed flora

Since the ability to degrade lignin with one kind of white-rot fungi or bacteria was very limited, superior mixed flora‘s ability to degrade lignin was investigated by an orthogonal experiment in this paper. The results showed that superior mixed flora reinforced the ability to degrade lignin, the degradation rates of both sample 9 and 10 were beyond 80% on the day 9. The cooperation between lignin peroxidase(LiP), Mn-dependent peroxidase(MnP) and laccase (Lac) for lignin degradation was also studied. By examining the activities of three enzymes produced by superior mixed flora, it was found that Lac was a key enzyme in the process of biological degradation of lignin but Lip was not; the enzyme activity ratios of Lac/MnP and Lac/LiP were significantly correlative with the degradation rate of lignin at the 0.01 level; and the ratio of MnP/LiP was an important factor affecting the degradation rate of lignin.     

木质素降解酶对四环素的降解可行性

选取了使用广泛、在环境中残留量大、生物毒性大、不易被生物降解的四环素类抗生素中的四环素,考察了其被木质素过氧化物酶(LiP)和锰过氧化物酶(MnP)粗酶液体外降解的效果,证明2种酶对四环素均有较强的降解作用,并在此基础上比较了3个不同酶活(15 U·L-1、40U·L-1、70 U·L-1)下的降解率.结果表明,同等酶活(40 U·L-1)下,LiP比MnP的降解能力强,在适宜的反应条件下,LiP对50 mg·L-1四环素的降解率在5min内可达95%以上,而MnP在2h后仅达70%.对于LiP,当酶活从15 U·L-1升高至40 U·L-1时,降解率提高约15%,当酶活进一步升高至70 U·L-1时,降解率变化不大,而提高酶活可明显提高MnP对四环素的降解率.此外,通过反应进程中补加H2O2,可以明显提高MnP对四环素的降解率.     

铜绿假单胞菌对白腐菌产木质纤维素降解酶的影响

采用固态发酵的方法,考察了两株不同型号的铜绿假单胞菌（AB93066和ATCC9027）对白腐菌（黄孢原毛平革菌）产木质纤维素降解酶（木素过氧化物酶LiP、锰过氧化物酶MnP和羧甲基纤维素酶CMCase）的影响,并对酶提取液的pH值、表面张力及稻草降解残渣中挥发性有机质含量的变化进行了分析.结果表明,添加5.72%的A...     

木质素过氧化物酶和锰过氧化物酶对染料脱色的性能比较

通过控制培养基中Mn2+的浓度获得只含有木质素过氧化物酶(LiP)或锰过氧化物酶(MnP)的单一酶培养物,用于比较LiP和MnP对染料降解脱色的条件和能力的差异.结果表明,LiP对橙Ⅰ降解脱色的最佳条件为h2O₂0.15 mmol/L,pH 3.0,温度40 ℃;MnP为h2O₂0.15 mmol/L,pH 3.5,温度40 ℃.最佳条件下,考察LiP和MnP作用下染料浓度和染料类型对脱色的影响,发现LiP作用下脱色率明显高于MnP作用下的脱色率,表明LiP可作用的染料浓度比MnP高,可作用的染料范围比MnP大.由此认为LiP具有比MnP更强的脱色能力.      

白腐真菌木质素降解酶的反应器发酵

为了获得更高的白腐真菌木质素降解酶的产量和相应的控制策略,应用5L搅拌罐生物反应器对氮限制下(C/N=56/2.2)P.chrysosporium产木质素降解酶进行了放大研究.结果表明,在分批发酵试验中,锰过氧化物酶(MnP)和漆酶(Lac)分别在培养第6d和第7d达到峰值,其酶活随时间的变化规律与摇瓶试验基本相同;而采用氮限制液体培养基进行补料没有获得更高的酶活,因此,可以得出采用发酵液体培养基作为补料液不利于白腐真菌持续产酶.另外,在分批发酵和分批补料发酵过程中,均发现体系pH值变化与白腐真菌生长和次生代谢产酶具有较好的相关性,当白腐真菌进入次生代谢阶段开始产酶时,体系pH值开始下降,随着发酵后期酶活的降低,pH值下降的幅度也逐渐变小,当体系酶活接近为0.0U/L时,pH值基本不再变化.因此,在实际发酵过程中,可以依据体系pH的变化间接了解白腐真菌的生长和产酶情况,而分批补料策略还有待于进一步研究.     

Influence of glucose feeding on the ligninolytic enzyme production of the white-rot fungus Phanerochaete chrysosporium

The present work studied the influence of glucose feeding on the ligninolytic enzyme production of Phanerochaete chrysosporium in a nitrogen-limited (C/N ratio is 56/8.8 mmol/L) medium. Several sets of shaking flask experiments were conducted. The results showed that 2 g/L glucose feeding on the first day of the culture (24 h after the inoculation) simulated both fungal biomass growth and enzyme production. The manganese peroxidase (MnP) activity was 2.5 times greater than that produced in cultures without glucose feeding. Furthermore, the glucose feeding mode in fed-batch culture was also investigated. Compared to cultures with glucose feeding every 48 h, cultures with glucose feeding of 1.5 g/L (final concentration) every 24 h produced more enzymes. The peak and total yield of MnP activity were 2.7 and 3 times greater compared to the contrast culture, respectively, and the enzyme was kept stable for 4 days with an activity of over 200 U/L. Translated from Acta Scientise Circumstantiae, 2007, 27(3): 363–368 [译自: 环境科学学报]     

Effects of culture conditions on ligninolytic enzymes and protease production by Phanerochaete chrysosporium in air

The production of ligninolytic enzymes and protease by Phanerochaete chrysosporium was investigated under different culture conditions. Different amounts of medium were employed in free and immobilized culture, together with two kinds of medium with different C/N ratios. Little lignin peroxidase （LIP） （〈 2 U/L） was detected in free culture with nitrogen-limited medium （C/N ratio： 56/2.2, in mmol/L）, while manganese peroxidase （MnP） maximum activity was 231 and 240 U/L in 50 and 100 ml medium culture, respectively. Immobilized culture with 50 ml nitrogen-limited medium gave the highest MnP and LiP production with the maximum values of 410 and 721 U/L separately on the day 5; however, flasks containing 100 ml nitrogen-limited medium only produced less MnP with a peak value of 290 U/L. Comparatively, carbon-limited medium （C/N ratio： 28/44, in mmol/L） was adopted in culture but produced little MnP and LiE Medium type had the greatest impact on protease production. Large amount of protease was produced due to glucose limitation. Culture type and medium volume influence protease activity corporately by affecting oxygen supply. The results implied shallow immobilized culture was a possible way to gain high production of ligninolytic enzymes.     

聚丙烯酰胺生物降解研究

研究了黄孢原毛平革菌对聚丙烯酰胺(PAM)的生物降解,从葡萄糖的加入量、pH、N浓度、Mn²⁺浓度和降解时间5个方面考察了对PAM降解的影响.结果表明,黄孢原毛平革菌对聚丙烯酰胺具有特殊的酶催化降解的能力,降解率可达50%,限氮条件(NH₄⁺=0.2g/L)和Mn2+浓度(Mn²⁺=0.017 5g/L)是菌株产聚丙烯酰胺降解酶的最佳条件.     

白腐真菌批式发酵动力学模型研究

为了进一步认识白腐真菌发酵产酶机理并指导相关发酵工艺设计,在一系列试验研究的基础上,建立了描述黄孢原毛平革菌批式发酵过程的动力学模型,并采用自由悬浮和固定化2种培养条件下的试验数据分别回归了模型各参数.模拟计算结果表明,生物量、葡萄糖、氨氮、木质素酶浓度的的计算值与实测值的偏差在15%以内.对比2种培养条件下的模型参数发现,二者得到的最大生物量浓度均为1.78 g/L;固定化培养菌体的比增长速率值(0.668 3 d-1)大于自由悬浮培养值(0.514 4d-1);自由悬浮培养中绝大部分葡萄糖消耗于非生长菌体代谢,固定化培养中则部分葡萄糖用于菌体生长,且氨氮消耗更快;菌体的产酶过程与生长无关;自由悬浮培养条件下,菌丝小球可合成MnP(231 U/L),其MnP合成能力为115.8 U·(g·d)-1,且高峰后依然可以保持26.1 U·(g·d)-1,适合采用补料-分批发酵的模式提高MnP的产量及其发酵效率;固定化培养条件下,菌体可同时合成MnP(410 U/L)和LiP(721 U/L),但其LiP和MnP合成能力在酶活高峰之后明显下降,分别由248.9 U·(g·d)-1和80.1 U·(g·d)-1下降到0 U·(g·d)-1和6.04 U·(g·d)-1,故而在考虑补料-分批发酵模式时,补料时机应选择在培养中期的LiP与MnP峰值之前.     

蚯蚓辅助微生物修复芘污染土壤

通过微宇宙实验,研究了蚯蚓对不同土壤中芘的微生物降解率的影响及蚯蚓对芘的富集率.结果表明,有机质较高土壤中芘的微生物降解率和蚯蚓富集率都低于有机质低的土壤,而老化作用降低了芘的生物有效性.无论在新鲜染毒还是老化的土壤中.蚯蚓均可提高土壤中芘的去除率和微生物降解率.在新鲜染毒的红壤和潮土中,蚯蚓可使芘的去除率由41.78%和27.02%分别提高到89.24%和57.53%,其中分别有51.48%和41.63%是蚯蚓促进的微生物降解作用引起的,37.76%和15.90%归因于蚯蚓富集作用.而在老化的红壤和潮土中,蚯蚓可使芘的去除率由36.53%和20.50%分别提高到91.93%和45.46%,其中分别有54.43%和32.83%是蚯蚓促进的微生物降解作用引起的,37.50%和12.63%归因于蚯蚓富集作用.     

碳质纳米材料与枝孢菌KR14的相互影响研究

碳质纳米材料(Carbon Nanomaterials, CNMs)因具有独特的电学及光学等性质而引起了人们的广泛关注,从而被大量使用并释放到环境中,进而影响生态系统环境及生物化学过程,但目前有关CNMs与环境微生物相互作用的研究鲜见报道.因此,本文研究了枝孢菌KR14(Cladosporium sp.)与3种CNMs(单壁碳纳米管(SWCNTs)、石墨烯(Graphene)和氧化石墨烯(GO))的相互作用.结果表明,CNMs的加入促进了3种非特异性酶(漆酶、锰过氧化物酶和木质素过氧化物酶)活性增加,其中,对锰过氧化物酶(MnP)活性的促进作用最为显著,18 d最高增加26.1%.在3种类型的CNMs中,SWCNTs对MnP活性刺激最佳,GO最弱.木质素降解实验和电化学分析表明,CNMs可作为电子导体提高真菌胞外电子传递效率,进而提高KR14对木质素的降解.X射线光电子能谱(XPS)结果表明,除GO外,SWCNTs和石墨烯的氧碳比(O/C)均上升,二者表面发生变化.拉曼光谱(Raman)和傅立叶变换红外光谱(FTIR)结果表明,SWCNTs的I_D/I_G显著提高,无序性增加;石墨烯出现2D峰,即与KR14相互作用后有一定程度堆叠;KR14可引起CNMs结构转变.本研究结果有助于深入理解和评价环境中CNMs与真菌之间的相互作用关系及CNMs对真菌降解木质素和环境碳循环的影响.     

锰过氧化物酶(MnP)对农药毒死蜱降解的初步研究

以广泛使用的有机磷杀虫剂毒死蜱为研究对象,利用白腐菌P.sordida YK-624菌株所产锰过氧化物酶(MnP)对其进行生物降解的初步研究。首先比较确定了MnP降解毒死蜱所需要的降解体系,即对于毒死蜱的降解,需要在有机酸(丙二酸)存在的缓冲体系中进行,此体系不需要表面活性剂Tween 80,但必须含有Mn2+和葡萄糖氧化酶氧化葡萄糖产生的H2O2。随后对主要影响因素的试验结果表明,MnP降解毒死蜱较好的条件是在pH 4.5的丙二酸缓冲体系中含7.5 mmol/L MnSO4、2.5 mmol/L葡萄糖以及40 U葡萄糖氧化酶,温度30℃。此时,400 U/L的MnP可将30 mg/L的毒死蜱经3 d时间降解77.51%。同时,利用GC-MS对毒死蜱酶降解后的产物进行了初步分析,得到1种可能的酶解产物O,O,O’,O’-四乙基二硫代焦磷酸酯,不同于水解和细菌降解途径,值得进行进一步研究。     

木质素过氧化物酶降解四环素机理的研究

为探究过氧化物酶对四环素类抗生素的降解机理,以白腐真菌Phanerochaete chrysosporium表达的木质素过氧化物酶(LiP)粗酶为研究对象,研究其产酶条件及其降解四环素(TC)的机理。结果表明:低浓度Mn2+可促进产酶,碳源、氮源限制是产酶的重要条件;降解反应进行10min后,H2O2消耗至阈值0.045mmol/L,LiP对TC的降解停止,其降解率达到82%;TC降解反应趋于平衡时,补加TC和H2O2后,LiP依然可以高效发挥作用,其二次降解率达到65%;LiP作用于不同初始浓度(10mg/L、25mg/L、50mg/L、100mg/L)TC,显示LiP对不同浓度TC均可有效降解,TC降解率分别为62%、80%、82%和90%;降解产物的抑菌性试验反映出降解产物较TC母体的抑菌性明显降低;LC/MS捕获到6种可能的降解产物E-TC、TP 461、E-TP 461、TP 477、TP 475和TP 445,并推测出LiP降解TC可能的降解途径。     

Noncovalent immobilization of manganese peroxidases from P. chrysosporium on carbon nanotubes

Manganese peroxidases (MnP) from Phanerochaete chrysosporium were adsorbed onto multi-walled carbon nanotubes (MWNT). Four different loadings of MnP on MWNTs were investigated, and the maximum enzyme loading of 47.5 μg/mg of MWNTs was obtained in 12 h. The adsorbed MnP showed a catalytic activity of up to 0.1 U/mg of the weight of the system of MnP/MWNTs, with 23% of its original activity retained. The AFM image of the adsorbed enzymes indicated that a layer of MnP covered the surface of the MWNTs and retained its original three-dimensional shape. Amino-based nonspecific interactions may play the dominant role in the adsorption of MnP on MWNTs.     

白腐菌锰过氧化物酶对2,2’,4,4’-四溴联苯醚的降解

多溴联苯醚(PBDEs)是一类在环境中普遍存在的全球性有机污染物,对这类污染物环境行为的研究虽然已有较多报道,但是微生物降解方面尤其是针对单一胞外酶降解的研究还比较少。文章以白腐菌模式菌种Phanerochaete chrysosporium为对象,研究其分泌的胞外酶锰过氧化物酶(MnP)对环境中最常检测到的2,2’,4,4’-四溴联苯醚(BDE47)的好氧降解,考察不同β-环糊精浓度对该酶降解BDE47的影响,并初步探讨了胞内细胞色素P450的作用。结果表明,MnP能有效降解BDE47,在培养15 d后,扣除空白损失,降解率达到70%左右。低浓度和高浓度的环糊精对MnP降解BDE47无显著性影响。胞内细胞色素P450对BDE47的降解没有明显贡献。     

Responses of butachlor degradation and microbial properties in a riparian soil to the cultivation of three different plants

A pot experiment was conducted to investigate the biodegradation dynamics and related microbial ecophysiological responses to butachlor addition in a riparian soil planted with different plants such as Phragmites australis, Zizania aquatica, and Acorus calamus. The results showed that there were significant differences in microbial degradation dynamics of butachlor in the rhizosphere soils among the three riparian plants. A. calamus displays a significantly higher degradation efficiency of butachlor in the rhizosphere soils, as compared with Z aquatica and P. australis. Half-life time of butachlor degradation in the rhizospheric soils of P. australis, Z aquatica, and A. calamus were 7.5, 9.8 and 5.4 days, respectively. Residual butachlor concentration in A. calamus rhizosphere soil was 35.2% and 21.7% lower than that in Z aquatica and P. australis rhizosphere soils, respectively, indicating that A. calamus showed a greater improvement effect on biodegradation of butachlor in rhizosphere soils than the other two riparian plant. In general, microbial biomass and biochemical activities in rhizosphere soils were depressed by butachlor addition, despite the riparian plant types. However, rhizospheric soil microbial ecophysiological responses to butachlor addition significantly (P < 0.05) differed between riparian plant species. Compared to Z aquatica and P. australis, A. calamus showed significantly larger microbial number, higher enzyme activities and soil respiration rates in the rhizosphere soils. The results indicated that A. calamus have a better alleviative effect on inhibition of microbial growth due to butachlor addition and can be used as a suitable riparian plant for detoxifying and remediating butachlor contamination from agricultural nonpoint pollution.     

木质素降解酶在不同堆肥基质中的吸附传输特性研究

为了深入探究木质素降解酶在不同堆肥基质中的吸附传输特性,通过批量实验对比了土壤、菜叶、稻草和米糠对木质素降解酶的吸附性能,并进行了动力学分析和等温吸附模型拟合,同时通过柱淋洗实验考察了木质素降解酶在4种堆肥基质中的迁移传输特性.结果表明,堆肥基质对木质素降解酶的吸附与基质种类有关,土壤、菜叶、稻草和米糠对木素过氧化物酶(LiP)和锰过氧化物酶(MnP)的吸附量分别为1.22、 1.27、 1.13、 1.22 U·g-1和5.09、 4.88、 4.43、 3.95 U·g-1.比较LiP和MnP吸附的动力学模型,准二级动力学方程为表征木质素降解酶吸附的最佳模型,其R2值为0.9732～0.9997, Elovich方程拟合较差,准一级动力学方程拟合最差;Langmuir模型对等温吸附数据进行拟合效果最好,而实验数据不适合用Freundlich方程表征.土壤、菜叶、稻草和米糠对LiP和MnP饱和吸附容量分别为1.23、1.30、1.17、1.14 U·g-1和5.70、 5.19、 4.73、 4.14 U·g-1.LiP和MnP在稻草和米糠基质中传输效果较好,可传输到最深层10 mL处,而在土壤和菜叶基质中则被滞留在浅层.     

Cometabolic degradation of veratryl alcohol and biphenyl by white rot fungus under nitrogen nutrition-rich condition

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真菌对磺胺二甲氧嘧啶降解过程的转录组分析及差异表达基因的功能分析

采用黄孢原毛平革菌降解磺胺二甲氧嘧啶（SDM）,4 d后SDM的去除率达74%,降解速率达3.24 mg·L^-1·d^-1.采用Illumina高通量测序平台对降解SDM后的菌株与对照组菌株进行测序,通过GO和KEGG富集分析,得到的差异表达基因能显著富集到与降解相关的“氧化还原酶活性”途径,及与应激反应有关的“ABC转运体”路径.通过对高表达高上调的差异表达基因的筛选与分析,得到了耐受和降解SDM的功能基因.水通道蛋白、ABC转运蛋白及甲基转运酶基因等在黄孢原毛平革菌对环境的耐受中起到重要的作用.乙醇氧化酶、细胞色素P450和糖苷水解酶等在黄孢原毛平革菌对SDM的降解中起着关键的作用.本文从转录组水平分析了SDM的降解机制,为黄孢原毛平革菌在环境修复中的应用提供一定的参考.