中国是农业大国,也是秸秆年产量最多的国家。统计数据显示,2015年中国秸秆资源总量约10.4亿吨,以水稻秸秆、小麦秸秆和玉米秸秆为主,在东北地区,玉米秸秆是主要的农业副产物（张强等 2005;蒋泓峰 2016）。秸秆作为一个重要的可再生资源,“用则利,弃则害”（毕于运 2010）。秸秆的主要成分包括木质素、纤维素和半纤维素,木质素和半纤维素以共价键的形式形成一种天然的屏障,将纤维素包裹在其中,防止纤维素被酶解。木质素紧密的碳水化合物结构是影响玉米秸秆利用效率的主要屏障（Buranov & Mazza 2008;宋丽丽 2013;左飒飒 2018）。利用微生物预处理秸秆因具有耗能小、成本低、无污染的特点引起研究者的广泛关注。其中白腐真菌对秸秆进行预处理,破坏秸秆的木质纤维素结构,对于秸秆资源的利用具有重要意义。
我国大型菌物资源丰富（李玉等 2015）,其中多孔菌种类繁多,目前达600多种,东北地区达300多种,作为白腐真菌的重要类群,多孔菌为其研究提供了宝贵的资源（戴玉成 2009;Dai 2010;司静等 2011a,2011b,2011c;王伟等 2011;韩美玲等 2017;郑飞等 2017;吴怡等 2019, 2020）。白腐真菌对木质素具有很强的降解能力,能够在纯培养的条件下将木质素完全降解为CO2和H2O（Reid 1995;李慧蓉 2005）。早期Kirk & Tien（1984）研究了白腐真菌模式菌株Phanerochaete chrysosporium Burds.对木质素的降解。此后Wan & Li（2010）利用虫拟蜡孔菌Ceriporiopsis subvermispora (Pilát) Gilb. & Ryvarden降解玉米秸秆,在接种42d时,其对木质素的降解效果达到39.2%,对纤维素的降解效果不足5%。Sun et al.（2011）利用毛栓孔菌Trametes hirsuta (Wulfen) Lloyd 对玉米秸秆进行降解,在预处理42d时,对木质素降解率达到71.91%。宋丽丽等（2017）利用硬毛粗盖孔菌Funalia trogii (Berk.) Bondartsev & Singer预处理玉米秸秆,木质素降解率为33.99%,纤维素降解率为8.77%。甄静等（2017）对T. hirsuta XYG422漆酶发酵条件进行优化,预处理玉米秸秆,木质素降解率达到83.54%。白腐真菌对木质纤维素的降解能力因属、种而异,比较理想的白腐真菌菌株对木质素具有特异性降解效果,微量降解纤维素（Blanchette et al. 1988）。
本研究从16株白腐真菌菌株中筛选得到有较强漆酶或过氧化物酶活性的2株菌,利用其对玉米秸秆进行预处理,发现它们对玉米秸秆中的酸不溶木质素具有较强的降解作用。研究结果为白腐真菌资源的开发利用以及对玉米秸秆木质素降解机制研究提供基础数据。
1 材料与方法
1.1 材料
1.1.1 供试菌株:本试验所用Phanerochaete chrysosporium CGMCC 5.0776购自中国科学院菌种保藏中心,其他16株菌株由作者采集、分离、纯化,现保藏于吉林农业大学菌种保藏中心（CCMJ）。
1.1.2 试验原料:玉米秸秆取自吉林农业大学试验田。
1.1.3 培养基:愈创木酚-PDA固体培养基:200g马铃薯提取液,琼脂20g,葡萄糖20g,愈创木酚0.4mL加水定容到1L,121℃灭菌3min,用于定性测定菌株的产漆酶能力（芦光新等 2014）。苯胺蓝-PDA固体培养基:200g马铃薯提取液,琼脂20g,葡萄糖20g,苯胺蓝0.1g,加水定容到1L,121℃灭菌30min,用于定性测定菌株的产过氧化物酶能力（蔡磊等 2002）。菌株活化培养基:葡萄糖20g,琼脂20g,酵母浸粉5.0g,KH2PO4 1g,MgSO4·7H2O 0.5g,ZnSO4·7H2O 0.05g,加水定容到1L,pH自然,121℃灭菌30min（司静 2014）。液体培养基:葡萄糖20g,酵母浸粉5g,KH2PO4 1g,MgSO4·7H2O 0.5g,ZnSO4·7H2O 50mg,加水定容到1L,pH自然,分装至250mL锥形瓶中,每瓶倒入液体培养基100mL,121℃灭菌30min后,加入维生素B1 40μL。玉米秸秆固体培养基:将玉米秸秆于60℃烘干至恒重后粉碎,过40目筛,按照玉米秸秆:水=1:2.5的比例加入到培养皿中,121℃灭菌30min。
1.2 方法
1.2.1 菌株活化:将于4℃低温保藏的白腐真菌菌株取出,置于室温过夜,接种至菌株活化培养基中,25℃恒温培养至菌丝铺满平板。
1.2.2 菌株筛选:取直径为1cm的菌块分别接种到愈创木酚-PDA固体培养基中25℃培养6d,以及苯胺蓝-PDA固体培养基中25℃培养5d,利用十字划线法,测量培养皿中的菌丝直径、显色圈/褪色圈直径、显色圈/褪色圈直径与菌丝直径的比值,每个菌株设置3个重复。选择菌丝生长较快且漆酶/过氧化物酶活性较高的菌株用于后续试验。
1.2.3 菌株鉴定:（1）形态学鉴定:采用传统分类学方法,观察菌株的宏观特征和微观特征,参照李玉等（2015）和赵继鼎等（1998）的研究进行鉴定。
（2）分子生物学鉴定:取一定量的新鲜菌丝体和子实体,采用基因组DNA提取试剂盒（康为世纪）提取总DNA。PCR扩增引物为ITS5和ITS4（White et al. 1990）,PCR反应体系:10×PCR buffer 2.5μL,dNTP（10mmol/L）2μL,引物ITS4和ITS5（10μmol/L）1μL,Taq DNA聚合酶（5U/μL）,模板DNA 2μL,补加去离子水至25μL。PCR反应条件:95℃预变性3min;94℃变性40s,54℃退火45s,72℃延伸1min,共35个循环;72℃延伸10min,4℃终止反应。产物送到生工生物工程（上海）股份有限公司测序,结合GenBank的一些已知序列并利用最大似然法和贝叶斯法分析构建系统发育树,选用Boletopsis leucomelaena (Pers.) Fayod和Hydnellum geogenium (Fr.) Banker作为外群,利用MAFFT进行序列的比对（Kazutaka et al. 2005）,Gblocks获取序列的保守区域（Talavera & Castresana 2007）,ModelFinder选择最佳模型和参数（Kalyaanamoorthy et al. 2017）,利用IQtree和MrBayes分别构建系统发育树（Ronquist et al. 2012;Nguyen et al. 2015）。
1.2.4 种子发酵:取直径为1cm的菌块5个,接种至液体培养基中,25℃、150r/min振荡培养6d。
1.2.5 玉米秸秆预处理:以种子发酵液体积:玉米秸秆质量=1:1的比例将种子发酵液接种到玉米秸秆固体培养基中,25℃培养33d后,将样品取出,于60℃烘干至恒重,装入自封袋中备用。测定预处理秸秆酸不溶木质素、纤维素含量,参照王玉万和徐文玉（1987）采用的方法,本方法主要测定酸不溶木质素的含量。
1.2.7 酶活性测定:采用试剂盒（苏州科铭生物技术有限公司）对预处理的玉米秸秆样品中漆酶、锰过氧化物酶（manganese peroxidase,MnP）和木质素过氧化物酶（lignin peroxidase,LiP）活性进行测定。漆酶活力单位的定义（U）:在波长420nm下,每分钟将1μmol 2,2′联氮双3乙基苯并噻唑啉6磺酸[2,2′azino bis(3ethylbenzothiazoline6sulfonic acid),ABTS]底物氧化所需的酶量。MnP活力单位的定义（U）:在波长465nm下,每分钟将1μmol愈创木酚氧化所需的酶量。LiP活力单位的定义（U）:在波长651nm下,每1mL反应体系中每分钟吸光度值变化0.01为一个酶活单位。
1.2.8 数据分析:采用软件SPSS 22.0计算平均值和标准差,使用Duncan’s分析各个处理之间的差异显著性。
2 结果与分析
2.1 菌株筛选
将16株菌接种于愈创木酚PDA固体培养基和苯胺蓝PDA固体培养基上,测量显色圈和变色圈直径（表1）。在愈创木酚PDA固体培养基上生长第6天时,除ZT002、ZT299外,均产生红褐色的显色圈。其中ZT153菌落直径与显色圈直径的比值最大,为1.40,除ZT-070、ZT-079外,菌落直径与显色圈直径的比值显著高于其他菌株,因此判断ZT-153具有较高的漆酶活性,用于后续实验。在苯胺蓝PDA固体培养基上,ZT-070接种第5天,菌丝铺满培养基,菌落直径为84.33mm,未出现褪色圈。其他菌株均产生褪色圈,其中ZT-307的菌落直径为61.36mm;褪色圈直径最大,为59.39mm;变色圈直径与显色圈直径比值为0.97。ZT-218的变色圈直径与显色圈直径比值最大为0.98,与ZT-307无显著性差异,根据褪色圈的大小判断ZT-218和ZT-307具有较强的产过氧化物酶能力,因ZT-307的菌丝直径和褪色圈直径显著高于ZT-218,因此选择ZT-307用于后续试验。
表1 愈创木酚平板显色和苯胺蓝平板褪色结果
Table 1
| 菌株 Strain | 愈创木酚平板显色 Guaiacol-containing plate coloration | 苯胺蓝平板褪色 Aniline blue-containing plate decolorization | ||||
|---|---|---|---|---|---|---|
| 菌落直径 Colony diameter (mm) | 显色圈直径 Coloration circle diameter (mm) | 显色圈直径/ 菌落直径 Coloration circle diameter/ Colony diameter | 菌落直径 Colony diameter (mm) | 褪色圈直径 Decolorization circle diameter (mm) | 褪色圈直径/ 菌落直径 Decolorization circle diameter/ Colony diameter | |
| ZT-002 | 51.33±3.35 cd | 0.00 | 0 | 57.93±1.39 fg | 51.05±3.50 b | 0.88±0.04 b |
| ZT-003 | 49.12±2.78 cd | 53.61±3.03 de | 1.09±0.02 cd | 58.91±4.11 f | 26.88±2.67 fg | 0.46±0.04 g |
| ZT-014 | 63.27±3.25 b | 65.15±3.85 b | 1.03±0.05 cde | 76.08±1.98 bc | 50.88±2.59 b | 0.67±0.05 d |
| ZT-018 | 52.64±3.74 c | 54.01±4.63 de | 1.03±0.03 cde | 68.26±1.07 e | 24.36±8.02 g | 0.36±0.11 h |
| ZT-021 | 43.89±0.31 de | 51.63±1.24 e | 1.18±0.03 bc | 66.51±4.84 e | 36.94±7.44 de | 0.55±0.07 ef |
| ZT-070 | 47.95±10.91 cd | 59.97±1.17 c | 1.30±0.32 ab | 84.33±0.66 a | 0.00 | 0 |
| ZT-079 | 32.52±3.87 f | 44.02±2.13 f | 1.36±0.16 a | 36.16±1.27 g | 9.68±0.27 h | 0.27±0.00 h |
| ZT-123 | 53.74±3.51 c | 46.89±1.76 f | 0.87±0.08 e | 66.90±3.97 e | 32.50±0.27 ef | 0.49±0.03 fg |
| ZT-153 | 33.30±5.13 f | 46.45±5.54 f | 1.40±0.05 a | 54.79±1.52 gh | 15.76±4.36 h | 0.29±0.07 h |
| ZT-156 | 51.50±2.48 cd | 56.92±2.19 cd | 1.10±0.02 cd | 69.45±1.59 de | 40.42±2.92 d | 0.58±0.04 de |
| ZT-157 | 50.21±2.10 cd | 55.21±1.75 cde | 1.10±0.02 cd | 54.86±0.82 gh | 42.15±0.28 cd | 0.77±0.01 c |
| ZT-178 | 71.24±1.05 a | 68.35±1.75 b | 0.96±0.03 de | 77.23±1.16 b | 12.33±0.16 h | 0.16±0.00 i |
| ZT-197 | 72.54±1.57 a | 76.18±0.68 a | 1.05±0.03 cde | 72.64±0.84 cd | 48.13±1.74 bc | 0.66±0.02 d |
| ZT-218 | 40.66±1.28 e | 45.10±0.40 f | 1.11±0.04 cd | 47.53±0.43 i | 46.70±0.67 bc | 0.98±0.01 a |
| ZT-299 | 10±0 g | 0.00 | 0 | 52.97±2.49 h | 14.19±3.53 h | 0.27±0.08 h |
| ZT-307 | 33.35±4.32 f | 31.54±0.95 g | 0.95±0.10 de | 61.36±1.12 f | 59.39±1.37 a | 0.97±0.01 ab |
注:同列数值后不同小写字母表示在P<0.05水平上差异显著
Note: Different lowercase letters after the same column indicate significant differences at P<0.05 level.
2.2 菌株鉴定
ZT-153、ZT-307的形态特征分别与T. betulina和B. fumosa在李玉等（2015）和赵继鼎等（1998）研究中的描述相似。
图1
图1
基于ITS序列构建ZT-153、ZT-307及其相关种的系统发育树
Fig. 1
Phylogenetic tree analysis of ZT-153 and ZT-307 tested strains and their related species based on ITS sequences.
表2 试验菌株
Table 2
| 菌株 Strain | 名称 Name | 采集地 Collecting site |
|---|---|---|
| ZT-153 | 桦栓孔菌Trametes betulina (L.) Pilát | 黑龙江漠河Mohe, Heilongjiang |
| ZT-307 | 亚黑管孔菌Bjerkandera fumosa (Pers.) P. Karst. | 辽宁本溪Benxi, Liaoning |
2.3 菌株对玉米秸秆的预处理效果
以P. chrysosporium CGMCC 5.0776为对照菌株,使用上述试验筛选的T. betulina ZT-153和B. fumosa ZT-307对玉米秸秆进行预处理（图2）。利用王玉万和徐文玉（1987）所采用的方法,测定在玉米秸秆预处理33d时,3株菌对玉米秸秆中酸不溶木质素和纤维素降解效率。结果显示,3株菌均可对玉米秸秆中的木质素和纤维素产生降解作用,P. chrysosporium CGMCC 5.0776对玉米秸秆木质素的降解率为12.02%,对纤维素的降解率为29.63%;B. fumosa ZT-307对木质素的降解率最高,为21.87%,对纤维素的降解率为4.50%;T. betulina ZT-153对木质素的降解率达到13.60%,对纤维素的降解率为4.10%;其中T. betulina ZT-153和B. fumosa ZT-307对木质素的降解率较对照P. chrysosporium CGMCC 5.0776高1.58%和9.85%,但3株菌对木质素的降解率差异不显著;T. betulina ZT-153和B. fumosa ZT-307对纤维素的降解率均小于5%,显著低于P. chrysosporium CGMCC 5.0776（表3）。
图2
图2
三株菌株预处理玉米秸秆33d时的菌丝长势
A:空白玉米秸秆;B:Trametes betulina ZT-153;C:Bjerkandera fumosa ZT-307;D:Phanerochaete chrysosporium CGMCC 5.0776
Fig. 2
Mycelial growth of three tested strains after cornstalk pretreatment for 33d.
A: CK; B: T. betulina ZT-153; C: B. fumosa ZT-307; D: P. chrysosporium CGMCC 5.0776.
表3 三株菌对玉米秸秆的降解效果
Table 3
| 菌株 Strain | 纤维素含量 Cellulose content (%) | 纤维素降解率 Degradation rate of cellulose (%) | 酸不溶木质素含量 Acid-insoluble lignin content (%) | 酸不溶木质素降解率 Degradation rate of acid-insoluble lignin (%) |
|---|---|---|---|---|
| Phanerochaete chrysosporium CGMCC 5.0776 | 12.56±0.42 a | 29.63±2.35 a | 5.61±0.42 a | 12.02±6.52 a |
| Trametes betulina ZT-153 | 17.12±0.54 b | 4.10±3.06 b | 5.51±0.20 a | 13.60±3.19 a |
| Bjerkandera fumosa ZT-307 | 17.05±2.33 b | 4.50±1.57 b | 4.98±0.26 a | 21.87±4.18 a |
注:同列数值后不同小写字母表示在P<0.05水平上差异显著
Note: Different lowercase letters after the same column indicate significant differences at P<0.05 level.
2.4 菌株预处理玉米秸秆过程中酶活性的测定
利用T. betulina ZT-153和B. fumosa ZT-307预处理玉米秸秆,每5d取一次样,提取样品粗酶液,测定其漆酶、MnP和LiP活性（图3）。测定T. betulina ZT-153在预处理玉米秸秆过程中表现出漆酶和MnP活性,B. fumosa ZT-307仅检测到LiP活性。T. betulina ZT-153在预处理玉米秸秆过程中,在第20天时漆酶活力急剧下降,之后迅速上升,在第30天时漆酶活性达到最高值,为21.36U/g;检测到较低的MnP活性,呈现先增高后降低的趋势,在预处理玉米秸秆20天时,达到峰值,为0.32U/g。B. fumosa ZT-307在预处理玉米秸秆过程中,在第10天和第20天时木质素过氧化物酶均达到峰值,为15U/g,测定结果显示在第10在时,其LiP活性比较稳定。
图3
图3
两株菌预处理玉米秸秆过程中酶活性的变化
A:Trametes betulina ZT-153;B:Bjerkandera fumosa ZT-307
Fig. 3
Variations of the enzyme activities during the course of cornstalk pretreatment by two fungal strains.
A: T. betulina ZT-153; B: B. fumosa ZT-307.
3 讨论
本研究从16株白腐真菌菌株中,通过王玉万和徐文玉（1987）所采用的方法测定筛选的2株菌株对玉米秸秆酸不溶木质素的降解效果,其中T. betulina ZT-153和B. fumosa ZT-307对玉米秸秆的木质素降解率高于模式菌株P. chrysosporium CGMCC 5.0776。
研究者利用T. betulina处理山毛榉（Fukasawa et al. 2005）、毛白杨（Wang et al. 2014）、小麦秸秆（Knežević et al. 2013）等基质时,取得了较好的降解木质素的效果,而利用其降解玉米秸秆基质的研究鲜见报道。本研究中,T. betulina ZT-153对玉米秸秆酸不溶木质素的降解率为13.60%,与该菌株对其他基质的降解效果相比,其对玉米秸秆酸不溶木质素的降解优势低于其对其他基质的降解作用,推断一方面是由于同一菌株在不同的培养体系和培养条件下,其降解能力和产酶能力不同（Singh & Chen 2008）;同一菌株对不同基质的木质素降解能力不同（Adejoye & Fasidi 2009）;同种不同菌株的产酶能力也存在差异（张桐等 2018）。另一方面由于本研究利用72%浓硫酸对秸秆进行处理,过滤后所剩余的残渣即为木质素含量,一些木质素可能因溶解在硫酸中而流失,所测定的木质素含量仅是酸不溶木质素含量（王玉万和徐文玉 1987）。B. fumosa ZT-307是本研究中对玉米秸秆木质素降解率最高的菌株,而利用B. fumosa对不同基质木质素降解的研究未见报道。根据现有的文献调查,较其他白腐真菌菌株相比,B. fumosa对玉米秸秆酸不溶木质素的降解效率较高。吴雪君（2017）利用绒毛栓孔菌T. pubescens Cui 7571预处理玉米秸秆42d,玉米秸秆酸不溶木质素含量由原来的21.65%转变为17.20%,计算后可知其对玉米秸秆酸不溶木质素的降解率为20.55%,而B. fumosa ZT-307在对玉米秸秆预处理33d时酸不溶木质素的降解率已达到21.87%。
在白腐真菌预处理玉米秸秆的过程中,测定T. betulina ZT-153分泌漆酶和MnP,B. fumosa ZT-307仅分泌LiP。木质素的生物降解是一个复杂的反应过程,其酶活性的分泌依赖于菌株、底物和环境条件等因素。在木质素降解过程中,漆酶可能在一个菌株或培养物中起中心作用,而一些菌株在木质素降解过程中,仅表现出MnP或LiP活性或同时表现出漆酶与MnP活性,并在木质素降解过程中发挥关键作用（Sun et al. 2011）。T. betulina ZT-153在预处理玉米秸秆30d时,漆酶仍具有较高活性,且达到其30d内的最大值,可能是由于真菌自溶导致细胞内的酶释放到培养基中所致（Arora et al. 2002）。
本研究筛选出的2株菌均对玉米秸秆中的木质素具有降解作用,其中B. fumosa ZT-307对玉米秸秆酸不溶木质素的降解率较模式菌株P. chrysosporium CGMCC 5.0776高9.85%,可见该菌株在玉米秸秆预处理方面具有较大的开发利用前景,后续还可以对其预处理条件进行优化,或利用此菌株与其他菌株或物理化学方法相结合预处理玉米秸秆,增加其对木质素的降解效果。
致谢:
感谢吉林农业大学饶固和汪阳在系统发育树构建方面提供的技术支持。
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