木质纤维素降解真菌菌株筛选及对玉米秸秆的生物降解研究
Screening of lignocellulose degrading fungal strains and their biodegradation of corn straw
收稿日期: 2022-06-21 接受日期: 2022-07-25
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Received: 2022-06-21 Accepted: 2022-07-25
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作者简介 About authors
ORCID:ANQi(0000-0001-5917-2497) 。
以8株野生白腐真菌为研究对象,用愈创木酚筛选培养基对这些菌株进行产木质素酶能力的初筛,并探究初筛后所得菌株在玉米秸秆固态发酵时的漆酶活性及其对玉米秸秆木质纤维素的降解能力。研究结果表明,8株菌株在愈创木酚筛选培养基上均表现出较好的木质素降解酶活性,仅菌株Han 577的菌丝圈直径d1与变色圈直径d2之比大于1。菌株An 369、Han 202和Han 474在玉米秸秆上的最大漆酶活性要远远高于其他菌株,分别为(901.11±42.83)、(698.89±42.17)和(843.61±78.82) U/L。白蜡范氏孔菌An 369、云芝栓孔菌An 174、肺形侧耳An 279和硬毛革孔菌Han 474对玉米秸秆的酸不溶木素降解率均大于20%。云芝栓孔菌An 174、肺形侧耳An 279、梨生多年卧孔菌Han 202对玉米秸秆的纤维素降解率均大于20%。迷宫栓孔菌An 360和肺形侧耳An 279对玉米秸秆半纤维素降解率则大于40%。整体来看,肺形侧耳An 279表现出较好的秸秆降解效率。
关键词:
The ligninase producing capacity of 8 wild white-rot fungal strains was screened by guaiacol screening medium, and the laccase activity and lignocellulose degradation capability of degrading corn straw of the strains initially screened were investigated during solid state fermentation. The 8 strains showed good lignin-degrading enzyme activities on guaiacol screening medium. Only the ratio of colony diameter d1 to photochramic laps d2 of strain Han 577 was greater than 1. The maximum laccase activity of strains An 369, Han 202 and Han 474 on degrading corn straw was (901.11±42.83), (698.89±42.17) and (843.61±78.82) U/L, respectively. The acid insoluble lignin degradation rates of corn straw by Vanderbylia fraxinea An 369, Trametes versicolor An 174, Pleurotus pulmonarius An 279 and Coriolopsis trogii Han 474 were all more than 20%. The cellulose degradation rates of corn straw by T. versicolor An 174, P. pulmonarius An 279 and Perenniporia pyricola Han 202 were all more than 20%. The hemicellulose degradation rates of corn straw by T. gibbosa An 360 and P. pulmonarius An 279 were all more than 40%. On the whole, P. pulmonarius An 279 exhibited the best corn straw degradation efficiency.
Keywords:
本文引用格式
安琪, 员瑗, 戴玉成, 韩美玲.
AN Qi, YUAN Yuan, DAI Yucheng, HAN Meiling.
农作物秸秆是农业生产过程中主要的废弃物,2019年我国秸秆总产量约8.5亿t,其中玉米秸秆的总产量约2.8亿t,占我国秸秆总量的32.94% (国家统计局 2019;常洪艳 2021)。如何有效地利用农作物秸秆,构建农作物秸秆高效降解和转化的循环体系,既是现代农业所必须面临的难题,也是目前许多学者所急切解决的问题。此前,农业秸秆最主要的处理方式是焚烧和随意丢弃任其腐烂,这种处理措施对生态环境造成严重的影响。为此,我国推出了秸秆还田这一举措,不仅能够避免秸秆焚烧所引起的大气污染,还能够增强土壤肥力(甄静等 2017;曹志宏等 2018;An et al. 2021;Han et al. 2021;杨娜等 2021;张芳芳等 2021)。秸秆主要成分为木质纤维素,是重要的可再生资源,其中,玉米秸秆的三大组分含量分别约为木质素(23.68%)、纤维素(41.93%)和半纤维素(24.71%) (Ding et al. 2019),其中木质素分散于纤维素纤维之间,而半纤维素在木质素和纤维素的纤维之间贯穿存在并起连接作用,进而形成了非常牢固的、类似于“钢筋混凝土结构”的纤维素-半纤维素-木质素的结构(图1) (Saha 2003;Mosier et al. 2005;毕于运 2010;安琪 2016;李赫龙 2016;刘霄 2019)。木质素与纤维素和半纤维素所形成的“钢筋混凝土结构”限制了水解酶与纤维素的接触以及木质素自身非水溶性和化学结构的复杂性是导致秸秆难以被降解的主要因素(甄静等 2017)。虽然采用物理或化学的方法能够有效处理农作物秸秆,但通常需要严苛且较为极端的条件(Singh et al. 2015;薛林贵等 2017;Singh et al. 2020;Ma et al. 2020)。而微生物能够通过分泌酶对农作物秸秆进行发酵与降解,在处理农作物秸秆时具有反应条件温和及对环境无二次污染的优点。因此,分离和筛选高效农作物秸秆降解菌具有重要的意义。近年来,许多研究人员陆续分离得到了一些对秸秆具降解能力的菌株,如细菌、真菌和放线菌(Fujimoto et al. 2005;Li et al. 2020;Mei et al. 2020)。张爽(2018)从土壤中分离并筛选获得具纤维素分解能力的3株木霉属的菌株。于慧娟和郭夏丽(2019)通过小麦秸秆粉末平板、刚果红染色法和小麦秸秆的降解率来筛选高效的秸秆降解菌,获得了4株具降解秸秆能力的真菌。黄青盈等(2022)通过食堂的餐厨垃圾、废水和被废水污染的土壤分离、驯化获得具降解秸秆能力的细菌。然而,Shrivastava et al. (2011)通过秸秆降解实验分析发现真菌对秸秆的降解效果是最优的。张斯童等(2016)分析了对不同种类的微生物对秸秆的降解效果,结果表明真菌对秸秆的降解能力要优于放线菌和细菌。
图1
有研究表明,木质素的降解可以有效地提高纤维素的降解率,而当纤维素被水解时,半纤维素才可能被全部水解(Bourquin et al. 2002;Pollgioni et al. 2015)。因此,筛选出对木质纤维素降解效果好的,尤其是对木质素具有较强降解能力以打破木质素对整个降解过程不利影响的菌株是非常必要的。白腐真菌是一类使木质腐烂成为淡色的海绵状团块的腐生于树木或木材上的真菌,能够通过分泌胞外的木质纤维素酶来降解木质纤维素,且白腐菌是目前为止最主要的、最为有效的降解木质素的真菌,是整个碳素循环的中心,是目前已知的唯一能够在纯培养体系里便可将木素降解为CO2和H2O的一大类真菌(Kirk & Fenn 1982;李慧蓉 2005;Sánchez 2009;Yingkamhaeng et al. 2022)。我国幅员辽阔、森林植被种类繁多,白腐真菌资源极为丰富(Cui et al. 2019)。传统的降解木质素的酶主要包括漆酶、锰过氧化物酶和木素过氧化物酶,其中,漆酶在木质素降解过程中起着非常重要的作用(Arora & Gill 2001)。而降解纤维素和半纤维素的酶主要是纤维素酶(如内切葡聚糖酶、β-葡萄糖苷酶)和半纤维素酶(如木聚糖酶、葡聚糖酶)。
1 材料与方法
1.1 材料
1.1.1 供试菌株
本试验所用8株白腐真菌均从野外采集、分离和纯化,全部菌株现均保藏于廊坊师范学院。
1.1.2 试剂
纤维素含量检测试剂盒AKSU007C和半纤维素含量检测试剂盒AKSU008C购自北京盒子生工科技有限公司,2,2ʹ-连氮-双(3-乙基苯并噻唑-6-磺酸)[ABTS]购自Sigma-Aldrich公司,CTAB植物基因组DNA快速提取试剂盒购自北京艾德莱生物科技有限公司。
麦芽浸粉、蛋白胨和酵母浸粉购自北京奥博星生物技术有限责任公司,其他药品购自天津市致远化学试剂有限公司。
1.1.3 试验原料
玉米秸秆(不含叶)取自廊坊市,风干后研磨粒径大小为20-60目。
1.1.4 培养基
完全培养基(CYM):葡萄糖20 g,蛋白胨2 g,酵母浸粉2 g,MgSO4·7H2O 0.5 g,琼脂20 g,K2HPO4·3H2O 1 g,KH2PO4 0.46 g,去离子水1 L。愈创木酚筛选培养基:葡萄糖20 g,蛋白胨2 g,酵母浸粉2 g,MgSO4·7H2O 0.5 g,琼脂20 g,K2HPO4·3H2O 1 g,KH2PO4 0.46 g,愈创木酚0.4 mL,去离子水1 L。苯胺蓝筛选培养基:葡萄糖20 g,蛋白胨2 g,酵母浸粉2 g,MgSO4·7H2O 0.5 g,K2HPO4·3H2O 1 g,KH2PO4 0.46 g,苯胺蓝0.1 g,去离子水1 L。液体培养基:葡萄糖20 g,蛋白胨2 g,酵母浸粉2 g,MgSO4·7H2O 0.5 g,K2HPO4·3H2O 1 g,KH2PO4 0.46 g,去离子水1 L。所有培养基均需121 ℃高温高压蒸汽灭菌30 min。
1.2 方法
1.2.1 菌种的活化
在CYM固体培养基平板上对供试菌株An 369、An 174、An 360、An 279、Han 1504、Han 202、Han 474和Han 577进行活化,于26 ℃避光培养7 d。
1.2.2 菌种的筛选
分别在平板上取直径为5 mm的菌块,接种至含愈创木酚筛选培养基的培养皿上,培养6 d,采用十字划线法,分别测量菌丝生长直径和变色圈直径,每个菌株设置3个重复。
1.2.3 菌种的分子生物学鉴定
1.2.4 种子液制备及均质体准备
在活化好的平板上,利用打孔器打取5块直径为1 cm的菌饼接种至内含100 mL液体培养基的250 mL三角瓶中,于26 ℃、150 r/min避光振荡培养7 d,然后用内切式匀浆机对培养物进行匀浆(8 000 r/min、2 min)处理以制备均质体。
1.2.5 玉米秸秆发酵
按照种子液均质体体积:玉米秸秆质量=1:1的比例制作玉米秸秆培养基并在其中接种均质体3 mL,于恒温培养箱中26 ℃避光培养28 d后,取出样品,于55 ℃烘干至恒重。
1.2.6 粗酶液提取
上述玉米秸秆发酵物于恒温培养箱中避光培养35 d,每隔7 d取出玉米秸秆样品3 g,添加50 mL 50 mmol/L的醋酸-醋酸钠缓冲液(pH 5.2)并于10 ℃、150 r/min避光振荡浸提4 h,经滤纸过滤后得到的液体即为粗酶液。
1.2.7 酶活性测定
粗酶液在10 ℃、12 000 r/min离心20 min,所得上清液用于酶活性测定。
1.2.8 玉米秸秆的木质纤维素含量测定
玉米秸秆的木质素含量测定采用美国可再生能源实验室(NERL)的方法(Sluiter et al. 2008),具体为:取0.3 g玉米秸秆粉末于95%乙醇中抽提6 h,抽提结束后加入72%硫酸3 mL并于30 ℃水浴1 h,再加入84 mL去离子水使得上述体系(粉末和硫酸)被稀释至4%,然后放入到高温高压灭菌锅于121 ℃处理1 h。静置条件下冷却至室温,待酸不溶木素沉积,用预先恒重的G3玻璃滤器进行过滤,并用热的去离子水对残渣进行洗涤,至向洗液中加数滴10%的BaCl2溶液也不会产生沉淀为止。所得滤液用于测定酸溶木素,测定在240 nm处吸光值并乘以25 L/(g·cm)计算酸溶木素含量;滤器和所得残渣于105 ℃烘干至恒重,得酸不溶木素的质量。
玉米秸秆的纤维素和半纤维素总含量测定方法和计算方法均参照纤维素含量检测试剂盒AKSU007C和半纤维素含量检测试剂盒AKSU008C的说明书进行。
2 结果与分析
2.1 菌株鉴定结果
菌株的采集地、寄主等信息见表1,经分子生物学鉴定,分别为白蜡范氏孔菌Vanderbylia fraxinea、云芝栓孔菌Trametes versicolor、迷宫栓孔菌Trametes gibbosa、肺形侧耳Pleurotus pulmonarius、云芝栓孔菌Trametes versicolor、梨生多年卧孔菌Perenniporia pyricola、硬毛革孔菌Coriolopsis trogii和烟管孔菌Bjerkandera adusta。
表1 八株白腐真菌的采集地点、寄主、鉴定物种名和对应的GenBank号
Table 1
菌株 Strain | 采集地点 Locality | 寄主 Host | 鉴定物种名 Name | GenBank号 GenBank number |
---|---|---|---|---|
An 174 | 河南云台山 Yuntai Mountain, Henan Province | 阔叶树树桩 Stump of angiosperm tree | 云芝栓孔菌 Trametes versicolor | ON796501 |
An 279 | 河北邯郸青崖寨 Qingyazhai, Handan, Hebei Province | 阔叶树活树 Living angiosperm tree | 肺形侧耳 Pleurotus pulmonarius | ON796502 |
An 360 | 湖北襄阳紫贞公园 Zizhen Park, Xiangyang, Hubei Province | 垂柳活树 Living tree of Salix babylonica | 迷宫栓孔菌 Trametes gibbosa | ON796503 |
An 369 | 湖北襄阳羊祜山 Yanghu Mountain, Xiangyang, Hubei Province | 阔叶树死树桩 Dead stump of angiosperm | 白蜡范氏孔菌 Vanderbylia fraxinea | ON796504 |
Han 202 | 河北承德老西营村 Laoxiying Village, Chengde, Hebei Province | 梨树活树 Living tree of Pyrus sp. | 梨生多年卧孔菌 Perenniporia pyricola | ON796505 |
Han 474 | 河北廊坊自然公园 Nature Park, Langfang, Hebei Province | 阔叶树落枝 Fallen angiosperm branch | 硬毛革孔菌 Coriolopsis trogii | ON796506 |
Han 577 | 河北石家庄驼梁山 Tuoliang Mountain, Shijiangzhuang, Hebei Province | 阔叶树落枝 Fallen angiosperm branch | 烟管孔菌 Bjerkandera adusta | ON796507 |
Han 1504 | 河北承德雾灵山 Wuling Mountain, Chengde, Hebei Province | 白桦活树 Living tree of Betula platyphylla | 云芝栓孔菌 Trametes versicolor | ON796508 |
2.2 菌株初筛结果
在愈创木酚选择培养基平板上分别培养8株白腐真菌6 d,平板的变色情况见表2。根据菌株在愈创木酚筛选培养基上生长和变色情况,可将8株白腐真菌分为2类,分别是:(1) 菌株在平板上生成红褐色的变色圈且变色圈直径小于菌丝圈直径(d1/d2>1),如烟管孔菌Bjerkandera adusta Han 577;(2) 菌株在平板上生成红褐色的变色圈且变色圈直径大于菌丝圈直径(d1/d2<1),如其他7种白腐真菌。初步定性表明这8种白腐真菌均具有降解木质素的能力,后续对这些白腐真菌进行了玉米秸秆降解试验,对其降解能力进行评估。
表2 八株白腐真菌在愈创木酚选择培养基上的显色测定
Table 2
菌株 Species strain | 愈创木酚筛选培养基显色 Coloration of guaiacol screening medium | ||
---|---|---|---|
菌落直径d1 Colony diameter d1 (cm) | 变色圈直径d2 Photochramic laps d2 (cm) | d1/d2 | |
An 174 | 4.9±0.1 | 5.9±0.1 | 0.83 |
An 279 | 4.5±0.1 | 4.6±0.1 | 0.98 |
An 360 | 3.5±0.1 | 4.3±0.0 | 0.81 |
An 369 | 4.6±0.1 | 5.4±0.1 | 0.85 |
Han 202 | 2.0±0.1 | 3.8±0.1 | 0.53 |
Han 474 | 4.8±0.0 | 5.9±0.1 | 0.81 |
Han 577 | 5.4±0.1 | 4.9±0.1 | 1.10 |
Han 1504 | 4.0±0.1 | 5.2±0.1 | 0.77 |
2.3 玉米秸秆降解过程中漆酶酶活性分析
供试菌株中,除烟管孔菌B. adusta Han 577外,其余7株在玉米秸秆上发酵的第7天、第14天、第21天和第28天均检测到漆酶活性(图2)。白腐真菌白蜡范氏孔菌V. fraxinea An 369、迷宫栓孔菌T. gibbosa An 360和云芝栓孔菌T. versicolor Han 1504产漆酶活性趋势相似,均在发酵的第7天漆酶活性最高,并于第21天达另一个漆酶活性小高峰;而云芝栓孔菌T. versicolor An 174、肺形侧耳P. pulmonarius An 279、梨生多年卧孔菌P. pyricola Han 202和硬毛革孔菌C. trogii Han 474的产漆酶活性趋势相似,均于发酵的第7天漆酶活性最高并在此后一直下降(图2)。白蜡范氏孔菌V. fraxinea An 369、云芝栓孔菌T. versicolor An 174、迷宫栓孔菌T. gibbosa An 360、肺形侧耳P. pulmonarius An 279、云芝栓孔菌T. versicolor Han 1504、梨生多年卧孔菌P. pyricola Han 202、硬毛革孔菌C. trogii Han 474和烟管孔菌B. adusta Han 577的最大漆酶活性分别为(901.11±42.83)、(207.13±1.40)、(282.92±15.16)、(171.89±10.22)、(70.95±6.19)、(698.89±42.17)、(843.61±78.82)和(0.50±0) U/L。由此可见,V. fraxinea An 369的最大漆酶活性高达(901.11±42.83) U/L,分别为T. versicolor An 174、T. gibbosa An 360、P. pulmonarius An 279、T. versicolor Han 1504、P. pyricola Han 202、C. trogii Han 474和B. adusta Han 577最大漆酶活性的4.35倍、3.19倍、5.24倍、12.70倍、1.29倍、1.07倍和1 802.22倍。相对而言,V. fraxinea An 369、P. pyricola Han 202和C. trogii Han 474最大漆酶活性均很高。整体而言,C. trogii Han 474在检测的整个阶段漆酶活性表现均较高,而烟管孔菌B. adusta Han 577在检测的整个阶段漆酶活性表现极差,几乎检测不到。
图2
图2
八株白腐真菌在玉米秸秆上发酵时漆酶活性
Fig. 2
Laccase activity of eight white rot fungal strains fermentated on corn straw.
2.4 八株白腐真菌对玉米秸秆的降解分析
供试菌株在玉米秸秆上培养28 d后,不同菌株对玉米秸秆木质纤维素降解情况见表3。这8株白腐真菌对木质素降解率可以分为3个类别,分别是:(1) 酸不溶木素降解率高于20%,包括V. fraxinea An 369 (23.23%)、T. versicolor An 174 (23.75%)、P. pulmonarius An 279 (22.49%)和C. trogii Han 474 (24.10%);T. versicolor Han 1504、P. pyricola Han 202和B. adusta Han 577的酸不溶木素降解率介于10%-20%之间,而T. gibbosa An 360的酸不溶木素降解率低于10%。(2) 纤维素降解率>20%,包括T. versicolor An 174 (26.14%)、P. pulmonarius An 279 (21.11%)、P. pyricola Han 202 (23.48%);V. fraxinea An 369、T. gibbosa An 360、T. versicolor Han 1504和C. trogii Han 474的纤维素降解率介于10%-20%之间,而B. adusta Han 577的纤维素降解率仅为4.79%。(3) 半纤维素的降解率:T. gibbosa An 360和P. pulmonarius An 279的半纤维素降解率最高,分别为40.82%和40.66%;B. adusta Han 577降解率为31.04%;V. fraxinea An 369、T. versicolor An 174、T. versicolor Han 1504和P. pyricola Han 202的半纤维素降解率介于10%-20%之间,而C. trogii Han 474的半纤维素降解率为8.28%。整体而言,P. pulmonarius An 279表现出最好的木质纤维素降解效果,其对玉米秸秆的纤维素、半纤维素和木质素的降解率分别为21.11%、40.66%和22.49%。
表3 八株白腐真菌对玉米秸秆中木质纤维素的降解率
Table 3
菌株 Strain | 酸不溶木素降解率 Degradation rate of acid insoluble lignin (%) | 纤维素降解率 Degradation rate of cellulose (%) | 半纤维素降解率 Degradation rate of hemicellulose (%) |
---|---|---|---|
白蜡范氏孔菌 V. fraxinea An 369 | 23.23 | 16.62 | 18.12 |
云芝栓孔菌 T. versicolor An 174 | 23.75 | 26.14 | 13.17 |
迷宫栓孔菌 T. gibbosa An 360 | 8.43 | 18.33 | 40.82 |
肺形侧耳 P. pulmonarius An 279 | 22.49 | 21.11 | 40.66 |
云芝栓孔菌 T. versicolor Han 1504 | 19.64 | 19.46 | 11.88 |
梨生多年卧孔菌 P. pyricola Han 202 | 18.90 | 23.48 | 12.18 |
硬毛革孔菌 C. trogii Han 474 | 24.10 | 12.36 | 8.28 |
烟管孔菌 B. adusta Han 577 | 14.80 | 4.79 | 31.04 |
3 讨论
木质纤维素的真菌生物降解是环境友好的农业废弃物利用方式,此前有较多研究关注真菌降解山毛榉、毛白杨和圆柏枝等基质,而对于作物秸秆的关注度整体较少(Fukasawa et al. 2005;Wang et al. 2014;康跃等 2019)。目前,涉及到秸秆降解的白腐真菌主要有Pleurotus ostreatus、Trametes versicolor、Phanerochaete chrysosporium、Coriolopsis gallica、Pleurotus sajor-caju、Lentinula edodes、Ceriporiopsis subvermispora、T. betulina、Bjerkandera fumosa和T. hirsute (Wan & Li 2010;Shrivastava et al. 2011;甄静等 2017;Ding et al. 2019;李灵灵等 2020;张芳芳等 2021),而本研究所使用的白腐真菌云芝栓孔菌Trametes versicolor An 174、肺形侧耳Pleurotus pulmonarius An 279、迷宫栓孔菌T. gibbosa An 360、白蜡范氏孔菌Vanderbylia fraxinea An 369、梨生多年卧孔菌Perenniporia pyricola Han 202、硬毛革孔菌Coriolopsis trogii Han 474、烟管孔菌B. adusta Han 577和云芝栓孔菌T. versicolor Han 1504有被此前研究使用的真菌物种,也添加了新的物种进行对比分析。Ceriporiopsis subvermispora在玉米秸上发酵42 d,对玉米秸的木质素和半纤维素的降解率分别为39.2%和27.0%,而对纤维素的降解率更是低于5% (Wan & Li 2010)。甄静等(2017)分析T. hirsuta XYG422对玉米秸秆的生物降解,发现T. hirsuta XYG422在培养60 d时对玉米秸秆中木质素、纤维素和半纤维素的降解率依次为83.54%、50.65%和19.53%。Ding et al. (2019)分析了T. versicolor CICC 50001、Pleurotus ostreatus CGMCCC 5.374、Phanerochaete chrysosporium CICC 40299、C. gallica CICC 2689、P. sajor-caju CGMCCC 5.593和L. edodes CGMCCC 5.697在玉米秸秆上发酵30 d的木质纤维素组分含量,发现对纤维素、半纤维素和木质素降解率最高的分别是C. gallica CICC 2689 (6.2%)、L. edodes CGMCCC 5.697 (5.34%)和P. sajor-caju CGMCCC 5.593 (4.49%)。T. betulina ZT-153、B. fumosa ZT-307和Phanerochaete chrysosporium CGMCC 5.0776在玉米秸秆上发酵33 d对玉米秸秆酸不溶木质素的降解率为13.60%、21.87%和12.02% (张芳芳等 2021)。而本研究中白蜡范氏孔菌V. fraxinea An 369、云芝栓孔菌T. versicolor An 174、肺形侧耳P. pulmonarius An 279和硬毛革孔菌C. trogii Han 474的酸不溶木素降解率分别为23.23%、23.75%、22.49%和24.10%,由此可见,这4种真菌的木质素降解率较好。T. versicolor BYL-7优化前对水稻秸秆纤维素降解率为12.0%,优化后对水稻秸秆纤维素降解率为13.2% (李灵灵等 2020)。T. betulina ZT-153、B. fumosa ZT-307和P. chrysosporium CGMCC 5.0776在玉米秸秆上发酵33 d对玉米秸秆纤维素降解率分别为17.12%、17.05%和12.56% (张芳芳等 2021)。而本研究中的云芝栓孔菌T. versicolor An 174、肺形侧耳P. pulmonarius An 279、梨生多年卧孔菌P. pyricola Han 202的纤维素降解率分别为26.14%、21.11%和23.48%,与此前研究相比,展现出优异的纤维素降解能力。T. versicolor BYL-7优化前对水稻秸秆半纤维素降解率为17.9%,优化后对水稻秸秆纤维素降解率为21.5% (李灵灵等 2020),而本研究中T. gibbosa An 360、P. pulmonarius An 279和B. adusta Han 577对玉米秸秆半纤维素降解率分别是40.82%、40.66%和31.04%,与之前研究所用菌株相比,半纤维素降解能力极为优异。由此可见,P. pulmonarius An 279对玉米秸秆木质纤维素的降解效果是这8株白腐真菌中最佳的,具高效降解玉米秸秆木质纤维素的能力。
白腐真菌分泌多种胞外酶来降解玉米秸秆的木质纤维素,漆酶是重要的木质素降解酶之一。本研究中,V. fraxinea An 369、C. trogii Han 474和P. pyricola Han 202的最大漆酶活性分别高达(901.11±42.83)、(843.61±78.82)和(698.89±42.17) U/L,而T. versicolor Han 1504和B. adusta Han 577的最大漆酶活性分别为(70.95±6.19) U/L和(0.50±0) U/L。同时,T. versicolor An 174和T. versicolor Han 1504为同种真菌的不同菌株,其最大漆酶活性分别为(207.13±1.40) U/L和(70.95±6.19) U/L。由此可见,不同真菌或同种真菌不同菌株的产漆酶能力存在较大差异。此前已有研究表明,同种真菌的不同菌株在同一培养条件下所分泌的漆酶活性并不相同(Janusz et al. 2015;An et al. 2016)。另一方面,V. fraxinea An 369、C. trogii Han 474和P. pyricola Han 202的最大漆酶活性虽然是最高的,但其对玉米秸秆的酸不溶木素降解率分别为23.23%、24.10%和18.90%,而T. versicolor Han 1504和B. adusta Han 577的最大漆酶活性虽然很低,但其对玉米秸秆的酸不溶木素降解率却分别高达19.64%和14.80%。同样地,T. gibbosa An 360的最大漆酶活性虽远大于B. adusta Han 577,但其对玉米秸秆的酸不溶木素降解率低于B. adusta Han 577。由此可见,通过漆酶活性和酸不溶木素降解率联合分析不难发现漆酶活性的高低并不与酸不溶木素降解率呈现相关性,这可能与其他木质素降解酶(如锰过氧化物酶和木素过氧化物酶等)也能够降解木质素有关。整体来看,本研究中所用大部分白腐真菌菌株对玉米秸秆木质纤维素均表现出较高的降解能力,研究结果为白腐真菌在农作物秸秆的综合利用奠定了良好的基础并展现了广阔的前景。
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一株纤维素降解菌的筛选与鉴定
DOI:10.11924/j.issn.1000-6850.casb2021-0131
[本文引用: 1]
为了解决玉米秸秆降解率低下问题,本研究从以氨基酸尾液为氮源的自然发酵堆肥中筛选稳定的纤维素降解菌,为制备高效的秸秆腐熟剂奠定基础。采用刚果红染色法进行初筛、采用纤维素酶活测定及玉米秸秆降解率测定来进行复筛,并通过形态学及分子生物学方法对高效降解菌株进行鉴定。结果表明筛选得到一株高效降解纤维素的菌株SC2,其滤纸酶活、内切葡聚糖酶活、外切葡聚糖酶活和β-葡聚糖苷酶活分别为17.70、58.97、16.85和79.26 U/mL,对玉米秸秆的降解率达到33.07%,根据其菌落特征、产孢结构、孢子形态及ITS序列鉴定SC2为黑曲霉(Aspergillus niger)。菌株SC2具有较好的纤维素降解能力,能够有效的促进秸秆的降解,可以用于制备玉米秸秆腐熟剂。
秸秆降解菌的筛选及其纤维素降解性能的研究
DOI:10.13560/j.cnki.biotech.bull.1985.2018-0731
[本文引用: 1]
通过秸秆粉平板、刚果红染色法以及小麦秸秆降解率来筛选高效秸秆降解菌,并测定菌株的胞外纤维素酶活来研究其秸秆纤维素的降解能力。结果表明,在所筛选的4株真菌中,菌株z-5的秸秆降解率最高,为47%;其次是菌株4-1、3-1和2-2,分别为45%、32%和31%,滤纸腐解实验结果与秸秆降解试验结果相似。4株秸秆降解菌均具有外切葡聚糖酶、内切葡聚糖酶和β-葡萄糖苷酶等3种纤维素酶,但变化趋势不同。菌株z-5和菌株4-1的三种酶活呈现出较好的协同性,菌株2-2和菌株3-1协同性较弱。通过秸秆粉平板,秸秆降解率测定以及滤纸孔洞法可以更准确快速地得到目的菌株;纤维素酶在秸秆降解中起着主导作用。
毛栓孔菌XYG422菌株产漆酶发酵条件优化及对玉米秸秆生物降解的研究
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