CN114806890A - 辣椒秸秆木质素降解菌剂组合及其应用 - Google Patents
辣椒秸秆木质素降解菌剂组合及其应用 Download PDFInfo
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Abstract
本发明公开了辣椒秸秆木质素降解菌剂组合及其应用,所述的菌剂组合包括黑管菌(Bjerkanderasp.)、哈茨木霉(Trichoderma harzianum)、漆斑菌属(Myrothecium sp.)、枝孢霉属(Cladsporiumsp.)以及无色杆菌(Achromobactersp.),这些菌株之间不存在拮抗作用,能够在降解的过程中互相协同充分实现原料物质转化与降解,在室内固态发酵对辣椒秸秆木质素的降解率达40.8%,对纤维素降解率达34.2%。
Description
技术领域
本发明属于微生物技术领域,具体涉及一种辣椒秸秆木质素高效降解菌剂组合,可用于辣椒秸秆中木质素快速降解以提高辣椒秸秆资源利用。
背景技术
国家大宗蔬菜产业技术体系统计,近年来我国辣椒年种植面积稳定在 2.1×106hm2以上,总产量达6.4×107t,相应的每年产生大量的辣椒秸秆。由于秸秆组分中的木质纤维素结构复杂,在自然条件下较难降解,其是一种由不同种类碳水化合物结合而成的复杂联合体,主要成分是木质素、纤维素和半纤维素;木质素包裹着纤维素和半纤维素,只有先将木质素降解才能释放纤维、半纤维素;且木质素是由苯基丙烷结构单元通过碳-碳键连接而成的三维空间高分子化合物,结构复杂难以降解,由此木质素成为木质纤维素降解的关键因素。
微生物法处理秸秆可有效地分解木质纤维素成分且菌剂中的有益微生物还能改善土壤微区环境以及生态群落结构,从而有助于土壤生态环境及土壤质量的改善与提高等,这一系列优点受到研究者的关注。目前对于以纤维素为主的玉米秸秆、水稻秸秆等研究较多而木质素含量高的辣椒秸秆研究较少,针对此类木质素降解的微生物法迫在眉睫,木质素降解菌剂的研发成为辣椒秸秆乃至农作物秸秆降解的技术瓶颈。
研究表明木质素的降解是通过胞外氧化酶和一些非特定的酶进行氧化反应,木质素被胞外酶降解为更小的单元后被吸收进入微生物细胞,然后被部分转化为酚类化合物,这些物质在氧化酶的作用下失去电子,随后因自由基间的相互作用自然聚合。该过程由多种微生物协同完成,真菌在降解过程中起主导作用。细菌对木质素的降解能力相对较弱,主要使木质素结构发生改性,增加水溶性,产生一种被修饰过的、可溶于水的、可酸沉淀的多聚木质素APPL(acid-precipitable polymeric lignin),使木质素更容易被真菌降解。本发明开发的降解辣椒秸秆木质素的复合菌系由真菌和细菌构成。
发明内容
为了解决辣椒秸秆废弃物不科学合理利用后导致资源浪费、环境污染等问题,本发明提供了一种降解辣椒秸秆木质素的复合菌剂,既能加快木质素的降解又能实现辣椒秸秆资源的高效利用。
为了实现上述目的,本发明采用以下技术方案:
辣椒秸秆木质素高效降解菌剂组合,包括黑管菌(Bjerkandera sp.),哈茨木霉(Trichoderma harzianum),漆斑菌属(Myrothecium sp.),枝孢霉属(Cladsporium sp.)以及无色杆菌(Achromobacter sp.)。
优选地,菌剂组合中活菌数比为1:1:1:1:1。
辣椒秸秆木质素的降解方法:将辣椒秸秆晒干后粉碎过筛至粉末状,并加入玉米秸秆粉末调节孔隙度,接种上述菌剂组合进行固态发酵。
与现有技术相比,本发明具有以下有益效果:
(1)黑管菌(Bjerkandera sp.)、哈茨木霉(Trichoderma harzianum)、漆斑菌属(Myrothecium sp.)、枝孢霉属(Cladsporium sp.)以及无色杆菌(Achromobacter sp.)均为有益微生物,且微生物之间互相不存在拮抗作用,能够在降解的过程中互相协同充分实现原料物质转化与降解。
(2)本发明的复合菌剂与单菌株Trichoderma harzianum相比,过氧化物酶酶活提高了17%、锰过氧化物酶酶活提高了15.8%、漆酶酶活提高了26.6%。
(3)组合菌剂进行室内固态发酵对辣椒秸秆木质素的降解率达40.8%,对纤维素降解率达34.2%。
附图说明
图1不同菌株木质素关键酶(锰过氧化物酶、过氧化物酶、漆酶)的酶活比较。
图2功能菌株的系统发育树。
图3不同复合菌系酶活比较。
图4不同pH对复合菌剂CS1酶活力的影响。
图5不同接种量复合菌剂CS1酶活力的影响。
图6不同处理辣椒秸秆木质素、纤维素降解率。
具体实施方式
实施例一 木质素降解菌株的筛选
1、木质素降解菌株酶活测定
在长沙保真生物科技有限公司购买的菌株:细菌为编号CB9的链霉菌(Streptomyces sp.)、编号CB12的无色杆菌(Achromobacter sp.)和编号CB38 的芽孢杆菌(Bacillus altitudinis);真菌分别是编号CF5的黑管菌(Bjerkandera sp.)、CF18哈茨木霉(Trichoderma harzianum)、CF30漆斑菌属(Myrothecium sp.)、 CF53枝孢霉属(Cladsporium sp.)、CF56烟曲霉(Aspergillus fumigatus)。连续检测7天测定其木质素过氧化物酶、锰过氧化物酶、漆酶的酶活,进一步筛选木质素降解能力强的菌株。将各个菌株在液态培养基(真菌液态培养基:称取新鲜去皮马铃薯200g切成小块用蒸馏水煮沸30分钟,纱布过滤,加入葡萄糖20g 定容至1L,pH自然,115℃,高压灭菌30min;细菌液态培养基:蛋白胨10g、酵母膏5g、NaCl 10g、蒸馏水1000mL,pH自然、115℃,灭菌30min)中进行发酵后得到菌悬液,以1%的接种量接种到产酶培养基中(碱木质素2g/L, K2HPO4 1g/L,MgSO4·7H2O0.1g/L,CaCl2 0.08g/L,FeSO4·7H2O 0.05g/L,MnCl2 0.02g/L,KH2PO4 1g/L,蛋白胨2g/L),30℃,180rpm摇床中培养,连续7天每天取1mL发酵培养液在4℃、离心10min,取其上清液作为粗酶液,测定木质素关键酶酶活(锰过氧化物酶、过氧化物酶、漆酶)。
漆酶(Lac)酶活测定:
通过在420nm处监测ABTS的氧化来测定。37℃,3mL反应体系,反应混合液含有2mL0.5mmol/L ABTS(溶于0.1mmol/L,pH 5.0的醋酸-醋酸钠缓冲溶液中),添加1mL粗酶液启动反应,在420nm下测定吸光度的变化值。一个酶活力单位(U)定义为每分钟氧化1μmol ABTS所需的酶量。
锰过氧化物酶(Mnp)酶活测定:
通过在468nm下监测2,6-DMP被氧化为3,3,5,5-四甲氧联对苯醌来测定。37℃,3mL反应体系,反应混合液含有50mmol/L pH 4.5的醋酸-醋酸钠缓冲液2.4mL,1.6mmol/LMnSO4溶液0.1mL,1.6mmol/L 2,6-DMP溶液0.1 mL,0.4mL粗酶液;37℃下加入1.6mmol/L的H2O2溶液0.1mL启动反应,测定最初3min内在468nm下吸光度值的变化。一个酶活力单位(U)定义为每分钟使1μmol的2,6-DMP氧化所需的酶量。
过氧化物酶(Lip)酶活测定:
通过在310nm下监测藜芦醇被氧化成藜芦醛来测定。3mL的反应体系,反应混合液含有0.24mmol/L黎芦醇1.85mL和粗酶液1.0mL,预热至37℃后加0.1mL 6mmol/L的H2O2启动反应,测定3min后在310nm下吸光度值的增加量。一个酶活力单位(U)定义为每分钟使1μmol藜芦醇氧化所需要的酶量。酶活计算方法:
ΔA:时间t内的吸光度变化值;b:比色皿厚度(cm);t:反应时间(min) V1:反应体系的体积(L);V2:粗酶液的体积(L);n:稀释倍数ε:摩尔消光系数(M-1·cm-1)
ABTS:ε420=36000M-1·cm-1;2,6-DMP:ε468=49600M-1·cm-1;VA:ε310=930000 M-1·cm-1
通过连续检测真菌、细菌的木质素关键酶,发现菌株CB9、CF56的酶活稳定性较差(见图1),故不在做后续研究,选定菌株CF5、CF18、CF30、CF53、 CB12、CB38作为复合菌群的备选菌株。
2、功能菌株的分子学鉴定
对组合中菌株提取DNA并扩增PCR,其中真菌PCR扩增选用18S rDNA通用引物;细菌PCR扩增选用16S rDNA通用引物;引物序列如下:
真菌18S rDNA引物序列:
ITS1:5'-TCCGTAGGTGAACCTGCGG-3'
ITS4:5'-TCCTCCGCTTATTGATATGC-3'
细菌16S rDNA引物序列:
27F:5'-AGAGTTTGATCCTGGCTCAG-3'
1492R:5'-TACGGYTACCTTGTTACGACTT-3'
PCR扩增片段送到公司测序,测序结果于NCBI数据库中进行Blast同源性比对,并在GenBank等基因数据库中找到相似性最大的菌株,获取其16S rDNA 基因序列和18S rDNA基因系列,使用MEGA7.0软件采用Neighbor-jioning法构建系统发育树,如图2所示,菌株CF5为黑管菌(Bjerkandera sp.),CF18为哈茨木霉(Trichoderma harzianum),CF30为漆斑菌属(Myrothecium sp.),CF53 为枝孢霉属(Cladsporium sp.),CB12为无色杆菌(Achromobacter sp.),CB38 为高地芽孢杆菌(Bacillus altitudinis)。
实施例二组合菌系的构建和优化
1、菌株之间拮抗实验
将菌株CF5、CF18、CF30、CF53、CB12、CB38两两接种于同一马铃薯葡萄糖培养基避光培养3天观察是否有菌株之间存在拮抗作用。表1表明各个菌株之间均不产生拮抗作用,可进行复合菌剂的构建。
表1菌株直接拮抗关系
+代表拮抗;-代表无拮抗
2、构建复合菌系
根据相关研究表明,在复合菌剂中只有5株菌是核心降解功能微生物,故将6株不拮抗的菌系进行随机组合构成一组含有5株菌的菌剂,组合如下:CS1: CF5、CF18、CF30、CF53、CB12;CS2:CF5、CF18、CF30、CF53、CB38; CS3:CF5、CF18、CF30、CB12、CB38;CS4:CF5、CF18、CF53、CB12、CB38; CS5:CF5、CF30、CF53、CB12、CB38;CS6:CF18、CF30、CF53、CB12、CB38。共6个组合,将组合中单菌株用液态培养基培养3天得到单菌株菌悬液,菌悬液过滤培养基,再将孢子液浓度为1×108cfu/ml的菌株按照体积比1:1:1: 1:1进行混合得到各组合菌剂。各组合菌剂接入液态产酶培养基,30℃,180rmp 摇床连续培养7天,每天取1mL粗酶液检测其酶活,评估各组合整体复合菌剂木质素酶活。
如图3所示,组合CS1(CF5、CF18、CF30、CF53、CB12)的锰过氧化物酶、过氧化物酶、漆酶显著高于其他组合,则选定CS1为最佳复合菌剂,作为后续研究。
3、复菌系的优化
利用单因素方法探究复合菌剂CS1在不同pH、接种量条件下的酶活,探索复合菌剂最佳产酶条件。先将复合菌剂按照不同的pH(5、6、7、8、9)进行培养,在第6天(复合菌剂CS1木质素酶活较高)取样测定木质素酶活;如图4所示,pH为7时酶活较高,则以此pH条件按照不同接种量(1%、3%、5%、 7%、9%)进行培养,如图5所示,接种量为3%时木质素酶活较其他有显著性差异。
实施例三 复合菌剂固态发酵验证
为了验证复合菌剂降解辣椒秸秆的能力,进行室内发酵试验,取样测定木质素、纤维素含量并计算降解率。根据复合菌系最佳的pH和接种量进行室内发酵培养,以辣椒秸秆和玉米秸秆为原材料,设置复合菌剂、复合菌剂的各个单菌株、市售堆肥菌剂(EM菌)进行28天的发酵、取样测定木质素、纤维素含量。其中辣椒秸秆提前晒干粉碎过20目筛子,玉米秸秆粉碎至1-2cm,两者按照2:1 的质量比混合,玉米秸秆在此起调节辣椒秸秆孔隙度的作用,用不含琼脂葡萄糖的MS培养基调节含水量至55%-65%,混匀称取30g置于250mL三角瓶,瓶口用6层纱布、封口膜进行包裹,设置115℃,30min进行灭菌,灭菌完成后放置55℃烘箱将三角瓶壁上水分烘干即可;在超净工作台接入3%菌剂,用纱布包裹瓶口,进行30℃避光培养28天,及时观察三角瓶内菌剂生长情况,用灭菌的MS培养基控制含水量。
木质素、纤维素测定计算方法如下:
选取有代表性的试样,105℃烘干并粉碎过20目,用四分法将样品保存至自封袋并编号。将自动纤维测定仪专用滤袋用2B铅笔编号后置于105℃烘箱中烘干,称重。称取风干秸秆样品0.5-0.6g,无损的转移至滤袋中。每个样品做2 个平行,另取2个空滤袋封口作为空白处理。将滤袋套在纤维仪玻璃支架上放入纤维仪洗涤室中,加入1.2L中性洗涤剂同时加入消泡剂。编制实验程序进行洗涤,完成后,将滤袋取出,放置于无粉吸水纸上晾干后。再用20mL丙酮冲洗2 次,热水冲洗2次,晾干后置于105℃烘箱中烘2h后,在干燥器中冷却30min 称重,直称至恒重(W1)。
称重后的滤袋,加入酸性洗涤剂和消泡剂。设置程序进行洗涤,晾干后置于 105℃烘箱中烘2h后,在干燥器中冷却30min称重,直称至恒重(W2,两次称量之差小于0.002g);将酸性洗涤纤维加入4℃预冷的72%硫酸,在20℃消化3h后过滤,并冲洗至中性。消化过程中溶解部分为纤维素,不溶解的残渣为酸性洗涤木质素和酸不溶灰分,将残渣烘干并再放入550±25℃高温炉中灼烧2 h,干燥器中冷却至室温后称重,直称至恒重(两次称量之差小于0.002g),即可得出酸性洗涤木质素和酸不溶灰分的含量。
中性洗涤纤维含量的计算:NDF(%)=W1/W×100;式中:W1-中性洗涤后残渣重(g);W-试样重(g);酸性洗涤纤维含量的计算:ADF(%)=W2/W×100;式中:W2-酸性洗涤后残渣重(g);W-试样重(g)。纤维素含量的计算:纤维素=ADF(%)-经72%硫酸处理后的残渣(%);酸性洗涤木质素(ADL)含量的计算:ADL(%)=残渣(%)-酸不溶灰分(硅酸盐,%)。
如图6可知,复合菌剂CS1对木质素的降解率达40.8%,对纤维素降解率达34.2%,其木质素降解与EM发酵菌相比提高了16%。
Claims (3)
1.辣椒秸秆木质素高效降解菌剂组合,其特征在于,包括黑管菌(Bjerkandera sp.),哈茨木霉(Trichoderma harzianum),漆斑菌属(Myrothecium sp.),枝孢霉属(Cladsporium sp.)以及无色杆菌(Achromobacter sp.)。
2.根据权利要求1所述的辣椒秸秆木质素高效降解菌剂组合,其特征在于,菌剂组合中活菌数比为1:1:1:1:1。
3.辣椒秸秆木质素的降解方法,其特征在于,将辣椒秸秆晒干后粉碎过筛至粉末状,并加入玉米秸秆粉末调节孔隙度,接种权利要求1所述的菌剂组合进行固态发酵。
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