芦笋老茎栽培食用菌和培养料堆制过程中微生物多样性的研究
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摘要
芦笋(Asparagus officinalis L.)为百合科天门冬属多年生草本植物。在每年的芦笋收获季节过后,大量的芦笋老茎没有加以充分利用,只是任其自然腐败,或直接焚烧,既造成资源的极大浪费,也带来了严重的环境污染问题。因此,如何合理利用芦笋老茎这种农业废弃物,是一个亟待解决的问题。由于多种农业废弃物可以用来栽培食用菌,所以作为食用菌培养料是利用芦笋老茎的有效途径之一。本论文计划进行利用芦笋老茎栽培鲍鱼菇、黄白侧耳和巴西蘑菇的实验研究。重点针对芦笋老茎培养料堆制发酵期间的微生物多样性展开研究。蘑菇堆料是一个复杂而又独特的微生态系统,内部栖息着多种多样的微生物。依靠微生物在适宜条件下的代谢作用,堆料中的生物大分子物质被分解、转化,堆料的腐熟速度和大分子物质的降解程度都与堆料内的微生物种类和数量直接相关。由于芦笋老茎是一种不同于稻草、麦秆等秸秆的原料,所以其堆制发酵期间的微生物群落及其演替变化规律也必然不同于其他原料的堆料。如果要建立和完善针对芦笋老茎的堆制发酵工艺,进而得到一种高质量的蘑菇培养料,关于芦笋老茎堆制发酵期间微生物多样性的研究是非常必要的。在研究方法上,本论文计划应用16S rDNA文库技术分析芦笋老茎堆料微生物的多样性,应用实时荧光定量PCR技术对堆料发酵期间微生物的菌群演替进行定量分析。本论文利用芦笋老茎这种农业废弃物栽培食用菌和对芦笋老茎堆料发酵过程中的微生物多样性进行研究,是一个全新的尝试,一方面可以填补关于芦笋老茎栽培食用菌和芦笋老茎堆料微生物多样性的研究空白,为下一步利用芦笋老茎栽培食用菌、制备芦笋老茎堆料发酵菌剂以及调控堆料过程提供理论依据,另一方面对于探讨一条规模化利用芦笋老茎资源的新途径,促进农村生态循环经济发展具有重要意义。
首先进行了用芦笋老茎栽培鲍鱼菇和黄白侧耳的初步研究,分析了芦笋老茎培养料对鲍鱼菇和黄白侧耳多糖含量和抗氧化性的影响。在没有添加其他辅料的芦笋老茎培养料上,黄白侧耳的产量(199g／袋)比鲍鱼菇的产量(140g／袋)更高；在芦笋老茎培养料中添加适量的葡萄糖、MgSO4和K2HPO4有利于黄白侧耳子实体的发育,能导致黄白侧耳产量的增加；而对于鲍鱼菇来说,在芦笋老茎培养料中添加辅助性碳源和无机盐反而会导致产量的降低。在没有添加其他辅料的芦笋老茎培养料上,鲍鱼菇子实体多糖含量明显比黄白侧耳高。与对照相比,在芦笋老茎培养料中添加辅助性碳源和无机盐对黄白侧耳子实体多糖含量没有明显的影响,但却能导致鲍鱼菇子实体多糖含量的显著降低。从DPPH自由基清除能力的ECso值和还原能力的ECso值可以看出,在没有添加其他辅料的芦笋老茎培养料上,鲍鱼菇的抗氧化性强于黄白侧耳；添加蔗糖和MgSO4有利于提高两种蘑菇的抗氧化性。
设计了以芦笋老茎为主料,棉籽壳、玉米芯、干牛粪和杂木屑为辅料的4种培养料来栽培巴西蘑菇,采用二次发酵法制各培养料,玉米秸秆培养料作为对照。结果表明巴西蘑菇在芦笋老茎和玉米秸秆培养料上的产量没有明显差别；添加棉籽壳或干牛粪的芦笋老茎培养料其巴西蘑菇产量明显高于添加杂木屑或玉米芯的芦笋老茎培养料。芦笋老茎培养料上栽培得到的巴西蘑菇子实体的抗氧化性明显高于对照培养料上栽培得到的子实体的抗氧化性,但多糖含量却明显低于对照；4种辅料相比较,棉籽壳和玉米芯比干牛粪和杂木屑更有利于提高巴西蘑菇子实体的多糖含量,棉籽壳比玉米芯、干牛粪和杂木屑更有利于提高巴西蘑菇的抗氧化性。
采用稀释涂布法对芦笋老茎堆料不同发酵阶段6个样品中的嗜热可培养微生物进行了分离,共分离到菌落形态有明显区别的26个细菌菌株、23个放线菌菌株和27个真菌菌株。根据16S rDNA序列分析结果,26株细菌中13株为芽孢杆菌属(Bacillus),4株为假黄色单胞菌属(Pseudoxanthomonas),1株为短芽孢杆菌属(Brevibacillus),1株为肠杆菌属(Enterobacter),1株为Paenibacillus属,1株为短波单胞菌属(Brevundimonas),1株为不动杆菌属(Acinetobacter),1株为Amaricoccus属,1株为副球菌属(Paracoccus),2个菌株在GenBank数据库中未找到与其相似的已知细菌属的序列,分类地位待定。根据16SrDNA序列分析结果,23株放线菌中,4株的序列与白浅灰链霉菌(Streptomyces albogriseolus)同源性最高,5株的序列与热普通链霉菌(Streptomyces thermovulgaris)同源性最高,2株的序列与Streptomyces pseudogriseolus同源性最高,1株的序列与Streptomyces espinosus同源性最高,有11株在GenBank数据库中未找到与其相似的已知链霉菌种的序列,分类地位待定。根据菌落形态和个体形态特征观察结果,27株真菌中12株为腐质霉属(Humicola),8株为青霉属(Penicillium),2株为链格孢霉属(Alternaria),1株为拟青霉属(Paecilomyces),4株为毛霉属(Mucor)。从这些结果可以看出,芦笋老茎堆料中的优势的嗜热可培养微生物主要是芽孢杆菌(Bacillus spp.)、假黄色单胞菌(Pseudoxanthomonas spp.)、白浅灰链霉菌(Streptomyces albogriseolus)、热普通链霉菌(Streptomyces thermovulgaris)、腐质霉(Humicola spp.)、青霉(Penicillium spp.)和毛霉(Mucor spp.)。
为了能高效提取芦笋老茎堆料样品中微生物的总基因组DNA,从DNA提取和PCR扩增效果等方面对4种DNA提取方法(即溶菌酶法、蛋白酶K-CTAB法、试剂盒法和改进的试剂盒法)进行了比较研究。琼脂糖凝胶电泳显示,溶菌酶法可以提取到样品中的基因组DNA,有一定程度的弥散,且基因组的含量较低,对蛋白质的去除率不高。蛋白酶K-CTAB法对于基因组DNA的损伤较小,提高了蛋白质的去除率,较好地提取到条带单一的基因组DNA,但是浓度仍然很低。试剂盒法成功地提取到了浓度很高的基因组DNA,稍有弥散。改进的试剂盒法也较好地提取到了基因组DNA,但是弥散得很厉害。用溶菌酶法提取的基因组DNA为模板对16S rDNA片段进行了PCR扩增,结果均无目的产物。用蛋白酶K-CTAB法得到的基因组DNA为模板进行PCR扩增后,在约1600bp处得到了目的产物,但是浓度却很低。用试剂盒法提取的DNA为模板进行PCR扩增时,在约1600bp处扩增到了较浓的单一条带,并且大小符合16SrDNA片段的理论值。用改进的试剂盒法提取的基因组DNA为模板进行PCR扩增时没有得到任何产物。这些实验结果表明4种方法均可以提取到芦笋老茎堆料样品中微生物的总基因组DNA,试剂盒法效果最好,其次是蛋白酶K-CTAB法,另外两种方法提取效果无明显差异；用试剂盒法提取到的总基因组DNA进行的16S rDNA片段的PCR扩增效果最好。
分别构建了芦笋老茎堆料不同发酵阶段6个样品的细菌16SrDNA克隆文库,并进行了文库中细菌多样性及其系统发育关系的分析。共获得228个细菌16SrDNA克隆,运用微生物分类学和生物信息学的方法对16SrDNA克隆文库进行了分析,结果表明：228个细菌16SrDNA克隆可分为5大类群,即变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)和未确定分类地位(Unidentified)的类群。第一优势类群——放线菌门的16SrDNA克隆在堆料发酵的全部阶段都能够被检出,并且占到总数的33%,表明放线菌在芦笋老茎堆料发酵过程中起着重要的作用。第二优势类群——变形菌门又分为a、p、γ-变形菌亚群,其中γ-变形菌亚群具有支配地位。拟杆菌16SrDNA序列在发酵中期(建堆后第11天)并没有被克隆出来。文库覆盖率(Coverage C)、稀疏曲线(Rarefaction curve)、香侬指数(Shannon-Wiener index)、物种多样性指数(Schaol)和文库相似度指数(Cs)等文库评价指数表明,除个别文库之外,所建文库能较好地反映芦笋老茎堆料中绝大部分细菌的多样性。
与传统方法及其它微生物定量方法相比,实时荧光定量PCR可以对样品中的DNA进行直接分析,从而达到对微生物定量的目的。该方法不仅减少了培养计数各个环节中的误差,同时还具有特异性强、定量准确、重复性好、无PCR后处理的污染等优点。本实验利用实时荧光PCR技术对芦笋老茎堆料不同发酵阶段的3种嗜热放线菌进行了定量分析。结果表明,Thermobifida fusca、白浅灰链霉菌(Streptomyces albogriseolus)和热普通链霉菌(Streptomyces thermovulgaris)在整个堆料发酵过程中始终存在。其中,Thermobifida fusca和热普通链霉菌在发酵过程中的数量变化近似一条钟形曲线,即发酵初期递增,发酵中后期逐渐降低,它们的动态变化非常符合一般堆料发酵中嗜热放线菌的菌群演替过程。特别是Thermobifida fusca表现得尤为明显,顶峰时期(发酵中期)的菌群数量达到3.45×107/g堆料,约为发酵初期和后期的103～104倍,在所考察的3种菌中是优势菌群。作为秸秆腐熟剂常用的菌种,白浅灰链霉菌也体现出其在芦笋老茎堆料发酵中的价值,该菌不仅贯穿于整个发酵过程,而且数量一直稳定,只是是在发酵末期,数量稍微有所上升,达到2.40×105/g堆料。
Asparagus officinalis L. is a well known healthy vegetable, and now widely cultivated as an important economic crop all over the temperate world. In Shanxi Province of China, approximately100,000tons per year of asparagus is produced, therewith, large amounts of asparagus old stem are also produced every year, and generally, these asparagus old stem are considered as useless residue and discarded, which not only causes environmental pollution but also is a waste of this resource. The asparagus old stem could be a basic component of the substratum formulation used to grow edible mushrooms in this region. Therefore, one aim of this work is about how to assess the potential of asparagus old stem as a major material for mushrooms production. The other aim of this work is to study the microbial diversity of the mushroom compost made from asparagus old stem. Composting is not only a way to reduce agricultural waste and recycle nutrients, but can also to produce useful components, e.g. compost for mushroom production, soil conditioners, or growth media for horticultural plants. Since bacteria, actinomycetes and fungi are the main organisms responsible for the decomposition, much effect has been put into understanding the changes in the microbial biomass, community structure and activity during the composting process. A variety of methods including16S rDNA cloning library technology have been used to investigate the microorganisms during composting. Real-time fluorescent quantitative PCR (qRT-PCR) is seldom used to analyze the flora succession in the mushroom compost, and is a new application in the study on microbial developments of the asparagus old stem compost. It is significant that qRT-PCR not only facilitates us to exlpore dynamically the fermentation of asparagus old stem, but also improves plenty of data to further develop the decomposing inoculants of asparagus old stem. This work is a new exploration on the utilization of asparagus old stem and the analysis of microbial diversity of the mushroom compost made from asparagus old stem.
The potential of asparagus old stem as a raw material for cultivating Pleurotus abalonus and Pleurotus geesteranus was evaluated. The effects of asparagus old stem substrate on yield, polysaccharides content and antioxidant properties of these two mushrooms were studied. On non-supplemented asparagus old stem substrate, the yield of P. geesteranus (199g/bag) was significantly higher than that of P. abalones (140g/bag) while the polysaccharides content of P. abalones was significantly higher than that of P. geesteranus. Addition of appropriate amounts of glucose, MgSO4and K2HPO4to the substrate increased the mushroom yield of P. geesteranus significantly. Addition of saccharides and inorganic salts to the substrate had no remarkable effect on the polysaccharides content of P. geesteranus fruit bodies, whereas caused the significant decrease of the polysaccharides content of P. abalones fruit bodies compared with the control. According to the scavenging effect on1,1-diphenyl-2-picrylhydrazyl radicals and reducing power of ethanolic extracts from two mushrooms, it was shown that the antioxidant properties of P. abalones was superior to that of P. geesteranus on non-supplemented asparagus old stem substrate, and the supplementation of sucrose and MgSO4to the substrate was favorable to the enhancement of antioxidant activity of two mushrooms.
Influences of different asparagus old stem composts on yield, polysaccharide content and antioxidant activity of Agaricus blazei Murill (ABM) fruit bodies were analyzed. The result showed that no significant differences were found in mushroom yields between asparagus old stem compost and maize straw compost. Addition of appropriate amounts of cottonseed hull or dried cow dung increased the mushroom yield significantly higher as compared with sawdust and corncob. These results manifested that the asparagus old stem was suitable for cultivating ABM. The antioxidant activity of fruit bodies cultivated on asparagus old stem compost was significantly stronger than that on maize straw compost. But the polysaccharide content of fruit bodies cultivated on maize straw compost was significantly higher. Among four auxiliary materials, cottonseed hull and corncob were favorable to the synthesis of polysaccharide, and cottonseed hull was more helpful to improve the antioxidant activity of ABM fruit bodies than dried cow dung, sawdust and corncob.
The culturable thermophiles (including bacteria, actinomycetes and fungi) in the asparagus old stem compost samples were investigated. By dilution-plate method, twenty-six strains of bacteria, twenty-three stains of actinomycetes and twenty-seven strains of fungi were isolated from the samples. According to16S rDNA sequence analysis, preliminary identification of these strains of both bacteria and actinomycetes was done. Among twenty-six strains of bacteria, thirteen strains were identified as Bacillus spp., four strains as Pseudoxanthomonas spp., one strain as Brevibacillus sp., one strain as Enterobacter sp., one strain as Paenibacillus sp., one strain as Brevundimonas sp., one strain as Acinetobacter sp., one strain as Amaricoccus sp., one strain as Paracoccus sp. Two strains of bacteria could not be identified because there were no matched gene sequences of bacterial species to be found in the GenBank. Among twenty-three stains of actinomycetes, four strains showed highest similarity to Streptomyces albogriseolus, five strains to Streptomyces thermovulgaris, two strains to Streptomyces pseudogriseolus, one strain to Streptomyces espinosus. By observing the morphological characteristics, twelve strains of fungi were identified as Humicola spp., eight strains as Penicillium spp., two strains as Alternaria spp., one strain as Paecilomyces sp., four strains as Mucor spp. The results indicated that Bacillus spp., Pseudoxanthomonas spp., Streptomyces albogriseolus, Streptomyces thermovulgaris, Humicola spp., Penicillium spp. and Mucor spp. were the most frequently isolated thermophilic microorganisms from the asparagus old stem compost.
Experiments were performed to determine the influence of four DNA extraction methods (i.e. lysozyme, protease K-CTAB, commercial reagent box and modified commercial reagent box methods) on microbial genomic DNA in asparagus old stem compost samples. The results of agarose gel electrophoresis of microbial genomic DNA showed that the four methods could all be used to extract the microbial DNA from asparagus old stem compost samples. But, the band of DNA extracts using the commercial reagent box method was clearer than those using other methods. In addition, the yield of DNA extracted using the commercial reagent box method was the highest. The results from the experiments indicated that the commercial reagent box method was the most efficient for DNA extraction from asparagus old stem compost, followed by the protease K-CTAB method. There were no distinct differences between the lysozyme method and the modified commercial reagent box method. The profiles of PCR products of16S rDNA, amplified from genomic DNA using the four DNA extraction methods were analyzed. The results of agarose gel electrophoresis showed that the PCR amplification based on the commercial reagent box method achieved the best result. We draw a conclusion that the DNA extraction method was an important factor affecting the extraction of genomic DNA and the PCR amplification of16S rDNA.
With protocols in molecular biology, six libraries of16S rDNA were constructed respectively on the basis of the total microbial DNA in six samples of asparagus old stem compost at different fermentation stages, and the microbial diversities and their phylogenesis were analyzed.228clones of bacterial16S rDNA were obtained in this study. Analysis of microbial bioinformatics showed that these clones could be divided into five groups (i.e. Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and unidentified bacteria). The16S rDNA clones of Actinobacteria were possessed of33%, and detected at all of stages of fermentation, suggesting that strains of Actinobacteria play an important role in the composting process of asparagus old stem. Proteobacteria includes α,β and γ-type, in which y-type was dominant one, and accounted for90%of all colonies of this group. In the five groups of bacteria, the proteobacterial colonies at the early and middle stages of fermentation (from4th to11th day) accounted for82%of all colonies.16S rDNA sequence of Bacteroidetes was not cloned at the middle stage of fermentation (on11th day). Evaluation indexes of library, such as Coverage C, Rarefaction Curve, Shannon-Wiener index, Schaoi and Sorensen similarity index, indicated that the libraries constructed were able to reflect the diversity of most of bacteria in asparagus old stem compost except for a few ones.
Compared with the traditional and other methods of quantitative determination of microbes, qRT-PCR is used to analyze directly DNA in the samples to measure the numbers of microbes. Use of qRT-PCR not only reduces the errors produced during the plate count, but also has lots of advantages, such as more specificity, accurate quantitative determination, good repeatability and no pollution like the dispose related with PCR. In this experiment, qRT-PCR was utilized to analyze quantitatively the numbers of three strains of thermophilic actinomycetes during the fermentation of asparagus old stem compost. The results showed that three strains of actinomycetes presented all the time during the fermentation, among which the change in number of Thermobifida fusca and Streptomyces thermovulgaris was close to a bell curve, namely, their number increased with time in early days of fermentation, and fell in the later period. Their dynamic variation accorded with the flora succession of thermophilic actinomycetes in the general fermentation of compost. Especially, the most value of Thermobifida fusca achieved3.45×107/g compost which was103to104times higher than that in early or later days of fermentation, and was the dominant population among the three strains of actinomycetes tested. As a common microbe used in the decomposing inoculants, Streptomyces albogriseolus existed in the whole fermentation, whose number was invariable, merely increased by2.40×105/g compost in the later stage of the fermentation.
首先进行了用芦笋老茎栽培鲍鱼菇和黄白侧耳的初步研究,分析了芦笋老茎培养料对鲍鱼菇和黄白侧耳多糖含量和抗氧化性的影响。在没有添加其他辅料的芦笋老茎培养料上,黄白侧耳的产量(199g／袋)比鲍鱼菇的产量(140g／袋)更高；在芦笋老茎培养料中添加适量的葡萄糖、MgSO4和K2HPO4有利于黄白侧耳子实体的发育,能导致黄白侧耳产量的增加；而对于鲍鱼菇来说,在芦笋老茎培养料中添加辅助性碳源和无机盐反而会导致产量的降低。在没有添加其他辅料的芦笋老茎培养料上,鲍鱼菇子实体多糖含量明显比黄白侧耳高。与对照相比,在芦笋老茎培养料中添加辅助性碳源和无机盐对黄白侧耳子实体多糖含量没有明显的影响,但却能导致鲍鱼菇子实体多糖含量的显著降低。从DPPH自由基清除能力的ECso值和还原能力的ECso值可以看出,在没有添加其他辅料的芦笋老茎培养料上,鲍鱼菇的抗氧化性强于黄白侧耳；添加蔗糖和MgSO4有利于提高两种蘑菇的抗氧化性。
设计了以芦笋老茎为主料,棉籽壳、玉米芯、干牛粪和杂木屑为辅料的4种培养料来栽培巴西蘑菇,采用二次发酵法制各培养料,玉米秸秆培养料作为对照。结果表明巴西蘑菇在芦笋老茎和玉米秸秆培养料上的产量没有明显差别；添加棉籽壳或干牛粪的芦笋老茎培养料其巴西蘑菇产量明显高于添加杂木屑或玉米芯的芦笋老茎培养料。芦笋老茎培养料上栽培得到的巴西蘑菇子实体的抗氧化性明显高于对照培养料上栽培得到的子实体的抗氧化性,但多糖含量却明显低于对照；4种辅料相比较,棉籽壳和玉米芯比干牛粪和杂木屑更有利于提高巴西蘑菇子实体的多糖含量,棉籽壳比玉米芯、干牛粪和杂木屑更有利于提高巴西蘑菇的抗氧化性。
采用稀释涂布法对芦笋老茎堆料不同发酵阶段6个样品中的嗜热可培养微生物进行了分离,共分离到菌落形态有明显区别的26个细菌菌株、23个放线菌菌株和27个真菌菌株。根据16S rDNA序列分析结果,26株细菌中13株为芽孢杆菌属(Bacillus),4株为假黄色单胞菌属(Pseudoxanthomonas),1株为短芽孢杆菌属(Brevibacillus),1株为肠杆菌属(Enterobacter),1株为Paenibacillus属,1株为短波单胞菌属(Brevundimonas),1株为不动杆菌属(Acinetobacter),1株为Amaricoccus属,1株为副球菌属(Paracoccus),2个菌株在GenBank数据库中未找到与其相似的已知细菌属的序列,分类地位待定。根据16SrDNA序列分析结果,23株放线菌中,4株的序列与白浅灰链霉菌(Streptomyces albogriseolus)同源性最高,5株的序列与热普通链霉菌(Streptomyces thermovulgaris)同源性最高,2株的序列与Streptomyces pseudogriseolus同源性最高,1株的序列与Streptomyces espinosus同源性最高,有11株在GenBank数据库中未找到与其相似的已知链霉菌种的序列,分类地位待定。根据菌落形态和个体形态特征观察结果,27株真菌中12株为腐质霉属(Humicola),8株为青霉属(Penicillium),2株为链格孢霉属(Alternaria),1株为拟青霉属(Paecilomyces),4株为毛霉属(Mucor)。从这些结果可以看出,芦笋老茎堆料中的优势的嗜热可培养微生物主要是芽孢杆菌(Bacillus spp.)、假黄色单胞菌(Pseudoxanthomonas spp.)、白浅灰链霉菌(Streptomyces albogriseolus)、热普通链霉菌(Streptomyces thermovulgaris)、腐质霉(Humicola spp.)、青霉(Penicillium spp.)和毛霉(Mucor spp.)。
为了能高效提取芦笋老茎堆料样品中微生物的总基因组DNA,从DNA提取和PCR扩增效果等方面对4种DNA提取方法(即溶菌酶法、蛋白酶K-CTAB法、试剂盒法和改进的试剂盒法)进行了比较研究。琼脂糖凝胶电泳显示,溶菌酶法可以提取到样品中的基因组DNA,有一定程度的弥散,且基因组的含量较低,对蛋白质的去除率不高。蛋白酶K-CTAB法对于基因组DNA的损伤较小,提高了蛋白质的去除率,较好地提取到条带单一的基因组DNA,但是浓度仍然很低。试剂盒法成功地提取到了浓度很高的基因组DNA,稍有弥散。改进的试剂盒法也较好地提取到了基因组DNA,但是弥散得很厉害。用溶菌酶法提取的基因组DNA为模板对16S rDNA片段进行了PCR扩增,结果均无目的产物。用蛋白酶K-CTAB法得到的基因组DNA为模板进行PCR扩增后,在约1600bp处得到了目的产物,但是浓度却很低。用试剂盒法提取的DNA为模板进行PCR扩增时,在约1600bp处扩增到了较浓的单一条带,并且大小符合16SrDNA片段的理论值。用改进的试剂盒法提取的基因组DNA为模板进行PCR扩增时没有得到任何产物。这些实验结果表明4种方法均可以提取到芦笋老茎堆料样品中微生物的总基因组DNA,试剂盒法效果最好,其次是蛋白酶K-CTAB法,另外两种方法提取效果无明显差异；用试剂盒法提取到的总基因组DNA进行的16S rDNA片段的PCR扩增效果最好。
分别构建了芦笋老茎堆料不同发酵阶段6个样品的细菌16SrDNA克隆文库,并进行了文库中细菌多样性及其系统发育关系的分析。共获得228个细菌16SrDNA克隆,运用微生物分类学和生物信息学的方法对16SrDNA克隆文库进行了分析,结果表明：228个细菌16SrDNA克隆可分为5大类群,即变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)和未确定分类地位(Unidentified)的类群。第一优势类群——放线菌门的16SrDNA克隆在堆料发酵的全部阶段都能够被检出,并且占到总数的33%,表明放线菌在芦笋老茎堆料发酵过程中起着重要的作用。第二优势类群——变形菌门又分为a、p、γ-变形菌亚群,其中γ-变形菌亚群具有支配地位。拟杆菌16SrDNA序列在发酵中期(建堆后第11天)并没有被克隆出来。文库覆盖率(Coverage C)、稀疏曲线(Rarefaction curve)、香侬指数(Shannon-Wiener index)、物种多样性指数(Schaol)和文库相似度指数(Cs)等文库评价指数表明,除个别文库之外,所建文库能较好地反映芦笋老茎堆料中绝大部分细菌的多样性。
与传统方法及其它微生物定量方法相比,实时荧光定量PCR可以对样品中的DNA进行直接分析,从而达到对微生物定量的目的。该方法不仅减少了培养计数各个环节中的误差,同时还具有特异性强、定量准确、重复性好、无PCR后处理的污染等优点。本实验利用实时荧光PCR技术对芦笋老茎堆料不同发酵阶段的3种嗜热放线菌进行了定量分析。结果表明,Thermobifida fusca、白浅灰链霉菌(Streptomyces albogriseolus)和热普通链霉菌(Streptomyces thermovulgaris)在整个堆料发酵过程中始终存在。其中,Thermobifida fusca和热普通链霉菌在发酵过程中的数量变化近似一条钟形曲线,即发酵初期递增,发酵中后期逐渐降低,它们的动态变化非常符合一般堆料发酵中嗜热放线菌的菌群演替过程。特别是Thermobifida fusca表现得尤为明显,顶峰时期(发酵中期)的菌群数量达到3.45×107/g堆料,约为发酵初期和后期的103～104倍,在所考察的3种菌中是优势菌群。作为秸秆腐熟剂常用的菌种,白浅灰链霉菌也体现出其在芦笋老茎堆料发酵中的价值,该菌不仅贯穿于整个发酵过程,而且数量一直稳定,只是是在发酵末期,数量稍微有所上升,达到2.40×105/g堆料。
Asparagus officinalis L. is a well known healthy vegetable, and now widely cultivated as an important economic crop all over the temperate world. In Shanxi Province of China, approximately100,000tons per year of asparagus is produced, therewith, large amounts of asparagus old stem are also produced every year, and generally, these asparagus old stem are considered as useless residue and discarded, which not only causes environmental pollution but also is a waste of this resource. The asparagus old stem could be a basic component of the substratum formulation used to grow edible mushrooms in this region. Therefore, one aim of this work is about how to assess the potential of asparagus old stem as a major material for mushrooms production. The other aim of this work is to study the microbial diversity of the mushroom compost made from asparagus old stem. Composting is not only a way to reduce agricultural waste and recycle nutrients, but can also to produce useful components, e.g. compost for mushroom production, soil conditioners, or growth media for horticultural plants. Since bacteria, actinomycetes and fungi are the main organisms responsible for the decomposition, much effect has been put into understanding the changes in the microbial biomass, community structure and activity during the composting process. A variety of methods including16S rDNA cloning library technology have been used to investigate the microorganisms during composting. Real-time fluorescent quantitative PCR (qRT-PCR) is seldom used to analyze the flora succession in the mushroom compost, and is a new application in the study on microbial developments of the asparagus old stem compost. It is significant that qRT-PCR not only facilitates us to exlpore dynamically the fermentation of asparagus old stem, but also improves plenty of data to further develop the decomposing inoculants of asparagus old stem. This work is a new exploration on the utilization of asparagus old stem and the analysis of microbial diversity of the mushroom compost made from asparagus old stem.
The potential of asparagus old stem as a raw material for cultivating Pleurotus abalonus and Pleurotus geesteranus was evaluated. The effects of asparagus old stem substrate on yield, polysaccharides content and antioxidant properties of these two mushrooms were studied. On non-supplemented asparagus old stem substrate, the yield of P. geesteranus (199g/bag) was significantly higher than that of P. abalones (140g/bag) while the polysaccharides content of P. abalones was significantly higher than that of P. geesteranus. Addition of appropriate amounts of glucose, MgSO4and K2HPO4to the substrate increased the mushroom yield of P. geesteranus significantly. Addition of saccharides and inorganic salts to the substrate had no remarkable effect on the polysaccharides content of P. geesteranus fruit bodies, whereas caused the significant decrease of the polysaccharides content of P. abalones fruit bodies compared with the control. According to the scavenging effect on1,1-diphenyl-2-picrylhydrazyl radicals and reducing power of ethanolic extracts from two mushrooms, it was shown that the antioxidant properties of P. abalones was superior to that of P. geesteranus on non-supplemented asparagus old stem substrate, and the supplementation of sucrose and MgSO4to the substrate was favorable to the enhancement of antioxidant activity of two mushrooms.
Influences of different asparagus old stem composts on yield, polysaccharide content and antioxidant activity of Agaricus blazei Murill (ABM) fruit bodies were analyzed. The result showed that no significant differences were found in mushroom yields between asparagus old stem compost and maize straw compost. Addition of appropriate amounts of cottonseed hull or dried cow dung increased the mushroom yield significantly higher as compared with sawdust and corncob. These results manifested that the asparagus old stem was suitable for cultivating ABM. The antioxidant activity of fruit bodies cultivated on asparagus old stem compost was significantly stronger than that on maize straw compost. But the polysaccharide content of fruit bodies cultivated on maize straw compost was significantly higher. Among four auxiliary materials, cottonseed hull and corncob were favorable to the synthesis of polysaccharide, and cottonseed hull was more helpful to improve the antioxidant activity of ABM fruit bodies than dried cow dung, sawdust and corncob.
The culturable thermophiles (including bacteria, actinomycetes and fungi) in the asparagus old stem compost samples were investigated. By dilution-plate method, twenty-six strains of bacteria, twenty-three stains of actinomycetes and twenty-seven strains of fungi were isolated from the samples. According to16S rDNA sequence analysis, preliminary identification of these strains of both bacteria and actinomycetes was done. Among twenty-six strains of bacteria, thirteen strains were identified as Bacillus spp., four strains as Pseudoxanthomonas spp., one strain as Brevibacillus sp., one strain as Enterobacter sp., one strain as Paenibacillus sp., one strain as Brevundimonas sp., one strain as Acinetobacter sp., one strain as Amaricoccus sp., one strain as Paracoccus sp. Two strains of bacteria could not be identified because there were no matched gene sequences of bacterial species to be found in the GenBank. Among twenty-three stains of actinomycetes, four strains showed highest similarity to Streptomyces albogriseolus, five strains to Streptomyces thermovulgaris, two strains to Streptomyces pseudogriseolus, one strain to Streptomyces espinosus. By observing the morphological characteristics, twelve strains of fungi were identified as Humicola spp., eight strains as Penicillium spp., two strains as Alternaria spp., one strain as Paecilomyces sp., four strains as Mucor spp. The results indicated that Bacillus spp., Pseudoxanthomonas spp., Streptomyces albogriseolus, Streptomyces thermovulgaris, Humicola spp., Penicillium spp. and Mucor spp. were the most frequently isolated thermophilic microorganisms from the asparagus old stem compost.
Experiments were performed to determine the influence of four DNA extraction methods (i.e. lysozyme, protease K-CTAB, commercial reagent box and modified commercial reagent box methods) on microbial genomic DNA in asparagus old stem compost samples. The results of agarose gel electrophoresis of microbial genomic DNA showed that the four methods could all be used to extract the microbial DNA from asparagus old stem compost samples. But, the band of DNA extracts using the commercial reagent box method was clearer than those using other methods. In addition, the yield of DNA extracted using the commercial reagent box method was the highest. The results from the experiments indicated that the commercial reagent box method was the most efficient for DNA extraction from asparagus old stem compost, followed by the protease K-CTAB method. There were no distinct differences between the lysozyme method and the modified commercial reagent box method. The profiles of PCR products of16S rDNA, amplified from genomic DNA using the four DNA extraction methods were analyzed. The results of agarose gel electrophoresis showed that the PCR amplification based on the commercial reagent box method achieved the best result. We draw a conclusion that the DNA extraction method was an important factor affecting the extraction of genomic DNA and the PCR amplification of16S rDNA.
With protocols in molecular biology, six libraries of16S rDNA were constructed respectively on the basis of the total microbial DNA in six samples of asparagus old stem compost at different fermentation stages, and the microbial diversities and their phylogenesis were analyzed.228clones of bacterial16S rDNA were obtained in this study. Analysis of microbial bioinformatics showed that these clones could be divided into five groups (i.e. Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and unidentified bacteria). The16S rDNA clones of Actinobacteria were possessed of33%, and detected at all of stages of fermentation, suggesting that strains of Actinobacteria play an important role in the composting process of asparagus old stem. Proteobacteria includes α,β and γ-type, in which y-type was dominant one, and accounted for90%of all colonies of this group. In the five groups of bacteria, the proteobacterial colonies at the early and middle stages of fermentation (from4th to11th day) accounted for82%of all colonies.16S rDNA sequence of Bacteroidetes was not cloned at the middle stage of fermentation (on11th day). Evaluation indexes of library, such as Coverage C, Rarefaction Curve, Shannon-Wiener index, Schaoi and Sorensen similarity index, indicated that the libraries constructed were able to reflect the diversity of most of bacteria in asparagus old stem compost except for a few ones.
Compared with the traditional and other methods of quantitative determination of microbes, qRT-PCR is used to analyze directly DNA in the samples to measure the numbers of microbes. Use of qRT-PCR not only reduces the errors produced during the plate count, but also has lots of advantages, such as more specificity, accurate quantitative determination, good repeatability and no pollution like the dispose related with PCR. In this experiment, qRT-PCR was utilized to analyze quantitatively the numbers of three strains of thermophilic actinomycetes during the fermentation of asparagus old stem compost. The results showed that three strains of actinomycetes presented all the time during the fermentation, among which the change in number of Thermobifida fusca and Streptomyces thermovulgaris was close to a bell curve, namely, their number increased with time in early days of fermentation, and fell in the later period. Their dynamic variation accorded with the flora succession of thermophilic actinomycetes in the general fermentation of compost. Especially, the most value of Thermobifida fusca achieved3.45×107/g compost which was103to104times higher than that in early or later days of fermentation, and was the dominant population among the three strains of actinomycetes tested. As a common microbe used in the decomposing inoculants, Streptomyces albogriseolus existed in the whole fermentation, whose number was invariable, merely increased by2.40×105/g compost in the later stage of the fermentation.
引文
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