| 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. |
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