本文關(guān)鍵詞： 菌劑 牛糞堆肥 微生物群落 多樣性 PCR-DGGGE　出處：《東北農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：畜禽糞便含有大量的微生物,其中包含病源微生物,直接排放到環(huán)境中,會(huì)對(duì)環(huán)境造成污染,集約化、規(guī)模化處理糞便最好的處理方法是堆肥化處理,而堆肥原材料中含有大量的難降解的木質(zhì)纖維素,向堆肥中添加菌劑是解決堆肥中難降解有機(jī)物是一種廣泛采用的方法。本研究使用木質(zhì)纖維素類復(fù)合菌劑作為外源菌劑進(jìn)行堆肥,通過(guò)測(cè)定堆體理化指標(biāo)、木質(zhì)纖維素降解率、木質(zhì)纖維素酶活研究添加外源菌劑對(duì)堆肥理化性質(zhì)及木質(zhì)纖維素的影響,并使用通用引物進(jìn)行聚合酶鏈?zhǔn)椒磻?yīng)-梯度變性凝膠電泳(PCR-DGGE)檢測(cè)堆肥中的細(xì)菌、真菌和放線菌,探討添加菌劑對(duì)堆肥微生物群落變化情況,研究結(jié)果如下:1.添加外源菌劑和未加菌劑的處理均在第2 d進(jìn)入高溫期(45℃),菌劑處理堆肥組于堆肥第3 d達(dá)到最高溫72℃,高于對(duì)照組堆肥(55℃),且添加菌劑處理的堆體高溫期持續(xù)21 d(2 d~22d),而對(duì)照組堆體僅持續(xù)15 d,而且高溫期的平均溫度菌劑堆肥明顯高于對(duì)照組;兩組堆肥的p H值均維持在7.8~9.0之間,在整個(gè)堆肥過(guò)程中菌劑處理堆肥的pH值均略低于對(duì)照堆肥,此結(jié)果顯示加入菌劑可使堆肥的p H值降低;兩種堆肥處理的含水率均呈下降趨勢(shì),堆肥第12 d含水率下降速率最大,堆肥結(jié)束后分別降至42%、48%;不同堆肥處理C/N均呈現(xiàn)下降趨勢(shì),菌劑堆肥在第31 d C/N達(dá)到19.5,率先達(dá)到腐熟標(biāo)準(zhǔn),而對(duì)照堆肥在第46 d C/N才達(dá)到20以下。因此,添加外源菌劑能夠提高堆肥溫度,延長(zhǎng)高溫期時(shí)間,并能夠促進(jìn)堆肥腐熟。2.接外源菌劑處理堆肥的木質(zhì)纖維素酶活在各個(gè)時(shí)期均高于對(duì)照堆肥,其中羧甲基纖維素鈉酶(CMCase)、濾紙酶和半纖維素酶最大值分別為1120 U·L-1、268 U·L-1、1681 U·L-1,而對(duì)照堆肥的木質(zhì)纖維素酶活最大值為959 U·L-1、209 U·L-1、1459 U·L-1;且木質(zhì)纖維素降解速率隨著酶活的變化而改變,除木質(zhì)素外,其他兩種木質(zhì)纖維素降解速率和酶活力隨堆肥p H值的減小而增大,當(dāng)堆肥第12 d時(shí),pH值(7.5)最小時(shí),纖維素和半纖維素的降解速率最大(19.07%,24%)。而經(jīng)過(guò)菌劑處理堆肥組的木質(zhì)素降解率和木質(zhì)素降解酶(漆酶)在堆肥過(guò)程中出現(xiàn)了雙峰值,菌劑對(duì)木質(zhì)纖維素中木質(zhì)素的降解有強(qiáng)效作用,整體上可以提高木質(zhì)纖維素降解速率和酶活。3.應(yīng)用PCR-DGGE技術(shù)分析菌劑堆肥過(guò)程中細(xì)菌、真菌和放線菌群落的變化。結(jié)果表明,隨著堆肥進(jìn)程,DGGE條帶表現(xiàn)出了明顯的動(dòng)態(tài)變化,高溫期和腐熟期出現(xiàn)了新的優(yōu)勢(shì)條帶。對(duì)DGGE條帶進(jìn)行UPGMA聚類分析結(jié)果表明:微生物群落多樣性變化規(guī)律為細(xì)菌最大,放線菌次之、真菌最小;細(xì)菌樣品之間的相似性在0.5~0.8之間,真菌樣品之間的相似性在0.14~0.84之間,放線菌樣品之間的相似性在0.5~0.9。對(duì)DGGE條帶進(jìn)行典型分析,提取兩因子進(jìn)行降維的主成成分分析,顯示菌劑組高溫期的條帶與自然組分別聚于不同組,兩者有明顯差異,綜合考慮,溫度和添加菌劑對(duì)微生物的群落演替起到了重要的限制作用,其對(duì)真菌群落演替的篩選作用大于細(xì)菌,對(duì)細(xì)菌篩選作用大于放線菌。對(duì)優(yōu)勢(shì)條帶回收測(cè)序的結(jié)果表明外源菌劑能夠成功定殖,在堆肥高溫期起主要優(yōu)勢(shì)細(xì)菌菌株可分為2類:厚壁菌門(Firmicutes)中的芽孢桿菌菌屬(Bacillus)和變形菌門(Proteobacteria)中的α-變形菌綱(α-proteobacteria)、β-變形菌綱(β-proteobacteria)、γ-變形菌綱(γ-proteobacteria);檢測(cè)到堆肥中真菌主要集中于2類,包括子囊菌門(Ascomycota)和擔(dān)子菌門(Basidiomycota),其中堆肥高溫期起主要優(yōu)勢(shì)作用的真菌菌株屬于子囊菌門(Ascomycota)的曲霉屬(Aspergillus)和短梗蠕孢屬(Trichocladium)、擔(dān)子菌門(Basidiomycota)的中的白腐真菌異擔(dān)孔菌屬(Heterobasidion);堆肥高溫期起主要作用的放線菌群落可分為3類:擬諾卡氏放線菌屬(Nocardiopsis sp.)、鏈霉菌屬(Streptomyces sp.)和節(jié)桿菌屬(Arthrobacter sp.)。檢測(cè)到三類微生物中高溫期優(yōu)勢(shì)菌株大部分為木質(zhì)纖維素降解菌,因此,添加外源菌劑能夠增加木質(zhì)纖維素降解菌的數(shù)量和種類。
[Abstract]:Animal manure contains a large number of microorganisms, including pathogenic microorganisms, directly discharged into the environment, will cause pollution to environment, intensive, large-scale processing method is the best manure composting, compost and wood fiber raw materials containing refractory large amounts of nutrients, to add compost compost inoculum is solved refractory organics is a widely used method. This study uses lignocellulosic composite agents as exogenous bacteria compost, through the determination of pile physicochemical indexes, lignocellulose degradation rate, study the effects of exogenous microbial agents on physicochemical properties and lignocellulose live lignocellulose enzyme, and the use of universal primers polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE) detection in composting of bacteria, fungi and actinomycetes, to investigate the effects of agents on composting microbial community changes, The results are as follows: 1. inoculant and without inoculum were second D into the period of high temperature (45 DEG C), treatment group in third compost compost inoculum D reached the highest temperature 72 degrees Celsius, higher than that of the control group (55 C), and the compost pile to add high temperature period agents processing for 21 d (2 D ~22d), while the control group pile lasted only 15 d, and the average temperature was high temperature composting period was significantly higher than the control group; the two group P compost H value was maintained at 7.8~9.0, in the whole composting process in composting agent treatment the pH value was slightly lower than that of the control compost, this results show that adding agents can make compost P H decreased; two aqueous composting rate decreased, compost twelfth D moisture content decreased the maximum rate, after composting were reduced to 42%, 48%; different composting C/N showed a downward trend in the thirty-first D C/N, microbial compost reached 19.5, has reached maturity and standard. The control was achieved at forty-sixth D C/N compost below 20. Therefore, inoculant can improve the composting temperature, extend the period of high temperature time, and.2. can promote the maturity of the compost treated with exogenous microbial agents compost wood cellulase activity in each period were higher than the control compost, the sodium carboxymethyl cellulose enzyme (CMCase), fpase and half the maximum value of cellulase were 1120 U - L-1268 U - L-11681 U - L-1, while the control compost wood cellulase maximum is 959 U - L-1209 U - L-11459 U - L-1; and lignocellulose degradation rate with the changes of enzyme activity change, in addition to lignin, the other two kinds of lignocellulose degradation rate and the enzyme activity decreases with the increase of P value with compost H, when compost twelfth D, pH value (7.5) of the hour, the cellulose and hemicellulose degradation rate (19.07%, 24%) maximum. After inoculated with compost group lignin degradation rate And the lignin degrading enzymes (Qi Mei) the Shuangfeng values appear in the process of composting inoculants have potent effects on lignin degradation of lignocellulose, the overall analysis could increase the activity of bacteria in composting process.3. the application of PCR-DGGE technology in lignocellulose degradation and enzyme, fungi and Actinomyces community changes. The results showed that and with the composting process, DGGE bands showed obvious dynamic change, high temperature period and maturity period showed an advantage of new belt. UPGMA cluster analysis results show that the DGGE band: the microbial community diversity of bacteria, actinomycetes and fungi bacteria samples between the minimum; the similarity between 0.5~0.8 fungi, the similarity between the samples between 0.14~0.84, put the similarity between actinomycetes samples were typical analysis of DGGE bands in 0.5~0.9. dimension extracted two principal component factor Analysis shows, agents group during the high temperature period bands and natural group were classified into different groups, there was significant difference between two groups, considering the temperature and additive limited effects on microbial community succession plays its role, screening of fungi community succession than bacteria, the bacteria screening effect than actinomycetes the dominant bands sequenced. Results show that exogenous microbial agents can successfully colonize in the high temperature period the main advantage of bacterial strains can be divided into 2 categories: Firmicutes (Firmicutes) in the genus Bacillus (Bacillus) and Proteobacteria (Proteobacteria) in alpha Proteobacteria (alpha -proteobacteria), beta Proteobacteria (beta -proteobacteria), gamma Proteobacteria (gamma -proteobacteria); detection of fungi in composting mainly concentrated in 2 categories, including Ascomycota and Basidiomycota (Ascomycota) (Basidiomycota), the main advantage of the high temperature period The role of fungi belonging to the Ascomycota (Ascomycota) Aspergillus (Aspergillus) and trichocladium (Trichocladium), Basidiomycota (Basidiomycota) of white rot fungi in the genus of bear hole (Heterobasidion); high temperature composting period plays a major role in the actinomycetes communities can be divided into 3 categories: quasi Nocardia Actinomyces (Nocardiopsis sp.), Streptomyces (Streptomyces sp.) and Arthrobacter sp. (Arthrobacter sp.). To detect three kinds of microbes in the high temperature period of dominant strains for the majority of lignocellulose degrading bacteria, therefore, exogenous agents could increase the number and types of lignocellulose degrading bacteria.

【學(xué)位授予單位】：東北農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S141.4

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