本文關(guān)鍵詞： 病死豬尸體 堆肥 PCR-DGGE 多樣性 微生物發(fā)酵　出處：《華中農(nóng)業(yè)大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

【摘要】：集約化養(yǎng)殖業(yè)迅猛發(fā)展的同時(shí),也造成來自畜禽糞便、污水以及病死畜禽殘?bào)w的嚴(yán)重污染。相對(duì)于畜禽糞便對(duì)環(huán)境的污染,死畜禽由于攜帶大量的病原微生物,如果不及時(shí)處理,對(duì)人和動(dòng)物的危害更大更直接。據(jù)統(tǒng)計(jì),養(yǎng)殖場(chǎng)的畜禽死亡率在5%~10%之間,而生豬的死亡率在8%~12%之間,遇到重大疫情,死亡率還會(huì)升高。因此,對(duì)死畜禽進(jìn)行無害化處理是解決環(huán)境污染,實(shí)現(xiàn)畜牧業(yè)良性發(fā)展的必由之路。堆肥是一個(gè)可被生物降解的有機(jī)質(zhì)堆,通過好氧微生物分解成穩(wěn)定的、類似土壤的腐殖質(zhì)過程。病死豬堆肥相對(duì)于傳統(tǒng)的死豬處理方法,如,掩埋、焚燒、高溫熬煮等,具有更加安全和環(huán)保的優(yōu)勢(shì)。堆肥過程的物質(zhì)轉(zhuǎn)化歸根到底是由堆肥體系中微生物的代謝活動(dòng)來完成。因此充分了解病死豬堆肥體系中微生物的菌群種類和動(dòng)態(tài)分布,挖掘病死豬堆肥體系的優(yōu)勢(shì)微生物,為開發(fā)病死豬堆肥微生物菌劑,促進(jìn)腐熟進(jìn)程,縮短堆肥周期奠定基礎(chǔ)。本研究通過開展6個(gè)月為一個(gè)堆肥周期的病死豬尸體自然發(fā)酵試驗(yàn),檢測(cè)了堆肥過程不同階段的理化參數(shù),利用PCR-DGGE技術(shù)分析了堆肥過程不同階段的微生物種類,構(gòu)建了系統(tǒng)樹。分離和篩選出5株高溫優(yōu)勢(shì)菌,并通過生理生化實(shí)驗(yàn)和16s r RNA測(cè)序?qū)@5株優(yōu)勢(shì)菌進(jìn)行了初步鑒定。主要研究結(jié)果如下:(1)實(shí)時(shí)監(jiān)測(cè)病死豬堆肥不同階段不同高度的溫度變化,對(duì)不同階段的樣品含氮量、含水率、p H值、有機(jī)質(zhì)含量、有效活菌數(shù)、蛔蟲死亡率、重金屬含量和類大腸桿菌數(shù)理化參數(shù)進(jìn)行測(cè)定和分析。堆肥試驗(yàn)中堆體溫度的變化經(jīng)歷了典型的升溫期、高溫期、降溫期三個(gè)階段,且堆體上、中、下三層溫度的變化趨勢(shì)基本是一致的,前3個(gè)月是有機(jī)質(zhì)降解階段,微生物活動(dòng)頻繁,中層溫度近70℃;p H變化遵循先下降-再升高-再下降的進(jìn)程趨勢(shì)。堆肥結(jié)束,病死豬尸體軟組織全部降解,堆料腐熟,外觀成黑色顆粒如腐殖質(zhì),依稀可見骨頭和牙齒,但這些難降解物質(zhì)很脆,容易捏碎。氮含量為5.3%左右,有機(jī)質(zhì)含量為53%,大腸桿菌值低于100個(gè)/g,蛔蟲卵殺滅率接近于100%,有效活菌數(shù)接近于0.35億/g,堆體周圍沒有活的蛆、蛹或新羽化的成蠅,達(dá)到了《糞便無害化衛(wèi)生標(biāo)準(zhǔn)(GB7959-87)》的規(guī)定。(2)提取了微生物基因組總DNA,完成了16s r RNA V6~V8區(qū)片段的PCR擴(kuò)增。通過變形梯度凝膠電泳,得到了18個(gè)樣品的DGGE圖譜。通過Quantity One軟件分析DGGE圖譜發(fā)現(xiàn),各個(gè)時(shí)期的細(xì)菌豐富度都比較高。前三個(gè)月的細(xì)菌的分布比較凌亂,后三個(gè)月的細(xì)菌分布趨于穩(wěn)定。致病菌只在前三個(gè)月存在,后三個(gè)月致病菌被殺死。分別對(duì)切膠回收后的45條優(yōu)勢(shì)條帶測(cè)序,并與已知序列在NCBI上進(jìn)行同源性比對(duì),得到了這45條序列的詳細(xì)結(jié)果并完成了它們的系統(tǒng)發(fā)育分析。結(jié)果發(fā)現(xiàn),堆肥過程中存在大量未知細(xì)菌,它們?cè)诙逊蔬^程中的作用還有待探究。堆肥過程的優(yōu)勢(shì)菌為枯草芽孢桿菌(Bacillus subtillis)、地衣芽孢桿菌(Bacillus lincheniformis)、枝芽孢桿菌(Virgibacillus)和蘇云金芽孢桿菌(Bacillus thuringiensis)。(3)傳統(tǒng)培養(yǎng)并結(jié)合高溫處理,分離獲得了5株優(yōu)勢(shì)菌。通過生理生化鑒定、顯微鏡形態(tài)觀察和16s r RNA測(cè)序,結(jié)果顯示,J1為未知菌與已知的Uncultured bacterium clone(JF155193)相似率為98%,J2、J3和J5為Bacillus subtillis strain,J4為Bacillus thuringiensis strain。產(chǎn)淀粉酶酶活性由強(qiáng)至弱依次為J4,J1,J2,J3,J5,產(chǎn)蛋白酶活性由強(qiáng)至弱依次為J1,J5,J2,J4,J3,產(chǎn)脂肪酶活性由強(qiáng)至弱依次為J3,J2,J5,其中J1和J4不產(chǎn)生脂肪酶。
[Abstract]:At the same time, the rapid development of intensive livestock farming, also caused from livestock manure, sewage and serious pollution of dead livestock and poultry residues. Compared with the pollution of livestock and poultry manure on the environment, dead livestock because carrying a lot of pathogens, if not timely treatment of human and animal harm more directly. According to statistics, farms livestock and poultry mortality between 5%~10% and mortality in pig 8%~12%, encountered a major epidemic, the mortality rate is also increased. Therefore, for the harmless treatment of dead livestock is to solve the environmental pollution, the only way which must be passed to achieve healthy development of animal husbandry. Composting is a organic matter biodegradation reactor, through aerobic microbial decomposition into stable the process is similar, humus soil. Compared with the dead pigs composting treatment methods, such as traditional, buried, burning, high-temperature boiling, with more safety and environmental protection. The advantages of composting The process of material transformation after all is by the metabolic activity of microorganisms in composting system to complete. Therefore fully understand microbial dead pig composting system bacteria species and the dynamic distribution of mining microorganisms pigs composting system, for the development of a dead pig compost microorganism, promote the composting process, lay the foundation for the study by shorten the composting period. Carry out 6 months for a composting cycle dead pig bodies test natural fermentation, detect the physicochemical parameters of different stages of the composting process, analysis of microbial species in different stages of the composting process by using PCR-DGGE technology, constructed a phylogenetic tree. Isolated and screened 5 strains of dominant bacteria in high temperature, and through physiological and biochemical tests and 16S R RNA sequencing of the 5 bacterial strains were identified. The main results are as follows: (1) temperature real-time monitoring of pigs in different composting stages In the different stages of the sample, nitrogen content, water content, P, H value, organic matter content, the effective number of living bacteria, Ascaris mortality, heavy metal content and coliform mathematical parameters were determined and analyzed. The changes of composting pile temperature experienced a typical heating period, high temperature period and cooling period in three stages, and the pile body, and the change trend of three layer temperature is basically the same, 3 months before the stage of organic matter degradation, microbial activity, middle temperature near 70 DEG C; P H changes follow the first drop process trend of increase decrease. The end of compost, mortality pig carcasses all soft tissue degradation, compost maturity, the appearance of black particles such as humus, faintly visible bones and teeth, but these refractory materials are brittle, easy to crush. The nitrogen content is about 5.3%, the content of organic matter was 53%, the Escherichia coli value below 100 / g, the killing rate of Ascaris eggs close to 100%, The effective number of living bacteria close to 35 million /g, no live maggot fly around the pile, or the newly emerged pupae, reached "manure harmless sanitary standard (GB7959-87) > rules. (2) the extraction of microbial genomic DNA, 16S R RNA completed the V6~V8 fragment by PCR amplification. The shape gradient gel electrophoresis, DGGE spectra of 18 samples were obtained by Quantity One software. DGGE atlas analysis showed that bacterial richness in each period are relatively high. The distribution of the first three months of the bacteria is messy, three months after the bacterial distribution tends to be stable. The pathogenic bacteria exist only in the first three months after. Three months of pathogens were killed. On gel cut after the 45 dominant bands of sequencing, homology and NCBI with known sequences, obtained the detailed results of the 45 sequences and the phylogenetic analysis of their systems. The results showed that there are large in the composting process The amount of unknown bacteria, their role in the composting process remains to be explored. The dominant bacteria during composting for Bacillus subtilis (Bacillus subtillis), Bacillus licheniformis (Bacillus lincheniformis), branch (Virgibacillus) and Bacillus thuringiensis (Bacillus thuringiensis). (3) the traditional culture combined with high temperature treatment isolated 5 strains of dominant bacteria. Through physiological and biochemical identification, microscopic observation and 16S R RNA sequencing results show that J1 is an unknown bacteria with known Uncultured bacterium clone (JF155193) similar rate was 98%, J2, J3 and J5 Bacillus subtillis strain J4 Bacillus thuringiensis strain., amylase enzyme activity is strong to the weak are J4, J1, J2, J3, J5, protease activity egories as J1, J5, J2, J4, J3, lipase activity from high to low according to the times of J3, J2, J5, J1 and J4 do not produce lipase.

【學(xué)位授予單位】：華中農(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

【參考文獻(xiàn)】

相關(guān)期刊論文 前7條

1 李華芝;李秀艷;韓波波;;高溫菌的生理生化及其16S rDNA序列分析[J];環(huán)境科學(xué)與技術(shù);2010年08期

2 聶志娟;徐鋼春;程起群;杜富寬;張勇;顧若波;朱文龍;;湖鱭體內(nèi)與生長(zhǎng)環(huán)境菌群PCR-DGGE指紋圖譜及多樣性分析[J];淡水漁業(yè);2015年01期

3 郭艷;張進(jìn)良;鄧昌彥;朱能武;;利用PCR-DGGE技術(shù)分析豬糞堆肥細(xì)菌群落結(jié)構(gòu)[J];河南師范大學(xué)學(xué)報(bào)(自然科學(xué)版);2010年05期

4 沈玉君;趙立欣;孟海波;;我國(guó)病死畜禽無害化處理現(xiàn)狀與對(duì)策建議[J];中國(guó)農(nóng)業(yè)科技導(dǎo)報(bào);2013年06期

5 沈德龍;曹鳳明;李力;;我國(guó)生物有機(jī)肥的發(fā)展現(xiàn)狀及展望[J];中國(guó)土壤與肥料;2007年06期

6 胡可;李華興;盧維盛;劉遠(yuǎn)金;王利賓;;生物有機(jī)肥對(duì)土壤微生物活性的影響[J];中國(guó)生態(tài)農(nóng)業(yè)學(xué)報(bào);2010年02期

7 劉曉璐;楊柳青;呂樂;郭濤;劉永智;趙鵬;董昕;閆海;;煙草根際固氮菌的篩選、鑒定及優(yōu)化培養(yǎng)[J];中國(guó)煙草學(xué)報(bào);2015年01期

相關(guān)碩士學(xué)位論文 前2條

1 周美紅;利用生活垃圾與污泥堆肥生產(chǎn)生物有機(jī)肥工藝研究[D];西北大學(xué);2007年

2 劉有勝;基于PCR-DGGE方法的餐廚垃圾堆肥微生物多樣性分析[D];湖南大學(xué);2008年

，

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