用于发酵床养殖的细菌分离鉴定及应用效果研究
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摘要
随着养殖业的发展,畜禽粪尿污染问题日益严重,发酵床垫料养殖技术的应运而生。筛选具有除臭功能的细菌,将其应用于畜禽的发酵床养殖中,从而达到健康养殖、提高动物产品质量和产量的目的。
本研究从土壤、堆肥样品中分离纯化获得105株细菌,筛选获得7株菌不产生氨气和硫化氢的菌株。对其生长pH值、温度、惟一碳、氮源利用,蛋白酶、淀粉酶活性,对动物的毒性试验等特性进行了分析,进而探讨了菌株的遗传特性。
在此基础上,进行了发酵床垫料处理,接种供试菌株后,进行了15d发酵处理。分别测定了垫料发酵过程中的温度、pH值,垫料中铵态氮和硝态氮的含量；细菌、放线菌、真菌数量的变化；最后,利用DGGE技术检测了不同发酵时间垫料中微生物群落动态。结果如下：
1.筛选获得7个菌株,菌属于革兰氏阳性芽孢杆菌,有耐高温、不耐低温的特点,在4℃和10℃不生长,全部菌株能够在45℃生长,其中4个菌株可在60℃生长。供试菌株在初始pH8-pH10培养基生长,不耐酸性,在初始pH4环境中不生长。
2.供试菌株理化特性分析表明,供试菌株均能水解淀粉,能够以甘露醇和蔗糖为惟一碳源；能够以大多数供试氨基酸为唯一氮源,能够利用铵态氮和硝态氮。
3. BOXAIR-PCR分析表明,菌株间遗传差异明显；在16S rDNA PCR-RFLP分析结果中,供试菌株分成了5个遗传群；代表菌株的16S rDNA基因序列比对结果显示,菌株B1是枯草芽孢杆菌(Bacillus stubtlis),菌株B5是栗褐芽孢杆菌(Bacillus badius),菌株B7是地衣芽孢杆菌(Bacillus licheniformis)。
4.毒性试验中,饲喂菌株B5的小白鼠生长正常,无死亡；通过解剖观察,肉眼未见任何器质性病变,表明供试菌株对小白鼠无致病性。
5.接种菌株B5的垫料发酵实验中,24h内垫料温度迅速升高至45℃左右,最高达70℃；发酵结束时,发酵床料温保持在50℃。发酵过程中,垫料中铵态氮和硝态氮含量迅速下降,由发酵初期的108.6mg/kg和138.72 mg/kg降至第8d的44.6mg/kg和52.32 mg/kg；随后,其含量略有上升。
6.对垫料细菌的PCR-DGGE分析表明,发酵过程中,垫料中细菌种群多样性变化不大,群落稳定。
Along with the development of livestock and poultry industry in China, the livestock and poultry manure pollution is becoming more and more serious, and the deep-litter system was introduced and applied. It is important to screen deodorizing bacteria and use in the local deep-litter system that can build a healthy livestock breeding system, and improve the quality and yield of animal products.
In this experiment,105 bacterial strains were isolated and purified, and seven strains which don not produce NH3 and H2S were screened. The original growth pH range, growth temperature, utilization of carbon and nitrogen sources, protease activity and amylase activity, toxicity test was done; furthermore, the genetic aspects of the strains were analyzed.
Base on the results, one deep-litter system was built, and the ferment time was 15d. During the course of fermentation, we detected variation of the temperature, pH value, ammonium and nitrate content, and the number of bacteria, fungi and actinomycetes. Finally, PCR-DGGE technique was used to reveal the microbial dynamics during the fermentation process. The results were as the follows:
1. Seven bacteria screened belong to gram-positive, can grow at high temperature, but can't grow at low temperature. These strains couldn't grow at 4℃and 10℃, however, they grew well at 45℃, and four of those can even grow at 60℃. These strains can also grow at original pH 8.0 to pH 10.0, but couldn't grow at original pH 4.0, which showed that these strains had low tolerant to acid environment.
2. Analysis of physiological and biochemical tests showed that, all the strains could digest starch, and used mannitol, glucose as sole carbon sources. These strains could use most tested ammonium acid, and NH4+-N, NO2-N as sole nitrogen source.
3. BOXAIR-PCR revealed that the genetic differentiation was existed, and 16S rDNA PCR-RFLP divided these strains into 5 genotypes,16S rDNA sequences of 3 representatives showed that, stain B1, B5 and B7 belonged to Bacillus stubtlis, Bacillus badius, and Bacillus licheniformis.
4. The results of mouse toxicity tests showed that mouse feed with strain B5 grew well, and there was no dead mouse, anatomic observation showed that no organic lesion was observed, which suggested that the tested strain had no pathogenicity to the trial mouse.
5. During the fermentation process, after inoculation of bacterial strain B5, the temperature in the fermenting bed increased to 45℃rapidly within 24h, and the highest was 70℃, when fermentation process completed, the temperature in the fermenting bed remained 50℃. At the same time, the content of ammonium nitrogen and nitrate nitrogen decreased quickly, and changed from 108.6mg/kg and 138.72 mg/kg at beginning to 44.6 mg/kg and 52.32 mg/kg on the 8th days, and then increased a little.
6. PCR-DGGE analysis showed that the bacterial communities in the litter material in the course of fermentation had little changes, and was stable.
本研究从土壤、堆肥样品中分离纯化获得105株细菌,筛选获得7株菌不产生氨气和硫化氢的菌株。对其生长pH值、温度、惟一碳、氮源利用,蛋白酶、淀粉酶活性,对动物的毒性试验等特性进行了分析,进而探讨了菌株的遗传特性。
在此基础上,进行了发酵床垫料处理,接种供试菌株后,进行了15d发酵处理。分别测定了垫料发酵过程中的温度、pH值,垫料中铵态氮和硝态氮的含量；细菌、放线菌、真菌数量的变化；最后,利用DGGE技术检测了不同发酵时间垫料中微生物群落动态。结果如下：
1.筛选获得7个菌株,菌属于革兰氏阳性芽孢杆菌,有耐高温、不耐低温的特点,在4℃和10℃不生长,全部菌株能够在45℃生长,其中4个菌株可在60℃生长。供试菌株在初始pH8-pH10培养基生长,不耐酸性,在初始pH4环境中不生长。
2.供试菌株理化特性分析表明,供试菌株均能水解淀粉,能够以甘露醇和蔗糖为惟一碳源；能够以大多数供试氨基酸为唯一氮源,能够利用铵态氮和硝态氮。
3. BOXAIR-PCR分析表明,菌株间遗传差异明显；在16S rDNA PCR-RFLP分析结果中,供试菌株分成了5个遗传群；代表菌株的16S rDNA基因序列比对结果显示,菌株B1是枯草芽孢杆菌(Bacillus stubtlis),菌株B5是栗褐芽孢杆菌(Bacillus badius),菌株B7是地衣芽孢杆菌(Bacillus licheniformis)。
4.毒性试验中,饲喂菌株B5的小白鼠生长正常,无死亡；通过解剖观察,肉眼未见任何器质性病变,表明供试菌株对小白鼠无致病性。
5.接种菌株B5的垫料发酵实验中,24h内垫料温度迅速升高至45℃左右,最高达70℃；发酵结束时,发酵床料温保持在50℃。发酵过程中,垫料中铵态氮和硝态氮含量迅速下降,由发酵初期的108.6mg/kg和138.72 mg/kg降至第8d的44.6mg/kg和52.32 mg/kg；随后,其含量略有上升。
6.对垫料细菌的PCR-DGGE分析表明,发酵过程中,垫料中细菌种群多样性变化不大,群落稳定。
Along with the development of livestock and poultry industry in China, the livestock and poultry manure pollution is becoming more and more serious, and the deep-litter system was introduced and applied. It is important to screen deodorizing bacteria and use in the local deep-litter system that can build a healthy livestock breeding system, and improve the quality and yield of animal products.
In this experiment,105 bacterial strains were isolated and purified, and seven strains which don not produce NH3 and H2S were screened. The original growth pH range, growth temperature, utilization of carbon and nitrogen sources, protease activity and amylase activity, toxicity test was done; furthermore, the genetic aspects of the strains were analyzed.
Base on the results, one deep-litter system was built, and the ferment time was 15d. During the course of fermentation, we detected variation of the temperature, pH value, ammonium and nitrate content, and the number of bacteria, fungi and actinomycetes. Finally, PCR-DGGE technique was used to reveal the microbial dynamics during the fermentation process. The results were as the follows:
1. Seven bacteria screened belong to gram-positive, can grow at high temperature, but can't grow at low temperature. These strains couldn't grow at 4℃and 10℃, however, they grew well at 45℃, and four of those can even grow at 60℃. These strains can also grow at original pH 8.0 to pH 10.0, but couldn't grow at original pH 4.0, which showed that these strains had low tolerant to acid environment.
2. Analysis of physiological and biochemical tests showed that, all the strains could digest starch, and used mannitol, glucose as sole carbon sources. These strains could use most tested ammonium acid, and NH4+-N, NO2-N as sole nitrogen source.
3. BOXAIR-PCR revealed that the genetic differentiation was existed, and 16S rDNA PCR-RFLP divided these strains into 5 genotypes,16S rDNA sequences of 3 representatives showed that, stain B1, B5 and B7 belonged to Bacillus stubtlis, Bacillus badius, and Bacillus licheniformis.
4. The results of mouse toxicity tests showed that mouse feed with strain B5 grew well, and there was no dead mouse, anatomic observation showed that no organic lesion was observed, which suggested that the tested strain had no pathogenicity to the trial mouse.
5. During the fermentation process, after inoculation of bacterial strain B5, the temperature in the fermenting bed increased to 45℃rapidly within 24h, and the highest was 70℃, when fermentation process completed, the temperature in the fermenting bed remained 50℃. At the same time, the content of ammonium nitrogen and nitrate nitrogen decreased quickly, and changed from 108.6mg/kg and 138.72 mg/kg at beginning to 44.6 mg/kg and 52.32 mg/kg on the 8th days, and then increased a little.
6. PCR-DGGE analysis showed that the bacterial communities in the litter material in the course of fermentation had little changes, and was stable.
引文
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