高温季节微生物发酵床基质垫层微生物群落结构变化动态的研究
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摘要
畜禽业生产规模的日益扩大,产生的废弃物成为许多城市和农村的新兴污染源,已成为一个亟待解决的新问题。近几年兴起的发酵床养殖技术,是根据微生态理论,借助于发酵床垫层中微生物的作用,分解畜禽排泄物,大大减少了污染,在生产中得到迅速推广、应用,生态效益显著。本研究采用传统平板分离法,结合磷脂脂肪酸(PLFAs)分析,对高温季节下微生物发酵床养猪基质垫层的微生物群落结构进行研究分析,结果如下:
(1)分离结果表明,各样品中含量最高为细菌(×10~5 cfu/g),其次是放线菌(×10~4 cfu/g),真菌含量最少(×10~3 cfu/g);根据微生物在各样品中含量可分为三个类群,类群Ⅰ特征为细菌含量大于70.00×10~5 cfu/g,真菌含量大于70.00×10~3 cfu/g,放线菌含量大于70.00×10~4 cfu/g;类群Ⅱ特征为细菌含量为30.00~70.00×10~5 cfu/g,真菌含量为30.00~70.00×10~3 cfu/g,放线菌含量为30.00~70.00×10~4 cfu/g;类群Ⅲ特征为细菌含量小于30.00×10~5 cfu/g,真菌含量小于30.00×10~3 cfu/g,放线菌含量小于30.00×10~4 cfu/g。在三十个采样点中,细菌的平均空间分布由高至低依次为类群Ⅱ(40.67%)、类群Ⅰ(30.67%)和类群Ⅲ(28.67%);真菌的空间分布平均比例由高至低依次是类群Ⅲ(66.00%)、类群Ⅱ(21.33%)和类群Ⅰ(12.67%);放线菌的空间分布平均比例由高至低依次是类群Ⅲ(67.33%)、类群Ⅱ(28.00%)和类群Ⅰ(4.67%)。
(2)微生物的数量-时间变化曲线可分为前峰型、中峰型、后峰型、双峰型等类型,细菌数量的时间变化曲线包含类型主要是中峰型和后峰型,真菌的数量变化曲线包括了全部四种类型,放线菌的数量变化曲线类型主要是前锋型和后峰型;细菌的平均含量随时间变化呈现先升后下降的趋势,真菌的平均含量则基本呈现下降趋势,而放线菌的平均含量变化平缓。
(3)以微生物发酵床基质垫层中三类微生物平均含量之间的相互比值作为衡量微生物结构比例变化的指数,细菌/真菌以B/F表示,细菌/放线菌以B/A表示,真菌/放线菌以F/A表示。分析表明,5、6、7三栏的细菌/真菌(B/F值)比值变化范围(0.32~8.73,1.29~4.1,0.26~6.89)比较小,细菌/真菌结构比例比较稳定;1栏、4栏、8栏和10栏的细菌/真菌(B/F值)变化范围(0.64~55.60,2.68~79.53,0.49~53.35,0.69~64.47)很大;其余三个栏2栏、3栏和9栏的变化范围(0.63~16.71,0.13~19.78、0.55~14.64)介于中间。2、3、5、6四栏的细菌/放线菌(B/A值)变化范围(0.74~6.16,0.18~4.88,0.54~4.13,0.55~8.94)比较小,细菌/放线菌结构比例比较稳定。1栏、4栏、7栏、8栏、9栏、10栏等六栏B/A值范围较大,变化范围最大的是10栏(1.48~88.39),细菌/放线菌结构比例变化较大。真菌/放线菌(F/A值)分析表明,10个栏的真菌放线菌(F/A)值都比较小(F/A值<10),4栏的F/A值变化范围最小(0.3~1.11),60天时10个栏的F/A值<1。
(4)根据聚集度指数负二项分布的K指标分析,细菌的聚集指数总体降低,由随机分布转变为聚集分布,且聚集分布的程度逐渐减弱,之后保持比较稳定的聚集分布程度;真菌属于聚集分布且聚集分布程度逐渐降低,但变化平缓;放线菌聚集指数变化比较大,由聚集分布转变为随机分布,又转变为聚集分布;在30-60天时细菌、真菌、放线菌聚集度指数K值都变化不大,趋于稳定,放线菌比细菌和真菌偏高,表明细菌、真菌、放线菌均处于比较稳定的聚集分布状态,放线菌的聚集分布程度比细菌和真菌偏高。
(5)不同栏位不同时间PLFA总量的比较结果表明:PLFA总量随时间变化呈相同的变化趋势“升—降—升—降”,其中2栏在15-45天时的变化范围(2227359.33~12464370.33)较大,10栏的变化范围(4581140.67~8981641.33)较小;在第1天时各栏微生物的PLFA总量相近(1358607.33~3345488.00 ) ,第60天时各栏微生物的PLFA总量也很相近(1458257.33~3078114.00)。PLFA特征标识分析表明:PLFA特征细菌标识16:0、特征真菌标识18:1ω9c、特征放线菌标识TBSA 10 Me 18:0和特征原生动物标识20:4ω6,9,12,15c数量在随时间变化中均呈现“降—升—降”的变化趋势,特征原生动物标识20:4ω6,9,12,15c数量较少,变化范围(16155.93~129881.93)较其他三者更小,变化平缓。
With the scale of livestock and poultry production expanding, waste generated as emerging pollution sources in many urban and rural had become a new problem demanding prompt solution. Based on micro-ecological theory of culture techniques of fermentation bed, the animal wastes were decomposed with the help of fermentation microorganisms, as result in pollution being greatly reduced and eco-efficiency being remarkable. The thesis focused on the microbial community structure analysis of microbial fermentation bed by used traditional plate separation combined with the Phospholipid Fatty Acids (PLFAs) analysis under the high temperature seasons. the results showed as follow:
(1) The result of traditional plate separation showed: in the microbial fermentation bed, the bacteria were the highest(×10~5 cfu/g),followed by the actinomyces (x10~4 cfu/g), and the fungi was minimum (x 10~3 cfu/g).The microorganisms were devided into three groups according to their quantity. The characteristics of groupⅠwere the bacteria content more than 70.00 x10~5 cfu/g, the fungal content more than 70.00 x10~3 cfu/g, and the actinomyces content more than 70.00x10~4 cfu/g. The characteristics of groupⅡwere the bacteria content of 30.00~70.00 x10~5 cfu/g, the fungi of 30.00 ~ 70.00 x10~3 cfu/g, the actinomyces of 30.00~70.00 x10~4 cfu/g. The characteristic of groupⅢwere the bacteria content less than 30.00 x10~5 cfu/ g , the fungi content less than 30.00 x10~3 cfu/g, the actinomyces content less than 30.00 x10~4 cfu/g. In 30 sampling sites, the bacteria averages in the space were distributed with the order from high to low were groupⅡ(40.67%), groupⅠ(30.67%),and groupⅢ(28.67%). The fungi averages were distributed as that groupⅢ(66.00%), groupⅡ(21.33%),andⅠgroup(12.67%). The actinomyces averages were distributed as groupⅢ(67.33%), groupⅡ(28.00%),andⅠgroup(4.67%).
(2) The microbial population dynamic curve with quantity-time could be divided into four types such as advanced peak type, central peak type, delayed peak type and petronas-peak type. The bacterial quantity dynamic curve mainly contained the two types of central peak type and delayed peak type, and the fungi curve included all the four types. For the actinomyces, there were main types of advanced peak type and delayed peak type. The averages of bacteria increased firstly, and then dropping the trends in the changes over time, the averages of fungi presented the downward trend basically, and the averages of actinomyces changed gently.
(3) The changes index of microbial structure was introduced into the research on treated ratio of content of three types microorganism in microbial fermentation bed. the B/F index refered to bacteria/fungi, the B/ A refered to bacteria/ actinomyces, and the F/A refered to fungi/actinomyces. The results showed that the ratios of bacteria /fung(iB/F value)in the 5th、6th、7th pigstys were narrow range(0.32~8.73,1.29~4.1,0.26~6.89), and the bacteria/fungi structure of them changed little. While the B/F value of 1st, 4th, 8th and 10th pigstys were wide-ranging (0.64~ 55.60,2.68 ~ 79.53, 0.49~ 53.35, 0.69~64.47).The rest three pigstys of 2nd,3rd and 9th range(0.63~16.71,0.13~19.78、0.55~14.64)changed in the middle. The ratio ranges of bacteria/ actinomyces (B/A value) in the four pigstys of 2nd, 3rd, 5th and 6th were small with the values 0.74 ~ 6.16, 0.18 ~ 4.88, 0.54 ~ 4.13, 0.55 ~ 8.94, and the bacteria/ actinomyces structural scale of them were more stable relatively. Whie the B/A value ranges of six pigstys in 1st, 4th, 7th, 8th, 9th and 10th were large, of which 10th pigsty change range (1.48:1 ~ 88.39) was the biggest, and bacteria/ actinomyces structural scale varied considerably. The fungi/ actinomyces (F/A value) analysis showed that, fungi/ actinomyces (F/A value) of 10 pigstys were comparatively small (F/A value < 10). The changing range of F/A value of 4th pigsty was minimum (0.3 ~ 1.11), and the F/A value of 10 pigstys were less than 1 on 60th day.
(4) According to analysis of aggregation index-negative binomial distribution K index, the aggregation index of bacteria reduced overall, it’s spatial distribution changed from random distribution to a aggregated distribution, and the extent of aggregated distribution weakened gradually, the distribution then remained relatively stable level of aggregated distribution. The fungal distribution was of aggregated distribution all along and the extent of aggregation gradually reduced gently. Actinomyces change of aggregated index was relatively bigger ,and it’s spatial distribution transformed from aggregated distribution into random distribution and then into aggregated distribution again. The aggregation index of bacteria, fungi and actinomyces were changed little during 30th-60th days, turning to be stable, and the index of actinomyces was higher than bacteria and fungi in this time.It suggested that bacteria, fungi, actinomyces were in relatively stable state of aggregated distribution, and actinomyces aggregated distribution degree was higher than bacteria or fungi.
(5) The comparison results of total amount PLFA in different pigstys of different time showed that total amount PLFA changes with time in the same changing trend of " increase - decrease - increase - decrease". Total microorganism PLFA amount of each pigstys on the 1st day (1358607. 33~ 3345488. 00) were similar, and it increased on 15th da(y8981641.33~12464370.33)while decreased on 30th day. On 45th day, total microorganism PLFA amount increased slightly in relative 30th day. And what happened on the 60th day(1458257.33~3078114.00)was as same as 1st day. 16:0 is characteristics identified PLFA of bacterial, 18:1ω9c of fungi, TBSA 10 Me 18:0 of actinomyces and 20:4ω6,9,12,15c of Protozoa. Analysis of identify characteristics PLFA showed that the content changes of them were significantly in the same trend of "decrease - increase - decrease". And the total amount of 20:4ω6, 9,12,15c was less and than the other three and so was the range. The rang of it was frome 16155.93 to 129881.93, changing gently.
(1)分离结果表明,各样品中含量最高为细菌(×10~5 cfu/g),其次是放线菌(×10~4 cfu/g),真菌含量最少(×10~3 cfu/g);根据微生物在各样品中含量可分为三个类群,类群Ⅰ特征为细菌含量大于70.00×10~5 cfu/g,真菌含量大于70.00×10~3 cfu/g,放线菌含量大于70.00×10~4 cfu/g;类群Ⅱ特征为细菌含量为30.00~70.00×10~5 cfu/g,真菌含量为30.00~70.00×10~3 cfu/g,放线菌含量为30.00~70.00×10~4 cfu/g;类群Ⅲ特征为细菌含量小于30.00×10~5 cfu/g,真菌含量小于30.00×10~3 cfu/g,放线菌含量小于30.00×10~4 cfu/g。在三十个采样点中,细菌的平均空间分布由高至低依次为类群Ⅱ(40.67%)、类群Ⅰ(30.67%)和类群Ⅲ(28.67%);真菌的空间分布平均比例由高至低依次是类群Ⅲ(66.00%)、类群Ⅱ(21.33%)和类群Ⅰ(12.67%);放线菌的空间分布平均比例由高至低依次是类群Ⅲ(67.33%)、类群Ⅱ(28.00%)和类群Ⅰ(4.67%)。
(2)微生物的数量-时间变化曲线可分为前峰型、中峰型、后峰型、双峰型等类型,细菌数量的时间变化曲线包含类型主要是中峰型和后峰型,真菌的数量变化曲线包括了全部四种类型,放线菌的数量变化曲线类型主要是前锋型和后峰型;细菌的平均含量随时间变化呈现先升后下降的趋势,真菌的平均含量则基本呈现下降趋势,而放线菌的平均含量变化平缓。
(3)以微生物发酵床基质垫层中三类微生物平均含量之间的相互比值作为衡量微生物结构比例变化的指数,细菌/真菌以B/F表示,细菌/放线菌以B/A表示,真菌/放线菌以F/A表示。分析表明,5、6、7三栏的细菌/真菌(B/F值)比值变化范围(0.32~8.73,1.29~4.1,0.26~6.89)比较小,细菌/真菌结构比例比较稳定;1栏、4栏、8栏和10栏的细菌/真菌(B/F值)变化范围(0.64~55.60,2.68~79.53,0.49~53.35,0.69~64.47)很大;其余三个栏2栏、3栏和9栏的变化范围(0.63~16.71,0.13~19.78、0.55~14.64)介于中间。2、3、5、6四栏的细菌/放线菌(B/A值)变化范围(0.74~6.16,0.18~4.88,0.54~4.13,0.55~8.94)比较小,细菌/放线菌结构比例比较稳定。1栏、4栏、7栏、8栏、9栏、10栏等六栏B/A值范围较大,变化范围最大的是10栏(1.48~88.39),细菌/放线菌结构比例变化较大。真菌/放线菌(F/A值)分析表明,10个栏的真菌放线菌(F/A)值都比较小(F/A值<10),4栏的F/A值变化范围最小(0.3~1.11),60天时10个栏的F/A值<1。
(4)根据聚集度指数负二项分布的K指标分析,细菌的聚集指数总体降低,由随机分布转变为聚集分布,且聚集分布的程度逐渐减弱,之后保持比较稳定的聚集分布程度;真菌属于聚集分布且聚集分布程度逐渐降低,但变化平缓;放线菌聚集指数变化比较大,由聚集分布转变为随机分布,又转变为聚集分布;在30-60天时细菌、真菌、放线菌聚集度指数K值都变化不大,趋于稳定,放线菌比细菌和真菌偏高,表明细菌、真菌、放线菌均处于比较稳定的聚集分布状态,放线菌的聚集分布程度比细菌和真菌偏高。
(5)不同栏位不同时间PLFA总量的比较结果表明:PLFA总量随时间变化呈相同的变化趋势“升—降—升—降”,其中2栏在15-45天时的变化范围(2227359.33~12464370.33)较大,10栏的变化范围(4581140.67~8981641.33)较小;在第1天时各栏微生物的PLFA总量相近(1358607.33~3345488.00 ) ,第60天时各栏微生物的PLFA总量也很相近(1458257.33~3078114.00)。PLFA特征标识分析表明:PLFA特征细菌标识16:0、特征真菌标识18:1ω9c、特征放线菌标识TBSA 10 Me 18:0和特征原生动物标识20:4ω6,9,12,15c数量在随时间变化中均呈现“降—升—降”的变化趋势,特征原生动物标识20:4ω6,9,12,15c数量较少,变化范围(16155.93~129881.93)较其他三者更小,变化平缓。
With the scale of livestock and poultry production expanding, waste generated as emerging pollution sources in many urban and rural had become a new problem demanding prompt solution. Based on micro-ecological theory of culture techniques of fermentation bed, the animal wastes were decomposed with the help of fermentation microorganisms, as result in pollution being greatly reduced and eco-efficiency being remarkable. The thesis focused on the microbial community structure analysis of microbial fermentation bed by used traditional plate separation combined with the Phospholipid Fatty Acids (PLFAs) analysis under the high temperature seasons. the results showed as follow:
(1) The result of traditional plate separation showed: in the microbial fermentation bed, the bacteria were the highest(×10~5 cfu/g),followed by the actinomyces (x10~4 cfu/g), and the fungi was minimum (x 10~3 cfu/g).The microorganisms were devided into three groups according to their quantity. The characteristics of groupⅠwere the bacteria content more than 70.00 x10~5 cfu/g, the fungal content more than 70.00 x10~3 cfu/g, and the actinomyces content more than 70.00x10~4 cfu/g. The characteristics of groupⅡwere the bacteria content of 30.00~70.00 x10~5 cfu/g, the fungi of 30.00 ~ 70.00 x10~3 cfu/g, the actinomyces of 30.00~70.00 x10~4 cfu/g. The characteristic of groupⅢwere the bacteria content less than 30.00 x10~5 cfu/ g , the fungi content less than 30.00 x10~3 cfu/g, the actinomyces content less than 30.00 x10~4 cfu/g. In 30 sampling sites, the bacteria averages in the space were distributed with the order from high to low were groupⅡ(40.67%), groupⅠ(30.67%),and groupⅢ(28.67%). The fungi averages were distributed as that groupⅢ(66.00%), groupⅡ(21.33%),andⅠgroup(12.67%). The actinomyces averages were distributed as groupⅢ(67.33%), groupⅡ(28.00%),andⅠgroup(4.67%).
(2) The microbial population dynamic curve with quantity-time could be divided into four types such as advanced peak type, central peak type, delayed peak type and petronas-peak type. The bacterial quantity dynamic curve mainly contained the two types of central peak type and delayed peak type, and the fungi curve included all the four types. For the actinomyces, there were main types of advanced peak type and delayed peak type. The averages of bacteria increased firstly, and then dropping the trends in the changes over time, the averages of fungi presented the downward trend basically, and the averages of actinomyces changed gently.
(3) The changes index of microbial structure was introduced into the research on treated ratio of content of three types microorganism in microbial fermentation bed. the B/F index refered to bacteria/fungi, the B/ A refered to bacteria/ actinomyces, and the F/A refered to fungi/actinomyces. The results showed that the ratios of bacteria /fung(iB/F value)in the 5th、6th、7th pigstys were narrow range(0.32~8.73,1.29~4.1,0.26~6.89), and the bacteria/fungi structure of them changed little. While the B/F value of 1st, 4th, 8th and 10th pigstys were wide-ranging (0.64~ 55.60,2.68 ~ 79.53, 0.49~ 53.35, 0.69~64.47).The rest three pigstys of 2nd,3rd and 9th range(0.63~16.71,0.13~19.78、0.55~14.64)changed in the middle. The ratio ranges of bacteria/ actinomyces (B/A value) in the four pigstys of 2nd, 3rd, 5th and 6th were small with the values 0.74 ~ 6.16, 0.18 ~ 4.88, 0.54 ~ 4.13, 0.55 ~ 8.94, and the bacteria/ actinomyces structural scale of them were more stable relatively. Whie the B/A value ranges of six pigstys in 1st, 4th, 7th, 8th, 9th and 10th were large, of which 10th pigsty change range (1.48:1 ~ 88.39) was the biggest, and bacteria/ actinomyces structural scale varied considerably. The fungi/ actinomyces (F/A value) analysis showed that, fungi/ actinomyces (F/A value) of 10 pigstys were comparatively small (F/A value < 10). The changing range of F/A value of 4th pigsty was minimum (0.3 ~ 1.11), and the F/A value of 10 pigstys were less than 1 on 60th day.
(4) According to analysis of aggregation index-negative binomial distribution K index, the aggregation index of bacteria reduced overall, it’s spatial distribution changed from random distribution to a aggregated distribution, and the extent of aggregated distribution weakened gradually, the distribution then remained relatively stable level of aggregated distribution. The fungal distribution was of aggregated distribution all along and the extent of aggregation gradually reduced gently. Actinomyces change of aggregated index was relatively bigger ,and it’s spatial distribution transformed from aggregated distribution into random distribution and then into aggregated distribution again. The aggregation index of bacteria, fungi and actinomyces were changed little during 30th-60th days, turning to be stable, and the index of actinomyces was higher than bacteria and fungi in this time.It suggested that bacteria, fungi, actinomyces were in relatively stable state of aggregated distribution, and actinomyces aggregated distribution degree was higher than bacteria or fungi.
(5) The comparison results of total amount PLFA in different pigstys of different time showed that total amount PLFA changes with time in the same changing trend of " increase - decrease - increase - decrease". Total microorganism PLFA amount of each pigstys on the 1st day (1358607. 33~ 3345488. 00) were similar, and it increased on 15th da(y8981641.33~12464370.33)while decreased on 30th day. On 45th day, total microorganism PLFA amount increased slightly in relative 30th day. And what happened on the 60th day(1458257.33~3078114.00)was as same as 1st day. 16:0 is characteristics identified PLFA of bacterial, 18:1ω9c of fungi, TBSA 10 Me 18:0 of actinomyces and 20:4ω6,9,12,15c of Protozoa. Analysis of identify characteristics PLFA showed that the content changes of them were significantly in the same trend of "decrease - increase - decrease". And the total amount of 20:4ω6, 9,12,15c was less and than the other three and so was the range. The rang of it was frome 16155.93 to 129881.93, changing gently.
引文
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