郑雪芳,
刘波^,,
朱育菁,
王阶平,
蓝江林,
陈倩倩
福建省农业科学院农业生物资源研究所 福州 350003
基金项目: 国家公益性行业(农业)科研专项201303094
福建省公益类科研院所专项2018R1017-1
福建省农业科学院科技创新团队项目STIT2017-1-11

详细信息

作者简介:郑雪芳, 主要从事农业微生物和生物防治研究。E-mail:zhengxuefangfz@163.com

通讯作者:刘波, 主要从事微生物生物技术和农业生物药物研究。E-mail:fzliubo@163.com

中图分类号:Q938.1

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出版历程

收稿日期:2018-06-12
录用日期:2018-09-28
刊出日期:2019-01-01

Analysis of microbial community structure of litter with different fermentation levels in pig-on-litter system using phospholipid fatty acid biomarkers

ZHENG Xuefang,
LIU Bo^,,
ZHU Yujing,
WANG Jieping,
LAN Jianglin,
CHEN Qianqian
Institute of Agricultural Bio-Resources, Fujian Academy of Agricultural Sciences, Fuzhou 350003, China
Funds: the Special Fund for Agro-scientific Research in the Public Interest of China201303094
Fujian Provincial Special Fund for Non-profit Institutions2018R1017-1
the Science and Technology Innovation Team Program of Fujian Academy of Agricultural SciencesSTIT2017-1-11

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Corresponding author:LIU Bo, E-mail:fzliubo@163.com

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摘要
摘要:发酵床养猪是一种新型的养殖技术，可有效缓解养猪的环境污染问题，微生物在其中起关键作用。为明确养猪发酵床发酵过程微生物群落的变化规律，为发酵床的科学管理提供依据，本研究采用磷脂脂肪酸生物标记（phospholipid fatty acids，PLFA）法分析养猪发酵床不同发酵等级垫料的微生物群落结构特征。采用色差法将垫料分为3个发酵程度等级：1级、2级和3级，采集不同发酵等级表层（0~15 cm）和里层（30~45 cm）垫料样本，测定各样本的PLFA。结果表明，共检测到61种PLFA，发酵2级垫料的PLFA种类最多，发酵3级垫料的PLFA种类最少。在各垫料中，PLFA分布量均表现为细菌>真菌>放线菌。指示细菌、真菌、放线菌、革兰氏阳性细菌（G⁺）、革兰氏阴性细菌（G^-）的PLFA及总PLFA在各发酵等级表层垫料的分布量均显著大于其在里层垫料的分布量，最大值出现在发酵1级表层垫料中。与对照（未发酵垫料）相比，发酵垫料总PLFA含量均显著增加（P < 0.05）。发酵3级表层垫料的真菌/细菌值最大，发酵2级表层垫料的G⁺/G^-值最大。多样性分析表明，Shannon指数和Pielou指数最大值出现在发酵2级垫料中，而Simpson指数最大值出现在发酵3级表层垫料中。聚类分析表明，当欧氏距离为233.15时，可将不同发酵等级垫料聚为3个类群，同一发酵级别的垫料聚在相同类群中；主成分分析表明，发酵1级表层和里层垫料单独归一类群，其他发酵等级垫料和对照垫料归另一类群中。综上，不同发酵等级垫料的微生物种群结构不同，发酵1级表层垫料微生物分布量最大，发酵2级垫料的微生物种类最多，相同发酵级别表层和里层垫料微生物群落结构相似。
Abstract:Pig-on-litter system is a new pig-raising technology that can reduce environmental pollution. In this system, micro-organisms are generally considered as the key factor. In order to determine the change in microbial community during fermentation and to set up basic data for scientific management of pig-on-litter system, microbial community of litter with different fermentation levels was analyzed using phospholipid fatty acid (PLFA) biomarkers. Fermentation levels of litters were divided into three grades (1^st, 2^nd and 3^rd) using the chromatic aberration (△E) method. Both surface (0-15 cm) and inner layer (30-45 cm) litters of each fermentation level were sampled. PLFA composition of each sample was determined by the Sherlock MIS 4.5 system. The results showed that the method used was sufficient to detect total 61 kinds of PLFA biomarkers. The most and least kinds of PLFA biomarkers occurred in litters with the 2^nd and 3^rd fermentation levels, respectively. PLFA biomarkers displayed the same order of distribution abundance in all samples-bacteria > fungi > actinomycetes. The contents of PLFAs that were referable to bacteria, fungi, actinomycetes, G⁺, G^- and total PLFA in the surface layer samples were all higher than those in the inner layer samples; being highest in surface layer litters with the 1^st fermentation level. Fermented litter had significantly higher content of total PLFA than unfermented litter (CK) (P < 0.05). The highest fungi/bacteria and G⁺/G^- ratios were for surface layer litter with the 3^rd and 2^nd fermentation levels, respectively. Diversity analyses indicated that the maximum values of the Shannon index and Pielou index were for litter of the 2^nd fermentation level and the maximum values of the Simpson index were for surface layer litter of the 3^rd fermentation level. Based on cluster analysis, the samples were clustered into three groups for Euclidean-distance of 223.15. Samples with the same fermentation level were clustered together. Also based on principal component analysis, surface layer and inner layer samples of the 1^st fermentation level were clustered into one lone group, while the other samples were clustered into other several groups. Put together, litters with different fermentation levels had different microbial community structures. The maximum values of microbial content and species were in surface layer litter with the 1^st and 2^nd fermentation levels, respectively. Moreover, surface layer and inner layer litters of the same fermentation level had similar microbial communities.

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图1养猪发酵床不同发酵等级垫料的PLFA组成
Figure1.Composition of different PLFA biomarkers in the litters of pig-on-litter system with different fermentation levels


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图2养猪发酵床不同发酵等级垫料真菌/细菌(A)和G⁺/G^-值(B)
柱状图中不同小写字母表示差异显著(P < 0.05)。
Figure2.Values of fungi/bacteria (A) and G⁺/G^- (B) of litters of pig-on-litter system with different fermentation levels
Different lowercase letters in the figure indicate significant differences at 0.05 level.


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图3养猪发酵床不同发酵等级垫料微生物群落结构的聚分类析
Figure3.Cluster analysis of microbial community of litters of pig-on-litter system with different fermentation levels


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图4养猪发酵床不同发酵等级垫料微生物群落结构的主成分分析(相同图案代表 3个重复样本)
Figure4.PCA analysis of microbial community of litters of pig-on-litter system with different fermentation levels (the same patterns represent three duplicate samples)


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表1养猪发酵床垫料不同发酵等级的样本特点
Table1.Characteristics of the tested litters of pig-on-litter system with different fermentation levels

样本 Sample	发酵级别 Fermentation level	深度 Depth (cm)	垫料色差(?E) Chromatic aberration	垫料发酵特点 Characteristics of litter
G1-1	1	0–15 (表层, Surface layer)	0 < ?E < 7.63	浅发酵, 垫料腐熟程度很浅, 呈灰黄色, 质地干燥松软 Weak fermentation, litters is gray-yellow color and dry soft.
G1-2	1	30–45 (里层, Inner layer)
G2-1	2	0–15 (表层, Surface layer)	7.63 < ?E < 39.06	中等发酵, 垫料腐熟程度较浅, 呈灰褐色, 潮湿 Medium degree of fermentation, litters is gray-brown color and wet.
G2-2	2	30–45 (里层, Inner layer)
G3-1	3	0–15 (表层, Surface layer)	39.06 < ?E < 70.49	深发酵, 垫料腐熟程度深, 呈深褐色, 潮湿 Deep degree of fermentation, litter is dark brown color and wet.
G3-2	3	30–45 (里层, Inner layer)
CK	未发酵 Unfermented	—	0	未发酵初始垫料, 呈浅黄色, 质地干燥松软 Unfermented litters, appearing pale yellow color and dry soft.

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表2养猪发酵床不同发酵等级垫料特征微生物PLFA含量
Table2.Contents of special microbial PLFA for the litters of pig-on-litter system with different fermentation levels

nmol·g-1
样本 Sample	特征微生物PLFA含量Content of special microbial PLFA					总PLFA Total PLFA
	细菌 Bacterium	真菌 Fungus	放线菌 Actinomycete	G+	G-
CK	467.79±5.72f	85.59±10.04de	6.13±1.02d	56.78±6.05f	49.85±7.00d	560.51±10.67e
G1-1	1 207.18±17.73a	122.91±16.36c	100.18±15.00a	210.70±13.64a	152.10±8.19a	1 435.05±80.53a
G1-2	836.95±22.57b	87.79±9.55de	6.54±2.38d	86.73±7.01d	84.68±8.76b	932.69±11.99b
G2-1	767.25±11.89c	70.82±4.48e	53.83±4.28c	113.13±4.81c	62.32±9.56c	897.14±13.12bc
G2-2	684.21±9.46d	102.06±9.21d	80.26±11.84b	168.19±12.13b	80.09±4.56b	868.29±8.18c
G3-1	534.53±20.93e	217.90±7.40a	15.53±3.14d	76.94±4.38de	46.28±3.92d	772.22±17.29d
G3-2	402.65±17.04g	149.52±11.28b	15.09±3.71d	64.54±3.96ef	53.30±0.91cd	574.94±20.98e
表中数值为平均值±标准差, 同列数值后不同小写字母表示差异显著(P < 0.05)。Data were means ± standard deviation. Data within the same column followed by different lowercase letters are significantly different (P < 0.05).

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表3养猪发酵床不同发酵等级垫料微生物群落多样性指数
Table3.Microbial community diversity indexes of litters of pig-on-litter system with different fermentation levels

样本Sample	Simpson指数Simpson index	Shannon指数Shannon index	Pielou指数Pielou index
CK	0.990 1±0.000 9ab	2.493 1±0.000 4d	0.606 5±0.000 7d
G1-1	0.989 5±0.003 2ab	2.627 9±0.001 0c	0.639 3±0.000 7c
G1-2	0.988 7±0.000 3b	2.829 9±0.000 5b	0.688 4±0.000 7b
G2-1	0.988 2±0.000 8b	2.964 8±0.000 9a	0.721 2±0.000 3a
G2-2	0.988 2±0.000 2b	2.964 8±0.000 5a	0.721 2±0.000 4a
G3-1	0.991 7±0.001 0a	2.088 8±0.000 7f	0.508 1±0.000 8f
G3-2	0.990 3±0.000 8ab	2.425 7±0.000 9e	0.590 1±0.000 9e
表中数值为平均值±标准差, 同列数值后不同小写字母表示差异显著(P < 0.05)。Data were means ± standard deviation. Data within the same column followed by different lowercase letters are significantly different (P < 0.05).

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