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整合宏组学方法研究番茄与玉米秸秆共堆肥生境中的关键微生物及其功能

朱屹 李俊良 焦博 朱倩倩 张小梅

朱屹,李俊良,焦博,等. 整合宏组学方法研究番茄与玉米秸秆共堆肥生境中的关键微生物及其功能 [J]. 福建农业学报,2020,35(7):764−772 doi: 10.19303/j.issn.1008-0384.2020.07.010
引用本文: 朱屹,李俊良,焦博,等. 整合宏组学方法研究番茄与玉米秸秆共堆肥生境中的关键微生物及其功能 [J]. 福建农业学报,2020,35(7):764−772 doi: 10.19303/j.issn.1008-0384.2020.07.010
ZHU Y, LI J L, JIAO B, et al. Functional Microorganisms in Tomato Stalks/Maize Straws Co-compost Unveiled by Integrated Meta-omics [J]. Fujian Journal of Agricultural Sciences,2020,35(7):764−772 doi: 10.19303/j.issn.1008-0384.2020.07.010
Citation: ZHU Y, LI J L, JIAO B, et al. Functional Microorganisms in Tomato Stalks/Maize Straws Co-compost Unveiled by Integrated Meta-omics [J]. Fujian Journal of Agricultural Sciences,2020,35(7):764−772 doi: 10.19303/j.issn.1008-0384.2020.07.010

整合宏组学方法研究番茄与玉米秸秆共堆肥生境中的关键微生物及其功能

doi: 10.19303/j.issn.1008-0384.2020.07.010
基金项目: 国家重点研发计划项目( 2017YFD0800403);国家自然科学基金项目( 31600084) ;山东省重大科技创新工程项目( 2019JZZY010721)
详细信息
    作者简介:

    朱屹(1996−),男,硕士研究生,主要从事农业资源的微生物利用研究(E-mail:876490555@qq.com

    通讯作者:

    张小梅(1986−),女,副教授,主要从事农业废弃物的资源化利用研究(E-mail:m0620@126.com

  • 中图分类号: X 712

Functional Microorganisms in Tomato Stalks/Maize Straws Co-compost Unveiled by Integrated Meta-omics

  • 摘要:   目的  探究番茄秸秆与玉米秸秆共堆肥发酵过程中的关键微生物及其功能,以进一步加速其发酵进程。  方法  以番茄秸秆和玉米秸秆为原料,二者按质量比3 1混合进行条垛式堆肥,每周采集发酵堆20~50 cm深度样品检测木质纤维素酶活力的变化,以酶活力最高时期的代表性样品为研究对象,对其进行高通量焦磷酸测序和Orbitrap宏蛋白质组学研究。  结果  多数真菌隶属于子囊菌门Ascomycota,其中嗜热丝孢菌Thermomyces的相对丰度最高,约占真菌全部序列的70.5%,该属真菌主要分泌内切-1,4-β-木聚糖酶,是主要的半纤维素降解真菌。主要细菌为放线菌门Actinobacteria、变形菌门Proteobacteria和厚壁菌门Firmicutes,三者共占细菌总序列数的87.0%。其中,嗜热裂孢菌属Thermobifida和糖单孢菌属Saccharomonospora是主要的放线菌属,其相对丰度分别占全部细菌序列的16.5%和1.36%。Thermobifida是唯一的纤维素降解菌,鉴定到4种内切纤维素酶组分和3种外切纤维素酶组分;同时产生一种果胶裂解酶和一种β-木聚糖酶。Saccharomonospora分别产生一种β-木聚糖酶、两种丝氨酸蛋白酶和两种胰蛋白酶,在半纤维素的降解和蛋白质降解方面发挥重要作用。Idiomarina是变形菌门下的主要细菌属,平均相对丰度15.6%,主要参与蛋白质的降解。而厚壁菌门下的清野氏菌属Planifilum虽基因丰度不高(仅为1.23%),但却在半纤维素的降解过程中发挥重要作用。  结论  将高通量焦磷酸测序和Orbitrap宏蛋白质组学结合的整合宏组学方法在探明复杂生境中关键微生物的群落结构及功能方面具有可行性。
  • 图  1  堆肥温度(a)和主要酶活力(b)的变化

    Figure  1.  Changes on temperature (a) and activities of primary enzymes (b) in compost

    图  2  第三周堆肥样品中细菌门(a)、真菌门(b)及主要属(平均相对丰度>1%,c)组成

    Figure  2.  Microbes by bacterial phylum (a), fungal phylum (b) and genera with average relative abundance greater than 1% (c) in 3-week compost sample

    图  3  不同时期所取堆肥样品在细菌(a)和真菌(b)属层次上的组成

    注:横坐标中的W1、W2,…,W5分别代表第1、2…5周的样品。仅列出了平均相对丰度>1%的属。

    Figure  3.  Genera of bacteria (a) and fungi (b) in samples collected at different times

    Note: W1-W5 on X-axis represent samples taken in 1st, 2nd, … and 5th week. Genera with average relative abundances >1% are listed.

    表  1  堆肥原料的理化性质

    Table  1.   Physiochemical properties of raw materials for composting

    原料
    Materials
    含水率
    Moisture content/%
    pH有机质
    Organic matter/%
    有机碳
    Organic carbon/%
    总氮
    Total nitrogen/%
    C/N
    番茄秸秆 Tomato stalk78.3± 1.58.56± 0.2196.7± 0.956.1± 0.52.83± 0.0119.8± 0.2
    玉米秸秆 Wheat straw10.5± 0.67.20± 0.0797.1± 1.156.3± 0.60.63± 0.0289.3± 1.7
    下载: 导出CSV

    表  2  第3周堆肥样品中主要细菌属和真菌属分泌蛋白的功能鉴定

    Table  2.   Functions of enzymes secreted by dominant bacterial and fungal genera in samples taken in 3rd week

    登录号
    Accession number
    家族
    Family
    描述
    Description
    微生物
    Microorganism
    光谱数
    Spectrum count
    分子量
    Molecular weight/kDa
    等电点
    Isoelectric point
    纤维素酶 Cellulase
    Q08166 GH9 内切纤维素酶 Thermobifida fusca +++ 104.5 4.40
    Q9KH72 GH6 纤维二糖水解酶 Thermobifida fusca +++ 48.6 4.72
    Q47NH7 GH48 纤维二糖水解酶 Thermobifida fusca ++ 107.1 4.59
    A0A147KMV8 GH9 内切纤维素酶 Thermobifida cellulosilytica +++ 102.4 4.32
    A0A147KIK5 GH48 纤维二糖水解酶 Thermobifida cellulosilytica + 106.3 4.51
    A0A068CD09 GH5 内切纤维素酶 Thermobifida fusca + 48.2 4.81
    Q7X2N2 GH5 内切纤维素酶 Thermobifida fusca + 67.7 4.54
    半纤维素酶 Hemicellulase
    A0A1I2PGX8 GH10 内切-1,4-β-木聚糖酶 Planifilum fulgidum + 52.8 5.58
    I1CXX6 GH10 β-木聚糖酶 Saccharomonospora glauca + 49.3 5.14
    P74912 GH10 β-木聚糖酶 Thermobifida alba + 52.4 5.66
    O43097 GH11 内切-1,4-β-木聚糖酶 Thermomyces lanuginosus + 24.3 4.92
    蛋白酶 Protease
    A0A094IT45 peptidase S1 类DegP周质丝氨酸内切蛋白酶 Idiomarina atlantica + 46.9 4.68
    C7MXV7 peptidase S8 类枯草菌素丝氨酸蛋白酶 Saccharomonospora viridis ++ 54.1 4.55
    C7MV18 Peptidase S1 胰酶 Saccharomonospora viridis + 39.4 4.96
    C7MTG2 Peptidase S1 胰酶 Saccharomonospora viridis + 36.7 4.53
    A0A1V9AC37 Peptidase S1 丝氨酸蛋白酶 Saccharomonospora sp. + 39.0 4.81
    其他 Others
    Q47MW8 PL1 果胶裂解酶 Thermobifida fusca ++ 53.6 6.86
    Q47PB9 AA10, CBM2 纤连蛋白 Thermobifida fusca + 46.8 4.65
    Q47QG3 AA10 推测分泌的纤维素结合蛋白 Thermobifida fusca + 25.4 7.64
    注:蛋白的相对丰度以平均“光谱数”来表征。符号+、++和+++分别代表平均光谱数<4、[4,7]和>7。
    Note: Relative abundance of enzymes as evaluated by mean value of "spectrum count". Symbols of +, ++ and +++ indicate mean "spectrum count" <4, ranging 4 to 7, and >7, respectively.
    下载: 导出CSV

    表  3  主要微生物属和酶活性的相关性

    Table  3.   Correlations between dominant genera and enzyme activities

    项目 Items内切纤维素酶 Endocellulase木聚糖酶 Xylanase蛋白酶 Protease
    芽孢杆菌 Bacillus 0.949* 0.582 0.921*
    嗜热真菌 Thermomyces 0.823 0.918* 0.507
    海源菌属 Idiomarina 0.565 0.120 0.904*
    注:表中仅列出了平均相对丰度>1%,且与酶活性显著正相关的微生物属。*代表显著相关。
    Note: Only genera with average relative abundance greater than 1% and significant correlation with enzymatic activities are listed. * indicates significant difference at P <0.05.
    下载: 导出CSV
  • [1] DOMI S NGO J L, NADAL M. Domestic waste composting facilities: a review of human health risks [J]. Environment International, 2009, 35(2): 382−389. doi: 10.1016/j.envint.2008.07.004
    [2] ARVANITOYANNIS I S, VARZAKAS T H. Vegetable waste treatment: comparison and critical presentation of methodologies [J]. Critical Reviews in Food Science and Nutrition, 2008, 48(3): 205−247. doi: 10.1080/10408390701279798
    [3] CHANDNA P, NAIN L, SINGH S, et al. Assessment of bacterial diversity during composting of agricultural byproducts [J]. BMC Microbiology, 2013, 13(1): 99−104. doi: 10.1186/1471-2180-13-99
    [4] JURADO M, LÓPEZ M J, SUÁREZ-ESTRELLA F, et al. Exploiting composting biodiversity: study of the persistent and biotechnologically relevant microorganisms from lignocellulose-based composting [J]. Bioresource Technology, 2014, 162: 283−293. doi: 10.1016/j.biortech.2014.03.145
    [5] 贾洋洋. 利用宏基因组方法分析堆肥生境中微生物区系的变化 [D]. 济南: 山东大学, 2012.

    JIA Y Y. Microbial diversity analysis in composting environment by metagenomic method [D]. Jinan: Shandong University, 2012. (in Chinese)
    [6] LIU W, WANG S T, ZHANG J, et al. Biochar influences the microbial community structure during tomato stalk composting with chicken manure [J]. Bioresource Technology, 2014, 154: 148−154. doi: 10.1016/j.biortech.2013.12.022
    [7] ZHANG L L, MA H X, ZHANG H Q, et al. Thermomyces lanuginosus is the dominant fungus in maize straw composts [J]. Bioresource Technology, 2015, 197: 266−275. doi: 10.1016/j.biortech.2015.08.089
    [8] ZHANG L L, ZHANG H Q, WANG Z H, et al. Dynamic changes of the dominant functioning microbial community in the compost of a 90-m3 aerobic solid state fermentor revealed by integrated meta-omics [J]. Bioresource Technology, 2016, 203: 1−10. doi: 10.1016/j.biortech.2015.12.040
    [9] JOHNSON-ROLLINGS A S, WRIGHT H, MASCIANDARO G, et al. Exploring the functional soil-microbe interface and exoenzymes through soil metaexoproteomics [J]. The ISME Journal, 2014, 8(10): 2148. doi: 10.1038/ismej.2014.130
    [10] URICH T, LANZÉN A, STOKKE R, et al. Microbial community structure and functioning in marine sediments associated with diffuse hydrothermal venting assessed by integrated meta-omics [J]. Environmental Microbiology, 2014, 16(9): 2699−2710. doi: 10.1111/1462-2920.12283
    [11] WALKLEY A, BLACK I A. An examination of the degtjareff method for determining soil organic matter, and a proposed modification of the chromic acid titration method [J]. Soil Science, 1934, 37(1): 29−38. doi: 10.1097/00010694-193401000-00003
    [12] LAWSON M J, KEELING A A. Production and physical characteristics of composted poultry carcases [J]. British Poultry Science, 1999, 40(5): 706−708. doi: 10.1080/00071669987133
    [13] NAVARRO A F, CEGARRA J, ROIG A, et al. Relationships between organic matter and carbon contents of organic wastes [J]. Bioresource Technology, 1993, 44(3): 203−207. doi: 10.1016/0960-8524(93)90153-3
    [14] LEE M H. Official methods of analysis of AOAC International (16th edition) [J]. Trends in Food Science & Technology, 1995, 6(11): 382.
    [15] DENNIS K L, WANG Y, BLATNER N R, et al. Adenomatous polyps are driven by microbe-instigated focal inflammation and are controlled by IL-10-producing T cells [J]. Cancer Research, 2013, 73(19): 5905−5913. doi: 10.1158/0008-5472.CAN-13-1511
    [16] GELFAND I M A, SNINSKY D H, WHITE J J. PCR protocols: a guide to methods and applications [M]. London: Academic Press, 1990: 315−322.
    [17] WEI H W, WANG L H, HASSAN M, et al. Succession of the functional microbial communities and the metabolic functions in maize straw composting process [J]. Bioresource Technology, 2018, 256: 333−341. doi: 10.1016/j.biortech.2018.02.050
    [18] YU R T, WANG L S, DUAN X Y, et al. Isolation of cellulolytic enzymes from moldy silage by new culture-independent strategy [J]. Biotechnology Letters, 2007, 29(7): 1037−1043. doi: 10.1007/s10529-007-9350-5
    [19] LOWRY O H, ROSEBROUGH N J, FARR A L, et al. Protein measurement with the Folin phenol reagent [J]. The Journal of Biological Chemistry, 1951, 193(1): 265−275.
    [20] ZHANG X M, LIU N, YANG F, et al. In situ demonstration and quantitative analysis of the intrinsic properties of glycoside hydrolases [J]. Electrophoresis, 2012, 33(2): 280−287. doi: 10.1002/elps.201100333
    [21] SAYKHEDKAR S, RAY A, AYOUBI-CANAAN P, et al. A time course analysis of the extracellular proteome of Aspergillus nidulans growing on Sorghum stover [J]. Biotechnology for Biofuels, 2012, 5(1): 1−17. doi: 10.1186/1754-6834-5-1
    [22] AHN H K, RICHARD T L, GLANVILLE T D. Laboratory determination of compost physical parameters for modeling of airflow characteristics [J]. Waste Management, 2008, 28(3): 660−670. doi: 10.1016/j.wasman.2007.04.008
    [23] 李季, 彭生平. 堆肥工程实用手册(第二版) [M]. 北京: 化学工业出版社, 2010: 79−83.
    [24] ZHANG X, ZHONG Y H, YANG S D, et al. Diversity and dynamics of the microbial community on decomposing wheat straw during mushroom compost production [J]. Bioresource Technology, 2014, 170: 183−195. doi: 10.1016/j.biortech.2014.07.093
    [25] WEBB M D, EWBANK G, PERKINS J, et al. Metabolism of pentachlorophenol by Saccharomonospora viridis strains isolated from mushroom compost [J]. Soil Biology and Biochemistry, 2001, 33(14): 1903−1914. doi: 10.1016/S0038-0717(01)00115-8
    [26] HATAYAMA K, SHOUN H, UEDA Y, et al. Planifilum fimeticola gen. nov., sp. nov. and Planifilum fulgidum sp. nov., novel members of the family ‘Thermoactinomycetaceae’ isolated from compost [J]. International Journal of Systematic and Evolutionary Microbiology, 2005, 55(5): 2101−2104. doi: 10.1099/ijs.0.63367-0
    [27] SONG J, WEON H Y, YOON S H, et al. Phylogenetic diversity of thermophilic actinomycetes and Thermoactinomyces spp. isolated from mushroom composts in Korea based on 16S rRNA gene sequence analysis [J]. FEMS Microbiology Letters, 2001, 202(1): 97−102. doi: 10.1111/j.1574-6968.2001.tb10786.x
    [28] WILLIAMS S T, SHARPE M E, HOLT J G. Bergey’s Manual of Systematic Bacteriology [M]. Beijing: Science Press, 1984: 2574−2585.
    [29] ANBARASAN S, JÄNIS J, PALOHEIMO M, et al. Effect of glycosylation and additional domains on the thermostability of a family 10 xylanase produced by Thermopolyspora flexuosa [J]. Applied and Environmental Microbiology, 2010, 76(1): 356−360. doi: 10.1128/AEM.00357-09
    [30] NEHER D A, WEICHT T R, BATES S T, et al. Changes in bacterial and fungal communities across compost recipes, preparation methods, and composting times [J]. PLoS One, 2013, 8(11): e79512. doi: 10.1371/journal.pone.0079512
    [31] CARINI P, MARSDEN P J, LEFF J W, et al. Relic DNA is abundant in soil and obscures estimates of soil microbial diversity [J]. Nature Microbiology, 2017, 2(3): 16242. doi: 10.1038/nmicrobiol.2016.242
    [32] ROCCA J D, HALL E K, LENNON J T, et al. Relationships between protein-encoding gene abundance and corresponding process are commonly assumed yet rarely observed [J]. The ISME Journal, 2015, 9(8): 1693−1699. doi: 10.1038/ismej.2014.252
    [33] ZHANG L L, LI L J, PAN X G, et al. Enhanced growth and activities of the dominant functional microbiota of chicken manure composts in the presence of maize straw [J]. Frontiers in Microbiology, 2018, 9: 1131. doi: 10.3389/fmicb.2018.01131
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  • 收稿日期:  2019-10-04
  • 修回日期:  2020-01-13
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