bioRxiv preprint doi: https://doi.org/10.1101/674069; this version posted June 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 The succession pattern of bacterial diversity in compost using pig 2 manure mixed with wood chips analyzed by 16S rRNA gene analysis 3 Zhengfeng Li4¶, Yan Yang1,2,3¶, Yuzhen Xia5, Tao Wu4, Jie Zhu4, Zhaobao Wang1,2,3*, Jianming 4 Yang1,2,3* 5 1 Energy-rich Compounds Production by Photosynthetic Carbon Fixation Research Center, 6 Qingdao Agricultural University, Qingdao, China 7 2 Shandong Province Key Laboratory of Applied Mycology, Qingdao Agricultural University, 8 Qingdao, China 9 3 College of Life Sciences, Qingdao Agricultural University, Qingdao, China 10 4 China Tobacco Yunnan Industrial Co., Ltd., Kunming, China 11 5 Hongta Tobacco (Group) Co., Ltd., Yuxi, China 12 13 14 * Corresponding authors 15 Email: [email protected] (ZW), 16 [email protected] (JY) 17 18 ¶ These authors are co-first authors on this work. 19 20 21 22 23 24 bioRxiv preprint doi: https://doi.org/10.1101/674069; this version posted June 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 25 Abstract 26 The pig manure mixed with wood chips and formed compost by means of 27 fermentation. We found that the protease activity, organic matter content and 28 ammonium nitrogen concentration were higher in the early stage of composting. 29 Meanwhile, the urease activity was highest in the high temperature period. The carbon 30 to nitrogen ratio of the compost decreased continuously with fermentation. The 31 dynamic change in the composition of bacterial overtime in the compost of a 180 kg 32 piles were explored using microbial diversity analysis. The results showed that the 33 microbial species increased with the compost fermentation. At the early stage of 34 composting, the phyla of Firmicutes and Actinomycetes were dominant. The microbes 35 in the high temperature period were mainly composed of Firmicutes and 36 Proteobacteria while the proportion of Bacteroides was increased during the cooling 37 period. In the compost of maturity stage, the proportion of Chloroflexi increased, 38 becoming dominant species with other microorganisms including Firmicutes, 39 Proteobacteria, Bacteroides, Chloroflexi but not Actinomycetes. Bacteria involved in 40 lignocellulose degradation, such as those of the Thermobifida, Cellvibrio, 41 Mycobacterium, Streptomyces and Rhodococcus, were concentrated in the maturity 42 stages of composting. Through correlation analysis, the environmental factors 43 including organic matter, ammonium nitrogen and temperature were consistent with 44 the succession of microbial including Rhodocyclaceae, Anaerolineaceae, 45 Thiopseudomonas, Sinibacillus and Tepidimicrobium. The change of urease activity 46 and carbon to nitrogen ratio corresponded to microbial communities, mainly bioRxiv preprint doi: https://doi.org/10.1101/674069; this version posted June 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 47 containing Anaerolineaceae, Rhodocyclaceae, Luteimoas, Bacillaceae, 48 Corynebacterium, Bacillus, Anaerococcus, Lactobacillus, Ignatzschineria, and 49 Bacillaceae. 50 51 Introduction 52 Aerobic composting of livestock manure and agricultural waste is the most 53 economical and environmentally friendly way of obtaining a fertilizer. During this 54 process, most microbes grow under aerobic conditions. Compared with anaerobic 55 fermentation, the aerobic fermentation cycle is shorter. The resulting composts can be 56 used in farmland as biological organic fertilizers, which is of great significance for 57 promoting ecological agriculture. 58 Aerobic composting generally undergoes four stages, including the heating period, 59 high temperature period, cooling period and maturity period Although the 60 microorganism communities of compost are highly complex, the succession of 61 microbial communities during composting obeys certain rules [1]. The traditional 62 research methods analyzing the microorganisms composition in compost mainly 63 include PLFA, DGGE, PCR-RFLP and plate culture method, etc.[2] However, 64 because of the limitations of culture conditions and the low resolution of 65 electrophoresis gels, these analyses of microorganisms have not been comprehensive. 66 Currently, the 16S RNA special fragment is amplified using the bacterial and archaeal 67 primers. By comparing amplification fragment of 16S RNA with online databases of 68 bacteria sequence, the microorganism’s type and quantity in the compost are bioRxiv preprint doi: https://doi.org/10.1101/674069; this version posted June 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 69 determined [3]. 70 Traditional composting methods include composting with single raw materials 71 and mixed raw materials. The fundamental reason for using various additives in 72 composting processes is to provide the best-growing environment for microbial 73 growth and achieve rapid composting, thus reducing the harmful gas emissions under 74 controlled conditions. For example, sawdust is an additive that reduces methane 75 emissions to a certain extent [4]. However, the quality of the fertilizer ultimately 76 depends on the microbial composition of the compost, and only a clear understanding 77 of the succession pattern of the microbial communities throughout the composting 78 stages will enable us to fully control this process. This knowledge would help us find 79 the best compost additives and microbial agents, laying the foundations for screening 80 microorganisms for special functions. 81 In this study, we studied the evolution of bacterial communities in a mixed 82 compost of pig manure and sawdust under the condition of a low carbon to nitrogen 83 ratio (20-25:1). Using 515F and 806R primers, the 16S RNA specific region of 84 prokaryotic genomes was amplified, and the whole process of composting bacteria 85 was systematically investigated. By analyzing the changes in compost of the dominant 86 bacteria and the correlation between microbial community and important 87 environmental parameters, we managed to explain the effects of environmental 88 factors on the changing microbial communities in compost and provide scientific 89 advice to control the fermentation process of the compost by adjusting the 90 physicochemical parameters. bioRxiv preprint doi: https://doi.org/10.1101/674069; this version posted June 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 91 Materials and methods 92 Compost composting and sampling 93 The compost study was performed in three piles (diameter, 1.5 m; height, 1.1 m). 94 An 800 cm length, 20 cm wide, 20 cm deep trench was dug at the surface and three 95 200 cm length , 20 cm wide and 20 cm deep trenches were used to vent at the bottom 96 of the compost. The branches and wheat straw were alternately put in the cross 97 position of each groove. Then the compost with pig manure and sawdust mixed with 98 C/N ratios of 25:1 was laid on top. A stick was inserted at the top of a pile of compost 99 to increase the overall aeration, and the piles were covered with a black perforated 100 plastic sheet to avoid heat and water loss. Three thermometers were inserted in 101 different directions into the 75 cm high compost. The real-time temperature of each 102 compost stack was the average value from three different sites. Water was added 103 during the experiment to maintain a moisture content level of 60 %. The oxygen was 104 supplied during the composting process by turning the pile once every five days. 105 Through long-term composting test, the 50 days of the natural fermentation 106 process samples in different fermentation stages were studied. Firstly, we determined 107 the midpoint of the diagonal as the central sampling point. Secondly, we selected four 108 additional samples equidistant from the center point. These five sub-samples were 109 then fully mixed to form one composting sample. Three samples from three piles were 110 obtained at each fermentation stage, and 12 samples were obtained at four 111 fermentation stages including the prime stage, high-temperature period, cooling 112 period, and maturity period. Samples obtained from each composting were labeled bioRxiv preprint doi: https://doi.org/10.1101/674069; this version posted June 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 113 Z31, Z32 and Z33 for third days after composting. The compost samples taken on day 114 seven were referred to as Z71, Z72 and Z73. The samples taken at day 20 were 115 labeled Z201, Z202 and Z203. On the fiftieth day, samples obtained from the 116 composts were labeled Z501, Z502 and Z503. All samples were stored at -80 °C until 117 use. 118 Compost physical and chemical analysis 119 The temperature was measured every day in the compost by thermometer place at 120 the same height and depth. Measurements were taken from three thermometers placed 121 at different angles. The water extract with a 1:4 ratio of the compost sample contrast 122 to distilled water was used for the measurement of pH.