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Identifying the key factors that affect the formation of humic substance during different materials composting
Accepted Manuscript Short Communication Identifying the key factors that affect the formation of humic substance during different materials composting Junqiu Wu, Yue Zhao, Haishi Qi, Xinyu Zhao, Tianxue Yang, Yingqiu Du, Hui Zhang, Zimin Wei PII: DOI: Reference:
S0960-8524(17)31405-0 http://dx.doi.org/10.1016/j.biortech.2017.08.100 BITE 18709
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
6 July 2017 15 August 2017 16 August 2017
Please cite this article as: Wu, J., Zhao, Y., Qi, H., Zhao, X., Yang, T., Du, Y., Zhang, H., Wei, Z., Identifying the key factors that affect the formation of humic substance during different materials composting, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.08.100
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Identifying the key factors that affect the formation of humic substance during different materials composting Junqiu Wua, Yue Zhaoa, Haishi Qia, Xinyu Zhaob, Tianxue Yangb, Yingqiu Duc, Hui Zhangd, Zimin Weia*
a
College of Life Science, Northeast Agricultural University, Harbin 150030, China
b
State key laboratory of environment criteria and risk assessment, Chinese Research
Academy of Environmental Sciences, Beijing 100012, China c
Agricultural Products Quality and Safety Research Institute, Heilongjiang Academy of
Agricultural Sciences, Harbin 150086, China d
Institute of Horticulture, Heilongjiang Academy of Agricultural Sciences, Harbin
150069, China Corresponding author Address: College of Life Science, Northeast Agricultural University, Harbin 150030, China. Tel/Fax: +86 45155190413 E-mail address: [email protected] or [email protected]
Abstract: The aim of this work was to identify the factors which can affect humic substance (HS) formation. Composting periods, HS precursors, bacteria communities and environment factors were recognized as the key factors and few studies explored the potential relationships among them. During composting, HS precursors were mainly formed in the heating and thermophilic phases, but HS were polymerized in the cooling
1
and mature phases. Moreover, bacterial species showed similar classification of community structure in the same composting period of different materials. Furthermore, structural equation model showed that NH4--N and NO3--N were the indirect environmental factors for regulating HS formation by the bacteria and precursors as the indirect and direct driver, respectively. Therefore, both environmental factors and HS precursors can be the regulating factors to promote HS formation. Given that, a new staging regulating method had been proposed to improve the amount of HS during different materials composting. Key Words: Composting; Precursors; Humic Substance; Redundancy Analysis; Structural Equation Model 1. Introduction Composting plays an important role in organic matter recycling and environment repairing (Lim et al., 2016; Xi et al., 2016). During composting, organic matter is decomposed and transformed by microorganisms to form humic substance (HS) precursors, for polyphenols, carboxyl acids, amino acids, reducing sugars and polysaccharide (Stevenson, 1994). These precursors are polymerized in various ways, including lignin-protein pathways, polyphenols pathways and Maillard reactions to form HS (Tan, 2014). HS as one of the most complex compounds in the composting has very important effect on improving soil fertility, because it is rich in mature organic matter. In addition, the complex supramolecular structure of HS has universal binding ability with heavy metal, polar aromatic compounds, organic dye (He et al., 2015; Smilek et al., 2015).
2
The formation of composting HS precursors can be divided into two aspects: decomposition and synthesis (Tan, 2014). The microorganism can degrade organic matter to satisfy its growth by releasing the precursors. As the growth of microorganisms, they began to synthesize precursors by the secondary metabolic pathways such as shikimic acid and malonic acid pathway. The precursors are polymerized under the action of enzymes or automatically to form HS (Tan, 2014). Therefore, the formation of precursors is inseparable from the action of microorganisms during composting. Composting is facilitated by microorganisms through community succession and abundance changes (Ali et al., 2014). In general, the composting has been divided into four periods according to the changes of temperature (Wei et al., 2016). However, the microbial activity is the main cause of compost temperature changes. This result illustrates that the microorganisms have different activities at varied composting periods, which may result in the different material changes and diverse precursors formation rules during composting. Although the key bacteria communities that influence the formation and polymerization of HS precursors have been investigated in our previous study of Wu et al. 2017, it is obscure about the specific period of the precursors formation and polymerization under the effect of bacteria during different materials composting. Additionally, Wu et al. (2017) showed that many indicators could influence HS formation. But few researches have explored the potential relationships among the various indicators for screening the key factor affecting HS formation. Structural equation models (SEM) as an a priori approach can visualize the casual relationships between variables by fitting data to the models representing casual
3
hypotheses (Eisenhauer et al., 2015). In this study, it has been conducted to detect the key factors affecting HS formation. In addition, according to the HS precursors changes and species information, the composting materials were clustered to analyze the differences among them. Accordingly, the purpose of this article is to combine with the previous study to provide a new regulating method to promote HS formation, which may be applicable to various types of composting materials. 2. Materials and methods 2.1 Composting test Five solid piles of cabbage waste (CW), lawn waste (LW), chicken manure (CM), garden waste (GW) and corn straw (CS) were conducted in the compost reactor and control the composting temperature which were according to the method of Zhao et al. (2016). In order to study the differences among the materials, the urea and sawdust (C/N = 61) have been chosen to adjust the C/N of raw materials to 30 to ensure the unicity of composting materials. The composting process has lasted 50 days. According to the changes of composting temperature (Zhao et al., 2016), the samples for day 0, 8, 20 and 32 were regarded as the samples of heating phase (HP), thermophilic phase (TP), cooling phase (CP) and maturity phase (MP), respectively. Samples from these phases were collected for HS precursors of polyphenols, carboxyl acids, amino acids, polysaccharide and reducing sugars concentration and bacteria analysis. Details of analytical methods have been reported by Wu et al. (2017). DNA extraction and the analysis of PCR-DGGE have also been reported by that study. Therefore, no more repetitive description is made herein.
4
2.2 Data analysis Redundancy analyses (RDA) was conducted to study the forming periods of precursors and HS by Canoco for Windows (Version 5.0). Non-metric multidimensional (NMDS) was utilized to analysis the differences in bacteria communities and precursors concentration changes during different materials composting. A principal coordinate analysis (PCoA) was conducted to reduce the n-dimensional of composting bacteria communities’ information into two linear axes explaining the maximum amount of variance (Flores-Rentería et a., 2016). SEM was constructed by the IBM SPSS AMOS 23.0. 3. Results and discussion 3.1 The formation and polymerization regularity of precursors during different materials composting It is important to investigate the generation of HS precursors for regulating HS formation, because precursors have significantly positive effect on HS polymerization (Wu et al., 2017). RDA was employed to study the formation and polymerization regularity of precursors under the effect of bacteria (Fig. 1a). Reducing sugars and polysaccharides positively correlate with species derived from LW1, LW2, LW4, CW2, CW3, CS1, CM2 and CM4 (p<0.05). Polyphenols and carboxyl are mainly promoted by communities in LW2, CM4, CM2, CS1, LW3 and GW1. The amino acids concentration was mainly increased by the bacteria species in CM1 and CM3. These results illustrated that the bacteria derived from the HP and TP mainly promote the formation of different kinds of precursors. As the composting process progressed, the species in the CP and
5
MP move to HS, which indicate that populations in these periods can promote the synthesis of HS. However, the communities derived from GW2 and CS2 can also enhance the HS formation. This result demonstrates that the HS formation exists throughout the composting process. In general, precursors formation is mainly concentrated in HP and TP and HS polymerized in CP and MP, which consisted with the dynamic of precursors and HS concentration (Wu et al., 2017). 3.2 The differences among various composting materials For guiding different materials composting, it is necessary to investigate the differences of precursors and bacteria communities among various materials. In this article, the simplex materials (not mixed) were chosen for better understand the differences. The NMDS of precursors concentration shows that the materials are divided into five groups (Fig. 1b), indicating that the HS precursors have their own formation regularity by the various property of raw materials during different composting, which may be caused by the various property of raw materials. Cellulose is the main structure of LW, CW and CS; however, they are not classified as the same group. This result may be due to that the changes of precursors concentration are different during composting. In addition, Wang et al. (2015) reported that the lignin in GW was hard to be degraded. Therefore, during the GW composting, protein and any other simple organic matters may primarily be decomposed by microbe. Whereas in CM, protein is more abundant than botanical materials. Therefore, it mainly produced amino acids (Fig. 1a), leading the CM adjacent to GW. The bacteria information in the five composting materials were mainly be divided into three categories (Fig. 1c): ① bacteria in HP; ② bacteria in TP
6
and CP, ③ bacteria in MP. This result demonstrates that the bacteria in the same period of different materials may perform similar functions. In HP, microbes degrade organic matter to satisfy their metabolism and release HS precursors. Until TP and CP, bacteria activity enters the secondary metabolic stage, begins to synthesis HS precursors and secrete polymerase. Therefore, the HS were mainly polymerized in the MP (Fig. 1a). As analyzed above, the bacteria function is similar to the compost in the same period, and according to the formation and polymerization regularity of different precursors, a new staging regulation method can be provided to improve the production of HS, which may apply to varied materials composting, at least the five substrates motioned in this article. But, first of all, the key factors which can direct and indirect influence HS formation must be identified for better understanding the HS formation. 3.3 Key factors of humic substance formation The environmental factors may be the most manageable factors affecting HS formation, and proposed a step-by-step regulating method to promote the production of HS (Wang et al., 2015). However, some other relationships can directly or indirectly affect the HS formation. Therefore, the SEM has been conducted to detect the casual relationships among the basic environmental factors, bacteria community information, precursors and HS (Fig. 2). During composting, the degradation of organic matter produces amino acids and reducing sugars, which directly promote HS formation. Amino acids and reducing sugars are also the energy and carbon source of bacteria metabolism, therefore, they can be produced during the whole process of composting (López-González et al., 2015). The greater the amount of amino acids and reducing
7
sugar production, the more obvious the promoting effect on HS synthesis. The polyphenols are mainly produced in HP and TP (Fig. 1), and in the other periods they are consumed as HS precursors, resulting in the negative relationship with HS production. However, the findings by incorporating all the data into SEM challenged the previous perceptions of bacteria significantly promote the formation of polyphenols, suggesting that the amino acids and reducing sugar, which related to the bacteria activity, are the dominant factors in forming HS and can be regulated during composting. Therefore, not only the environmental factors, the HS precursors can also be utilized to promote the formation of HS. Given that, the precursors and environmental factors are used as controlling factors to propose a new regulating method for HS formation. 3.4 A new staging regulating method to promote the formation of HS precursors Large of efforts availably improved HS production by adding lignin, biochar and any other waste materials, their fundamental theory is to provide precursors for HS formation (Dias et al., 2010). But little attention is paid on the key factors which have latent effect on the production of HS. In conjunction with all the conclusions have received above (Fig. 1 and 2), a staging regulating method has been provided to improve HS production (Fig. 4). The HS production may be promoted by adding HS precursors or environment factor on the hollow arrows. In HP, the amino acids and reducing sugars have been selected to stimulate the bacteria activity, because they are the small molecule organic matter that easy to be used by bacteria to quickly adapt to the composting environment (Berggren et al., 2010). The crop residues (e.g., bean dregs) or sugar-moll wastes may be a good choice to be the regulating materials. As the
8
composting step into TP and CP, the bacteria activity is intense and organic matter is degraded considerably. Adding NH4+-N and NO3--N is to supplement the depleted nitrogen sources and further promotes bacteria activities to release more HS precursors. Polyphenols, as one of the HS precursors that may not be affected by bacteria, but can significantly increase the concentration of carboxyl groups and thus may play an important role in improving the aromatization of HS. Furthermore, NO3--N may promote the polymerization of precursors to form HS, therefore, amino acids, reducing sugar and NO3--N may significantly improve the amount of HS by the Maillard reaction at MP. Anyhow, the new staging regulating method be provided in this article and refined the adjustment process and simplified the regulating factors, which will more effectively in improving the production of HS. 4. Conclusion This study revealed that the bacteria may decompose and transform organic matter into HS precursors in HP and TP. Thereafter, these precursors will polymerize to form HS in CP and MP. Therefore, a staging regulating method has been proposed to promote HS production by screening the key factors affecting HS formation. The ammonia and polyphenols wastewater, crop residues and sugar-mall wastes which contain sufficient NH4+-N, NO3--N, polyphenols, amino acids and reducing sugars, respectively, can be utilized as the wastes to promote HS production according to the method. The increased HS production provides more possibilities for the environmental remediation. E-supplementary data for this work can be found in e-version of this paper online.
9
Acknowledgement This work was financially supported by the National Natural Science Foundation of China (No. 51178090), National Natural Science Foundation of China (No. 51378097), and the National Key Technology R&D Program (No. 2012BAJ21B02-02). Reference [1] Ali, M., Kazmi, A.A., Ahmed, N., 2014. Study on effects of temperature, moisture and pH in degradation and degradation kinetics of aldrin, endosulfan, lindane pesticides during full-scale continuous rotary drum composting. Chemosphere. 102, 68-75. [2] Berggren, M., Laudon, H., Haei, M., Ström, L., Jansson, M., 2010. Efficient aquatic bacterial metabolism of dissolved low-molecular-weight compounds from terrestrial sources. ISME J. 4, 408-416. [3] Eisenhauer, N., Bowker, M.A., Grace, J.B., Powell, J.R., 2015. From patterns to causal understanding: structural equation modeling (SEM) in soil ecology. PEDOBIOLOGIA. 58, 65-72. [4] Flores-Rentería, D., Rincón, A., Valladares, F., Yuste, J.C., 2016. Agricultural matrix affects differently the alpha and beta structural and functional diversity of soil microbial communities in a fragmented Mediterranean holm oak forest. Soil Biol. Biochem. 92, 79-90. [5] He, X.S., Xi, B.D., Zhang, Z.Y., Gao, R.T., Tan, W.B., Cui, D.Y., Yuan, Y., 2015. Composition, removal, redox, and metal complexation properties of dissolved organic nitrogen in composting leachates. J. Hazard. Mater. 2015, 283: 227-233.
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[6] Lim, S.L., Lee, L.H., Wu, T.Y., 2016. Sustainability of using composting and vermicomposting technologies for organic solid waste biotransformation: recent overview, greenhouse gases emissions and economic analysis. J. Cleaner Prod. 111, 262–278. [7] López-González, J.A., Suárez-Estrella, F., Vargas-García, M.C., López, M.J., Jurado, M.M., Moreno, J., 2015. Dynamics of bacterial microbiota during lignocellulosic waste composting: Studies upon its structure, functionality and biodiversity. [8] Smilek, J., Sedláček, P., Kalina, M., & Klučáková, M., 2015. On the role of humic acids’ carboxyl groups in the binding of charged organic compounds. Chemosphere. 138, 503-510. [9] Stevenson F.J., 1994 Humic Chemistry: Genesis, composition, Reactions. John Wiley and Sons, New York. [10] Tan KH., 2014 Humic matter in soil and the environment: principles and controversies. CRC Press. [11] Wang, K., Li, X.K., He, C., Chen, C.L., Bai, J.W., Ren, N.Q., Wang, J.Y., 2014. Transformation of dissolved organic matters in swine, cow and chicken manures during composting. Bioresour. Technol. 168, 222-228. [12] Wang, X., Cui, H., Shi, J., Zhao, X., Zhao, Y., Wei, Z., 2015. Relationship between bacterial diversity and environmental parameters during composting of different raw materials. Bioresour. Technol. 198, 395–402. [13] Wei, Y.Q., Wei, Z.M., Cao, Z.Y., Zhao, Y., Zhao, X.Y., Lu, Q., Wang, X.Q., Zhang,
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X., 2016. A regulating method for the distribution of phosphorus fractions based on environmental parameters related to the key phosphate-solubilizing bacteria during composting. Bioresour. Technol., 211, 610-617. [14] Wu, J.O., Zhao, Y., Zhao, W., Yang, T.X., Zhang, X., Xie, X.Y., Cui, H.Y., Wei, Z.M., 2017. Effect of precursors combined with bacteria communities on the formation of humic substances during different materials composting. Bioresour. Technol. 226, 191-199. [15] Xi, B.D, Zhao, X.Y, He, X.S, Huang, C.H, Tan, W.B, Gao, R.T, Zhang, H., Li, D., 2016. Successions and diversity of humic-reducing microorganisms and their association with physical-chemical parameters during composting[J]. Bioresour. Technol. 219, 204-211. [16] Zhao, Y., Lu, Q., Wei, Y.Q., Cui, H.Y., Zhang, X., Wang, X.Q., Wei, Z.M., 2016b. Effect of actinobacteria agent inoculation methods on cellulose degradation during composting based on redundancy analysis. Bioresour. Technol. 219, 196-203. Figure captions Fig. 1 The formation and polymerization of HS precursors during different materials composting. a: RDA between bacteria community, HS and HS precursors; b: NMDS of precursors concentration in different materials composting; c: NMDS of bacteria community (1-HP: heating phase; 2-TP: thermophilic phase; 3-CP: cooling phase; 4-MP: manure phase). Different colors represent different categories. Fig. 2 Structural equation model showing the direct and indirect effects of the key factors on the formation of HS. PCO1 and PCO2 represent the data derived from
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the principal coordinate analysis of bacteria community. The path coefficients are adjacent to the arrows, the width of the arrows represent the strength of the path. Fig. 3. A new staging regulating method for promoting HS production. The same color presented one period. The words above the hollow arrows meant the wastes containing HS precursors or environmental factors, which can be added into the composting piles to promote the production of HS. This method can be both utilized in lab and factory scale.
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Highlights
Precursors formed in heating and thermophilic phase during different composting.
The functions of bacteria have been identified in different composting stages.
Precursors are the direct factors affecting humic substance (HS) formation.
Environment factor and bacteria are the indirect factors to influence HS formation.
A new staging regulating method has been proposed to increase the amount of HS.
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Graphical abstract
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S0960-8524(17)31405-0 http://dx.doi.org/10.1016/j.biortech.2017.08.100 BITE 18709
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
6 July 2017 15 August 2017 16 August 2017
Please cite this article as: Wu, J., Zhao, Y., Qi, H., Zhao, X., Yang, T., Du, Y., Zhang, H., Wei, Z., Identifying the key factors that affect the formation of humic substance during different materials composting, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.08.100
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Identifying the key factors that affect the formation of humic substance during different materials composting Junqiu Wua, Yue Zhaoa, Haishi Qia, Xinyu Zhaob, Tianxue Yangb, Yingqiu Duc, Hui Zhangd, Zimin Weia*
a
College of Life Science, Northeast Agricultural University, Harbin 150030, China
b
State key laboratory of environment criteria and risk assessment, Chinese Research
Academy of Environmental Sciences, Beijing 100012, China c
Agricultural Products Quality and Safety Research Institute, Heilongjiang Academy of
Agricultural Sciences, Harbin 150086, China d
Institute of Horticulture, Heilongjiang Academy of Agricultural Sciences, Harbin
150069, China Corresponding author Address: College of Life Science, Northeast Agricultural University, Harbin 150030, China. Tel/Fax: +86 45155190413 E-mail address: [email protected] or [email protected]
Abstract: The aim of this work was to identify the factors which can affect humic substance (HS) formation. Composting periods, HS precursors, bacteria communities and environment factors were recognized as the key factors and few studies explored the potential relationships among them. During composting, HS precursors were mainly formed in the heating and thermophilic phases, but HS were polymerized in the cooling
1
and mature phases. Moreover, bacterial species showed similar classification of community structure in the same composting period of different materials. Furthermore, structural equation model showed that NH4--N and NO3--N were the indirect environmental factors for regulating HS formation by the bacteria and precursors as the indirect and direct driver, respectively. Therefore, both environmental factors and HS precursors can be the regulating factors to promote HS formation. Given that, a new staging regulating method had been proposed to improve the amount of HS during different materials composting. Key Words: Composting; Precursors; Humic Substance; Redundancy Analysis; Structural Equation Model 1. Introduction Composting plays an important role in organic matter recycling and environment repairing (Lim et al., 2016; Xi et al., 2016). During composting, organic matter is decomposed and transformed by microorganisms to form humic substance (HS) precursors, for polyphenols, carboxyl acids, amino acids, reducing sugars and polysaccharide (Stevenson, 1994). These precursors are polymerized in various ways, including lignin-protein pathways, polyphenols pathways and Maillard reactions to form HS (Tan, 2014). HS as one of the most complex compounds in the composting has very important effect on improving soil fertility, because it is rich in mature organic matter. In addition, the complex supramolecular structure of HS has universal binding ability with heavy metal, polar aromatic compounds, organic dye (He et al., 2015; Smilek et al., 2015).
2
The formation of composting HS precursors can be divided into two aspects: decomposition and synthesis (Tan, 2014). The microorganism can degrade organic matter to satisfy its growth by releasing the precursors. As the growth of microorganisms, they began to synthesize precursors by the secondary metabolic pathways such as shikimic acid and malonic acid pathway. The precursors are polymerized under the action of enzymes or automatically to form HS (Tan, 2014). Therefore, the formation of precursors is inseparable from the action of microorganisms during composting. Composting is facilitated by microorganisms through community succession and abundance changes (Ali et al., 2014). In general, the composting has been divided into four periods according to the changes of temperature (Wei et al., 2016). However, the microbial activity is the main cause of compost temperature changes. This result illustrates that the microorganisms have different activities at varied composting periods, which may result in the different material changes and diverse precursors formation rules during composting. Although the key bacteria communities that influence the formation and polymerization of HS precursors have been investigated in our previous study of Wu et al. 2017, it is obscure about the specific period of the precursors formation and polymerization under the effect of bacteria during different materials composting. Additionally, Wu et al. (2017) showed that many indicators could influence HS formation. But few researches have explored the potential relationships among the various indicators for screening the key factor affecting HS formation. Structural equation models (SEM) as an a priori approach can visualize the casual relationships between variables by fitting data to the models representing casual
3
hypotheses (Eisenhauer et al., 2015). In this study, it has been conducted to detect the key factors affecting HS formation. In addition, according to the HS precursors changes and species information, the composting materials were clustered to analyze the differences among them. Accordingly, the purpose of this article is to combine with the previous study to provide a new regulating method to promote HS formation, which may be applicable to various types of composting materials. 2. Materials and methods 2.1 Composting test Five solid piles of cabbage waste (CW), lawn waste (LW), chicken manure (CM), garden waste (GW) and corn straw (CS) were conducted in the compost reactor and control the composting temperature which were according to the method of Zhao et al. (2016). In order to study the differences among the materials, the urea and sawdust (C/N = 61) have been chosen to adjust the C/N of raw materials to 30 to ensure the unicity of composting materials. The composting process has lasted 50 days. According to the changes of composting temperature (Zhao et al., 2016), the samples for day 0, 8, 20 and 32 were regarded as the samples of heating phase (HP), thermophilic phase (TP), cooling phase (CP) and maturity phase (MP), respectively. Samples from these phases were collected for HS precursors of polyphenols, carboxyl acids, amino acids, polysaccharide and reducing sugars concentration and bacteria analysis. Details of analytical methods have been reported by Wu et al. (2017). DNA extraction and the analysis of PCR-DGGE have also been reported by that study. Therefore, no more repetitive description is made herein.
4
2.2 Data analysis Redundancy analyses (RDA) was conducted to study the forming periods of precursors and HS by Canoco for Windows (Version 5.0). Non-metric multidimensional (NMDS) was utilized to analysis the differences in bacteria communities and precursors concentration changes during different materials composting. A principal coordinate analysis (PCoA) was conducted to reduce the n-dimensional of composting bacteria communities’ information into two linear axes explaining the maximum amount of variance (Flores-Rentería et a., 2016). SEM was constructed by the IBM SPSS AMOS 23.0. 3. Results and discussion 3.1 The formation and polymerization regularity of precursors during different materials composting It is important to investigate the generation of HS precursors for regulating HS formation, because precursors have significantly positive effect on HS polymerization (Wu et al., 2017). RDA was employed to study the formation and polymerization regularity of precursors under the effect of bacteria (Fig. 1a). Reducing sugars and polysaccharides positively correlate with species derived from LW1, LW2, LW4, CW2, CW3, CS1, CM2 and CM4 (p<0.05). Polyphenols and carboxyl are mainly promoted by communities in LW2, CM4, CM2, CS1, LW3 and GW1. The amino acids concentration was mainly increased by the bacteria species in CM1 and CM3. These results illustrated that the bacteria derived from the HP and TP mainly promote the formation of different kinds of precursors. As the composting process progressed, the species in the CP and
5
MP move to HS, which indicate that populations in these periods can promote the synthesis of HS. However, the communities derived from GW2 and CS2 can also enhance the HS formation. This result demonstrates that the HS formation exists throughout the composting process. In general, precursors formation is mainly concentrated in HP and TP and HS polymerized in CP and MP, which consisted with the dynamic of precursors and HS concentration (Wu et al., 2017). 3.2 The differences among various composting materials For guiding different materials composting, it is necessary to investigate the differences of precursors and bacteria communities among various materials. In this article, the simplex materials (not mixed) were chosen for better understand the differences. The NMDS of precursors concentration shows that the materials are divided into five groups (Fig. 1b), indicating that the HS precursors have their own formation regularity by the various property of raw materials during different composting, which may be caused by the various property of raw materials. Cellulose is the main structure of LW, CW and CS; however, they are not classified as the same group. This result may be due to that the changes of precursors concentration are different during composting. In addition, Wang et al. (2015) reported that the lignin in GW was hard to be degraded. Therefore, during the GW composting, protein and any other simple organic matters may primarily be decomposed by microbe. Whereas in CM, protein is more abundant than botanical materials. Therefore, it mainly produced amino acids (Fig. 1a), leading the CM adjacent to GW. The bacteria information in the five composting materials were mainly be divided into three categories (Fig. 1c): ① bacteria in HP; ② bacteria in TP
6
and CP, ③ bacteria in MP. This result demonstrates that the bacteria in the same period of different materials may perform similar functions. In HP, microbes degrade organic matter to satisfy their metabolism and release HS precursors. Until TP and CP, bacteria activity enters the secondary metabolic stage, begins to synthesis HS precursors and secrete polymerase. Therefore, the HS were mainly polymerized in the MP (Fig. 1a). As analyzed above, the bacteria function is similar to the compost in the same period, and according to the formation and polymerization regularity of different precursors, a new staging regulation method can be provided to improve the production of HS, which may apply to varied materials composting, at least the five substrates motioned in this article. But, first of all, the key factors which can direct and indirect influence HS formation must be identified for better understanding the HS formation. 3.3 Key factors of humic substance formation The environmental factors may be the most manageable factors affecting HS formation, and proposed a step-by-step regulating method to promote the production of HS (Wang et al., 2015). However, some other relationships can directly or indirectly affect the HS formation. Therefore, the SEM has been conducted to detect the casual relationships among the basic environmental factors, bacteria community information, precursors and HS (Fig. 2). During composting, the degradation of organic matter produces amino acids and reducing sugars, which directly promote HS formation. Amino acids and reducing sugars are also the energy and carbon source of bacteria metabolism, therefore, they can be produced during the whole process of composting (López-González et al., 2015). The greater the amount of amino acids and reducing
7
sugar production, the more obvious the promoting effect on HS synthesis. The polyphenols are mainly produced in HP and TP (Fig. 1), and in the other periods they are consumed as HS precursors, resulting in the negative relationship with HS production. However, the findings by incorporating all the data into SEM challenged the previous perceptions of bacteria significantly promote the formation of polyphenols, suggesting that the amino acids and reducing sugar, which related to the bacteria activity, are the dominant factors in forming HS and can be regulated during composting. Therefore, not only the environmental factors, the HS precursors can also be utilized to promote the formation of HS. Given that, the precursors and environmental factors are used as controlling factors to propose a new regulating method for HS formation. 3.4 A new staging regulating method to promote the formation of HS precursors Large of efforts availably improved HS production by adding lignin, biochar and any other waste materials, their fundamental theory is to provide precursors for HS formation (Dias et al., 2010). But little attention is paid on the key factors which have latent effect on the production of HS. In conjunction with all the conclusions have received above (Fig. 1 and 2), a staging regulating method has been provided to improve HS production (Fig. 4). The HS production may be promoted by adding HS precursors or environment factor on the hollow arrows. In HP, the amino acids and reducing sugars have been selected to stimulate the bacteria activity, because they are the small molecule organic matter that easy to be used by bacteria to quickly adapt to the composting environment (Berggren et al., 2010). The crop residues (e.g., bean dregs) or sugar-moll wastes may be a good choice to be the regulating materials. As the
8
composting step into TP and CP, the bacteria activity is intense and organic matter is degraded considerably. Adding NH4+-N and NO3--N is to supplement the depleted nitrogen sources and further promotes bacteria activities to release more HS precursors. Polyphenols, as one of the HS precursors that may not be affected by bacteria, but can significantly increase the concentration of carboxyl groups and thus may play an important role in improving the aromatization of HS. Furthermore, NO3--N may promote the polymerization of precursors to form HS, therefore, amino acids, reducing sugar and NO3--N may significantly improve the amount of HS by the Maillard reaction at MP. Anyhow, the new staging regulating method be provided in this article and refined the adjustment process and simplified the regulating factors, which will more effectively in improving the production of HS. 4. Conclusion This study revealed that the bacteria may decompose and transform organic matter into HS precursors in HP and TP. Thereafter, these precursors will polymerize to form HS in CP and MP. Therefore, a staging regulating method has been proposed to promote HS production by screening the key factors affecting HS formation. The ammonia and polyphenols wastewater, crop residues and sugar-mall wastes which contain sufficient NH4+-N, NO3--N, polyphenols, amino acids and reducing sugars, respectively, can be utilized as the wastes to promote HS production according to the method. The increased HS production provides more possibilities for the environmental remediation. E-supplementary data for this work can be found in e-version of this paper online.
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Acknowledgement This work was financially supported by the National Natural Science Foundation of China (No. 51178090), National Natural Science Foundation of China (No. 51378097), and the National Key Technology R&D Program (No. 2012BAJ21B02-02). Reference [1] Ali, M., Kazmi, A.A., Ahmed, N., 2014. Study on effects of temperature, moisture and pH in degradation and degradation kinetics of aldrin, endosulfan, lindane pesticides during full-scale continuous rotary drum composting. Chemosphere. 102, 68-75. [2] Berggren, M., Laudon, H., Haei, M., Ström, L., Jansson, M., 2010. Efficient aquatic bacterial metabolism of dissolved low-molecular-weight compounds from terrestrial sources. ISME J. 4, 408-416. [3] Eisenhauer, N., Bowker, M.A., Grace, J.B., Powell, J.R., 2015. From patterns to causal understanding: structural equation modeling (SEM) in soil ecology. PEDOBIOLOGIA. 58, 65-72. [4] Flores-Rentería, D., Rincón, A., Valladares, F., Yuste, J.C., 2016. Agricultural matrix affects differently the alpha and beta structural and functional diversity of soil microbial communities in a fragmented Mediterranean holm oak forest. Soil Biol. Biochem. 92, 79-90. [5] He, X.S., Xi, B.D., Zhang, Z.Y., Gao, R.T., Tan, W.B., Cui, D.Y., Yuan, Y., 2015. Composition, removal, redox, and metal complexation properties of dissolved organic nitrogen in composting leachates. J. Hazard. Mater. 2015, 283: 227-233.
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[6] Lim, S.L., Lee, L.H., Wu, T.Y., 2016. Sustainability of using composting and vermicomposting technologies for organic solid waste biotransformation: recent overview, greenhouse gases emissions and economic analysis. J. Cleaner Prod. 111, 262–278. [7] López-González, J.A., Suárez-Estrella, F., Vargas-García, M.C., López, M.J., Jurado, M.M., Moreno, J., 2015. Dynamics of bacterial microbiota during lignocellulosic waste composting: Studies upon its structure, functionality and biodiversity. [8] Smilek, J., Sedláček, P., Kalina, M., & Klučáková, M., 2015. On the role of humic acids’ carboxyl groups in the binding of charged organic compounds. Chemosphere. 138, 503-510. [9] Stevenson F.J., 1994 Humic Chemistry: Genesis, composition, Reactions. John Wiley and Sons, New York. [10] Tan KH., 2014 Humic matter in soil and the environment: principles and controversies. CRC Press. [11] Wang, K., Li, X.K., He, C., Chen, C.L., Bai, J.W., Ren, N.Q., Wang, J.Y., 2014. Transformation of dissolved organic matters in swine, cow and chicken manures during composting. Bioresour. Technol. 168, 222-228. [12] Wang, X., Cui, H., Shi, J., Zhao, X., Zhao, Y., Wei, Z., 2015. Relationship between bacterial diversity and environmental parameters during composting of different raw materials. Bioresour. Technol. 198, 395–402. [13] Wei, Y.Q., Wei, Z.M., Cao, Z.Y., Zhao, Y., Zhao, X.Y., Lu, Q., Wang, X.Q., Zhang,
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X., 2016. A regulating method for the distribution of phosphorus fractions based on environmental parameters related to the key phosphate-solubilizing bacteria during composting. Bioresour. Technol., 211, 610-617. [14] Wu, J.O., Zhao, Y., Zhao, W., Yang, T.X., Zhang, X., Xie, X.Y., Cui, H.Y., Wei, Z.M., 2017. Effect of precursors combined with bacteria communities on the formation of humic substances during different materials composting. Bioresour. Technol. 226, 191-199. [15] Xi, B.D, Zhao, X.Y, He, X.S, Huang, C.H, Tan, W.B, Gao, R.T, Zhang, H., Li, D., 2016. Successions and diversity of humic-reducing microorganisms and their association with physical-chemical parameters during composting[J]. Bioresour. Technol. 219, 204-211. [16] Zhao, Y., Lu, Q., Wei, Y.Q., Cui, H.Y., Zhang, X., Wang, X.Q., Wei, Z.M., 2016b. Effect of actinobacteria agent inoculation methods on cellulose degradation during composting based on redundancy analysis. Bioresour. Technol. 219, 196-203. Figure captions Fig. 1 The formation and polymerization of HS precursors during different materials composting. a: RDA between bacteria community, HS and HS precursors; b: NMDS of precursors concentration in different materials composting; c: NMDS of bacteria community (1-HP: heating phase; 2-TP: thermophilic phase; 3-CP: cooling phase; 4-MP: manure phase). Different colors represent different categories. Fig. 2 Structural equation model showing the direct and indirect effects of the key factors on the formation of HS. PCO1 and PCO2 represent the data derived from
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the principal coordinate analysis of bacteria community. The path coefficients are adjacent to the arrows, the width of the arrows represent the strength of the path. Fig. 3. A new staging regulating method for promoting HS production. The same color presented one period. The words above the hollow arrows meant the wastes containing HS precursors or environmental factors, which can be added into the composting piles to promote the production of HS. This method can be both utilized in lab and factory scale.
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Highlights
Precursors formed in heating and thermophilic phase during different composting.
The functions of bacteria have been identified in different composting stages.
Precursors are the direct factors affecting humic substance (HS) formation.
Environment factor and bacteria are the indirect factors to influence HS formation.
A new staging regulating method has been proposed to increase the amount of HS.
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Graphical abstract
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