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Biochar combined with montmorillonite amendments increase bioavailable organic nitrogen and reduce nitrogen loss during composting
Bioresource Technology 294 (2019) 122224
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Biochar combined with montmorillonite amendments increase bioavailable organic nitrogen and reduce nitrogen loss during composting
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Longji Zhua,1, Hongyu Yanga,1, Yue Zhaoa, Kejia Kangb, Yan Liub, Pingping Heb, Zhenting Wua, ⁎ Zimin Weia, a b
College of Life Science, Northeast Agricultural University, Harbin 150030, China Heilongjiang Province Environmental Science Research Institute, Harbin 150030, China
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Composting Biochar Montmorillonite Bioavailable organic nitrogen Nitrogen loss
This study aimed to compare the effects of biochar, montmorillonite and their mixture on nitrogen availability and nitrogen loss during chicken manure composting. Four lab-scale composting experiments, the control (CK), 5% biochar addition (BC), 5% montmorillonite addition (M) and 2.5% biochar + 2.5% montmorillonite addition (BCM), were established. Results showed that the addition of BC, M and BCM significantly improved the contents of bioavailable organic nitrogen and NH4+-N in composts. In addition, BC and BCM reduced N loss by 19.2% and 12.2%, respectively, in comparison with CK. Significant shift of key bacterial communities associated with N transformation were also found in four treatments. Redundancy analysis and structural equation models indicated different additives changed the correlation among bacterial communities, environmental factors and organic N fractions. Comparison of N availability and N loss indicated that the combination of biochar and montmorillonite are more effective than that of separate application during composting.
1. Introduction With the increasing global population and urbanization, the quantity of solid waste increases rapidly. The daily production of solid waste would be up to 11 million tons by the end of 21st century in the world
(Hoornweg et al., 2013). Organic solid waste is the largest proportion of solid waste, which includes agricultural waste, kitchen waste, sludge and garden waste etc. (Zhao et al., 2018, 2019a). Composting is a complex bio-oxidative process involving various microorganisms which could transform organic wastes into valuable end-products and reduce
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Corresponding author. E-mail address: [email protected] (Z. Wei). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.biortech.2019.122224 Received 5 September 2019; Received in revised form 26 September 2019; Accepted 28 September 2019 Available online 03 October 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
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2. Materials and methods
environmental risk (Awasthi et al., 2018; Wang et al., 2018d). The endproducts of composting with abundant humic substances and nutrients have been used as soil fertilizer and conditioner to improve soil fertility (Zhao et al., 2019b; Gao et al., 2019). Nitrogen (N) is one of the crucial elements for the survival and growth of biology (Wang et al., 2018c). Compost is usually rich in nitrogen and nutrients, for example, chicken manure compost is rich in proteins and ammonium, which is a good organic nitrogen fertilizer. However, there are also some challenges for the composting of nitrogenous wastes, such as N loss (Zhang et al., 2016; Wang et al., 2019b). It has been reported that 16–76% of nitrogen would be lost during composting of different materials (Barrington et al., 2002). Many studies were concentrated on inorganic nitrogen dynamics during composting of organic wastes (Shi et al., 2018; Wang et al., 2018b). Nevertheless, organic N plays a dominant role in the N transformation during composting. Microorganisms can use different forms of organic N as N sources or C sources. Especially, bioavailable organic N (BON) including amino acid, amino sugar and amine etc., is easily to be utilized by microorganisms and transformed into inorganic N (e.g., NH4+, NO3−) (Zhu et al., 2019a). BON contents in composts could reflect the ability of N supply and the value of composts. Therefore, it is necessary to develop efficient composting technology to improve BON contents in composts and reduce N loss. Recently, novel materials are becoming more and more popular in various filed (Ma et al., 2015; Nguyen et al., 2015). Meanwhile, the application of additives in composting has also received increasing attention, which could improve the composting efficiency and the quality of compost products (Yang et al., 2019). Among the all additives, biochar and montmorillonite are two of the common amendments and have been widely applied to improve the organic matter degradation, promote the humic substance formation, reduced the bioavailability of heavy metals and reduce N loss (Wang et al., 2017a; Chen et al., 2017; Hao et al., 2019; Yu et al., 2019). Biochar plays an important role in organic matter sequestration, due to the developed porous structure, the large surface area and the enriched functional groups (Crombie et al., 2013; Li et al., 2018). Wang et al. (2017a) found that biochar amendment improved the available nutrient contents and reduced nitrogen loss during pig manure composting. Montmorillonite, an irregular lamellar crystal, is composed of layers of one octahedral and two tetrahedral sheets (Van Olphen, 1963), which can adsorb organic matter by van der Waals interactions, ligand exchange and cation bridging etc. (Arnarson and Keil, 2000). A recent study also found that montmorillonite had the good adsorption capacity of methane and nitrogen (Luo et al., 2019). In addition, additives could impact the fates of microbial community and volatile organic compounds in the composting system (Wang et al., 2018a; Turan et al., 2009). The effect of biochar amendment on organic nitrogen fractions transformation during composting is largely unknown, although there were many reports about biochar application in composting. In addition, there are few studies on the application of montmorillonite in composting. Thus, if montmorillonite can absorb nitrogen in compost is unknown. In addition, the effect of combined application of biochar and montmorillonite on organic nitrogen fractions during composting is rarely reported. In this study, chicken manure (CM) was selected as a typical nitrogenous organic waste for composting experiment. The aims of this study were to (1) compare the effects of biochar, montmorillonite and their mixture amendments on the BON contents and N loss during CM composting; (2) investigate the effects of three additives on microbial communities; (3) explore potential mechanisms of three additives in organic N transformation based on key microorganisms and environmental factors. This study may provide a new strategy for improving the availability of N and reducing N loss of composting.
2.1. Composting materials and experimental design Fresh CM and rice husk were collected from Xiangfang farm in Harbin, China. Biochar was produced from corn stalks wastes via slow pyrolysis (300–450 °C) by the Heilongjiang Academy of Agricultural Sciences in Harbin, China. Montmorillonite (> 95%, wt) was purchased from Inner Mongolia Hemingsheng Chemical Co., Ltd., China. Both of biochar and montmorillonite were crushed into fine particles (200–800 mesh) before adding into composting. Four lab-scale composting experiments were set-up from CM mixed with rice husk and then added 5% biochar (BC), 5% montmorillonite (M) and 2.5% biochar + 2.5% montmorillonite (BCM) in to the composting mixture (dry weight basis). There were three replicates for each treatment, so a total of twelve piles in this experiment. The initial C/N ratio of the composting mixture was adjusted to about 25 and the moisture was around 60%. All composting materials were put into the special cylinder compost reactor described in the previous study (Zhao et al., 2016). Composting experiments were carried out for 60 days, and samples were collected on day 0, 4, 9, 13, 21, 40 and 60 for physicochemical and microbial analysis. To obtain representative samples, fivepoint sampling method was used in this study, and then one representative sample was obtained by mixing five sub-samples (Chen et al., 2019a,b). The samples of 0d in four treatments were collected before composting, and only one sample of 0d was used representing the samples from four treatments due to no significant differences of properties among four treatments at the 0th day (p < 0.05). Each of the sample was divided into two parts, where one was air-dried and milled to 0.15 mm to analyze physicochemical properties, and the other was freeze-dried, ground to 1 mm, and stored at −80 °C for DNA extraction. 2.2. Physicochemical properties measurement Composting temperature was measured by a digital thermometer every day. The pH value was measured with aqueous of compost samples (sample-water ratio 1:10 w/v) by a digital pH meter. The moisture content (MC) was estimated based on the weight loss after drying samples at 105 °C for 24 h until a constant weight. Total organic carbon (TOC) was measured by the total organic carbon analyzer (TOCVCPH, Shimadzu, Japan) with a solid sample module (SSM-5000A), which can directly measure TOC contents of solid samples after sieving with 200 mesh (Zhu et al., 2019b). Organic matter was determined by weight loss after ignition at 550 °C for 4 h in a muffle furnace (Wei et al., 2019). To measure ammonia nitrogen (NH4+) concentration, the sample was shaken with 2 mol·L−1 KCl (1:10 ratio) at 200 rpm for 1 h and filtered and then the filtrates were assayed for ammonium using the NaRSH’S colorimetry. Nitrogen loss (N loss) was calculated based on the following formula:
N loss (%) = 1 − (X1N2)/(X2N1) where, X1, X2 are initial and final ash contents, and N1, N2 are initial and final nitrogen contents (Wang et al., 2017b). 2.3. Extraction and determination of organic N fractions Total nitrogen (TN) was measured according to the Kjeldahl method. Total organic nitrogen (TON) was identified by subtracting NH4+ from TN. The acid hydrolysis method was used to separate organic N forms (Keeney & Bremner, 1964), including amino acid N (AAN), amino sugar N (ASN), amine N (AN) and hydrolyzable unknown N (HUN). The procedure was briefly introduced as follows: each compost sample (1 g), 6 mol·L−1 HCl (50 mL) and octanol (100 µL) were combined in a 150 mL flask. Afterwards, flasks were sealed and placed in an oven at 105 °C for 12 h. Then, various organic N forms were 2
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of NH4+ in compost products, thereby improved the N availability for plants. The distribution of organic N fractions is shown in Fig. 1c. Compared with inorganic N, organic N had richer amounts and forms in composts. In general, AN, AAN and HUN were the major forms of organic N in all treatments, while ASN was only appeared in uncertain days, which was consistent with the previous work (Zhou et al., 2018). Zhou et al. (2018) also found that the contents of ASN showed an irregular trend during composting with different wastes due to rapid cycling of ASN in microorganisms. The relative contents of organic N fractions were fluctuant during composting, due to the complex transformation relationships among different N fractions (Zhou et al., 2018). Among the four forms of organic N, AN, AAN and ASN are recognized as the main contributors of BON (Li et al., 2014), which can be utilized and transformed by microorganisms and a few plants. Importantly, at the end of composting (60th day), the contents of BON in BC, M and BCM were significantly higher than that in CK (p < 0.05) and the order was as follows: M (11.2 g/kg) > BCM (10.5 g/kg) > BC (7.33 g/ kg) > CK (6.24 k/kg). This result indicates biochar and montmorillonite all contributed to the increase of BON contents in composts, especially for M and BCM. Awasthi et al. (2017) also found that biochar amendment increased the content of water-soluble organic N in composts. In addition, amino acid is an important precursor of humic substance (Wu et al., 2017a; Zhang et al., 2019). Thus, the increase of AAN contents may be contributed to the formation of humic substance. However, the N loss in M was significantly higher in comparison with three other treatments (p < 0.05), which may be due to the fact that montmorillonite overly promoted the N mineralization and the nitrification was week at the thermophilic phase (Fig. 1d). In comparison with CK, the N loss in BC and BCM reduced by 19.2% and 12.2%, respectively (p < 0.01). Based on the above results, it can be concluded that the mixture addition of biochar and montmorillonite can both increase the BON contents and reduce N loss during CM composting.
measured by steam distillation with different additives, where total hydrolyzable organic N (THN) with NaOH after Kjeldahl digestion with H2SO4 and catalyst (CuSO4 and K2SO4) mixture; AN with MgO; AN + ASN with phosphate-borate buffer; AAN with phosphate-borate buffer after treating with 0.5 mol·L−1 NaOH and ninhydrin (pH = 2.5) at 100 °C to convert AAN to NH4+. The contents of organic N fractions were calculated as follows (Zhu et al., 2019b): ASN = (AN + ASN) − AN BON = AN + AAN + ASN HUN = THN − BON 2.4. DNA extraction and PCR-DGGE analysis DNA was extracted from 3 g of each sample using soil DNA kit (Omega Biotek, Inc.) according to the manufacturer’s instructions. The concentration and quality of extracted DNA were detected with the Nanodrop One/Onec (Thermo Fisher Scientific, Wilmington, USA). The DNA extracts were stored at −20 °C for further analysis. 16S rRNA gene was amplified using the bacterial universal primers 357F (5′-CC TAC GGG AGG CAG CAG-3′) and 534R (5′-ATT ACC GCG GCT GCT GG-3′). PCR products were used for DGGE analysis by the Gene Mutation Detection System (Bio-Rad, Hertfordshire, UK). Gels contained 8% (w/ v) poly-acrylamide with a linear gradient of 35–60% denaturant (Wu et al., 2017b). Gels were electrophoresed at 60 °C for 6 h at 130 V. Finally, Gel Doc™ imaging system (Bio-Rad, Hertfordshire, UK) was used for scanning gels after stained with EB. Representative bands of DGGE was sequenced and the results were compared with the GenBank database. 2.5. Statistical analysis All the data of physicochemical properties were analyzed using SPSS 23.0 and OriginPro 2017. Differences among samples were analyzed using the LSD All-Pairwise Comparisons Test in Statistix 8. Differences among samples were declared at the p < 0.05 probability level of significance. The intensity of DGGE bands was qualified with Quantity One (version 5.0, Bio-Rad, USA). Cluster heatmap was conducted using R 3.6.1. Redundancy analysis (RDA) was performed using CANOCO 5.0. Structural equation models (SEMs) were conducted by AMOS 23.0 software (IBM Corporation Software Group, Somers, NY) using the maximum-likelihood estimation method. Non-significant chisquare test (p > 0.05), high goodness-of-fit index (GFI > 0.90), and low root mean square errors of approximation (RMSEA < 0.05) were used to show the overall goodness of fit for SEMs.
3.2. Comparison of bacterial community composition in different treatments PCR-DGGE was performed to compare the bacterial community composition in different treatments. A total of forty-eight different bands were detected in the DGGE profiles in different treatments. Sequencing results showed that these bands were mainly belonging to four phyla: Proteobacteria (26.8%), Firmicutes (22.0%), Bacteroidetes (19.5%) and Actinobacteria (14.6%), which is consistent with many previous studies (Zhu et al., 2019a,b). There were twenty-four bands shown in Fig. 2a, which were significantly correlated with N fractions (Fig. 3). The bacterial communities exhibited a significant difference among different treatments. For example, the relative abundance of bands 2, 23, 26, 27, 44 in BC were higher than those in CK. These bands belonged to Firmicutes, Bacteroidetes and Actinobacteria, suggesting that biochar could promote different microbial taxa. Previous study indicated that biochar can serve as a suitable habitat for microorganisms due to its porous structure and high surface area (Xiao et al., 2017). In addition, biochar contained lots of labile aliphatic compounds and inorganic nutrients, which can be used as carbon source or nutrient source for microorganisms (Steiner, et al., 2016). Thus, the abundance of above bacteria increased in BC treatment. Meanwhile, the relative abundance of bands 14, 27 and 44 in M were also higher than those in CK. In comparison, more than half of the bands showed higher abundance in BCM than CK. These results indicate that biochar and montmorillonite could enhance the activity of some bacteria during composting, especially for the mixture addition of biochar and montmorillonite, which is consistent with the variation of N fractions contents in different treatments. The NMDS analysis suggests that the samples of each treatment tended to cluster together (Fig. 2b). In addition, the samples of BCM were completely separated from that of CK, suggesting the significant difference in bacterial community composition between these two
3. Results and discussion 3.1. Comparison of the contents of N fractions in different treatments The contents of TON ranged from 12.8 to 25.7 g/kg during composting (Fig. 1a). In general, TON contents in all treatments decreased at the first four days and then showed an increased trend, eventually, leveled off. The initial decreasing trend of TON was due to the rapid degradation of complex nitrogenous organic compounds and loss of NH3 (Awasthi et al., 2017). During the first four days, the TON contents decreased faster in BC, M and BCM than that in CK. At the final stage of composting, the TON contents of three treatments were also lower than that of CK, especially for the M group, suggesting that biochar, montmorillonite and their mixture addition promoted the decomposition of TON during composting. The contents of NH4+ showed a significantly decreasing trend in all treatments at the first 21 day of composting, and then increased until the 60th day of composting (Fig. 1b). The NH4+ contents in BC, M and BCM were higher than that in CK throughout the composting process. The above results indicate that biochar, montmorillonite and their mixture addition contributed to the sequestration 3
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Fig. 1. Distribution of (a) total organic nitrogen, (b) NH4+, (c) organic N fractions and (d) nitrogen loss in different treatments.
bacterial communities and N fractions will contribute to understand the transformation mechanism of N in different treatments.
treatments. Although both BC and M partly overlapped with CK, there were still visible differences among them. The Shannon-Wiener index of BCM was also significantly higher than that of CK at the mature phase of composting (the 40th and 60th days) (Fig. 2c). These above results together indicate the obviously changes of bacterial communities caused by the addition of biochar and montmorillonite, especially for their mixture. Biochar and montmorillonite amendments may provide a suitable environment for microorganisms to transform organic N during composting. Therefore, further studying the relationship between
3.3. Roles of bacterial communities in organic N transformation There were different relationships between bacterial communities and N fractions in different treatments (Fig. 3). In CK, the bacteria in group D1, including Pseudomonas, Acinetobacter and Gramella etc., showed significantly positive correlations with BON, AN and AAN,
Fig. 2. Composition of bacterial community in different treatments. (a) Abundance of bacterial bands derived from DGGE (only show the bands significantly correlated with N fractions in Fig. 3), and the size of the circle represents the relative abundance of the DGGE band; (b) NMDS of bacterial community composition; (c) Shannon-Wiener index of bacterial community. 4
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have the BON sequestration ability (negatively correlated with BON) in CK, while the addition of montmorillonite and their mixture with biochar made D4 bacteria developed the ability of BON sequestration (positively correlated with BON). These results indicate that biochar and montmorillonite could strengthen different types of microorganisms associated with N transformation, which may be due to the fact that biochar and montmorillonite provide different habitat environment for microorganisms. The porous structure of biochar provides sufficient oxygen and carbon source for almost all aerobic bacteria. By contrast, the surface of montmorillonite is rich in both positive and negative charges, but only positive charges could adsorb bacteria due to the electronegativity of bacteria in compost. Thus, biochar provides a more suitable habitat environment for microorganisms than montmorillonite. It is similar to the previous found that microorganisms were more prone to colonize on biochar surfaces during composting, compared with other bulking agents (e.g., peat bog and zeolite) (Wang et al., 2017a).
3.4. Relationships between bacterial communities and environmental factors in different treatments As described in Section 3.3., bacterial communities associated with N transformation could also be influenced by the environmental conditions during composting. Thus, to explore the relationship between key bacterial communities (e.g., groups D1–D4) and environmental factors in different treatments, RDA was performed (Fig. 4). All of the canonical axes were significant (p < 0.05), indicating that these environmental factors are important in explaining the variation of bacterial communities. 70.10%, 67.42%, 69.45%, and 61.52% of the variations of species-environmental factors relation were explained by the first two canonical axes in CK, BC, M and BCM, respectively. Considering the critical role of group D1 bacteria in the accumulation of BON, different treatments had variable effects on group D1 bacteria and environmental factors. In CK treatment, MC and OM were positively correlated with group D1 bacteria (Fig. 4a). By contrast, MC and OM was still positively correlated with group D1 bacteria in BC (Fig. 4b), but the correlation between each band (i.e., group D1) and environmental factor (i.e., OM and MC) changed slightly. This result indicates that MC and OM may strengthen group D1 bacteria to increase the BON contents in BC treatment. Sánchez-García et al. (2015) found that the addition of biochar could prevent the formation of large clumps and promote O2 diffusion into the composting mixture, thereby facilitate the consumption of water and organic matter by microorganisms. In addition, pH also played a more important role in BC treatment. The pH was significantly positively correlated with bands 2, 3, 10, 22, 23, 25, 31 and 35 which contributed to the accumulation of TON. Previous studies also found that biochar addition slightly increased the initial pH of composting (Vandecasteele et al., 2016; Liu et al., 2017). In M treatment, except for MC and OM, the group D1 bacteria also positively correlated with C/N, suggesting that higher C/N could promote N transformation, especially for BON (Fig. 4c). In addition, high C/N was contributed to the decomposition of organic N by microorganisms. From this view, high C/N would increase the contents of inorganic N in compost, which may be a potential reason for N loss in M treatment. This is because increasing C/N properly will provide carbon source for microorganisms to depolymerize nitrogenous compounds. However, the correlation between group D1 bacteria and environmental factors are weaker in BCM than that in CK (Fig. 4d). Some of the DGGE bands of group D4 (e.g., 2, 3, 36 and 46) were negatively correlated with C/N, while band 25 showed a positive correlation to the temperature. Based on the above results, we find that different treatments could change various environmental factors to affect the key bacteria associated with N transformation during composting.
Fig. 3. Relationships between bacterial taxa and nitrogen fractions in different treatments. Group D1–D4 represent four typical bacterial taxa associated with N transformation. The numbers from 1 to 48 represent 48 DGGE bands, respectively.
suggesting that these bacteria may contribute to the accumulation of organic N fractions during composting. By contrast, the relationship between D1 bacteria and organic N fractions in BC were higher than that in CK. This result indicates that the addition of biochar promoted the role of D1 bacteria in the organic N transformation, which may be a main reason for the accumulation of organic N, especially for BON fractions (e.g., AN, AAN). In contrast, the relationship between D1 bacteria and organic N fractions (i.e., BON, AN, AAN) in M was not significantly different from that in CK. Nevertheless, the relationship between D1 bacteria and organic N fractions in BCM became more negatively in comparison with CK. This result indicates that D1 may not play a critical role in BON accumulation in M and BCM, which is not similar to BC. In comparison with CK, group D2 bacteria in M became negatively correlated with BON, AN and AAN, while D3 bacteria became positively correlated with ASN and HUN. This result indicates that D2 bacteria may be related to BON mineralization, while D3 bacteria played an important role in the accumulation of ASN and HUN in M treatment. In addition, group D4 bacteria were negatively correlated with BON, AN and AAN in CK, while the relationship became more positively in M and BCM. This result indicates that group D4 bacteria may have a stronger ability for the storage of BON than group D1. In total, group D1, D3 and D4 bacteria were all contributed to the storage of BON, rather than mineralization. In addition, there were more bacteria (e.g., bands 36, 44 and 47) significantly negatively correlated with NH4+ in M in comparison with CK, which is consistent with the serious N loss in M. These results further indicate that different treatments had varying impacts on bacterial communities associated with N transformation during composting. For instance, D1 bacteria had the BON sequestration ability (positively correlated with BON) in CK, and biochar just promoted the ability of D1 bacteria. However, D4 bacteria did not
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Fig. 4. RDA of the relationships between environmental factors and bacterial communities (p < 0.05) in (a) CK, (b) BC, (c) M and (d) BCM. The yellow arrows represent bacterial taxa significantly correlated with nitrogen fractions in Fig. 3. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
In M, neither environmental factors nor bacterial communities had a positive effect on AAN (Fig. 5f). Nevertheless, AAN had a directly positive effect on NH4+, suggesting that AAN were greatly mineralized into inorganic N. In addition, OM, C/N and MC together had positive correlations with NH4+. This result is consistent with the higher N loss in M treatment. In BCM treatment, C/N and OM all showed an indirectly positive effect on AAN by changing bacterial community, and AAN did not have an effect on NH4+, suggesting that BCM could reduce AAN mineralization into NH4+ in comparison with M, thereby reducing the risk of N loss. These above results confirm that all the treatments in this study contributed to increase AAN contents, but montmorillonite amendments could cause a serious N loss in comparison with CK. In addition, results found that the bacterial community (e.g., ammonifier) played an important role in the transformation of AAN to NH4+. For example, Flavobacterium (band 1) and Pseudomonas fluorescence (band 12) could depolymerize the high molecular weights N-containing compounds (e.g., proteins), which is the rate-limiting step of microbial decomposition of organic N (Bach and Munch, 2000). AN is one form of organic N with the simplest structure, which is easily used by microorganisms (Zhou et al., 2018). Some of AN are from the deamination of amino acids and amino sugars, and some of AN are from amide compounds. OM had an indirectly positive effect on AN, mediated by bacterial community in CK (Fig. 5b). Some bacillus, such as Bacillus subtilis and Bacillus cereus could convert simple nitrogenous compounds to ammonium by deamination. OM could provide carbon sources for microorganisms to decompose nitrogenous compounds. By contrast, OM and bacterial community together showed a directly positive effect on AN in BC treatment. In addition, indirectly positive effect of the temperature (T) on AN was also found in BC treatment (Fig. 5e). Only OM was found to have a directly positive effect on AN in the M treatment (Fig. 5g). MC and C/N had an indirectly positive effect
3.5. Possible mechanism of different additives to increase BON contents Based on the results in Sections 3.2–3.4, biochar and montmorillonite amendments can not only change the functional bacterial communities, but also the environmental factors during composting. However, it is still largely unknown if these complex factors of composts directly or indirectly affected N transformation in different treatments. SEM, an a prior method to visualize the causal relationships between variables by fitting data to the model representing causal hypotheses, was used to further confirm the causal relationships between different environmental factors, bacterial communities (data of NMDS) and N bioavailability (data of N fractions) in CK, BC, M and BCM. SEMs have been widely to explore the causal relationships between environmental conditions and microbial communities in the area of environmental science, especially for composting (Chen et al., 2019b; Wang et al., 2019a). The hypothesis of SEMs was based on organic N mineralization process mediated by microorganisms (e.g., ammoniation). The factors used in SEMs were selected based on the results of RDA. SEMs results showed that environmental factors and bacterial communities affected BON fractions and their mineralization into NH4+, but key environmental factors and bacterial taxa changed in different treatments (Fig. 5). AAN as the main forms of BON, occupied a large proportion of organic N in composts, which is usually found in proteins, polypeptide and microbial residue. Only bacterial communities had a negative effect on AAN in CK (Fig. 5a), while OM indirectly positively affected AAN mediated by bacterial communities in BC (Fig. 5d). This result indicates that biochar amendments may increase OM contents, thereby it would provide abundant carbon source for microorganisms to depolymerize high molecular weight nitrogenous compounds into AAN. This result may be explained the higher contents of AAN in BC (15.3%) than that in CK (10.7%) at the end of composting. 6
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Fig. 5. Effects of environmental factors and bacterial communities on organic nitrogen transformation (based on structural equation models). Red and black arrows represent significantly positive and negative relationships, respectively (p < 0.05). Dashed lines represent non-significant relationship (p > 0.05). Numbers adjacent to arrows are standardized path coefficients, analogous to relative regression weights and indicative of the effect size of the relationship. *p < 0.05, **p < 0.01, ***p < 0.001. df, degree of freedom. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
of ASN during composting. In M treatment, T had an indirectly positive effect on NH4+ by affecting ASN (Fig. 5h). ASN was rapidly mineralized into NH4+ at the thermophilic phase of composting, and the nitrification and denitrification were inhibited at high temperature, so nitrogen would be loss as the form of NH3. In BCM treatment, the mineralization of ASN into NH4+ was significantly inhibited in comparison with M (Fig. 5k).
on AN by changing bacterial community in BCM (Fig. 5j). Meanwhile, HUN and AN had a positive effect on NH4+ in M and BCM treatments, respectively, suggesting that the mineralization of organic N fractions in M and BCM were more serious than those in CK and BC. As for the ASN, it was only appeared in CK, M and BCM treatments. T, C/N and OM all had significant effects on ASN in CK (Fig. 5c). These multiple effects on ASN may be a reason causing the irregularly variable contents 7
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Based on the above results, it can be found that there were different mechanisms on N transformation in different treatments. On the one hand, biochar can promote the accumulation of BON (e.g., AN, AAN) by changing organic matter contents, moisture contents and T of composts. These key environmental factors may be contributed to the direct adsorption of BON by biochar. On the other hand, biochar could also promote microbial storage and transformation of BON by creating a proper living environment for microorganisms. However, compared with biochar, montmorillonite amendment had weaker effect on microorganisms of composts. And microorganisms had no significant contributions on the sequestration of BON in M treatment. The Montmorillonite might directly adsorb BON on its surface. Although BON contents in M treatment was higher than that in CK and BC treatments, the N loss in M was also the most serious. Previous study indicated that van der Waals interaction was a major mechanism of adsorption to montmorillonite of organic matter (Arnarson and Keil, 2000). It is acknowledged that van der Waals interaction is usually weaker than the chemical bond. Thus, when lots of BON molecular were adsorbed on the surface of montmorillonite, some of them may be mineralized to inorganic nitrogen due to the incompact combination between montmorillonite and BON. Based on the above results, the mixture of biochar and montmorillonite amendments is a better choice for real compositing production, which can not only increase the bioavailability of nitrogen, but also reduce nitrogen loss. The retained BON in compost products will be transformed inorganic N for plant growth, when plants are short of N. Thus, biochar and montmorillonite amendments can also improve the durable nitrogen supply capacity of compost products in practical application.
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4. Conclusion This study confirmed that the addition of functional additives (i.e., BC, M and BCM) significantly improved the contents of bioavailable organic N and NH4+ in composts. In addition, BC and BCM reduced N loss by 19.2% and 12.2%, respectively. BC and BCM affected N transformation by changing key bacterial communities and environmental factors during composting, while M only changed environmental factors related to N adsorption. Comparison of N availability and N loss indicated that the combination of biochar and montmorillonite could be more effective for practical composting production. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement This work was financially supported by the National Natural Science Foundation of China (No. 51778116, No. 51878132 and No. 51978131) Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.122224. References Arnarson, T.S., Keil, R.G., 2000. Mechanisms of pore water organic matter adsorption to montmorillonite. Mar. Chem. 71 (3), 309–320. Awasthi, M.K., Chen, H., Wang, Q., Liu, T., Duan, Y., Awasthi, S.K., Ren, X., Tu, Z., Li, J., Zhao, J., Zhang, Z., 2018. Succession of bacteria diversity in the poultry manure composted mixed with clay: studies upon its dynamics and associations with physicochemical and gaseous parameters. Bioresour. Technol. 267, 618–625. Awasthi, M.K., Wang, Q., Chen, H., Wang, M., Ren, X., Zhao, J., Li, J., Guo, D., Li, D., Awasthi, S.K., Sun, X., Zhang, Z., 2017. Evaluation of biochar amended biosolids co-
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Role of NH3 recycling on nitrogen fractions during sludge composting. Bioresour. Technol. https://doi.org/10.1016/j.biortech.2019.122175. Wang, X., Zhao, Y., Wang, H., Zhao, X., Cui, H., Wei, Z., 2017b. Reducing nitrogen loss and phytotoxicity during beer vinasse composting with biochar addition. Waste Manage. 61, 150–156. Wei, Y., Wu, D., Wei, D., Zhao, Y., Wu, J., Xie, X., Zhang, R., Wei, Z., 2019. Improved lignocellulose-degrading performance during straw composting from diverse sources with actinomycetes inoculation by regulating the key enzyme activities. Bioresour. Technol. 271, 66–74. Wu, J., Zhao, Y., Qi, H., Zhao, X., Yang, T., Du, Y., Zhang, H., Wei, Z., 2017a. Identifying the key factors that affect the formation of humic substance during different materials composting. Bioresour. Technol. 244, 1193–1196. Wu, J., Zhao, Y., Zhao, W., Yang, T., Zhang, X., Xie, X., Cui, H., Wei, Z., 2017b. Effect of precursors combined with bacteria communities on the formation of humic substances during different materials composting. Bioresour. Technol. 226, 191–199. Xiao, R., Awasthi, M.K., Li, R., Park, J., Pensky, S.M., Wang, Q., Wang, J.J., Zhang, Z., 2017. Recent developments in biochar utilization as an additive in organic solid waste composting: a review. Bioresour. Technol. 246, 203–213. Yang, K., Zhu, L., Zhao, Y., Wei, Z., Chen, X., Yao, C., Meng, Q., Zhao, R., 2019. A novel method for removing heavy metals from composting system: the combination of functional bacteria and adsorbent materials. Bioresour. Technol., 122095. Yu, H., Zhao, Y., Zhang, C., Wei, D., Wu, J., Zhao, X., Hao, J., Wei, Z., 2019. Driving effects of minerals on humic acid formation during chicken manure composting: emphasis on the carrier role of bacterial community. Bioresour. Technol. https://doi. org/10.1016/j.biortech.2019.122239. Zhang, Y., Zhao, Y., Chen, Y., Lu, Q., Li, M., Wang, X., Wei, Y., Xie, X., Wei, Z., 2016. A regulating method for reducing nitrogen loss based on enriched ammonia-oxidizing
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Biochar combined with montmorillonite amendments increase bioavailable organic nitrogen and reduce nitrogen loss during composting
T
Longji Zhua,1, Hongyu Yanga,1, Yue Zhaoa, Kejia Kangb, Yan Liub, Pingping Heb, Zhenting Wua, ⁎ Zimin Weia, a b
College of Life Science, Northeast Agricultural University, Harbin 150030, China Heilongjiang Province Environmental Science Research Institute, Harbin 150030, China
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Composting Biochar Montmorillonite Bioavailable organic nitrogen Nitrogen loss
This study aimed to compare the effects of biochar, montmorillonite and their mixture on nitrogen availability and nitrogen loss during chicken manure composting. Four lab-scale composting experiments, the control (CK), 5% biochar addition (BC), 5% montmorillonite addition (M) and 2.5% biochar + 2.5% montmorillonite addition (BCM), were established. Results showed that the addition of BC, M and BCM significantly improved the contents of bioavailable organic nitrogen and NH4+-N in composts. In addition, BC and BCM reduced N loss by 19.2% and 12.2%, respectively, in comparison with CK. Significant shift of key bacterial communities associated with N transformation were also found in four treatments. Redundancy analysis and structural equation models indicated different additives changed the correlation among bacterial communities, environmental factors and organic N fractions. Comparison of N availability and N loss indicated that the combination of biochar and montmorillonite are more effective than that of separate application during composting.
1. Introduction With the increasing global population and urbanization, the quantity of solid waste increases rapidly. The daily production of solid waste would be up to 11 million tons by the end of 21st century in the world
(Hoornweg et al., 2013). Organic solid waste is the largest proportion of solid waste, which includes agricultural waste, kitchen waste, sludge and garden waste etc. (Zhao et al., 2018, 2019a). Composting is a complex bio-oxidative process involving various microorganisms which could transform organic wastes into valuable end-products and reduce
⁎
Corresponding author. E-mail address: [email protected] (Z. Wei). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.biortech.2019.122224 Received 5 September 2019; Received in revised form 26 September 2019; Accepted 28 September 2019 Available online 03 October 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
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2. Materials and methods
environmental risk (Awasthi et al., 2018; Wang et al., 2018d). The endproducts of composting with abundant humic substances and nutrients have been used as soil fertilizer and conditioner to improve soil fertility (Zhao et al., 2019b; Gao et al., 2019). Nitrogen (N) is one of the crucial elements for the survival and growth of biology (Wang et al., 2018c). Compost is usually rich in nitrogen and nutrients, for example, chicken manure compost is rich in proteins and ammonium, which is a good organic nitrogen fertilizer. However, there are also some challenges for the composting of nitrogenous wastes, such as N loss (Zhang et al., 2016; Wang et al., 2019b). It has been reported that 16–76% of nitrogen would be lost during composting of different materials (Barrington et al., 2002). Many studies were concentrated on inorganic nitrogen dynamics during composting of organic wastes (Shi et al., 2018; Wang et al., 2018b). Nevertheless, organic N plays a dominant role in the N transformation during composting. Microorganisms can use different forms of organic N as N sources or C sources. Especially, bioavailable organic N (BON) including amino acid, amino sugar and amine etc., is easily to be utilized by microorganisms and transformed into inorganic N (e.g., NH4+, NO3−) (Zhu et al., 2019a). BON contents in composts could reflect the ability of N supply and the value of composts. Therefore, it is necessary to develop efficient composting technology to improve BON contents in composts and reduce N loss. Recently, novel materials are becoming more and more popular in various filed (Ma et al., 2015; Nguyen et al., 2015). Meanwhile, the application of additives in composting has also received increasing attention, which could improve the composting efficiency and the quality of compost products (Yang et al., 2019). Among the all additives, biochar and montmorillonite are two of the common amendments and have been widely applied to improve the organic matter degradation, promote the humic substance formation, reduced the bioavailability of heavy metals and reduce N loss (Wang et al., 2017a; Chen et al., 2017; Hao et al., 2019; Yu et al., 2019). Biochar plays an important role in organic matter sequestration, due to the developed porous structure, the large surface area and the enriched functional groups (Crombie et al., 2013; Li et al., 2018). Wang et al. (2017a) found that biochar amendment improved the available nutrient contents and reduced nitrogen loss during pig manure composting. Montmorillonite, an irregular lamellar crystal, is composed of layers of one octahedral and two tetrahedral sheets (Van Olphen, 1963), which can adsorb organic matter by van der Waals interactions, ligand exchange and cation bridging etc. (Arnarson and Keil, 2000). A recent study also found that montmorillonite had the good adsorption capacity of methane and nitrogen (Luo et al., 2019). In addition, additives could impact the fates of microbial community and volatile organic compounds in the composting system (Wang et al., 2018a; Turan et al., 2009). The effect of biochar amendment on organic nitrogen fractions transformation during composting is largely unknown, although there were many reports about biochar application in composting. In addition, there are few studies on the application of montmorillonite in composting. Thus, if montmorillonite can absorb nitrogen in compost is unknown. In addition, the effect of combined application of biochar and montmorillonite on organic nitrogen fractions during composting is rarely reported. In this study, chicken manure (CM) was selected as a typical nitrogenous organic waste for composting experiment. The aims of this study were to (1) compare the effects of biochar, montmorillonite and their mixture amendments on the BON contents and N loss during CM composting; (2) investigate the effects of three additives on microbial communities; (3) explore potential mechanisms of three additives in organic N transformation based on key microorganisms and environmental factors. This study may provide a new strategy for improving the availability of N and reducing N loss of composting.
2.1. Composting materials and experimental design Fresh CM and rice husk were collected from Xiangfang farm in Harbin, China. Biochar was produced from corn stalks wastes via slow pyrolysis (300–450 °C) by the Heilongjiang Academy of Agricultural Sciences in Harbin, China. Montmorillonite (> 95%, wt) was purchased from Inner Mongolia Hemingsheng Chemical Co., Ltd., China. Both of biochar and montmorillonite were crushed into fine particles (200–800 mesh) before adding into composting. Four lab-scale composting experiments were set-up from CM mixed with rice husk and then added 5% biochar (BC), 5% montmorillonite (M) and 2.5% biochar + 2.5% montmorillonite (BCM) in to the composting mixture (dry weight basis). There were three replicates for each treatment, so a total of twelve piles in this experiment. The initial C/N ratio of the composting mixture was adjusted to about 25 and the moisture was around 60%. All composting materials were put into the special cylinder compost reactor described in the previous study (Zhao et al., 2016). Composting experiments were carried out for 60 days, and samples were collected on day 0, 4, 9, 13, 21, 40 and 60 for physicochemical and microbial analysis. To obtain representative samples, fivepoint sampling method was used in this study, and then one representative sample was obtained by mixing five sub-samples (Chen et al., 2019a,b). The samples of 0d in four treatments were collected before composting, and only one sample of 0d was used representing the samples from four treatments due to no significant differences of properties among four treatments at the 0th day (p < 0.05). Each of the sample was divided into two parts, where one was air-dried and milled to 0.15 mm to analyze physicochemical properties, and the other was freeze-dried, ground to 1 mm, and stored at −80 °C for DNA extraction. 2.2. Physicochemical properties measurement Composting temperature was measured by a digital thermometer every day. The pH value was measured with aqueous of compost samples (sample-water ratio 1:10 w/v) by a digital pH meter. The moisture content (MC) was estimated based on the weight loss after drying samples at 105 °C for 24 h until a constant weight. Total organic carbon (TOC) was measured by the total organic carbon analyzer (TOCVCPH, Shimadzu, Japan) with a solid sample module (SSM-5000A), which can directly measure TOC contents of solid samples after sieving with 200 mesh (Zhu et al., 2019b). Organic matter was determined by weight loss after ignition at 550 °C for 4 h in a muffle furnace (Wei et al., 2019). To measure ammonia nitrogen (NH4+) concentration, the sample was shaken with 2 mol·L−1 KCl (1:10 ratio) at 200 rpm for 1 h and filtered and then the filtrates were assayed for ammonium using the NaRSH’S colorimetry. Nitrogen loss (N loss) was calculated based on the following formula:
N loss (%) = 1 − (X1N2)/(X2N1) where, X1, X2 are initial and final ash contents, and N1, N2 are initial and final nitrogen contents (Wang et al., 2017b). 2.3. Extraction and determination of organic N fractions Total nitrogen (TN) was measured according to the Kjeldahl method. Total organic nitrogen (TON) was identified by subtracting NH4+ from TN. The acid hydrolysis method was used to separate organic N forms (Keeney & Bremner, 1964), including amino acid N (AAN), amino sugar N (ASN), amine N (AN) and hydrolyzable unknown N (HUN). The procedure was briefly introduced as follows: each compost sample (1 g), 6 mol·L−1 HCl (50 mL) and octanol (100 µL) were combined in a 150 mL flask. Afterwards, flasks were sealed and placed in an oven at 105 °C for 12 h. Then, various organic N forms were 2
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of NH4+ in compost products, thereby improved the N availability for plants. The distribution of organic N fractions is shown in Fig. 1c. Compared with inorganic N, organic N had richer amounts and forms in composts. In general, AN, AAN and HUN were the major forms of organic N in all treatments, while ASN was only appeared in uncertain days, which was consistent with the previous work (Zhou et al., 2018). Zhou et al. (2018) also found that the contents of ASN showed an irregular trend during composting with different wastes due to rapid cycling of ASN in microorganisms. The relative contents of organic N fractions were fluctuant during composting, due to the complex transformation relationships among different N fractions (Zhou et al., 2018). Among the four forms of organic N, AN, AAN and ASN are recognized as the main contributors of BON (Li et al., 2014), which can be utilized and transformed by microorganisms and a few plants. Importantly, at the end of composting (60th day), the contents of BON in BC, M and BCM were significantly higher than that in CK (p < 0.05) and the order was as follows: M (11.2 g/kg) > BCM (10.5 g/kg) > BC (7.33 g/ kg) > CK (6.24 k/kg). This result indicates biochar and montmorillonite all contributed to the increase of BON contents in composts, especially for M and BCM. Awasthi et al. (2017) also found that biochar amendment increased the content of water-soluble organic N in composts. In addition, amino acid is an important precursor of humic substance (Wu et al., 2017a; Zhang et al., 2019). Thus, the increase of AAN contents may be contributed to the formation of humic substance. However, the N loss in M was significantly higher in comparison with three other treatments (p < 0.05), which may be due to the fact that montmorillonite overly promoted the N mineralization and the nitrification was week at the thermophilic phase (Fig. 1d). In comparison with CK, the N loss in BC and BCM reduced by 19.2% and 12.2%, respectively (p < 0.01). Based on the above results, it can be concluded that the mixture addition of biochar and montmorillonite can both increase the BON contents and reduce N loss during CM composting.
measured by steam distillation with different additives, where total hydrolyzable organic N (THN) with NaOH after Kjeldahl digestion with H2SO4 and catalyst (CuSO4 and K2SO4) mixture; AN with MgO; AN + ASN with phosphate-borate buffer; AAN with phosphate-borate buffer after treating with 0.5 mol·L−1 NaOH and ninhydrin (pH = 2.5) at 100 °C to convert AAN to NH4+. The contents of organic N fractions were calculated as follows (Zhu et al., 2019b): ASN = (AN + ASN) − AN BON = AN + AAN + ASN HUN = THN − BON 2.4. DNA extraction and PCR-DGGE analysis DNA was extracted from 3 g of each sample using soil DNA kit (Omega Biotek, Inc.) according to the manufacturer’s instructions. The concentration and quality of extracted DNA were detected with the Nanodrop One/Onec (Thermo Fisher Scientific, Wilmington, USA). The DNA extracts were stored at −20 °C for further analysis. 16S rRNA gene was amplified using the bacterial universal primers 357F (5′-CC TAC GGG AGG CAG CAG-3′) and 534R (5′-ATT ACC GCG GCT GCT GG-3′). PCR products were used for DGGE analysis by the Gene Mutation Detection System (Bio-Rad, Hertfordshire, UK). Gels contained 8% (w/ v) poly-acrylamide with a linear gradient of 35–60% denaturant (Wu et al., 2017b). Gels were electrophoresed at 60 °C for 6 h at 130 V. Finally, Gel Doc™ imaging system (Bio-Rad, Hertfordshire, UK) was used for scanning gels after stained with EB. Representative bands of DGGE was sequenced and the results were compared with the GenBank database. 2.5. Statistical analysis All the data of physicochemical properties were analyzed using SPSS 23.0 and OriginPro 2017. Differences among samples were analyzed using the LSD All-Pairwise Comparisons Test in Statistix 8. Differences among samples were declared at the p < 0.05 probability level of significance. The intensity of DGGE bands was qualified with Quantity One (version 5.0, Bio-Rad, USA). Cluster heatmap was conducted using R 3.6.1. Redundancy analysis (RDA) was performed using CANOCO 5.0. Structural equation models (SEMs) were conducted by AMOS 23.0 software (IBM Corporation Software Group, Somers, NY) using the maximum-likelihood estimation method. Non-significant chisquare test (p > 0.05), high goodness-of-fit index (GFI > 0.90), and low root mean square errors of approximation (RMSEA < 0.05) were used to show the overall goodness of fit for SEMs.
3.2. Comparison of bacterial community composition in different treatments PCR-DGGE was performed to compare the bacterial community composition in different treatments. A total of forty-eight different bands were detected in the DGGE profiles in different treatments. Sequencing results showed that these bands were mainly belonging to four phyla: Proteobacteria (26.8%), Firmicutes (22.0%), Bacteroidetes (19.5%) and Actinobacteria (14.6%), which is consistent with many previous studies (Zhu et al., 2019a,b). There were twenty-four bands shown in Fig. 2a, which were significantly correlated with N fractions (Fig. 3). The bacterial communities exhibited a significant difference among different treatments. For example, the relative abundance of bands 2, 23, 26, 27, 44 in BC were higher than those in CK. These bands belonged to Firmicutes, Bacteroidetes and Actinobacteria, suggesting that biochar could promote different microbial taxa. Previous study indicated that biochar can serve as a suitable habitat for microorganisms due to its porous structure and high surface area (Xiao et al., 2017). In addition, biochar contained lots of labile aliphatic compounds and inorganic nutrients, which can be used as carbon source or nutrient source for microorganisms (Steiner, et al., 2016). Thus, the abundance of above bacteria increased in BC treatment. Meanwhile, the relative abundance of bands 14, 27 and 44 in M were also higher than those in CK. In comparison, more than half of the bands showed higher abundance in BCM than CK. These results indicate that biochar and montmorillonite could enhance the activity of some bacteria during composting, especially for the mixture addition of biochar and montmorillonite, which is consistent with the variation of N fractions contents in different treatments. The NMDS analysis suggests that the samples of each treatment tended to cluster together (Fig. 2b). In addition, the samples of BCM were completely separated from that of CK, suggesting the significant difference in bacterial community composition between these two
3. Results and discussion 3.1. Comparison of the contents of N fractions in different treatments The contents of TON ranged from 12.8 to 25.7 g/kg during composting (Fig. 1a). In general, TON contents in all treatments decreased at the first four days and then showed an increased trend, eventually, leveled off. The initial decreasing trend of TON was due to the rapid degradation of complex nitrogenous organic compounds and loss of NH3 (Awasthi et al., 2017). During the first four days, the TON contents decreased faster in BC, M and BCM than that in CK. At the final stage of composting, the TON contents of three treatments were also lower than that of CK, especially for the M group, suggesting that biochar, montmorillonite and their mixture addition promoted the decomposition of TON during composting. The contents of NH4+ showed a significantly decreasing trend in all treatments at the first 21 day of composting, and then increased until the 60th day of composting (Fig. 1b). The NH4+ contents in BC, M and BCM were higher than that in CK throughout the composting process. The above results indicate that biochar, montmorillonite and their mixture addition contributed to the sequestration 3
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Fig. 1. Distribution of (a) total organic nitrogen, (b) NH4+, (c) organic N fractions and (d) nitrogen loss in different treatments.
bacterial communities and N fractions will contribute to understand the transformation mechanism of N in different treatments.
treatments. Although both BC and M partly overlapped with CK, there were still visible differences among them. The Shannon-Wiener index of BCM was also significantly higher than that of CK at the mature phase of composting (the 40th and 60th days) (Fig. 2c). These above results together indicate the obviously changes of bacterial communities caused by the addition of biochar and montmorillonite, especially for their mixture. Biochar and montmorillonite amendments may provide a suitable environment for microorganisms to transform organic N during composting. Therefore, further studying the relationship between
3.3. Roles of bacterial communities in organic N transformation There were different relationships between bacterial communities and N fractions in different treatments (Fig. 3). In CK, the bacteria in group D1, including Pseudomonas, Acinetobacter and Gramella etc., showed significantly positive correlations with BON, AN and AAN,
Fig. 2. Composition of bacterial community in different treatments. (a) Abundance of bacterial bands derived from DGGE (only show the bands significantly correlated with N fractions in Fig. 3), and the size of the circle represents the relative abundance of the DGGE band; (b) NMDS of bacterial community composition; (c) Shannon-Wiener index of bacterial community. 4
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have the BON sequestration ability (negatively correlated with BON) in CK, while the addition of montmorillonite and their mixture with biochar made D4 bacteria developed the ability of BON sequestration (positively correlated with BON). These results indicate that biochar and montmorillonite could strengthen different types of microorganisms associated with N transformation, which may be due to the fact that biochar and montmorillonite provide different habitat environment for microorganisms. The porous structure of biochar provides sufficient oxygen and carbon source for almost all aerobic bacteria. By contrast, the surface of montmorillonite is rich in both positive and negative charges, but only positive charges could adsorb bacteria due to the electronegativity of bacteria in compost. Thus, biochar provides a more suitable habitat environment for microorganisms than montmorillonite. It is similar to the previous found that microorganisms were more prone to colonize on biochar surfaces during composting, compared with other bulking agents (e.g., peat bog and zeolite) (Wang et al., 2017a).
3.4. Relationships between bacterial communities and environmental factors in different treatments As described in Section 3.3., bacterial communities associated with N transformation could also be influenced by the environmental conditions during composting. Thus, to explore the relationship between key bacterial communities (e.g., groups D1–D4) and environmental factors in different treatments, RDA was performed (Fig. 4). All of the canonical axes were significant (p < 0.05), indicating that these environmental factors are important in explaining the variation of bacterial communities. 70.10%, 67.42%, 69.45%, and 61.52% of the variations of species-environmental factors relation were explained by the first two canonical axes in CK, BC, M and BCM, respectively. Considering the critical role of group D1 bacteria in the accumulation of BON, different treatments had variable effects on group D1 bacteria and environmental factors. In CK treatment, MC and OM were positively correlated with group D1 bacteria (Fig. 4a). By contrast, MC and OM was still positively correlated with group D1 bacteria in BC (Fig. 4b), but the correlation between each band (i.e., group D1) and environmental factor (i.e., OM and MC) changed slightly. This result indicates that MC and OM may strengthen group D1 bacteria to increase the BON contents in BC treatment. Sánchez-García et al. (2015) found that the addition of biochar could prevent the formation of large clumps and promote O2 diffusion into the composting mixture, thereby facilitate the consumption of water and organic matter by microorganisms. In addition, pH also played a more important role in BC treatment. The pH was significantly positively correlated with bands 2, 3, 10, 22, 23, 25, 31 and 35 which contributed to the accumulation of TON. Previous studies also found that biochar addition slightly increased the initial pH of composting (Vandecasteele et al., 2016; Liu et al., 2017). In M treatment, except for MC and OM, the group D1 bacteria also positively correlated with C/N, suggesting that higher C/N could promote N transformation, especially for BON (Fig. 4c). In addition, high C/N was contributed to the decomposition of organic N by microorganisms. From this view, high C/N would increase the contents of inorganic N in compost, which may be a potential reason for N loss in M treatment. This is because increasing C/N properly will provide carbon source for microorganisms to depolymerize nitrogenous compounds. However, the correlation between group D1 bacteria and environmental factors are weaker in BCM than that in CK (Fig. 4d). Some of the DGGE bands of group D4 (e.g., 2, 3, 36 and 46) were negatively correlated with C/N, while band 25 showed a positive correlation to the temperature. Based on the above results, we find that different treatments could change various environmental factors to affect the key bacteria associated with N transformation during composting.
Fig. 3. Relationships between bacterial taxa and nitrogen fractions in different treatments. Group D1–D4 represent four typical bacterial taxa associated with N transformation. The numbers from 1 to 48 represent 48 DGGE bands, respectively.
suggesting that these bacteria may contribute to the accumulation of organic N fractions during composting. By contrast, the relationship between D1 bacteria and organic N fractions in BC were higher than that in CK. This result indicates that the addition of biochar promoted the role of D1 bacteria in the organic N transformation, which may be a main reason for the accumulation of organic N, especially for BON fractions (e.g., AN, AAN). In contrast, the relationship between D1 bacteria and organic N fractions (i.e., BON, AN, AAN) in M was not significantly different from that in CK. Nevertheless, the relationship between D1 bacteria and organic N fractions in BCM became more negatively in comparison with CK. This result indicates that D1 may not play a critical role in BON accumulation in M and BCM, which is not similar to BC. In comparison with CK, group D2 bacteria in M became negatively correlated with BON, AN and AAN, while D3 bacteria became positively correlated with ASN and HUN. This result indicates that D2 bacteria may be related to BON mineralization, while D3 bacteria played an important role in the accumulation of ASN and HUN in M treatment. In addition, group D4 bacteria were negatively correlated with BON, AN and AAN in CK, while the relationship became more positively in M and BCM. This result indicates that group D4 bacteria may have a stronger ability for the storage of BON than group D1. In total, group D1, D3 and D4 bacteria were all contributed to the storage of BON, rather than mineralization. In addition, there were more bacteria (e.g., bands 36, 44 and 47) significantly negatively correlated with NH4+ in M in comparison with CK, which is consistent with the serious N loss in M. These results further indicate that different treatments had varying impacts on bacterial communities associated with N transformation during composting. For instance, D1 bacteria had the BON sequestration ability (positively correlated with BON) in CK, and biochar just promoted the ability of D1 bacteria. However, D4 bacteria did not
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Fig. 4. RDA of the relationships between environmental factors and bacterial communities (p < 0.05) in (a) CK, (b) BC, (c) M and (d) BCM. The yellow arrows represent bacterial taxa significantly correlated with nitrogen fractions in Fig. 3. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
In M, neither environmental factors nor bacterial communities had a positive effect on AAN (Fig. 5f). Nevertheless, AAN had a directly positive effect on NH4+, suggesting that AAN were greatly mineralized into inorganic N. In addition, OM, C/N and MC together had positive correlations with NH4+. This result is consistent with the higher N loss in M treatment. In BCM treatment, C/N and OM all showed an indirectly positive effect on AAN by changing bacterial community, and AAN did not have an effect on NH4+, suggesting that BCM could reduce AAN mineralization into NH4+ in comparison with M, thereby reducing the risk of N loss. These above results confirm that all the treatments in this study contributed to increase AAN contents, but montmorillonite amendments could cause a serious N loss in comparison with CK. In addition, results found that the bacterial community (e.g., ammonifier) played an important role in the transformation of AAN to NH4+. For example, Flavobacterium (band 1) and Pseudomonas fluorescence (band 12) could depolymerize the high molecular weights N-containing compounds (e.g., proteins), which is the rate-limiting step of microbial decomposition of organic N (Bach and Munch, 2000). AN is one form of organic N with the simplest structure, which is easily used by microorganisms (Zhou et al., 2018). Some of AN are from the deamination of amino acids and amino sugars, and some of AN are from amide compounds. OM had an indirectly positive effect on AN, mediated by bacterial community in CK (Fig. 5b). Some bacillus, such as Bacillus subtilis and Bacillus cereus could convert simple nitrogenous compounds to ammonium by deamination. OM could provide carbon sources for microorganisms to decompose nitrogenous compounds. By contrast, OM and bacterial community together showed a directly positive effect on AN in BC treatment. In addition, indirectly positive effect of the temperature (T) on AN was also found in BC treatment (Fig. 5e). Only OM was found to have a directly positive effect on AN in the M treatment (Fig. 5g). MC and C/N had an indirectly positive effect
3.5. Possible mechanism of different additives to increase BON contents Based on the results in Sections 3.2–3.4, biochar and montmorillonite amendments can not only change the functional bacterial communities, but also the environmental factors during composting. However, it is still largely unknown if these complex factors of composts directly or indirectly affected N transformation in different treatments. SEM, an a prior method to visualize the causal relationships between variables by fitting data to the model representing causal hypotheses, was used to further confirm the causal relationships between different environmental factors, bacterial communities (data of NMDS) and N bioavailability (data of N fractions) in CK, BC, M and BCM. SEMs have been widely to explore the causal relationships between environmental conditions and microbial communities in the area of environmental science, especially for composting (Chen et al., 2019b; Wang et al., 2019a). The hypothesis of SEMs was based on organic N mineralization process mediated by microorganisms (e.g., ammoniation). The factors used in SEMs were selected based on the results of RDA. SEMs results showed that environmental factors and bacterial communities affected BON fractions and their mineralization into NH4+, but key environmental factors and bacterial taxa changed in different treatments (Fig. 5). AAN as the main forms of BON, occupied a large proportion of organic N in composts, which is usually found in proteins, polypeptide and microbial residue. Only bacterial communities had a negative effect on AAN in CK (Fig. 5a), while OM indirectly positively affected AAN mediated by bacterial communities in BC (Fig. 5d). This result indicates that biochar amendments may increase OM contents, thereby it would provide abundant carbon source for microorganisms to depolymerize high molecular weight nitrogenous compounds into AAN. This result may be explained the higher contents of AAN in BC (15.3%) than that in CK (10.7%) at the end of composting. 6
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Fig. 5. Effects of environmental factors and bacterial communities on organic nitrogen transformation (based on structural equation models). Red and black arrows represent significantly positive and negative relationships, respectively (p < 0.05). Dashed lines represent non-significant relationship (p > 0.05). Numbers adjacent to arrows are standardized path coefficients, analogous to relative regression weights and indicative of the effect size of the relationship. *p < 0.05, **p < 0.01, ***p < 0.001. df, degree of freedom. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
of ASN during composting. In M treatment, T had an indirectly positive effect on NH4+ by affecting ASN (Fig. 5h). ASN was rapidly mineralized into NH4+ at the thermophilic phase of composting, and the nitrification and denitrification were inhibited at high temperature, so nitrogen would be loss as the form of NH3. In BCM treatment, the mineralization of ASN into NH4+ was significantly inhibited in comparison with M (Fig. 5k).
on AN by changing bacterial community in BCM (Fig. 5j). Meanwhile, HUN and AN had a positive effect on NH4+ in M and BCM treatments, respectively, suggesting that the mineralization of organic N fractions in M and BCM were more serious than those in CK and BC. As for the ASN, it was only appeared in CK, M and BCM treatments. T, C/N and OM all had significant effects on ASN in CK (Fig. 5c). These multiple effects on ASN may be a reason causing the irregularly variable contents 7
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Based on the above results, it can be found that there were different mechanisms on N transformation in different treatments. On the one hand, biochar can promote the accumulation of BON (e.g., AN, AAN) by changing organic matter contents, moisture contents and T of composts. These key environmental factors may be contributed to the direct adsorption of BON by biochar. On the other hand, biochar could also promote microbial storage and transformation of BON by creating a proper living environment for microorganisms. However, compared with biochar, montmorillonite amendment had weaker effect on microorganisms of composts. And microorganisms had no significant contributions on the sequestration of BON in M treatment. The Montmorillonite might directly adsorb BON on its surface. Although BON contents in M treatment was higher than that in CK and BC treatments, the N loss in M was also the most serious. Previous study indicated that van der Waals interaction was a major mechanism of adsorption to montmorillonite of organic matter (Arnarson and Keil, 2000). It is acknowledged that van der Waals interaction is usually weaker than the chemical bond. Thus, when lots of BON molecular were adsorbed on the surface of montmorillonite, some of them may be mineralized to inorganic nitrogen due to the incompact combination between montmorillonite and BON. Based on the above results, the mixture of biochar and montmorillonite amendments is a better choice for real compositing production, which can not only increase the bioavailability of nitrogen, but also reduce nitrogen loss. The retained BON in compost products will be transformed inorganic N for plant growth, when plants are short of N. Thus, biochar and montmorillonite amendments can also improve the durable nitrogen supply capacity of compost products in practical application.
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4. Conclusion This study confirmed that the addition of functional additives (i.e., BC, M and BCM) significantly improved the contents of bioavailable organic N and NH4+ in composts. In addition, BC and BCM reduced N loss by 19.2% and 12.2%, respectively. BC and BCM affected N transformation by changing key bacterial communities and environmental factors during composting, while M only changed environmental factors related to N adsorption. Comparison of N availability and N loss indicated that the combination of biochar and montmorillonite could be more effective for practical composting production. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement This work was financially supported by the National Natural Science Foundation of China (No. 51778116, No. 51878132 and No. 51978131) Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.122224. References Arnarson, T.S., Keil, R.G., 2000. Mechanisms of pore water organic matter adsorption to montmorillonite. Mar. Chem. 71 (3), 309–320. Awasthi, M.K., Chen, H., Wang, Q., Liu, T., Duan, Y., Awasthi, S.K., Ren, X., Tu, Z., Li, J., Zhao, J., Zhang, Z., 2018. Succession of bacteria diversity in the poultry manure composted mixed with clay: studies upon its dynamics and associations with physicochemical and gaseous parameters. Bioresour. Technol. 267, 618–625. Awasthi, M.K., Wang, Q., Chen, H., Wang, M., Ren, X., Zhao, J., Li, J., Guo, D., Li, D., Awasthi, S.K., Sun, X., Zhang, Z., 2017. Evaluation of biochar amended biosolids co-
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