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Insight to key diazotrophic community during composting of dairy manure with biochar and its role in nitrogen transformation
Waste Management 105 (2020) 190–197
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Waste Management journal homepage: www.elsevier.com/locate/wasman
Insight to key diazotrophic community during composting of dairy manure with biochar and its role in nitrogen transformation Xiaotong Wu, Yu Sun, Liting Deng, Qingxin Meng, Xin Jiang, Ayodeji Bello, Siyuan Sheng, Yue Han, Haifeng Zhu, Xiuhong Xu ⇑ College of Resources and Environment, Northeast Agricultural University, Harbin 150030, China
a r t i c l e
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Article history: Received 9 October 2019 Revised 14 January 2020 Accepted 10 February 2020
Keywords: Composting Biochar High-throughput sequencing Diazotroph Nitrogen transformation
a b s t r a c t Analyzing diazotrophic community may help to understand nitrogen transformation in composting and improves the final compost quality. In this study, diazotrophic community dynamics were investigated in terms of nifH gene during dairy manure and corn straw composting with biochar addition using highthroughput sequencing. Biochar decreased the diversity of diazotrophic community and altered diazotroph community structure during composting. At phylum level, Proteobacteria, Actinobacteria and Firmicutes were dominant diazotrophic communities throughout composting process. Biochar addition registered higher correlation coefficient (R) between physicochemical factors (temperature, ammonium (NH+4-N) and nitrate (NO 3 -N)) and diazotroph community composition. Rhodopseudomonas and Pseudoxanthomonas was the key diazotrophic communities influencing NH+4-N transformation in control (CK) and biochar compost (BC), respectively, while for NO 3 -N transformation Clostridium and Bradyrhizobium in CK, Azospira and Methylocystis in BC served as predominant factors. These results indicated that addition of biochar altered the key diazotroph communities influencing nitrogen transformation. Furthermore, some diazotrophs (e.g. Rhodopseudomonas, Bradyrhizobium and Azospira) affecting NH+4-N and NO 3 -N transformation were also observed to be mediating total nitrogen (TN). Interestingly, interactions between diazotrophic communities were observed and these interactions could also influence nitrogen transformation. Ó 2020 Elsevier Ltd. All rights reserved.
1. Introduction Composting, an effective method used in degrading agricultural wastes leading to production of organic fertilizer by microbial metabolism, is widely recognized (Jain et al., 2018; Wang et al., 2017a). Nitrogen transformation was crucial for composting processes and compost quality, including ammonification, nitrification, denitrification and biological nitrogen fixation, and specific microorganisms contributed to each nitrogen transformation process are sensitive to environmental changes (Levy-Booth et al., 2014; Liu et al., 2019; Yin et al., 2018). Biochar as a pivotal factor in regulating physicochemical factors promoted nitrogen transformation and reduced ammonia loss during composting by their abundant pore structure and complex functional groups (Hale and Crowley (2015); Malinowski et al., 2019; Jain et al., 2018; Sánchez-García et al., 2015). Also, biochar addition can enhance the abundance of microorganisms and microbial activities during
⇑ Corresponding author. E-mail address: [email protected] (X. Xu). https://doi.org/10.1016/j.wasman.2020.02.010 0956-053X/Ó 2020 Elsevier Ltd. All rights reserved.
composting (Du et al., 2019; Zhou et al., 2019). Therefore, adding biochar to compost may create favorable conditions for microbial communities and improve compost quality. High-throughput sequencing can comprehensively explain microbial community composition and has gradually become the main method of investigating microbial communities in various environments including compost (Duan et al., 2019; Meng et al., 2019a). Among the nitrogen transformation processes during composting, biological nitrogen fixation process, during which N2 is converted into organic nitrogen, increases nitrogen content (Pepe et al., 2013; Sun et al., 2016). Diazotrophs are special functional group of microorganisms responsible for nitrogen fixation including free-living bacteria (e.g., Azospirillum, Rhodospirillum and Azotobacter), symbiotic nitrogen fixiation bacteria (e.g., Methylobacterium, Bradyrhizobium, and Rhizobium) and a part of sulfate-reducing bacteria (e.g., Desulfobulbus) (Yin et al., 2018; Marques et al., 2017; Burow et al., 2014). Diazotrophs are supposed to increase nitrogen in environment and exert influences on nitrogen transformation (Beauchamp et al., 2006; Sun et al., 2016). Therefore, diazotrophs are interesting microbial communities in
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compost, which may have great impact on nitrogen transformation and compost quality. However, to our knowledge, few researches have been conducted on diazotrophic community in composting especially under the influence of biochar (Che et al., 2018; Pereira et al., 2011), and its role in nitrogen transformation are rarely explored. The lack of information on diazotrophs in composting may limit the understanding of the whole microbial communities and nitrogen transformation. This study aimed to: (1) explore diazotroph community succession in dairy manure and corn stalk composting affected by biochar using high-throughput technology; (2) examine the correlation between diazotrophic community structure and physicochemical factors by mantel test and Spearman correlation analysis; (3) quantify the relationships between nitrogen transformation and diazotrophs genera by stepwise regression and Path analyses; (4) identify key diazotrophs genera affecting nitrogen transformation. 2. Material and methods 2.1. Composting Dairy manure and corn stalk were used for composting. Corn stalk was cut into 1–2 cm and was homogeneously mixed with dairy manure at ratio 1:5 (Meng et al., 2019a; Sun et al., 2019b). Initial physicochemical factors of dairy manure, corn stalk and biochar were listed in Supplementary Table 1. Two compost mixtures (treatments); first treatment (dairy manure + corn stalk) as control (CK) and the second treatment (CK + 10% rice biochar) (BC) were set-up and replicated three times in a trapezoid shape (2.5 m 1. 5 m 1 m). Composting lasted for 43 days, and piles were turned on day 11, day 19 and day 28 according to the temperature variation. 2.2. Sample collection Samples were collected on day 0 (initial phase), day 2 (mesophilic phase), day 4 (thermophilic phase), day 28 (cooling phase) and day 43 (maturation phase), each were taken at three depths (10 cm, 30 cm, 50 cm from pile surface) and mixed. Samples of these composting phases from CK and BC treatments were named as CK1, CK2, CK3, CK4, CK5, and BC1, BC2, BC3, BC4, BC5, respectively. The samples were divided into two parts, one was airdried for the determination of physicochemical parameters and the other was stored at 80 °C for molecular experiments. 2.3. Determination of physicochemical parameters Temperature of compost piles was measured with electronic thermometers. Moisture content (MC) was determined by oven drying the samples at 105 °C to constant weight, and pH was determined using the method of previous study (Abid and Sayadi, 2006). Ammonia nitrogen (NH+4-N) and nitrate nitrogen (NO 3 -N) were measured using leach liquor (2 molL1 KCL at the ratio of 1:10 with 30 min oscillation at 150 rpm) by flow injection analysis (FIAstar 5000, FOSS, Denmark). Total carbon (TC) and total nitrogen (TN) were determined using the combustion method by automatic elemental analysis (LECO CHNS-932, 120 USA). 2.4. High-throughput sequencing of NifH gene in diazotrophs Total DNA was extracted using MP Biomedicals Fast DNA Soil Sample Extraction Kit (MoBio Laboratory, Carlsbad, CA, USA) according to the instructions, and then stored at 20 °C. NifH gene was sequenced using primers nifHF (50 -AAAGGYGGWATCG GYAARTCCACCAC 30 ) and nifHR (50 -TTGTTSGCSGCRTACATSGC
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CATCAT 30 ), and PCR amplification was conducted with the method of Rösch et al. (2002). PCR products were detected by 2% agarose gel electrophoresis and sequenced using Illumina MiSeq platform. The raw data was archived in NCBI Sequence Read Archive with accession No. PRJNA546109. 2.5. Statistical analysis Qiime 1.9.1 was used for quality optimization of raw sequences (filtering the reads below 200 bp and containing N base), and optimized sequences were clustered into operational taxonomic units (OTUs) with 97% similarity using UPARSE 7.0.1090, then effective sequences were aligned against NCBI database to identify species information (Caporaso et al., 2010; Edgar, 2013). Mothur 1.30.2 and Vegan package in R 2.4 were used to calculate Alpha diversity (Shannon and Chao1 indices), rarefaction curve and mantel test analysis, while Qiime 1.9.1 was used to evaluate Beta diversity (Non-metric Multidimensional Scaling) by Bray-Curtis distance. Values of physicochemical factors were statistically analyzed by One-way ANOVA using SPSS 21.0 (IBM, USA) and Microsoft Excel 2016. Spearman correlation, stepwise regression and Path analyses were done to examine direct and indirect effects of key diazotrophs on nitrogen transformation using SPSS Statistics 21.0 and Data Processing System (DPS) software (Sun et al., 2019a; Tang and Zhang, 2013). 3. Results and discussion 3.1. Variation of physicochemical factors Data of physicochemical factors were shown in Supplementary Materials. Temperature rose rapidly at the beginning of composting and temperature values above 50 °C were recorded for 22 days in CK and 27 days in BC, indicating that pathogens were eliminated in compost (Meng et al., 2019b). Little difference was observed between BC and CK temperature, and similar results were reported by Jain et al. (2018) who explored the effects of biochar on dairy cow manure and Hydrilla verticillate composting. Moisture content of CK decreased from 71.14% to 29.98%, while that of BC declined from 62.03% to 29.45%. pH shown an upward trend during composting and final pH values of CK (8.16) and BC (8.17) were within the range (7.00–8.50) for mature compost (Sun et al., 2019b). Increase in pH may due to decomposition of organic matter by microorganisms and production of NH+4-N during composting (Qian et al., 2014; Ren et al., 2016). C/N ratios were 15.75 and 15.33 in CK and BC, respectively, indicating that the final compost was mature. Similar patterns of NH+4-N variation were observed in CK and BC, which increased in the first 4 days and then declined. This phenomenon may be attributed to the fact that mineralization of organic nitrogen by microorganisms increased the level of NH+4-N in mesophilic phase (Ren et al., 2016), and then volatilization and nitrification led to its reduction during in thermophilic and cooling phases of composting (Rashad et al., 2010; Raj and Antil, 2011). Concentration of NO 3 -N showed more rapid rising in CK than that of BC in cooling and maturation phases, indicating that biochar might negatively affect the concentration of NO 3 -N in cooling and maturation phases, which may be related to the excessive adsorption by biochar causing the reduction of effective nutrients (Liang et al., 2019; Wang et al., 2013). TN declined within the first 4 days of composting and later increased in both treatments. Several studies have confirmed that nitrogen fixation activities of diazotroph community could increase TN content in cooling and maturation phase of composting (Kalamdhad et al., 2009; Yin et al., 2018). So, the rising of TN in this study might partially be
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contributed by diazotroph community. Final concentration of TN in BC (22.26 gkg1) was significantly higher than that of CK (19.01 gkg1), which consistent with previous study (Malinowski et al., 2019), suggesting that biochar may improve nitrogen conservation in composting.
3.2. Community structure analyses A total of 633863 nifH sequences in CK and 591573 sequences in BC were obtained after optimized screening, and 1712 and 1806 operational taxonomic units (OTUs) were identified in CK and BC respectively based on 97% nucleotide similarity. Effective species diversity in this study was determined by saturated Shannon rarefaction curves (Supplementary Material), which revealed that sufficient sequences of diazotrophs was achieved. Shannon and Chao1 indices increased in CK but dropped in BC during thermophilic phase (Fig. 1a and b). The diversity indices gradually decreased after thermophilic phase in CK, while they showed irregular decreasing patterns in BC. At the end of composting, the diversity indices decreased significantly in both treatments, which could be attributed to screening of microorganisms by high temperature (Huhe et al., 2017). These results indicated that biochar addition limited the diversity of diazotrophs and strengthened the inhibiting effect of high temperature on nifH gene. Previous studies reported that high concentration of biochar (above 5%) has deleterious effects on microbes because it may contain some toxic compounds and creating an undesirable C/N stoichiometric ratio (Liang et al., 2019; Liao et al., 2014).
Non-metric multidimensional scaling (NMDS) analysis interpreted the difference of diazotrophic community composition between samples using Bray-Curtis dissimilarity on OTU level. The distance between the positions of early composting stage (mesophilic and thermophilic phase) and late stages (cooling and maturation phase) was observed (Fig. 2). This suggests that composition of diazotroph community varied significantly during composting. Piceno et al. (2017) also reported that composting phase exhibited significant influence on the succession of microbial communities. The main reason for this result was that the temperatures, pH and nutrients varied in each phase and microorganisms were sensitive to the environment (Malinowski et al., 2019). Contracting distance was registered with BC and CK samples during composting, indicating that biochar could definitely affect the community composition of diazotroph during the whole composting process. This phenomenon was consistent with the study of Duan et al. (2019) who found that bacterial community was altered by biochar during cow manure composting. 3.3. Diazotrophic community composition As shown in Fig. 3, relative abundance of diazotroph community varied significantly during composting in both treatments. On both phyla and genus level, relative abundance of major diazotrophic communities in BC was different from those in CK. At phylum level (Fig. 3A), Proteobacteria were dominant during composting, followed by Actinobacteria and Firmicutes. Other phyla with relative abundance less than 1%, were classified as Others. The unclassified community occupied 10.72% of total diazotrophic
Fig. 1. Changes of Shannon and Chao1 index of diazotroph community.
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Fig. 2. Non-metric multidimensional scaling (NMDS) of simples using Bray-Curtis dissimilarity and the stress value is 0.112. The ellipse is used to divide the sample, three parallel in one ellipse, each color represents a composting period.
Fig. 3. Relative abundance of phylum level (A) and genus level (B). CK and BC were shown in the same coordinate system (average value of parallel samples was taken as the final relative abundance). Phylum and genera which less than 1% in relative abundance were classified as others.
communities. In the initial phase, Proteobacteria accounted for 75.10% and 81.66% of diazotrophs in CK and BC, respectively. Proportion of Proteobacteria decreased drastically in mesophilic and thermophilic phase of both treatments, however it rose rapidly after thermophilic phase, indicating that thermophilic conditions were not favorable for Proteobacteria. Proteobacteria played a vital role in carbon cycle and nitrogen mineralization (Awasthi et al., 2017; Liang et al., 2019), indicating that Proteobacteria were active in nitrogen transformation in later phase of composting. Actinobacteria were dominant at thermophilic phase in CK (32.14%) and BC (57.36%), but dropped at maturation phase of CK (3.37%) and BC (1.43%). These results suggested that Actinobacteria were tolerant to high temperature. Actinobacteria could form spore under high temperature and this property was beneficial for this phylum to survive and sustain higher abundance during thermophilic composting (Jurado et al., 2014; Zhao et al., 2016). Relative abundance of Firmicutes was increased at mesophilic (7.15%) and thermophilic phase (26.33%) in CK, and this phenomenon might be related to the ability of Firmicutes in forming endospores that can withstanding high temperature (Zhou et al., 2019). In BC, relative abundance of Firmicutes increased throughout the whole process of composting and this was in accordance with the study by Duan et al. (2019)
who found that the relative abundance of Firmicutes increased during wheat straw and dairy manure composting with 12% biochar addition. Relative abundance of the aforementioned three phyla showed obvious variation at mesophilic and thermophilic phases, suggesting that succession of the diazotroph community might be affected by temperature. On genus level, diazotroph community also changed significantly during composting (Fig. 3B). Psychrobacter were the most abundant genus of Proteobacteria phylum and it decreased significantly at mesophilic and thermophilic phase in this study, owing to the fact that Psychrobacter prefer low temperature environment (Joseph et al., 2008). However, higher abundance of Psychrobacter was observed in BC (26.76%) than in CK (10.35%) at mesophilic phase, suggesting that addition of biochar might create an environment favorable for the survival of Psychrobacter under higher temperature, which was consistent with the report of Wang et al. (2017a). The relative abundance of Streptomyces increased at thermophilic phase in CK (31.61%) and BC (55.27%). Streptomyces, belonging Actinobacteria, has been reported as free-living nitrogen fixation bacteria (Dahal et al., 2017). Higher proportion of Streptomyces in BC might indicate more contribution to nitrogen fixation (Prakash and Cummings, 1988). The relative abundance of
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BC. These results suggested that biochar could enhance the sensitivity of diazotroph community to NH+4-N and NO 3 -N. Previous studies have reported that NH+4-N and NO 3 -N were the key factors for shaping microbial community composition (Meng et al., 2019a; Zhou et al., 2019). In this study, abundance of Streptomyces increased at thermophilic phase of composting (31.61% in CK and 55.27% in BC) and interestingly the NH+4-N content also increased in this period (Supplementary Table S2). Therefore, a positive correlation between Streptomyces and NH+4-N was observed (Supplementary Table S4), indicating NH+4-N accumulation was likely to be promoted by Streptomyces. Similar findings were reported in previous studies (Meng et al., 2019a; Fang et al., 2019). Besides, the positive correlations between some diazotrophs (e.g., Streptomyces, Azospira and Rhodopseudomonas) and NH+4-N were enhanced by biochar as shown by Spearman analysis, suggesting that adding biochar may facilitate NH+4-N accumulation in composting. Spearman analysis showed that Azotobacter, Azoarcus and Pseudoxanthomonas were positively correlated with TN in both treatments (Supplementary Table S4), but no significant correlation was found between TN and diazotroph community composition (Table 1). The results indicated that the presence of some diazotrophs (e.g., Azotobacter) could increase nitrogen availability in compost matrix, which is of significance for improving compost quality. This finding was in accordance with previous studies which reported that diazotrophs were able to fix nitrogen and increase nitrogen content in environments (Orr et al., 2011; Pepe et al., 2013). Furthermore, abundance of Azotobacter was higher in BC, suggesting that biochar addition could promote the growth of this genus, which might contribute to the higher level of nitrogen in compost.
Bradyrhizobium, belonging Bradyrhizobiaceae and Rhizobiales, gradually increased from initial phase (3.35%) to thermophilic phase (21.28%) in CK, and decreased significantly thereafter. Bradyrhizobium were also found in BC, and its population increased during mesophilic phase. Bradyrhizobium are common symbiotic nitrogen fixing bacteria and they can only fix nitrogen only after inoculating legume plants (Liu et al., 2019). In this study, Bradyrhizobium were detected and its relative abundance varied during composting, suggesting that this group of diazotroph could not only survive but also grow under the harsh condition of composting. So, it is interesting to explore their ability of nitrogen fixation during composting in future study. Rhodopseudomonas, another genus belongs to Bradyrhizobiaceae and Rhizobiales, shown an upward and then downward trend during composting in both treatments, however the relative abundance of Rhodopseudomonas in BC was higher than that of CK in all stages of composting. These results indicated that the addition of biochar was beneficial to the survival of this group and might increase the chance of symbiotic nitrogen fixation in soil after fertilizer application in legume field. Azotobacter and Azospira are free-living nitrogen fixation bacteria (Marques et al., 2017; Zhan and Sun, 2011). The proportion of Azotobacter increased at cooling phase in CK (13.72%) and BC (15.84%). Azospira increased at mesophilic phase of CK (11.17%) and BC (5.42%), and then disappeared at cooling phase and maturation phase in both treatments. The relative abundance of Clostridium reached maximum at thermophilic phase in CK (19.63%) and at maturation phase in BC (11.79%). Clostridium are ubiquitous in natural environment and has ability to fix nitrogen under anaerobic conditions (Woodhouse et al., 2013). Thus, it is reasonable to predict that Clostridium might carry out the function of nitrogen fixation in various depths of composting pile. Relative abundance of Pseudoxanthomonas increased at the end of composting with 5.55% in CK and 12.14% in BC. In addition to nitrogen fixation, Pseudoxanthomonas were reported to be capable of degrading macromolecular organic matter and producing soluble phosphorus (Li et al., 2013), indicating that the high relative abundance of Pseudoxanthomonas might contribute to compost quality improvement.
3.5. Key diazotroph communities affecting nitrogen transformation Stepwise regression model indicated that Rhodopseudomonas was the key diazotroph community affecting NH+4-N variation positively in CK (Table 2), suggesting that the increase of Rhodopseudomonas abundance might indicate the accumulation of NH+4-N, which can be attributed to the phenomenon that growth of Rhodopseudomonas was inhibited by low NH+4-N concentration (Wang et al., 2017b). The other two variables (Azoarcus and Azotobacter) were negatively related to NH+4-N transformation, which was supported by Spearman analysis (Supplementary Table S4). According to path analysis diagram (Fig. 4A), Rhodopseudomonas (0.4938*) had higher direct influence than Azoarcus (0.3541*) and Azotobacter (0.3815*) on NH+4-N in CK. This finding suggested that Rhodopseudomonas was the better predictive variables determining NH+4-N transformation and corroborated with result presented in stepwise regression model (Table 2) in CK. Although the addition of biochar altered the key diazotroph communities affecting NH+4-N, Rhodopseudomonas and Azoarcus were still
3.4. Relationship between physicochemical factors and diazotroph community Mantel test and partial mantel test analyses showed that pH and MC correlated significantly with diazotrophic community composition in CK (Table 1), while significant correlation was observed between temperature and diazotrophic community composition in BC. NH+4-N was significantly correlated with diazotroph community composition in both treatments (Table 1), and higher correlation was observed in BC (0.672) than in CK (0.417). The variation of NO 3 -N was associated with diazotrophic community dynamic in
Table 1 Mantel test and partial mantel test of correlation between physicochemical factors and community composition. Factor
CK
BC
Mantel test
T pH MC TN NH+4-N NO 3 -N
Partial mantel test
Mantel test
Partial mantel test
R
P
R
P
R
P
R
P
– 0.457 0.745 – 0.738 0.658
– 0.001 0.001 – 0.001 0.001
– 0.169 0.242 – 0.417 –
– 0.018 0.017 – 0.001 –
0.236 0.336 0.773 0.396 0.773 0.395
0.034 0.015 0.001 0.002 0.001 0.007
0.263 – 0.144 – 0.672 0.205
0.016 – 0.034 – 0.001 0.023
Note: R statistic closer to 1 represents the stronger positive correlation, and the minus represents negative correlation. Partial Mantel test was used to estimate relationship between the physicochemical factor and diazotrophs community structure with excluding other relevant factors. T: temperature; MC: moisture content; TC: total carbon; TN: total nitrogen.
X. Wu et al. / Waste Management 105 (2020) 190–197 Table 2 Stepwise regression models with NH+4-N, NO 3 -N and TN as dependent variables and genus as independent variables (n = 5). Compost
Standardization regression equations
R2
P
CK
NH+4-N
0.9994
0.0306
0.9999
0.0108
0.9998
0.0180
0.9993
0.0332
0.9998
0.0166
0.9995
0.0278
BC
= 1165.6827 + 2.5599 Rhodopseudomonas 0.0809 Azoarcus 0.0428 Azotobacter NO 3 -N = 139.0069 + 0.0159 Azospira + 0.0428 Clostridium 0.0513 Bradyrhizobium TN = 19.41158 + 0.0016 Rhodopseudomonas 0.0001 Azoarcus 0.0006 Bradyrhizobium NH+4-N = 1773.7553 + 0.8667 Rhodopseudomonas 0.2339 Pseudoxanthomonas 0.0557 Azoarcus NO 3 -N = 87.2901 – 0.0315 Methylocystis 0.0170 Azospira 0.0012 Azoarcus TN = 22.2709 – 0.0023 Methylocystis + 0.0020 Rhodopseudomonas 0.0018 Azospira
Note: Nitrogen index (NH+4-N, NO 3 -N, TN) were the dependent variable and diazotroph genera were the independent variable. High R2 (approaching 1.0000) and significant P value (<0.05) indicated that the equations were successfully established.
observed closely correlated with NH+4-N transformation in BC (Table 2). Another genus affecting NH+4-N variation was Pseudoxanthomonas. The path analysis diagram (Fig. 4B) shown that direct effect of Rhodopseudomonas and Azoarcus on NH+4-N variation were
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0.2647* and 0.2574*, respectively, and Pseudoxanthomonas had a higher direct effect (0.6247*). These results indicated that Pseudoxanthomonas was the key genus influencing NH+4-N variation. However, biochar addition might reduce the effect of Rhodopseudomonas on NH+4-N transformation, this might be due to that biochar improving the ventilation of composting matrix and also the nitrogenase activity of Rhodopseudomonas was then inhibited by the increased level of oxygen (Arashida et al., 2019). In the control pile, Azospira, Clostridium and Bradyrhizobium were major communities influencing NO 3 -N transformation (Table 2). Compared with Azospira (0.7473), Clostridium (3.5427*) and Bradyrhizobium (4.8495*) were observed higher direct influence on NO 3 -N transformation (Fig. 4C). Clostridium and Bradyrhizobium have been previously reported to be involved in denitrification process (Sánchez et al., 2011; Thakur and Medhi, 2019), indicating that Clostridium and Bradyrhizobium could be the key diazotroph communities regulating NO 3 -N transformation. Bradyrhizobium via Clostridium had indirect negative impact (4.6143) on NO 3 -N transformation. On the contrary, positive indirect effects of Bradyrhizobium was mediated through Clostridium with path coefficient value 3.3006. These results indicated that interactions between Clostridium and Bradyrhizobium played an important role on NO 3 -N transformation. In BC, NO3 -N equation altered in BC, which was established by Methylocystis, Azospira and Azoarcus (Table 2). Direct effects of Methylocystis, Azospira
Fig. 4. Path diagram of direct and indirect effects of genus on NH+4-N (A, B), NO 3 -N (C, D) and TN (E, F). A, C, E represent CK piles, and B, D, F represent BC piles.
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and Azoarcus on NO 3 -N transformation were 0.5630*, 0.7649* and 0.1880, respectively (Fig. 4D), inferring that the influence of Azospira on NO 3 -N variation was strengthened by biochar addition. Based on path analysis, Azospira via Methylocystis had indirect negative impact (0.3522*) on NO 3 -N transformation, and Methylocystis via Azospira had indirect negative impact (0.4384*) on NO 3 -N transformation. These results suggested that interaction between different diazotroph communities also had significant influence on NO 3 -N transformation in BC. Rhodopseudomonas, Azoarcus and Bradyrhizobium were the key genera affecting TN in CK (Table 2). Genera Rhodopseudomonas shown positive relationship with TN, whereas Azoarcus shown negative relationship, which was consistent with the result of NH+4-N stepwise regression model in this study. Additionally, Bradyrhizobium were negatively related to TN and denoted as NO 3 -N consumption (Table 2), which might suggest that diazotroph communities could affect TN by regulating NH+4-N and NO 3 -N transformation. Higher direct effect (1.2256*) of Bradyrhizobium on TN variation was observed (Fig. 4E), compared with Rhodopseudomonas (0.1593) and Azoarcus (0.2689*). Different from CK, equations in BC suggested that Methylocystis, Rhodopseudomonas and Azospira were the key communities influencing TN (Table 2). Path analysis diagram (Fig. 4F) shown that the direct influence of Azospira, Methylocystis and Rhodopseudomonas on TN were 0.8903*, 0.4807* and 0.2567, respectively. Besides, negative indirect effect of Methylocystis via Azospira was higher (0.5314*) than that of Azospira via Methylocystis (0.3044*). This result indicated that Azospira and Methylocystis were key factors determining TN, and further confirmed that the interaction between diazotroph communities could influence nitrogen transformation. Based on the above results it is inferred that the key diazotroph communities could serve as integrative variables in regulating nitrogen transformation simultaneously during composting.
4. Conclusion Addition of biochar changed the structure of diazotroph community and reduced the diversity of diazotroph community during composting. The main phyla in both treatments were Proteobacteria, Actinobacteria and Firmicutes. According to partial Mantel test analysis, biochar enhanced the interaction between temperature, NH+4-N, NO 3 -N and diazotroph community composition. Diazotroph communities could regulate NH+4-N and NO 3 -N transformation and the interactions between diazotroph communities had influence on nitrogen transformation. Additionally, biochar altered the key diazotrophic communities and their influence on nitrogen transformation.
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.
Acknowledgments This study is financially supported by National Natural Science Fund of China (31672469).
Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi.org/10.1016/j.wasman.2020.02.010.
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Waste Management journal homepage: www.elsevier.com/locate/wasman
Insight to key diazotrophic community during composting of dairy manure with biochar and its role in nitrogen transformation Xiaotong Wu, Yu Sun, Liting Deng, Qingxin Meng, Xin Jiang, Ayodeji Bello, Siyuan Sheng, Yue Han, Haifeng Zhu, Xiuhong Xu ⇑ College of Resources and Environment, Northeast Agricultural University, Harbin 150030, China
a r t i c l e
i n f o
Article history: Received 9 October 2019 Revised 14 January 2020 Accepted 10 February 2020
Keywords: Composting Biochar High-throughput sequencing Diazotroph Nitrogen transformation
a b s t r a c t Analyzing diazotrophic community may help to understand nitrogen transformation in composting and improves the final compost quality. In this study, diazotrophic community dynamics were investigated in terms of nifH gene during dairy manure and corn straw composting with biochar addition using highthroughput sequencing. Biochar decreased the diversity of diazotrophic community and altered diazotroph community structure during composting. At phylum level, Proteobacteria, Actinobacteria and Firmicutes were dominant diazotrophic communities throughout composting process. Biochar addition registered higher correlation coefficient (R) between physicochemical factors (temperature, ammonium (NH+4-N) and nitrate (NO 3 -N)) and diazotroph community composition. Rhodopseudomonas and Pseudoxanthomonas was the key diazotrophic communities influencing NH+4-N transformation in control (CK) and biochar compost (BC), respectively, while for NO 3 -N transformation Clostridium and Bradyrhizobium in CK, Azospira and Methylocystis in BC served as predominant factors. These results indicated that addition of biochar altered the key diazotroph communities influencing nitrogen transformation. Furthermore, some diazotrophs (e.g. Rhodopseudomonas, Bradyrhizobium and Azospira) affecting NH+4-N and NO 3 -N transformation were also observed to be mediating total nitrogen (TN). Interestingly, interactions between diazotrophic communities were observed and these interactions could also influence nitrogen transformation. Ó 2020 Elsevier Ltd. All rights reserved.
1. Introduction Composting, an effective method used in degrading agricultural wastes leading to production of organic fertilizer by microbial metabolism, is widely recognized (Jain et al., 2018; Wang et al., 2017a). Nitrogen transformation was crucial for composting processes and compost quality, including ammonification, nitrification, denitrification and biological nitrogen fixation, and specific microorganisms contributed to each nitrogen transformation process are sensitive to environmental changes (Levy-Booth et al., 2014; Liu et al., 2019; Yin et al., 2018). Biochar as a pivotal factor in regulating physicochemical factors promoted nitrogen transformation and reduced ammonia loss during composting by their abundant pore structure and complex functional groups (Hale and Crowley (2015); Malinowski et al., 2019; Jain et al., 2018; Sánchez-García et al., 2015). Also, biochar addition can enhance the abundance of microorganisms and microbial activities during
⇑ Corresponding author. E-mail address: [email protected] (X. Xu). https://doi.org/10.1016/j.wasman.2020.02.010 0956-053X/Ó 2020 Elsevier Ltd. All rights reserved.
composting (Du et al., 2019; Zhou et al., 2019). Therefore, adding biochar to compost may create favorable conditions for microbial communities and improve compost quality. High-throughput sequencing can comprehensively explain microbial community composition and has gradually become the main method of investigating microbial communities in various environments including compost (Duan et al., 2019; Meng et al., 2019a). Among the nitrogen transformation processes during composting, biological nitrogen fixation process, during which N2 is converted into organic nitrogen, increases nitrogen content (Pepe et al., 2013; Sun et al., 2016). Diazotrophs are special functional group of microorganisms responsible for nitrogen fixation including free-living bacteria (e.g., Azospirillum, Rhodospirillum and Azotobacter), symbiotic nitrogen fixiation bacteria (e.g., Methylobacterium, Bradyrhizobium, and Rhizobium) and a part of sulfate-reducing bacteria (e.g., Desulfobulbus) (Yin et al., 2018; Marques et al., 2017; Burow et al., 2014). Diazotrophs are supposed to increase nitrogen in environment and exert influences on nitrogen transformation (Beauchamp et al., 2006; Sun et al., 2016). Therefore, diazotrophs are interesting microbial communities in
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compost, which may have great impact on nitrogen transformation and compost quality. However, to our knowledge, few researches have been conducted on diazotrophic community in composting especially under the influence of biochar (Che et al., 2018; Pereira et al., 2011), and its role in nitrogen transformation are rarely explored. The lack of information on diazotrophs in composting may limit the understanding of the whole microbial communities and nitrogen transformation. This study aimed to: (1) explore diazotroph community succession in dairy manure and corn stalk composting affected by biochar using high-throughput technology; (2) examine the correlation between diazotrophic community structure and physicochemical factors by mantel test and Spearman correlation analysis; (3) quantify the relationships between nitrogen transformation and diazotrophs genera by stepwise regression and Path analyses; (4) identify key diazotrophs genera affecting nitrogen transformation. 2. Material and methods 2.1. Composting Dairy manure and corn stalk were used for composting. Corn stalk was cut into 1–2 cm and was homogeneously mixed with dairy manure at ratio 1:5 (Meng et al., 2019a; Sun et al., 2019b). Initial physicochemical factors of dairy manure, corn stalk and biochar were listed in Supplementary Table 1. Two compost mixtures (treatments); first treatment (dairy manure + corn stalk) as control (CK) and the second treatment (CK + 10% rice biochar) (BC) were set-up and replicated three times in a trapezoid shape (2.5 m 1. 5 m 1 m). Composting lasted for 43 days, and piles were turned on day 11, day 19 and day 28 according to the temperature variation. 2.2. Sample collection Samples were collected on day 0 (initial phase), day 2 (mesophilic phase), day 4 (thermophilic phase), day 28 (cooling phase) and day 43 (maturation phase), each were taken at three depths (10 cm, 30 cm, 50 cm from pile surface) and mixed. Samples of these composting phases from CK and BC treatments were named as CK1, CK2, CK3, CK4, CK5, and BC1, BC2, BC3, BC4, BC5, respectively. The samples were divided into two parts, one was airdried for the determination of physicochemical parameters and the other was stored at 80 °C for molecular experiments. 2.3. Determination of physicochemical parameters Temperature of compost piles was measured with electronic thermometers. Moisture content (MC) was determined by oven drying the samples at 105 °C to constant weight, and pH was determined using the method of previous study (Abid and Sayadi, 2006). Ammonia nitrogen (NH+4-N) and nitrate nitrogen (NO 3 -N) were measured using leach liquor (2 molL1 KCL at the ratio of 1:10 with 30 min oscillation at 150 rpm) by flow injection analysis (FIAstar 5000, FOSS, Denmark). Total carbon (TC) and total nitrogen (TN) were determined using the combustion method by automatic elemental analysis (LECO CHNS-932, 120 USA). 2.4. High-throughput sequencing of NifH gene in diazotrophs Total DNA was extracted using MP Biomedicals Fast DNA Soil Sample Extraction Kit (MoBio Laboratory, Carlsbad, CA, USA) according to the instructions, and then stored at 20 °C. NifH gene was sequenced using primers nifHF (50 -AAAGGYGGWATCG GYAARTCCACCAC 30 ) and nifHR (50 -TTGTTSGCSGCRTACATSGC
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CATCAT 30 ), and PCR amplification was conducted with the method of Rösch et al. (2002). PCR products were detected by 2% agarose gel electrophoresis and sequenced using Illumina MiSeq platform. The raw data was archived in NCBI Sequence Read Archive with accession No. PRJNA546109. 2.5. Statistical analysis Qiime 1.9.1 was used for quality optimization of raw sequences (filtering the reads below 200 bp and containing N base), and optimized sequences were clustered into operational taxonomic units (OTUs) with 97% similarity using UPARSE 7.0.1090, then effective sequences were aligned against NCBI database to identify species information (Caporaso et al., 2010; Edgar, 2013). Mothur 1.30.2 and Vegan package in R 2.4 were used to calculate Alpha diversity (Shannon and Chao1 indices), rarefaction curve and mantel test analysis, while Qiime 1.9.1 was used to evaluate Beta diversity (Non-metric Multidimensional Scaling) by Bray-Curtis distance. Values of physicochemical factors were statistically analyzed by One-way ANOVA using SPSS 21.0 (IBM, USA) and Microsoft Excel 2016. Spearman correlation, stepwise regression and Path analyses were done to examine direct and indirect effects of key diazotrophs on nitrogen transformation using SPSS Statistics 21.0 and Data Processing System (DPS) software (Sun et al., 2019a; Tang and Zhang, 2013). 3. Results and discussion 3.1. Variation of physicochemical factors Data of physicochemical factors were shown in Supplementary Materials. Temperature rose rapidly at the beginning of composting and temperature values above 50 °C were recorded for 22 days in CK and 27 days in BC, indicating that pathogens were eliminated in compost (Meng et al., 2019b). Little difference was observed between BC and CK temperature, and similar results were reported by Jain et al. (2018) who explored the effects of biochar on dairy cow manure and Hydrilla verticillate composting. Moisture content of CK decreased from 71.14% to 29.98%, while that of BC declined from 62.03% to 29.45%. pH shown an upward trend during composting and final pH values of CK (8.16) and BC (8.17) were within the range (7.00–8.50) for mature compost (Sun et al., 2019b). Increase in pH may due to decomposition of organic matter by microorganisms and production of NH+4-N during composting (Qian et al., 2014; Ren et al., 2016). C/N ratios were 15.75 and 15.33 in CK and BC, respectively, indicating that the final compost was mature. Similar patterns of NH+4-N variation were observed in CK and BC, which increased in the first 4 days and then declined. This phenomenon may be attributed to the fact that mineralization of organic nitrogen by microorganisms increased the level of NH+4-N in mesophilic phase (Ren et al., 2016), and then volatilization and nitrification led to its reduction during in thermophilic and cooling phases of composting (Rashad et al., 2010; Raj and Antil, 2011). Concentration of NO 3 -N showed more rapid rising in CK than that of BC in cooling and maturation phases, indicating that biochar might negatively affect the concentration of NO 3 -N in cooling and maturation phases, which may be related to the excessive adsorption by biochar causing the reduction of effective nutrients (Liang et al., 2019; Wang et al., 2013). TN declined within the first 4 days of composting and later increased in both treatments. Several studies have confirmed that nitrogen fixation activities of diazotroph community could increase TN content in cooling and maturation phase of composting (Kalamdhad et al., 2009; Yin et al., 2018). So, the rising of TN in this study might partially be
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contributed by diazotroph community. Final concentration of TN in BC (22.26 gkg1) was significantly higher than that of CK (19.01 gkg1), which consistent with previous study (Malinowski et al., 2019), suggesting that biochar may improve nitrogen conservation in composting.
3.2. Community structure analyses A total of 633863 nifH sequences in CK and 591573 sequences in BC were obtained after optimized screening, and 1712 and 1806 operational taxonomic units (OTUs) were identified in CK and BC respectively based on 97% nucleotide similarity. Effective species diversity in this study was determined by saturated Shannon rarefaction curves (Supplementary Material), which revealed that sufficient sequences of diazotrophs was achieved. Shannon and Chao1 indices increased in CK but dropped in BC during thermophilic phase (Fig. 1a and b). The diversity indices gradually decreased after thermophilic phase in CK, while they showed irregular decreasing patterns in BC. At the end of composting, the diversity indices decreased significantly in both treatments, which could be attributed to screening of microorganisms by high temperature (Huhe et al., 2017). These results indicated that biochar addition limited the diversity of diazotrophs and strengthened the inhibiting effect of high temperature on nifH gene. Previous studies reported that high concentration of biochar (above 5%) has deleterious effects on microbes because it may contain some toxic compounds and creating an undesirable C/N stoichiometric ratio (Liang et al., 2019; Liao et al., 2014).
Non-metric multidimensional scaling (NMDS) analysis interpreted the difference of diazotrophic community composition between samples using Bray-Curtis dissimilarity on OTU level. The distance between the positions of early composting stage (mesophilic and thermophilic phase) and late stages (cooling and maturation phase) was observed (Fig. 2). This suggests that composition of diazotroph community varied significantly during composting. Piceno et al. (2017) also reported that composting phase exhibited significant influence on the succession of microbial communities. The main reason for this result was that the temperatures, pH and nutrients varied in each phase and microorganisms were sensitive to the environment (Malinowski et al., 2019). Contracting distance was registered with BC and CK samples during composting, indicating that biochar could definitely affect the community composition of diazotroph during the whole composting process. This phenomenon was consistent with the study of Duan et al. (2019) who found that bacterial community was altered by biochar during cow manure composting. 3.3. Diazotrophic community composition As shown in Fig. 3, relative abundance of diazotroph community varied significantly during composting in both treatments. On both phyla and genus level, relative abundance of major diazotrophic communities in BC was different from those in CK. At phylum level (Fig. 3A), Proteobacteria were dominant during composting, followed by Actinobacteria and Firmicutes. Other phyla with relative abundance less than 1%, were classified as Others. The unclassified community occupied 10.72% of total diazotrophic
Fig. 1. Changes of Shannon and Chao1 index of diazotroph community.
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Fig. 2. Non-metric multidimensional scaling (NMDS) of simples using Bray-Curtis dissimilarity and the stress value is 0.112. The ellipse is used to divide the sample, three parallel in one ellipse, each color represents a composting period.
Fig. 3. Relative abundance of phylum level (A) and genus level (B). CK and BC were shown in the same coordinate system (average value of parallel samples was taken as the final relative abundance). Phylum and genera which less than 1% in relative abundance were classified as others.
communities. In the initial phase, Proteobacteria accounted for 75.10% and 81.66% of diazotrophs in CK and BC, respectively. Proportion of Proteobacteria decreased drastically in mesophilic and thermophilic phase of both treatments, however it rose rapidly after thermophilic phase, indicating that thermophilic conditions were not favorable for Proteobacteria. Proteobacteria played a vital role in carbon cycle and nitrogen mineralization (Awasthi et al., 2017; Liang et al., 2019), indicating that Proteobacteria were active in nitrogen transformation in later phase of composting. Actinobacteria were dominant at thermophilic phase in CK (32.14%) and BC (57.36%), but dropped at maturation phase of CK (3.37%) and BC (1.43%). These results suggested that Actinobacteria were tolerant to high temperature. Actinobacteria could form spore under high temperature and this property was beneficial for this phylum to survive and sustain higher abundance during thermophilic composting (Jurado et al., 2014; Zhao et al., 2016). Relative abundance of Firmicutes was increased at mesophilic (7.15%) and thermophilic phase (26.33%) in CK, and this phenomenon might be related to the ability of Firmicutes in forming endospores that can withstanding high temperature (Zhou et al., 2019). In BC, relative abundance of Firmicutes increased throughout the whole process of composting and this was in accordance with the study by Duan et al. (2019)
who found that the relative abundance of Firmicutes increased during wheat straw and dairy manure composting with 12% biochar addition. Relative abundance of the aforementioned three phyla showed obvious variation at mesophilic and thermophilic phases, suggesting that succession of the diazotroph community might be affected by temperature. On genus level, diazotroph community also changed significantly during composting (Fig. 3B). Psychrobacter were the most abundant genus of Proteobacteria phylum and it decreased significantly at mesophilic and thermophilic phase in this study, owing to the fact that Psychrobacter prefer low temperature environment (Joseph et al., 2008). However, higher abundance of Psychrobacter was observed in BC (26.76%) than in CK (10.35%) at mesophilic phase, suggesting that addition of biochar might create an environment favorable for the survival of Psychrobacter under higher temperature, which was consistent with the report of Wang et al. (2017a). The relative abundance of Streptomyces increased at thermophilic phase in CK (31.61%) and BC (55.27%). Streptomyces, belonging Actinobacteria, has been reported as free-living nitrogen fixation bacteria (Dahal et al., 2017). Higher proportion of Streptomyces in BC might indicate more contribution to nitrogen fixation (Prakash and Cummings, 1988). The relative abundance of
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BC. These results suggested that biochar could enhance the sensitivity of diazotroph community to NH+4-N and NO 3 -N. Previous studies have reported that NH+4-N and NO 3 -N were the key factors for shaping microbial community composition (Meng et al., 2019a; Zhou et al., 2019). In this study, abundance of Streptomyces increased at thermophilic phase of composting (31.61% in CK and 55.27% in BC) and interestingly the NH+4-N content also increased in this period (Supplementary Table S2). Therefore, a positive correlation between Streptomyces and NH+4-N was observed (Supplementary Table S4), indicating NH+4-N accumulation was likely to be promoted by Streptomyces. Similar findings were reported in previous studies (Meng et al., 2019a; Fang et al., 2019). Besides, the positive correlations between some diazotrophs (e.g., Streptomyces, Azospira and Rhodopseudomonas) and NH+4-N were enhanced by biochar as shown by Spearman analysis, suggesting that adding biochar may facilitate NH+4-N accumulation in composting. Spearman analysis showed that Azotobacter, Azoarcus and Pseudoxanthomonas were positively correlated with TN in both treatments (Supplementary Table S4), but no significant correlation was found between TN and diazotroph community composition (Table 1). The results indicated that the presence of some diazotrophs (e.g., Azotobacter) could increase nitrogen availability in compost matrix, which is of significance for improving compost quality. This finding was in accordance with previous studies which reported that diazotrophs were able to fix nitrogen and increase nitrogen content in environments (Orr et al., 2011; Pepe et al., 2013). Furthermore, abundance of Azotobacter was higher in BC, suggesting that biochar addition could promote the growth of this genus, which might contribute to the higher level of nitrogen in compost.
Bradyrhizobium, belonging Bradyrhizobiaceae and Rhizobiales, gradually increased from initial phase (3.35%) to thermophilic phase (21.28%) in CK, and decreased significantly thereafter. Bradyrhizobium were also found in BC, and its population increased during mesophilic phase. Bradyrhizobium are common symbiotic nitrogen fixing bacteria and they can only fix nitrogen only after inoculating legume plants (Liu et al., 2019). In this study, Bradyrhizobium were detected and its relative abundance varied during composting, suggesting that this group of diazotroph could not only survive but also grow under the harsh condition of composting. So, it is interesting to explore their ability of nitrogen fixation during composting in future study. Rhodopseudomonas, another genus belongs to Bradyrhizobiaceae and Rhizobiales, shown an upward and then downward trend during composting in both treatments, however the relative abundance of Rhodopseudomonas in BC was higher than that of CK in all stages of composting. These results indicated that the addition of biochar was beneficial to the survival of this group and might increase the chance of symbiotic nitrogen fixation in soil after fertilizer application in legume field. Azotobacter and Azospira are free-living nitrogen fixation bacteria (Marques et al., 2017; Zhan and Sun, 2011). The proportion of Azotobacter increased at cooling phase in CK (13.72%) and BC (15.84%). Azospira increased at mesophilic phase of CK (11.17%) and BC (5.42%), and then disappeared at cooling phase and maturation phase in both treatments. The relative abundance of Clostridium reached maximum at thermophilic phase in CK (19.63%) and at maturation phase in BC (11.79%). Clostridium are ubiquitous in natural environment and has ability to fix nitrogen under anaerobic conditions (Woodhouse et al., 2013). Thus, it is reasonable to predict that Clostridium might carry out the function of nitrogen fixation in various depths of composting pile. Relative abundance of Pseudoxanthomonas increased at the end of composting with 5.55% in CK and 12.14% in BC. In addition to nitrogen fixation, Pseudoxanthomonas were reported to be capable of degrading macromolecular organic matter and producing soluble phosphorus (Li et al., 2013), indicating that the high relative abundance of Pseudoxanthomonas might contribute to compost quality improvement.
3.5. Key diazotroph communities affecting nitrogen transformation Stepwise regression model indicated that Rhodopseudomonas was the key diazotroph community affecting NH+4-N variation positively in CK (Table 2), suggesting that the increase of Rhodopseudomonas abundance might indicate the accumulation of NH+4-N, which can be attributed to the phenomenon that growth of Rhodopseudomonas was inhibited by low NH+4-N concentration (Wang et al., 2017b). The other two variables (Azoarcus and Azotobacter) were negatively related to NH+4-N transformation, which was supported by Spearman analysis (Supplementary Table S4). According to path analysis diagram (Fig. 4A), Rhodopseudomonas (0.4938*) had higher direct influence than Azoarcus (0.3541*) and Azotobacter (0.3815*) on NH+4-N in CK. This finding suggested that Rhodopseudomonas was the better predictive variables determining NH+4-N transformation and corroborated with result presented in stepwise regression model (Table 2) in CK. Although the addition of biochar altered the key diazotroph communities affecting NH+4-N, Rhodopseudomonas and Azoarcus were still
3.4. Relationship between physicochemical factors and diazotroph community Mantel test and partial mantel test analyses showed that pH and MC correlated significantly with diazotrophic community composition in CK (Table 1), while significant correlation was observed between temperature and diazotrophic community composition in BC. NH+4-N was significantly correlated with diazotroph community composition in both treatments (Table 1), and higher correlation was observed in BC (0.672) than in CK (0.417). The variation of NO 3 -N was associated with diazotrophic community dynamic in
Table 1 Mantel test and partial mantel test of correlation between physicochemical factors and community composition. Factor
CK
BC
Mantel test
T pH MC TN NH+4-N NO 3 -N
Partial mantel test
Mantel test
Partial mantel test
R
P
R
P
R
P
R
P
– 0.457 0.745 – 0.738 0.658
– 0.001 0.001 – 0.001 0.001
– 0.169 0.242 – 0.417 –
– 0.018 0.017 – 0.001 –
0.236 0.336 0.773 0.396 0.773 0.395
0.034 0.015 0.001 0.002 0.001 0.007
0.263 – 0.144 – 0.672 0.205
0.016 – 0.034 – 0.001 0.023
Note: R statistic closer to 1 represents the stronger positive correlation, and the minus represents negative correlation. Partial Mantel test was used to estimate relationship between the physicochemical factor and diazotrophs community structure with excluding other relevant factors. T: temperature; MC: moisture content; TC: total carbon; TN: total nitrogen.
X. Wu et al. / Waste Management 105 (2020) 190–197 Table 2 Stepwise regression models with NH+4-N, NO 3 -N and TN as dependent variables and genus as independent variables (n = 5). Compost
Standardization regression equations
R2
P
CK
NH+4-N
0.9994
0.0306
0.9999
0.0108
0.9998
0.0180
0.9993
0.0332
0.9998
0.0166
0.9995
0.0278
BC
= 1165.6827 + 2.5599 Rhodopseudomonas 0.0809 Azoarcus 0.0428 Azotobacter NO 3 -N = 139.0069 + 0.0159 Azospira + 0.0428 Clostridium 0.0513 Bradyrhizobium TN = 19.41158 + 0.0016 Rhodopseudomonas 0.0001 Azoarcus 0.0006 Bradyrhizobium NH+4-N = 1773.7553 + 0.8667 Rhodopseudomonas 0.2339 Pseudoxanthomonas 0.0557 Azoarcus NO 3 -N = 87.2901 – 0.0315 Methylocystis 0.0170 Azospira 0.0012 Azoarcus TN = 22.2709 – 0.0023 Methylocystis + 0.0020 Rhodopseudomonas 0.0018 Azospira
Note: Nitrogen index (NH+4-N, NO 3 -N, TN) were the dependent variable and diazotroph genera were the independent variable. High R2 (approaching 1.0000) and significant P value (<0.05) indicated that the equations were successfully established.
observed closely correlated with NH+4-N transformation in BC (Table 2). Another genus affecting NH+4-N variation was Pseudoxanthomonas. The path analysis diagram (Fig. 4B) shown that direct effect of Rhodopseudomonas and Azoarcus on NH+4-N variation were
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0.2647* and 0.2574*, respectively, and Pseudoxanthomonas had a higher direct effect (0.6247*). These results indicated that Pseudoxanthomonas was the key genus influencing NH+4-N variation. However, biochar addition might reduce the effect of Rhodopseudomonas on NH+4-N transformation, this might be due to that biochar improving the ventilation of composting matrix and also the nitrogenase activity of Rhodopseudomonas was then inhibited by the increased level of oxygen (Arashida et al., 2019). In the control pile, Azospira, Clostridium and Bradyrhizobium were major communities influencing NO 3 -N transformation (Table 2). Compared with Azospira (0.7473), Clostridium (3.5427*) and Bradyrhizobium (4.8495*) were observed higher direct influence on NO 3 -N transformation (Fig. 4C). Clostridium and Bradyrhizobium have been previously reported to be involved in denitrification process (Sánchez et al., 2011; Thakur and Medhi, 2019), indicating that Clostridium and Bradyrhizobium could be the key diazotroph communities regulating NO 3 -N transformation. Bradyrhizobium via Clostridium had indirect negative impact (4.6143) on NO 3 -N transformation. On the contrary, positive indirect effects of Bradyrhizobium was mediated through Clostridium with path coefficient value 3.3006. These results indicated that interactions between Clostridium and Bradyrhizobium played an important role on NO 3 -N transformation. In BC, NO3 -N equation altered in BC, which was established by Methylocystis, Azospira and Azoarcus (Table 2). Direct effects of Methylocystis, Azospira
Fig. 4. Path diagram of direct and indirect effects of genus on NH+4-N (A, B), NO 3 -N (C, D) and TN (E, F). A, C, E represent CK piles, and B, D, F represent BC piles.
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and Azoarcus on NO 3 -N transformation were 0.5630*, 0.7649* and 0.1880, respectively (Fig. 4D), inferring that the influence of Azospira on NO 3 -N variation was strengthened by biochar addition. Based on path analysis, Azospira via Methylocystis had indirect negative impact (0.3522*) on NO 3 -N transformation, and Methylocystis via Azospira had indirect negative impact (0.4384*) on NO 3 -N transformation. These results suggested that interaction between different diazotroph communities also had significant influence on NO 3 -N transformation in BC. Rhodopseudomonas, Azoarcus and Bradyrhizobium were the key genera affecting TN in CK (Table 2). Genera Rhodopseudomonas shown positive relationship with TN, whereas Azoarcus shown negative relationship, which was consistent with the result of NH+4-N stepwise regression model in this study. Additionally, Bradyrhizobium were negatively related to TN and denoted as NO 3 -N consumption (Table 2), which might suggest that diazotroph communities could affect TN by regulating NH+4-N and NO 3 -N transformation. Higher direct effect (1.2256*) of Bradyrhizobium on TN variation was observed (Fig. 4E), compared with Rhodopseudomonas (0.1593) and Azoarcus (0.2689*). Different from CK, equations in BC suggested that Methylocystis, Rhodopseudomonas and Azospira were the key communities influencing TN (Table 2). Path analysis diagram (Fig. 4F) shown that the direct influence of Azospira, Methylocystis and Rhodopseudomonas on TN were 0.8903*, 0.4807* and 0.2567, respectively. Besides, negative indirect effect of Methylocystis via Azospira was higher (0.5314*) than that of Azospira via Methylocystis (0.3044*). This result indicated that Azospira and Methylocystis were key factors determining TN, and further confirmed that the interaction between diazotroph communities could influence nitrogen transformation. Based on the above results it is inferred that the key diazotroph communities could serve as integrative variables in regulating nitrogen transformation simultaneously during composting.
4. Conclusion Addition of biochar changed the structure of diazotroph community and reduced the diversity of diazotroph community during composting. The main phyla in both treatments were Proteobacteria, Actinobacteria and Firmicutes. According to partial Mantel test analysis, biochar enhanced the interaction between temperature, NH+4-N, NO 3 -N and diazotroph community composition. Diazotroph communities could regulate NH+4-N and NO 3 -N transformation and the interactions between diazotroph communities had influence on nitrogen transformation. Additionally, biochar altered the key diazotrophic communities and their influence on nitrogen transformation.
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.
Acknowledgments This study is financially supported by National Natural Science Fund of China (31672469).
Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi.org/10.1016/j.wasman.2020.02.010.
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