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Effect of organic acids production and bacterial community on the possible mechanism of phosphorus solubilization during composting with enriched phosphate-solubilizing bacteria inoculation
Bioresource Technology 247 (2018) 190–199
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Effect of organic acids production and bacterial community on the possible mechanism of phosphorus solubilization during composting with enriched phosphate-solubilizing bacteria inoculation
MARK
Yuquan Weia,1, Yue Zhaoa,1, Mingzi Shia, Zhenyu Caoa, Qian Lua, Tianxue Yangb, Yuying Fana, ⁎ Zimin Weia, a b
College of Life Science, Northeast Agricultural University, Harbin 150030, China Laboratory of Water Environmental System Engineering, Chinese Research Academy of Environmental Science, Beijing 100012, China
G RA P H I C A L AB S T R A C T
A R T I C L E I N F O
A B S T R A C T
Keywords: Phosphate-solubilizing bacteria (PSB) Composting Inoculation Organic acids Tricalcium phosphate solubilization
Enriched phosphate-solubilizing bacteria (PSB) agent were acquired by domesticated cultivation, and inoculated into kitchen waste composting in different stages. The effect of different treatments on organic acids production, tricalcium phosphate (TCP) solubilization and their relationship with bacterial community were investigated during composting. Our results pointed out that inoculation affected pH, total acidity and the production of oxalic, lactic, citric, succinic, acetic and formic acids. We also found a strong advantage in the solubilization of TCP and phosphorus (P) availability for PSB inoculation especially in the cooling stage. Redundancy analysis and structural equation models demonstrated inoculation by different methods changed the correlation of the bacterial community composition with P fractions as well as organic acids, and strengthened the cooperative function related to P transformation among species during composting. Finally, we proposed a possible mechanism of P solubilization with enriched PSB inoculation, which was induced by bacterial community and organic acids production.
⁎
1
Corresponding author. E-mail addresses: [email protected], [email protected] (Z. Wei). Authors contributed equally to this work.
http://dx.doi.org/10.1016/j.biortech.2017.09.092 Received 5 August 2017; Received in revised form 12 September 2017; Accepted 15 September 2017 Available online 19 September 2017 0960-8524/ © 2017 Elsevier Ltd. All rights reserved.
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1. Introduction
2016b; Chen et al., 2006), this study was essentially focused to the effect of bacterial community and different organic acids on TCP-solubilization during KW composting. Besides, different stages for inoculation were employed. Direct multivariate analyses, such as redundancy analysis (RDA), could be used as a preliminary estimate for the correlation between changes in microbial community and environmental variables, and structural equation modeling (SEM) could further construct complex relationships among composting biotic and abiotic factors (Liang et al., 2017; Flores-Renteria et al., 2016; Wei et al., 2016a). Nevertheless, there is little consideration whether changes of bacterial community and organic acids production could be associated with P-solubilization during composting by these relationship model. Our intent was to evaluate our hypothesis by RDA and SEM to link bacterial community, organic acids production and TCP-solubilization in different inoculation treatments, which may provide a better understanding of the P-solubilization process of phosphate-solubilizing inoculant during composting and lead to new perspectives on strategies to improve P availability of composts with precipitated inorganic P.
Although the total amount of phosphorus (P) is high in many lands, P deficiency is a constraint to plant growth worldwide due to the low availability in soil (Wei et al., 2016b). Therefore, P-fertilizers is frequently applied, particularly in conventional intensive agricultural soils to overcome P-limitation. Unfortunately, most of chemical P-fertilizers can be immobilized by Ca2+, Fe3+ and Al3+, which lead to the rapid formation of poorly available P for plants after applied (Malik et al., 2012). Given that demand for improving the availability of P-fertilizers continues to increase and phosphate-solubilizing bacteria (PSB) has a key role in the mobilization and transformation of soil P, more and more attention was paid on the understanding of the mechanisms for solubilizing of mineral phosphates and how to improve the efficiency of PSB application. Many studies have shown that PSB can transform the insoluble P to soluble forms by acidification, chelation, exchange reactions, and polymeric substances formation, which may be related to the production of organic acids, the release of protons (H+), the redox-active metals, etc. (Delvasto et al., 2006). Among them, the mechanism that phosphate solubilizing ability of PSB is associated with the release of low molecular weight organic acids is generally accepted. Low molecular weight organic acids may play an important role the detoxification of pollutants, the bioavailability of heavy metals and even microbial activity in soil conditions (Onireti et al., 2017). On the other hand, these organic acids (e.g., oxalic acid, citric acid, acetic acid, succinic acid, etc.) could also lower pH, chelate the cations (mainly calcium) bound to phosphate through their hydroxyl and carboxyl groups or compete with phosphate for adsorption sites, leading to the increased solubility and availability of mineral phosphates (Mander et al., 2012; Khan et al., 2007). However, the predominant organic acids produced by different phosphate-solubilizing microorganisms were diverse distinctly (Panhwar et al., 2014; Nakasaki et al., 2013). Moreover, the process of P-solubilization is a complex phenomenon, which are severely affected by many factors. The impacts of environmental factors, such as temperature, pH, oxygen concentration, moisture, etc. on microbial P-solubilizing capability have been widely investigated (Zhu et al., 2012; Chen et al., 2006), but there is no studies specifically on the influence of the interaction of microbial communities and PSB on solubilizing insoluble phosphate and production of organic acids. Composting is a controlled biological decomposition process of organic matter, accompanying with a dynamic activity of various microbial populations (Xi et al., 2016; Zhao et al., 2016a; He et al., 2014). Given that composting can affect the distribution of P fractions, a large number of studies has been oriented to the addition of mineral phosphates with poor availability and inoculation of phosphate solubilizing microbes during composting to develop P-enriched compost (Wei et al., 2016b; Chang and Yang, 2009). Many microorganisms have been utilized as solubilizing insoluble P inoculants in composts. However, considering that the inoculation during composting may have different performances due to the influence of the source of microbial inoculum, the scale of composting, and the stage of inoculation etc. (Zhao et al., 2016b), we hypothesized that: (1) compared to uninoculated compost, changes in the time of inoculation of phosphate solubilizing microbes during composting may cause different biological microenvironment and bacterial community structure due to the competition or collaborative symbiosis between inoculants and indigenous bacteria, (2) compared to uninoculated compost, inoculation of PSB in different stages will stimulate microbial activity related to organic acids production more and therefore lead to more solubilization of precipitated inorganic P, and (3) different bacterial community relationship may change the type and amount of organic acid involved in the P-solubilization process. Given that tricalcium phosphate (TCP) is mainly used as a model material as they represent the vast majority of precipitated inorganic P salts and kitchen waste (KW) has a relatively low P content (Wei et al.,
2. Materials and methods 2.1. Preparation of phosphate-solubilizing inoculant The enriched PSB was obtained from different solid waste composting samples as described by Wei et al. (2016a). The enrichment process was similar to the method described as Zhang et al. (2016), but the enriched medium was sterile National Botanical Research Institute's phosphate growth medium (NBRIP) broth (Wei et al., 2016a). The liquids with enriched PSB was cultivated for 3 days at 40 °C until the population up to 108 CFU mL−1 to measure the phosphate-solubilizing activity in broth culture including water soluble phosphorus (WSP) and microbial biomass phosphorus (MBP) according to Panhwar et al. (2014), and pH of the medium was recorded with a pH meter. The phosphate-solubilizing content of the PSB agent increased after the process of domestication culture for 10 cycles, and the 10th cycle of phosphate-solubilizing inoculant with high P-solubilization capacity was centrifuged and suspended in sterile water, then sprayed on the composting mixture. The concentrations of the strain were 1 × 108 CFU mL−1. 2.2. Composting experiment design KW was prepared as the main material, which was collected from the cafeteria in Northeast Agricultural University (Harbin, China), mainly containing the cooked food residues, such as steamed rice, noodles, steamed bread, and cooked vegetable, meat, fish. The indigestible materials, such as plastics, bones, and egg shell, were selected before the KW were crushed and homogenized. Sawdust was added to adjust the C/N radio of 25, which was obtained from a timber mill in Harbin, China. Details of raw materials were described in Table 1. The initial moisture was adjusted to 60%. Five lab-scale composting experiments were carried out for 40 days in the special compost reactor (the working volume was 12.5 L) as described by Zhao et al. (2016b) and various treatments with three replicates each were applied as shown in Table 2. Considering the phosphate-solubilizing inoculant was enriched at the temperature below 50 °C, it was not inoculated at hightemperature stage of composting. The KW with a relatively low P content was chosen as the major raw material and therefore allowed the impact of enriched phosphate-solubilizing inoculant with different stages inoculation on the solubilization of TCP in composting to be readily observed. During composting, moisture was maintained at 50–60% and the ventilation rate was 0.5 L min−1 kg−1 in order to maintain oxygen concentration inside the composts above 10%. Composting materials were turned over manually every four days to ensure homogenized. Representative compost samples of each treatment were 191
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Table 1 The physical and chemical properties of raw materials for composting. Materials
pH
TOC (%)
TN (%)
C/N
TP (g kg−1)
Bulk density (g cm3)
KW Sawdust Mixed materials
4.99 ± 0.40 7.02 ± 0.26 5.40 ± 0.33
57.42 ± 2.45 43.29 ± 2.25 54.15 ± 2.31
3.36 ± 0.09 0.91 ± 0.03 2.37 ± 0.08
17.08 ± 0.88 47.25 ± 1.94 24.59 ± 1.05
8.62 ± 0.27 0.95 ± 0.04 7.84 ± 0.18
0.51 ± 0.02 0.20 ± 0.01 0.45 ± 0.01
TOC: total organic carbon; TN: total nitrogen; TP: total phosphorus.
2.4. DNA extraction and PCR-DGGE
Table 2 Different treatments of composting. Treatment
CK CP CMP1 CMP2 CMP3
Basic Materials in composts KW
Sawdust
+ + + + +
+ + + + +
TCP (w/ w)
– 2.5 2.5 2.5 2.5
DNA kit (Omega Biotek, Inc.) was used to extract the total DNA of compost samples. Touch-down PCR was performed with the templates of the extracted DNA and the specific bacterial primers of 534r and 357f with a GC-clamp. The system setting and method of polymerase chain reaction referenced to Wei et al. (2016b). Each PCR concoction was set in a polyacrylamide (8%) gels at a 35–60% denaturant gradient. A Gene Mutation Detection System (Bio-Rad Laboratories, Inc.) was run at 150 V for 4 h at 60 °C to separate the fragments. After electrophoresis, gels were stained in 3 × GelRed™ nuclear acid gel stain (Biotium, USA) and photographed with a UVP Imaging System (UVP Inc., USA). This procedure was performed in triplicate for each sample with different gels. Representatives of bands that were clear and had high intensity were excised from DGGE gels and transferred to 30-μL Milli-Q water (Millipore, USA), incubated overnight for elution of DNA at 4 °C. Then, PCR was performed to re-amplify the DNA using primers 534r and 357f without a GC-clamp. Finally, the sequences were analyzed, and the PCR fragments were identified according to Wei et al. (2016a). After sequencing, the results were compared with the GenBank from the National Center of Biotechnology Information (NCBI) using BLAST.
Composite inoculum (v/w)
Initial stage (Day 0)
Cooling stage (Day 12)
– – 5 – 2.5
– – – 5 2.5
taken on days 0, 3, 7, 12, 17, 23, 30 and 40 following the initiation of the composting process. Approximately 200 g of each compost were collected each time. One part of experimental samples was immediately stored at −20 °C for DNA extraction and others were air-dried, passed through a 0.25 mm sieve, and stored in a desiccator for physical-chemical analysis.
2.3. Analysis of physicochemical indexes, P fractions and organic acids 2.5. Statistical analysis Temperature was monitored with a digital thermometer on a daily basis and pH was measured using a digital pH meter as described by Wei et al. (2016b). Total acidity was measured according to the methods described by Huang et al. (2006). The determination of total phosphorus (TP), inorganic phosphorus (IP), organic phosphorus (OP), citric acid phosphorus (CAP), Olsen P and MBP were determined colorimetrically following the procedure of Wei et al. (2015). Potential available P (PAP) during composting was estimated as: ∑Olsen P + CAP + MBP in this study. The level of TCP solubilization (STCP) was estimated as the ratio of ΔAP to the content of added TCP, where available P fractions (AP) could be estimated as Olsen P, CAP, MBP or PAP and ΔAP was calculated as the increased amount of P fraction in day40 relative to day0. Organic acids in composting samples were extracted according to the method described by Nakasaki et al. (2013). The amount and composition of organic acids were analyzed by high performance liquid chromatography (HPLC). To prepare the liquid samples, the compost samples (3 g) were extracted using 10 ml 0.1% phosphoric acid at 4 °C for 10 min, and the mixtures were centrifuged at 12,000g for 10 min, then the supernatants were filtered through 0.20-μm PTFE syringe filters. The resulting filtrates were used to measure organic acids using an HPLC. The HPLC quantitative analysis was carried out on Agilent 1260, fitted with Agilent Zorbax Eclipse plus C18 reverse-phase column (250 mm × 4.6 mm). Organic acids were detected with a UV diodearray detector (Agilent 1260) set at 210 nm. The mobile phase was 10 mM diammonium hydrogen phosphate in Milli-Q water and 1 M phosphoric acid was applied to adjust the pH to 2.7 at a flow rate of 1 ml/min. The column temperature was 30 °C. Peaks were identified against a set of standard peaks obtained from nine known organic acids, i.e., succinic, citric, acetic, oxalic, formic, lactic, tartaric, malic, and propionic acids. All experiments were repeated three times.
All data obtained were subjected to statistical analysis using Origin8.0 and SPSS19.0. Analyses of variance (ANOVA) and multiple comparison tests (LSD All-Pairwise Comparisons Test) were performed to compare mean values for the different factors and variables analyzed (P < 0.05) using Statistic 8. DGGE banding profiles, including band numbers and relative intensity (within lane), were digitized using Quantity one software (version 4.5, Bio-Rad laboratories, USA). Shannon-Wiener index was determined as described by Wei et al. (2016a). Nonmetric multidimensional scaling ordination (NMDS) was used to identify differences in bacterial communities between different treatments based on the relative abundance of DGGE bands. The relationships between different organic acids and P fractions as well as microbes in composting were analyzed using the RDA from Canoco for Windows (Version 5.0). The “robust” maximum likelihood evaluation program of AMOS 7.0 software was used to analyze the model of SEM. Prior to SEM analysis, we examined the normal distributions of data for heteroscedasticity as well as all bivariate relationships for signs of nonlinearities. The model fit was evaluated using a chi-square (χ2) test where a non-significant P-value indicates a good fit of the model to the data (Morrissey and Franklin, 2015). The reduced path model was confirmed with stepwise multiple regression analysis. SEM models were individually performed for each composting monitoring physicochemical parameters, bacterial indicator (NMDS axis), organic acids and P fractions, but only the best fitted ones are presented. Data were reported as the means of triplicates. 3. Results and discussion 3.1. Evolution of process monitoring parameters 3.1.1. Characteristics of temperature, pH and total acidity Changes in temperature and pH are crucial to describe the microbial 192
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Fig. 1. Dynamic of (a) temperature, (b) pH, (c) total acidity, (d) TP, (e) Olsen P, (f) CAP, and (g) MBP during composting.
be observed from day 0 day to day 16, followed by a decrease but later than CP. This fact highlights the intense microbial acid-producing activity of phosphate-solubilizing inoculant, which may accelerate the solubilization of insoluble phosphate TCP during composting.
activity related to P transformation during the composting process (Wei et al., 2016a). In general, temperature in all treatments followed the typical evolution of composting processes (Fig. 1a). After an initial heating period of 3–4 days, the temperature quickly rose to 50–60 °C due to the degradation of abundant low molecular weight substances (e.g., organic matter, protein materials, sugars, etc.) in KW by microbial activity. As it was expected, a thermal activation was promoted after PSB inoculations. The pH values of the five treatments evolved in the range from 4.86 to 7.92 during composting (Fig. 1b), which declined slowly from the initial stage to day 7–12 and then showed an increased trend. The lower pH in CMP1, CMP2 and CMP3 compared to CP might be attributed to organic acids produced by strengthened microbial reactions accompanied with the degradation of organic matter (Yang et al., 2013). The values of total acidity ranged from 1.2 mmol/g to 6.9 mmol/g during composting and showed a decreased trend (Fig. 1c). A noteworthy increase of total acidity in three inoculated groups could
3.1.2. Variation in P solubilization and availability The content of TP gradually increased during composting (Fig. 1d). Initially, TP content was nearly 22.0 g/kg in four TCP-addition groups and 7.0 g/kg in CK. Composting process led to 45% increase of TP in CK above their initial values, while only led to 24% increase of TP in CP. This may be due to the fact that the high TCP addition significantly reduced the degradation of organic compounds by inhibiting the microbial growth and enzymatic activity during composting (Zeng et al., 2013; Malik et al., 2012). On the other hand, TP content of CMP2 and CMP3 on the 40th day were all significantly higher than that in CP and CMP1 (P < 0.05), suggesting that PSB inoculation in cooling stage 193
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could be more beneficial for the “concentration effect’’ than that in the beginning of composting. The content of IP and OP increased during composting, and IP was the major component of TP in all the groups (63–94%), which was the result of the addition of TCP with a high IP content. The OP content in CMP2 and CMP3 increased significantly after the 16th day relative to that in CK, CP and CMP1 (P < 0.05), which indicated that PSB inoculation in the cooling stage was suitable to accelerate effectively the TCP transformation to OP during composting. Fig. 1(e–g) illustrates the contents of Olsen P, CAP and MBP during composting in different treatments. Overall, the Olsen P, CAP and MBP in the end of composting were significantly higher than the values at the beginning of composting (P < 0.05). Olsen P and CAP content of four TCP-addition groups were all significantly higher than that in CK at the beginning of composting (P < 0.05), whereas the content of MBP was similar in all groups. The content of CAP had an obvious increase during composting, especially in CMP2 and CMP3, which might be related to the suitable condition after the thermophilic phase of composting for the acid-producing activity of PSB inoculant to solubilize TCP. The values of Olsen P decreased rapidly in the initial stage and the thermophilic phase. Thereafter, a large increase was observed in the cooling stage in four TCP-addition treatments, the most significant increase being found in CMP2 and CMP3. In contrast, there is a large increase in the concentration of MBP before the 7th day of composting, and a gradual decrease after the 23rd day of composting for all treatments. Combined with the results of OP, the change of CAP may be associated with the synthesis of OP with a corresponding decrease in MBP, suggesting that microbes might be important mediators in the transformation of P forms. A post hoc test for the all composts showed that there was a significant difference in the level of STCP between CP, CMP1, CMP2 and CMP3 (Table 3). The higher STCP of PAP in CMP2 (19.5%) and CMP3 (14.9%) compared to CP (5.8%) and CMP1 (7.3%) suggested that PSB inoculation in the cooling stage had a clear advantage to enhance the solubilization and availability of TCP. Moreover, compared to the results of P availability in the earlier report of Wei et al. (2016b), the enriched phosphate-solubilizing inoculant could have better adaptability to KW composting environments than the PSB screened from different composting samples when directly applied into KW.
The concentration of major organic acids presented varied changes during composting in five treatments (Fig. 2). The concentration of all the detected organic acids except succinic acid were increased in the early 3 days of composting, which may be caused by the rapid decomposition of organic matter. All these dominant acids were sharply increased in the cooling stage, then had a descending trend until the end of composting, which might be the result of microbial secretion associated with solubilizing phosphate (TCP) (Mander et al., 2012; Miller et al., 2010). It was reported that solubilizing rock phosphate by PSB was dependent with higher concentrations of organic acids required to mobilize major quantities of insoluble phosphate into the solution by direct oxidation of phosphates that occurs on the outer face of the cytoplasmic membrane (Wei et al., 2017; Chen et al., 2014; Delvasto et al., 2006). In this study, the average concentration of different organic acids in the five treatments during composting increased in the order: acetic acid in CMP1 (3.0 mg/g) < succinic acid in CMP1 (3.6 mg/g) < oxalic acid in CMP2 (3.8 mg/g) < lactic acid in CMP1 (6.2 mg/g) < citric acid in CMP2 (6.9 mg/g) < formic acid in CMP2 (28.0 mg/g). It was evident that the inoculation of enriched PSB agent increased the production of the six major organic acids compared with CP with only TCP (Fig. 2). Moreover, the content of formic acid in CMP2 on the 23rd day was the highest among the six detected organic acids in all treatments (54.5 mg/g) and its concentration during composting was significantly higher than others, which was positively related to pH (r = 0.507, P < 0.01) and the content of OP (r = 0.926, P < 0.01), Olsen P (r = 0.431, P < 0.01), CAP (r = 0.434, P < 0.01) and MBP (r = 0.563, P < 0.01) (Fig. 3). Besides, a negative correlation between the Olsen P and the content of acetic acid was observed (P < 0.05). MBP was correlated with the content of oxalic acid (r = 0.563, P < 0.01), citric acid (r = 0.886, P < 0.01), and succinic acid (r = 0.418, P < 0.01), but not with lactic acid, suggesting that a part of organic acids is meaningful to enhance the biological fixation of phosphates, i.e., sorption of phosphates into the cellular material of microorganisms (Wei et al., 2017). Therefore, oxalic acid, citric acid, succinic acid, acetic acid and formic acid seem to play more important roles than lactic acid in the improvement of available P fractions during composting, and inoculation of the enriched phosphate-solubilizing agent, especially in the cooling stage, could further improve the production of these key organic acids in KW compost. 3.2. DGGE analysis
3.1.3. Changes in organic acids HPLC analysis showed the presence of multiple organic acids during composting, and six different major organic acids with low molecular weight, i.e., oxalic, lactic, citric, succinic, acetic and formic acids, were detected in all treatments. The change of the total organic acids content in composting may be caused by a range of processes including the decomposition of organic matter, microbial synthesis, and degradation by microorganisms, etc. (Nakasaki et al., 2013; Chen et al., 2006). Concentration of total organic acids increased significantly from day 12 to day 23 (P < 0.01) and reached the maximum at the cooling stage in all treatments during composting, ranging from 45.7 mg/g to 82.7 mg/ g.
The DGGE diagram illustrated the bacterial communities observed through the whole composting process in five treatments (Fig. 4). The bacterial communities during composting, especially in the days when PSB was inoculated, exhibited a significant difference among different treatments, suggesting that phosphate-solubilizing bacterial agent was successfully inoculated into the composts. The Shannon-Wiener index which presented the dynamic of bacteria community were low before the cooling stage, ranging from 1.72 to 2.55 in all groups, then increased until the end of composting. The highest values of ShannonWiener index (i.e. the highest diversity) was presented in CMP1, however, more increase of Shannon-Wiener index in CMP2 was found than other inoculated treatments during composting (P < 0.05), indicating that inoculation seemed more effective to affect bacterial community after the cooling stage. In this study, thirty-one bands were detected in the DGGE profile, and six bands were detected in the PSB inoculant. Twenty-two different dominant 16S rDNA gene fragments were isolated and identified, belonging to the following three domains: Firmicutes (50%), Proteobacteria (41%) and Actinobacteria (9%). Most of the bands in the enriched PSB were classified as Proteobacteria. A part of bands in the PSB inoculant were also present in CK and CP. It might be caused by the source of the PSB inoculants, which were enriched from indigenous microbes during composting. These bands might be ubiquitous in different organic waste composts as the results of Wei et al. (2016a), but have the phosphate-
Table 3 The level of TCP solubilization (STCP) in different treatment groups after composting for 40 days (values are mean ± standard deviation, n = 3). Treatment
CP CMP1 CMP2 CMP3
STCP (%) Olsen P
CAP
2.36 ± 0.19d 3.65 ± 0.14c 13.80 ± 0.74a 9.69 ± 0.81b
2.26 2.54 4.57 3.99
MBP ± ± ± ±
0.14c 0.16c 0.27a 0.32b
1.22 1.36 0.98 1.36
PAP ± ± ± ±
0.08b 0.07a 0.01c 0.09a
5.83 ± 0.49d 7.30 ± 0.58c 19.48 ± 0.80a 14.94 ± 0.82b
Within a column, values labeled with the same letter are not significantly different (P < 0.05).
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Fig. 2. The content of different organic acids produced during composting.
Fig. 3. Pearson correlation matrix between different P fractions, organic acids and physicochemical parameters in different treatments (n = 40). *P < 0.05;
195
**
P < 0.01.
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Fig. 4. The diagram of dynamic changes in bacterial community by DGGE fingerprint during composting in different treatments. M is the Marker made from inoculant.
bacterial populations and Olsen P as well as MBP especially in CMP2, indicating that added TCP was subject to solubilization by more composting microorganisms and inoculation could strengthen the cooperative function related to P transformation among these species during composting especially inoculating in the cooling stage. In addition, the relationship between P-solubilization and organic acids production was probably influenced by the changes of diversity and composition of microorganism communities during composting (Mander et al., 2012; Miller et al., 2010). Little correlations were found between the majority of organic acids and different P fractions (e.g., Olsen P and CAP) in CP, indicating that organic acids production may be not intrinsic to the P-solubilization process during composting, while available P fractions tended to correlate more strongly with six major organic acids in the inoculated groups, either positive or negative. The positively significant correlation between Olsen P, OP and formic acid was obtained in CMP2. The above results indicated that inoculation in different stage could change the mechanism of species and P solubilization by organic acids production, and PSB dependence of formic acid to transform TCP was increased obviously in the cooling stage. Interestingly, both the enriched PSB inoculant in this study or the screened PSB described in Wei et al. (2017) could perfectly adapt the composting conditions, but different inoculants have a better function to transform insoluble phosphate in different inoculation stages, which may result from diverse assemblages of microbial communities may have unknown potential consequences on the capacity of the microbial metabolism (Flores-Renteria et al., 2016). Besides, the variation in the nutrient composition of the substrates and structural (taxonomic) composition of composting microbes may influence the relationship between inoculants and indigenous bacteria (Zhao et al., 2016b). Though the organic acids could be produced from cells to facilitate solubilization of P from mineral phosphates in high amounts by supplying both protons and metal complexing organic acid anions (Yadav et al., 2017), they might also influenced microbial community in turn. Considering that SEM is an a priori approach allowing for an intuitive graphical representation of complex networks of relationships found in ecosystems (Hu et al., 2016; Morrissey and Franklin, 2015), we
solubilizing phenotype under the special environmental pressure with insoluble P addition. Besides, two bands in enriched PSB inoculant only emerged in the inoculated piles, i.e., band 17 and 18. Non-metric multidimensional scaling ordination indicated that the bacterial communities of composting in different treatments could be divided into five groups. Samples in the same day clustered together, indicating that composting process is more crucial that could lead to the changes of bacterial communities, which is consistent with the earlier report of Wei et al. (2017). In the same cluster, the distance between CMP2 and CMP3 was short especially in the end of composting, suggesting that bacterial community composition was similar between CMP2 and CMP3, in contrast, bacterial community of CMP1 was more dispersed among three inoculated groups. The dispersion among three inoculated groups in the same day may be associated with the different microenvironment due to the segregation of organic acids from key PSB.
3.3. The possible mechanism of bacterial community and organic acids production affecting P solubilization and availability Based on the data in CP, CMP1, CMP2 and CMP3, RDA was performed to analyze the interactions between the PSB inoculants, indigenous bacteria in DGGE profile, different P fractions and organic acids (Fig. 5). All of the canonical axes were extremely significant (P < 0.05), demonstrating that these bands may be important in explaining P transformation and organic acids production. The variation of the species-environmental factors relation explained by the first two canonical axes was 40.9%, 45.5%, 47.6% and 43.4% for CP, CMP1, CMP2 and CMP3, respectively. Each species had its own correlation on different organic acids to participate in the transformation of P fractions. As for the bands in PSB inoculant, band 12, 14 and 19 were also indigenous bacteria in CP but correlated slightly with MBP and Olsen P and not with organic acids, meanwhile, there were few indigenous species nearby the arrows of different organic acids, MBP and Olsen P. On the other hand, different treatment based on inoculation methods had obviously variable effects on bacteria community and available P fractions. Positive correlations were found between the majority of 196
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Fig. 5. RDA of the correlation between species, different P fractions and organic acids. Numbers represented the bacteria bands described in Fig. 4. The blue arrows represented indigenous bacteria; purple arrows represented DGGE bands derived from phosphate-solubilizing inoculant; the P fractions were indicated by red arrows and the green arrows meant different organic acids. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Moreover, acetic acid exerted a directly negative impact on bacterial composition but an indirectly positive impact on TCP solubilization in CMP2, suggesting that acetic acid could influence the microbial community by modifying the micro-environmental conditions and in turn strongly influenced the ability of microbial communities to solubilize insoluble phosphate. SEMs further confirm the relationship between the bacteria community, organic acids and P transformation, which allowed us to disentangle the complexity of the direct and indirect effects of the organic acids and roles of microbial communities, especially the enriched PSB inoculant, within the composting system. Therefore, one possible mechanism of TCP solubilization was proposed that the inoculation of enriched PSB agent could improve the production of organic acids, such as formic and acetic acid, to transform P fractions directly, which could obviously increase P availability. Meanwhile, a part of organic acids, e.g., acetic acid, could also act as a regulator of composting microenvironment to modify the microbial diversity and functional redundancy during composting, which had better response on microbial overall P-solubilization functioning in the cooling stage. Further study is needed to evaluate the adaptability and P-solubilization ability of the enriched PSB agent in different composts in composting plant and get
constructed SEMs to further test the casual relationships between different major organic acids, bacterial community composition (data of NMDS), TCP solubilization (data of STCP) and P availability (data of Olsen P) in CP, CMP1, CMP2 and CMP3 (Fig. 6). Squared multiple correlations showed that over 95% of the variance of P availability could be explained by the SEMs. Organic acids, pH and bacterial community affected both TCP solubilization and P availability, but the key organic acids changed during composting in different inoculation groups. Although formic acid had the highest content among all the major organic acids during composting, it exerted indirect influence over Olsen P by driving the changes of bacterial composition in CMP1. The result that citric acid affected TCP solubilization and Olsen P directly could be found in both CP and CMP1, but disappeared in CMP2 and CMP3, showing that the inoculation of enriched PSB in the cooling stage of composting have more effect on the changes of P-solubilizing mechanism. On the other hand, the path analysis in CMP2 supported pH as a driver of both bacterial composition and TCP solubilization had indirect effects on TCP solubilization mediated through bacterial composition, which had bad model fitness for other treatments, suggesting inoculation in the cooling stage may change the potential roles of microbial communities and pH in composting P-solubilizing process. 197
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Fig. 6. Structural equation models of the treatments (CP, CMP1, CMP2 and CMP3), representing hypothesized causal relationships among different major organic acids, bacterial community composition, TCP solubilization and Olsen P. Arrows depict casual relationships: red lines indicate positive effects, and black lines indicate negative effects. Continuous and dashed arrows indicate significant and nonsignificant relationships, respectively. Arrow widths are proportional to r values. The proportion of variance explained for each variable are denoted by r2 values. Significance levels are indicated: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Foundation of China (No. 51378097, No. 51178090, and No. 51778116) as well as the National Key Technology R & D Program (No. 2012BAJ21B02-02).
an increased understanding of its potential applicability at real conditions. 4. Conclusions
Appendix A. Supplementary data Inoculation with enriched PSB agent decreased pH, increased the total acidity, and enhanced the production of oxalic, lactic, citric, succinic, acetic and formic acids during KW composting compared with the control groups. Meanwhile, there was a clear advantage in the solubilization of TCP and P availability for inoculation in the cooling stage and in both initial and cooling stages. Inoculation changed the composition and diversity of bacterial community and strengthened the cooperative function related to P transformation among species during composting. Finally, one possible mechanism of P solubilization induced by bacterial community and organic acids production was proposed.
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2017.09.092. References Chang, C.H., Yang, S.S., 2009. Thermo-tolerant phosphate-solubilizing microbes for multi-functional biofertilizer preparation. Bioresour. Technol. 100, 1648–1658. Chen, Y.P., Rekha, P.D., Arun, A.B., Shen, F.T., Lai, W.A., Young, C.C., 2006. Phosphate solubilizing bacteria from subtropical soil and their tricalcium phosphate solubilizing abilities. Appl. Soil Ecol. 34, 33–41. Chen, Y., Fan, J.B., Du, L., Xu, H., Zhang, Q.H., He, Y.Q., 2014. The application of phosphate solubilizing endophyte Pantoea dispersa triggers the microbial community in red acidic soil. Appl. Soil Ecol. 84, 235–244. Delvasto, P., Valverde, A., Ballester, A., Igual, J.M., Munoz, J.A., Gonzalez, F., Blazquez, M.L., Garcia, C., 2006. Characterization of brushite as a re-crystallization product formed during bacterial solubilization of hydroxyapatite in batch cultures. Soil Biol.
Acknowledgements This work was financially supported by the National Natural Science 198
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bacteria in acid sulfate soils and their beneficial effects on rice growth. PLoS One 9 (10), e97241. Wei, Y., Zhao, Y., Fan, Y., Lu, Q., Li, M., Wei, Q., Zhao, Y., Cao, Z., Wei, Z., 2017. Impact of phosphate-solubilizing bacteria inoculation methods on phosphorus transformation and long-term utilization in composting. Bioresour. Technol. 241, 134–141. Wei, Y., Wei, Z., Cao, Z., Zhao, Y., Zhao, X., Lu, Q., Wang, X., Zhang, X., 2016a. A regulating method for the distribution of phosphorus fractions based on environmental parameters related to the key phosphate-solubilizing bacteria during composting. Bioresour. Technol. 211, 610–617. Wei, Y., Zhao, Y., Wang, H., Lu, Q., Cao, Z., Cui, H., Zhu, L., Wei, Z., 2016b. An optimized regulating method for composting phosphorus fractions transformation based on biochar addition and phosphate-solubilizing bacteria inoculation. Bioresour. Technol. 221, 139–146. Wei, Y.Q., Zhao, Y., Xi, B.D., Wei, Z.M., Li, X., Cao, Z.Y., 2015. Changes in phosphorus fractions during organic wastes composting from different sources. Bioresour. Technol. 189, 349–356. Xi, B., Zhao, X., He, X., Huang, C., Tan, W., Gao, R., Zhang, H., Li, D., 2016. Successions and diversity of humic-reducing microorganisms and their association with physicalchemical parameters during composting. Bioresour. Technol. 219, 204–211. Yadav, H., Fatima, R., Sharma, A., Mathur, S., 2017. Enhancement of applicability of rock phosphate in alkaline soils by organic compost. Appl. Soil Ecol. 113, 80–85. Yang, F., Li, G.X., Yang, Q.Y., Luo, W.H., 2013. Effect of bulking agents on maturity and gaseous emissions during kitchen waste composting. Chemosphere 93, 1393–1399. Zeng, Y., De Guardia, A., Ziebal, C., De Macedo, F.J., Dabert, P., 2013. Impact of biodegradation of organic matters on ammonia oxidation in compost. Bioresour. Technol. 136, 49–57. 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 bacteria during composting. Bioresour. Technol. 221, 276–283. Zhao, X., He, X., Xi, B., Gao, R., Tan, W., Zhang, H., Li, D., 2016a. The evolution of water extractable organic matter and its association with microbial community dynamics during municipal solid waste composting. Waste Manage. 56, 79–87. Zhao, Y., Lu, Q., Wei, Y., Cui, H., Zhang, X., Wang, X., Shan, S., Wei, Z., 2016b. Effect of actinobacteria agent inoculation methods on cellulose degradation during composting based on redundancy analysis. Bioresour. Technol. 219, 196–203. Zhu, H., Sun, L., Zhang, Y., Zhang, X., Qiao, J., 2012. Conversion of spent mushroom substrate to biofertilizer using a stress-tolerant phosphate-solubilizing Pichia farinose FL7. Bioresour. Technol. 111, 410–416.
Biochem. 38, 2645–2654. Flores-Renteria, D., Rincon, A., Valladares, F., Curiel Yuste, J., 2016. Agricultural matrix affects differently the alpha and beta structural and functional diversity of soil microbial communities in a fragmented Mediterranean holm oak forest. Soil Biol. Biochem. 92, 79–90. He, X., Xi, B., Cui, D., Liu, Y., Tan, W., Pan, H., Li, D., 2014. Influence of chemical and structural evolution of dissolved organic matter on electron transfer capacity during composting. J. Hazard. Mater. 268, 256–263. Hu, H., Wang, J., Li, J., Li, J., Ma, Y., Chen, D., He, J., 2016. Field-based evidence for copper contamination induced changes of antibiotic resistance in agricultural soils. Environ. Microbiol. 18, 3896–3909. Huang, G.F., Wu, Q.T., Wong, J.W.C., Nagar, B.B., 2006. Transformation of organic matter during co-composting of pig manure with sawdust. Bioresour. Technol. 97, 1834–1842. Khan, M.S., Zaidi, A., Wani, P.A., 2007. Role of phosphate-solubilizing microorganisms in sustainable agriculture – a review. Agron. Sustain. Dev. 27, 29–43. Liang, Y., Pei, M., Wang, D., Cao, S., Xiao, X., Sun, B., 2017. Improvement of soil ecosystem multifunctionality by dissipating manure-induced antibiotics and resistance genes. Environ. Sci. Technol. 51, 4988–4998. Malik, M.A., Marschner, P., Khan, K.S., 2012. Addition of organic and inorganic P sources to soil – effects on P pools and microorganisms. Soil Biol. Biochem. 49, 106–113. Mander, C., Wakelin, S., Young, S., Condron, L., O'Callaghan, M., 2012. Incidence and diversity of phosphate-solubilising bacteria are linked to phosphorus status in grassland soils. Soil Biol. Biochem. 44, 93–101. Miller, S.H., Browne, P., Prigent-Combaret, C., Combes-Meynet, E., Morrissey, J.P., O'Gara, F., 2010. Biochemical and genomic comparison of inorganic phosphate solubilization in Pseudomonas species. Env. Microbiol. Rep. 2, 403–411. Morrissey, E., Franklin, R., 2015. Resource effects on denitrification are mediated by community composition in tidal freshwater wetlands soils. Environ. Microbiol. 17, 1520–1532. Nakasaki, K., Araya, S., Mimoto, H., 2013. Inoculation of Pichia kudriavzevii RB1 degrades the organic acids present in raw compost material and accelerates composting. Bioresour. Technol. 144, 521–528. Onireti, O.O., Lin, C., Qin, J., 2017. Combined effects of low-molecular-weight organic acids on mobilization of arsenic and lead from multi-contaminated soils. Chemosphere 170, 161–168. Panhwar, Q.A., Naher, U.A., Shamshuddin, J., Othman, R., Latif, M.A., Ismail, M.R., 2014. Biochemical and molecular characterization of potential phosphate-solubilizing
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Effect of organic acids production and bacterial community on the possible mechanism of phosphorus solubilization during composting with enriched phosphate-solubilizing bacteria inoculation
MARK
Yuquan Weia,1, Yue Zhaoa,1, Mingzi Shia, Zhenyu Caoa, Qian Lua, Tianxue Yangb, Yuying Fana, ⁎ Zimin Weia, a b
College of Life Science, Northeast Agricultural University, Harbin 150030, China Laboratory of Water Environmental System Engineering, Chinese Research Academy of Environmental Science, Beijing 100012, China
G RA P H I C A L AB S T R A C T
A R T I C L E I N F O
A B S T R A C T
Keywords: Phosphate-solubilizing bacteria (PSB) Composting Inoculation Organic acids Tricalcium phosphate solubilization
Enriched phosphate-solubilizing bacteria (PSB) agent were acquired by domesticated cultivation, and inoculated into kitchen waste composting in different stages. The effect of different treatments on organic acids production, tricalcium phosphate (TCP) solubilization and their relationship with bacterial community were investigated during composting. Our results pointed out that inoculation affected pH, total acidity and the production of oxalic, lactic, citric, succinic, acetic and formic acids. We also found a strong advantage in the solubilization of TCP and phosphorus (P) availability for PSB inoculation especially in the cooling stage. Redundancy analysis and structural equation models demonstrated inoculation by different methods changed the correlation of the bacterial community composition with P fractions as well as organic acids, and strengthened the cooperative function related to P transformation among species during composting. Finally, we proposed a possible mechanism of P solubilization with enriched PSB inoculation, which was induced by bacterial community and organic acids production.
⁎
1
Corresponding author. E-mail addresses: [email protected], [email protected] (Z. Wei). Authors contributed equally to this work.
http://dx.doi.org/10.1016/j.biortech.2017.09.092 Received 5 August 2017; Received in revised form 12 September 2017; Accepted 15 September 2017 Available online 19 September 2017 0960-8524/ © 2017 Elsevier Ltd. All rights reserved.
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1. Introduction
2016b; Chen et al., 2006), this study was essentially focused to the effect of bacterial community and different organic acids on TCP-solubilization during KW composting. Besides, different stages for inoculation were employed. Direct multivariate analyses, such as redundancy analysis (RDA), could be used as a preliminary estimate for the correlation between changes in microbial community and environmental variables, and structural equation modeling (SEM) could further construct complex relationships among composting biotic and abiotic factors (Liang et al., 2017; Flores-Renteria et al., 2016; Wei et al., 2016a). Nevertheless, there is little consideration whether changes of bacterial community and organic acids production could be associated with P-solubilization during composting by these relationship model. Our intent was to evaluate our hypothesis by RDA and SEM to link bacterial community, organic acids production and TCP-solubilization in different inoculation treatments, which may provide a better understanding of the P-solubilization process of phosphate-solubilizing inoculant during composting and lead to new perspectives on strategies to improve P availability of composts with precipitated inorganic P.
Although the total amount of phosphorus (P) is high in many lands, P deficiency is a constraint to plant growth worldwide due to the low availability in soil (Wei et al., 2016b). Therefore, P-fertilizers is frequently applied, particularly in conventional intensive agricultural soils to overcome P-limitation. Unfortunately, most of chemical P-fertilizers can be immobilized by Ca2+, Fe3+ and Al3+, which lead to the rapid formation of poorly available P for plants after applied (Malik et al., 2012). Given that demand for improving the availability of P-fertilizers continues to increase and phosphate-solubilizing bacteria (PSB) has a key role in the mobilization and transformation of soil P, more and more attention was paid on the understanding of the mechanisms for solubilizing of mineral phosphates and how to improve the efficiency of PSB application. Many studies have shown that PSB can transform the insoluble P to soluble forms by acidification, chelation, exchange reactions, and polymeric substances formation, which may be related to the production of organic acids, the release of protons (H+), the redox-active metals, etc. (Delvasto et al., 2006). Among them, the mechanism that phosphate solubilizing ability of PSB is associated with the release of low molecular weight organic acids is generally accepted. Low molecular weight organic acids may play an important role the detoxification of pollutants, the bioavailability of heavy metals and even microbial activity in soil conditions (Onireti et al., 2017). On the other hand, these organic acids (e.g., oxalic acid, citric acid, acetic acid, succinic acid, etc.) could also lower pH, chelate the cations (mainly calcium) bound to phosphate through their hydroxyl and carboxyl groups or compete with phosphate for adsorption sites, leading to the increased solubility and availability of mineral phosphates (Mander et al., 2012; Khan et al., 2007). However, the predominant organic acids produced by different phosphate-solubilizing microorganisms were diverse distinctly (Panhwar et al., 2014; Nakasaki et al., 2013). Moreover, the process of P-solubilization is a complex phenomenon, which are severely affected by many factors. The impacts of environmental factors, such as temperature, pH, oxygen concentration, moisture, etc. on microbial P-solubilizing capability have been widely investigated (Zhu et al., 2012; Chen et al., 2006), but there is no studies specifically on the influence of the interaction of microbial communities and PSB on solubilizing insoluble phosphate and production of organic acids. Composting is a controlled biological decomposition process of organic matter, accompanying with a dynamic activity of various microbial populations (Xi et al., 2016; Zhao et al., 2016a; He et al., 2014). Given that composting can affect the distribution of P fractions, a large number of studies has been oriented to the addition of mineral phosphates with poor availability and inoculation of phosphate solubilizing microbes during composting to develop P-enriched compost (Wei et al., 2016b; Chang and Yang, 2009). Many microorganisms have been utilized as solubilizing insoluble P inoculants in composts. However, considering that the inoculation during composting may have different performances due to the influence of the source of microbial inoculum, the scale of composting, and the stage of inoculation etc. (Zhao et al., 2016b), we hypothesized that: (1) compared to uninoculated compost, changes in the time of inoculation of phosphate solubilizing microbes during composting may cause different biological microenvironment and bacterial community structure due to the competition or collaborative symbiosis between inoculants and indigenous bacteria, (2) compared to uninoculated compost, inoculation of PSB in different stages will stimulate microbial activity related to organic acids production more and therefore lead to more solubilization of precipitated inorganic P, and (3) different bacterial community relationship may change the type and amount of organic acid involved in the P-solubilization process. Given that tricalcium phosphate (TCP) is mainly used as a model material as they represent the vast majority of precipitated inorganic P salts and kitchen waste (KW) has a relatively low P content (Wei et al.,
2. Materials and methods 2.1. Preparation of phosphate-solubilizing inoculant The enriched PSB was obtained from different solid waste composting samples as described by Wei et al. (2016a). The enrichment process was similar to the method described as Zhang et al. (2016), but the enriched medium was sterile National Botanical Research Institute's phosphate growth medium (NBRIP) broth (Wei et al., 2016a). The liquids with enriched PSB was cultivated for 3 days at 40 °C until the population up to 108 CFU mL−1 to measure the phosphate-solubilizing activity in broth culture including water soluble phosphorus (WSP) and microbial biomass phosphorus (MBP) according to Panhwar et al. (2014), and pH of the medium was recorded with a pH meter. The phosphate-solubilizing content of the PSB agent increased after the process of domestication culture for 10 cycles, and the 10th cycle of phosphate-solubilizing inoculant with high P-solubilization capacity was centrifuged and suspended in sterile water, then sprayed on the composting mixture. The concentrations of the strain were 1 × 108 CFU mL−1. 2.2. Composting experiment design KW was prepared as the main material, which was collected from the cafeteria in Northeast Agricultural University (Harbin, China), mainly containing the cooked food residues, such as steamed rice, noodles, steamed bread, and cooked vegetable, meat, fish. The indigestible materials, such as plastics, bones, and egg shell, were selected before the KW were crushed and homogenized. Sawdust was added to adjust the C/N radio of 25, which was obtained from a timber mill in Harbin, China. Details of raw materials were described in Table 1. The initial moisture was adjusted to 60%. Five lab-scale composting experiments were carried out for 40 days in the special compost reactor (the working volume was 12.5 L) as described by Zhao et al. (2016b) and various treatments with three replicates each were applied as shown in Table 2. Considering the phosphate-solubilizing inoculant was enriched at the temperature below 50 °C, it was not inoculated at hightemperature stage of composting. The KW with a relatively low P content was chosen as the major raw material and therefore allowed the impact of enriched phosphate-solubilizing inoculant with different stages inoculation on the solubilization of TCP in composting to be readily observed. During composting, moisture was maintained at 50–60% and the ventilation rate was 0.5 L min−1 kg−1 in order to maintain oxygen concentration inside the composts above 10%. Composting materials were turned over manually every four days to ensure homogenized. Representative compost samples of each treatment were 191
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Table 1 The physical and chemical properties of raw materials for composting. Materials
pH
TOC (%)
TN (%)
C/N
TP (g kg−1)
Bulk density (g cm3)
KW Sawdust Mixed materials
4.99 ± 0.40 7.02 ± 0.26 5.40 ± 0.33
57.42 ± 2.45 43.29 ± 2.25 54.15 ± 2.31
3.36 ± 0.09 0.91 ± 0.03 2.37 ± 0.08
17.08 ± 0.88 47.25 ± 1.94 24.59 ± 1.05
8.62 ± 0.27 0.95 ± 0.04 7.84 ± 0.18
0.51 ± 0.02 0.20 ± 0.01 0.45 ± 0.01
TOC: total organic carbon; TN: total nitrogen; TP: total phosphorus.
2.4. DNA extraction and PCR-DGGE
Table 2 Different treatments of composting. Treatment
CK CP CMP1 CMP2 CMP3
Basic Materials in composts KW
Sawdust
+ + + + +
+ + + + +
TCP (w/ w)
– 2.5 2.5 2.5 2.5
DNA kit (Omega Biotek, Inc.) was used to extract the total DNA of compost samples. Touch-down PCR was performed with the templates of the extracted DNA and the specific bacterial primers of 534r and 357f with a GC-clamp. The system setting and method of polymerase chain reaction referenced to Wei et al. (2016b). Each PCR concoction was set in a polyacrylamide (8%) gels at a 35–60% denaturant gradient. A Gene Mutation Detection System (Bio-Rad Laboratories, Inc.) was run at 150 V for 4 h at 60 °C to separate the fragments. After electrophoresis, gels were stained in 3 × GelRed™ nuclear acid gel stain (Biotium, USA) and photographed with a UVP Imaging System (UVP Inc., USA). This procedure was performed in triplicate for each sample with different gels. Representatives of bands that were clear and had high intensity were excised from DGGE gels and transferred to 30-μL Milli-Q water (Millipore, USA), incubated overnight for elution of DNA at 4 °C. Then, PCR was performed to re-amplify the DNA using primers 534r and 357f without a GC-clamp. Finally, the sequences were analyzed, and the PCR fragments were identified according to Wei et al. (2016a). After sequencing, the results were compared with the GenBank from the National Center of Biotechnology Information (NCBI) using BLAST.
Composite inoculum (v/w)
Initial stage (Day 0)
Cooling stage (Day 12)
– – 5 – 2.5
– – – 5 2.5
taken on days 0, 3, 7, 12, 17, 23, 30 and 40 following the initiation of the composting process. Approximately 200 g of each compost were collected each time. One part of experimental samples was immediately stored at −20 °C for DNA extraction and others were air-dried, passed through a 0.25 mm sieve, and stored in a desiccator for physical-chemical analysis.
2.3. Analysis of physicochemical indexes, P fractions and organic acids 2.5. Statistical analysis Temperature was monitored with a digital thermometer on a daily basis and pH was measured using a digital pH meter as described by Wei et al. (2016b). Total acidity was measured according to the methods described by Huang et al. (2006). The determination of total phosphorus (TP), inorganic phosphorus (IP), organic phosphorus (OP), citric acid phosphorus (CAP), Olsen P and MBP were determined colorimetrically following the procedure of Wei et al. (2015). Potential available P (PAP) during composting was estimated as: ∑Olsen P + CAP + MBP in this study. The level of TCP solubilization (STCP) was estimated as the ratio of ΔAP to the content of added TCP, where available P fractions (AP) could be estimated as Olsen P, CAP, MBP or PAP and ΔAP was calculated as the increased amount of P fraction in day40 relative to day0. Organic acids in composting samples were extracted according to the method described by Nakasaki et al. (2013). The amount and composition of organic acids were analyzed by high performance liquid chromatography (HPLC). To prepare the liquid samples, the compost samples (3 g) were extracted using 10 ml 0.1% phosphoric acid at 4 °C for 10 min, and the mixtures were centrifuged at 12,000g for 10 min, then the supernatants were filtered through 0.20-μm PTFE syringe filters. The resulting filtrates were used to measure organic acids using an HPLC. The HPLC quantitative analysis was carried out on Agilent 1260, fitted with Agilent Zorbax Eclipse plus C18 reverse-phase column (250 mm × 4.6 mm). Organic acids were detected with a UV diodearray detector (Agilent 1260) set at 210 nm. The mobile phase was 10 mM diammonium hydrogen phosphate in Milli-Q water and 1 M phosphoric acid was applied to adjust the pH to 2.7 at a flow rate of 1 ml/min. The column temperature was 30 °C. Peaks were identified against a set of standard peaks obtained from nine known organic acids, i.e., succinic, citric, acetic, oxalic, formic, lactic, tartaric, malic, and propionic acids. All experiments were repeated three times.
All data obtained were subjected to statistical analysis using Origin8.0 and SPSS19.0. Analyses of variance (ANOVA) and multiple comparison tests (LSD All-Pairwise Comparisons Test) were performed to compare mean values for the different factors and variables analyzed (P < 0.05) using Statistic 8. DGGE banding profiles, including band numbers and relative intensity (within lane), were digitized using Quantity one software (version 4.5, Bio-Rad laboratories, USA). Shannon-Wiener index was determined as described by Wei et al. (2016a). Nonmetric multidimensional scaling ordination (NMDS) was used to identify differences in bacterial communities between different treatments based on the relative abundance of DGGE bands. The relationships between different organic acids and P fractions as well as microbes in composting were analyzed using the RDA from Canoco for Windows (Version 5.0). The “robust” maximum likelihood evaluation program of AMOS 7.0 software was used to analyze the model of SEM. Prior to SEM analysis, we examined the normal distributions of data for heteroscedasticity as well as all bivariate relationships for signs of nonlinearities. The model fit was evaluated using a chi-square (χ2) test where a non-significant P-value indicates a good fit of the model to the data (Morrissey and Franklin, 2015). The reduced path model was confirmed with stepwise multiple regression analysis. SEM models were individually performed for each composting monitoring physicochemical parameters, bacterial indicator (NMDS axis), organic acids and P fractions, but only the best fitted ones are presented. Data were reported as the means of triplicates. 3. Results and discussion 3.1. Evolution of process monitoring parameters 3.1.1. Characteristics of temperature, pH and total acidity Changes in temperature and pH are crucial to describe the microbial 192
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Fig. 1. Dynamic of (a) temperature, (b) pH, (c) total acidity, (d) TP, (e) Olsen P, (f) CAP, and (g) MBP during composting.
be observed from day 0 day to day 16, followed by a decrease but later than CP. This fact highlights the intense microbial acid-producing activity of phosphate-solubilizing inoculant, which may accelerate the solubilization of insoluble phosphate TCP during composting.
activity related to P transformation during the composting process (Wei et al., 2016a). In general, temperature in all treatments followed the typical evolution of composting processes (Fig. 1a). After an initial heating period of 3–4 days, the temperature quickly rose to 50–60 °C due to the degradation of abundant low molecular weight substances (e.g., organic matter, protein materials, sugars, etc.) in KW by microbial activity. As it was expected, a thermal activation was promoted after PSB inoculations. The pH values of the five treatments evolved in the range from 4.86 to 7.92 during composting (Fig. 1b), which declined slowly from the initial stage to day 7–12 and then showed an increased trend. The lower pH in CMP1, CMP2 and CMP3 compared to CP might be attributed to organic acids produced by strengthened microbial reactions accompanied with the degradation of organic matter (Yang et al., 2013). The values of total acidity ranged from 1.2 mmol/g to 6.9 mmol/g during composting and showed a decreased trend (Fig. 1c). A noteworthy increase of total acidity in three inoculated groups could
3.1.2. Variation in P solubilization and availability The content of TP gradually increased during composting (Fig. 1d). Initially, TP content was nearly 22.0 g/kg in four TCP-addition groups and 7.0 g/kg in CK. Composting process led to 45% increase of TP in CK above their initial values, while only led to 24% increase of TP in CP. This may be due to the fact that the high TCP addition significantly reduced the degradation of organic compounds by inhibiting the microbial growth and enzymatic activity during composting (Zeng et al., 2013; Malik et al., 2012). On the other hand, TP content of CMP2 and CMP3 on the 40th day were all significantly higher than that in CP and CMP1 (P < 0.05), suggesting that PSB inoculation in cooling stage 193
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could be more beneficial for the “concentration effect’’ than that in the beginning of composting. The content of IP and OP increased during composting, and IP was the major component of TP in all the groups (63–94%), which was the result of the addition of TCP with a high IP content. The OP content in CMP2 and CMP3 increased significantly after the 16th day relative to that in CK, CP and CMP1 (P < 0.05), which indicated that PSB inoculation in the cooling stage was suitable to accelerate effectively the TCP transformation to OP during composting. Fig. 1(e–g) illustrates the contents of Olsen P, CAP and MBP during composting in different treatments. Overall, the Olsen P, CAP and MBP in the end of composting were significantly higher than the values at the beginning of composting (P < 0.05). Olsen P and CAP content of four TCP-addition groups were all significantly higher than that in CK at the beginning of composting (P < 0.05), whereas the content of MBP was similar in all groups. The content of CAP had an obvious increase during composting, especially in CMP2 and CMP3, which might be related to the suitable condition after the thermophilic phase of composting for the acid-producing activity of PSB inoculant to solubilize TCP. The values of Olsen P decreased rapidly in the initial stage and the thermophilic phase. Thereafter, a large increase was observed in the cooling stage in four TCP-addition treatments, the most significant increase being found in CMP2 and CMP3. In contrast, there is a large increase in the concentration of MBP before the 7th day of composting, and a gradual decrease after the 23rd day of composting for all treatments. Combined with the results of OP, the change of CAP may be associated with the synthesis of OP with a corresponding decrease in MBP, suggesting that microbes might be important mediators in the transformation of P forms. A post hoc test for the all composts showed that there was a significant difference in the level of STCP between CP, CMP1, CMP2 and CMP3 (Table 3). The higher STCP of PAP in CMP2 (19.5%) and CMP3 (14.9%) compared to CP (5.8%) and CMP1 (7.3%) suggested that PSB inoculation in the cooling stage had a clear advantage to enhance the solubilization and availability of TCP. Moreover, compared to the results of P availability in the earlier report of Wei et al. (2016b), the enriched phosphate-solubilizing inoculant could have better adaptability to KW composting environments than the PSB screened from different composting samples when directly applied into KW.
The concentration of major organic acids presented varied changes during composting in five treatments (Fig. 2). The concentration of all the detected organic acids except succinic acid were increased in the early 3 days of composting, which may be caused by the rapid decomposition of organic matter. All these dominant acids were sharply increased in the cooling stage, then had a descending trend until the end of composting, which might be the result of microbial secretion associated with solubilizing phosphate (TCP) (Mander et al., 2012; Miller et al., 2010). It was reported that solubilizing rock phosphate by PSB was dependent with higher concentrations of organic acids required to mobilize major quantities of insoluble phosphate into the solution by direct oxidation of phosphates that occurs on the outer face of the cytoplasmic membrane (Wei et al., 2017; Chen et al., 2014; Delvasto et al., 2006). In this study, the average concentration of different organic acids in the five treatments during composting increased in the order: acetic acid in CMP1 (3.0 mg/g) < succinic acid in CMP1 (3.6 mg/g) < oxalic acid in CMP2 (3.8 mg/g) < lactic acid in CMP1 (6.2 mg/g) < citric acid in CMP2 (6.9 mg/g) < formic acid in CMP2 (28.0 mg/g). It was evident that the inoculation of enriched PSB agent increased the production of the six major organic acids compared with CP with only TCP (Fig. 2). Moreover, the content of formic acid in CMP2 on the 23rd day was the highest among the six detected organic acids in all treatments (54.5 mg/g) and its concentration during composting was significantly higher than others, which was positively related to pH (r = 0.507, P < 0.01) and the content of OP (r = 0.926, P < 0.01), Olsen P (r = 0.431, P < 0.01), CAP (r = 0.434, P < 0.01) and MBP (r = 0.563, P < 0.01) (Fig. 3). Besides, a negative correlation between the Olsen P and the content of acetic acid was observed (P < 0.05). MBP was correlated with the content of oxalic acid (r = 0.563, P < 0.01), citric acid (r = 0.886, P < 0.01), and succinic acid (r = 0.418, P < 0.01), but not with lactic acid, suggesting that a part of organic acids is meaningful to enhance the biological fixation of phosphates, i.e., sorption of phosphates into the cellular material of microorganisms (Wei et al., 2017). Therefore, oxalic acid, citric acid, succinic acid, acetic acid and formic acid seem to play more important roles than lactic acid in the improvement of available P fractions during composting, and inoculation of the enriched phosphate-solubilizing agent, especially in the cooling stage, could further improve the production of these key organic acids in KW compost. 3.2. DGGE analysis
3.1.3. Changes in organic acids HPLC analysis showed the presence of multiple organic acids during composting, and six different major organic acids with low molecular weight, i.e., oxalic, lactic, citric, succinic, acetic and formic acids, were detected in all treatments. The change of the total organic acids content in composting may be caused by a range of processes including the decomposition of organic matter, microbial synthesis, and degradation by microorganisms, etc. (Nakasaki et al., 2013; Chen et al., 2006). Concentration of total organic acids increased significantly from day 12 to day 23 (P < 0.01) and reached the maximum at the cooling stage in all treatments during composting, ranging from 45.7 mg/g to 82.7 mg/ g.
The DGGE diagram illustrated the bacterial communities observed through the whole composting process in five treatments (Fig. 4). The bacterial communities during composting, especially in the days when PSB was inoculated, exhibited a significant difference among different treatments, suggesting that phosphate-solubilizing bacterial agent was successfully inoculated into the composts. The Shannon-Wiener index which presented the dynamic of bacteria community were low before the cooling stage, ranging from 1.72 to 2.55 in all groups, then increased until the end of composting. The highest values of ShannonWiener index (i.e. the highest diversity) was presented in CMP1, however, more increase of Shannon-Wiener index in CMP2 was found than other inoculated treatments during composting (P < 0.05), indicating that inoculation seemed more effective to affect bacterial community after the cooling stage. In this study, thirty-one bands were detected in the DGGE profile, and six bands were detected in the PSB inoculant. Twenty-two different dominant 16S rDNA gene fragments were isolated and identified, belonging to the following three domains: Firmicutes (50%), Proteobacteria (41%) and Actinobacteria (9%). Most of the bands in the enriched PSB were classified as Proteobacteria. A part of bands in the PSB inoculant were also present in CK and CP. It might be caused by the source of the PSB inoculants, which were enriched from indigenous microbes during composting. These bands might be ubiquitous in different organic waste composts as the results of Wei et al. (2016a), but have the phosphate-
Table 3 The level of TCP solubilization (STCP) in different treatment groups after composting for 40 days (values are mean ± standard deviation, n = 3). Treatment
CP CMP1 CMP2 CMP3
STCP (%) Olsen P
CAP
2.36 ± 0.19d 3.65 ± 0.14c 13.80 ± 0.74a 9.69 ± 0.81b
2.26 2.54 4.57 3.99
MBP ± ± ± ±
0.14c 0.16c 0.27a 0.32b
1.22 1.36 0.98 1.36
PAP ± ± ± ±
0.08b 0.07a 0.01c 0.09a
5.83 ± 0.49d 7.30 ± 0.58c 19.48 ± 0.80a 14.94 ± 0.82b
Within a column, values labeled with the same letter are not significantly different (P < 0.05).
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Fig. 2. The content of different organic acids produced during composting.
Fig. 3. Pearson correlation matrix between different P fractions, organic acids and physicochemical parameters in different treatments (n = 40). *P < 0.05;
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**
P < 0.01.
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Fig. 4. The diagram of dynamic changes in bacterial community by DGGE fingerprint during composting in different treatments. M is the Marker made from inoculant.
bacterial populations and Olsen P as well as MBP especially in CMP2, indicating that added TCP was subject to solubilization by more composting microorganisms and inoculation could strengthen the cooperative function related to P transformation among these species during composting especially inoculating in the cooling stage. In addition, the relationship between P-solubilization and organic acids production was probably influenced by the changes of diversity and composition of microorganism communities during composting (Mander et al., 2012; Miller et al., 2010). Little correlations were found between the majority of organic acids and different P fractions (e.g., Olsen P and CAP) in CP, indicating that organic acids production may be not intrinsic to the P-solubilization process during composting, while available P fractions tended to correlate more strongly with six major organic acids in the inoculated groups, either positive or negative. The positively significant correlation between Olsen P, OP and formic acid was obtained in CMP2. The above results indicated that inoculation in different stage could change the mechanism of species and P solubilization by organic acids production, and PSB dependence of formic acid to transform TCP was increased obviously in the cooling stage. Interestingly, both the enriched PSB inoculant in this study or the screened PSB described in Wei et al. (2017) could perfectly adapt the composting conditions, but different inoculants have a better function to transform insoluble phosphate in different inoculation stages, which may result from diverse assemblages of microbial communities may have unknown potential consequences on the capacity of the microbial metabolism (Flores-Renteria et al., 2016). Besides, the variation in the nutrient composition of the substrates and structural (taxonomic) composition of composting microbes may influence the relationship between inoculants and indigenous bacteria (Zhao et al., 2016b). Though the organic acids could be produced from cells to facilitate solubilization of P from mineral phosphates in high amounts by supplying both protons and metal complexing organic acid anions (Yadav et al., 2017), they might also influenced microbial community in turn. Considering that SEM is an a priori approach allowing for an intuitive graphical representation of complex networks of relationships found in ecosystems (Hu et al., 2016; Morrissey and Franklin, 2015), we
solubilizing phenotype under the special environmental pressure with insoluble P addition. Besides, two bands in enriched PSB inoculant only emerged in the inoculated piles, i.e., band 17 and 18. Non-metric multidimensional scaling ordination indicated that the bacterial communities of composting in different treatments could be divided into five groups. Samples in the same day clustered together, indicating that composting process is more crucial that could lead to the changes of bacterial communities, which is consistent with the earlier report of Wei et al. (2017). In the same cluster, the distance between CMP2 and CMP3 was short especially in the end of composting, suggesting that bacterial community composition was similar between CMP2 and CMP3, in contrast, bacterial community of CMP1 was more dispersed among three inoculated groups. The dispersion among three inoculated groups in the same day may be associated with the different microenvironment due to the segregation of organic acids from key PSB.
3.3. The possible mechanism of bacterial community and organic acids production affecting P solubilization and availability Based on the data in CP, CMP1, CMP2 and CMP3, RDA was performed to analyze the interactions between the PSB inoculants, indigenous bacteria in DGGE profile, different P fractions and organic acids (Fig. 5). All of the canonical axes were extremely significant (P < 0.05), demonstrating that these bands may be important in explaining P transformation and organic acids production. The variation of the species-environmental factors relation explained by the first two canonical axes was 40.9%, 45.5%, 47.6% and 43.4% for CP, CMP1, CMP2 and CMP3, respectively. Each species had its own correlation on different organic acids to participate in the transformation of P fractions. As for the bands in PSB inoculant, band 12, 14 and 19 were also indigenous bacteria in CP but correlated slightly with MBP and Olsen P and not with organic acids, meanwhile, there were few indigenous species nearby the arrows of different organic acids, MBP and Olsen P. On the other hand, different treatment based on inoculation methods had obviously variable effects on bacteria community and available P fractions. Positive correlations were found between the majority of 196
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Fig. 5. RDA of the correlation between species, different P fractions and organic acids. Numbers represented the bacteria bands described in Fig. 4. The blue arrows represented indigenous bacteria; purple arrows represented DGGE bands derived from phosphate-solubilizing inoculant; the P fractions were indicated by red arrows and the green arrows meant different organic acids. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Moreover, acetic acid exerted a directly negative impact on bacterial composition but an indirectly positive impact on TCP solubilization in CMP2, suggesting that acetic acid could influence the microbial community by modifying the micro-environmental conditions and in turn strongly influenced the ability of microbial communities to solubilize insoluble phosphate. SEMs further confirm the relationship between the bacteria community, organic acids and P transformation, which allowed us to disentangle the complexity of the direct and indirect effects of the organic acids and roles of microbial communities, especially the enriched PSB inoculant, within the composting system. Therefore, one possible mechanism of TCP solubilization was proposed that the inoculation of enriched PSB agent could improve the production of organic acids, such as formic and acetic acid, to transform P fractions directly, which could obviously increase P availability. Meanwhile, a part of organic acids, e.g., acetic acid, could also act as a regulator of composting microenvironment to modify the microbial diversity and functional redundancy during composting, which had better response on microbial overall P-solubilization functioning in the cooling stage. Further study is needed to evaluate the adaptability and P-solubilization ability of the enriched PSB agent in different composts in composting plant and get
constructed SEMs to further test the casual relationships between different major organic acids, bacterial community composition (data of NMDS), TCP solubilization (data of STCP) and P availability (data of Olsen P) in CP, CMP1, CMP2 and CMP3 (Fig. 6). Squared multiple correlations showed that over 95% of the variance of P availability could be explained by the SEMs. Organic acids, pH and bacterial community affected both TCP solubilization and P availability, but the key organic acids changed during composting in different inoculation groups. Although formic acid had the highest content among all the major organic acids during composting, it exerted indirect influence over Olsen P by driving the changes of bacterial composition in CMP1. The result that citric acid affected TCP solubilization and Olsen P directly could be found in both CP and CMP1, but disappeared in CMP2 and CMP3, showing that the inoculation of enriched PSB in the cooling stage of composting have more effect on the changes of P-solubilizing mechanism. On the other hand, the path analysis in CMP2 supported pH as a driver of both bacterial composition and TCP solubilization had indirect effects on TCP solubilization mediated through bacterial composition, which had bad model fitness for other treatments, suggesting inoculation in the cooling stage may change the potential roles of microbial communities and pH in composting P-solubilizing process. 197
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Fig. 6. Structural equation models of the treatments (CP, CMP1, CMP2 and CMP3), representing hypothesized causal relationships among different major organic acids, bacterial community composition, TCP solubilization and Olsen P. Arrows depict casual relationships: red lines indicate positive effects, and black lines indicate negative effects. Continuous and dashed arrows indicate significant and nonsignificant relationships, respectively. Arrow widths are proportional to r values. The proportion of variance explained for each variable are denoted by r2 values. Significance levels are indicated: ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Foundation of China (No. 51378097, No. 51178090, and No. 51778116) as well as the National Key Technology R & D Program (No. 2012BAJ21B02-02).
an increased understanding of its potential applicability at real conditions. 4. Conclusions
Appendix A. Supplementary data Inoculation with enriched PSB agent decreased pH, increased the total acidity, and enhanced the production of oxalic, lactic, citric, succinic, acetic and formic acids during KW composting compared with the control groups. Meanwhile, there was a clear advantage in the solubilization of TCP and P availability for inoculation in the cooling stage and in both initial and cooling stages. Inoculation changed the composition and diversity of bacterial community and strengthened the cooperative function related to P transformation among species during composting. Finally, one possible mechanism of P solubilization induced by bacterial community and organic acids production was proposed.
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2017.09.092. References Chang, C.H., Yang, S.S., 2009. Thermo-tolerant phosphate-solubilizing microbes for multi-functional biofertilizer preparation. Bioresour. Technol. 100, 1648–1658. Chen, Y.P., Rekha, P.D., Arun, A.B., Shen, F.T., Lai, W.A., Young, C.C., 2006. Phosphate solubilizing bacteria from subtropical soil and their tricalcium phosphate solubilizing abilities. Appl. Soil Ecol. 34, 33–41. Chen, Y., Fan, J.B., Du, L., Xu, H., Zhang, Q.H., He, Y.Q., 2014. The application of phosphate solubilizing endophyte Pantoea dispersa triggers the microbial community in red acidic soil. Appl. Soil Ecol. 84, 235–244. Delvasto, P., Valverde, A., Ballester, A., Igual, J.M., Munoz, J.A., Gonzalez, F., Blazquez, M.L., Garcia, C., 2006. Characterization of brushite as a re-crystallization product formed during bacterial solubilization of hydroxyapatite in batch cultures. Soil Biol.
Acknowledgements This work was financially supported by the National Natural Science 198
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bacteria in acid sulfate soils and their beneficial effects on rice growth. PLoS One 9 (10), e97241. Wei, Y., Zhao, Y., Fan, Y., Lu, Q., Li, M., Wei, Q., Zhao, Y., Cao, Z., Wei, Z., 2017. Impact of phosphate-solubilizing bacteria inoculation methods on phosphorus transformation and long-term utilization in composting. Bioresour. Technol. 241, 134–141. Wei, Y., Wei, Z., Cao, Z., Zhao, Y., Zhao, X., Lu, Q., Wang, X., Zhang, X., 2016a. A regulating method for the distribution of phosphorus fractions based on environmental parameters related to the key phosphate-solubilizing bacteria during composting. Bioresour. Technol. 211, 610–617. Wei, Y., Zhao, Y., Wang, H., Lu, Q., Cao, Z., Cui, H., Zhu, L., Wei, Z., 2016b. An optimized regulating method for composting phosphorus fractions transformation based on biochar addition and phosphate-solubilizing bacteria inoculation. Bioresour. Technol. 221, 139–146. Wei, Y.Q., Zhao, Y., Xi, B.D., Wei, Z.M., Li, X., Cao, Z.Y., 2015. Changes in phosphorus fractions during organic wastes composting from different sources. Bioresour. Technol. 189, 349–356. Xi, B., Zhao, X., He, X., Huang, C., Tan, W., Gao, R., Zhang, H., Li, D., 2016. Successions and diversity of humic-reducing microorganisms and their association with physicalchemical parameters during composting. Bioresour. Technol. 219, 204–211. Yadav, H., Fatima, R., Sharma, A., Mathur, S., 2017. Enhancement of applicability of rock phosphate in alkaline soils by organic compost. Appl. Soil Ecol. 113, 80–85. Yang, F., Li, G.X., Yang, Q.Y., Luo, W.H., 2013. Effect of bulking agents on maturity and gaseous emissions during kitchen waste composting. Chemosphere 93, 1393–1399. Zeng, Y., De Guardia, A., Ziebal, C., De Macedo, F.J., Dabert, P., 2013. Impact of biodegradation of organic matters on ammonia oxidation in compost. Bioresour. Technol. 136, 49–57. 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 bacteria during composting. Bioresour. Technol. 221, 276–283. Zhao, X., He, X., Xi, B., Gao, R., Tan, W., Zhang, H., Li, D., 2016a. The evolution of water extractable organic matter and its association with microbial community dynamics during municipal solid waste composting. Waste Manage. 56, 79–87. Zhao, Y., Lu, Q., Wei, Y., Cui, H., Zhang, X., Wang, X., Shan, S., Wei, Z., 2016b. Effect of actinobacteria agent inoculation methods on cellulose degradation during composting based on redundancy analysis. Bioresour. Technol. 219, 196–203. Zhu, H., Sun, L., Zhang, Y., Zhang, X., Qiao, J., 2012. Conversion of spent mushroom substrate to biofertilizer using a stress-tolerant phosphate-solubilizing Pichia farinose FL7. Bioresour. Technol. 111, 410–416.
Biochem. 38, 2645–2654. Flores-Renteria, D., Rincon, A., Valladares, F., Curiel Yuste, J., 2016. Agricultural matrix affects differently the alpha and beta structural and functional diversity of soil microbial communities in a fragmented Mediterranean holm oak forest. Soil Biol. Biochem. 92, 79–90. He, X., Xi, B., Cui, D., Liu, Y., Tan, W., Pan, H., Li, D., 2014. Influence of chemical and structural evolution of dissolved organic matter on electron transfer capacity during composting. J. Hazard. Mater. 268, 256–263. Hu, H., Wang, J., Li, J., Li, J., Ma, Y., Chen, D., He, J., 2016. Field-based evidence for copper contamination induced changes of antibiotic resistance in agricultural soils. Environ. Microbiol. 18, 3896–3909. Huang, G.F., Wu, Q.T., Wong, J.W.C., Nagar, B.B., 2006. Transformation of organic matter during co-composting of pig manure with sawdust. Bioresour. Technol. 97, 1834–1842. Khan, M.S., Zaidi, A., Wani, P.A., 2007. Role of phosphate-solubilizing microorganisms in sustainable agriculture – a review. Agron. Sustain. Dev. 27, 29–43. Liang, Y., Pei, M., Wang, D., Cao, S., Xiao, X., Sun, B., 2017. Improvement of soil ecosystem multifunctionality by dissipating manure-induced antibiotics and resistance genes. Environ. Sci. Technol. 51, 4988–4998. Malik, M.A., Marschner, P., Khan, K.S., 2012. Addition of organic and inorganic P sources to soil – effects on P pools and microorganisms. Soil Biol. Biochem. 49, 106–113. Mander, C., Wakelin, S., Young, S., Condron, L., O'Callaghan, M., 2012. Incidence and diversity of phosphate-solubilising bacteria are linked to phosphorus status in grassland soils. Soil Biol. Biochem. 44, 93–101. Miller, S.H., Browne, P., Prigent-Combaret, C., Combes-Meynet, E., Morrissey, J.P., O'Gara, F., 2010. Biochemical and genomic comparison of inorganic phosphate solubilization in Pseudomonas species. Env. Microbiol. Rep. 2, 403–411. Morrissey, E., Franklin, R., 2015. Resource effects on denitrification are mediated by community composition in tidal freshwater wetlands soils. Environ. Microbiol. 17, 1520–1532. Nakasaki, K., Araya, S., Mimoto, H., 2013. Inoculation of Pichia kudriavzevii RB1 degrades the organic acids present in raw compost material and accelerates composting. Bioresour. Technol. 144, 521–528. Onireti, O.O., Lin, C., Qin, J., 2017. Combined effects of low-molecular-weight organic acids on mobilization of arsenic and lead from multi-contaminated soils. Chemosphere 170, 161–168. Panhwar, Q.A., Naher, U.A., Shamshuddin, J., Othman, R., Latif, M.A., Ismail, M.R., 2014. Biochemical and molecular characterization of potential phosphate-solubilizing
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