Assessing the Performance and the Microbial Dynamics of Co-composting System Using Recycled Cow Manure and Bedding Material Waste 


 Co-composting of recycled cow manure and waste bedding material has been used to convert both agricultural wastes to biofertilizers. This study explored the succession of microbial community, metabolic function and substances conversion capacities during 60 days’ co-composting using high throughput sequencing technology. The study revealed that co-composting of cow manure and bedding material waste at a ratio of 1.32 (CM+B) had the highest efficiency among four treatments. The bacterial and fungal community diversity changed significantly during the co-composting of CM+B group, and the major phyla included Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria and Ascomycota. PICRUSt and FUNGuild analysis showed that carbohydrate, lipid metabolism and especially nitrogen fixation were enhanced in the thermophilic phase, while animal and plant pathogens were not detected after the co-composting. Wood saprotrophs became the dominant fungal group (89.1%) in the maturation phase. Canonical correlation analysis (CCA) and redundancy analysis (RDA) confirmed that temperature influenced bacterial community succession more than it influenced fungal community succession. Ruminiclostridium had a significantly positive relationship with temperature (p_value < 0.05), while pH and C/N had significant effect on the fungal (p_value < 0.05), and Penicillium and Mortierella were significantly related to moisture (p_value < 0.05). This work describes an efficient methodology to deal with co-composting systems that had been successfully applied in agricultural wastes treatment, enabling further understanding in mechanisms underlying the substance conversion and the involved microbial community succession in sophisticated composting system.


Introduction
Livestock production makes up approximately 40% of the global value of agricultural products and provides livelihoods for almost 1.3 billion people worldwide, while also results in Concern of manure generation in substantial quantity (Organization, 2017). Currently, more than half of the total manure generated from all livestock is cow manure. In China, approximately 380 million tons of cow manure are produced each year, mostly from centralized large-scale farms (Wang et al., 2018a). Generally, cow manure is rich in nutrients that are essential for crops, including nitrogen (N), phosphorus (P), potassium (K), etc., and has been considered a good source of long-term biofertilizer ( Composting is a microorganism-mediated, cost-e cient method to convert various unstable and complex organic matters into stable and humus-like substances. The major components of organic wastes include carbohydrates, including cellulose, proteins, lipids and lignin, and the biodegradation process of organic wastes is carried out by mesophilic and thermophilic microorganisms in sequence (Duan et al., 2019; Xu et al., 2019). The composting products can be further utilized to improve the physical properties of soil (Bello et al., 2020;Esmaeili et al., 2020;Guo et al., 2019). Dissolved organic matters (DOM), as the direct material and energy resource for microorganisms, re ect compost stability and maturity (Said-Pullicino et al., 2007). Notably, the composting process is dramatically affected by physiochemical properties of feedstock (e.g. moisture and C/N ratio) and environmental factors (e.g., temperature and pH) (Awasthi et al., 2018;Guo et al., 2012;Wang et al., 2018b). For this reason, fresh cow manure is unsuitable feedstock for composting as its moisture content is generally higher than 90%. However, studies have shown that when amending with co-compost materials such as wood shavings, sawdust, and cornstalks, the composting process of livestock manure was enhanced because those diverse additives altered the physiochemical properties of feedstock to a more appropriate level (Chang et  manure can also effectively reduce the moisture content and obtain the recycled cow manure. Due to the signi cant difference in physicochemical properties between recycled cow manure and bedding material waste, it is proposed that co-composting using the mixture of both wastes shall be able to promote biotransformation e ciency comparing to the composing of recycled cow manure alone. In this study, an e cient method for bedding material waste was developed to serve as additives for composting of recycled cow manure. The effects of bedding material waste supplementation were investigated systematically and dynamically regarding physiochemical properties of compost samples and degradation of organic matters. To obtain a comprehensive understanding of the co-composting process, the microbial community of composting system (included both bacterial and fungal community) and metabolic function pro les were analyzed. The correlations among bacterial and fungal communities, physicochemical properties, and metabolic function distribution were explored. The degradation and humi cation of organic matters were also analyzed to evaluate the potential application value in agricultural and landscape industry.

Composting process and sampling
Recycled cow manure (obtained after fresh cow manure dewatering) and waste bedding material were collected from the research farm at Jixiang Livestock Co., Ltd., Fujian, China and used as feedstock for co-composting. The basic features of these two materials were shown in Table 1. Four different treatments were performed, including pure cow manure (CM), cow manure with bedding material waste (CM + B; weight ratio = 1.32:1), cow manure with bedding material waste and urea (CM + B + N; weight ratio = 1:1; nitrogen loading = 80 kg), and cow manure with bedding material waste, urea and sheeting (CM + B + N + S; weight ratio = 1:1; nitrogen loading = 80 kg). Urea was used as an inorganic nitrogen source (Awasthi et al., 2018) and sheeting with small pores was used to maintain temperature of compost. The co-composting piles were built after thoroughly mixing raw materials using a mechanized mixer, in a pyramid-shape of approximately 3 m × 3 m × 1.5 m (length × width × height). Each pile was turned by forklift every 5 day to facilitate oxygen supplementation and avoid anaerobic reactions. The cocomposting process lasted for 60 days. For each pile, samples were taken from ve different positions of the middle layers (30-50 cm deep) on day 0, 3,7,10,15,20,30,40 and 60, and mixed homogeneously as a representative sample at each time point before each turning. Each sample was about approximately 1kg and used for analysis of physiochemical parameters in triplicate. According to the temperature change, mixed samples from three replicates on day 0, 7, 20, 40 and 60 were used for microbial community analysis.

Physicochemical analysis
The moisture content was determined as the weight loss after drying samples at 105°C for 24 h in an electric oven in the laboratory. The dried samples were then crushed to pass through a 0.25 mm sieve.
Measurements of pH and electrical conductivity (EC) were conducted by portable pH and EC meters after mixing each sample with distilled water at a ratio of 1:10 (w/w). The total organic carbon (TOC) and total Kjeldahl nitrogen (TKN) were measured using the Kjeldahl method and the C/N ratio was calculated accordingly. The germination index (GI) was tested according to the methods in previous report (Alwaneen, 2016; Li et al., 2019).

Spectroscopy analysis of DOM
The three-dimensional uorescence EEM spectra, characterized by the excitation/emission (Ex/Em) wavelength pairs and the speci c uorescence intensity, were able to qualitatively reveal the DOM composition. Five grams of dried cow manure from each sample was extracted with 50 mL deionized water for 24 h at 25°C, and the aqueous extract was harvested by centrifugation at 10,000 rpm for 10 min and ltration through a 0.45 mm membrane lter. A fraction of the aqueous extract of the samples was freeze-dried to obtain solid DOM for spectroscopy analysis. The chemical changes in the DOM were quantitatively assessed by spectral methods, including excitation-emission matrix (EEM) uorescence (Wang et al., 2013). The uorescence measurement was conducted on the aqueous extract using an FP-6500 uorescent spectrometer equipped with a xenon excitation source. To obtain the uorescence spectra of the EEM, the excitation wavelengths were increased from 220 to 450 nm at 5 nm steps, and the emission wavelengths were detected from 280 to 550 nm at 2 nm steps.

DNA extraction and high-throughput sequencing
Genomic DNA was extracted from 0.5 g of each sample (dry weight) by using a PowerSoil Kit (MO BIO Laboratories, Carlsbad, CA, USA). The concentration of extracted DNA was determined by using a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scienti c, USA). The DNA extract was pooled and kept at -80°C until use. The variable V3-V4 region of the 16S rRNA gene was selected for the construction of the bacterial community library for MiSeq sequencing. ITS1-F (5′-C TTGGTCATTTAGAGGAAGTAA-3′) and ITS2-R (5'-GCTGCGTTCTTCATCGATGC-3') were used to analyse the fungal community. The PCRs were as described in

Changes in physicochemical characteristics during cocomposting
The main physicochemical parameters of the co-composting piles are shown in Fig. 1. Compost temperature is used as an indicator of microbial activity throughout the composting process, and also an indicator of the maturity and stability of the compost product . Among the four treatments, CM + B group arrived the highest temperature very fast (Fig. 1a). There were four phases in CM + B composting, mesophilic phase (< 55°C, day 0-7), thermophilic phase (55-62°C, day 7-32), cooling phase (40-55°C, day 32-41), mature phase (< 40°C, day 41-60). The temperature rose rapidly to 55°C on day 7 and reached 62°C after 20 days, re ecting active microbial degradation during the mesophilic and thermophilic phase. Diverse labile substances (e.g., carbohydrates and proteins) were decomposed rapidly, which released a large amount of heat (Zhou et al., 2018). The thermophilic phase (> 55°C) lasted for approximately 25 days, followed by a cooling phase and a maturation phase during which the temperature gradually declined to approximately 36°C upon the end of the composting. The temperature of the other groups arrived the highest on day 30-45, prolonging the composting period.
Water content in all the treatments decreased because of the increasing temperature and ventilation in the composting process (Fig. 1b). The water content of CM + B group dropped the fastest from 70-80-55% during the thermophilic phase because of the sharp temperature change, and gradually stabilized to about 50% in the cooling and mature stage. Compared with CM + B group, the variation of moisture content in other treatments was less dramatic because the temperature increase was slow. Similar to temperature, the pH showed an increasing trend possibly due to the release of NH 3 from microbial metabolism (Reyes-Torres et al., 2018), and then fell gradually (Fig. 1c). The highest pH of CM + B, CM + B + N, CM + B + N + S was around 8.99-9.20, which re ected a balance of acid production and ammonia accumulation in the composting piles. The nal EC value was observed to be higher than the original value in all treatments (Fig. 1d), which was consistent with the previous studies . They also fell in the desired range for a mature compost product and unlikely to be phytotoxic (Wang et al., 2020a). Treatment of CM + B also exhibited the highest EC value, increasing from initially 0.55 mS cm -1 to 2.8 mS cm -1 . As Fig. 1e and 1f, the contents of TOC and TKN in all treatments declined in the mesophilic phase because of the aerobic degradation of macromolecules and the rapid loss of CO 2 and NH 3 . Then an increase of TOC and TKN was observed in CM and CM + B during thermophilic phase, which may be caused by decomposition of organic matter and N 2 xation (Zhou et al., 2018). Consequently, C/N ratio declined rapidly in the mesophilic stage of composting and stabilized in the cooling and maturation phase (Fig. 1g). GI value greater than 80% is generally considered an indicator that compost is mature and phytotoxicity-free GI in CM and CM + B groups kept increasing and met the standard of mature compost product, while GI in CM + B + N and CM + B + N + S was too low to be satisfactory maybe because of the addition of different nitrogen source compared to the other two groups (Fig. 1h).

Analysis of humic substances
The uorescence of organic matter would be in uenced by the presence of condensed aromatic rings and/or unsaturated aliphatic carbon chains (Yu et al., 2019). Previous studies revealed that the redshift in the maximum uorescence intensity could be attributed to an increase in aromatic group condensation in these molecules (Lv et (Fig. 2). Peak D and E (Em < 380 nm, Ex < 250 nm) in the early composting process represents aromatic proteins. The peak C (Em > 380 nm, Ex < 250 nm), peak B (Em < 380 nm, Ex > 250 nm) and peak A (Em > 380 nm, Ex > 250 nm) are attributed to fulvic acid, water-soluble microbial metabolites and humic acid, respectively. The uorescence intensity of peak B decreased from thermophilic stage followed by an increase. The uorescence intensity of peak D and peak E showed a downtrend while the uorescence intensity of peak A and peak C showed an uptrend. That suggested during the composting process, the primary reactions were the transformation of protein and water-soluble microbial metabolites to fulvic acid and humic acid, especially during mesophilic phase. After that, the tendency gradually shifted to the stabilization of the newly formed humic acid-like and fulvic acid-like organic materials during the curing and mature phases. Compared with the other treatments, CM + B treatment exhibited an extremely higher uorescence intensity of peak A. That was an indicator of high DOM conversion e ciency during composting process, possibly due to the appropriate nutrient ratio and fast temperature rise in CM + B. The e cient transformation of DOM was also consistent with GI data because humic substances bene t the seed germination.

Similarity and diversity of microbial communities
A total of 31, 081 bacterial sequences and 36, 761 fungal sequences from all samples were analyzed after quality ltering. Venn diagrams was used to analyze the similarity of the microbial communities in the samples of the four treatments (Fig. 3a&b). Specially, in CM + B there were 1826 bacterial OTUs and 434 fungal OTUs identi ed respectively, of which 346 and 168 were unique. That indicated the highest diversity of microbial community in CM + B and signi cant differences from other treatments. A dramatic variation of bacterial community was also in CM + B observed suggested by the sobs index (Fig. S1A), while change of fungal community was expected to be similar in CM + B, CM + B + N and CM + B + N + S (Fig. S1B). Based on the above results, the treatment CM + B was selected for dynamic analysis of microbial community in compost samples.
The similarity of samples at different time points of CM + B composting process was also analyzed by Venn diagram and PCoA. Bacterial diversity increased signi cantly in the mesophilic phase and gradually decreased in the cooling phase, while fungal diversity decreased continuously as composting progressed (Fig. 3c&d). Samples of day 7 and day 20 exhibited the most unique bacterial OTUs of 544 and 269 respectively. The fungal taxonomic richness was found to be much lower than the bacteria. Only 6 fungal OTUs were identi ed from fungal OTU libraries, including Pseudeurotium, Mycothermus, and some other unclassi ed_k_Fungi. According to PCoA, the close distance between day 40 and day 60 for both bacteria and fungi indicated the stable status of the microbial community after the cooling phase. The short distance between day 7 and day 20 in the bacterial analysis indicated that the bacterial community had a signi cant change in the mesophilic phase (Fig. 3e), while there was an obvious change in the fungal community on day 20 according to the PCoA (Fig. 3f), indicating that the fungal community was signi cantly in uenced by thermophilic phase in the co-composting process.

Bacterial community succession and predicted bacterial functions
At phylum level, a closer observation of the bacterial community revealed eight dominant groups in the four composting systems (Fig. 4a).   (Ganguly & Chakraborty, 2018). It can also act as an indicator of mature product as its dominant abundance only appeared in the maturation phase. Solibacillus has a high capacity to degrade lignocelluloses and lignin (Huang et al., 2019). Acinetobacter harbinensis, a heterotrophic nitrifying bacterium, has the ability to remove ammonium (Qin et al., 2017). The bacterial community structure of other composting groups in genera level was showed in Fig. S2. A-C.
The metabolic potential of bacterial communities during CM + B composting was evaluated by PICRUSt based on the Clusters of Orthologous Groups (COG) database. As predicted, there were three main functional groups, including metabolism, information storage and processing, and cellular processes and signaling (Fig. 4c). The metabolism group (49.87-52.76%) was the largest of the three groups during cocomposting. We noticed that the proportion of the lipid and carbohydrate metabolism group generally increased from day 0 to day 60, which may be related to the high biodegradability of those substrates.
However, amino acid metabolism experienced a signi cant decline during the rst week of composting and then increased in the cooling phase and mature phase. Amino acid metabolism is known to enhance microbial growth and activity since amino acids can serve as sources of both carbon and energy for microbes during composting (Bello et al., 2020). Therefore, the energy production and conversion subsystem were further analysed by PICRUSt based on the KEGG database (Fig. S3A). The oxidative phosphorylation metabolism and carbon xation pathway were two main categories during the composting of CM + B. The relative abundances of genes involved in carbon xation exhibited an increase during the thermophilic phase. As for nitrogen metabolism, the functional enzymes that showed signi cant change were associated with nitri cation, denitri cation and nitrogen xation (Fig. S3B). The relative abundance of nitrogenase (nif) increased during composting and became the dominant functional enzyme in the thermophilic phase.

Fungal community succession and predicted fungal functions
For fungal community, six main phyla were detected during co-composting (Fig. 5a). The similarities of fungal community between CM + B and CM + B + N groups indicated that the ratio of cow manure and bedding material possibly have a minor impact at least at phylum level. During the composting of CM + B, Ascomycota made up the greatest proportion of the classi ed OTUs, and its relative abundance increased from initial value of 47.04-88.05% on day 40 and 90.07% on day 60. At the genus level, the relative abundances of the 20 most abundant classi ed fungal genera showed obvious variation over the 60-day composting period (Fig. 5b). Orpinomyces, which was detected only in the original cow manure, includes anaerobic fungi that inhabit the gastrointestinal tract of mammalian herbivores and cannot survive in the aerobic compost matrix (Zavrel et al., 2013). The thermophilic genus Mycothermus was dominant during the composting process, especially on day 40 (97.32%) and day 60 (98.77%). The microbial consortium consisting of the thermophilic fungus Mycothermus thermophilus (Scytalidium thermophilum) and a range of thermophilic Proteobacteria and Actinobacteria was primarily responsible to biodegradation of feedstock and release of ammonia . Candida and Aspergillus were detected in all samples, indicating their high adaptability toward diverse environmental conditions (e.g. temperature, moisture and pH). Both of their relative abundance reached the highest value during thermophilic phase, consistent with their thermophilic features. Under relatively high temperature, they were able to degrade various components of lignocelluloses, promote the formation of precursor substances and thus accelerate the synthesis of humic substances (Huang et al., 2019). The fungal community structure of other composting groups was showed in Fig. S2D-F. The fungi in the co-composting of CM + B system, including the 35 most abundant OTUs, were classi ed by ecological guild and by trophic mode (Fig. 5c). A neighbor-joining phylogenetic tree was constructed to demonstrate the phylogenetic relationships among the main fungal communities in the CM + B co-composting system which showed that pathotrophs and saprotrophs were the dominant fungal trophic modes in this co-composting system and saprotrophic fungi were the most commonly detected taxa, with 13 OTUs belonging to 11 known genera of Ascomycota (e.g., Aspergillus and Candia) and Basidiomycota (e.g., Wallemia). The wood saprotrophs and many unde ned saprotrophs were the groups that showed the most signi cant changes during the co-composting. Notably, the relative abundances of the OTUs of wood saprotrophs started at only 3.5% on day 0 but increased quickly, and they became the dominant fungal group (89.1%) in the mature phase. These data con rmed the e cient conversion of cellulose and hemicellulose during the co-composting of CM + B. Animal and plant pathogens were not detected after 60 days of co-composting in this experiment, indicating that the CM + B co-composting of CM + B improved the safety of raw materials as an agricultural fertilizer. High temperature was reported to be the most important factor in the elimination of pathogens during aerobic composting (Duan et al., 2019). Compared to bacteria, fungi was more advantageous on lignocellulose degradation because of their mycelial structure (Wang et al., 2018c). Ascomycota and Basidiomycota are the dominant fungal phyla for lignocellulose degradation, and high abundances of these phyla promote the degradation of organic waste during composting . Mycothermus, Penicillium, and Aspergillus were the core functional genera to produce lignocellulose-degrading enzymes during composting (Kertesz & Thai, 2018b 3.6. Relationships of bacterial/fungal communities to physicochemical characteristics and microbial metabolism CCA, RDA and heatmaps were used to describe the relationship between microbial community during CM + B composting process and environmental factors, including temperature, moisture, pH, and C/N (Fig. 6   &S4). All the p_value data was showed in tables in supplementary le (Table. S1 -4). CCA showed the dynamics of the genera data for the top 20 bacterial genera in the different phases of co-composting process (Fig. 6a). Romboutsia, Paeniclostridium and Symbiobacterium were observed mainly on days 7-20 and were positively correlated with temperature (Fig. S4A). Entering thermophilic phase, Ruminiclostridium turned to be the dominant genus and also had a signi cantly positive relationship with temperature (p_value < 0.05). Bacteroides, Solibacillus and bacillus showed signi cantly positive relationships with moisture (p_value < 0.05). In contrast, Lysinibacillus, Solibacillus, and Thermobacillus showed negative correlations with moisture and C/N. Ruminiclostridium and Symbiobacterium had a signi cantly positive relationship with pH (p_value < 0.05). In addition, the ve most abundant genera, including Acinetobacter and Bacillus, were all positively correlated with pH at 40 and 60 days. Interestingly, the RDA revealed that only four fungal genera showed positive correlations with temperature not signi cantly ( Fig. 6b and Fig. S4B), including Candida which was dominant on day 20, and Pyrenochaetopsis which was detected only on day 20 (Fig. S4B). Both genera Penicillium and Mortierella detected mainly in the early phase of co-composting, were signi cantly related to moisture (p_value < 0.05). Unclassi ed_f__Neocallimastigaceae and unclassi ed_p__Ascomycota were signi cantly related to the C/N. In addition, Aspergillus, and Ascochyta were negatively correlated with pH and temperature.
These results indicated that temperature had less in uence on the succession of fungal communities than on the succession of bacterial communities. CCA and RDA revealed the dominant bacterial/fungal genera in the original cow manure co-compost mixture was affected mainly by moisture and C/N. Thermophilic genera became dominant producers with increasing temperature. PH was a key factor affecting the bacterial community structure during the curing and mature phases of cow manure cocomposting.

Conclusion
This work designed a co-composting system to deal with diverse kinds of agricultural waste simultaneously. Recycled cow manure and bedding material waste were used as feedstock at an optimal mass ratio of 1.32:1. With the addition of bedding material waste, the temperature of composting pile rose quickly and achieved e cient degradation of organic matters. The EEM spectroscopy analysis indicated that co-composting helped convert degradable organic components in raw materials into humus-like substances, which increased the productivity of compost products and enhanced the value for agricultural and landscaping applications. Bacterial and fungal community diversity changed signi cantly during co-composting. High-throughput 16S rRNA/ITS gene sequencing indicated that the dominant phyla included Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria of bacteria and Ascomycota of fungal. Lysinibacillus and Mycothermus were the indicators of bacterial and fungal community in the maturation phase, respectively. The carbohydrate and lipid utilization capacity as well as the metabolic diversity within the bacterial community displayed an increase in this co-composting process, and animal and plant pathogens were not detected after co-composting. Ruminiclostridium had a signi cantly positive relationship with temperature (p_value < 0.05), while pH and C/N had signi cant effect on the fungal (p_value < 0.05). The relative abundances of the OTUs of wood saprotrophs increased quickly and became the dominant fungal group (89.1%) in the mature phase. The results proved the effectiveness of co-composting as a method for treatment of diverse agricultural and livestock industry waste.    Predicted COG database using PICRUSt method in the co-composting of cow manure and bedding materials (CM+B).