sustainability Article Planifilum fulgidum Is the Dominant Functional Microorganism in Compost Containing Spent Mushroom Substrate Hong Zhang 1, Wenying Wang 2, Zaixue Li 3, Chuanlun Yang 3, Shuang Liang 4 and Lushan Wang 1,* 1 State Key Laboratory of Microbial Technology, Shandong University, Qingdao 266237, China; [email protected] 2 School of Life Sciences, Qinghai Normal University, Xining 810016, China; [email protected] 3 Shandong Chambroad Holding Group Co., Ltd., Boxing 256599, China; [email protected] (Z.L.); [email protected] (C.Y.) 4 School of Environmental Science and Engineering, Shandong University, Qingdao 266237, China; [email protected] * Correspondence: [email protected] Abstract: The extensive accumulation of spent mushroom substrate (SMS) owing to the large-scale production of edible fungi is causing environmental problems that cannot be ignored. Co-composting is a promising method for agricultural and animal husbandry waste disposal. In this study, the composition and function of microbial communities in the process of cattle manure–maize straw composting with SMS addition were compared through an integrated meta-omics approach. The results showed that irrespective of SMS addition, the predominant fungi were Ascomycota, while the dominant bacteria were Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes. High temperature promoted the evolution from Gram-negative bacteria (Bacteroides, Proteobacteria) to Gram-positive bacteria (Firmicutes, Actinomycetes). The composting process was accelerated Citation: Zhang, H.; Wang, W.; Li, Z.; by SMS addition, and the substrate was effectively degraded in 14 days. Metaproteomics results Yang, C.; Liang, S.; Wang, L. showed that the dominant microorganism, Planifilum fulgidum, secreted large amounts of S8, M17, Planifilum fulgidum Is the Dominant and M32 proteases that could degrade macromolecular protein substrates in the presence of SMS. Functional Microorganism in Planifilum fulgidum, along with Thermobifida fusca and Melanocarpus albomyces, synergistically degraded Compost Containing Spent Mushroom Substrate. Sustainability hemicellulose, cellulose, and protein. In addition, the dominant microorganisms related to the initial 2021, 13, 10002. https://doi.org/ raw materials such as Pichia, Lactobacillus in the microbial agent and Hypsizygus in SMS could not ◦ 10.3390/su131810002 adapt to the high-temperature environment (>60 C) and were replaced by thermophilic bacteria after 5 days of composting. Academic Editor: Chunjiang An Keywords: co-composting; microbial agent; microbial diversity; metaproteomics; Planifilum Received: 10 July 2021 Accepted: 3 September 2021 Published: 7 September 2021 1. Introduction Publisher’s Note: MDPI stays neutral Spent mushroom substrate (SMS) is a solid culture material discarded after the harvest with regard to jurisdictional claims in of edible fungi. In general, the raw materials used for the cultivation of edible fungi published maps and institutional affil- include agricultural and forestry wastes, such as corn cob, cottonseed shell, wood chips, iations. and other nutrition-rich organic matter [1]. It is estimated that more than 13 million tons of SMS are generated in China each year, causing environmental problems that cannot be ignored [2–4]. SMS contains numerous mycelia and edible fungal metabolites, such as carbohydrates, proteins, organic acids, and bioactive substances, which have the Copyright: © 2021 by the authors. potential of ecological high-value utilization [5,6]. In addition, with large-scale planting and Licensee MDPI, Basel, Switzerland. intensive breeding, the substantial increase in straw and livestock manure has become a This article is an open access article major problem for the sustainable development of agriculture and ecological environmental distributed under the terms and protection in China [7,8]. Reasonable use of these wastes can improve the economic benefits, conditions of the Creative Commons protect the environment, and accomplish recycling of wastes and sustainable development Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ of agriculture. 4.0/). Sustainability 2021, 13, 10002. https://doi.org/10.3390/su131810002 https://www.mdpi.com/journal/sustainability Sustainability 2021, 13, 10002 2 of 17 Aerobic composting is considered as an efficient and sustainable technology for or- ganic wastes disposal, because it limits the overall environmental pollution, and its end product is suitable for use as a fertilizer and soil amendment [9,10]. Co-composting uses a combination of different organic wastes and appropriate parameters to provide ideal conditions for the composting process [11]. This approach can not only treat different organic wastes simultaneously but it also can make comprehensive use of different waste properties, shorten the fermentation period, and improve the quality of compost [12,13]. In a previous study, Zhang et al. [14] confirmed the rationality of co-composting with straw and cow dung. The high temperature generated by the accumulation of microbial metabolism can effectively kill harmful mesophilic organisms, such as Acinetobacter, Pseu- domonas, and nematodes. Meanwhile, the dominant microflora (Firmicutes, Actinomycetes) in the composts was beneficial to crop plants in eliminating pathogens (through the release of some antibiotics) and environmental stress tolerance (through the secretion of signal compounds) [15,16]. The antifungal and protease activities of dominant composting bac- teria Bacillus can reduce the infection rate of bacterial wilt and other fungal diseases of tomatoes and cucumbers [17]. In order to maintain the beneficial microorganisms, tomato root could exudate lactic acid and hexanoic acid to favor the growth of Bacillus cereus [18]. Composting is a process in which a variety of microorganisms participate in the degra- dation and transformation of organic matter. Owing to the complexity of co-composting substrates, the composting process is mainly dependent on the interaction of microor- ganisms and their secreted degradative enzyme systems [14]. With the development of genomics technology, the dominant microbial community and its dynamics in the process of high-temperature aerobic composting with various substrates have been examined [19–21]. By using metaproteomic methods, the dominant microorganisms and their functional enzymes involved in the degradation of organic matter can be located and identified [22]. Integrated “omics” approaches enable linking the microbial community structure to func- tion, and they elucidate the degradation mechanism of lignocellulosic composting. With the advancement in microbial technology, more and more microbial agents are widely used in practical application to improve composting. Previous studies have indi- cated that the addition of single or mixed microbial agents can increase the temperature of thermophilic stage during composting as well as prolong the thermophilic stage [8,23,24]. Wei et al. [25] reported that the inoculation of thermophilic actinomycetes increased the cellulase activity and promoted lignocellulosic degradation. Furthermore, the addition of functional bacteria has been observed to accelerate nitrogen conversion and decrease ammonia–nitrogen emissions, thus reducing nitrogen loss [26–28]. However, some studies have shown that the addition of inoculants had no significant effect on shortening the com- posting duration [29]. Ballardo et al. [30] observed that the presence of Bacillus thuringiensis did not alter the global structure of the dominant microbial community associated with the final product and did not affect its potential use as a fertilizer. Thus, the mechanism of the effect of microbial agent addition on the composting process and microbial community is still unclear. In the present study, two composting processes with and without SMS addition were performed in parallel to evaluate the effects of changes in the substrate on the structure and function of microorganisms. Concurrently, to investigate the role of microbial agents in the composting process, groups of microbial agents were added to the composting processes. By using 16S rRNA and ITS rRNA high-throughput sequencing, the composition and dynamic changes of microbial communities in bacteria and fungi during the composting process were monitored, and a metaproteomic approach was employed to directly observe the functions of the dominant communities during the composting process. The results of this study provided novel insights into the degradation mechanism of co-composting and practical application of microbial agents. Sustainability 2021, 13, 10002 3 of 17 2. Materials and Methods 2.1. Materials and Compost Sampling The composting process was conducted at Shandong Bohua High-efficient Ecological Agriculture Science & Technology Co., Ltd., Binzhou, Shandong Province, China (37◦210 N, 118◦260 E, measured by ASHTECHGPSZ-X). Aerobic composting started on 6 January 2019 and lasted for 29 days. The experimental materials were collected from the surrounding cow farms and mushroom factories. The microbial agent was purchased from Shandong Chambroad Holding Group Co., Ltd., China, and high-throughput sequencing revealed that the inoculum was a combination of three genera: Acetobacter, Lactobacillus, and Pichia. The four composting systems were (1) Corn straw and cow dung mixed at a mass ratio of 1:2 (SC); (2) Corn straw and cow dung mixture (mass ratio of 1:2) inoculated with 0.3% microbial inoculum (SCI),