Metagenomics analysis of the effects of Agaricus bisporus mycelia on microbial diversity and CAZymes in compost

Sample collection and DNA extraction

Commercial strain A15 of A. bisporus from Sylvan (USA) was used in this study and stored in the Engineering Research Center of Chinese Ministry of Education for Edible and Medicinal Fungi (ERCCMEEMF) at Jilin Agricultural University (Changchun, China). The compost fermentation and mycelial culture experiments were performed at Zhejiang Longchen Modern Agricultural Science and Technology Co. Ltd, Jiaxing City, Zhejiang Province, China. The compost comprised wheat straw (90 t), chicken manure (83 t), peanut meal (3 t), and gypsum (9 t). Peanut meal was added as an auxiliary nitrogen source because the components of wheat straw and chicken manure in China were different from those in Europe and America (Jun et al., 2021). Before commencing compost fermentation, the initial C/N ratio of the compost was adjusted to 25:1. The straws were completely dampened and piled up for 1–3 days, and then the other materials were mixed and piled again for 2–3 days. The pre-compost was placed into the tunnel for primary fermentation (PI) (Straatsma et al., 2000; Mouthier et al., 2017). The pile was turned three times on days 2, 4, and 7, respectively. The treatment parameters were adjusted based on compost temperature to allow the material temperature to reach 70 °C–80 °C and remain constant for 6 days (PI). Immediately after that, secondary fermentation (Compost-PII) was conducted for 6–7 days by pasteurization (Vieira & Pecchia, 2018). After secondary fermentation, inoculation was performed when the temperature decreased to 24 °C and NH₃ level was ≤10 mg/L (Sharma, Lyons & Chambers, 2005). The temperature, humidity, air pressure, air volume, and other environmental factors were adjusted using Dutch Christiaens Group equipment for the intelligent control system. Under optimal environmental conditions, A. bisporus mycelia could grow all over the compost after 18 days (Mycelium-PIII) (Iiyama, Stone & Macauley, 1994).

Subsequently, samples were collected from the uninoculated compost and mycelia-filled compost, respectively. Before sample collection, the composts in the top, middle, and bottom layers of the reactor were fully mixed (Meng et al., 2021). All the samples were divided into two parts: one part was stored at 4 °C for physicochemical analysis and the other was frozen at −80 °C for DNA extraction. The genomic DNA was extracted from the samples using an Omega EZNA soil DNA kit, and the integrity, purity, and concentration of the extracted genomic DNA were examined by 1% agarose gel electrophoresis (100 V, 1.5 h), NanoDrop 2000, and Qubit 3.0, respectively. The extracted DNA was stored in an ultra-low-temperature freezer at −20 °C and transported to OE Biomedical Technology (Shanghai, China) for sequencing.

Analysis of compost physicochemical properties

The compost samples were dried in an oven at 105 °C for 5 h to assess the moisture content. The dried samples were crushed and placed in the control box of a resistance furnace at 600 °C for 2 h to determine the ash content. The total nitrogen (Total-N) and carbon (Total-C) content in the samples were determined using the Kjeldahl method and K₂Cr₂O₃ oxidation. Determination of the pH values with an electronic pH meter (Mettler-Toledo Instruments Co., Ltd., Shanghai, China) using 10%(w/v) sample suspensions were used. Laccase and xylanase activities in the samples were evaluated using a Solarbio kit, and lignin, cellulose, and hemicellulose components were determined according to Van Soes method.

Metagenome sequencing, assembly, and annotation

Metagenome sequencing was accomplished using the Illumina HiSeq platform with a 500 bp sequencing library. The raw data (raw reads) quality was pre-processed using Trimmomatic (Bolger, Lohse & Usadel, 2014) software, and optimized sequences were spliced and assembled using MEGAHIT (Li et al., 2015; Li et al., 2016) software based on De-Bruijn graph principle, and contigs with length <500 bp were filtered out for subsequent analysis. The open reading frame (ORF) of the spliced contigs was predicted with Prodigal software (Hyatt et al., 2010). CD-HIT software was adopted to remove redundant and non-redundant initial unigenes. The clustering parameters included 95% identity and 90% coverage. The clean reads of each sample were aligned to the non-redundant genes set (95% identity) using bowtie 2 software to calculate the gene abundance in the corresponding samples. The representative sequences in the non-redundant unigenes set were annotated to the obtained species information according to the best alignment attained by BLASTP (E value<1e−5) to National Center for Bio-technology Information (NCBI) Non-Redundant Database (Nr). Then, the sum of gene abundances for the corresponding species was used to calculate species abundance.

Identification of carbohydrate-active enzymes

To evaluate the carbon utilization potential of microbial communities during A. bisporus mycelial growth, the non-redundant genes were compared with the carbohydrate-active enzymes database (CAZy) using DIAMOND software (e<1e⁻⁵) (Buchfink, Xie & Huson, 2015). First, all proteins with the highest sequence similarity were screened and subjected to CAZy to search against sequence libraries with the families of glycoside hydrolases (GHs), auxiliary activities (AAs), carbohydrate-binding modules (CBMs), glycosyltransferases (GTs), polysaccharide lyases (PLs), and carbohydrate esterases (CEs). Then, the differences in the CAZyme family between the two samples (uninoculated compost and mycelia-filled compost) were compared and analyzed (Donhauser et al., 2021).

Data and statistical analyses

Raw data were entered and stored in Excel. The differences among the samples were examined by independent samples t-tests with statistical significance at p < 0.05 and p < 0.01. The data are presented as mean ± standard deviation (SD). GraphPad Prism 8.0 software and Origin 2021 were applied for statistical analysis and plotting, and a cloud platform (http://www.cloudtutu.com/) was employed for plotting.

Physicochemical properties of Compost-PII and Mycelium-PIII

After completion of PII, the material temperature was reduced using fans. Subsequently, A. bisporus was inoculated (4‰ (w/w)) and the compost substrate was filled with mycelial growth after 18 days of incubation at 22 °C–24 °C. During this period, the water content in the compost decreased from 66.48% to 61.77% with the increase in mycelial growth, whereas the ash content increased by 1.71% (Table 1). It must be noted that the water content can affect microbial activities, which in turn can influence enzyme activities. During A. bisporus mycelial growth, carbon consumption predominantly increased, whereas nitrogen utilization was relatively less maintained at 2.12–2.13% and no significant difference (Table 1). After mycelial growth, the pH of the compost decreased from 7.80 to 6.27. Analysis of the cellulose and lignin contents in the compost by Van Soes method revealed that the cellulose content and lignin content significantly decreased (Fig. 1). Furthermore, evaluation of the activities of several known carbon source degradation-related enzyme families indicated a moderate increase in xylanase and laccase activities after A. bisporus mycelial growth, three-fold and 15-fold increase respectively (Figs. 2B & 2C). The solubility of lignin in aqueous solutions was low, and the decrease in the pH of the culture material may have a significant effect on lignin solubility. In addition, the increase in protease (Fig. 2D) also promotes better development of the mycelium (Wang et al., 2021a and Wang et al., 2021b).

Diversity of microbial communities in Compost-PII and Mycelium-PIII

The effective data volume of each sample in this experiment was 11.23–17.22 G. The N50 statistics of Contigs were distributed between 1631–2349 bp, and the number of OFR in the set of non-redundant genes was 1029162 after redundancy. The annotation rates were 89.44%, 74.62%, 42.21% and 2.57% for the non-redundant genes compared with NR, eggNOG, KEGG and CAZy databases respectively. Metagenomics analysis indicated the dominance of bacterial community in Compost-PII and Mycelium-PIII samples (93.17% and 94.27%, respectively), followed by fungi (0.25% and 0.29%, respectively), whereas the archaeal abundance remained almost unchanged. However, the abundance of viruses declined with A. bisporus mycelial growth (1.18% in Compost-PII and 0.13% in Mycelium-PIII). A total of 181 phyla, 157 classes, 805 families, 3460 genera, and 22,567 species were detected in the samples. The six most prominent bacterial phyla were Proteobacteria, Actinobacteria, Chloroflexi, Planctomycetes, Bacteroidetes, and Firmicutes (Zhang et al., 2014), and the abundances of Actinobacteria and Planctomycetes significantly increased after A. bisporus mycelial growth (Fig. 3A). Intriguingly, numerous lignocellulose-decomposing bacteria have been reported to belong to Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes (Lewin, Wentzel & Valla, 2013; Pankratov et al., 2011). Proteobacteria and Bacteroidetes are known to play a major role in organic matter degradation and C cycling (Wang, Mao & Li, 2018), and Bacteroidetes can break down lignocellulose into short-chain fatty acids (Dodd, Mackie & Cann, 2011). It is noteworthy that although the relative abundance of the Basidiomycota phylum was low, it showed a great increase (Fig. 3B).

Analysis of bacterial and fungal communities

In the present study, Thermobifida, Thermostaphylospora, Sphaerobacter, Thermopolyspora, Pseudoxanthomonas, and Rhodothermus were the predominant bacterial genera in the Compost-PII and Mycelium-PIII samples (Cao et al., 2019; Durrant, Wood & Cain, 1991). When compared with the Compost-PII samples, the relative abundances of Thermobifida, Thermostaphylospora, Sphaerobacter, Thermomonospora, and Chelatococcus were significantly increased in Mycelium-PIII samples; in contrast, the relative abundances of Thermopolyspora, Rhodothermus, and Pseudoxanthomonas presented the opposite trend (Fig. 4A). Analyses of the microbial community composition confirmed significant shifts in the microbial community structure between the two groups, and many of the enriched genera also co-varied with function. Moreover, the variability of these microbial communities may be correlated with nutrients, compost temperature, moisture content, and pH.

At the species level, the relative abundance of A. bisporus presented the highest increase among fungi, exhibiting a 77.57-fold increase after complete mycelial growth (Fig. 4D). In addition to A. bisporus, the activities of other microorganisms, such as the bacteria (Fig. 4C) Thermostaphylospora_chromogena, Thermomonospora_sp._CIF_1, Sandaracinaceae_bacterium, and Chelatococcus_composti, and fungi (Fig. 4D) Spizellomyces_punctatus, Rozella_allomycis, and Basidiobolus_meristosporus, were also enhanced during A. bisporus mycelial growth.

Analysis of CAZymes

The carbon-utilization potential of the microbial communities in the compost was assessed to evaluate the effects of altered substrate quantity and quality resulting from the shift in microbial activity during A. bisporus mycelial growth. Among the genes annotated with CAZy, in total, 431 different CAZyme families (229 GHs, 81 GTs, 48 PLs, 17 AAs, 16 CEs, and 40 CBMs) were detected in the samples. The most abundant enzyme classes at all temperatures were GHs and GTs, whereas those with the lowest abundance were PLs and AAs. At the family level, GTs were especially abundant in all the samples, with GT2 (cellulose/chitin synthase and other functions), GT4 (sucrose synthase and other functions), and GT83 (galacturonosyl transferase and other functions) being the most abundant (Paixão et al., 2021; Leadbeater et al., 2021).

Cellulose, hemicellulose, and lignin are major constituents of lignocellulose-containing raw materials (Stech et al., 2014). In the present study, cellulose- and hemicellulose-degrading enzymes exhibited the highest activities in Compost-PII and Mycelium-PIII samples, with GH5, GH8, and GH9 families being the predominant cellulose-degrading enzymes and GH2, GH10, GH11, GH26, and GH53 families being the major hemicellulose-degrading enzymes (Table 2). The GH2 family includes multiple enzymes, and it has been demonstrated that α-1,3-L-arabinofuranosidase activity on substituted xylan does not improve compost degradation by A. bisporus. In nature, it is generally attributed to the metabolism of basidiomycetes white-rot fungi, since they degrade lignin more rapidly and extensively than other microorganisms (Woiciechowski et al., 2013). Although not a wood-rotting fungi, the Agaricus bisporus still plays a key role in the degradation of lignin as a grass-rotting fungi. Although AA7 family genes have been reported to play a role in lignin degradation (Andlar et al., 2018) and the content of these genes was relatively high in the lignin-degradation-related enzymes family in the present study, this enzymes family was not significantly different. There is a large content of the AA3 and AA6 enzyme families associated with lignin breakdown (Table 2).

Variations in enzyme families between Compost-PII and Mycelium-PIII

The effect of A. bisporus mycelium on the compost substrate was mainly reflected in the enzyme families with low relative abundance at the population level. For instance, the abundances of GT32 and GH24 significantly decreased, whereas those of GH42, AA10, and CBM13 significantly increased. Furthermore, the abundance of GH8 presented a slight decrease (Fig. 5A). GH24 is known to act in association with lysozyme, GH8 is the main family of enzymes involved in cellulose degradation, and GH42 plays an important role in hemicellulose degradation. Thermostaphylospora belonging to Actinobacteria mainly causes an increase in the activities of β-galactosidase and α-L-arabyranosidase of the GH42 family in the galactose metabolism pathway during the mycelial growth stage, and CBM67 has been reported to exhibit α-L-rhamnose-binding activity (Fujimoto et al., 2013).

In the present study, the increase in the laccase content during the mycelial growth stage was mainly related to the AA10 family (Table 2). The major strains that cause differential laccase production are Thermobifida, Thermostaphylospora, and Cellulomonas, and in the present study, the relative abundances of Thermobifida and Cellulomonas decreased, while that of Thermostaphylospora increased (Figs. 5B & 5C). Furthermore, the increase in the xylanase content during A. bisporus mycelial growth stage was mainly related to the CBM13 family, and Thermostaphylospora was also the dominant genus that caused this increase (Figs. 5D & 5E).