Analysis of the Bacterial Flora of Substrates Used for the Cultivation of Agaricus bisporus: Relationship between Clostridia and Yield

Agaricus bisporus has a high nutritional value and health benefits and its popularity is increasing among vegans and health-conscious consumers, indicating the need for its stable production. Therefore, we examined the bacterial flora of the substrates used to produce A. bisporus using a 16S rRNA gene analysis and discussed the relationship between the bacterial flora and yield. The results obtained showed that A. bisporus yield slightly decreased with an increase in the abundance of Clostridia in substrates after primary fermentation. Lactobacillus showed little or no relationship with A. bisporus yield. Clostridia was identified as an indicator of A. bisporus yield.

Agaricus bisporus is one of the most widely cultivated mushrooms in the world (Ohga et al., 1998).Although there are more than 3,000 edible mushroom species, A. bisporus, which has a high nutritional value and health benefits, is becoming increasingly popular among vegans and healthconscious consumers and may dominate the global market by 2025 (Sassine, 2021).It is also regarded as a meat substitute with similar nutritional value to several vegetables (Chang and Miles, 2004).Therefore, the stable production of A. bisporus is required to meet future demands.
Previous studies reported that substrates prepared from fermented horse manure for the cultivation of A. bisporus were of low quality due to poor fermentation, which negatively affected its yield (Sharma, 1991;Zhang et al., 2014;Sassine, 2021).Therefore, the underlying reasons need to be examined.
A similar study reported the poor fermentation of silage.Silage is a livestock feed prepared from forage crops or grasses and produced by microbial fermentation (Yimi, 2002).The poor fermentation of silage is caused by bacteria belonging to the genus Clostridium (Yimi, 2002;Inoue et al., 2012).Lactobacillus has been used to prevent the poor fermentation of silage (Kumai et al., 1990).Although silage and the substrates of A. bisporus cultivation differ in their intended applications and materials used, both share the need for microbial fermentation.Based on this background, we hypothesized that the mechanisms responsible for the poor fermentation of substrates used in the cultivation of A. bisporus may be similar to those of silage.However, the impact of Clostridium and Lactobacillus on A. bisporus cul-tivation has not yet been examined in detail.
Substrates used for A. bisporus cultivation include horse manure, chicken manure, and cow manure.A. bisporus has traditionally been grown on a mixed substrate of horse manure and wheat straw (Farnet et al., 2013), and the present study focused on horse manure.The preparation of this substrate is divided into primary and secondary fermentation phases.During primary fermentation, heat is generated by the activity of thermophilic bacteria, and the temperature of the growing substrate increases to >60°C (Sassine, 2021).Microorganisms break down carbohydrates and proteins, enabling the availability of nutrients in the substrate to A. bisporus (Ekman, 2017).During secondary fermentation, microorganisms break down refractory organic matter, such as cellulose and lignin (Zhang et al., 2014;Kertesz and Thai, 2018;Cao et al., 2019).Secondary fermentation occurs indoors and the substrate is not stirred.The substrate is then aged at 48°C to kill pathogens and pests and remove free ammonia (Omori and Koide, 2019;Suwannarach et al., 2022).Based on these findings, the reasons for the poor fermentation of substrates used for A. bisporus cultivation may be similar to those of silage; however, the stage of poor fermentation that adversely affects the cultivation and yield of A. bisporus remains unclear.Therefore, the present study examined the bacterial flora of primary and secondary postfermentation substrates and investigated the relationships between the relative abundance of Clostridium, Lactobacillus, and other bacteria involved in cultivation of A. bisporus and its yield.
The substrates used for A. bisporus production that underwent primary and secondary fermentation were supplied by A. bisporus farmers A, B, and C in Chiba Prefecture, Japan.Farmers A and B have high, stable yields, whereas farmer C has low, unstable yields.All farmers used horse manure, rice straw, and also the same processes in primary and secondary fermentation.In primary fermentation, farmers added water while stirring the substrate used for A. bisporus production until its color changed from brown to black.
After the completion of primary fermentation, each sample was collected from three locations of the substrate (farmer A: n=3, farmer B: n=3, farmer C: n=3).In secondary fermentation, farmers packed the substrate used for A. bisporus production into a shelf in-house.After aging the substrate, each sample was collected from three locations on one shelf covered with the substrate (farmer A: n=3, farmer B: n=3, farmer C: n=3).All samples were collected from approximately 5 cm inside the substrate surface around June in 2021 and immediately frozen.Each sample weight was approximately 30 g.The yields of farmers A, B, and C were the total amount of A. bisporus harvested from the substrates.Yield was expressed as the total A. bisporus weight per cultivated area.After thawing frozen samples and removing rice straw that remained in shape, 0.2 g of the substrate was used for DNA extraction.DNA was extracted from each sample using the NucleoSpin®DNA Stool Kit, and the gene amplicon sequencing of extracted DNA was contracted to Genome-Lead Inc.A composite pair of primers comprising unique 17-or 21-base adapters was used to amplify the V3-4 region of the bacterial 16S rRNA gene from each sample.The forward primer sequence was 5′-TC GTCGGCAGCGTCAGATGTGTATAAGAGACAGNCCTA CGGGNGGCWGCAG-3′, with the italicized sequence corresponding to the global broadly conserved bacterial primer F341.The reverse primer sequence was 5′-GTCTCGT GGGCTCGGAGATGTGTATAAGAGACAGNGACTACHV GGGTATCTAATCC-3′, with the italicized sequence representing the universal broadly conserved bacterial primer 806R.A second PCR amplification was performed using primers containing adapter and index sequences.Gene amplicon sequencing was performed under a 2×301-bp paired-end run on the Miseq system (Illumina).The MiSeq sequencer was used to sequence a library of the bacterial 16S rRNA gene V3-4 region.The resulting sequences were then analyzed using QIIME 2.
The relative abundance of bacteria is represented in the figure as the mean±standard error.An analysis of variance with the Kruskal-Wallis test was performed to compare the mean relative abundance of bacteria among substrate samples collected from the farmers.Statistical analyses were conducted using SPSS version 28.0.1.1 ( 14).
We compared the relative abundance of the bacterial phyla present in the substrates used to produce A. bisporus after primary and secondary fermentation (Fig. 1A and B).The relative abundance of Firmicutes was high in the substrates from all farmers after primary fermentation, but decreased to less than 10% after secondary fermentation.The bacterial flora at the phylum level of the primary and secondary post-fermentation substrates did not significantly differ among the farmers.
We compared the mean relative abundance of bacterial classes or orders present in the substrates used to produce A. bisporus after primary fermentation (Fig. 2A and Table 1).The average relative abundance of Clostridia, the bacteria responsible for the poor fermentation of silage, was high in the substrate from farmer C. We performed the Kruskal-Wallis test using the relative abundance of Clostridia present after primary fermentation in the substrates from farmers A, B, and C, which revealed a near-significant trend (p = 0.076) between farmers A and C. Bacilli and Actinobacteria are substrates used to produce A. bisporus (Yamauchi et al., 2017;Omori and Koide, 2019;Sassine, 2021).In the present study, the mean relative abundance of Bacilli was >14% in all substrates, while that of Actinobacteria was <3%.We considered the stirring of substrates during primary fermentation to increase the abundance of aerobic bacteria, such as Bacilli.The mean relative abundance of Lactobacillales, which inhibit the growth of Clostridia in silage (Kumai et al., 1990), was <1% in all substrates.Pseudomonadales are pathogens that cause brown blotch disease in A. bisporus (Miyazaki, 2018;Kuroda et al., 2019), and the mean relative abundance of Pseudomonadales in the present study was <4% in all substrates.
We then compared the mean relative abundance of bacterial classes or orders present after secondary fermentation in the substrates (Fig. 2B and Table 1).The mean relative abundance of Clostridia in all substrates was <0.3%, which was significantly different from that present after primary fermentation.The mean relative abundance of Bacilli was lower and that of Actinobacteria was higher after secondary fermentation than after primary fermentation.Although the lack of stirring during secondary fermentation may have affected the abundance of aerobic bacteria in the substrates tested, these species remained predominant.The mean relative abundance of Lactobacillales was <0.02%, while that of Pseudomonadales was <0.7% in all the substrates.The mean relative abundance of Lactobacillales in the primary and secondary post-fermentation substrates was low and similar for all farmers.Secondary fermentation, including sterilization, markedly reduced the mean relative abundance of pathogens, such as Pseudomonadales.This may also account for Lactobacillales and Pseudomonadales showing little or no relationship with the yield of A. bisporus.The relationship between the abundance of Clostridia in the substrates after fermentation and the yields of A. bisporus are shown in Fig. 3.These yields were obtained from cultivation with the substrates used for the analysis of the bacterial flora.The higher mean relative abundance of Clostridia present in the substrates after primary fermentation slightly decreased the yield of A. bisporus, suggesting a relationship between the yield of A. bisporus and the relative abundance of Clostridia after primary fermentation.Therefore, even if numerous Bacilli and Actinobacteria exist, which are useful for cultivation with the substrates, the yield of A. bisporus may decrease if there is an excess of Clostridia in the substrate after primary fermentation.The relative abundance of Clostridia in the substrate after second fermentation was low and similar in all farmers, whereas yields differed.Therefore, the yield of A. bisporus did not appear to correlate with the relative abundance of Clostridia in the substrates after secondary fermentation.

Compost Bacterial Flora Analysis
We examined the bacterial flora of the substrates used for A. bisporus cultivation and found a relationship between the abundance of Clostridia and the yield of A. bisporus.The yield of A. bisporus slightly decreased with increases in the abundance of Clostridia present in the substrates after primary fermentation.Lactobacillus, which inhibits the growth of Clostridia in the fermentation of silage, was virtually absent in the substrates.A relationship was not observed between the abundance of Bacilli or Actinobacteria present in the substrates and the yield of A. bisporus.Collectively, the present results may be useful for improving the productivity of A. bisporus using fermented substrates.All novel sequence raw data have been deposited in DDBJ (accession number DRA016706)

Fig. 3 .Fig. 2 .
Fig. 3. Relationship between Clostridia and the yield of Agaricus bisporus in post-fermentation substrates.The vertical axis is yield per 100 m 2 and the horizontal axis is the relative abundance of Clostridia.The r-squared value of the graph for primary fermentation was 0.9982.The r-squared value of the graph for secondary fermentation was 0.0086.

Table 1 .
Bacteria present in substrates after primary and secondary fermentation (order level).