Ann Microbiol (2017) 67:751–761 https://doi.org/10.1007/s13213-017-1303-1 ORIGINAL ARTICLE Diversity and dynamics of the DNA and cDNA-derived bacterial compost communities throughout the Agaricus bisporus mushroom cropping process Conor Francis Mcgee1 & Helen Byrne1 & Aisling Irvine1 & Jude Wilson1 Received: 2 August 2017 /Accepted: 29 September 2017 /Published online: 11 October 2017 # Springer-Verlag GmbH Germany and the University of Milan 2017 Abstract The cultivation of Agaricus bisporus involves the bacterial community is present throughout the mushroom conversion of agricultural materials via fermentation into cropping process. utilisable simple sugars as a nutrient source for the fungal crop during mushroom cropping. However, little is currently Keywords Mushroom cropping . Microbial ecology . known about the role of the bacterial community contributing Fermentation . Nitrification . cDNA . DNA to the fermentation process. In this investigation we characterised the composition and dynamics of the DNA and cDNA-derived bacterial populations throughout a com- Introduction mercial mushroom cropping process using MiSeq sequencing. Both methods indicated substantial changes in the bacterial Agaricus bisporus has become one of the most widely culti- community structure after the first flush of the mushroom vated mushroom species, with an estimated worldwide annual crop. However, differences were observed between the com- farm gate worth of $4.7 billion (Sonnenberg et al. 2011). position of the bacterial community determined by each of the Commercial cultivation of A. bisporus is undertaken using two methods. The cDNA-derived community indicated that mushroom compost, a partially composted organic substrate, thermotolerant genera with known sulphur-reducing charac- which fulfils a dual functional role as both a growing medium teristics were highly active up to the first flush. Activity of the and a nutrient source (van Griensven and van Roestel 2004). phyla Actinobacteria and Firmicutes was observed to increase Mushroom compost is a specially prepared semi-pasteurised as fermentation progressed, indicating that the members of composted organic material in which wheat straw acts as the these phyla played prominent roles in the conversion of wheat main carbohydrate source for the mushroom crop (Straatsma straw into utilisable sugars. The cDNA-derived community et al. 1994). Nutrition for the A. bisporus mycelium is released comprised genera with roles in the nitrification process that by the continual microbial fermentation of complex lignocel- became highly active at post flush 1. Subsequent chemical lulosic carbohydrates (cellulose, hemicellulose and lignin) analysis of extractable nitrate indicated that substantial nitrifi- present in the wheat straw of the mushroom compost through- cation occurred up until the termination of the cropping pro- out the cropping process (Zhang et al. 2014). Elucidation of cess. This study has demonstrated that a highly dynamic the breakdown of these complex carbohydrate polymers is of particular interest not only to the mushroom industry, but also Electronic supplementary material The online version of this article to a range of other sectors, such as the biofuel and paper and (https://doi.org/10.1007/s13213-017-1303-1) contains supplementary pulp industries (Perez et al. 2002). Currently, little knowledge material, which is available to authorized users. is available regarding the composition and dynamics of the microbial communities involved in compost fermentation * Conor Francis Mcgee throughout the mushroom cropping process. [email protected] Agaricus bisporus mushroom compost undergoes three distinct phases during its production and preparation prior to 1 Monaghan Mushrooms R&D Department, Group Headquarters, being suitable for use in the mushroom cropping process Tyholland, County Monaghan, Ireland (Kabel et al. 2017). Phase 1 consists of a short partial 752 Ann Microbiol (2017) 67:751–761 composting process, the main components of which are wheat result, these authors also found that the fermentation of hemi- straw, gypsum and horse/chicken manure (Straatsma et al. cellulose and cellulose via β-xylanase and β-glucanase en- 1994). Phase 2 involves semi-pasteurisation of the composted zyme activities was more associated with the uninoculated Phase 1 product by heat treatment prior to inoculation with a Phase 3 compost, indicating that the wider mushroom com- spawn of A. bisporus (Iiyama et al. 1994). The inoculated post microbial community was affiliated with these activities Phase 2 material then undergoes a period termed the Bspawn (Savoie 1998). run/Phase 3^ wherein the mycelium colonises the semi- The current literature suggests that little diversity is present pasteurised Phase 2 material (van Griensven and van Roestel in the active portion of the compost fungal community despite 2004). By Phase 3, the dominant sources of carbohydrates the bacterial community being highly diverse (Zhang et al. present in the compost are the complex fibres of lignin, hemi- 2014; Székely et al. 2009; Siyoum et al. 2016; McGee et al. cellulose and cellulose in the wheat straw (Patyshakuliyeva 2017). To date, few studies have been conducted that follow et al. 2015). Traditionally, the mushroom cropping process the changes in microbial communities in mushroom compost consists of transferring the Phase 3 material to a commercial throughout the entire cropping process using advanced molec- cropping house and applying a peat-based layer, referred to as ular techniques. The advent of next-generation sequencing Bcasing soil^, on top of the Phase 3 compost substrate to technologies has resulted in powerful techniques for determin- induce mushroom fruiting (Berendsen et al. 2012). The initial ing the depth and composition of microbial communities. mushroom crop, termed a Bflush^, is induced by controlling Most studies following microbial communities in mushroom environmental conditions such as temperature, CO2 and mois- compost have focused on Phase 1 material and included rela- ture inside the cropping house (Sharma et al. 2005). Typically, tively few samples from cropping (Zhang et al. 2014;Siyoum a series of two to three flushes are harvested from the compost et al. 2016). This has resulted in a knowledge gap regarding bed over the mushroom cropping process before the substrate the dynamics and composition of microbial communities dur- is considered spent (Royse et al. 2008). However, successive ing the mushroom cropping cycle. mushroom flushes produce diminishing mushroom crop The aim of this study was to perform an in-depth investi- yields despite the substantial levels of nutrition remaining in gation of the bacterial community present in mushroom com- the compost (Beyer and Muthersbaugh 1996; Kabel et al. post throughout the cropping cycle targeting both DNA and 2017). Pecchia et al. (2014) highlighted several theories that cDNA-derived communities. The purpose of using both DNA have been proposed over the years to explain why diminishing and cDNA targets was to distinguish between the dormant and crop yields may occur, such as changes in the composition of active microbial communities during the cropping process. microbial communities present in the compost, accumulation DNA-based studies of microbial communities are sometimes of metabolites on the surface of the mycelium and depletion of compromised due to legacy DNA and the influence of dor- available nutrients present in the compost. mant species. Characterisation of the active portion of the Carbohydrate levels in spent compost have been previously bacterial community throughout the cropping process may reported to consist of between 11 and 16% of the total com- shed better understanding on those bacterial groups that may post weight (Iiyama et al. 1994; Jurak et al. 2015). Microbial play important functional roles in the fermentation of the com- fermentation in the later stages of the mushroom cropping plex organic material present and reveal the composition of process has attracted much interest as it represents potentially bacterial communities throughout mushroom cropping for the unreleased nutrition for the mycelium (Jurak et al. 2015; first time. Kabel et al. 2017) as well as being an interesting study for investigating the bioconversion of organic biomass (Zhang et al. 2014). Materials and methods Fermentation of the complex carbohydrates in compost is undertaken by the release of exo-enzymes by the total micro- Compost and sampling bial community present in the compost environment (Zhang et al. 2014). To date, several studies have examined the activ- Compost samples were obtained from a commercial mush- ity of carbohydrate-degrading exo-enzymes present in the room production house based on a farm in County mushroom compost throughout the cropping process to un- Monaghan, Ireland, which operated under normal commercial derstand nutrient release (Bonnen et al. 1994; Savoie 1998; cultivation conditions. The compost used in this trial was pro- Jurak et al. 2015). The study of Bonnen et al. (1994)compared duced by an Irish commercial compost production company, enzyme activity at the community level in Phase 3 compost and the peat casing layer applied on top of the compost was either inoculated and uninoculated with produced by an Irish casing supplier. The compost used in the A. bisporus throughout a cropping process and found that growing house was composed of a combination of straw, gyp- the lignin-degrading enzymes laccase and manganese