Diversity and Dynamics of the DNA and Cdna-Derived Bacterial Compost Communities Throughout the Agaricus Bisporus Mushroom Cropping Process

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 peroxi- sum and poultry manure which was inoculated with a white dase were associated with A. bisporus. In contrast to this button strain of Agaricus bisporus. Compost samples were Ann Microbiol (2017) 67:751–761 753 obtained at 11 time points during the 40-day mushroom Table 1 Sampling times throughout the mushroom cropping process cropping process, namely on days 0, 4, 10, 17, 21, 22, 28, and corresponding compost nitrate and nitrite levels 31, 32, 35 and 39. The sampling days correspond to distinct Day Stage Nitrite level (mg kg−1) Nitrate level (mg kg−1) stages in the cropping process, as outlined in Table 1. Compost was destructively sampled at three separate points 0 Filling 0.13 2.78 along the mushroom production bed on sampling dates and 4 Casing Run 0.21 4.55 homogenised by hand. Sampling of compost consisted of re- 10 Pinning 0.15 4.64 moving 300 cm2 of the peat casing layer covering the mush- 17 Flush 1 0.25 2.33 room bed and taking compost to the bottom of the bed 21 End of Flush 1 0.35 10.86 (approx. 30 cm). Approximately 5 kg of compost was obtain- 22 Pinning ND ND ed from each sampling point, which was then homogenised 28 Flush 2 0.33 12.2 together by hand. A 10-g sample of homogenised compost 31 End of Flush 2 0.33 9.1 was stored in LifeGuard (Mo Bio Laboratories, Carlsbad, 32 Pinning ND ND CA) preservation solution at −20 °C until further processing. 35 Flush 3 0.28 17.4 39 End ND ND

Characterisation ND Not determined

Compost physico-chemical characterisation [pH, electrical PCR amplification and purification conductivity (EC), moisture content, ash and nitrogen] was conducted by the Monaghan Mushrooms Compost Analysis The V4 region of the bacterial 16S rRNA gene was amplified Laboratory (Tyholland, Co.. Monaghan, Ireland). by PCR using the paired end universal bacterial primers Determination of compost nitrate and nitrite levels was con- established by Kozich et al. (2013) for MiSeq sequencing. ducted by Southern Scientific Services Ltd. (Farranfore, Co. PCR was performed using the Q5 High Fidelity PCR kit Kerry, Ireland). Analysis of extractable compost nitrite and (NEB, Ipswich, MA) in a 25-μl reaction volume consisting nitrate levels was performed by Southern Scientific Services of 12.5 μl Q5, 6.3 μl water, 0.2 μl bovine serum albumin Ltd. on freeze-dried samples collected on days 0, 4, 10, 17, 21, − (50 mg ml 1; Invitrogen, Thermo Fisher Scientific), 2.5 μl 28, 31 and 35 of the cropping process. of each primer and 1 μl of template DNA or cDNA. The PCR reaction consisted of an initial denaturation step at 95 °C for 120 s, followed by 30 cycles of 95 °C for 20 s, Extraction of DNA and RNA from compost 55 °C for 15 s and 72 °C for 45 s, with a final elongation step at 72 °C for 5 min. The products of the PCR reactions were Compost–LifeGuard suspensions were thawed on ice and cen- visualised on a 1% gel. The PCR analysis was conducted in trifuged at 10,000 rpm for 2 min to pellet the compost. The duplicate for each sample, and these duplicates were then supernatant was discarded and the compost was grinded in combined in a 1.5-ml Eppendorf tube. Bacterial genomic liquid nitrogen using a mortar and pestle into a fine homoge- DNA obtained from a pure bacterial culture was used as a nous powder. Extractions for genomic DNA from compost positive control, while water was used as a negative control. were performed in triplicate for each sample (n = 3). DNA Pooled duplicate PCR reactions had a volume of 40 μland was extracted from samples by transferring 250 mg of com- were purified with ethanol purification by adding 10 μlof post powder to bead beating tubes supplied with a DNA ex- NaCl (5 M), followed by 100 μl of 96% ethanol and then traction kit (PowerSoil DNA Extraction kit, Mo Bio incubation of the mixture at −20 °C overnight. Each sample Laboratories) and extracted following the manufacturer’spro- was then centrifuged at 13, 000 rpm for 15 min and the super- tocol. Purification of the DNA was performed using a natant discarded. The pellet was washed with 70% ethanol and PowerClean DNA Clean-Up kit (Mo Bio laboratories) follow- centrifuged again at 13,000 rpm for 15 min, following which it ing the manufacturer’s protocol and visualised on a 1% gel. was then air dried in a laminar flow cabinet and resuspended DNA quantity and quality was checked using a NanoDrop in 50 μl of nuclease-free sterile water. ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA). RNA was extracted in duplicate (n =2)from samples using a Total Soil RNA Purification kit (Norgen Illumina MiSeq sequencing Biotek Corp., Thorold, ONT, Canada) following the manufacturer’s protocols. RNA was converted to cDNA using an Samples were prepared for Illumina sequencing following Invitrogen SuperScript Vilo cDNA synthesis kit (Thermo a modified workflow protocol established by Kozich et al. Fisher Scientific). (2013). Sequencing was performed on an Illumina MiSeq 754 Ann Microbiol (2017) 67:751–761 sequencer based in TrinSeq (Trinity Genome Sequencing Results Laboratory, Trinity College Dublin, Ireland) using a v2 sequencing kit (Illumina Co., San Diego, CA). Physicochemical characterisation of compost Concentrations of PCR samples were quantified using a Qubit 3.0 fluorometer (Thermo Fisher Scientific) and di- The compost substrate at filling was found to be slightly acidic luted to 4 nM using Illumina elution buffer. Five microli- (pH 5.96) and highly saline (EC 5.82 μScm−1). The moisture ters of each sample was transferred to a 1.5-ml Eppendorf content was recorded as 64.7%, while the remaining dry tube to make a pooled library, following which 5 μlofthe weight consisted of 21.7% ash. Total Kjeldahl nitrogen was pooled library was transferred to a clean 1.5-ml found to be 2.7%, of which 0.05% was ammonia. The ratio of Eppendorf tube and 5 μlofNaOH(0.2N)addedtode- total carbon to nitrogen (C:N) was found to be 15.25. Analysis nature the library by incubating it for 5 min at room tem- of extractable compost nitrate levels revealed a substantial perature. Cooled HT1 solution from the v3 sequencing kit increase occurring from day 21 (post flush 1) until the termi- (999 μl) was then added to the denatured library. A nation of the cropping process (Table 1). Compost nitrite 300-μl sample of the library preparation was transferred levels were found to be much lower than nitrate levels and to a clean 1.5-ml Eppendorf tube, and 6 μlofPhiXwas were found to increase only slightly from day 21 onwards. then added to the pooled library to give a final PhiX concentration of 12.5 pM. Another 294 μl of cooled Analysis of sequence data using the Mothur process HT1 was added to the library to give the final sample for loading onto the MiSeq system. The sequencing run MiSeq sequencing data from the amplified 16S amplicons was performed following the steps set out by Kozich et al. was quality filtered using the Mothur process, generating (2013). 8,347,643 good sequence reads from the 55 pooled samples. The sequences were classified into 671 unique genera belong- ing to 32 bacterial phyla. For the Bray–Curtis-based non- Analysis metric multidimensional scaling (NMDS) plots (Fig. 1), stacked bar charts (Fig. 2) and PERMANOVA-only phyla Output files from the Illumina MiSeq sequencing were and/or genera composing more than 0.1% of the total se- processed for analysis using the Mothur program devel- quences were used, although this restriction was not found oped by Schloss et al. (2009). Bacterial 16S sequencing to affect the overall conclusions of the study. After the 0.1% data were analysed following the protocol developed by cutoff threshold was applied to the DNA- and cDNA-derived Kozich et al. (2013). Paired-end reads were merged and communities, the number of unique genera was reduced to sequences were quality controlled filtered to remove se- 273 and 274, respectively, highlighting the high level of lowly quence lengths of > 275 bp prior to clustering using a abundant taxa. A large proportion of genera accounting for ≤ 97% threshold against a reference alignment with a set 0.1% of total community abundance is typical of microbial homoploymer length of 8. To reduce the noise in the se- communities (Shade et al. 2014). quences, pre-clustering was utilised that allowed up to two differences between sequences, and the resulting sequences were screened for chimeras using VSEARCH. A DNA-derived community Bayesianclassifierwasthenusedtoclassifythosese- quences against the Ribosomal Database Project 16S Analysis of the DNA-derived bacterial communities revealed rRNA gene training set, and archaeal, eukaryotic, mito- a trend of constant change throughout the cropping process. chondria and chloroplast 16S fragments were removed. NMDS plots of the DNA-derived bacterial community at the Bacterial taxonomic data were normalised as a propor- taxonomic level of phylum (Fig. 1a) and genus (Fig. 1b) vi- tion of the total number of sequences prior to analysis. sually demonstrated separation between bacterial community Taxonomic data were analysed using the software pack- structures throughout the cropping process. PERMANOVA age Primer (version 6.1.9) with the PERMANOVA add-on determined that community structures were significantly dif- (version 1.0.1) (Primer-E Ltd., Plymouth, UK). Data were ferent between each subsequent sampling day at the genus initially fourth root transformed, and a resemblance ma- level (Electronic Supplementary Material Table 1). trix was constructed using the Bray–Curtis dissimilarity Community structures at both the phylum and genus levels measure. Permutational multivariate analysis of variance appeared to change the most between days 0 (filling) and days (PERMANOVA) was performed using unrestricted per- 21–22 (end of flush 1), prior to the separation distances be- mutation of raw data with 9999 permutations. Analysis tween community replicates appearing to be somewhat dimin- of variance was carried out using Microsoft Excel 2010 ished between days 28 (flush 2) to 39 (termination of cropping (Microsoft Corp., Redmond, WA). process). Ann Microbiol (2017) 67:751–761 755

Fig. 1 Bray–Curtis based non-metric multidimensional scaling (NMDS) Symbols represent individual replicate communities structures (n =3)on plot of bacterial community structures derived from sequenced 16S DNA the sampled days throughout the cropping process. Numbers above fragments based on the taxonomic level of phylum (a) and genera (b). symbols refer to sampling day

The composition of the DNA-derived community was certain particular phyla were observed. The RA of the found to be mainly dominated by six phyla in particular Bacteriodetes, Chloroflexi and Proteobacteria gradually de- (Actinobacteria, Bacteriodetes, Firmicutes, Proteobacteria, creased throughout the cropping process, as inversely, the Planctomycetes and an Unclassified phylum), which together RA of the Actinobacteria, Firmicutes, Planctomycetes and generally accounted for > 90% of the total community relative Verrucomicrobia increased (Fig. 2a). The phyla abundance (RA) on each cropping day. Three additional phy- Deinococcus-Thermus and the Unclassified bacterial phylum la, the Chloroflexi, Deinococcus-Thermus and were found to remain relatively stable throughout cropping. Verrucomicrobia, were found to be present on each sampling Changes in phyla abundance could usually be attributed to day, although they were not consistently as highly abundant as several genera, found to be highly abundant throughout the the six dominant phyla over the course of the cropping pro- cropping process. The top 20 most abundant bacterial genera cess. The community was found to be initially (day 0) com- in the DNA-derived community throughout the cropping proposed of Proteobacteria (43.2%), Actinobacteria (11.8%), cess are presented in Table 2. Of the 250 unique genera of Unclassified (10.4%), Chloroflexi (9.3%), Bacteriodetes Proteobacteria detected throughout the cropping process, the (7.9%), Firmicutes (4.8%), Planctomycetes (4.6%) and decrease in RA was primarily driven by the genera Deinococcus-Thermus (3.7%). Over the course of the Chelatococcus and Pseudoxanthomonas (Table 2). These cropping process several trends in changing abundances of two genera accounted for 21.4% of the total community RA

Fig. 2 Stacked bar chart of average relative abundances of bacterial phyla derived from DNA (a) and cDNA (b) analyses throughout the cropping process. Relative abundance was determined from the average of three (n =3)andtwo(n = 2) samples in DNA and cDNA analyses, respectively 756 Ann Microbiol (2017) 67:751–761

Table 2 The 20 most abundant bacterial genera derived from sequenced DNA 16S amplicons throughout the cropping process, ranked as percentage of total abundance across all days

Number Phylum Genus Sampling times during the 40-day mushroom cropping process (days)

0 4 10 17 21 22 28 31 32 35 39

1 Planctomycetes Unclassified 1 3.5 3.1 5.2 7.9 11.4 11.9 15.1 13.1 15.6 17.3 15.8 2 unclassified Unclassified 2 10.4 8.2 7.6 10.4 8.7 9.1 12 9.4 8.7 10.8 9.5 3 Actinobacteria Thermobifida 4.8 3.3 4.6 3.8 11.2 9.7 12.3 6.8 6 8.7 10.3 4 Proteobacteria Unclassified 3 0 13.4 10.5 7.1 8.1 2.9 0.9 11.5 9.3 3.4 0.7 5 Firmicutes Unclassified 4 1.6 1.4 1.8 3.2 6.6 7.5 8.3 6.3 6.4 7.4 10.9 6 Planctomycetes Pirellula 1 0.8 1.7 3.3 3.7 4.7 5.5 3.9 3.8 5.9 4.9 7 Proteobacteria Pseudoxanthomonas 13.9 11 3.7 2.2 0.6 0.3 0 0.5 0.4 0.1 0.2 8 Firmicutes Unclassified 5 1.2 1.1 1.4 1.7 3.6 3.4 3.7 3.2 3.5 4 5.9 9 Proteobacteria Chelatococcus 7.5 7.3 4.2 2.6 2.1 2.1 0.6 0.8 1.2 0.7 0.7 10 Actinobacteria Thermomonospora 1.3 1 1 1.4 2.5 3.3 4.8 3.1 3.5 4 3.4 11 Proteobacteria Unclassified 6 1.7 1 1.8 1.8 2.7 2.9 3.4 2.2 2.7 4.3 3.7 12 Verrucomicrobia Unclassified 7 0.6 0.5 0.8 2.1 1.7 1.9 2.3 3.5 4.8 2.7 2.3 13 Bacteroidetes Unclassified 8 1.1 14.4 0.4 0.9 0.5 0.1 0 0.9 0.1 0 0 14 Deinococcus-Thermus Truepera 3.6 1.2 2.4 0.9 0.8 0.7 1.3 1.9 2.6 1 1.6 15 Firmicutes Unclassified 9 0.4 0.4 0.5 0.7 1.9 2.2 1.5 2.1 3.7 1.5 2.7 16 Chloroflexi Sphaerobacter 2.2 1.2 2.9 3.6 0.8 0.7 1.3 1 0.7 0.9 0.3 17 Proteobacteria Pseudomonas 0 1.5 4.2 5.8 0.8 0.4 0.1 0.8 0.9 0.1 0.1 18 Actinobacteria Unclassified 10 2.4 1.1 1.6 1.5 1.3 1.1 0.9 1 0.9 1.2 1 19 Chloroflexi Unclassified 11 6 1.9 1.6 1.6 0.3 0.4 0.4 0.8 0.4 0.3 0.1 20 Actinobacteria Unclassified 12 0.7 0.5 0.5 0.6 1.3 1.6 2.3 1 1 1.8 1.4 Accumulative abundance (%) 63.9 74.3 58.4 63.1 70.6 66.9 76.7 73.8 76.2 76.1 75.5

on day 0 (filling), but then steadily decreased gradually to 4.5% of the community RA on day 0, subsequently increasing 0.9% by the termination of the cropping process (day 39). to 20.7% by day 39. Of the 16 genera composing the The composition of the Bacteriodete community was found Verrucomicrobial community, only one, an unclassified genus to be evenly spread over 64 lowly abundant genera, and the of the class Spartobacteria, was found to be highly abundant. abundance of this community gradually decreased from 7.9% The Spartobacterial genus was found to account for the in- (day 0) throughout the process to 0.5% (day 39). No particular crease in Verrucomicrobial RA observed from day 10 Bacteriodete genus was shown to dominate apart from the (0.9%) to day 39 (2.5%). genus Unclassified 8, which was highly abundant on cropping day 4. A community of 154 unique genera of Firmicutes was cDNA-derived community detected during the cropping process. The increase in Firmicute RA was found to be driven by the three genera The cDNA-derived community was generated from duplicate Unclassified 4 (Order Bacillales), Unclassified 5 (Family replicates (n =2)onsamplingdays.Duetotheuseofonlytwo Paenibacillaceae) and Unclassified 9 (Family replicates, cDNA data were not subjected to multivariate Planococcaceae), which collectively accounted for 3.2% of NMDS analysis for examining community structures. cDNA the community RA on day 0, increasing to 19.5% by day data were instead analysed for the RAs of the 16S transcripts 39. The RA increase in the phylum Actinobacteria (106 dif- as an indication of the active bacterial community and to con- ferent genera) could be mainly attributed to the genera trast and compare the results to the corresponding DNA- Thermobifida and Thermomonospora, which together derived community. accounted for 6.1% RA on day 0 and steadily increased to The bacterial cDNA-derived community was found to be 13.7% by day 39. Only eight genera of Planctomycetes were highly changeable throughout the cropping process and to be detected throughout the cropping process, of which two were dominated by several phyla in particular, namely found to be highly abundant members in the DNA-derived Actinobacteria, Bacteriodetes, Chloroflexi, Deinococcus- community. The RA of Unclassified 1 (Family Thermus, Firmicutes, Proteobacteria and the Unclassified bac- Planctomycetaceae) and Pirellula together accounted for terial phylum (Fig. 2b). The collective RA of these seven Ann Microbiol (2017) 67:751–761 757 phyla typically accounted for > 90% of the total community Deinococcus-Thermus community composition. The activity on all sampling days. Certain trends in bacterial activity, as of Thermus was found to be highest in the early stages of the determined by cDNA RA, were also observed for the abun- cropping process leading up to the first mushroom flush when dance of particular phyla throughout the cropping process. In compost temperatures are the highest (generally > 20 °C). particular, the phyla Bacteriodetes and Deinococcus-Thermus Another thermotolerant genus, Thermodesulfobacterium of were observed to decrease in abundance/activity throughout the phylum Thermodesulfobacteria, was found to be highly the cropping process, while the abundance/activity of the abundant in the early stages of the cropping process (days 0 Actinobacteria and Firmicutes were observed to increase. and 4). The Bacteriodete community was dominated by the Several phyla displayed no clear trends and remained more genus Unclassified 8, which peaked in activity between days 0 or less consistently active throughout the cropping process, and 10 (1.1–11.2% total RA). The genus Pirellula was the such as the Chloroflexi, Proteobacteria and the Unclassified most active Planctomycete, followed by the genus phylum. Unclassified 1; the activity of both genera was found to spike The Bacteriodetes had an average RA of 7.7% ± 3.4 across on days 10 and 17 and then to return to < 1% for the remainder sampling days 0–21, which subsequently decreased to of the cropping process. Sphaerobacter was found to be the 2.3% ± 1.3 between sampling days 22 and 39. The phylum active genus of the phylum Chloroflexi, although its activity Deinococcus-Thermus was also observed to follow a similar was not found to follow a clear trend. trend of decreasing RA throughout cropping process, with an average RA of 15.6% ± 10.3 across days 0–21, which fell to 3.9% ± 2.5 between days 22 and 39. The Firmicutes were Discussion initially observed to account for 10.8% of the total community RA on day 0, after which their abundance increased quite This study has shown the bacterial community to be diverse steadily to 44.6% over the course of the cropping process. A and highly dynamic throughout the mushroom cropping pro- slight increase in Actinobacteria abundance was observed cess. PERMANOVA analysis of the bacterial DNA-derived throughout the cropping process, with the exception of days community structures revealed significant differences be- 31 and 32 when the Proteobacterial RA peaked. The tween each time point investigated during the cropping pro- Planctomycetes were found to be quite lowly abundant, gen- cess, indicating constant change in the bacterial community erally < 1%, with the exception of days 10 and 17 when throughout the cropping process. Observations of the RAs in abundance peaked at 13.3 and 10%, respectively. The RA of DNA and cDNA-derived communities during the cropping the top 20 most abundant genera in the cDNA-derived com- process showed clear trends in changing abundances of par- munity is shown in Table 3. ticular bacterial phyla and genera that were driving the chang- The spike in Proteobacteria activity observed on days 31– es in community structures detected with PERMANOVA. 32 was due to three genera, Brevundimonas, Rhizobium and Comparison of the composition of the DNA- and cDNA- Stenotrophomonas. In particular, the Proteobacterial genus derived communities revealed distinctly different community Rhizobium was found to contribute highly to observed profiles throughout the cropping process. The Proteobacteria Proteobacterial activity during the later stages of the cropping were found to be the dominant bacterial phylum in the DNA- process. Rhizobium cDNA RA was found to rapidly increase derived community at filling (43%), while the phylum between sampling days 21 and 39 and became the most dom- Deinococcus-Thermus accounted for only 3.7%. In contrast, inant bacterial genus in the spent mushroom compost. analysis of the cDNA-derived community found activity of Pseudomonas was found to be a Proteobacterial genus that the phyla Deinococcus-Thermus and Proteobacteria to be 29 rapidly decreased in terms of RA throughout the cropping and 23%, respectively. There are two explanations, or a com- process. Pseudomonas RAwas highly abundant between sam- bination of both, which likely explain these differences: (1) pling days 4 and 17, after which the RA decreased rapidly for the DNA-derived community is being influenced by the pres- the remainder of the cropping process. ence of DNA from dormant species, whereas the cDNA com- The increase in Firmicute RA towards the latter half of the munity reflects the active component of the total community cropping process was found to be driven by several genera, (Schostag et al. 2015); (2) the extracellular preservation of most notably Ureibacillus and Unclassified 1 which DNA in the substrate (Dlott et al. 2015). Differences between accounted for 7.4% of the total RA on day 0 but increased the DNA- and cDNA-derived communities were found to to 16% by day 39. The increase in Actinobacteria RA was extend throughout the cropping process. In particular, distinct- mainly due to the genera Microbispora and Unclassified 13, ly contrasting community profiles were observed between the while the genus Thermobifida was found to decrease in RA DNA- and cDNA-derived communities for the phyla throughout the cropping process. The decrease in RA of the Deinococcus-Thermus, Planctomycetes, Proteobacteria and active Deinococcus-Thermus phylum was driven by the genus Verrucomicrobia throughout the cropping process. These re- Thermus, which typically accounted for > 90% of the sults highlight the vastly different portrayals of the bacterial 758 Ann Microbiol (2017) 67:751–761

Table 3 The 20 most abundant bacterial genera derived from sequenced cDNA 16S amplicons throughout the cropping process, ranked as percentage of total abundance across all days

Number Phylum Genus Sampling times during the 40-day mushroom cropping process (days)

0 4 10 17 21 22 28 31 32 35 39

1 Proteobacteria Rhizobium 0.7 0 0.1 0.7 6.2 10 6.5 30.2 30.1 9 10.9 2Firmicutes Unclassified 1 5.8 4.1 3.7 3.5 7.3 11.6 15.9 7.3 8.9 16.1 12.6 3 Deinococcus-Thermus Thermus 27.1 12.9 2.4 22.1 8.4 4.8 4.7 1.5 0.1 4 6.3 4 Actinobacteria Unclassified 13 0.5 1.7 0.2 1.1 8.2 12.7 20.7 3.4 0 13.2 5.2 5 Proteobacteria Pseudomonas 0.1 21 21.3 12.2 2.4 0.9 0.7 1.6 0.8 0.6 3.8 6unclassified Unclassified 2 9.5 5.4 6 5.2 10 5.9 2.6 3.7 2.5 3.1 2.2 7 Proteobacteria Stenotrophomonas 0.2 0 0.8 0.3 3.7 2.7 4.8 10.1 16.3 2.3 3.3 8Firmicutes Ureibacillus 1.6 0.8 0.3 0.9 1.9 4.7 4.3 2.8 3.5 4.9 3.4 9 Actinobacteria Unclassified 10 4.6 2.2 1.9 3.1 2.1 2.3 2 1.5 0.4 4.9 3.2 10 Chloroflexi Sphaerobacter 0.6 0.8 2.1 1.2 4.6 6.8 6 0.6 0 3.2 1.6 11 Proteobacteria Unclassified 3 0 9.8 8.7 2.6 1.4 0.5 0.3 0.6 0.6 0.3 0.4 12 Planctomycetes Pirellula 0.5 0.6 9.3 7.9 0.3 0.7 0.7 0.1 0 0.8 0.7 13 Firmicutes Unclassified 9 0.4 0.9 2.1 1.2 0.6 1.1 1.3 3 6.1 2.2 1.4 14 Bacteroidetes Unclassified 8 1.1 11.2 1.4 2 1.9 1.1 0.2 0.7 0.1 0.1 0.2 15 Actinobacteria Microbispora 0.5 0.4 0.5 0.5 1.7 3 2.7 1.4 0.2 4.7 3.5 16 Firmicutes Staphylococcus 0 0 0 0 0.4 0.4 1.1 0.6 0.3 0.9 12.6 17 Actinobacteria Thermobifida 2.1 1.1 7 2.4 1 0.8 1 0.3 0.1 0.3 0.2 18 Firmicutes Thermoflavimicrobium 0.1 0.7 0.2 0.2 1 2 2.9 1.2 1.4 3 0.9 19 Proteobacteria Brevundimonas 0.1 0 0.3 0.3 0.8 1 0.9 3.1 3.7 1.4 1.1 20 Thermodesulfobacteria Thermodesulfobacterium 1.1 5.6 0 0.5 4.8 0.3 0.1 0.1 0 0.2 0 Accumulative abundance (%) 56.6 79.2 68.3 67.9 68.7 73.3 79.4 73.8 75.1 75.2 73.5

community presented by these differing technical molecular Pleurotus ostreatus (Vajna et al. 2012)andAgaricus bisporus, approaches. possibly playing a role in cellulose degradation (Zhang et al. The phylum Deinococcus-Thermus was found to be lowly 2014). However, the effect of Thermus during the cropping abundant in the DNA-derived community throughout the process is currently unknown, although interestingly, it was cropping process. However, its abundance in the cDNA- not the only thermophilic genus we observed throughout the derived community activity was found to be much higher, cropping process. The genus Thermodesulfobacterium of the typically consisting of > 8% of the total community at each phylum Thermodesulfobacteria was found to be sporadically sampling point. The phylum Deinococcus-Thermus contains active up until the end of the early stages of cropping. The many thermotolerant genera, some of which are highly toler- genus Thermodesulfobacterium consists of thermophilic spe- ant of hazardous environmental conditions (Griffiths and cies with known sulphur-reducing properties (Muyzer and Gupta 2007). Truepera was the most abundant genus of the Stams 2008). Thermodesulfobacterium have been shown to Deinococcus-Thermus in the DNA-derived community, al- be highly active in the thermophilic phases of composting though Thermus was found to be the dominant active genus and have been linked to the release of hydrogen sulphide of the phylum throughout the cropping cycle. The abundance (H2S) gas from a range of substrates, particularly between of the Truepera in the DNA-derived community was possibly pH5.5and6(Callietal.2008). a legacy of their role in the thermophilic phase of the We believe that this is the first report indicating high activ- composting process when they are known to be highly abun- ity of thermo-tolerant bacteria during the mushroom cropping dant (Bishop et al. 2016). Thermus species are typically process. In this cropping system, the compost substrate tem- thermotolerant aerobes that possess sulphur- and arsenic- peratures never exceeded 30 °C. However, the activity of reducing properties and are the source of several enzymes thermophilic/thermotolerant bacteria in temperate environ- with commercial applications at high temperatures (Gihring ments has been observed previously (Portillo et al. 2012). et al. 2001; Skirnisdottir et al. 2001; Pantazaki et al. 2002). The activity of these species may have been influenced by Thermus species have been previously associated with Phase the high levels of sulphur typically found in mushroom com-

1 of the composting processes for the edible mushrooms post. Emissions of H2S gas from spent mushroom compost are Ann Microbiol (2017) 67:751–761 759 known to be high as a result of the use of sulphur-based nu- characterised (Lee and Lee 2014;Zhouetal.2015), and the trients during production and conditioning of the compost observed increase in the activity of this genus appears to fit substrate (Wever et al. 2005; Velusami et al. 2013). The higher well with the increase in nitrate levels in the compost. activity of thermophilic/thermotolerant genera leading up to A common theme determined between both the DNA- and the first flush (day 21) of the cropping process may be ex- cDNA-derived communities was the gradual increase in ac- plained as a combination of their common sulphur-reducing tivity of the Actinobacteria and Firmicutes as the mushroom properties and the higher compost temperatures during this cropping process progressed. In the cDNA-derived communi- period. These temperatures are usually close to 25 °C prior ty two unclassified genera of Firmicutes (Unclassified 1 and to falling back and remaining at approximately 20 °C for the Unclassified 9) along with Ureibacillus and remainder of the cropping process. The composition of the Thermoflavimicrobium contributed to increasing activity at cDNA-derived community highlights the activity of the post flush 1. The increase in Actinobacteria activity was Deinococcus-Thermus and Thermodesulfobacteria in the spread broadly over a number of genera. As the cropping compost environment, which was not as pronounced as in process progresses the only sources of sugars are from com- the DNA-derived community, indicating that it is possibly a plex carbohydrates (Jurak et al. 2015). The increasing activity more powerful tool than DNA analysis for determining the of the Actinobacteria and Firmicutes during cropping is po- functional roles of microbial taxa in active environments. tentially due to the fermentative characteristics of members of These results shed light on a potentially important role of these phyla. Metagenomic studies have shown both phyla to thermophilic bacteria in the mushroom cropping process and be amongst the most dominant bacterial phyla during the fer- highlight our current lack of knowledge regarding the bacte- mentation of douchi (Yang et al. 2016), and both are regarded rial community in the cropping process. as highly active glucose fermenters (Nielsen et al. 2012). The The Proteobacteria were the most diverse phylum in the Firmicutes have also been shown to be the dominant phylum compost environment. However, Proteobacterial activity was during processing of complex carbohydrate sources, such as for the most part dominated by three genera. Activity of the sugar-cane (Sharmin et al. 2012). It is possible that the con- genus Pseudomonas was particularly high between the days sortia of Firmicutes and Actinobacteria present in the cropping of mushroom pinning (day 10) and onset of the first flush of compost contribute substantially to the bacterial fermentation mushrooms (day 17). Several Pseudomonas species are of complex carbohydrates. known to parasitize A. bisporus fruiting bodies, inducing Bacteriodetes were found to decrease in both abundance blotch diseases of mushroom crops. Blotch diseases are and activity throughout the cropping cycle. The Bacteriodete characterised by pits and discoloration on fruiting bodies in- genus Unclassified 8 was found to be the most abundant and duced by the causative agents Pseudomas tolaasii, P. agarici active genus in both the DNA- and cDNA-derived communi- (blotch disease) and P. gingeri (ginger blotch) (Geels et al. ties. The wider Bacteriodete community appeared to have 2008; Largeteau and Savoie 2010; Abou-Zeid 2012). little activity throughout the cropping process, indicating lim- Although Pseudomonas species induce diseases on mush- ited functions of this phylum during the cropping process. room fruiting bodies, the increase in activity of the genus A discrepancy was found between the DNA- and cDNA- Pseudomonas in the compost layer during the pinning–flush derived communities for the phylum Planctomycetes. The 1 period may in some way be linked to the development of the abundance of the Planctomycetes in the DNA-derived com- mushroom crop. As activity of the Pseudomonas species de- munity was found to increase over the course of flush 1, sub- creased, the activity of the genera Rhizobium and sequently remaining steady until the termination of the Stenotrophomonas began to increase post flush 1 (day 21 cropping process. In contrast, the activity of the onwards). The observed increase in the activity of Planctomycetes in the cDNA-derived community was found Rhizobium and Stenotrophomonas corresponded with higher to be high during the pinning–flush 1 period, then decreasing levels of extractable nitrate in the compost environment. The substantially for the remainder of the cropping process. It is presence of Rhizobium has previously been associated with possible that the increase in activity of the Planctomycetes the mushroom cropping process, having been shown to be observed in the cDNA-derived community between pinning present in the casing layer (Siyoum et al. 2016). Rhizobium and flush 1 led to the substantial increase in DNA abundance species are known for their ability to differentiate into bacte- observed from this point. Interestingly, the results of our anal- roids when forming endosymbiotic nitrogen-fixing associa- ysis of the DNA-derived community would indicate a possible tions, releasing ammonia within the roots of leguminous prominent role of the Planctomycetes during mushroom plants (Mus et al. 2016). However, no nitrogen fixation is cropping, but their actual role appears to be quite limited based currently known to occur in their free-living state, and they on cDNA transcripts. are currently not known to have any nitrification properties Although the majority of 16S bacterial sequences could be (Mus et al. 2016). On the other hand, the nitrifying and nitri- classified to the taxonomic level of phylum (> 85%), many fication properties of Stenotrophomonas are well individual genera of these phyla remain unclassified to date. 760 Ann Microbiol (2017) 67:751–761

This study has highlighted the current limitations of 16S stud- Geels FP, Hesen LPW, Van Griensven LJLD (2008) Brown ies in determining the role these taxa play in the cropping discolouration of mushrooms caused by Pseudomonas agarici.J Phytopathol 140(3):249–259 process. It also highlights the need for further transcriptomic Gihring TM, Druschel GK, McCleskey BR, Hamers RJ, Banfield JF approaches in order to develop a more complete understand- (2001) Rapid arsenite oxidation by Thermus aquaticus and ing of the cropping process and determine the functional roles Thermus thermophilus: field and laboratory investigations. – of different taxa. Environ Sci Technol 35(19):3857 3862 Griffiths E, Gupta RS (2007) Identification of signature proteins that are distinctive of the Deinococcus-Thermus phylum. Int Microbiol 10: 201–208 Conclusion Kabel MA, Jurak E, Makela MR, de Vries RP (2017) Occurrence and function of enzymes for lignocellulose degradation in commercial Agaricus bisporus cultivation. Appl Microbiol Biotechnol 101(11): The bacterial community present throughout the mushroom 4363–4369. https://doi.org/10.1007/s00253-017-8294-5 cropping process was found to be highly dynamic, with mi- Kozich JJ, Westcott SL, Baxter NT, Highlander SK, Schloss PD (2013) crobial succession displaying consistent trends at the level of Development of a dual index sequencing strategy and cuaration phyla and genera. Thermotolerant and sulphur-reducing bac- pipeline for analyzing amplicon sequence data on the MiSeq teria appeared to play a potentially important role during the Illumina sequencing platform. Appl Environ Microbiol 79(17): 5112–5120 initial phase of the cropping process. As fermentation Jurak E, Patyshakuliyeva A, de Vries RP, Gruppen H, Kabel MA (2015) progressed, the phyla Actinobacteria and Firmicute increased Compost grown Agaricus bisporus lacks the ability to degrade and in both abundance (DNA) and activity (cDNA), indicating consume highly substituted xylan fragments. PLoS One 10(8): roles in the breakdown of wheat straw. The post flush 1 envi- e0134169 Largeteau M, Savoie JM (2010) Microbially induced diseases of ronment appears to favour bacteria with nitrifying properties Agaricus bisporus: biochemical mechanisms and impact on com- and resulted in a nitrate-rich substrate at the end of the mercial mushroom production. Appl Microbiol Biotechnol 86(1): cropping process. 63–73 Lee EY, Lee CW (2014) Isolation and nitrogen removal characteristics of heterotrophic nitrification-aerobic denitrifying bacteria, Acknowledgements Funding for this project was jointly provided by Stenotrophomonas sp. CW-4Y. 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