ORIGINAL ARTICLE

DGGE and T-RFLP Analysis of Bacterial Successionduring Mushroom Compost Production and Sequence-aidedT-RFLP Profile of Mature Compost

Anna J. Székely & Rita Sipos & Brigitta Berta &

Balázs Vajna & Csaba Hajdú & Károly Márialigeti

Received: 22 April 2008 /Accepted: 30 June 2008# Springer Science + Business Media, LLC 2008

Abstract The amount of button mushroom (Agaricusbisporus) harvested from compost is largely affected bythe microbial processes taking place during compostingand the microbes inhabiting the mature compost. In thisstudy, the microbial changes during the stages of this spe-cific composting process were monitored, and the domi-nant bacteria of the mature compost were identified toreveal the microbiological background of the favorableproperties of the heat-treated phase II mushroom compost.16S ribosomal deoxyribonucleic acid (rDNA)-based dena-turing gradient gel electrophoresis (DGGE) and terminalrestriction fragment length polymorphism (T-RFLP) mo-lecular fingerprinting methods were used to track the suc-cession of microbial communities in summer and wintercomposting cycles. DNA from individual DGGE bands werereamplified and subjected to sequence analysis. Principalcomponent analysis of fingerprints of the compostingprocesses showed intensive changes in bacterial communityduring the 22-day procedure. Peak temperature samplesgrouped together and were dominated by Thermus thermo-philus. Mature compost patterns were almost identical by

both methods (DGGE, T-RFLP). To get an in-depthanalysis of the mature compost bacterial community, thesequence data from cultivation of the bacteria and cloningof environmental 16S rDNA were uniquely coupled withthe output of the environmental T-RFLP fingerprints(sequence-aided T-RFLP). This method revealed the dom-inance of a supposedly cellulose-degrading consortiumcomposed of phylotypes related to Pseudoxanthomonas,Thermobifida, and Thermomonospora.

Introduction

Composting is a self-heating, aerobic, solid-phase processdriven by the microbiological decomposition of organicmaterials [8]. Microbiologically, composting can be inter-preted as a succession of microbiota, which is continuouslyadapting to the changing nutrient supply and alteringenvironmental conditions (temperature, moisture content,carbon dioxide, oxygen, and ammonium content, etc.) [40].Initially, mesophilic decomposer organisms dominate andutilize the easily degradable carbon sources. The intensivemetabolic activity of these microorganisms generates heat,which results in a rapid temperature increase and thetakeover of the declining mesophilic community by ther-mophilic microbes. Thermophiles already play a major partin the breakdown of more complex substances such aspolysaccharides and lignin. At the end, during maturation,mesophilic microorganisms reappear and form a newmicrobial community [8].

The microbial succession of composting and the diver-sity of the different stages have been studied by severalmethods. Cultivation-based analysis of hot and maturingcompost recovered mainly bacterial isolates belonging toActinobacteria and Bacilli [4, 35, 42], but plating at higher

Microb EcolDOI 10.1007/s00248-008-9424-5

R. Sipos : B. Berta : B. Vajna :K. MárialigetiDepartment of Microbiology, Eötvös Loránd University,Pázmány Péter sétány 1/C,1117 Budapest, Hungary

C. HajdúStrain Research and Molecular Biological Laboratory,Korona Spawn Factory,3395 Demjén, Hungary

Present address:A. J. Székely (*)Department of Microbiology, Eötvös Loránd University,Pázmány Péter sétány 1/C,1117 Budapest, Hungarye-mail: mikromara@gmail.com

A. J. Székely I

temperatures (75°C) revealed the presence of Thermusthermophilus too [2], and from the end of the hot-composting stage, mesophilic Pseudomonas and Xantho-monas strains have also been cultured [26]. The alternatingmicrobial communities of the different composting stepshave been detected by several culture-independent techni-ques, such as community-level physiological profiles [3,31], phospholipid fatty acid analyses [3, 10, 11], andamplified environmental deoxyribonucleic acid (DNA)-based methods [4, 22, 26, 30, 37, 39], as the most common.Sequence analysis of 16S ribosomal DNA (rDNA) clonesand excised gel bands supported the idea of a mesophilic–thermophilic–mesophilic succession of microbial commu-nities [22, 26, 37].

However, limited information is available for themicrobiology of such a special composting methodologyas that used for the production of substrate for growingAgaricus bisporus (button mushroom) since most of ourknowledge about compost microbial diversity is derivedfrom studies tracking microbes of waste managementcomposting [2, 4, 22, 30, 31, 35, 37], preparation of soilamendments [39, 42], or unconditioned phase I mushroomcompost [26]. These composting strategies are slower andless regulated than high-quality, phase II mushroomcompost production, where the thermophilic step isfollowed by a relatively short indoor heat treatment(maturing) to prepare a growth media free from pathogensand selective for A. bisporus [8]. To the best of ourknowledge, the effect of this special curing on the bac-terial community of compost has not been studied earlier.However, this community is of overriding importance asits members will coexist with the growing mushroommycelia and presumably—like the well-known growth-promoting fungi, Scytalidium thermophilum [34]—havean impact on mushroom yields.

The communities of the previous composting steps alsohave a significant effect on mushroom production as theydrive the biodegradation responsible for the final composi-tion of the substrate. These microbial processes are affectedby several factors, such as the properties of raw materials[5] or the environmental conditions during the outdoorsteps. Seasonal changes have an undoubted impact not onlyon ambient temperature but the quality of raw materials, asfresh straw is not available in the winter.

Methodological difficulties of microbiological studies ofcompost include distortions arising from solely cultivation-based microbial diversity studies [14]. However, polymerasechain reaction (PCR)-amplified community 16S rDNA-based analyses are also biased, and precautions [18, 32, 41]have to be taken during DNA isolation and PCR amplifi-cation in order to suitably reflect bacterial composition.Clone libraries are appropriate to get good-quality sequenceinformation, but rarefaction analyses [28, 43] demonstrate

that species abundance and distribution can hardly bemeasured. Molecular fingerprints like denaturing gradientgel electrophoresis (DGGE) and terminal restriction frag-ment length polymorphism (T-RFLP) better reflect thecommunity composition. Excision and sequencing ofDGGE bands provide information about the actual speciesresponsible for the pattern, but separation of PCR productsby DGGE is imperfect [23], and the method is consideredto be less sensitive than T-RFLP [21, 25]. T-RFLP analysis,as a semiquantitative and highly reproducible method [13,32], is adequate to deduce species diversity and evenness,but phylogenetic identification of T-RFLP peaks basedexclusively on comparison of terminal restriction fragment(T-RF) lengths with database information is misleadingbecause of incomplete sequence databases [39], imprecisesize calling of peaks, and the possible presence of pseudo-T-RFs [29].

Accordingly, in this study, the monitoring of microbialchanges during two mushroom compost production cyclesoccurring in different seasons (summer and winter) hasbeen evaluated by parallel DGGE and T-RFLP analysesexploiting the advantages of both techniques. In addition,the effect of the spatial location of the samples in the open-air thermophilic step was compared. Finally, identificationof key members of the most important mature, ready-for-spawning compost bacterial community was done by anovel approach called sequence-aided T-RFLP analysis, inwhich the precise sequences of clones and cultures and thesemiquantitative T-RFLP fingerprint peaks supplementedeach other.

Materials and Methods

Composting Methodology and Sampling

The compost samples used in this investigation originatedfrom a commercial mushroom compost plant, where thecomposting process was carried out following the recom-mendations of Gerrits [8].

Phase I of the composting cycles started on day 0when 1,000 kg wheat straw, 890 kg broiler chickenmanure, 200 kg horse manure, and 80 kg gypsum weremixed and wetted with approximately 5 m3 tap water toadjust the moisture content to 75±2%. The initial pHvalue of the mixed raw materials varied between 8.1 and8.6, and the total Kjeldahl nitrogen (TKN) was 1.8±0.2%of the dry matter content. On day 1, the mixed raw materialswere stacked in a 5×2.2×80 m (width×height×length)heap. The windrow was turned, mixed, and wetted everysecond day for 5 days. On the sixth day, the compost wastransferred into a 6×4×40 m open-air bunker equippedwith forced aeration. The compost stack was put into a new

A. J. Székely et al.

bunker every second day. On day 15, the compost wastaken out of the bunker, left resting for 1 day, andtransferred into a 4×3×36 m indoor composting tunnelfor heat treatment (phase II). The thermal profile oftreatment was the following: thermal equalization, 8 h at58°C (pasteurization), 48°C for approximately 3 days untilammonium content fell under 10 ppm (conditioning), andcooling to 25°C. After this treatment, the pH of the phase IIcompost was 7.6±0.2, the moisture content fell under 70%,and the nitrogen content (TKN) increased slightly to 2.1±0.2% indicating the decomposition of the raw materials andthe maturity and readiness of the compost for spawningwith A. bisporus.

In our study, one cycle in the summer and winter periodswas followed through each stage of composting. Theambient temperature during the cycles changed between+15°C and +27°C (average +20°C) and −1°C and +5°C(average +2°C), respectively. The temperature of thecompost stacks was measured 1.5 m deep and also 30 cmdeep in the case of edge sampling. The sampling date andsite of each composite sample and the measured temper-atures are summarized in Table 1. The core samples(approximately 3 kg) were taken during the turning of thewindrow and consisted of a mix of five to six subsamplesoriginating from the center of the compost stacks 6–10 mwide apart. The edge samples were taken from the outersurface of the bunkers to a depth of 30 cm. Subsamples(20 g) designated for DNA isolation were stored at −20°C.

Isolation of Cultures

Ten grams of the mature summer compost sample (day 22)was homogenized in 100 mL physiological salt solution.Tenfold serial dilution was prepared, and 0.1 mL of thesuspension was aliquoted onto three replicate plates of

tryptone soya agar (TSA; CM0131, Oxoid, Basingstoke,UK) and compost extract medium (CEM) solidified withagar [24]. CEM was made adding 50 g of dry maturemushroom compost granulated in a Cyclotec mill (Model1093, Rose Scientific, Edmonton, Canada) and 1.5 g ofsorghum grits to 1 L distilled water, boiled for 30 min, andfiltered. Both TSA and CEM plates were incubated at 45°Cfor 72 h. Fifty discrete bacterial colonies were isolated fromthe appropriate dilutions (Petri dishes containing 40–100colonies).

DNA Extraction and PCR Amplification

Genomic DNA from cultures was extracted with V-geneBacterial Genomic DNA Mini-prep Kit (V-Gene Biotechnol-ogy, Hangzhou, China) following the manufacturer’s instruc-tions. For environmental DNA isolation, 500 mg of frozencompost samples were homogenized and pulverized by quickgrinding with liquid nitrogen in sterile mortar, withoutallowing the sample to thaw. One milliliter of CLS-TC lysisbuffer (Bio 101, La Jolla, CA, USA) was added to the frozenmaterial. Following melting, the lysate was transferred to amicrocentrifuge tube, and the debris was pelleted by centri-fugation at 14,000×g for 5 min. The crude DNA extract waspurified by Wizard® SV Genomic DNA Purification System(Promega, Madison, WI, USA) according to the manufac-turer’s protocol excluding the first lysis steps.

PCR reactions took place in a solution containing 1 U ofTaq polymerase (Fermentas, Vilnius, Lithuania), 1× PCRbuffer with (NH4)2SO4 supplied by the manufacturer, 2 mMMgCl2, 0.2 mM of each deoxynucleoside triphosphate,0.3 μM of each 16S rDNA primer, and 1 μL of templateDNA (10–100 ng) with a final volume of 50 μL on aGeneAmp PCR System (Model 2400, Applied Biosystems,Foster City, CA, USA). All of the primers used in this study

Table 1 Origin and processing of samples

Sample Season of cycle Day of composting Position Composting step Temperature (°C) Processing

Cultivation Cloning DGGE T-RFLP

W1 Winter 1 Core Heap 8 + +W3 Winter 3 Core Heap 35 + +W9E Winter 9 Edge Bunker 47 + +W9C Winter 9 Core Bunker 73 + +W15E Winter 15 Edge Bunker 53 + +W15C Winter 15 Core Bunker 78 + +W22 Winter 22 Core Mature compost 25 + +S3 Summer 3 Core Heap 44 + +S6 Summer 6 Core Bunker 56 + +S9 Summer 9 Core Bunker 81 + +S15 Summer 15 Core Bunker 79 + +S22 Summer 22 Core Mature compost 26 + + + +

Succession and Diversity of Mushroom Compost Bacterial Community

were widely applied eubacterial primers binding to phylo-genetically highly conserved regions of the 16S rDNA. 27Fforward [19] and 1492R reverse [27] primers were used forcultures and for DGGE and 27F labeled with tetrachloro-fluorescein phosphoramidite (TET) and 519R reverse [19]for T-RFLP. The PCR program with these primer pairsconsisted of an initial 95°C denaturation step for 5 min,followed by 32 amplification cycles of denaturation for 30 sat 94°C, 30 s at 52°C, and 1 min at 72°C and the finalelongation step at 72°C for 10 min. In the case of DGGE, anested PCR with 968F forward (with a 40-bp GC clamp),and 1401R reverse primers [12] was carried out to gainsufficient amount of amplified DNA from all samples. Thethermal profile of the second PCR was the following: 5 minat 95°C, 15 cycles for 30 s at 94°C, 30 s at 57°C, 45 s at72°C, then 20 cycles of 30 s at 94°C, 30 s at 48°C, 45 s at72°C; and final elongantion for 10 min at 72°C. Prior to T-RFLP, cloning, or sequence analysis, purification of PCRproducts was carried out using the Viogene PCR-M CleanUp System (Proteogenix, Illkirch Cedex, France).

DGGE and T-RFLP Analysis

DGGE separation of the GC-clamped PCR products wascarried out as described previously [23]. For furthersequencing of the dominant community members, follow-ing visualization, discrete bands were excised, resuspendedin 20 μL sterile diethylpyrocarbonate (DEPC)-treated water(Carl Roth, Karlsruhe, Germany), and eluted overnight at4°C. One-microliter aliquot of the supernatant was used forPCR reamplification with the same primer set without GCclamp and thermal profile as in the case of the nested PCR.For statistical analysis, the DGGE-banding pattern of thesamples was aligned and converted into binary similaritymatrix using Phoretix 1D software V4.0 (NonlinearDynamics, Newcastle upon Tyne, UK).

For T-RFLP analysis, aliquots of amplified 16S rDNA ofcompost samples (10 μL) or reamplified clones (3 μL) weredigested in a 20-μL reaction volume with 3 U restrictionendonucleases Hin6I and BsuRI, separately (Fermentas) for3 h at 37°C. Purification of enzymatic digests, electropho-resis of labeled fragments, and analyses of T-RFLP profileswere done as reported in an earlier study [32]. The sum ofthe peaks higher than 50 relative fluorescence units—except the primer peaks—was calculated. For statisticalanalysis, only chromatograms with total peak area between150,000 and 350,000 fluorescent units were considered.Consensus profiles of parallel samples and alignment of T-RFs of different samples were done by the T-Align program[33]. Only T-RFs with relative abundance greater than orequal to 1% in at least one of the samples were included insimilarity matrices. Each individual T-RF was scored aspresent or absent and analyzed as binary data.

Principal component analyses (PCA) were conductedusing the STATISTICA data analysis software system V7.1(StatSoft, Tulsa, OK, USA).

Sequence-aided T-RFLP

Final mature compost sample of the summer cycle (S22)was subjected to a more detailed analysis, in which T-RFpeaks were compared with the T-RFLP profile of individualclones and cultures for accurate identification.

For cloning, the PCR product was ligated into pGEM®-TEasy Vector System and then transformed into E. coliJM109 cells in accordance with manufacturer’s instructions(Promega). Single-cell colonies were suspended in 50 μLsterile DEPC-treated water (Carl Roth) and incubated at98°C for 5 min to lyse the cells. After chilling on ice andcentrifugation for 2 min at 13,000 rpm, the supernatantwas used in subsequent PCRs.

Amplification with TET-labeled 27F and 519R primerswas carried out for each strain and 200 positive clones. TheT-RFLP chromatogram of the reamplified clones and strainswas created as described for environmental samples. Theclones with identical Hin6I T-RFs were further groupedwith BsuRI. After comparison of the T-RFs of the clonesand strains with the T-RFLP pattern of sample S22, one tothree representative clones of clone clusters correspondingto T-RFs of sample S22 with relative abundance greaterthan or equal to 0.2% were submitted to sequence analysis.Relative abundance was calculated from individual peakarea and total peak area excluding primer peaks.

DNA Sequencing and Phylogenetic Analysis

Big Dye Terminator Cycle Sequencing Kit V3.1 (AppliedBiosystems) was used to determine the nucleotide sequen-ces of purified PCR products of each pure culture, selectedclone, and reamplified excised DGGE band. The sequenc-ing reactions were performed according to the manufac-turer’s protocol with the primers 27F (pure cultures, clones)and 1401R (DGGE bands). Sequencing products wereseparated on a Model 310 Genetic Analyzer (AppliedBiosystems). Sequences were analyzed and checked forquality using DNA Sequencing Analysis Software V5.2(Applied Biosystems).

Phylogenetic and molecular evolutionary analyses wereconducted using MEGAV4.0 [38]. Similarity searches in theNational Center for Biotechnology Information databasewere carried out using the basic local alignment search toolprogram [1]. The closest identified relatives were includedin further phylogenetic analyses. Alignment of the sequenceswas carried out using ClustalW (http://www.ebi.ac.uk/clustalw/). Evolutionary distances were calculated using themethod of maximum composite likelihood.

A. J. Székely et al.

The DNA sequences obtained in this study weresubmitted to European Molecular Biology LaboratoryNucleotide Sequence Database with accession numbersAM932206 to AM932226 (DGGE bands), AM932227 toAM932256 (clone library), and AM932257 to AM932282(isolated strains).

Results

DGGE Analysis

DGGE banding pattern changed greatly during heapcomposting (days 1–6). Only few bands present in theinitial composting samples remained through the ther-mophilic bunker stage (days 6–15). At the end of thetunnel process (day 22), the banding pattern changedagain, resulting in a profile characteristic for both thesummer and winter mature compost samples (S22 andW22, Fig. 1).

PCA of the binary data of DGGE profiles confirmed thedifferences between mesophilic and thermophilic samples(Fig. 2). Composting steps can be separated into threedistinct groups. Heap composting (day 3) and phase IImature compost (day 22) can be easily distinguished fromeach other and from the other samples. Samples of thethermophilic phase (days 6–15) are also grouped together

by the first two principal components regardless of theseason of the composting cycle. The spatial origin ofbunker samples had no essential influence on clustering.

Partial sequencing of 21 bands excised from the DGGEprofiles was determined. The recovered sequences hadhigh similarity to known bacteria or environmental sam-ples (≥96% identity). Many sequenced bands had closestrelatives originating from different waste-treating facil-ities such as compost, aerobic, and anaerobic digestors(Table 2).

Phylogenetically, the sequences were distributed in threegroups: Deinococcus–Thermus, Firmicutes, and γ-Proteo-bacteria. The dominant bands of the initial heap compost-ing samples (days 1–3) were related to γ-Proteobacteriaand aerobic Firmicutes groups, while the sequencesrecovered from thermophilic samples belonged to Dein-ococcus–Thermus and anaerobic Firmicutes groups. Basedon the sequences of the bands excided from the wintersamples, the two characteristic bands present in the higherdenaturant concentration area of all thermophilic samples(days 6–15) supposedly corresponded to the species T.thermophilus (Fig. 1; broken line). In the winter process,bands related to the anaerobic species Thermacetogoniumphaeum became prominent at the end of phase I compost-ing (Fig. 1; W15, solid line), showing the presence ofanaerobism even in the edge samples. The two intensivebands of the final mature compost samples were the speciesPseudoxanthomonas taiwanensis (Fig 1; S22 and W22,dotted line).

Figure 1 DGGE fingerprints of 16S rDNA from compost samples oftwo composting cycles (summer and winter). For explanation oflabels, see Table 1. The arrows and letters on the gel indicate theexcided and sequenced bands (Table 2.). Broken lines mark the bandscorresponding to Thermus thermophilus, solid line marks theThermacetogenium phaeum-related bands, and Pseudoxanthomonastaiwanensis bands are ringed around by dotted lines

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Figure 2 Principal component analysis (PCA) of DGGE profiles ofcompost samples. PC1 accounts for 21%, PC2 for 16%, and PC3 for15% of the variation. Heap samples are marked by circles (winter,filled; summer, empty), thermophilic samples by triangles (winter,filled; summer, empty), and mature compost samples by squares(winter, filled; summer, empty). For further explanation of labels, seeTable 1

Succession and Diversity of Mushroom Compost Bacterial Community

T-RFLP Analysis

Based on preliminary experiments with restriction endonu-cleases AluI, BsuRI, Hin6I, MspI, and TaqI (data notshown), Hin6I and BsuRI—as the enzymes giving the mostcomplex chromatograms—were chosen for the 16S ribo-somal ribonucleic acid (rRNA)-based T-RFLP profiling ofthe composting process. The changes of bacterial commu-nity during composting were analyzed using PCA of T-RFLP chromatograms (Fig. 3).

PCA of BsuRI digestion showed a similar clustering ofsamples as those of DGGE (Fig. 3a). Three groups weredistinguished: phase II compost samples (S22 and W22),winter heap samples (W1 and W3), and thermophilicsamples. The effect of spatial origin of the bunker sampleswas perceptible by PCA but only due to PC3.

The PCA of Hin6I digestion also grouped the maturecompost samples together (Fig. 3b, S22 and W22).However, in the case of the remaining samples, differenti-ation between the summer and winter composting cycles is

Table 2 Phylogenetic affiliation of sequences retrieved from DGGE bands

Sample name Band Closest phylogenetic relative

Namea,b Similarity (%) Phylogenyc Habitat—ecological niche Accession number

S9 A U. b. clone CFB-7 97.2 Reactor-degrading garbage AB274496Clostridium aldrichii DSM 6159 92.1 Firmicutes Wood-fermenting anaerobic digester X71846

S22 A Pseudoxanthomonas taiwanensis 99.7 γ-Pb. Hot springs AF427039B Thioalcalovibrio denitrificans 98.5 γ-Pb. Highly alkaline soda lakes AF126545

W1 A U. b. clone A-B-07 98.9 Thermophilic anaerobic reactor AB259980Exiguobacterium aurantiacum 93.7 Firmicutes Effluent of potato-processing factory X70316

B Ureibacillus suwonensis 99.7 Firmicutes Oyster mushroom compost AY850379

W3 A Psychrobacter faecalis 99.2 γ-Pb. Bioaerosol from pigeon feces AJ421528B Acinetobacter junii 100.0 γ-Pb. Diseased ornamental carp EF669479C Pseudomonas sp. BBTR25 99.2 γ-Pb. Swine effluent-impacted soil DQ337603

Pseudomonas lindanilytica 96.8 γ-Pb. Marine coastal water EF633314

W9 E A Thermus thermophilus 99.7 D-T. Hydrothermal vent AY554280B Thermus thermophilus 100.0 D-T. Aerobic organic waste digestor AY788091

W9 C A U. b. clone A55_D21_L_B_D03 100.0 Anaerobic solid waste reactor EF559044Halocella cellulolsilytica 93.4 Firmicutes Anaerobic sediment X89072

B Thermus thermophilus 99.8 D-T. Hydrothermal vent AY554280

W15 E A Thermacetogenium phaeum 96.9 Firmicutes Thermophilic methanogenic reactor AB020336B Thermus thermophilus 100.0 D-T. Aerobic organic waste digestor AY788091

W15 C A U. b. DGGE band M26711 99.5 Household waste compost DQ179086Thermacetogenium phaeum 96.1 Firmicutes Thermophilic methanogenic reactor AB020336

B U. b. DGGE band M26711 98.4 Household waste compost DQ179086Thermacetogenium phaeum 91.1 Firmicutes Thermophilic methanogenic reactor AB020336

C Thermus thermophilus 98.1 D-T. Hydrothermal vent AY554280D Thermus thermophilus 100.0 D-T. Aerobic organic waste digestor AY788091

W22 A Pseudoxanthomonas taiwanensis 100.0 γ-Pb. Hot springs AF427039B Pseudoxanthomonas taiwanensis 100.0 γ-Pb. Hot springs AF427039C Xylella fastidiosa OSL92–3 96.4 γ-Pb. Plant pathogenic bacterium AF159577

a For sequences with uncultured or nondescribed closest relatives, the closest described species is also stated.bU. b. Uncultured bacteriumcγ-Pb. γ-Proteobacteria, D-T. Deinococcus–Thermus

A. J. Székely et al.

clearly visible (mainly by PC3). The winter samples couldfurther be divided into heap (W1 and W3) and thermophilicsamples.

Culturing of Strains from Mature Compost

As DGGE and both T-RFLP analyses showed a verysimilar bacterial community developing toward the end of

the composting process (day 22) in both seasons, themature compost sample of the summer composting process(S22) was chosen for a more detailed bacterial communityanalysis.

Partial sequencing of 16S rRNA of 46 strains isolatedfrom the S22 sample was assessed (Table 3). Similarityanalysis of the sequences showed that the strains belongedto 20 different operational taxonomic units (OTUs; ≥97%identity) and represented five phylogenetic groups: Actino-bacteria (19 strains), Bacteroidetes (three strains), Firmi-cutes (seven strains), α-Proteobacteria (three strains), andγ-Proteobacteria (14 strains). The most common specieswere P. taiwanensis (ten strains) and Thermobifida cellulo-silytica (five strains). For three strains, no cultured relatives(≤93% similarity) could be found in the database; therefore,they supposedly belong to new, yet nondescribed species.

Cloning and Sequence-aided T-RFLP Analysis of MatureCompost

T-RFLP screening of the 200 clones of the clone librarygenerated from sample S22 resulted in 66 clone clusterswith different T-RFs. Comparison of the T-RFs of theclones with the environmental T-RFLP profile of sampleS22 resulted in the identification of 27 dominant T-RFs(relative peak area ≥0.2%). Sequence analysis of the repre-sentatives of these clone clusters showed that they corre-sponded to seven different phylogenetic groups (Table 3):Actinobacteria (six clusters), Firmicutes (two clusters),Gemmatimonadetes (one cluster), α-Proteobacteria (threeclusters), β-Proteobacteria (one cluster), γ-Proteobacteria(nine clusters), and δ-Proteobacteria (two clusters).

Based on the sequence data of the strains and clones,82% of the total peak area of the T-RFLP profile of sampleS22 could be identified, indicated by solid T-RF peaks inFig. 4, meaning that most of the dominant bacterialcommunity members were identified, under usual reserveabout methodological biases [32]. More than half of thecommunity (55% of total peak area) belonged to Proteo-bacteria-related sequences, of which 73% (40% of totalpeak area) corresponded to Xanthomonadaceae (mainlyPseudoxanthomonas genus-related) phylotypes. Based onthe T-RFLP pattern, the second biggest phylogenetic groupin phase II mushroom compost was Actinobacteria (18%),with Thermobifida (12%) and Thermomonospora (5%) asthe most common genera. Firmicutes species were only thethird most frequent group (4%) followed by Gemmatimo-nadetes (3%) and Bacteroidetes (1%). OTUs withoutknown previously isolated relatives (≤93% similarity)represent a remarkable part of total peak area (28%). Theseclones and strains belonged to Bacteroidetes, Firmicutes,Gemmatimonadetes, and Proteobacteria (Table 3).

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A

B

Figure 3 Principal component analysis of T-RFLP data sets derivedfrom BsuRI (a) and Hin6I (b) digestion of 16S rRNA amplified fromcompost samples. On graph a, PC1 represents 19%, PC2 13%, andPC3 11% variation; on graph b, PC1 accounts for 18%, PC2 for 15%,and PC3 for 11% of variation. Heap samples are marked by circles(winter, filled; summer, empty), thermophilic samples by triangles(winter, filled; summer, empty), and mature compost samples bysquares (winter, filled; summer, empty). For further explanation oflabels, see Table 1

Succession and Diversity of Mushroom Compost Bacterial Community

Table 3 Sequence identity and T-RF size of representative strains and 16S rDNA clones generated from mature mushroom compost sample (S22)

Strain/clonenumbera

Sizes ofT-RFs (bp)

Ratio of totalpeak area (%)b

Closest phylogenetic relative

Hin6I BsuRI Namec,d Similarity (%) Habitat—ecologicalniche

Accessionnumber

ActinobacteriaC 267 496 235 1.0 Arthrobacter flavus 94.0 Pond, Antarctica AB299278CEM 2 357 67 <0.2 Nonomuraea fastidiosa 98.9 Silt of hot spring MFU48844CEM 13 35 220 <0.2 Pseudonocardia thermophila 99.7 Mushroom compost AJ252830C 165 358 67 3.4 Thermobifida alba 99.4 Soil, dry-heated AF028247TSA 27 358 67 3.4 Thermobifida alba 100.0 Soil, dry-heated AF028247TSA 46 362 67 <0.2 Thermobifida cellulosilytica 99.9 Overheated compost AJ298058CEM 1 359 67 8.1 Thermobifida fusca 100.0 Soil CP000088C 235 360 67 8.1 Thermobifida fusca 100.0 Sludge compost AB211018CEM 19 363 181 <0.2 Thermocrispum agreste 98.8 Compost X79183C 223 68 251 2.6 U. b. DOK_BIODYN_clone199 99.6 Agricultural soil DQ827907

Thermomonospora chromogena 97.3 Soil AF116558CEM 23 370 67 0.7 Thermomonospora chromogena 95.9 Soil AF116558C 215 381 251 0.3 Thermomonospora chromogena 97.6 Soil AF116559CEM 16 381 251 0.3 Thermomonospora chromogena 98.9 Soil AF116558C 270 79 67 1.1 Thermomonospora curvata 94.4 Straw AF002262CEM 6 369 67 <0.2 Thermopolyspora flexuosa 99.1 Soil AY039253

BacteroidetesTSA 34 100 39 1.1 U. b. clone Niitsu9–3. 96.4 Garbage composting AB187722

Sphingobacterium kitahiroense 88.1 Soil AB361248TSA 35 100 39 1.1 U. b. clone WET-E-G23 99.4 Urban creek freshwater EF659287

Sphingobacterium kitahiroense 88.3 Soil AB361248FirmicutesCEM 41 239 310 0.5 Bacillus mojavensis 100.0 Desert soil EF433405TSA 52 240 311 0.5 Bacillus licheniformis 100.0 Unpublished BAC16SRRC 220 220 100 1.4 U. b. clone 1B07 97.3 Compost DQ346486

Moorella glycerini 91.1 Hot spring MGU82327CEM 17 220 98 1.4 Thermoactinomyces thalpophilus 100.0 Sheep manure AF138738C 282 490 67 2.2 U. b. clone JAB SMS 57 89.8 Soil AY694602

Thermobacillus composti 87.5 Composting reactor AB254031TSA 30 242 235 <0.2 Ureibacillus thermosphaericus 96.5 Sewage sludge compost AB210995

GemmatimonadetesC 214 374 180 2.6 U. b. clone Niitsu4–39. 93.3 Garbage composting AB187698

α-ProteobacteriaC 263 59 39 0.9 U. b. clone 2B13 96.9 Compost DQ346497

Caenimicrobium bisanense 89.9 Sludge of a textiledye works

EF100695

C 245 330 39 1.5 Chelatococcus daeguensis 92.1 Sludge of a textiledye works

EF584507

TSA 38 340 39 0.8 Chelatococcus daeguensis 98.9 Sludge of a textiledye works

EF584507

C 173 339 39 0.8 U. b. clone C7-DM-12 97.1 Mushroom compost DQ082889Devosia terrae 92.2 Soil EF067859

TSA 37 343 39 4.1 Rhodobium orientis 92.2 Soil and waterincluding salt

AM696713

β-ProteobacteriaC 257 62 154 2.9 Bacterium SM-5–6 93.7 Swine manure biofilm AY773137

Pusillimonas noertemannii 91.9 Aminonaphthalene-degradingcommunity

AY695828

γ-ProteobacteriaTSA 40 65 39 2.8 Luteimonas composti 95.5 Food waste compost DQ846687

A. J. Székely et al.

Discussion

In this study, microbial succession during mushroomcompost elaboration was monitored with DGGE and T-RFLP. In agreement with earlier studies comparing thesemethods [21, 25], DGGE revealed less diversity (15–22bands per sample) than T-RFLP (20–59 peaks greater thanor equal to 1% of total peak area per sample), but the evalua-tion of the results by PCA showed that both molecularfingerprinting techniques gave a similar clustering of thesamples. However, moderate discrepancies between DGGEand T-RFLP and between BsuRI and Hin6I digestion-basedT-RFLP profiles were apparent. DGGE and BsuRI diges-tion separated the mesophilic, thermophilic, and maturecompost samples regardless of the season (Fig. 2 and 3a),while Hin6I digestion also differentiated between thecomposting cycles (Fig. 3b). Spatial origin of the sampleshad no remarkable effect on their clustering (Fig. 2 and 3),denoting the efficacy of the mixing procedure.

A quick change in dominance relations of the microbialcommunity could lead to differences in PCA clustering.During heap composting, temperature increases intensively,and therefore, a pronounced shift between dominantphylotypes happens (Fig. 1), although presumably lessdominant species do not disappear completely. According-

ly, less sensitive DGGE detected a tight clustering ofsamples of the same day (S3, W3), where supposedly theenvironmental conditions were similar favoring the domi-nance of similar taxa, while T-RFLP clustered the winterheap samples tightly (W1, W3), showing the effect of theless dominant but still present microbes of the rawmaterials. The summer sample S3 in the case of T-RFLPgrouped with the thermophilic samples (Fig. 3), whichcould be explained by the faster temperature increase due tohigher ambient temperature in the summer (Table 1) and thesubsequent earlier appearance of thermophilic species.

A possible explanation for the dissimilarities of PCA ofBsuRI and Hin6I digestions is the diverse phylogeneticdistribution of the first recognition site of these enzymes.BsuRI differentiates between higher phylogenetic groups,such as Actinobacteria and Proteobacteria, while Hin6Igives a finer resolution of microdiversity of the mostcommon compost taxa, such as Pseudoxanthomonas,Thermobifida, and Thermomonospora (Table 3). The mainphylogenetic structure of the communities present at thedifferent steps of composting may not vary as much fromcycle to cycle, explaining the clustering of samples bythermal phases rather than by season according to BsuRIdigestion. However, as mushroom compost elaboration is amainly spontaneous process lacking any inoculation step,

Table 3 (continued)

Strain/clonenumbera

Sizes ofT-RFs (bp)

Ratio of totalpeak area (%)b

Closest phylogenetic relative

Hin6I BsuRI Namec,d Similarity (%) Habitat—ecologicalniche

Accessionnumber

C 268 205 69 3.8 U.b. clone EV221H2111601SAH73 91.9 Subsurface water DQ223225Natronocella acetinitrilica 90.2 Soda lake sediment EF103127

C 170 66 208 5.5 Sulfur-oxidizing bacterium NDII1.1 88.7 Hydrothermal vent AF170424Pseudoxanthomonas taiwanensis 85.8 Hot springs AF427039

C 102 67 208 3.9 U. b. clone Niitsu31–45. 92.9 Garbage composting AB188010Pseudoxanthomonas taiwanensis 88.3 Hot springs AF427039

C 200 90 39 1.2 Pseudoxanthomonas taiwanensis 97.2 Hot springs AF427039C 188 115 39 7.2 Pseudoxanthomonas taiwanensis 98.7 Hot springs AF427039TSA 31 115 39 7.2 Pseudoxanthomonas taiwanensis 99.9 Hot springs AF427039C 221 213 39 7.8 Pseudoxanthomonas taiwanensis 92 Hot springs AF427039TSA 49 373 39 <0.2 Pseudoxanthomonas byssovorax 99.9 Cotton waste composts AY928806C 144 526 39 11.4 Pseudoxanthomonas taiwanensis 99.6 Hot springs AF427039TSA 48 532 39 <0.2 Pseudoxanthomonas taiwanensis 99.7 Hot springs AF427039

δ-ProteobacteriaC 225 88 39 0.8 U. b. clone H50–814 93.0 Maturing compost EF174280

Chondromyces robustus 83.2 AJ233941C 231 57 75 0.8 U. b. clone FAC48 85.8 Forest soil DQ451487

Sorangium cellulosum 82.3 AF421890

aC Clone, CEM strains cultivated on compost extract media, TSA strains cultivated on tryptone soya agarb Calculated from Hin6I digestion-based T-RFLP chromatogramc For sequences with uncultured or nondescribed closest relatives, the closest described species is also stated.dU. b. Uncultured bacterium

Succession and Diversity of Mushroom Compost Bacterial Community

the microbes and the quality of the raw materials must havean effect on the finer composition of the microbialcommunities developing by way of composting, explainingthe result of Hin6I PCA. All of these findings suppose asimilar mechanism as described in the plant succession ofstudy of Fukami et al. [6], where the final composition ofthe experimental plots at the species level depended on thestartup sowing, but in respect of the trait groups, conver-gence to a common structure was evident.

Sequence analysis of the bands excided from the DGGEgel gave an insight into the dominant taxa of the microbialsuccession of composting (Table 2). Members of themesophilic Acinetobacter and Pseudomonas genera werefound in the third-day samples. These genera are wellknown for their fast-growing species with broad substratespecificities [15]; therefore, their detection in the rapidlychanging heap-composting period is not surprising. Inaddition, some of the species responsible for the intensivelignocellulose decomposition have also been identified byDGGE. The presence of T. thermophilus-related bandscould be demonstrated in the thermophilic samples (Fig. 1).This species is considered to take part in the breakdown ofhemicellulose by its xylanolytic activity [20]. Members ofthe spore-forming Ureibacillus genus have been detectedfrom the day 1 sample (Table 2, W1 band B) and from thephase II compost sample (S22) by cultivation (Table 3,strain 30), supposing the survival of this genus through theentire composting process. Similarly to an earlier study[22], DGGE bands related to Actinobacteria were notdetected. In summary, these results support the conceptionof a mesophilic–thermophilic–mesophilic succession ofbacterial species during composting.

All of the fingerprinting methods used in this studypresented high similarity between the phase II compost

samples (S22, W22), suggesting that, irrespective of thefreshness of the straw or the external temperature, the finalready-for-spawning compost had a characteristic microbialstructure. Accordingly, a detailed, polyphasic approach(culturing, cloning, T-RFLP) was used for the analysis ofS22 sample by comparing the community T-RFLP patternwith the sequence data of clones and cultivated strains fromthe same sample (sequence-aided T-RFLP; Fig. 4).

The result of this analysis presented a significant numberof OTUs of mature compost without cultured relatives.Many were related to environmental sequences originatingfrom compost or soil environments. The majority of thesesequences belonged to the Proteobacteria, but the mostabundant Firmicutes clone cluster (clone 282) also lacked aclose cultured relative (Table 3, Fig. 4). Opposed to thecloning-based garbage composting study of Takaku et al.[37], Bacteroidetes phylotypes were not dominant in phaseII mushroom compost. Although, one of the isolated strainswithout close cultured relatives (strain 34) was similar to aclone of the latter study.

Our combined analysis in accordance with DGGErevealed the unambiguous dominant presence of P. taiwa-nensis-related phylotypes in the mature compost. Strainsbelonging to this moderately thermophilic species havebeen detected earlier from cellulose-degrading communities[9, 36]. The study of a cellulose-degrading model commu-nity showed that Pseudoxanthomonas spp. may enhancecellulose degradation by their acetate-consuming effect andconsequent pH neutralization [16]. On the other hand, themost common genera of Actinobacteria in the examinedphase II compost were Thermobifida and Thermomono-spora, which are well known for their cellulose- andhemicellulose-degrading ability [7, 17]. The temperatureof the conditioning process in the tunnel (48°C) and the

68: Thermomonospora sp.

90:Pseudoxanthomonas sp.

66: Xanthomonadaceae67: Xanthomonadaceae

65: Xanthomonadaceae

115: Pseudoxanthomonas sp.

205: -Pb.

330: -Pb.

339-43: -Pb. 374: .Gemmatimonadetes

370: Thermomonospora sp.

496: Actinobacteria

490: Firmicutes220: Firmicutes

79: Actinobacteria

62: -Pb.

526: Pseudoxanthomonas sp.

213: Xanthomonadaceae

358-60: Thermobifida sp.

γ

β

α

α

Figure 4 T-RFLP profile of Hin6I-digested 16S rRNA genesamplified from the mature mushroom compost sample (S22). Thesolid T-RF peaks have been identified by sequencing of clones and/orstrains. The length (bp) of the T-RFs and the name of the major group

(≥1% of total peak area) corresponding to the peak are labeled (seeTable 3 for detailed identification). α-Pb. α-Proteobacteria, β-Pb. β-Proteobacteria, γ-Pb. γ-Proteobacteria

A. J. Székely et al.

nature of the compost explain the dominant presence ofthese bacteria. Presumably, these groups together form anextremely efficient cellulose-degrading community andenhance the development of the adequate mushroom-selective compost, which is characterized by high amountsof lignin compared to cellulose [8]. As such a consortiumhas not been found in unconditioned phase I mushroomcompost [26], their dominant presence supports the bene-ficial effect of heat treatment on compost resulting in highermushroom yields.

In conclusion, tracking of microbial succession duringmushroom compost production by 16S rDNA DGGE andT-RFLP analysis showed pronounced shifts in speciescomposition between early mesophilic and thermophilicsteps and mature, ready-for-spawning compost. The seasonof the composting process and the spatial origin of thesamples had only minor effect on the bacterial communityfingerprints, while the phase II compost samples (W22,S22) were tightly clustered by all PCAs regardless of themethod of fingerprinting. By sequence-aided T-RFLP ofmature compost, sequence data of cultivation and cloningwas uniquely coupled with the outcome of T-RFLPanalysis, resulting in a novel, more realistic semiquantita-tive description of the species diversity than cloning orcultivation by itself. Besides several OTUs without closecultured relatives, the final compost microbiota wasdominated by Pseudoxanthomonas-, Thermobifida-, andThermomonospora-related phylotypes, which were sup-posed to form an intensive cellulose-degrading consortiumand, to the best of our knowledge, for the first time havebeen identified together as dominant members of maturecompost community.

Acknowledgments We are thankful to the staff of Quality Cham-pignons, Korona Spawn Factory, and Mr. László Bujdosó forsampling and operating the compost system. This work was supportedby grant BIO 0029/2002 from the Ministry of Agriculture and RuralDevelopment.

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