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An investigation of the biodiversity of thermophilic

and thermotolerant fungal species in composts
using culture-based and molecular techniques

Adrian LANGARICA-FUENTES, Pauline S. HANDLEY, Ashley HOULDEN,

Graeme FOX, Geoffrey D. ROBSON*
Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road, Manchester M13 9PT, UK

article info abstract

Article history: In this study, the biodiversity of thermophilous fungi in two different commercial com-
Received 17 July 2013 posts was investigated using culture-based methods, denaturing gradient gel electro-
Revision received 26 March 2014 phoresis (DGGE) and tag-encoded pyrosequencing. 454 pyrosequencing of the internal
Accepted 19 May 2014 transcribed spacer (ITS) region recovered a total of 175 OTUs between the two composts.
Available online The Ascomycota was the dominant phylum in both composts (90 % of all sequences
Corresponding editor: recovered) with the thermophilic-rich orders Sordariales and Eurotiales being the most
Kevin Newsham numerous. Molecular studies demonstrated the frequent presence of several thermophilic
(Scytalidium thermophilum, Myriococcum thermophilum) and thermotolerant (Pseudallescheria
Keywords: boydii, Corynascus verrucosus and Coprinopsis sp.) fungi in the composts, despite the absence
Biodiversity of these species from the culture-based analysis. Conversely, Aspergillus fumigatus and
Composting Mycocladus corymbifer, which were the dominant species in cultivation analyses, had very
DGGE low representation in molecular studies. The results show that the previous picture of the
Tag-encoded pyrosequencing dominant thermophilous fungi in compost communities derived from culture-based
Thermophilous fungi analysis has been biased, and that composting environments represent a potentially rich
resource of novel fungi.
ª 2014 Elsevier Ltd and The British Mycological Society. All rights reserved.

Introduction composting is an exceptionally complex process in which

Composting is a self-heating, aerobic process in which organic
matter is decomposed under controlled conditions by the
action of micro-organisms (Finstein & Morris 1975; Kutzner
2000). In recent years, this practice has gained worldwide
importance as a way of managing large-scale waste and
decomposing it into a beneficial humus-like product that can
be used for improving soil quality and fertility (Epstein 1997;
Kumar 2011). From a microbiological perspective,
many different micro-organisms participate. The composting
process generally proceeds in predictable stages (mesophilic,
thermophilic and curing) that can be recognised by rises and
declines in temperature (de Bertoldi et al. 1983; Ryckeboer
et al. 2003). These temperature phases reflect the activities
of successive microbial populations performing the degrada-
tion of increasingly recalcitrant organic matter. As different
environmental factors (nutrient availability, water activity,
temperature, pH, etc.) change during the course of

* Corresponding author. Tel.: þ44 (0)161 275 5048; fax: þ44 (0)161 275 5082.
E-mail address: geoff.robson@manchester.ac.uk (G.D. Robson).
http://dx.doi.org/10.1016/j.funeco.2014.05.007
1754-5048/ª 2014 Elsevier Ltd and The British Mycological Society. All rights reserved.
An investigation of thermophilic fungi in composts
composting, the microbial populations responsible for the
degradative activities also change (Epstein 1997; Kutzner
2000).
The main groups of micro-organisms participating in the
composting process are bacteria and fungi (Kutzner 2000;
Hultman et al. 2010). Fungi are particularly important as
they play a central role in composting due to their ability to
attack organic residues that are too dry, acidic or low in
nitrogen for bacterial decomposition (Finstein & Morris 1975;
Ryckeboer et al. 2003). This activity is carried out as a result
of extensive hyphal networks that penetrate throughout the
composting material and decompose the more recalcitrant
organic fractions chemically and mechanically. In addition,
the hyphal network physically stabilises the compost, pro-
viding improved aeration and drainage (Singer et al. 2000).
A large number of fungal species can be found in com-
posting materials. During the mesophilic stage, most of the
fungal species appear to be those already present in the
original substratum prior to the composting process (Kutzner
2000). By contrast, thermophilic and thermotolerant fungi
from compost are usually well adapted to the process con-
ditions and can be found in composting piles from different
sources (Johri et al. 1999; Kutzner 2000). Thermophilic fungi
have been defined as those fungi that show no growth below
20  C and have a maximum growth temperature at or above
50  C (Cooney & Emerson 1964; Maheshwari et al. 2000). The
optimum growth temperature for these fungi usually ranges
between 40 and 50  C (Morgenstern et al. 2012). In contrast,
thermotolerant fungi can grow at temperatures below 20  C
but can also grow at temperatures above 50  C (Cooney &
Emerson 1964; Mouchacca 2000). The term “thermophilous”
has been used to designate both thermophilic and thermoto-
lerant fungi (Mouchacca 1997; Johri et al. 1999). Thermophi-
lous fungi have been suggested as important biodegradation
agents in composts due to the high temperatures occurring in
these systems (Ryckeboer et al. 2003) and they have been
linked to the degradation of recalcitrant substrates such as
cellulose, hemicellulose and lignin during the process
(Sharma 1989; Tuomela et al. 2000).
Despite the clear importance of thermophilic and ther-
motolerant fungi in composting, most of the research on
these species was conducted over thirty years ago. Due to
the limited availability of molecular techniques at that time,
enumeration and isolation of thermophilic fungi was mostly
performed using classical culture-based methods on rich
organic complex media (Cooney & Emerson 1964; Tansey
1971; Kane & Mullins 1973; Tansey & Jack 1977). Moreover,
only a few publications have concentrated on their diversity
and role in composting environments (Kane & Mullins 1973;
Klamer & Sochting 1998). Even though culture-based
approaches have been useful in identifying several species
of thermophilic fungi in compost, they are known to detect
only a small proportion of all fungi present in environ-
mental samples (Ishii et al. 2000; Ryckeboer et al. 2003;
Novinscak et al. 2009). Thus, thermophilic fungi playing
important roles in the degradation process and producing
enzymes of potential commercial interest may have
remained hitherto undetected. The need to investigate
previously studied substrata to unearth overlooked fungal
diversity has been highlighted several times (Hawksworth &
133
Rossman 1997; Blackwell 2011) and recent estimates suggest
that only about 100 000 fungal species are known from at
least 1.5 million, but possibly as many as 3 million, total
fungal species on Earth (Hawksworth 2012).
Recent studies on microbial diversity in composts have
begun to utilise culture-independent approaches such as
phospholipid fatty acid analysis (PLFA), denaturing gradient
gel electrophoresis (DGGE) of PCR-amplified DNA fragments
combined with the sequencing of relevant bands, terminal
restriction fragment length polymorphism analysis (T-RFLP)
and more recently, next-generation sequencing (Anderson &
Cairney 2004; Hultman et al. 2010; Lindahl et al. 2013). These
techniques have successfully determined microbial diversity
by targeting taxonomically relevant molecular markers such
as the 16S rRNA gene in bacteria and the 18S rRNA gene or the
internal transcribed spacers (ITS region) in fungi (Mitchell &
Zuccaro 2006; Novinscak et al. 2009). Next-generation
sequencing technologies (such as 454 pyrosequencing, Illu-
mina, SOLiD and IonTorrent) offer the opportunity to analyse
microbial communities at a very high level of detail (Tedersoo
et al. 2010; Monard et al. 2013).
To date, studies of microbial diversity in composts using
molecular techniques have focused on the bacterial com-
munity (Ishii et al. 2000; Poulsen et al. 2008; Szekely et al.
2009; De Gannes et al. 2013), with only a few recent studies
investigating the fungal populations using DGGE or clone
libraries (Hultman et al. 2009; Novinscak et al. 2009; Bonito
et al. 2010). No studies, however, have focused on thermo-
philic fungi or attempted to compare compost biodiversity
using culture-based techniques and molecular method-
ologies. The aim of this study was to use culture-based
methods, DGGE and tag-encoded pyrosequencing to assess
the biodiversity of thermophilic fungi that participate in the
composting process and compare the results obtained using
these techniques.
Materials and methods
Compost samples
Mature compost samples were obtained from two different
commercial composting facilities (A and B). The compost
obtained from site A (The Compost Shop, Hightown, Mersey-
side, UK) utilised forestry and botanical cuttings as starting
material. Compost from site B (Colima city council’s com-
posting site, Colima, Mexico) used a mix of botanical cuttings
and municipal solid waste. Both composts were produced
using the open windrow method with occasional turning to
ensure aeration of the process. Compost samples were
obtained at the curing (final) stage of the process and were
considered by their manufacturers as ready-to-use. Composts
were sieved (4 mm), mixed thoroughly to ensure homogeneity
before chemical analyses, culture-based experiments and
DNA extractions were performed.
Compost chemical analysis
Percentage water content (% w/w) in compost samples was
determined by measuring the difference in mass of a 5 g sample
134
before and after drying to constant weight at 110  C. The pH of
the composts was measured using a water extract with a
compost:distilled water ratio of 1:2 (w/v) using a pH meter.
Total nitrogen and carbon content (% w/w dry weight) were
determined simultaneously by dry combustion using approx-
imately 0.1 g of 0.5 mm sieved air-dried compost in a LECO
TruSpecTM CN autoanalyzer (LECO Corporation, MI, USA).
Fungal viable counts and isolation of thermophilous fungi
from compost
To estimate the fungal viable load of each compost at dif-
ferent incubation temperatures, 100 ml samples of a 1:20
dilution from a compost suspension (1 g of compost mixed
with 10 ml of PBS buffer) were used to inoculate Petri dishes
that contained either potato dextrose agar (PDA), soil extract
agar (SEA) or carboxymethyl cellulose agar (CMCA). The
compost suspension used was thoroughly homogenised and
compost aggregates disintegrated by vortexing for 2 min.
Before inoculating each plate, the suspension was vortexed
again for 10 s to ensure compost particles were suspended
in the supernatant, which was then spread onto the agar
medium surface. Chloramphenicol (50 mg ml1) and deoxy-
cholic acid (500 mg ml1) were added to the media to mini-
mise the growth of bacteria and to obtain more compact
fungal colonies, respectively. Five dishes of each medium
were incubated at five different temperatures (25  C, 37  C,
45  C, 50  C and 55  C) immediately after inoculation and
monitored at daily intervals for 7 d. Colony-forming units
per gram (CFU g1) dry weight of compost were determined
at the five temperatures on PDA, SEA and CMCA.
For the isolation of culturable thermophilic and thermo-
tolerant species, a larger number of plates (20 of each
medium) were inoculated using the technique described
above and incubated only at 45  C, 50  C and 55  C. Morpho-
logically distinct colonies from these plates were subcultured
and purified for their subsequent identification via ITS
sequencing.
Identification of thermophilous fungal isolates via ITS-rDNA
barcoding
For DNA extraction from fungal isolates, 250 ml conical flasks
containing 50 ml of potato dextrose broth (PDB) were inoculated
using a loop of spores or mycelia from pure cultures and
incubated at 45  C for 96 hr at 150 rpm. Biomass was harvested
using vacuum filtration, flash frozen in liquid nitrogen and
ground to a powder using a sterile mortar and pestle. Genomic
DNA was extracted from ground mycelia (0.5 g) using a
DNeasy Plant Mini extraction kit (Qiagen, West Sussex, UK),
following the manufacturer’s instructions. The ITS1-5.8S-ITS2
region of the fungal rRNA gene complex was amplified using
the universal fungal primers ITS1 (50 -TCCGTAGGT-
GAACCTGCGG-30 ) and ITS4 (50 -TCCTCCGCTTATTGATATGC-30 )
(White et al. 1990). The ITS region is widely used to identify
fungal species owing to its variability in both sequence and
length (Webb et al. 2000; Barratt et al. 2003; Kennedy & Clipson
2003; Cosgrove et al. 2007). Each 50 ml PCR reaction mix con-
tained the following: 1X NH4 reaction buffer (Bioline, London,
UK), 1.5 mM MgCl2, 0.4 mM of each primer, 0.2 mM of each dNTP,
A. Langarica-Fuentes et al.
2.5 U of BIOTAQ DNA Polymerase (Bioline, London, UK) and
2.5e5.0 ml of genomic DNA. Amplification was performed using
the following conditions: 94  C for 5 min; 35 cycles with dena-
turation at 94  C for 1 min, annealing at 56  C for 1 min, and
extension at 72  C for 1 min, with a final extension at 72  C for
5 min. In cases where the ITS1/ITS4 primer set failed to amplify
a product, the alternative primer set NL1 (50 -GCATATCAA-
TAAGCGGAGGAAAAG-30 ) and NL4 (50 -GGTCCGTGTTTCAA-
GACGG-30 ) (O’Donnell 1993) was used to amplify the D1/D2
variable region at the 50 end of the 28S rDNA gene. The PCR
reaction using this primer set contained the same reagents as
used for the ITS1/ITS4 reaction but with MgCl2 at a concen-
tration of 2.5 mM. Amplification was performed using the fol-
lowing conditions: 94  C for 5 min; 40 cycles with denaturation
at 94  C for 1 min, annealing at 60  C for 1 min, and extension at
72  C for 1 min, with a final extension at 72  C for 5 min. PCR
products were separated by gel electrophoresis along with a
known-size marker (Hyperladder IV, Bioline, UK) in order to
verify that the amplified products were present and were of the
expected size (w500e600 bp). Successfully amplified DNA was
purified using a QIAquick PCR Purification Kit (Qiagen, West
Sussex, UK) according to the manufacturer’s instructions and
sequenced in-house using an ABI Prism 3100 Genetic Analyser
(Applied Biosystems Inc., CA, USA). Sequences were used to
interrogate the NCBI nucleotide database using the BLAST
algorithm (http://www.ncbi.nlm.nih.gov/blast/).
DNA extraction from compost
The PowerSoil DNA Isolation Kit (MO-BIO Laboratories, CA,
USA) was used to extract DNA from samples of compost
(0.25 g) according to the manufacturer’s instructions before
and after incubation at 50  C for 2 weeks. The incubation
treatment was performed to promote the enrichment and
survival of thermophilous organisms in the samples. For the
DNA extraction of the non-incubated samples (I), compost
material obtained immediately after the sieving and homog-
enisation steps was used. For the incubated samples (þI),
compost (500 g) was adjusted to 30 % moisture content and
incubated at 50  C for 2 weeks prior to DNA extraction. During
the incubation period, water lost due to evaporation was
added at 3-d intervals and proper aeration was maintained.
Extractions were carried out in triplicate for each sample and
then pooled into a single aliquot to obtain a representative
DNA sample. DNA concentrations in extracts were deter-
mined at 260 nm using a NanoDrop ND-1000 Spectropho-
tometer and stored at 20  C until required.
DNA metabarcoding of compost fungal communities using
454 pyrosequencing
The ITS1 region of the fungal rRNA gene complex was amplified
using the ITS5/ITS2 primer set and sequenced using the 454
pyrosequencing GS Junior technology (Roche 454 Life Sciences,
Branford, CT, USA). The ITS1 region has been used successfully
to examine fungal communities in other next-generation
sequencing studies (Buee et al. 2009; Jumpponen & Jones
2009; Tedersoo et al. 2010). Barcodes and adapter regions were
added to the ITS5 and ITS2 primers to construct fusion primers
appropriate for 454 sequencing (Supplementary Table S1).
An investigation of thermophilic fungi in composts
For PCR amplification, each 50 ml PCR reaction mix con-
tained the following reagents: 1X FastStart High Fidelity
reaction buffer (Roche Diagnostics GmbH, Mannheim, Ger-
many), 1.5 mM MgCl2, 0.4 mM of each primer, 0.2 mM of each
dNTP, 3 % (v/v) DMSO, 0.3 mg/ml of BSA, 5 U of FastStart High
Fidelity polymerase (Roche Diagnostics GmbH, Manheim,
Germany) and 5 ml of genomic DNA. The PCR was performed
under the following conditions: an initial denaturation step at
95  C for 5 min; 25 cycles of denaturation at 95  C for 45 s,
annealing at 54  C for 45 s, and extension at 72  C for 1 min,
with a final extension at 72  C for 10 min.
PCR products were run on a FlashGel cassette (Lonza,
Basel, Switzerland). Bands of the expected size (300e400 bp)
were recovered. DNA was further purified using two rounds of
Agencourt AMPure XP (Beckman Coulter, Inc., CA, USA) bead
clean-up using a DNA:bead volume ratio of 0.7:1. Fragment
size and efficient removal of primer-dimers or other small
DNA fragments were verified using a 2100 Bioanalyzer (Agilent
Technologies, Inc., CA, USA). PCR products were pooled
together at equimolar ratios to prepare for pyrosequencing.
The concentration of double-stranded DNA was accurately
quantified using Picogreen dsDNA reagent and a Qubit 2.0
fluorometer (Invitrogen, Ltd, Paisley, UK) prior to pooling of
samples. Emulsion PCR and sequencing of the pooled samples
in a single 454 GS Junior run were carried out according to the
manufacturer’s instructions.
Bioinformatic processing of pyrosequencing data
Sequence processing, clustering, taxonomic assignment and
biodiversity calculations were performed using the bio-
informatic pipeline QIIME v. 1.6.0 (Caporaso et al. 2010) in con-
junction with the UNITE/QIIME 12_11 ITS reference database (all
fungal rDNA ITS sequences in the current UNITE þ INSD release
(z300 000 sequences)) (http://unite.ut.ee/repository.php).
Firstly, sequences were de-multiplexed and primer and barcode
sequences were removed. During the quality-filtering step,
sequences that were too short or too long (<200 bp or >1000 bp),
those with homopolymer runs longer than eight nucleotides or
with a mean quality score of <25 were removed. The remaining
sequences were denoised using Denoiser (Reeder & Knight
2010) and OTUs were then picked using an open-reference
OTU picking protocol, where sequences were clustered
against the UNITE/QIIME database using the UCLUST
Ref algorithm at 97 % similarity (Edgar 2010) and the sequences
that did not match the database were clustered de novo. Sus-
pected chimeras, PCR artefacts and sequences belonging to
other eukaryotic kingdoms were removed manually after this
step. A representative set of OTUs was generated and then the
taxonomy for each of the OTUs was assigned using the UNITE/
QIIME database. Statistical analyses, such as rarefaction curves
and alpha diversity metrics (Chao1, Shannon’s Index (H) and
Equitability Index (J)) were produced for each of the samples in
QIIME using an OTU table rarefied to 4 500 sequences.
DGGE analysis of compost fungal communities
DGGE analysis was performed on the same samples used for
pyrosequencing to cross-validate and complement the results
of the metabarcoding approach. The ITS1 region of the fungal
135
rRNA gene complex was amplified using the primers ITS5-GC
(50 -CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGG-
GAAGTA-AAAGTCGTAACAAGG-30 ) and ITS2 (50 -
CTGCGTTCTTCATCGAT-30 ) (White et al. 1990). The PCR mix
and cycle regime that were used are described by Cosgrove
et al. (2007). DGGE analyses were carried out using the D-
Code Universal Mutation Detection System (Bio-Rad, Hert-
fordshire, UK). Acrylamide gels (10 % w/v) were prepared with
a urea and formamide denaturing gradient ranging from 25 %
to 45 % in 1X TAE using a 50 ml gradient mixer (Scie-Plas Ltd,
Cambridge, UK). Purified PCR products (500 ng) were loaded
into each lane of the gel and run at 65  C and 42 V for 16 hr in
1X TAE (pH z 8.5). Gels were stained with SybrGold Nucleic
acid gel stain (Molecular Probes, Leidien, The Netherlands) for
1 hr and photographed using a UV gel documentation system
(Wolf Laboratories Ltd, York, UK). Prominent DGGE bands
were excised from the gel using a surgical blade and sub-
sequently extracted and purified using a QIAquick Gel
Extraction Kit (Qiagen, West Sussex, UK) according to the
manufacturer’s instructions. Purified DNA was reamplified
using the ITS5-GC/ITS2 primer set and resolved by DGGE to
verify the position of the original band. DNA fragments were
then sequenced and the results were used to interrogate the
NCBI nucleotide database using the BLAST algorithm (http://
www.ncbi.nlm.nih.gov/blast/).
Nucleotide sequence accession numbers
DNA sequences belonging to the different morphotypes
recovered during the culture-based analyses were deposited
in the GenBank (NCBI) sequence database under accession
numbers JQ898121eJQ898132. The nucleotide sequences from
the key DGGE bands recovered are also available in GenBank
under accession numbers JQ898133eJQ898142. The de-multi-
plexed ITS dataset obtained using pyrosequencing was
deposited on MG-RAST under project number 6291 (accession
numbers 4539036.3e4539039.3).
Results
Compost chemical analysis
The results of the chemical analysis performed on the two
composts samples (A and B) are summarised in Table 1.
Compost A had a water content of 31.5 % (w/w) and a pH of
7.54, while compost B had a lower water content of 21.8 % (w/
w) and a pH of 8.14. Total carbon and nitrogen content was
higher in compost A than in compost B, indicating that com-
post A was richer in nutrients. However, the C/N ratios of the
composts were similar (11.28 and 9.10, respectively).
Fungal viable counts and culture-based isolation of
thermophilic fungi from compost
Cultivation experiments were performed to evaluate the via-
ble fungal load at temperatures within the mesophilic (25  C
and 37  C) and thermophilic (45  C, 50  C and 55  C) temper-
ature range (Fig 1). The highest total fungal viable count
observed in compost A was 3.30  104 (0.47  104) CFU g1 dry
136
Table 1 e Chemical analysis of the two composts used in
the biodiversity assessment
Compost A Compost B
Water content (%) 31.5 21.8
pH 7.54 8.14
Total carbon (%) 13.66  0.29 % 6.82  0.51 %
Total nitrogen (%) 1.21  0.06 % 0.75  0.02 %
C/N ratio 11.28  0.60 9.10  0.30
weight when incubated on SEA at 25  C, while the highest total
fungal viable count in compost B was 3.80  104 (0.34  104)
CFU g1 dry weight on SEA at 45  C (Fig 1). Compost A had a
greater mesophilic than thermophilic fungal load, as CFU
counts at 25  C and 37  C were the highest for this compost. By
contrast, compost B had a greater fungal load at 37  C, 45  C
and 50  C, suggesting the presence of a higher number of
thermophilic and/or thermotolerant species (Fig 1).
Six and seven morphologically different fungal colony
types capable of growing at temperatures of 45e55  C were
observed in compost A and B respectively and were identified
by phylogenetic analysis of the ITS/28S region (Table 2).
Aspergillus fumigatus accounted for ca. 75 % of all colonies
recovered from compost A with a viable count of 4.0  103
(0.14  103) and 6.5  103 (1.26  103) CFU g1 dry weight at
45  C on PDA and SEA, respectively. Thermomyces lanuginosus
Fig 1 e Mean total fungal viable count (CFU/g dry weight) of
compost A and B when incubated at different temperatures
on PDA, SEA and CMCA media. Data represent the mean of
three replicates ± SEM.
A. Langarica-Fuentes et al.
was the dominant species found in compost B, with a viable
count of 2.76  104 (0.39  104) CFU g1 dry weight at 45  C on
SEA (>70 % of total colonies recovered).
DNA metabarcoding of compost fungal communities using
454 pyrosequencing
31 069 sequences were obtained in the pyrosequencing run for
the four samples analysed in this study. After demultiplexing,
removal of short and poor quality sequences and reverse
primer trimming, 26 188 high-quality sequences remained.
After OTU clustering was performed and non-fungal taxa and
suspected artefacts were removed, the final OTU table con-
tained 175 OTUs that accounted for 24 461 sequences from the
four samples. The average number of sequences per sample
was 6115  381. The samples with the highest and lowest
number of sequences had 6607 and 4997 sequences, respec-
tively. These data were used subsequently for all further
taxonomic and diversity analyses (Supplementary Table S2).
Rarefaction analysis of the OTUs assigned at 97 % similarity
(Fig 2) revealed a high fungal richness in the compost samples.
Although not completely saturated, the rarefaction curves of
the four libraries were close to reaching the plateau zone and
indicate that the majority of the OTUs present in the samples
were recovered with the sequencing depth attained. Of the 175
OTUs obtained in the study, 114 were present in compost A
prior to incubation, 93 in the compost A after incubation, 79 in
compost B before incubation and 88 in compost B after incu-
bation. Of the 175 OTUs, 84 OTUs were present in the two
composts, 59 were unique to compost A and 32 were unique to
compost B. Diversity indices calculated at a sampling depth of
4500 sequences (Table 3) revealed that prior to incubation,
compost B had a higher Shannon’s Index value than compost
A, despite having a lower number of OTUs.
Taxonomic assignment of fungal OTUs using QIIME
revealed that in compost A prior to incubation, the phylum
with the highest number of sequences was the Ascomycota
(95.1 %), followed by the Basidiomycota (1.0 %) and the sub-
phylum Mucoromycotina (formerly of the Zygomycota) (0.7 %).
Three percent of the sequences from this sample could not be
assigned to a phylum. When OTUs were analysed at the order
level, the most abundant orders of fungi in compost A prior to
incubation were the Sordariales (53.7 %), Orbiliales (21.6 %),
Microascales (5.4 %), Eurotiales (3.9 %) and Hypocreales (1.8 %)
(Fig 3). The most abundant OTUs in this compost before incu-
bation, (Table 4) included thermophilic or thermotolerant
fungi (Scytalidium sp. (48.6 %) and Talaromyces thermophilus
(1.2 %)), but we also detected other species that are generally
considered to be mesophilic (e.g. Arthrobotrys amerospora
(21.7 %), Pseudallescheria fimeti (2.6 %), Thielavia sp. (1.7 %) and
Arthrographis kalrae (1.2 %)) and OTUs with poor similarity to
known sequences such as an uncultured Ascomycota OTU
(5.4 %) and an uncultured fungal OTU (1.3 %). Other thermo-
philic/thermotolerant species detected at lower frequencies
were T. lanuginosus (1.1 %), A. fumigatus (0.6 %), Pseudallescheria
boydii (0.6 %), Corynascus verrucosus (0.4 %), Coprinopsis sp.
(0.2 %), Mortierella wolfii (0.1 %), Myceliophthora sp. (0.08 %),
Myriococcum thermophilum (0.06 %) and Rhizomucor miehei
(0.02 %). In terms of unknown or poorly characterised species
present in compost A, 16.9 % of all sequences belonging to 64
An investigation of thermophilic fungi in composts 137

Table 2 e Fungi isolated from composts A and B as identified by ITS/28S sequence similarity
Isolate Media of Putative identity based on % Similarity NCBI accession number Observed frequency
cultivation closest match in NCBI database of morphotypea

Compost A
A-BG PDA Emericella nidulans 99.2 FJ878645.1 þ
A-DG PDA, SEA, CMCA Aspergillus fumigatus 99.7 JQ356539.1 þþþ
A-S PDA, SEA Mycocladus corymbifer (Absidia ramosa) 100 JQ912660.1 þþ
A-W PDA, SEA Talaromyces thermophilus 100 JF412001.1 þ
A-P PDA, SEA Myceliophthora thermophila 99.6 JN659479.1 þ
A-R PDA, SEA Thermomyces lanuginosus 100 GU441538.1 þ
Compost B
B-P PDA, SEA Myceliophthora thermophila 92.2 HQ871765.1 þ
B-R PDA, SEA Thermomyces lanuginosus 100 JN600618.1 þþþ
B-BG PDA Emericella nidulans 99.1 FJ878645.1 þ
B-S PDA, SEA Mycocladus corymbifer (Absidia ramosa) 99.8 HQ285687.1 þ
B-DG PDA, SEA, CMCA Aspergillus fumigatus 99.7 JQ356539.1 þþ
B-Y PDA, SEA Malbranchea cinnamomea 100 JF922020.1 þ
B-CO PDA Emericella sp. 100 EF025927.1 þ

a Subjective measure of the frequency with which each morphotype was observed on the SEA and PDA plates : þ, morphology of <5 % of the
colonies; þþ, morphologies of 20e30 % of the colonies present; þþþ, the dominant morphotype, representing >70 % of colonies present on the
plates.

different OTUs could only be assigned up to the family level or
above (Supplementary Table S2).
In compost B, the dominant phylum was again the Asco-
mycota (83.2 % of sequences). The Basiodiomycota (11.7 %)
and the subphylum Mucoromycotina (4.1 %) had higher per-
centage in this compost in comparison with compost A,
while 1 % of sequences in sample B could not be assigned to a
phylum. At the order level, the most abundant groups were
the Sordariales (29.1 %), Eurotiales (21.3 %), Agaricales (11.6 %)
and Mortierellales (4.1 %) (Fig 3). Sequences that could only be
assigned to orders in the Sordariomycetes (14.0 %) or other
Ascomycota (16.9 %) also accounted for high percentages of
the population (Fig 3). The most abundant OTUs in compost B
before incubation are shown in Table 5. In this compost, the
most abundant OTUs were mainly the thermophilic and
thermotolerant species T. lanuginosus (20.7 %), M. thermophi-
lum (13.4 %), Coprinopsis sp. (11.5 %), C. verrucosus (8.6 %),
Scytalidium sp. (5.2 %) and M. wolfii (4.0 %). Among the 10 most
abundant OTUs, P. fimeti (0.53 %) was the only species that
has not been previously associated with thermotolerant
behaviour. OTUs with poor similarity were again abundant in
this compost, such as an uncultured soil fungus OTU (16.6 %)
and an OTU whose closest match (82 % similarity) was P.
boydii (13.8 %). Other thermophilic/thermotolerant species
detected at lower frequencies were A. fumigatus (0.15 %),
Emericella sp. (0.05 %), Thielavia heterothallica (0.03 %), Ther-
moascus aurantiacus (0.03 %), P. boydii (0.02 %) and Mycelioph-
thora sp. (0.02 %). In terms of unknown or poorly
characterised species present in compost B, 32.8 % of all
sequences belonging to 43 different OTUs could only be
assigned to the family level or above (Supplementary
Table S2).
Effect of incubation at 50  C on species diversity and selection of
thermophiles
Taxonomic assignment of the OTUs from compost A showed
that the percentage of sequences belonging to the different
phyla after incubation at 50  C for 2 weeks remained relatively
unchanged. The frequencies of the ascomycetes at the start
and end of the incubation were 95.1 % and 97.8 %, those of the

Table 3 e Diversity indices calculated for the non-

incubated samples (LI) and incubated samples (DI) of
composts A and B using the 454 sequencing dataset of the
fungal ITS region rarefied to 4,500 sequences
Compost Number Chao1 Shannon Equitability
Fig 2 e Calculated rarefaction curves of observed OTU sample of OTUs estimate diversity index (J)
index (H)
richness for the different composts at 97% sequence
similarity. Compost A, room temperature (A L I); compost AI 113 129.2 3.03 0.44
A, after incubation at 50  C (A D I); compost B, room AþI 81 167.1 0.99 0.16
temperature (B L I); Compost B, after incubation at 50  C BI 65 86.2 3.29 0.54
BþI 79 96.1 3.38 0.54
(B D I).
138 A. Langarica-Fuentes et al.

Fig 3 e Phylogenetic assignment (order level or above) of fungal sequences at recovered from the different compost samples
using 454 pyrosequencing. Compost A, room temperature (A L I); compost A, after incubation at 50  C (A D I); compost B,
room temperature (B L I); compost B, after incubation at 50  C (B D I). Letter between brackets indicates the phylum to which
the order belongs: (A), Ascomycota; (B), Basidiomycota; (M), subphylum Mucoromycotina. Data shown next to each
taxonomical category indicates relative abundance (%) in the different samples.

basidiomycetes were 1.0 % and 0.8 %, and those of the sub- different orders showing substantial changes. The frequency
phylum Mucoromycotina were 0.7 % and 0.6 %, respectively. of the Sordariales increased dramatically, with the relative
However, at the order level, a shift in the community com- abundances of this order at the start and end of the incubation
position was observed, with the relative abundance of several being 53.7 % and 91.5 %, respectively. Members of the order
 An investigation of thermophilic fungi in composts
Table 4 e List of the 10 most abundant operational taxonomic units (OTUs) found in compost A at room temperature (LI) and after incubation at 50  C (DI)
OTU Putative identity based on closest match in NCBI database Taxonomic assignment in QIIME using RDP classifier Abundance

Closest NCBI Coverage Similarity Accession Phylum Order Family Genus No. of sequences % Total
database match (%) (%) no.

Before incubation (A L I)
1 Uncultured Scytalidiuma 100 99 JF905969.1 Ascomycota Sordariales Chaetomiaceae Scytalidium 2430 48.6 %
2 Arthrobotrys amerospora 100 99 AF106533.1 Ascomycota Orbiliales Orbiliaceae Arthrobotrys 1082 21.7 %
3 Uncultured Ascomycota 89 90 FR682424.1 Ascomycota e e e 269 5.4 %
4 Pseudallescheria fimeti 100 97 KC461507.1 Ascomycota Microascales Microascaceae Pseudallescheria 130 2.6 %
5 Thielavia sp./Chrysosporium 100 99 EU620166.1/ Ascomycota e e e 87 1.7 %
synchronum AM943023.1
6 Scedosporium prolificans 100 91 AB362936.1 Ascomycota Microascales Microascaceae e 78 1.6 %
7 Sordaria sp. 100 99 JN207345.1 Ascomycota Sordariales Sordariaceae e 72 1.4 %
8 Uncultured fungus 45 100 JF433024.1 e e e e 64 1.3 %
9 Arthrographis kalrae 100 97 AB213441.1 Ascomycota Incertae sedis Eremomycetaceae Arthrographis 61 1.2 %
10 Talaromyces thermophilusa 100 99 JF412001.1 Ascomycota Eurotiales Trichocomaceae Talaromyces 60 1.2 %
After incubation (A D I)
1 Uncultured Scytalidiuma 100 99 JF905963.1 Ascomycota Sordariales Chaetomiaceae Scytalidium 5605 89.6 %
2 Thermomyces lanuginosusa 100 99 JQ639282.1 Ascomycota Eurotiales Incertae sedis Thermomyces 160 2.6 %
3 Pseudallescheria boydiib 100 100 AB489088.1 Ascomycota Microascales Microascaceae Pseudallescheria 37 0.6 %
4 Pseudallescheria fimeti 100 97 KC461507.1 Ascomycota Microascales Microascaceae Pseudallescheria 34 0.5 %
5 Myriococcum thermophiluma 100 100 JF412008.1 Ascomycota Sordariales Chaetomiaceae Myriococcum 34 0.5 %
6 Coprinopsis sp.b 100 99 FJ548835.1 Basidiomycota Agaricales Psathyrellaceae Coprinopsis 32 0.5 %
7 Uncultured soil fungus 100 100 DQ980586.1 Ascomycota e e e 28 0.4 %
8 Aspergillus fumigatusb 100 100 KC492453.1 Ascomycota Eurotiales Trichocomaceae Aspergillus 25 0.4 %
9 Corynascus verrucosusb 100 100 FJ537093.1 Ascomycota Sordariales Chaetomiaceae Corynascus 22 0.4 %
10 Uncultured fungus 45 100 JF433024.1 e e e e 20 0.3 %

a Indicates a species regarded as thermophilic.

b Indicates a species generally regarded as thermotolerant.

139
 140
Table 5 e List of the 10 most abundant operational taxonomic units (OTUs) found in compost B at room temperature (LI) and after incubation at 50  C (DI)
OTU Putative identity based on closest match in NCBI database Taxonomic assignment in QIIME using RDP classifier Abundance

Closest NCBI database match Coverage (%) Similarity (%) Accession no. Phylum Order Family Genus No. of sequences % Total

Before incubation (B L I)
1 Thermomyces lanuginosusa 100 99 JQ639282.1 Ascomycota Eurotiales Incertae sedis Thermomyces 1367 20.7 %
2 Uncultured soil fungus 100 100 DQ980586.1 Ascomycota e e e 1092 16.6 %
3 Pseudallescheria boydiib 100 82 EF639869.1 Ascomycota e e e 911 13.8 %
4 Myriococcum thermophiluma 100 100 JF412008.1 Ascomycota Sordariales Chaetomiaceae Myriococcum 886 13.4 %
5 Coprinopsis sp.b 100 99 FJ548835.1 Basidiomycota Agaricales Psathyrellaceae Coprinopsis 761 11.5 %
6 Corynascus verrucosusb 100 100 FJ537093.1 Ascomycota Sordariales Chaetomiaceae Corynascus 566 8.6 %
7 Uncultured Scytalidiuma 100 99 JF905969.1 Ascomycota Sordariales Chaetomiaceae Scytalidium 342 5.2 %
8 Mortierella wolfiib 94 100 JN943806.1 Mucormycotina Mortierellales Mortierellaceae Mortierella 266 4.0 %
9 Scytalidium thermophiluma 100 100 AB085928.1 Ascomycota Sordariales Chaetomiaceae Scytalidium 91 1.4 %
10 Pseudallescheria fimeti 100 97 KC461507.1 Ascomycota Microascales Microascaceae Pseudallescheria 35 0.5 %
After incubation (B D I)
1 Thermomyces lanuginosusa 100 99 JQ639282.1 Ascomycota Eurotiales Incertae sedis Thermomyces 1222 18.5 %
2 Myriococcum thermophiluma 100 100 JF412008.1 Ascomycota Sordariales Chaetomiaceae Myriococcum 1175 17.8 %
3 Coprinopsis sp.b 100 99 FJ548835.1 Basidiomycota Agaricales Psathyrellaceae Coprinopsis 1099 16.6 %
4 Uncultured soil fungus 100 100 DQ980586.1 Ascomycota e e e 873 13.2 %
5 Pseudallescheria boydii 100 82 EF639869.1 Ascomycota e e e 786 11.9 %
6 Uncultured Scytalidiuma 100 99 JF905969.1 Ascomycota Helotiales Incertae sedis Scytalidium 516 7.8 %
7 Corynascus verrucosusb 100 100 FJ537093.1 Ascomycota Sordariales Chaetomiaceae Corynascus 216 3.3 %
8 Pseudallescheria fimeti 100 97 KC461507.1 Ascomycota Microascales Microascaceae Pseudallescheria 127 1.9 %
9 Uncultured fungus 45 100 JF433024.1 e e e e 80 1.2 %
10 Scytalidium thermophiluma 100 100 AB085928.1 Ascomycota Sordariales Chaetomiaceae Scytalidium 63 1.0 %

a Indicates a species regarded as thermophilic.

b Indicates a species generally regarded as thermotolerant.

A. Langarica-Fuentes et al.
An investigation of thermophilic fungi in composts
Orbiliales, which was the second most abundant order before
incubation (21.6 %) were not detected following incubation.
Other fungal orders showing considerable variation were the
Microascales, the relative abundance of which decreased from
5.4 % to 1.3 %, and the Hypocreales, which decreased in fre-
quency from 1.8 % to 0.5 % (Fig 3). Closer examination at the
OTU level revealed that several mesophilic organisms that
were among the most abundant OTUs before incubation (e.g.
A. amerospora, Thielavia sp., A. kalrae, Sordaria sp. and Scedo-
sporium prolificans) decreased substantially (>90 % reduction in
relative abundance) or were not detectable following incuba-
tion. In contrast, some thermophilic fungi, such as Scytalidium
sp., T. lanuginosus and M. thermophilum, showed substantial
increases in relative abundance in comparison with the
unincubated sample (82 %, 124 % and 800 % increases,
respectively). However, not all thermophilous species showed
similar increases in abundances after incubation, with the
frequencies of some remaining constant or decreasing (e.g. A.
fumigatus, C. verrucosus, M. wolfii, Myceliophthora sp., R. miehei
and T. thermophilus). Incubation at 50  C for 2 weeks caused an
apparent decrease in the evenness of species (from 0.44 to
0.15) and in Shannon’s diversity (from 3.03 to 0.98; Table 3),
largely owing to the substantial increase in the population of
Scytalidium.
For compost B, incubation at 50  C for 2 weeks appeared to
have a weaker impact on fungal community composition. The
most abundant phylum continued to be the Ascomycota, the
frequency of which was 83.2 % and 79.6 % pre- and post-incu-
bation. The frequencies of the Mucoromycotina before and
after incubation were 4.1 % and 0.8 %, while those of the Basi-
diomycota were 11.7 % and 17.1 % and those of unknown fungi
were 1.0 % and 2.4 %, respectively. At the order level, the most
abundant groups continued to be the Sordariales (30.7 %),
Eurotiales (19.6 %), Agaricales (16.7 %), other Ascomycota
(13.2 %) and other unassigned orders in the Sordariomycetes
(12 %). The relative abundances of these orders varied slightly
when compared with those observed before incubation (20 %
change). The most abundant OTUs in compost B after incu-
bation were similar to those observed before the treatment,
with most of these fungi being thermophiles (Table 5). As with
compost A, the relative abundance of some of these thermo-
philous species apparently increased. The frequencies of Scy-
talidium sp., M. thermophilum and Coprinopsis sp. apparently
increased by 50, 44 and 32 %, respectively, while others, such as
T. lanuginosus, Emericella sp., P. boydii and T. heterothallica,
remained unchanged or decreased slightly. Unlike the fungal
community in compost A, that in compost B remained largely
balanced, with no dominant species following incubation. For
compost B, the Shannon’s diversity Index and Equitability
Index (evenness of species) remained largely unchanged after
the incubation at 50  C (Table 3).
When comparing the 10 most abundant OTUs of the two
compost samples (Tables 4 and 5), we observed that before
incubation there were only two species in common between
the two composts. Among the 10 most abundant OTUs in
compost A were a large fraction of mesophilic species, while
thermophiles seemed more abundant in compost B. However,
after 2 weeks of incubation at 50  C, eight of the 10 most
abundant species were found in both composts. Most meso-
philic species in compost A (Scytalidium thermophilum, T.
141
lanuginosus, M. thermophilum, Coprinopsis sp., P. fimeti and two
uncultured fungi) either decreased substantially or were not
detectable after incubation.
Fingerprinting of the fungal compost community by DGGE
analysis
Sequence-dependent separation of amplified ITS PCR prod-
ucts using DGGE was used successfully to examine the
diversity of fungal species present in compost A and B and
cross-validate the results of the pyrosequencing approach
(Supplementary Fig S1). While the overall resolution of the
technique was low (on average 12.2  0.5 bands were detected
in the DGGE fingerprints), excision and sequencing of the most
prominent bands identified the same dominant species in the
454 pyrosequencing analyses (Supplementary Table S3).
Discussion
In this study, the fungal communities of two different com-
mercial composts were analysed using culture-based techni-
ques and two culture-independent molecular methods in
order to evaluate their overall diversity and characterise the
most abundant thermophilous species present in them.
Samples came from separate geographical regions and had
different feedstocks, with C/N ratios within the range reported
for other mature composts (Epstein 1997). Previous culture-
based analyses of commercial composts have found total
fungal load values of between 1.0  103 and 1.0  106 CFU g1
dry weight (Johri et al. 1999; Ryckeboer et al. 2003; Anastasi
et al. 2005; Vaz-Moreira et al. 2008). Although our culture-
based approach showed comparatively low total numbers of
fungi, several thermophilic and thermotolerant species were
readily isolated from both samples. The use of several media
to isolate a number of different fungi was important as some
species were only recovered on a particular medium. Most of
the species cultured in this study have been previously iso-
lated from composts composed of similar feedstocks, but, to
the best of our knowledge, this is the first time that Myce-
liophthora thermophila and T. thermophilus have been cultured
from composts produced using green waste.
Tag-encoded pyrosequencing revealed high fungal species
richness in the composts. However, the diversity found was
lower than that previously reported for other terrestrial
environments such as soils or leaf litter (Buee et al. 2009;
Baldrian et al. 2012; Kerekes et al. 2013), in which degrada-
tion of organic material occurs more slowly without reaching
high temperatures. The sequences obtained in this study
suggest that the fungal populations present in composts
belong to the phyla Ascomycota, Basiodiomycota and the
subphylum Mucoromycotina. No sequences belonging to the
phyla Chytridiomycota or Glomeromycota were found. The
Ascomycota was by far the dominant phylum, accounting for
90 % of all sequences in our study. The two most frequent
orders of Ascomycota recorded in this study, the Sordariales
(51.2 %) and Eurotiales (12.0 %), are orders to which the
majority of thermophilic fungi belong (Morgenstern et al.
2012). Other orders of Ascomycota that were frequent in this
study (e.g. the Microascales and Hypocreales) are generally
known as wood-inhabiting or soil-borne saprotrophs and
142
plant pathogens (Moore et al. 2011). They are highly versatile,
able to utilise a wide range of carbon sources and are often
reported to be efficient cellulolytic organisms (Webster &
Weber, 2007; Schoch et al. 2009). The Basidiomycota do not
seem to be as abundant in the composts that we studied as
they are in soils, in which next-generation sequencing studies
of fungal communities have revealed this group to be frequent
(Buee et al. 2009; Lim et al. 2010). Most basidiomycetes found
in the compost samples studied belonged to the orders
Tremellales and Cystofilobasidiales (basidiomycetous yeasts)
and the genera Coprinopsis, suggesting that only a few specific
members of this phylum are adapted to the environmental
conditions present during composting.
Eighty four out of 175 OTUs found in this study were present
in both composts, confirming that thermophilic and thermo-
tolerant fungi are ubiquitous in composts irrespective of their
geographical location (Ellis 1980; Johri et al. 1999). However, as
expected, there were OTUs unique to each location and the
community structures were different between the two com-
posts. Fungal populations have been observed to vary between
composts in culture-based studies (Finstein & Morris 1975;
Ryckeboer et al. 2003; Anastasi et al. 2005), as several different
factors impact on the community profile of the final compost
product. These factors include the composting method used,
the mix of feedstock utilised, the duration of curing period and
several other local environmental variables that make each
composting pile unique (Kutzner 2000). Further next-generation
sequencing studies in which different environmental factors
are monitored throughout the composting process would be
necessary to provide more information on which of these fac-
tors has the largest effect on fungal communities.
OTUs belonging to known thermophilic and thermotoler-
ant organisms were found in the pyrosequencing dataset in
high numbers, suggesting that they are likely to play a major
role in the degradation process when the temperature of the
composting pile is high. The incubation of the mature com-
post samples at a temperature of 50  C for 2 weeks proved to
be a useful tool for identifying the thermophilous species that
are likely to contribute to the composting process during the
thermophilic stage (Tables 4 and 5). The changes in the com-
munity structure observed after the incubation treatment
(more markedly observed in compost A) indicated the occur-
rence of biological activity during this period. These changes
appear to have been caused by both an increase in the abun-
dance of thermophiles and a reduction in the numbers of
mesophiles. The heat treatment to which these composts was
subjected would have been sufficient to inactivate mesophilic
fungi, as previous experiments have demonstrated that a
short spell of heat exposure will kill mycelia and spores of
several mesophiles (Jensen 1948; Pullman et al. 1981; Hong
et al. 1997). In contrast, thermophilic organisms grow well at
this temperature and their spores germinate at 40e50  C
(Deploey 1990; Maheshwari et al. 2000). DNA from dead cells
can be more resistant to high temperatures, and there is a
chance that the molecular techniques used in this study
recovered DNA from both live and dead micro-organisms
(Ranjard et al. 2000; Kennedy & Clipson 2003). However, pre-
vious studies of similar composting environments (Hansgate
et al. 2005; Hultman et al. 2009) have shown that DNA is
turned over relatively quickly by microbial activity.
A. Langarica-Fuentes et al.
The majority of the most abundant OTUs recovered after
the incubation treatment have been previously related to
thermophilic and thermotolerant properties (Tansey & Jack
1977; Maheshwari et al. 2000). Interestingly, however, other
fungi that have been previously identified in composts, but
have not been suggested to be active in the thermophilic
stages, such as P. boydii, P. fimeti and Coprinopsis sp., were also
found to remain abundant after the incubation period. These
results suggest that the complex interactions occurring in the
compost environment might allow them to grow or at least
tolerate the high temperatures that occur during composting.
Pyrosequencing results also demonstrated that there might be
some strong competition between thermophiles, as not all
thermophilic species increased their representation in the
community during the incubation period.
The use of culture-independent techniques allowed us to
identify the dominant thermophilic species in these com-
posts, irrespective of their abilities to grow on agar media.
Although we found that some of these species were difficult to
culture or could not be cultured (e.g. S. thermophilum, M. ther-
mophilum, T. thermophilus and C. verrucosus), their high fre-
quencies indicate that they are likely to play an important role
in these environments. Myriococcum thermophilum is thought
to be one of the less common thermophilic fungi in composts
as its cultivation has only previously been reported from
mushroom composts (Fergus 1971; Straatsma et al. 1994) but
pyrosequencing data indicated that it was present in both
composts in this study at relatively high abundance. On the
other hand, the culture-based experiments overestimated the
relative importance of several species. For example, A. fumi-
gatus, which accounted for over 75 % of all colonies from
compost A, only accounted for 0.64 % of the pyrosequencing
reads from this sample. In contrast, Mycocladus corymbifer,
which was cultured from both composts, was not detected in
pyrosequencing studies. Acknowledging these discrepancies
is important as fungal populations in composts have been
extensively studied previously using culture-based techni-
ques (Kutzner, 2000; Ryckeboer et al. 2003) and in several
studies, A. fumigatus, Rhizomucor pusillus and M. corymbifer
(Absidia ramosa) were thought to be the dominant thermo-
philic and thermotolerant organisms in composts (Finstein &
Morris 1975; Anastasi et al. 2005). Thus, the picture of the
dominant thermophilic fungi in compost communities
derived from culture-based analyses has apparently been
biased. In addition, many of the OTUs detected (16.9 and
32.8 % of all sequences in composts A and B, respectively)
could not be assigned to any known fungal genera with a high
degree of confidence. Hence, despite being a highly studied
environment using culture-based techniques, molecular
methods indicate that there are likely to be a high number of
new fungal species present in composts, many of which are
likely to be thermophilic. Composting environments repre-
sent a potentially rich resource of novel fungi that may have
important applications in the biotechnology industry.
Acknowledgements
We would like to thank Graeme Fox and Paul Fullwood
(Manchester University Sequencing Facility) for their help and
An investigation of thermophilic fungi in composts
assistance in this project. This work was supported by Consejo
Nacional de Ciencia y Tecnologıa, Mexico (Grant No. 307891).
Two anonymous reviewers supplied helpful comments on the
manuscript.
Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.funeco.2014.05.007.
references
Anastasi, A., Varese, G.C., Filipello Marchisio, V., 2005. Isolation
and identification of fungal communities in compost and
vermicompost. Mycologia 97, 33e44.
Anderson, I.C., Cairney, J.W.G., 2004. Diversity and ecology of soil
fungal communities: increased understanding through the
application of molecular techniques. Environmental
Microbiology 6, 769e779.
Baldrian, P., Kolarik, M., Stursova, M., Kopecky, J.1, Valaskova, V.,
et al., 2012. Active and total microbial communities in forest
soil are largely different and highly stratified during
decomposition. ISME Journal 6, 248e258.
Barratt, S.R., Ennos, A.R., Greenhalgh, M., Robson, G.D.,
Handley, P.S., 2003. Fungi are the predominant micro-
organisms responsible for degradation of soil-buried polyester
polyurethane over a range of soil water holding capacities.
Journal of Applied Microbiology 95, 78e85.
Blackwell, M., 2011. The fungi: 1, 2, 3... 5.1 million species?
American Journal of Botany 98, 426e438.
Bonito, G., Isikhuemhen, O.S., Vilgalys, R., 2010. Identification of
fungi associated with municipal compost using DNA-based
techniques. Bioresource Technology 101, 1021e1027.
Buee, M., Reich, M., Murat, C., Morin, E., Nilsson, R.H., et al., 2009. 454
pyrosequencing analyses of forest soils reveal an unexpectedly
high fungal diversity. New Phytologist 184, 449e456.
Caporaso, J.G., Kuczynski, J., Stombaugh, J., Bittinger, K.,
Bushman, F.D., et al., 2010. QIIME allows analysis of high-
throughput community sequencing data. Nature Methods 7,
335e336.
Cooney, D.G., Emerson, R., 1964. Thermophilic Fungi: An Account
of Their Biology, Activities and Classification. W.H. Freeman
and Co., San Francisco; London.
Cosgrove, L., McGeechan, P.L., Robson, G.D., Handley, P.S., 2007.
Fungal communities associated with degradation of polyester
polyurethane in soil. Applied Environmental Microbiology 73,
5817e5824.
de Bertoldi, M., Vallini, G., Pera, A., 1983. The biology of
composting: a review. Waste Management Research 1, 157e176.
De Gannes, V., Eudoxie, G., Hickey, W.J., 2013. Prokaryotic
successions and diversity in composts as revealed by 454-
pyrosequencing. Bioresource Technology 133, 573e580.
Deploey, J.J., 1990. The effects of temperature, nutrients, and
spore concentration on the germination of conidia from
Dactylomyces thermophilus. Biodeterioration Research 3, 617e626.
Edgar, R.C., 2010. Search and clustering orders of magnitude
faster than BLAST. Bioinformatics 26, 2460e2461.
Ellis, D.H., 1980. Thermophilous fungi isolated from some
Antarctic and Sub-Antarctic soils. Mycologia 72, 1033e1036.
Epstein, E., 1997. The Science of Composting. CRC Press.
Technomic, Lancaster, Pa.
Fergus, C.L., 1971. The temperature relationships and thermal
resistance of a new thermophilic Papulaspora from mushroom
compost. Mycologia 63, 426e431.
143
Finstein, M.S., Morris, M.L., 1975. Microbiology of municipal solid
waste composting. In: Perlman, D. (Ed.), Advances in Applied
Microbiology. Academic Press, pp. 113e151.
Hansgate, A.M., Schloss, P.D., Hay, A.G., Walker, L.P., 2005.
Molecular characterization of fungal, community dynamics in
the initial stages of composting. FEMS Microbiology Ecology 51,
209e214.
Hawksworth, D., 2012. Global species numbers of fungi: are tropical
studies and molecular approaches contributing to a more
robust estimate? Biodiversity and Conservation 21, 2425e2433.
Hawksworth, D.L., Rossman, A.Y., 1997. Where are all the
undescribed fungi? Phytopathology 87, 888e891.
Hong, T.D., Ellis, R.H., Moore, D., 1997. Development of a model to
predict the effect of temperature and moisture on fungal spore
longevity. Annals of Botany 79, 121e128.
Hultman, J., Kurola, J., Rainisalo, A., Kontro, M., Romantschuk, M.,
2010. Utility of molecular tools in monitoring large scale
composting. In: Insam, H., Franke-Whittle, I., Goberna, M.
(Eds.), Microbes at Work. Springer Berlin Heidelberg,
pp. 135e151.
Hultman, J., Vasara, T., Partanen, P., Kurola, J., Kontro, M.H., et al.,
2009. Determination of fungal succession during municipal
solid waste composting using a cloning-based analysis. Journal
of Applied Microbiology 108, 472e487.
Ishii, K., Fukui, M., Takii, S., 2000. Microbial succession during a
composting process as evaluated by denaturing gradient gel
electrophoresis analysis. Journal of Applied Microbiology 89,
768e777.
Jensen, H., 1948. Thermal death points for spores and mycelia of
moulds on fermented tobacco. Physiologia Plantarum 1,
255e264.
Johri, B.N., Satyanarayana, T., Olsen, J., 1999. Thermophilic
Moulds in Biotechnology. Kluwer Academic Publishers,
Dordrecht; London.
Jumpponen, A., Jones, K.L., 2009. Massively parallel 454
sequencing indicates hyperdiverse fungal communities in
temperate Quercus macrocarpa phyllosphere. New Phytologist
184, 438e448.
Kane, B.E., Mullins, J.T., 1973. Thermophilic fungi in a municipal
waste compost system. Mycologia 65, 1087e1100.
Kennedy, N., Clipson, N., 2003. Fingerprinting the fungal
community. Mycologist 17, 158e164.
Kerekes, J., Kaspari, M., Stevenson, B., Nilsson, R.H.,
Hartmann, M., et al., 2013. Nutrient enrichment increased
species richness of leaf litter fungal assemblages in a tropical
forest. Molecular Ecology 22, 2827e2838.
Klamer, M., Sochting, U., 1998. Fungi in a controlled compost
system with special emphasis on the thermophilic fungi. In:
International Symposium on Composting and Use of
Composted Materials for Horticulture, pp. 405e413.
Kumar, S., 2011. Composting of municipal solid waste. Critical
Reviews in Biotechnology 31, 112e136.
Kutzner, H.J., 2000. Microbiology of composting. In: Klein, J.,
Winter, J. (Eds.), Biotechnology, Environmental Processes III e
Solid Waste and Waste Gas Treatment, Preparation of
Drinking Water, second edn, vol. 11c. Wiley-VCH, Weinheim.
Lim, Y., Kim, B., Kim, C., Jung, H., Kim, B.-S., et al., 2010.
Assessment of soil fungal communities using pyrosequencing.
The Journal of Microbiology 48, 284e289.
Lindahl, B.D., Nilsson, R.H., Tedersoo, L., Abarenkov, K.,
Carlsen, T., et al., 2013. Fungal community analysis by high-
throughput sequencing of amplified markers e a user’s guide.
New Phytologist 199, 288e299.
Maheshwari, R., Bharadwaj, G., Bhat, M.K., 2000. Thermophilic
fungi: their physiology and enzymes. Microbiology and
Molecular Biology Reviews 64, 461e488.
Mitchell, J.I., Zuccaro, A., 2006. Sequences, the environment and
fungi. Mycologist 20, 62e74.
144
Monard, C., Gantner, S., Stenlid, J., 2013. Utilizing ITS1 and ITS2 to
study environmental fungal diversity using pyrosequencing.
FEMS Microbiology Ecology 84, 165e175.
Moore, D., Robson, G.D., Trinci, A.P.J., 2011. 21st Century
Guidebook to Fungi. Cambridge University Press.
Morgenstern, I., Powlowski, J., Ishmael, N., Darmond, C.,
Marqueteau, S., et al., 2012. A molecular phylogeny of
thermophilic fungi. Fungal Biology 116, 489e502.
Mouchacca, J., 1997. Thermophilic fungi: biodiversity and
taxonomic status. Cryptogamie Mycologie 18, 19e69.
Mouchacca, J., 2000. Thermotolerant fungi erroneously reported
in applied research work as possessing thermophilic
attributes. World Journal of Microbiology and Biotechnology 16,
869e880.
Novinscak, A., DeCoste, N.J., Surette, C., Filion, M., 2009.
Characterization of bacterial and fungal communities in
composted biosolids over a 2 year period using denaturing
gradient gel electrophoresis. Canadian Journal of Microbiology
55, 375e387.
O’Donnell, K., 1993. Fusarium and its near relatives. In:
Reynolds, D.R., Taylor, J.W. (Eds.), The Fungal Holomorph:
Mitotic, Meiotic and Pleomorphic Speciation in Fungal
Systematics. CAB International, Wallingford, UK, pp. 225e233.
Poulsen, P.H.B., Moller, J., Magid, J., 2008. Determination of a
relationship between chitinase activity and microbial
diversity in chitin amended compost. Bioresource Technology 99,
4355e4359.
Pullman, G.S., Devay, J.E., Garber, R.H., 1981. Soil solarization and
thermal death e a logarithmic relationship between time and
temperature for 4 soilborne plant-pathogens. Phytopathology
71, 959e964.
Ranjard, L., Poly, F., Nazaret, S., 2000. Monitoring complex
bacterial communities using culture-independent molecular
techniques: application to soil environment. Research in
Microbiology 151, 167e177.
Reeder, J., Knight, R., 2010. Rapidly denoising pyrosequencing
amplicon reads by exploiting rank-abundance distributions.
Nature Methods 7, 668e669.
Ryckeboer, J., Mergaert, J., Vaes, K., Klammer, S., De Clercq, D.,
et al., 2003. A survey of bacteria and fungi occurring during
composting and self-heating processes. Annals of Microbiology
53, 349e410.
Schoch, C.L., Sung, G.-H., Lopez-Giraldez, F., Townsend, J.P.,
Miadlikowska, J., et al., 2009. The Ascomycota tree of life: a
phylum-wide phylogeny clarifies the origin and evolution of
A. Langarica-Fuentes et al.
fundamental reproductive and ecological traits. Systematic
Biology 58, 224e239.
Sharma, H.S.S., 1989. Economic importance of thermophilous
fungi. Applied Microbiology and Biotechnology 31, 1e10.
Singer, A., Crohn, D., Humpert, C.P., 2000. Compost Microbiology
and the Soil Food Web. California Environmental Protection
Agency, Integrated Waste Management Board, Sacramento,
pp. 1e6.
Straatsma, G., Samson, R.A., Olijnsma, T.W., Dencamp, H.J.M.O.,
Gerrits, J.P.G., et al., 1994. Ecology of thermophilic fungi in
mushroom compost, with emphasis on Scytalidium
thermophilum and growth-stimulation of Agaricus bisporus
mycelium. Applied and Environmental Microbiology 60, 454e458.
Szekely, A., Sipos, R., Berta, B., Vajna, B., Hajdu, C., et al., 2009.
DGGE and T-RFLP analysis of bacterial succession during
mushroom compost production and sequence-aided T-RFLP
profile of mature compost. Microbial Ecology 57, 522e533.
Tansey, M.R., 1971. Isolation of thermophilic fungi from self-
heated, industrial wood chip piles. Mycologia 63, 537e547.
Tansey, M.R., Jack, M.A., 1977. Growth of thermophilic and
thermotolerant fungi in soil in situ and in vitro. Mycologia 69,
563e578.
Tedersoo, L., Nilsson, R.H., Abarenkov, K., Jairus, T., Sadam, A.,
et al., 2010. 454 Pyrosequencing and Sanger sequencing of
tropical mycorrhizal fungi provide similar results but reveal
substantial methodological biases. New Phytologist 188,
291e301.
Tuomela, M., Vikman, M., Hatakka, A., Itavaara, M., 2000.
Biodegradation of lignin in a compost environment: a review.
Bioresource Technology 72, 169e183.
Vaz-Moreira, I., Silva, M.E., Manaia, C.M., Nunes, O.C., 2008.
Diversity of bacterial isolates from commercial and
homemade composts. Microbial Ecology 55, 714e722.
Webb, J.S., Nixon, M., Eastwood, I.M., Greenhalgh, M.,
Robson, G.D., et al., 2000. Fungal colonization and
biodeterioration of plasticized polyvinyl chloride. Applied and
Environmental Microbiology 66, 3194e3200.
Webster, J., Weber, R., 2007. Introduction to Fungi. Cambridge
University Press, Cambridge.
White, T.J., Bruns, T., Lee, S., Taylor, J., 1990. Amplification and
direct sequencing of fungal ribosomal RNA genes for
phylogenetics. In: Innis, M.A., Gelfand, D.H., Sninsky, J.J.,
White, T.J. (Eds.), PCR Protocols: A Guide to Methods and
Applications. Academic Press, New York, pp. 315e332.