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Microbiology of Composting

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RESEARCH ARTICLE

Molecular analysis of bacterial community succession during

prolonged compost curing
Michael Danon1, Ingrid H. Franke-Whittle2, Heribert Insam2, Yona Chen1 & Yitzhak Hadar1
1
Faculty of Agricultural, Food and Environmental Quality Sciences, The Hebrew University of Jerusalem, Rehovot, Israel; and 2Institute for Microbiology,
University of Innsbruck, Technikerstraße 25d, A-6020 Innsbruck, Austria

Correspondence: Yitzhak Hadar, Abstract

Department of Plant Pathology and
Microbiology, Faculty of Agricultural, Food
and Environmental Quality Sciences, The
Hebrew University of Jerusalem, PO Box 12,
Rehovot 76100, Israel. Tel.: 1972 8 9489935;
fax: 1972 8 9468785; e-mail:
hadar@agri.huji.ac.il
Received 20 December 2007; revised 11 March
2008; accepted 10 April 2008.
First published online 4 June 2008.
DOI:10.1111/j.1574-6941.2008.00506.x
Editor: Alfons Stams
Keywords
biosolids compost; clone library; community
composition; oligonucleotide microarray;
organic matter degradation; PCR-DGGE.
The compost environment consists of complex organic materials that form a
habitat for a rich and diverse microbial community. The aim of this research was to
study the dynamics of microbial communities during the compost-curing phase.
Three different methods based on 16S rRNA gene sequence were applied to
monitor changes in the microbial communities: (1) denaturing gradient gel
electrophoresis of PCR-generated rRNA gene fragments; (2) partial rRNA gene
clone libraries; and (3) a microarray of oligonucleotide probes targeting rRNA
gene sequences. All three methods indicated distinctive community shifts during
curing and the dominant species prevailing during the different curing stages were
identified. We found a successional transition of different bacterial phylogenetic
groups during compost curing. The Proteobacteria were the most abundant
phylum in all cases. The Bacteroidetes and the Gammaproteobacteria were
ubiquitous. During the midcuring stage, Actinobacteria were dominant. Different
members of nitrifying bacteria and cellulose and macromolecule-degrading
bacteria were found throughout the curing process. In contrast, pathogens were
not detected. In the cured compost, bacterial population shifts were still observed
after the compost organic matter and other biochemical properties had seemingly
stabilized.

toward Sclerotium rolfsii has been demonstrated during

Introduction
Composting is an aerobic process by which organic materi-
als are degraded through the activities of successive groups
of microorganisms. Soil amendment with composted or-
ganic material is an ancient practice that is applied through-
out the world, and the long-term benefits of compost
application to fields are well documented (Ros et al., 2006).
Previous studies have emphasized the importance of achiev-
ing compost maturity to ensure balanced plant nutrition
and for the biological control of soil-borne plant disease
(Fuchs, 2002; Noble & Coventry, 2005). Compost maturity
is achieved during the curing process. The duration of
curing in the industry varies according to a number of
factors, including source materials, composting process and
facility, climate, and planned utilization of the final product.
Noncured composts may be phytotoxic, whereas extensively
cured composts may lose their plant-disease-suppressive
properties. For instance, loss of compost suppressiveness
prolonged curing time (Danon et al., 2007). Compost of an
appropriate age should therefore be used to control disease
in infested soils before sowing. The successful application of
compost is considerably dependent on the selection of an
appropriate curing period.
The biochemistry of the compost-curing process has been
studied extensively. Chen et al. (1989) found that the
original cellulose and hemicellulose contents of cattle man-
ure were reduced by one-third during a 5-month compost-
ing and maturing process. Hemicellulose and cellulose may
be the main substrates for microorganisms during the
maturation process because these components are present
in large quantities in cattle manure and the less complex
carbon sources are consumed early on in the process (Chen
et al., 1989). Recently, Tang et al. (2006) reported that
organic matter decomposition during the maturation of
cattle-manure compost resulted in decreased C/N ratios,
microbial biomass, and microbial diversity. The C/N ratio in

FEMS Microbiol Ecol 65 (2008) 133–144

c2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
134
the compost may level off much before the compost
stabilizes (Chefetz et al., 1996) and although the concentra-
tion of the dissolved organic carbon (DOC) of municipal
solid-waste compost did not change at maturation, the
relative concentrations of different hydrophilic and hydro-
phobic fractions in the dissolved organic matter continued
to change concomitant with the decrease in phytotoxicity
(Chefetz et al., 1998). Tang et al. (2006) showed that during
the maturation period, the proportion of Actinobacteria
increases slightly, and this was associated with the disap-
pearance of phytotoxicity.
Considerable effort and a variety of techniques have been
applied to the study of compost microbial populations
(Cahyani et al., 2003; Schloss et al., 2003; Ryckeboer et al.,
2003b). The initial phase of composting is thought to be the
most dynamic part of the process and is characterized by
rapid increases in temperature, large changes in pH, and the
degradation of simple organic compounds. Schloss et al.
(2003) reported two significant shifts in the composition of
the microbial community: one occurring between 12 and
24 h and the other between 60 and 72 h into the process.
Ryckeboer et al. (2003b) attempted to determine the micro-
bial succession of the dominating taxa and functional
groups of microorganisms, as well as the total microbial
activity during composting of biowaste, using incubation,
isolation, and enumeration techniques. They reported that
bacteria dominated during the thermophilic phase while
fungi, Streptomycetes, and yeasts were below detection
limits. Different bacterial populations were found in the
thermophilic and mesophilic composting phases. During
the peak-heating phase of fresh wastes, the only bacteria
isolated were bacilli; however, during the cooling and
maturation phases, the bacterial diversity of both Gram-
positive and Gram-negative bacteria increased (Ryckeboer
et al., 2003b). Using cultivation-independent methods,
Cahyani et al. (2003) studied the bacterial communities in
composting of rice straw. They reported a successional
transition of bacterial community members: Alphaproteo-
bacteria in the raw materials, Bacillus and Actinomycetes at
the thermophilic stage, and Cytophaga and clostridial mem-
bers at the middle and curing stages. These authors reported
that the microbial community remained stable during the
curing phase. In contrast, Steger et al. (2007) revealed
compositional changes within the Actinobacteria commu-
nity in a full-scale composting process of organic household
waste over a period of 57 weeks.
Despite the important role of microbial populations in
compost quality, only a few studies have thoroughly exam-
ined the dynamics of the total microbial populations during
compost curing. The aim of this study was to characterize
changes in the microbial community structure during
prolonged curing of biosolids compost, using molecular
techniques. For this purpose, we used three different culti-
c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
M. Danon et al.
vation-independent techniques based on 16S rRNA gene
sequences: PCR-denaturing gradient gel electrophoresis
(DGGE), clone libraries, and an oligonucleotide microarray
(COMPOCHIP). Molecular techniques are becoming in-
creasingly useful for the detection of different microorgan-
isms without first having to isolate and cultivate them (Dees
& Ghiorse, 2001; Green et al., 2004; Franke-Whittle et al.,
2005; Kelly et al., 2005). Although they share the same basis,
i.e. exploiting conserved and variable regions in the riboso-
mal small subunit sequence, each of the techniques used in
our study has its inherent advantages and disadvantages. A
combined analysis was therefore expected to produce more
reliable information on the qualitative and quantitative
succession of bacterial populations during compost curing.
Materials and methods
Prolonged compost-curing process and compost
sampling
Compost samples were obtained from a commercial com-
posting facility (Shacham, Givaat Ada, Dlila Facility, Israel)
that prepared compost from a mixture of sewage sludge and
yard waste (1 : 1, v/v) in windrows (2.5 m wide and 2 m
high). Aeration was achieved by bi-weekly turning for the
entire 12-week composting process. The biosolids compost,
considered to be mature by the producers, i.e. appropriate
for field application, was sampled and further treated as
follows: c. 700 L of bulk compost was collected and placed in
a cubic, 700-L bin, rewetted and turned every 2 weeks for the
first month, then every month for the next 4 months. The
resultant compost pile was then left unturned for seven
additional months (in total, 1 year of prolonged curing).
Compost temperature in the container increased after
rewetting to 42 1C in the first week, and then decreased over
the next 2 weeks to 32 1C (c. 5 1C above ambient). Approxi-
mately 20 L of compost was sampled from the bin at time 0
(beginning of prolonged curing), then after each turning,
and stored at 4 1C until analysis.
DNA extraction, general molecular procedures,
and replications
DNA from 0.25 g of compost subsamples was extracted
using a ‘PowerSoil’ kit (Mobio, Solana Beach, CA). The
DNA extracts were used as templates for PCR amplification
of bacterial partial 16S rRNA genes for DGGE, clone
libraries, and oligonucleotide microarray analysis. For
DGGE, we used two replicate DNA samples. The DNA was
amplified using the 341-907 primer set with a GC clamp
(Muyzer et al., 1998) and the products were run in separate
lanes. For clone libraries we used three replicate DNA
samples. The DNA was amplified using the 341-907 primer
FEMS Microbiol Ecol 65 (2008) 133–144
Bacterial succession during compost curing
set without a GC clamp. The PCR products were pooled and
mixed before ligation to a vector.
PCR-DGGE analysis
PCR amplification and DGGE were conducted as described
previously (Danon et al., 2007). Briefly, DGGE was prepared
according to Muyzer et al. (1993) using an IngenyPhor-U2
system (Ingeny, Goes, The Netherlands) with 6% (w/v)
polyacrylamide gel [acrylamide/bisacrylamide (37 : 1)] in
1  Tris acetate–EDTA (TAE) buffer and a 20–60% dena-
turing gradient (80% denaturant corresponding to 7 M urea
and 32%, v/v formamide). Dominant DGGE bands were
excised and reamplified. Reamplified bands that migrated
identically to the excised bands in the DGGE were cloned
and sequenced.
Cloning, sequencing, and phylogenetic analysis
Two clone libraries, for noncured (0 days) and cured (336
days) compost samples, were constructed using the pGEM-
T easy vector system (Promega, Madison, WI). Ligation and
transformation were performed according to the manufac-
turer’s directions. Colonies were screened for the presence of
the correctly sized insert, and plasmid DNA was reamplified
before sequencing. Plasmids from cloned PCR-DGGE bands
were extracted and purified using a miniprep DNA purifica-
tion kit (Genomed, Löhne, Germany).
Sequencing was performed at the Macrogen Inc. Sequen-
cing Center (Seoul, Korea). Sequences were examined using
the CHECK_CHIMERA program located at the Ribosomal Data-
base Project (Cole et al., 2005), and chimeric sequences were
removed from phylogenetic analyses.
Phylogenetic analysis of sequences derived from clones
and from DNA of excised bands was performed by align-
ment to known bacterial sequences using the ‘greengenes’
16S rRNA gene database and alignment tool (DeSantis et al.,
2003) (http://greengenes.lbl.gov/). Aligned sequences and
close relatives were imported to the MEGA software package
version 3.1 (Kumar et al., 2004). Similarity was tested to
sequences available at the National Center for Biotechnology
Information (NCBI) using BLAST analysis (Altschul et al.,
1997). The phylogenetic tree was constructed using the MEGA
software with the Kimura two-parameter method for dis-
tance matrix calculations and the neighbor-joining method
for tree design. Tree topologies were evaluated by perform-
ing bootstrap analysis of 1000 data sets. The rRNA gene
sequences were submitted to the GenBank database under
accession numbers EU215227–EU215310.
Oligonucleotide microarray analysis
The COMPOCHIP microarray, spotted with 369 probes
targeting microorganisms that have been reported pre-
FEMS Microbiol Ecol 65 (2008) 133–144
135
viously in the composting process, as well as plant, animal,
and human pathogens, and plant-disease-suppressive
bacteria, was applied to DNA extracted from different
composts. The specificity of all probes was assessed in silico,
using the ARB program (Ludwig et al., 2004), and the
array was tested with pure cultures of microorganisms,
and was shown to work well with only a low percentage
of nonspecific hybridizations (Franke-Whittle et al., 2005).
For most target organisms, at least three probes were
spotted on the slide, and for a few organisms there were
only two probes. All the probes included on the COMPO
CHIP microarray were designed so as to have similar
melting temperatures, and probe sequences ranged in
length from 17 to 25 nucleotides. Five or six replicate
DNA samples were analyzed. Fluorescence labeling of
target DNA, hybridization, scanning of arrays, and image
analysis were conducted as described by Franke-Whittle
et al. (2005).
Statistical analysis
Statistical analysis of the DGGE data was conducted using
Dice correlation coefficients and the unweighted-pair-group
method with arithmetic averages (UPGMA) to form a
complete linkage dendrogram (Fingerprinting II Informa-
tix, BioRad Laboratories, Hercules, CA).
Statistical analysis of clone libraries and coverage deter-
mination were conducted using the procedure developed by
Kemp & Aller (2004) to confirm that an asymptotic accu-
mulation curve in both libraries had been reached. Unifrac
(Lozupone et al., 2006) was used to define phylotypes at 3%
similarity cutoff.
Statistical analyses of microarray data were performed
using the program CANOCO 4.5 (ter Braak & Šmilauer, 2002).
Data from the microarray analysis, the physicochemical
analysis, and the cloning of DGGE bands were subjected to
principal component analysis (PCA). As differing numbers
of replicates were used for each set of experiments, an
average was used for each set of replicates in the statistical
analysis. Because a strong correlation between SD and
means of replicate samples was found, a log-transformation
of microarray data was conducted to equalize variances. For
the covariance-based redundancy analysis (RDA), the fol-
lowing settings were used: inter-sample distance scaling, no
post-transformation of scores, log data transformation (no
offset), and center by species.
Results
PCR-DGGE
The community composition of bacteria in biosolids com-
post samples subjected to different curing times is shown in
c2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
136 M. Danon et al.

40 50 60 70 80 90 100 Compost curing time (days)

0
0
19
19
41
41
67
67
130
167 Fig. 1. PCR-DGGE analysis of the bacterial
205 community in composts from different stages of
167 curing. A UPGMA algorithm was applied to a
205 similarity matrix of Dice and Pearson correlation
coefficients generated from the DGGE banding
130
patterns. The numbers correspond to bands
336
identified by 16S rRNA gene sequence analysis
336 (Table 1).

Table 1. Identification of PCR-DGGE bands by 16S rRNA gene sequence Clone libraries
analysis
Clone libraries were constructed from cured (0 days) and
Band Accession noncured (336 days) composts, each library including a
number number Phylum/class Family
total of 41 and 43 clones, respectively. The complete
8 EU215311 Bacteroidetes Cryomorphaceae phylogenetic analysis is given in the supplementary infor-
10 EU215312 Bacteroidetes Sphingobacteriaceae
mation. Figure 2 illustrates the phylogeny of compost
13 EU215313 Alphaproteobacteria Phyllobacteriaceae
15 EU215314 Alphaproteobacteria Caulobacteraceae
bacteria members affiliated with (a) Actinobacteria and (b)
19 EU215315 Gammaproteobacteria Xanthomonadaceae Betaproteobacteria. The clone libraries were found to differ
21 EU215316 Gammaproteobacteria Xanthomonadaceae significantly with respect to phylotype composition (P value
23 EU215317 Gammaproteobacteria Xanthomonadaceae  0.01). Clone rRNA gene sequences were found to belong
26 EU215318 Gammaproteobacteria Xanthomonadaceae to seven different phyla: Actinobacteria, Bacteroidetes, Chlor-
29 EU215319 Bacteroidetes Flavobacteriaceae oflexi, Deinococci, Gemmatimonadetes, Firmicutes, and Pro-
30 EU215320 Actinobacteria Corynebacterineae
teobacteria. Most of the clones, derived from both the cured
37 EU215321 Alphaproteobacteria Caulobacteraceae
38 EU215322 Actinobacteria Promicromonosporaceae
and noncured composts, were classified as belonging to the
Alpha-, Beta-, Gamma-, and Deltaproteobacteria classes. The
Dominant DGGE bands were excised, reamplified and cloned. Ampli-
Gammaproteobacteria class contained the highest number of
cons and clones were compared with the original bands in DGGE. clones: five from the noncured compost and 12 from the
Amplicons and clones that did not migrate to the identical positions of
cured samples. Eight clones from the noncured compost and
the excised bands in the DGGE were not identified.
five clones from the cured compost were found to belong to
Alphaproteobacteria. Only clones from the noncured com-
post grouped within the Caulobacteraceae family. Other
Fig. 1. UPGMA analysis of the DGGE profiles generated
groups found only in the noncured compost were Promicro-
from individual PCR products showed that the duplicate
monosporaceae (Actinobacteria) (Fig. 2a), Comamonadaceae
samples of specific ages clustered together. The noncured (0
(Betaproteobacteria) (Fig. 2b) and members of the Chloro-
days) samples were distinctly different from those represent-
flexi phylum. Members of the Deinococcus phylum were
ing longer curing times. Among the cured compost samples,
present only in the cured compost clone library (Supple-
the 19- and 41-day samples clustered separately from the
mentary Fig. S1).
longer curing times. The oldest sample (336 days) was
distinctly different from the 67- to 205-day samples. The
Microarray
bands were numbered (1–44) and identified according to
similarity to sequences in the GenBank and the greengenes The COMPOCHIP 16S rRNA gene microarray was applied
database. The identifications of DGGE bands are listed in in order to directly determine which bacteria were present in
Table 1. the different compost samples analyzed. Because a linear

c 2008 Federation of European Microbiological Societies FEMS Microbiol Ecol 65 (2008) 133–144
Published by Blackwell Publishing Ltd. All rights reserved
Bacterial succession during compost curing 137

60 Actinomadura pelletieri AF163119.1

(a) Actinobacteria 99 Actinomadura str. JCM 3308 AF134067.1
Cured compost clone M37 EU215289
91 Cured compost clone M46 EU215297
Thermomonosporaceae
Noncured compost clone M3 EU215229
Cured compost clone M41 EU215293

97 Microbacteriaceae str. Ellin145 AF408987

65
89 Leifsonia str. PTX1 DQ901014.1

99 Noncured compost clone M11 EU215237

Noncured compost clone M65 EU215238

56 Promicromonospora sukumoe AB023375.1

58 Promicromonosporaceae
Cellulomonas sp. str. X7 AF060791.1
95 Compost PCR-DGGE band 38 EU215322
89 Noncured compost clone M62 EU215242
Cured compost clone M17 EU215270

81 Cured compost clone M23 EU215276

Cured compost clone M25 EU215278

Compost PCR-DGGE band 30 EU215320

91
Cured compost clone M36 EU215288
79
60 Cured compost clone M52 EU215301
Cured compost clone M42 EU215294
Corynebacterineae
98 Mycobacterium terrae X52925.1
Mycobacterium tokaiense AF480590.1

77 Mycobacterium thermoresistible M29570.1

93 Noncured compost clone M76 EU215252

0.01
Noncured compost clone M86 EU215261
67
(b) Betaproteobacteria Cured compost clone M85 EU215307

Noncured compost clone M87 EU215262

71 Oxalobacteraceae
Cured compost clone M32 EU215284
99
Oxalobacter str. HI-D2 DQ196473.1

Oxalobacteraceae isolate D8-13b AM403210

Variovorax paradoxus str. WP1 AF208386.1

Noncured compost clone M2 EU215228

99
Fungal ascocarp clone AY599736.1
Sludge clone H21 AF234687.1
61
Denitrifying reactor clone 81 AJ412678.1
97 Noncured compost clone M6 EU215232 Comamonadaceae

Ottowia thiooxydans str. K11 AJ537466.1

91
Acidovorax avenae AY512827.1
55
Acidovorax sp. AHL 5 AY379977.1
Noncured compost clone M9 EU215235
69 Noncured compost clone M75 EU215251

0.01

Fig. 2. 16S rRNA gene phylogeny of bacteria found in composts at different curing stages (a) Actinobacteria and (b) Betaproteobacteria phyla.
Neighbor-joining phylogenetic tree drawn to scale, with branch lengths computed using the maximum composite likelihood method. The scale bar
represents 0.01 base substitutions per site. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000
replicates) is shown next to the branches. Sequences of PCR-DGGE bands are marked by triangles. Sequences from noncured and cured compost are
marked by black and white circles, respectively.

FEMS Microbiol Ecol 65 (2008) 133–144

c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
138 M. Danon et al.

1.0

0d 0-c
0.8
0-e
0-b

0.6
0-d
0-a

0.4 41 d
RDA Axis 2

41-d
0.2
67-b
41-b
41-f
130 d 67-e
41-c
67-d
0.0 67-f
336 d 41-e 41-a
67-a
336-d 336-e 130-e
–0.2 67-c
336-b 130-f
336-a 130-d
336-f 130-c
336-c 67 d
–0.4
130-a Fig. 3. Two dimensional ordination plot of com-
post communities from different curing stages
–0.6 analyzed by the COMPOCHIP microarray and
–1.0 –0.5 0.0 0.5 1.0
canonical analysis (redundancy analysis). Indivi-
RDA Axis 1 dual replicates are represented by letters (a–f).

correlation between target concentrations and signal inten- the 336-day samples. These results were supported by strong
sities of the various probes has been reported by others hybridization signals for probe 350, specific for A. faecalis.
(Taroncher-Oldenburg et al., 2003; Tiquia et al., 2004), Despite its name, A. faecalis is not typical to feces, but is a
the results obtained in this study were analyzed semi- common, nonpathogenic, environmental bacterium that
quantitatively. has been reported to be present in composts by other
Figure 3 shows an ordination graph: the two relevant axes authors (Droffner et al., 1995; Ryckeboer et al., 2003a).
explained 34.1% of the variance. Multivariate analysis The 295 and 296 probes, specific for members of the
showed that the bacterial-community compositions of the genera Nitrosovibrio and Nitrosospira, indicated the presence
composts changed with curing time, namely, the noncured of these bacteria in composts throughout the curing process.
and cured composts of different ages clustered separately. However, in the noncured samples, the probes exhibited
Bacteria belonging to the low G1C and Alphaproteobacteria higher signal levels.
groups were found in high numbers in composts from all
curing stages, as well as noncured compost. The probe
targeting the genus Actinomyces also revealed high levels of
Discussion
these organisms in both cured and noncured compost The microbial community in compost during prolonged
samples. The microarray included probes specific for mem- curing was studied using three different culture-indepen-
bers of the genus Chryseobacterium. Higher levels of this dent techniques. We followed the dynamics of the microbial
genus were found in the younger composts than in the more population from the point at which the commercial produ-
mature ones. Higher signals for the probes targeting Micro- cer considered the compost appropriately mature for field
bacterium and Sphingobacterium were detected in the non- application and at which point it was found to be suppres-
cured composts. The EUB 338II probe specific for the sive to plant pathogens (Danon et al., 2007). The results
Verrucomicrobia group was found to correlate well with the obtained from analysis using DGGE, clone libraries, and a
more cured composts. The Pseudomonas genus-specific microarray revealed that under the conditions tested here,
probes gave signals above the threshold values for samples the compost community continued to change during the
from days 0, 41, and 336, but not for those from days 67 curing phase. Recognizing the inherent advantages and
and 130. disadvantages of each technique, we attempted to integrate
High signal-to-noise ratios were obtained for composts the data analysis to produce a comprehensive picture of the
from all curing stages with probe 270 specific for Alcaligenes microbial community dynamics. Microarrays allow the
faecalis/defragrans. Although the signal intensities were parallel detection of up to several thousand microbial
found to decrease with time, Alcaligenes was still present in strains, species, genera, or higher taxonomic groups in a

c 2008 Federation of European Microbiological Societies FEMS Microbiol Ecol 65 (2008) 133–144
Published by Blackwell Publishing Ltd. All rights reserved
Bacterial succession during compost curing
single experiment, depending on the availability of the
probe set used on the microarray. However, because differ-
ent probes have different affinities for their targets (Loy &
Bodrossy, 2006), the information on the relative abundance
of the different microorganisms derived from the micro-
array analysis needs to be interpreted with caution. We
found higher signals (indicative of better hybridization) for
most probes (both higher taxonomic group level probes as
well as genus- and species-specific probes) upon hybridiza-
tion with the noncured compost samples when compared
with the cured compost samples. This would indicate that
the longer the curing time, the less likely it is that the
resulting compost product will have a bacterial community
that matches the expected typical compost bacteria targeted
by the microarray. As a result of this, clone libraries were
more reliable for the comparison of higher taxonomic
groups comprising the cured compost microbial commu-
nity (Fig. 4).
In a previous report (Danon et al., 2007), we have
described how biosolids compost undergoes a transition
from a highly active to a stabilized biosolid during pro-
longed curing. Samples of noncured compost were charac-
terized by intense activity, with rapid decomposition of
organic matter, as reflected by a higher respiration rate, a
faster utilization of glucose, and a higher concentration of
DOC. The solubilized material became depleted in carbohy-
drates and enriched in aromatic compounds. As the avail-
able carbon was depleted, the microbial activity, indicated
by respiration, decreased, the response to added glucose was
slower, and competition for NH1 4 decreased, allowing an
increase in nitrification. It was proposed that as curing
progresses, the remaining substrate in the compost is
more lignocellulosic in nature (Danon et al., 2007). The
biochemical data of the previous report were used for
comparison of the same samples with microbial population
analysis conducted in the current study (Fig. 5). Some
of the bacteria associated with the changes in the chemical
properties of compost during prolonged curing were then
identified.
Noncured compost populations
Differences between the noncured and cured composts in
relative abundances of bacterial groups are illustrated in
Fig. 4. Using both the cloning and the microarray ap-
proaches, we found that the members of the Bacteroidetes
phylum were more abundant in the noncured composts
than in the cured composts (Fig. 4). With a more detailed
principal component analysis including DGGE and micro-
array data as well as process parameters (NH14 , NO3 , DOC,
and sugar concentration), we found that the dominant
population in the noncured compost consisted of a variety
of Bacteroidetes species, including Chryseobacterium, Sphin-
FEMS Microbiol Ecol 65 (2008) 133–144
139
gobacterium, and Flavobacterium (Fig. 5). We found mem-
bers of Sphingobacterium, both by DGGE (band 10) and
with the microarray (probe 550) in the noncured compost.
The phylum Bacteroidetes includes a wide variety of
bacteria known for their utilization of macromolecules such
as proteins, starch, cellulose, and chitin (Manz et al., 1996),
and its members have been detected previously by mole-
cular methods in various composts (Alfreider et al., 2002;
Michel et al., 2002; Verkhovtseva et al., 2002). Green et al.
(2004) reported that the most frequently detected
sequences in compost-amended potting mixes belong to
Chryseobacteria.
Nitrosovibrio and Nitrosospira were predominantly found
in the noncured composts in close proximity to the NH4
vector (Fig. 5). These bacteria were detected by the micro-
array only, indicating that they represent a minor popula-
tion in the compost total microbial community
(Fig. 4). Nevertheless, minor populations may play impor-
tant roles in their environment, and ammonium-oxidizing
bacteria, including members of the above genera, are often
reported in composts (Kowalchuk et al., 1999; Innerebner
et al., 2006).
We found bacteria belonging to the Betaproteobacteria to
be more abundant in noncured than cured composts (Fig.
4). Members of the Comamonadaceae family were detected
only in the noncured compost clone library (Fig. 2b). These
bacteria may originate from the activated sludge process
during wastewater treatment, as they were related to Acid-
ovorax sequences isolated from wastewater-treatment plants.
Interestingly, Valle et al. (2004) reported that in a phenol-
degrading activated sludge system, the population size of
Acidovorax sp. AHL-5 (AY379977.1) changes in association
with the phenol-degradation rate. Acidovorax species have
also been shown to be the dominant 3-hydroxybutyrate
(PHB)-degrading bacteria in soils, composts, and freshwater
(Mergaert & Swings, 1996). Other Betaproteobacteria,
namely members of the Oxalobacteraceae, were found in
both libraries (Fig. 2b).
We found bacteria of the phylum Chloroflexi only in the
noncured compost clone library (Fig. S1). Filamentous
members of the phylum Chloroflexi have also been found in
activated sludge, and they have occasionally been associated
with incidences of bulking (Bjornsson et al., 2002). As the
compost used in this study was derived from sewage sludge,
the detection of these bacteria is not surprising. Chloroflexi
filaments appear to be specialized in polysaccharide
degradation.
Members of the genus Brevundimonas (Alphaproteobac-
teria; Caulobacteraceae) were present and dominant only in
the noncured compost clone library (Fig. S1). However,
PCA of the DGGE bands (Fig. 5) showed that band 15,
identified as Caulobacteraceae, appears to be a dominant
microorganism in the compost sample with a curing time of
c2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
140 M. Danon et al.

100

Clones in library 90

Proteobacteria 80

Gammaproteobacteria
% of cured compost bacteria
Alphaproteobacteria 70

Betaproteobacteria 60

Gammaproteobacteria 50 Proteobacteria

Actinobacteria
40
Deltaproteobacteria
30
Actinobacteria
20
Bacteroidetes Alphaproteobacteria
10
other Bacteria Bacteroidetes
Betaproteobacteria
0 10 20 30 40 50 60 70 80 90 100

100
Universal
Probes in microarray 90

Proteobacteria 80
% of cured compost bacteria

Alphaproteobacteria Low G+C

70
Actinobacteria
Gammaproteobacteria

Betaproteobacteria
60 Proteobacteria
Gammaproteobacteria
Epsilonproteobacteria 50
Actinobacteria
40
Bacteroidetes
30 Alphaproteobacteria
Low G+C
Nitrospirae 20
Planctomycetes Bacteroidetes
10
Universal
Betaproteobacteria
Other Bacteria 0
0 10 20 30 40 50 60 70 80 90 100
% of noncured compost bacteria

Fig. 4. Percentage contribution of various bacterial phyla to the total bacterial community in cured and noncured compost, as detected by clones in
libraries vs. microarray probes. Groups below the diagonal line were more abundant in the noncured compost, and those above it were more abundant
in the cured compost.

41 days. Pedro et al. (2001) found Brevundimonas present in Populations at intermediate compost-curing
the mesophilic phase of industrial and agricultural waste times
composting. These bacteria may play an important role in
the composting process, and it is possible that they origi- PCA of microarray data showed that the Actinobacterium
nated from the biosolids, as Caulobacteria members have Microbacterium dominated composts with intermediate
been isolated previously from sewage and activated sludge curing times of 41–130 days (Fig. 5). Manickam et al.
(Baker et al., 1983). (2006) reported the ability of Microbacterium isolated from

c 2008 Federation of European Microbiological Societies FEMS Microbiol Ecol 65 (2008) 133–144
Published by Blackwell Publishing Ltd. All rights reserved
Bacterial succession during compost curing
1.0
0.5
0.0
–0.5
–1.0
–1.0 –0.5 0.0
Fig. 5. Principal component analysis of chemical and molecular data. Circles indicate the clustering time of curing in days. The principal DGGE bands
and microarray probes are highlighted (in red and blue, respectively).
contaminated soil to degrade the persistent and toxic hexa-
chlorocyclohexane. Members of the Actinomycetes genus have
been reported to develop more slowly than most other micro-
organisms and are comparatively ineffective competitors under
high-nutrient conditions. The proportion of Actinomycetes
relative to other bacteria has been suggested as an indicator of
compost maturity (Palmisano & Barlaz, 1996). Thus, it is not
surprising that microarray analysis revealed high levels of
Actinomycetes in the composts from longer curing times.
DGGE analysis showed the presence of Promicromono-
sporaceae members between 130 and 205 days of curing. We
also found Promicromonosporaceae in the noncured com-
posts clone library (Fig. 2a). Promicromonospora sukumoe
and related organisms of the Cellulomonas group have been
characterized as cellulose-degrading Actinobacteria (Bakali-
dou et al., 2002). Cellulomonas has been reported previously
in mature-compost bacterial populations (Ryckeboer et al.,
2003b). Cellulolytic bacteria are common in compost
(Herrmann & Shann, 1997) and are known to occur mainly
FEMS Microbiol Ecol 65 (2008) 133–144
141
0.5 1.0 1.5
at the end of the thermophilic stage (Gray et al., 1971). At
the end of the composting process, however, the cellulose is
often inaccessible to enzymatic attack because of low water
content or association with protective substances such as
lignin (Stutzenberger et al., 1970). This results in a decrease
in the number of cellulolytic organisms. It has also been
reported that in materials with high cellulose content,
thermotolerant microorganisms with the ability to degrade
cellulose can dominate the end stages of the composting
process (Ryckeboer et al., 2003b). Actinobacteria may sur-
vive the thermophilic phase of composting and become
active during compost maturation. The decomposition of
cellulose by cellulolytic bacteria such as Cellulomonas occurs
during the maturing process (Tang et al., 2006).
Cured compost populations
Gammaproteobacteria were also abundant in the cured
compost, and were detected in the clone library as well as
c2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
142
by microarray (Fig. 4). It is possible that different species of
Pseudomonas were responsible for hybridization with the
genus-level probe, and that certain species were present in
higher numbers at the start of the curing process, while
other species were present later in the curing process.
Pseudomonas species are widely distributed in the environ-
ment and have been reported previously in mature-compost
bacterial communities (Ryckeboer et al., 2003a). Pseudomo-
nas strains are known to be able to participate in N2 fixation,
denitrification, and degradation of pollutants or interaction
with toxic metals (Lalucat et al., 2006), and several strains
are known to confer plant-disease suppressiveness (Haas &
Défago, 2005). We found sequences affiliated to the Gam-
maproteobacteria, Xanthomonadaceae families both in the
cured compost clone library and among the DGGE bands of
cured compost samples (Table 1).
The dominant Actinobacteria found in the cured compost
were Corynebacterineae (i.e. Mycobacterium species), as
detected in the clone library and by DGGE. Many of the
Mycobacteria are potential pathogens. Mycobacterium
avium, an environmental opportunistic pathogen, has been
isolated from environmental water samples. Furthermore,
growth of M. avium in natural water was stimulated by the
addition of humic and fulvic acids in acidic, microaerobic
environments (Kirschner et al., 1999).
We found only a few Firmicutes sequences in the compost
clone libraries; we did not detect Firmicutes in DGGE and
their detection rate in the microarray was low. This finding
is surprising considering the dominance previously attrib-
uted to this phylum in compost. Ryckeboer et al. (2003b)
reported Bacillus as the most dominant bacterial taxon
recovered from compost feedstock, and it was also the most
abundant group of bacteria recovered from compost during
the thermophilic phase and throughout the entire compost-
ing process. Dees & Ghiorse (2001) reported that the two
most abundant cultivated isolates from hot synthetic com-
post belong to the genera Aneurinibacillus and Brevibacillus.
We suggest that Firmicutes members do not always persist in
compost after the thermophilic phase has ended.
It is apparent that members of different bacterial species
capable of similar functions were found throughout most of
the process. For example, at the beginning of the curing
process, the NH4 concentration was high (Fig. 5) and
supported a population specializing in nitrification (i.e.
Nitrosovibrio, Nitrosospira); thus, NH4 concentrations de-
creased rapidly and NO3 increased until 41 days. From 41 to
130 days, NO3 levels continued to increase, suggesting that
nitrification activity was maintained by more versatile
bacteria (Danon et al., 2007). Biodegradation of macromo-
lecules, especially of complex materials such as lignocellu-
lose, is a much slower process. A population specializing in
cellulolytic activity (i.e. Promicromonosporaceae) became
established during the intermediate curing time (130–205
c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
M. Danon et al.
days). A retarded response to added glucose in the cured
compost (Fig. 5) indicates a shift from microbial r-strate-
gists to K-strategists. The r-strategists are adapted to inter-
vals of rapid growth, depending on the availability of their
substrate, as opposed to the K-strategists, which typically
have slow growth rates, presumably benefiting from poly-
merized substrates that have a long residence time (Fontaine
et al., 2003).
In conclusion, we found a successional transition of
bacterial populations during compost curing. Comparison
of the results obtained by the three methods was used for
identification of different bacterial phylogenetic groups that
changed during prolonged compost curing. The Proteobacter-
ia were the most abundant phylum in all cases. The Bacter-
oidetes and the Gammaproteobacteria were ubiquitous but
opposite in their relative dominance. At the beginning of the
compost-curing process, Bacteroidetes were dominant. Later,
during the midcuring stage, Actinobacteria were dominant.
Finally, in the cured compost, the Gammaproteobacteria were
more abundant. Despite using a microarray that targets many
pathogens (Franke-Whittle et al., 2005; Franke-Whittle et al.,
2005), we did not detect any human, animal, or plant
pathogens by this method. This suggests efficient eradication
of pathogens during the composting. Furthermore, this
observation indicates that no reinfestation with pathogens
occurred during the prolonged curing.
The initial phase of composting is thought to be the most
dynamic part of the process (Schloss et al., 2003). Never-
theless, we found that the microbial community continued
to change long after the compost organic matter and other
biochemical properties had stabilized. It is assumed that
bacterial populations, playing their specific role in changing
the chemical environment, were replaced in turn by selec-
tion pressures driven by these changes in the microhabitat.
Changes in complex lignocellulose-rich organic matter may
have been too minute to detect using standard physico-
chemical analysis. However, bacterial community response
to these changes was more easily observed.
Acknowledgements
We gratefully acknowledge the assistance of Dana Levinson,
and thank Sharon Nahum-Zmora for her significant role in
carrying out the compost-curing process and providing the
samples, the chemical data, and critical remarks. This
research was supported by The Negev Foundation and the
Israeli Ministry of Agriculture and Rural Development, the
Wastewater Treatment Program.
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Supplementary material
The following supplementary material is available for this
article:
Fig. S1. 16S rRNA gene phylogeny of bacteria found in
composts at different curing stages.
This material is available as part of the online article from:
http://www.blackwell-synergy.com/doi/abs/10.1111/j.1574-
6941.2008.00506.x (This link will take you to the article
abstract).
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