Eng. Life Sci. 2010, 10, No.

6, 528536

528

Vladimir V. Zverlov1
Wolfgang Hiegl1
Daniela E. Kock1
Josef Kellermann2
Tanja Kollmeier1

Research Article

Hydrolytic bacteria in mesophilic and

thermophilic degradation of plant biomass

Wolfgang H. Schwarz1
1

Department of
Microbiology, Technische
Universit.at Munchen,
.
Freising-Weihenstephan,
Germany

Max Planck Institute for

Biochemistry, Am
Klopferspitz, Martinsried,
Germany

Adding plant biomass to a biogas reactor, hydrolysis is the first reaction step in the
chain of biological events towards methane production. Maize silage was used to
enrich efficient hydrolytic bacterial consortia from natural environments under
conditions imitating those in a biogas plant. At 55601C a more efficient hydrolyzing culture could be isolated than at 371C. The composition of the optimal
thermophilic bacterial consortium was revealed by sequencing clones from a 16S
rRNA gene library. A modified PCR-RFLP pre-screening method was used to group
the clones. Pure anaerobic cultures were isolated. 70% of the isolates were related to
Clostridium thermocellum. A new culture-independent method for identification of
cellulolytic enzymes was developed using the isolation of cellulose-binding proteins.
MALDI-TOF/TOF analysis and end-sequencing of peptides from prominent
protein bands revealed cellulases from the cellulosome of C. thermocellum and from
a major cellulase of Clostridium stercorarium. A combined culture of C. thermocellum and C. stercorarium was shown to excellently degrade maize silage. A spore
preparation method suitable for inoculation of maize silage and optimal hydrolysis
was developed for the thermophilic bacterial consortium. This method allows for
concentration and long-term storage of the mixed culture for instance for inoculation of biogas fermenters.
Keywords: Biogas / Cellulase / Clostridia / Enrichment / Hydrolytic bacterial consortium
Received: March 18, 2010; revised: July 20, 2010; accepted: August 2, 2010
DOI: 10.1002/elsc.201000059

Introduction

Hydrolysis of plant biomass is the first step in the chain of

interacting bacterial processes, from the decomposition of
organic materials to their metabolic conversion for example
into biogas. Degradation of the polysaccharides in the biomass
must be as complete as possible to enable an economically and
energetically efficient biogas production process. Reaction rate
and degree of substrate degradation in the hydrolysis step are
therefore the most important factors in the efficiency and yield
of the entire biogas production process. This reaction step in
plant biomass fed biogas fermenters is performed entirely by a
consortium of hydrolytic bacteria with still largely unknown
composition. They produce the monosaccharides and oligosaccharides, the short chain fatty acids and alcohols, and the
gasses necessary for the microbial processes downstream in the
biogas production chain, which are performed by the

Correspondence: Dr. Wolfgang H. Schwarz (wschwarz@wzw.tum.de),

Department of Microbiology, Technische Universit.at Munchen,
Emil.
Ramann-Str. 4, 85350 Freising-Weihenstephan, Germany

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

syntrophic bacteria and finally the aceticlastic and hydrogenotrophic methanogenic archaea [1, 2]. In contrast to the
rumen of cattle and other ruminants, anaerobic and microaerophilic fungi and ciliates do not play a prominent role in
plant biomass fed biogas reactors.
A number of mesophilic and thermophilic bacteria, mainly
anaerobic but also aerobic, with the ability to degrade biomass
have been isolated. Most are able to hydrolyze the relatively
easily accessible polysaccharides such as starch, pectin, b-1,3glucans, mixed-linkage glucans, hemicellulose or gums. The
extracellular enzymes of these bacteria are usually not very
active on natural cellulose although some of the strains have
been called cellulolytic due to their activity on carboxymethyl cellulose or mixed-linkage b-glucan [3, 4]. Many
environmental bacteria (especially soil dwellers) produce
endo-glucanases which are active on carboxymethyl cellulose
but not on crystalline (natural) cellulose. In contrast, true
cellulose degraders such as Clostridium thermocellum are
specialized and very efficient in the degradation of crystalline
cellulose. Although C. thermocellum produces enzymes for
the degradation of hemicellulose, it does not make use of the
released sugars (xylose) as a carbon source. The role of the

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Eng. Life Sci. 2010, 10, No. 6, 528536

hemicellulases seems rather to be to hydrolyse the hemicellulose which embeds the cellulose microfibrils, and, thus to
expose the cellulose substrates and make them accessible for
the cellulases. Accompanying bacteria in the consortium may
utilize the released sugars.
Another isolated thermophilic cellulolytic bacterium that
seems to specialize in the degradation of hemicellulose is Clostridium stercorarium [4, 5]. This bacterium differs from C. thermocellum in that it secretes only two cellulolytic exoenzymes,
namely an endo-glucanase and an exo-glucanase, CelZ (GH9) and
CelY (GH48), respectively. These cellulases are not organized in
the highly specialized and very effective cellulosome as was
identified in C. thermocellum [3, 6]. In contrast to C. thermocellum, C. stercorarium produces a multitude of soluble and highly
active hemicellulases. A complete enzyme system for the degradation of arabinoxylan has been reconstituted from recombinantly produced components [7], but many more enzymes
related to the decay of hemicellulose have been identified.
Different bacterial species are specialized on the degradation of certain polysaccharides in the substrate. Their number
within a bacterial community may vary with the type of
substrate present. More quantitative investigations of a
number of communities on various substrates will be needed
to understand the systems biology of plant biomass degradation.
The composition of hydrolytic bacterial communities in
purely plant-fed biogas plants, and the optimal conditions for
hydrolysis are to date essentially uncharted territory. The aim
of this study was to obtain insight into the underlying
processes and the methods to investigate them by mimicking
the hydrolytic stage of a biogas plant in a laboratory environment. Mesophilic and thermophilic processes are both
regarded.
A synthetic mixture of two bacteria commonly involved in
the natural degradation of lignocellulosic material was set up.
Both bacteria are also frequently found in thermophilic biogas
reactors (unpublished data). Their efficiency was compared
with mesophilic and thermophilic enrichments of hydrolytic
bacterial cultures having optimal performance on the same
substrate. Bacteria in an optimally hydrolyzing thermophilic
mixed culture were investigated on a molecular basis for their
phylogenetic heterogeneity to identify the composition of the
consortium. Potentially hydrolytic bacterial groups were
identified by homology of 16S rRNA sequence determination,
by isolation and identification of culturable cellulolytic
bacteria, and by identification of proteins binding to cellulose.

Material and methods

2.1

Strains and media

Type cultures of C. thermocellum (DSM 1237) and C. stercorarium (DSM 8532) were obtained from the German Collection
of Microorganisms and Cell Cultures (DSMZ). They were
grown in GS2 medium with 0.5% w/v cellobiose as carbon
source [8]. Escherichia coli clones were grown in LB-medium
with the appropriate antibiotics at 371C in accordance with
standard laboratory procedures.

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Hydrolytic bacteria for biomass degradation

529

In addition to samples from biogas plants from throughout

the state of Bavaria, Germany, samples were collected in the
area of Freising and Munich (both in Southern Bavaria) from
decomposing plant material, such as compost heaps, a
commercial compost fermenter (BM), cattle manure, garden
soil, bushes and agriculturally used plots (C18c, F34, G40), and
biogas fermenters (E32, E33, D5, D9). Samples were pasteurized (801C for 20 min) to inactivate vegetative cells, fungi,
nematodes and ciliates in order to select for hyperthermophilic
and spore-forming bacteria, which contain the majority of the
polysaccharide hydrolyzing bacteria. Bacterial mixed cocultures were grown in GS2 medium in pre-reduced, rubber
stoppered 50 mL bottles containing 2% (dry w/v) silage (airdried maize silage from whole plant or grass silage from
permanent grassland). This mixture was ground to a final
grain size of 2 mm with a cutting mill (Retsch, Germany) to
ensure representative composition in each sample. The flasks
were incubated at 37, 55 and 601C as indicated. They were not
constantly shaken but degassed and shaken manually once a
day.
To isolate purified bacterial cultures, samples were streaked
to single colonies under strictly anaerobic conditions on GS2agar plates with a 5 mL-overlay of GS2-agar containing 2% w/v
of crystalline cellulose MN301 as an indicator for cellulolysis.
Single colony purification was repeated three times.

2.2

Sporulation

To 50 mL GS2 medium 0.2% w/v filterpaper Whatman No. 1

was added as carbon source. Cultures were incubated at 551C
for 57 days until cellulose was degraded completely. As a
reference, a non-incubated bottle was included in every series.
The bottles were then placed in a cold room (461C) for
2448 h. Spore formation was controlled microscopically. The
bottle content was centrifuged at 41C, 5000 rpm for 25 min,
and washed twice with 50 mL H2O (bidest). The spore
containing pellet was resuspended in 5 mL H2O (bidest) and
stored at 41C. Spores were reactivated by incubation with
substrate in a buffered medium (usually GS2) and incubation
at 551C. Other media tested for sporulation were those
described by Monot et al. [9] and Tortora et al. [10], and the
CAMM [11] and CM3 medium [12].

2.3

Determination of biomass

About 0.5 g of finely ground air-dried silage (2 mm sieve) was

dispersed in 50 mL pre-reduced medium (GS2). Bottles were
sealed with butyl-rubber stoppers. After sterilization, all bottles
except the control were inoculated and incubated at the
respective temperature for 7 days, unless indicated otherwise.
To measure the biomass content, bottles were emptied into
Fibre Caps (nylon filter by Foss, Fibercap system 2023, pore
width 23 mm). The caps were dried at 1001C over night and the
dry weight was determined. In accordance with the protocol of
the manufacturer (Foss, Germany) the fiber material was
subjected to van Soest analysis to determine neutral detergent
fiber, acid detergent fiber and acid detergent lignin [13, 14].

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530

V. V. Zverlov et al.

The content of cellulose, hemicellulose and ash1lignin was

calculated from these values. All tests were performed at least
in triplicate.

2.4

Recombinant DNA techniques

Preparation of chromosomal and plasmid DNA, endonuclease

digestion, ligation and transformation was carried out by
standard procedures or in accordance with supplier protocols.
Chromosomal DNA from culture samples was prepared using
the Presto Spin D Bug DNA Purification Kit (Molzym,
Germany) or using the standard chloroformphenol extraction
method. Plasmid DNA was prepared with the QIAprep Spin
Miniprep Kit (Qiagen, Germany). Restriction digests of DNA
were performed as per the manufacturer recommendations
(MBI Fermentas or Boehringer Ingelheim Bioproducts).

2.5

Sequence analysis

PCR amplification, cloning and sequencing of the 16S rRNA

gene were performed with bacteria-specific oligonucleotide
primers as described in Berezina et al. [15]. PCR fragments of
the 16S rRNA gene were digested with restriction endonucleases BsuRI and Hin6I and the band pattern was analyzed by
electrophoresis in 12% polyacrylamide gel slabs. PCR fragments were cloned in Topo TA Cloning vector DNA (Invitrogen) and transformed in One Shots TOP10 Chemically
Competent E. coli cells (Invitrogen). At least two clones from
each PCR-RFLP group were sequenced from both ends, and
manually connected and aligned with the ARB software
program [17, 16]. Sequences were deposited at the EMBL
Nucleotide Sequence Database (successive accession numbers
from FN868398 to FN868437).

2.6

Eng. Life Sci. 2010, 10, No. 6, 528536

plants with optimal performance from throughout the German

state of Bavaria were included. Mixed bacterial cultures were
enriched on maize and grass silage. The biological process
undergone in these bottles represents the hydrolytic step in a
biogas plant. Hydrolysis was determined and cultures with
efficient hydrolysis performance were selected for further
investigation.
The enriched cultures were tested for growth, degradation
of the biomass and content of cellulose, hemicellulose and
mixed lignin1ash fractions. In comparison to an uninoculated
control (K), some enrichment cultures degraded about half of
the available biomass under the conditions of small batch
cultures. However, the kinetic experiments revealed that
degradation stopped following about day 5, presumably due to
a decrease in pH, and an accumulation of unfavorable metabolic end products (data not shown). The results from the best
hydrolyzing cultures are shown in Fig. 1. The best results were
obtained with the two cultures G40 and C18c at 601C. It is
interesting to note that higher temperatures allowed for the
isolation of substantially better cultures. It should also be
noted that a mixed culture of the bacterial species C. thermocellum and C. stercorarium performed similarly to the bestenriched bacterial consortium in the test at 601C (Fig. 1 and
below).
The most stable and well-performing cultures for the
mesophilic (E32 at 371C) and the thermophilic conditions
(G40 at 601C) on maize silage were selected and investigated
further (Fig. 1). From the data in Fig. 1 it is apparent that in all
cultures, including a mixture of pure cultures of C. thermocellum and C. stercorarium, hemicellulose as well as cellulose
were degraded simultaneously alongside cellulose, and that the

Preparation of cellulose-binding proteins

A bacterial culture of 500 mL was centrifuged (5000  g,

15 min, 41C) and the supernatant was incubated overnight at
41C with 100 mg/mL phosphoric acid swollen cellulose. After
centrifugation (13 000  g, 15 min, 41C) the pellet was resuspended in protein loading buffer. The protein composition was
analyzed by denaturing SDS-PAGE and staining with
Coomassie Brilliant Blue G-250.
Protein identification by MALDI-TOF/TOF was performed
as previously described in Zverlov et al. [17]. All sequenced
peptides were 100% identical with proteins from the protein
database entries.

Results

3.1

Enrichment of hydrolytic bacterial cultures

More than 20 samples were collected from different natural

sources where plant biomass degradation takes place. In
addition, samples from mesophilic and thermophilic biogas

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Figure 1. Degradation of maize silage by different bacterial

cultures. Incubation for 7 days at the temperature (1C) in
brackets; remaining biomass (dry weight in % of original
biomass) was fractionated using van Soest analysis. K, uninoculated control (incubated at 601C); CTH, C. thermocellum;
CST, C. stercorarium. Each data set is represented by three
columns, which, from left to right, designate soluble fraction
(white bars), hemicellulose (hatched bars), and cellulose1
lignin1ash (crosshatched bars). Standard deviation between
multiple measurements is indicated.

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Eng. Life Sci. 2010, 10, No. 6, 528536

reduction of these two polysaccharide classes was approximately proportional (Fig. 2). This is an indication that cellulose degradation is naturally coupled to hemicellulose
degradation and that hydrolysis of either one of these two
polysaccharides cannot occur efficiently in isolation. This can
be explained by the tight structural connection between
hemicellulose and the embedded cellulose in the plant cell wall.

3.2

Hydrolytic bacteria for biomass degradation

531

the finding that C. thermocellum degrades cellulose down to a

pH of 6.0 in pure culture (data not shown). However, the
hydrolysis is slowed down to the background level measured
for loss of fibers through the nylon mesh even without
inoculation. Weight loss of 38% at and below pH 5.8 was
observed, whereas degradation was 62% at the initial pH of 6.4
and 82% above pH 7.1. Effective cellulose hydrolysis thus
implies the need for pH regulation by the addition of alkali or
by concurrent methanogenesis.

Influence of pH on cellulolytic cultures

Cultures were enriched from the natural samples on maize

silage in media adjusted to a range of initial pH values from 5.2
to 8.0, conditions that can occur in a biogas plant. A strip of
Whatman filter paper No 1 was added to the maize silage as an
indicator for cellulolysis. No cellulose-hydrolytic culture could
be enriched below an initial pH of 6.3 (data not shown). This
does not necessarily indicate that cellulolysis did not take place
at a pH below 6.3. However, in a mixed culture the easily
degradable polysaccharides like pectin, starch, or hemicellulose
are metabolized first and lower the pH of the culture fluid
below a detrimental pH level, which prevents the much slower
degradation of cellulose. This observation is corroborated by

3.3

Phylogenetic analysis of hydrolytic culture

The culture exhibiting the best and most stable hydrolysis of

maize silage (G40 at 601C) was investigated by molecular
methods for its bacterial composition. DNA from single 16S
rRNA clones was subjected to PCR-RFLP analysis to avoid
sequencing of multiple copies of identical DNA. Operational
taxonomic units for each sequenced DNA were determined by
16S rRNA sequence similarity to known bacteria in
the ARB database [17, 16]. Clones with identical PCR-RFLP
pattern were added to the number of clones given in
Table 1.

Figure 2. Degradation of maize silage by the

best hydrolytic cultures selected at 371C for
culture E32 (A) and 551C for culture G40 (B)
respectively in 110 days (0 is inoculated
control). The results of van Soest analysis
are represented by three columns which,
from left to right, designate hemicellulose
(white bars), cellulose (hatched bars) and
lignin1ash fractions (crosshatched bars) in
percentage of the input material (dry weight)
before inoculation. Standard deviation
between multiple measurements is indicated.

Table 1. Operational taxonomic units by 16S rRNA gene sequence similarity to known bacteria in the ARB database.
Next related bacterium
Clostridium cellulosi
Clostridium thermocellum
Clostridium thermopalmarium/thermobutyricum
Clostridium thermosuccinogenes
Clostridiaceae bacterium Aso3-CS349
Clostridiaceae bacterium Aso3-CS342
Tepidimicrobium sp. (Firmicutes)
Tepidanaerobacter sp. (Firmicutes)
Symbiobacterium thermophilum
Uncultured Bacillus sp. clone 96
Uncultured Bacillus sp. clone 54
Total

% identity

Clones

% of clones

9192
99
9799
9699
99
95
98/99
9596
9799
98/99
9598

1
4
13
5
26
1
2
10
6
2
17
87

1.1
4.6
14.9
5.7
29.9
1.1
2.3
11.5
6.9
2.3
19.5
100

The sequence identity (in %) and the number of clones falling into this group are indicated. Last lane: portion of clones analyzed by PCR-RFLP (in %).

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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532

All sequenced clones given in Table 1 were related to

members of the phylum Firmicutes. This is not surprising
given the initial pasteurization step which excluded non-sporeforming bacteria. The only bacterial species described as
cellulolytic and related to the sequences obtained are
C. cellulosi and C. thermocellum. Both species are described as
thermophilic anaerobic degraders of cellulose [5, 18]. It is not
sure from the literature, if the two other OTUs belonging to
the Clostridiaceae (C. thermopalmarium and C. thermosuccinogenes) contain cellulolytic bacteria. However, they have
been isolated from compost and thus from a natural environment with conditions similar to a biogas plant [19], and
ferment sugars to mainly butyrate [20, 21].
The other sequences belong to clearly non-cellulolytic
bacterial groups. The two OTUs of the Bacillus group are most
probably saccharolytic but not cellulolytic bacteria. They most
certainly contribute to the overall hydrolysis of starch, pectin,
gums and various hemicelluloses, or even produce endo-glucanases, which reinforce the cellulolytic activity of the clostridia.
Tepidimicrobium is a thermophilic, peptolytic and strictly nonsaccharolytic bacterium related to the clostridia [22]. Although
spore formation for the members of the genus Tepidimicrobium
has not been observed so far, this is probably due to their genetic
similarity to clostridia. Tepidanaerobacter is an anaerobic,
moderately thermophilic, syntrophic, primary alcohol and
lactate degrading bacterium which grows well in co-culture with
the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus. Without an externally added electron acceptor it
utilizes ethanol, glycerol and lactate syntrophically [23].
Symbiobacterium thermophilum is also a thermophilic,
syntrophic bacterium [24]. These bacteria may add to the overall
conversion of plant biomass into chemical products favorable for
methanogenesis such as acetate, CO2 and H2.

3.4

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V. V. Zverlov et al.

Isolation of new cellulolytic bacteria

To identify hydrolytic bacteria, cultures from the well hydrolyzing

mixed culture G40 were streaked to single colonies for further
characterization in pure form. Bacteria producing cellulolytic
exo-enzymes form colonies surrounded by a halo in the turbid
cellulose overlay. The 16S rRNA gene sequence of these purified
cultures was compared to a database of bacterial 16S rRNA
sequences (ARB database). According to the 16S rRNA sequence,
10% of the isolates were unknown bacteria, 10% were
a-proteobacteria, 70% were closer than 98% sequence identity to
C. thermocellum, and 10% were other Clostridium species.
The a-proteobacteria were most probably contaminating
bacteria occluding a cellulolytic strain, and may have resulted
from partially impure cultures. It is a general problem with the
purification of thermophilic anaerobic bacteria and especially
with C. thermocellum that they are not easily separated from
each other by single colony streaking (J. Wiegel, personal
communication). However, it is possible that cellulolytic
bacteria are among the Clostridium isolates and the
unknown bacteria. C. thermocellum was isolated repeatedly
from different cellulolytic samples in our studies as well as in
other labs, and all isolates were cellulolytic. These strains will
be further investigated in a later study.

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

The isolation of bacteria in pure culture is hampered by the

necessity of culturing, which restricts the result to culturable
strains. Non-culturable bacteria probably constitute the
majority of the bacteria in such environmental samples and are
not picked up by this method. It is thus possible that the
distribution of cellulolytic strains found here is not representative of the distribution of cellulolytic strains in the mixed
culture. However, a point of interest is that the majority of
isolated cellulolytic bacteria from the hydrolytic mixed culture
G40 were related to C. thermocellum. This was corroborated by
the data from 16S rRNA sequence analysis and the protein
isolation data described below.

3.5

Isolation of proteins binding to cellulose

As an additional, methodically unrelated and new technique

for determining the major hydrolyzing agents in fermentation
sludge, proteomics methodology was applied to identify
cellulose hydrolyzing proteins and their most probable
producing hosts. This technique is largely independent at least
of culturing biases. The intrinsic cellulose adsorption of
cellulose hydrolyzing enzymes the so-called true cellulases
was exploited to enrich these proteins from the multitude of
proteins present in the cleared supernatant, i.e. from the
bacterial cells and the residual substrate [3]. To avoid the loss
of binding cellulases together with residual cellulose fibers in
the centrifugation pellet, a necessary step in excluding the
bacterial cells in the culture, the last round in growing the
mixed culture was performed in a synthetic medium, which
had been shown to grow all bacteria in the blend and which
contained a completely degradable cellulosic substrate. After
adsorption to a homogeneous cellulose matrix the bound
enzymes could be washed from the non-adsorbed proteins of
the clarified culture supernatant.
The SDS-PAGE of the concentrated proteins (Fig. 3) shows
discrete bands of a surprisingly limited number of major
polypeptides. Proteins with only one hit were not taken into
account in this analysis. The four most prominent major bands
were analyzed. Most unusual is a band at 230 kDa, which
reminds the scaffoldin band (CipA) of clostridial cellulosomes.
After MALDI-TOF/TOF and peptide sequencing this M1 band
gave multiple hits with the CipA protein of C. thermocellum
type strain ATCC 27405, the cellulosome integrating scaffoldin [25]. This indicated that the CipA protein was one of the
major proteins in the preparation.
Similarly, proteins from the bands M2 to M4 could be
assigned to proteins of the C. thermocellum cellulosome: pectate
lyase PL1 (CTHE 2179) and cellobiohydrolase CbhA (GHF9) in
band M2, cellobiohydrolase CelK (GHF9) in band M3, the major
cellobiohydrolase CelS (GHF48), and endoglucanase CelQ
(GHF9) in band M4 [2630]. These proteins have been shown
earlier to be among the major proteins of the cellulosome
[17, 31]. Only one protein from another bacterium was
obtained, cellulase CelY (GHF48) from C. stercorarium, which
was identified in band M4. This processive cellulase is one of the
two cellulases from the two-enzyme cellulase system of C. stercorarium, which had also been shown to be involved in hydrolysis of crystalline cellulose [32].

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Eng. Life Sci. 2010, 10, No. 6, 528536

M1

M2
M3
M4

Figure 3. SDS-PAGE of concentrated samples of cellulose-binding proteins from the thermophilic hydrolytic bacterial mixture.
Marker proteins with defined molecular mass are on the right
lane (170, 130, 95, 72, 55, 43, 34 kDa). Bands extruded for
MALDI-TOF-TOF analysis are marked M1 to M4.

3.6

Growth of hydrolytic reference cultures on silage

From the earlier studies it was known that C. thermocellum and

C. stercorarium are ubiquitarily occurring bacterial species
commonly involved in cellulose and hemicellulose degradation
during the natural decay of plant biomass [5]. In this study it
was shown that extracellular cellulases of these two species
were the predominant hydrolytic proteins attaching to cellulose. Hence it was investigated how efficiently a combination of
these two thermophilic anaerobic species alone and in coculture can degrade the polysaccharides in maize silage.
Whereas C. thermocellum is able to degrade and utilize cellulose with high efficiency, it degrades hemicellulose as a side
activity of its enzyme complex (cellulosome). However, it
cannot utilize the sugars produced from hemicellulose, which
consist mainly of xylose. In contrast, C. stercorarium is less
efficient at cellulose hydrolysis and possesses only two genes
for cellulases. It does, however, have a great number of genes
for the hydrolysis of hemicellulose. It hydrolyzes hemicellulose
efficiently, aside from contributing to cellulose degradation,
and utilizes the degradation products, the hexoses as well as the
pentoses [5, 7, 33].
Both bacteria produce the typical end products of clostridial fermentation: ethanol, lactate, acetate, butyrate and
other short chain products in addition to CO2 and H2 [5].
These products fit well into the scheme of typical substrates for
the syntrophic and acetogenic bacteria, as well as for the
aceticlastic and hydrogenotrophic methanogenic archaea in the
biogas process. These bacteria conclude the disproportioniza-

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Hydrolytic bacteria for biomass degradation

533

tion process from the biomass polysaccharides to the reduced

and oxidized final products, methane and CO2, respectively.
Both bacterial species, C. thermocellum and C. stercorarium,
grow well on maize silage (not shown). Dense growth with
about 109 cells/mL was obtained within 50 h with vigorous
formation of hydrolysis gas and of fermentation products,
mainly acetate and ethanol (GC data not shown). C. stercorarium grew at least initially faster on the substrate but did not
ultimately degrade as much of the substrate (65 versus 78%)
(Fig. 4). In contrast, C. thermocellum alone initially did not
grow as quickly, but degraded as much of the substrate as the
synthetic mix of both species after more than 60 h incubation.
The mixed culture of both strains was able to degrade the
substrate within 2 days by 68%. After 80 h, ca. 76% of the
substrate (dry weight) was degraded, which corresponds
almost to the maximum possible degradation achievable with
an optimal enriched culture.
A synergistic effect of the two bacterial species in the
kinetics of substrate degradation is evident. This may be a
result of the combined action of hemicellulases and cellulases,
to which both bacteria contribute to a different degree.
However, the overall degradation seems to be limited by a
decrease in pH (down to a pH value of 5.5) as well as by
an accumulation of fermentative products in the culture
supernatant.

3.7

Preparation of spores for inoculation

Various media described in the literature for the preparation of

bacterial spores were tested and all resulted in varying but
always efficient degrees of spore formation. GS2 medium with
filter paper as sole carbon source was chosen as optimal for the
thermophilic culture. The spores were reactivated and tested
on maize silage to prove that the degradation of plant fibers
was still as effective as in the original culture, and that the
hydrolytic quality of the culture was not diminished: degradation levels of 44.8 (71.0) and 45.5 (71.0)% were achieved
after the addition of 0.1 and 0.5 mL of spore solution,
respectively, compared to 40.8 (70.1)% degradation with the
addition of 1.0 mL fresh culture to maize silage in GS2
medium. This result shows that spore-forming bacteria are
sufficient for plant fiber hydrolysis.
With this method the enriched hydrolytic culture can now
be stored for longer periods and reactivated on demand. A
concentrated addition of spores for instance to a not well
hydrolyzing biogas fermenter is now possible, without adding
much volume to a vessel and disturbing the measurement of
degradation efficiency.

Discussion

To determine the degradation efficiency of bacterial cultures, a

sieving and washing technique that measures the reduction in dry
weight of particles held back by a fine nylon mesh was chosen. In
addition to pure hydrolysis of polysaccharides in plant fiber, i.e.
the saccharification by enzymatic degradation, the loss of small
particles was accepted as a result of hydrolysis. Consequently, the

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V. V. Zverlov et al.

Figure 4. Time course for degradation of maize silage by

C. stercorarium (CST), C. thermocellum (CTH), and a mixture of
both (MC), resp., measured as decrease of dry substance in
substrate after washing (T 5 601C). Filled circle, C. stercorarium;
circle, C. thermocellum; filled triangle, mixed culture of C. stercorarium and C. thermocellum; triangle, no inoculation (control).

contribution of hydrolysis to the solubilization (i.e. the release of

sugars) may be overestimated. The reduction in particle size may
be a consequence of the removal of hemicellulose between lignin
structures and cellulose microfibers allowing the loosened fibers
to slip through the mesh. Fiber size reduction is thus one of the
effects of hydrolytic attack of the enzymes produced by the
bacteria and loss of fiber dry mass does seem to be a reliable
measurement for hydrolytic action and is at least proportional to
the true hydrolytic action.
Another pre-condition of the type of hydrolysis determination in these experiments was that hydrolysis was determined in
batch without removing the fermentation products. Only the
produced hydrolysis gas (CO2 and H2) was released on a daily
basis. The accumulation of alcohols and acids may result in
unfavorable conditions for further growth of the hydrolytic
bacteria and thus stop enzyme production, also leading to
reduction or even cessation of enzymatic activity at acidic pH
levels. Cultures that produce less toxic or acidic products, or that
are more resistant to their harmful effects will grow longer and
ultimately have a higher hydrolysis rate. Cultures producing
more acid resistant hydrolytic enzymes will also be more active.
Understanding of these effects is important for handling the
biogas process in an optimal way and eventually isolating a crash
resistant culture to be added to a biogas fermenter.
With the described method an optimally hydrolyzing
culture was obtained from dozens of natural samples. Various
conditions have been studied with this culture and their
influence on the hydrolysis rate was determined. The optimal
hydrolytic culture was most efficient at a temperature of 601C.
A culture with a similarly high degradation rate could not be
obtained at 371C and even cultures from well-performing
mesophilic biogas plants could not achieve such high results in
the test. However, the total degradation value of the best
mesophilic culture albeit after a considerably elongated

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Eng. Life Sci. 2010, 10, No. 6, 528536

incubation time was about the same as with the optimal

thermophilic culture.
The optimally degrading culture showed comparable but
slightly better degradation as a synthetic two-component
mixture of purified hydrolytic bacteria, C. thermocellum and
C. stercorarium as well in end degradation value as in degradation rate. This was unexpected as the hydrolytic bacteria
were only a minority in the investigated mixed cultures,
outnumbered by the saccharolytic/acidogenic and syntrophic
bacteria (Table 1) [1]. These bacteria, which are not present in
the synthetic mix, assist the hydrolytic bacteria in further
processing the fermentation products and hence in creating a
suitable environment for productive hydrolysis. This means
production of hydrolytic enzymes as well as maintenance of
the optimum pH for hydrolytic activity of the enzymes.
The major hydrolytic bacteria in one fermentation mix
resembling cultures in biogas fermenters could be identified as
C. thermocellum, and C. stercorarium and/or closely related
bacteria. Both, C. thermocellum and C. stercorarium have been
previously identified as superior biomass fiber degraders
[3, 34, 35]. The experiments here show that they can be present
in relatively high numbers in natural mixed cultures. Unpublished results demonstrate that at least C. thermocellum is
present in a wide range of natural habitats where plant biomass
is degraded, almost independent of the average temperature.
This may be a source for these bacteria which find favorable
conditions for multiplication and activity in biogas plants.
C. thermocellum was identified in the selected thermophilic
culture by three independent methods: 16S rRNA gene
sequencing, strain isolation and exo-protein analysis. Whereas
DNA isolation, PCR amplification with general bacterial
primers and DNA sequencing of PCR-RFLP selected clones are
relatively independent on the bacterial species, strain isolation
from natural samples is strongly biased by the culturability
of the bacteria. More than 90% of soil bacteria cannot be
cultivated in pure culture either because they depend on cocultivated bacteria or on as yet unknown growth factors or
medium conditions. The new protein isolation and analysis
approach used here for the first time is probably biased by the
pre-selection of cellulose-binding proteins. However, the two
biased methods rely on completely different selection criteria
and should achieve independent results. However, all cellulases
of great importance for the hydrolysis of natural (i.e. crystalline) cellulose contain a cellulose-binding module, either
covalently attached to each enzyme as in Caldicellulosiruptor
sp. or in some clostridia such as C. stercorarium, or through
the complex-integrating protein as is the case in cellulosomeforming bacteria such as C. thermocellum. The method seems
to have isolated cellulosomes or parts of it, in addition to
abundant soluble cellulose-binding enzymes. The isolation of
complete cellulosomes explains why even enzymes which do
not have a functional cellulose-binding module such as CelS or
the pectate lyase were identified with this method.
The proteomic method based approach preferentially
identifies sequences of proteins from bacteria with sequenced
genomes, such as C. thermocellum, or of cellulases for which
sequences are in the databases such as for C. stercorarium.
Completely new and to date unknown sequences will not be
recognized. However, the redundancy of cellulase sequences in

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Eng. Life Sci. 2010, 10, No. 6, 528536

the databases is overwhelming, and completely new cellulase

sequences are probably not to be expected. It can thus be
assumed that all major cellulases present in the protein
preparation have been identified and due to the high
confidence of the partial sequences assigned to the two
species C. thermocellum and C. stercorarium, respectively.
The optimal cellulolytic pH of the cellulase system of
C. thermocellum has been determined to be above pH 6.5. This
has consequences for biogas plants in that, although good growth
of cellulolytic bacteria can be observed, effective cellulose hydrolysis cannot be expected below pH 6.5. Such low pH values would
limit the substrate for growth and hence the hydrolysis to
easily accessible polysaccharides in the plant material, such as
starch, pectin or some types of hemicellulose [36]. The overall
hydrolysis efficiency of a biogas fermenter would thus be
predicted to be far below optimum if the pH is too low. Running
two stage biogas plants with a separate hydrolysis stage without
simultaneous acid utilization by aceticlastic methanogenic bacteria
must be considered with caution in light of these findings.
The results give insights into the hydrolysis in biogas plants
or other plant fiber digesters and open possibilities for
monitoring the healthy hydrolysis state of a reactor. Optimal
conditions for hydrolysis have not been found to be incompatible with conditions for optimal methanogenesis. This
explains the high performance of one-stage fermenters widely
used in the biogas industry. However, further research is
needed to define optimal conditions for a separate hydrolysis
step in biogas formation from plant biomass for enhanced
hydrolysis rates. C. thermocellum and C. stercorarium were
identified as major players in the hydrolysis of plant biomass,
even though their number in the mixed culture is low. This is
in contrast with the activity of the co-culture of C. stercorarium
and C. thermocellum, which is not better than that of the mixed
culture G40. This discrepancy in activity and obvious cell
number may at least partially be explained by the fast utilization of potentially inhibitory hydrolysis products such as
cellobiose, and by a synergistic effect of other still unknown
hydrolytic bacteria in the mixed culture. Some of those may
have escaped 16S rRNA gene sequence analysis due to low
abundance as was seen for C. stercorarium.
Sporulation conditions for effective inoculation of hydrolysis fermenters have been developed, which allow the addition
of culture concentrates without disturbing the mass balance of
a monitored fermenter. This will enable future application of
the addition of hydrolytic bacterial cultures to biogas
fermenters with suboptimal substrate turnover.

Acknowledgements
The authors were supported by grant no. FKZ 22011705 from
the German Federal Ministry of Food, Agriculture and
Consumer Protection attended by the Fachagentur Nachwachsende Rohstoffe (FNR, Agency for Renewable Resources,
Guelzow, Germany), grant no. FKZ 03SF0346C from the
Federal Ministry of Education and Research, and by project
N/07/45 from the Bavarian State Ministry for Food, Agriculture and Forestry (StMELF). The technical assistance of
Sabine Dummert is gratefully acknowledged. We acknowledge

& 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Hydrolytic bacteria for biomass degradation

535

samples from biogas plants in Bavaria (Germany), which were

provided by LfL-ILT (Bavarian State Research Center of Agriculture, Institute for Agricultural Engineering and Animal
Husbandry, Freising, Germany).
Conflict of interest
The authors have declared no conflict of interest.

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