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Sugarcane celluloseutilizationbyade¢nedmicrobial consortiumClaudia Guevara & Marıa Mercedes Zambrano
Corpogen, Bogota, Colombia
Correspondence: Marıa Mercedes
Zambrano, Corpogen, Carrera 5 No. 66A-34,
Bogota, Colombia. Tel.: 1571 348-4610;
fax: 1571 348-4607;
e-mail: [email protected]
Received 9 June 2005; revised 8 November
2005; accepted 9 November 2005.
First published online January 2006.
doi:10.1111/j.1574-6968.2005.00050.x
Editor: Elizabeth Baggs
Keywords
cellulose; sugarcane; enzymatic potentiation;
microbial consortia.
Abstract
Microorganisms isolated from diverse environmental sources were initially
screened for carboxymethylcellulase activity. Nine strains that grew at elevated
temperatures and which presented the highest activity were characterized further.
Culture supernatants were assayed for potentiation of the enzymatic activity and,
based on these results, consortia of four or nine microorganisms were tested for
their capacity to grow on, and degrade a sugarcane leaf substrate. As predicted by
the supernatant mixes, both consortia assayed were capable of degrading the
cellulosic substrate provided. The group comprising of four strains was as efficient
as the mix of all nine strains.
Introduction
Cellulose, the major component of plant biomass, is the
most abundant biopolymer in nature and is therefore
attractive as a sustainable source of fuel and material for
industrial processes (Mansfield & Meder, 2003). Cellulose
utilization, usually carried out by enzymes that act synergis-
tically and in a co-ordinated manner, is mediated mostly by
microorganisms in a complex process that involves cellu-
lases present in fungi and bacteria. These microorganisms
are therefore important in terms of global carbon cycling
and as a source of enzymes potentially useful in both
industry and biotechnology (Lynd et al., 2002).
The large volumes of cellulosic waste generated annually
because of forestry, agricultural and industrial activities are
difficult to degrade and cause imbalances in the ecosystem.
Utilization of this biomass, which could provide a low-cost,
renewable source of carbon and energy, is difficult because
of its recalcitrance to being broken down into more readily
utilizable components (Bhat & Bhat, 1997). Treatment of
agricultural residues with either cellulolytic enzymes or
microorganisms could lead to more efficient degradation of
this waste material, promote cycling of nutrients in the
environment and reduce the impact of waste accumulation
on terrestrial and aquatic ecosystems (de Vries & Visser,
2001). In the sugarcane industry, the current practice in
many places is to burn the sugarcane before harvesting to
eliminate foliage that interferes with manual processing. The
Colombian sugarcane industry, which generates between 36
and 54.4 tons of waste material per hectare annually
(Victoria et al., 2003), will require new strategies for the
removal of this unused biomass that can no longer be
burned, starting in the year 2005.
With the aim of identifying microorganisms with cellulo-
lytic activity and which would thus be potentially useful for
hydrolysis of sugarcane foliage waste, we screened a collec-
tion of microorganisms for carboxymethylcellulase
(CMCase) activity. The identified isolates were then ana-
lyzed in an in vitro potentiation assay and mixed cultures
were assayed for their ability to degrade a sugarcane
cellulose substrate under controlled laboratory conditions.
Materials andmethods
Microorganisms,mediaand culture conditions
Soil samples were serially diluted in 0.9% NaCl, plated on
0.1� tryptic soy agar (TSA, BD Difco, Sparks, MD) and
potato dextrose agar (PDA, BD Difco) and incubated at
30 1C. Single isolates were distinguished by morphology,
analyzed by Gram staining and stored at � 80 1C in 15%
glycerol. Escherichia coli DH5a was used as a control.
Carboxymethylcellulose (CMC) (Sigma, St Louis, MO)
medium viscosity, broth contained (per litre): 1 g NaNO3,
1 g K2HPO4, 1 g KCl, 0.5 g MgSO4, 0.5 g yeast extract, 1 g
glucose and 5 g CMC. CMC agar consisted of CMC broth
containing 1.7% agar (BD Difco). SSC medium contained
(per litre): 2.5 g (NH4)2 SO4, 5.5 g NaCl, 5.5 g KH2PO4, 0.1 g
FEMS Microbiol Lett 255 (2006) 52–58c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
MgSO4, 0.1 g CaCl2, 3% treated sugarcane leaves (Ponce-
Noyola & De la Torre, 1993; Lee et al., 2000). ASC medium
consisted (per litre) of 3% treated sugarcane leaves in
distilled H2O. Growth on crystalline cellulose was carried
out in M63 medium (Miller, 1992) containing 1% crystal-
line cellulose (Merck, Darmstadt, Germany). All liquid
cultures were grown shaken at 150 rpm. at 40 1C, unless
otherwise specified. For growth curves and consortia, strains
were first grown in 5 mL CMC broth to an OD600 nm of 0.5
or 106 conidia per mL for actinomycetes, and 2 mL were
used to inoculate 48 mL in 250 mL flasks. Growth was
followed by plating dilutions on solid media to determine
colony-forming units (CFU). Total protein was determined
using the Bradford reagent (BioRad, Hercules, CA), after
centrifugation of cultures and lysis of cells with 1 N NaOH,
as described (Pavlostathis et al., 1988). For consortia,
cells were washed and resuspended in 0.9% NaCl before
mixing in equal proportions and inoculating into SSC or
ASC medium.
CMCagardiffusionassay
Cells were tested for cellulolytic activity by picking isolated
colonies onto CMC agar plates. After overnight growth,
clearing zones indicative of extracellular cellulase activity
were identified by staining with Congo red (Sigma), as
described (Teather & Wood, 1982).
Enzymeactivity
Individual strains were grown in triplicate at 40 1C in CMC
broth. Cultures were centrifuged 10 min at 8000 g and
0.5 mL of the supernatant were mixed with 0.5 mL of a 1%
CMC solution in 0.1 M sodium phosphate buffer (pH 7),
incubated at 40 1C for 30 min and the reaction stopped at
4 1C for 10 min. After centrifuging for 10 min at 4000 g, the
amount of reducing sugar in the supernatant was deter-
mined colorimetrically using dinitrosalycilic acid (DNS)
and glucose as a standard (Miller et al., 1960). Essentially,
250 mL of the supernatant were mixed with an equal volume
of 3,5 DNS solution (1 g DNS, 1.6 g NaOH, 43.8 g potassium
sodium tartrate, H2O to 100 mL), boiled for 5 min, placed
on ice for 10 min, mixed with 2.5 mL H2O, and read at
540 nm. Enzyme activity is given as international units (IU)
where 1 U of activity is defined as the amount of enzyme
required to liberate 1 mmol of glucose equivalent per min
under the assay conditions. All values were normalized
against the background activity detected using buffer only.
Enzyme activity was assayed at different temperatures (20,
30, 40, 50, 60, 70, 90 and 100 1C) to determine the optimum
temperature. The following buffers were used to determine
optimum pH at 40 1C: 0.05 M sodium citrate, pH 4; 0.05 M
sodium acetate, pH 5; 0.1 M sodium phosphate, pH 6, pH 7
and pH 8; 0.1 M Tris, pH 9 and 0.1 M glycine, pH 10 (Cao &
Tan, 2002).
Potentiationassays
Cultures grown in CMC broth were centrifuged at 8000 g for
10 min, the supernatants were filter-sterilized using 0.22mm
filters (Sartorius AG, Goettingen, Germany) and then mixed in
equal proportions, to a final volume of 500mL. Enzyme activity
was determined as described above, using CMC as a substrate.
Preparationof sugarcane leavesand cellulosedetermination
Fresh sugarcane leaves were processed as described by Lee
et al. (2000). Briefly, leaves were cut into small pieces, boiled
for 1 h in 1% sodium dodecyl sulfate (SDS), washed
extensively with water, dried at 55 1C and autoclaved before
use. To determine cellulose concentration, the cellulose
material was first washed with a nitric acid–acetic acid
reagent and water to remove noncellulosic material (Upde-
graff, 1969). Cellulose was then quantified using the phe-
nol–sulfuric acid method (Dubois et al., 1956), using
glucose as a standard (Desvaux et al., 2000).
Molecular techniques
Single colonies were boiled for 10 min in 200mL 1% Tween
20, 2� TE (Sambrook et al., 1989) and 5 mL of the super-
natant were used for PCR amplification of the 16S rRNA
gene using universal primers 1492R and 27F (Dojka et al.,
2000) in a 50 mL reaction volume containing 2.5 mM MgCl2,
0.2 mM dNTPs (Promega, Madison, WI), 300 nM primers
and 1.25 U Taq DNA polymerase (Corpogen, Bogota, Co-
lombia). The PCR was carried out in a PTC-100 thermo-
cycler (MJ Research, Waltham MA) for 5 min at 94 1C, 35
cycles of 30 s at 95 1C, 45 s at 55 1C, 1 min at 72 1C, and a
12 min extension at 72 1C. PCR products were analyzed by
agarose gel electrophoresis (Sambrook et al., 1989), purified
using QIAquick PCR Purification columns (Qiagen, Valen-
cia, CA) and sequenced on a ABI 3730xl DNA Analyzer
using primers 27F, 1492R and X91R (Fox et al., 1995; Dojka
et al., 2000). Sequences were analyzed against the databases
using BLAST (Altschul et al., 1990). The nucleotide se-
quences for strains CG311, CG312, CG323 and CG325 have
been deposited in the NCBI database under accession
numbers AY929250–AY929253.
Results anddiscussion
Isolationand characterizationofmicroorganisms
Microorganisms were isolated from a variety of environ-
mental sources that included decomposing agricultural
material, sugarcane waste and bagasse residue from a paper
FEMS Microbiol Lett 255 (2006) 52–58 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
53Microbial degradation of cellulosic residues
mill. A collection of 178 strains was initially analyzed for
production of halos of hydrolysis on CMC agar plates,
detected using Congo red. Fifty-five bacterial strains that
produced clearing zones greater than 0.5 cm in diameter
were selected for additional studies. When analyzed for their
capacity to withstand elevated temperatures, only 23 of
these 55 strains were able to grow up to 60 1C. Most of these
strains (21/23) were Gram positive and five had a morphol-
ogy suggestive of actinomycete bacteria. Whereas all 23
isolates had detectable cellulolytic activity at 37 and 40 1C,
as evidenced by the formation of clearing zones on CMC
agar, only 13 and 5 showed halo production at 50 and 60 1C,
respectively. This initial screen for cellulolytic activity by
identifying clearing zones on CMC agar media was quick
and easy to perform and allowed us to reduce the number of
strains to be further analyzed.
These 23 strains were further characterized by carrying
out enzymatic assays using CMC as substrate and DNS to
determine reducing sugars (Miller et al., 1960). Culture
supernatants were assayed for enzymatic activity at 37 and
40 1C, temperatures at which all the strains grew and showed
activity on solid medium. Based on these activities, nine
strains, all of which had higher CMCase activity at 40 1C
than at 37 1C, were selected for additional studies (Table 1).
Although variations were evident when these nine strains
were assayed for enzymatic activity at temperatures ranging
from 20 to 90 1C, enzymatic activity was observed consis-
tently at 40 1C and this temperature was therefore chosen for
all subsequent assays. In order to determine the optimum
pH, enzyme activity was assayed at 40 1C using different
buffers. Most of the strains (8/9) showed higher activity at a
pH above 7, indicating a preference for slightly alkaline
conditions (Table 1).
In order to see if the strains identified in this study could
utilize alternative cellulose substrates, they were grown in
medium containing crystalline cellulose as a sole carbon
source. All strains, except the control noncellulolytic E. coli
DH5a strain, were able to grow with only crystalline
cellulose as substrate as indicated by the increase in protein
content over a 12-day incubation period (Fig. 1). These
values represent actual growth and not differences in
recovery of total protein, as controls indicated that lysis of
all strains was equally efficient and the amount of protein
recovered was directly proportional to the number of cells
present as determined by CFUs (data not shown). Thus
these strains, particularly strains 6–9 (CG323–CG326)
which showed a substantial increase in biomass, were able
to degrade the more recalcitrant crystalline cellulose used
here. This suggests that these strains apparently contained
more than one type of enzyme, despite the fact that the
initial screen was carried out using CMC, a substrate that is
not necessarily representative of the structurally heteroge-
neous nature of cellulosic substrates. In contrast to CMC,
which is soluble and preferentially degraded by endogluca-
nases, efficient crystalline cellulose utilization probably
requires the activity of exoglucanases (Teeri, 1997). Thus,
Table 1. Characteristics of cellulolytic strains
Strain no�
CMC agarw assay Enzymatic assayz
Strain typeHalo size (cm) Activity (IU mL�1) Optimum temperature ( 1C) Optimum pH
1. (CG308) 3 0.056 40 8 Bacillus subtilis
2. (CG309) 2.5 0.190 50 8–9 Bacillus subtilis
3. (CG310) 3 0.134 40 8 Bacillus subtilis
4. (CG311) 2 0.171 40 8 Cellulomonas sp.
5. (CG312) 2.5 0.115 40 8 Bacillus subtilis
6. (CG323) 1.5 0.078 40 8–9 Streptomyces sp.
7. (CG325) 2 0.176 50 8 Streptomyces sp.
8. (CG324) 2.5 0.139 40 8 Streptomyces sp.
9. (CG326) 1.5 0.142 40 7 Streptomyces sp.
�The designation according to the laboratory strain collection is given in parentheses.wEach strain’s maximal enzyme activity during growth.zAssays were undertaken using CMC as substrate.
CMC, carboxymethylcellulose.
Fig. 1. Growth on crystalline cellulose. Growth of the nine strains (1–9)
and a control noncellulolytic strain of Escherichia coli DH5a (C) in liquid
medium containing crystalline cellulose was followed by determining
protein concentration at day 0 (white bars) and day 12 (black bars).
FEMS Microbiol Lett 255 (2006) 52–58c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
54 C. Guevara & M.M. Zambrano
our approach allowed the identification of different species
of microorganisms with cellulose-degrading capacity in
vitro, both on soluble CMC and on the more recalcitrant
crystalline cellulose.
Strain identification
Sequence analysis of the 16S rRNA gene revealed that four of
the strains were Bacillus subtilis, four were Streptomyces and
one was a Cellulomonas (Table 1). Whereas the sequences
obtained from the four Streptomyces species had identities
ranging between 95% and 99%, the four B. subtilis strains
had 16S rRNA gene sequences that were 99% identical,
despite the fact that some of them were obtained from
different geographical locations. This is not surprising since
it has been reported that 16S rRNA gene analysis has
limitations in terms of discriminating among strains of B.
subtilis (Chun & Bae, 2000; de Clerck et al., 2004). However,
the different morphologies and phenotypes observed in the
various assays strongly suggest that these strains are not
clonal but rather distinct isolates. The narrow range of
species identified in this screen could be attributed to the
high prevalence of these bacterial groups in soils and to
their known cellulolytic activity (Crawford, 1978; Cantwell
et al., 1986; Warren, 1996; Spiridonov & Wilson, 2000;
Boraston et al., 2003). Both Actinomycetes and Bacillus
strains have been identified as predominant aerobic cellulo-
lytic species isolated from soils and waste sites with high
cellulose content (Ulrich & Wirth, 1999; Pourcher et al.,
2001). In addition, one of these strains was a Cellulomonas
sp., an important and extensively studied aerobic cellulose-
degrading microorganism (Cazemier et al., 1999; Lynd
et al., 2002; Gutierrez-Nava et al., 2003). It is interesting
to note that Bacillus and Actinomycetes strains are known
for their ability to enter into resting states (spores) and are
good producers of secondary metabolites such as anti-
biotics, strategies that could provide them with an addi-
tional advantage over competitors under conditions of
slow growth on cellulosic substrates (Lynd et al., 2002).
However, when these nine strains were tested for possible
antagonistic activity by spotting one strain over a lawn of
another strain on solid medium (Loessner et al., 2003),
none of them was found to inhibit growth of the other
strains (data not shown). Although it is possible that they
might produce metabolites antagonistic against other
microorganisms common in soils, this result indicated that
it could be possible to grow these strains together as a
consortium.
Potentiationassays
Given that cellulolytic enzymes work best synergistically, we
next devised a potentiation assay to screen for potential
synergistic effects that could identify the best possible
combination of cellulolytic bacteria. Initially, each strain
was grown in CMC broth and assayed at different time
points to determine the point of maximal enzyme activity,
which was between 4 and 6 h for strains 1–5 (CG308-312),
and around 24 h for strains 6–9 (CG323-326) (data not
shown). Culture supernatants harvested at the time point of
maximal enzyme activity were filter-sterilized and used for
potentiation assays. In this assay, supernatants were ana-
lyzed for enzyme activity individually and after being mixed
in equal proportions with other supernatants. Initially, pairs
of supernatants were mixed in all possible combinations and
the activity of each mix was compared with the expected
activity, which was taken as the average of both individual
activities. Out of 36 possible combinations, 11 mixes showed
activity higher than that expected from both individual
activities (Table 2). Activity was then determined for com-
binations of three supernatants. These combinations were
made based on results from previous mixes and combined
strains from different species. Although in this case only 28
out of 86 possible combinations were tested, to reduce the
number of assays, a larger proportion (19/28) of the mixes
tested showed an increase in enzymatic activity when
compared with the expected value based on individual
activities. Based on these results, additional assays were
carried out with a few combinations of four supernatants.
In addition, mixes were undertaken with the faster growing
strains (1–5), the slower growing actinomycetes (6–9) and
with all nine strains (Table 2). Although in this case all of the
combinations tested showed potentiation of activity, mixes
containing supernatants from both fast and slow-growing
bacteria (1, 2, 6, 7/3, 5, 8, 9/4, 5, 6, 7; Table 2) showed more
activity than a mix composed of all nine samples. Two of
these combinations (1, 2, 6, 7 and 4, 5, 6, 7) resulted in
activity that was over five times the respective expected
activities.
These results show that combinations of culture super-
natants in some cases resulted in higher enzymatic activity
than expected from individual activities. This observation is
consistent with the idea that degradation of natural cellu-
losic substrates usually requires the co-ordinated action of
multiple enzymes that work synergistically, such that the
collective activity is higher than the sum of the individual
activities (Lynd et al., 2002; Boraston et al., 2003). The
potentiation effect observed here, particularly evident when
mixes of three or four supernatants were assayed, can be
attributed to extracellular, diffusible cellulase enzymes. It is
important to note that not all combinations tested resulted
in a potentiation of the activity, indicating that those that
did show an increase were probably because of a real effect.
In some cases, and despite the fact that supernatants were
mixed based on previous potentiation results, the
incorporation of additional supernatants did not necessarily
result in an increment in activity (Table 2). This may be
FEMS Microbiol Lett 255 (2006) 52–58 c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
55Microbial degradation of cellulosic residues
explained by competition over substrate that somehow
inhibits the synergistic effect observed previously. Alterna-
tively, it is possible that the relative amount of enzymes
present in each supernatant or the ratio of protein to
substrate is important for the observed potentiation effect.
Consortiumactivity
Having determined that mixes of various supernatants led to
an increased enzymatic activity, the next step was to see if
these strain combinations resulted in an efficient breakdown
of cellulosic material. Sugarcane leaves were therefore pro-
cessed by treatment with SDS and added either to minimal
salts medium (SSC) or to water (ASC) as a sole carbon
source. Microorganisms were then inoculated into these
media and growth was followed over time by determining
protein concentration. In this case, only one group of four
strains (4, 5, 6, 7) was chosen because it showed high activity
in the previous assay and it included the only Cellulomonas
strain in addition to B. subtilis and Streptomyces species. As
Table 2. Enzyme activity in mixed supernatants
Strains
Enzyme activityRelative activity
Observed
[10�2 IU mL�1 (� SD)] Expected (Obs/Exp)
1, 9 10.65 (� 0.73) 9.88 1.08
2, 8 18.73 (� 4.83) 16.43 1.14
3, 5 15.63 (� 2.53) 12.46 1.25
4, 6 23.53 (� 5.81) 12.45 1.89
4, 8 28.94 (� 4.48) 15.49 1.87
4, 9 31.45 (� 6.00) 15.65 2.01
5, 8 25.41 (� 2.32) 12.69 2.00
5, 9 12.93 (� 3.75) 12.85 1.01
6, 7 19.09 (� 6.60) 12.70 1.50
6, 8 14.45 (� 2.53) 10.84 1.33
7, 8 31.92 (� 0.96) 15.74 2.03
1, 2, 3 19.89 (� 2.61) 12.65 1.57
1, 2, 4 18.98 (� 1.71) 13.88 1.37
1, 2, 5 18.98 (� 0.17) 12.01 1.58
1, 2, 6 13.88 (� 0.97) 10.78 1.29
1, 2, 7 8.88 (� 0.94) 14.04 0.63
1, 2, 8 20.83 (� 2.32) 12.80 1.63
1, 2, 9 15.20 (� 0.82) 12.91 1.18
1, 4, 5 15.20 (� 4.52) 11.38 1.34
1, 6, 7 13.88 (� 0.92) 10.31 1.35
1, 8, 9 20.37 (� 8.28) 11.21 1.82
2, 4, 5 12.96 (� 1.86) 15.86 0.82
2, 6, 7 21.77 (� 0.93) 14.79 1.47
2, 8, 9 8.33 (� 1.36) 15.69 0.53
3, 4, 5 18.05 (� 2.45) 14.01 1.29
3, 4, 6 15.20 (� 1.19) 12.77 1.19
3, 4, 7 15.73 (� 2.25) 16.04 0.98
3, 4, 8 8.51 (� 0.18) 14.80 0.58
3, 4, 9 18.98 (� 0.10) 14.91 1.27
3, 6, 7 17.59 (� 3.98) 12.94 1.36
3, 8, 9 6.66 (� 0.33) 13.83 0.48
4, 6, 7 9.73 (� 0.89) 14.16 0.69
4, 8, 9 17.10 (� 6.40) 15.06 1.14
5, 6, 7 12.77 (� 4.60) 12.30 1.04
5, 6, 8 8.79 (� 0.26) 11.06 0.79
5, 6, 9 12.96 (� 0.93) 11.17 1.16
5, 8, 9 12.95 (� 1.61) 13.19 0.98
6, 8, 9 24.92 (� 3.69) 11.96 2.08
7, 8, 9 33.60 (� 2.41) 15.22 2.21
1, 2, 6, 7 69.44 (� 6.22) 12.48 5.56
1, 2, 8, 9 18.98 (� 0.20) 13.16 1.44
3, 4, 6, 7 29.17 (� 4.11) 13.98 2.09
3, 4, 8, 9 21.75 (� 3.65) 14.67 1.48
3, 5, 8, 9 50.83 (� 17.04) 13.26 3.83
4, 5, 6, 7 68.05 (� 24.10) 13.51 5.04
1, 2, 3, 4, 5 31.44 (� 2.35) 13.32 2.36
6, 7, 8, 9 27.30 (� 7.11) 13.38 2.04
All 31.17 (� 4.54) 13.34 2.34
Enzyme activity in mixes from various strain supernatants (1–9) was
determined using carboxymethylcellulose as substrate (Observed) and
compared with the activity calculated from individual activities (Ex-
pected). The results are the mean� standard deviation from three
independent replicates.
Fig. 2. Microbial consortia on sugarcane leaf cellulosic material. Growth
as determined by protein concentration (a) and enzymatic activity using
carboxymethylcellulose as substrate (b) of consortia consisting of four
microorganisms, 4, 5, 6, 7 (triangles), and all nine microorganisms
(squares) was followed over time in ASC (empty symbols) or SSC (filled
symbols) media. Controls (circles) were inoculated with the noncelluloly-
tic strain Escherichia coli DH5a.
FEMS Microbiol Lett 255 (2006) 52–58c� 2005 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
56 C. Guevara & M.M. Zambrano
shown in Fig. 2a, both consortia were able to grow over the
21-day incubation period, whereas growth of the control
noncellulolytic strain was negligible in both media. CMCase
activity also increased over time for the two consortia,
especially for the group of four strains (4, 5, 6, 7) in SSC
medium, but not for the control strain (Fig. 2b). Although it
is difficult to make a direct comparison between these
CMCase activities and those presented in Table 2, it is
evident that at the point of maximal enzyme activity (day
21) the group of four strains also had greater CMCase
activity than the group containing all strains for each
medium analyzed. However, only in SSC medium did the
activity of the consortium of four strains double the activity
of the consortium of all strains (36.0 and
18.5� 10�2 IU mL�1, respectively), similar to the superna-
tant activities (68.0 and 31.1� 10�2 IU mL�1, respectively)
in Table 2. In order to determine if, in fact, the observed
increase in protein concentration was due to the capacity of
these microbial consortia to utilize the cellulose present in
the medium, the amount of cellulose was determined at the
beginning and at the end of the incubation period. In
contrast to the control culture, which showed little or no
degradation of cellulose, both microbial consortia were able
to degrade the sugarcane cellulose provided (Fig. 3). Most
interesting was the observation that the group composed of
four strains (4, 5, 6, 7) showed more degradation in SSC
medium than a consortium consisting of all nine micro-
organisms, consistent with the observed increase in enzyme
activity (Fig. 2b). In contrast, the consortium of all nine
strains was more efficient at degrading cellulose in ASC
medium. This result was unexpected because ASC medium
consists only of leaf cellulose substrate and water. However,
there could have been residual nutrients present along with
the prepared cellulose, despite the fact that it was washed
extensively prior to use.
The two consortia analyzed in this study, one composed
of all nine strains and the other of strains 4, 5, 6 and 7, were
capable of growing and degrading a complex sugarcane
cellulose substrate under the experimental culture condi-
tions provided. Although these strains showed good
CMCase activity when assayed individually, they were not
necessarily the ones with highest activity, suggesting that
specific combinations of microorganisms can result in high-
er activity than expected from the observation of individual
strains. Thus this approach, the determination of CMCase
activity in mixed culture supernatants, allowed us to identify
a microbial consortium that was efficient at utilizing a
cellulose substrate in vitro. The fact that the group of only
four microorganisms proved as efficient, in terms of cellu-
lose breakdown, as the consortium consisting of all nine
strains suggests that specific combinations of strains can be
important for activity. The difference observed for both
consortia is indicative, however, of the complexity of mixed
bacterial cultures where it is difficult to predict the outcome
of interactions among the various species. An analysis of the
population dynamics in the different media used, which
might help to understand differences in terms of enzyme
activities and degradation of cellulosic material, will be
carried out in the future in order to determine if all species
in these mixed cultures are present during the entire
incubation period or if the relative abundances of each vary
over time. It might also be interesting to evaluate the
capacity of these strains to degrade sugarcane leaf residues
in the presence of native microflora, as an alternative for
reducing the environmental impact of accumulated cellulo-
sic waste material.
Acknowledgement
We would like to thank A.V. Suescun and W. Ocampo for
their help in this work.
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Fig. 3. Sugarcane cellulose degradation by microbial consortia. Cellulose
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58 C. Guevara & M.M. Zambrano