Sugarcane cellulose utilization by a defined microbial consortium

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).


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



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.



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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Sugarcane cellulose utilization by a defined microbial consortium