Download Physiological interactions between a mesophilic cellulolytic

FEMS Microbiology Ecology 86 (1992) 237-245
0 1992 Federation of European Microbiological Societies 0168-6496/92/$05.00
Published by Elsevier
237
FEMSEC 00369
Physiological interactions between a mesophilic cellulolytic
Clostridium and a non-cellulolytic bacterium
Katherine Cavedon
*
and Ercole Canale-Parola
Department of Microbiology, University of Massachusetts, Amherst, Massachusetfs, U.S.A.
Received 25 March 1991
Revision received and accepted 8 October 1991
Key words: Cellulose; Cellulolytic co-culture; Cross-feeding; Interactions; Mutualism; N, fixation
1. SUMMARY
A mesophilic cellulolytic bacterium (Cfostridium strain C7) capable of N, fixation and a
non-cellulolytic bacterium ( KZebsielfQstrain W 1),
both isolated from freshwater environments rich
in decaying plant material, were co-cultured in a
chemically defined, vitamin-deficient medium
containing cellulose as the carbon and energy
source. In the co-culture, an extracellular cellulase complex produced by the Clostridium hydrolyzed cellulose to soluble sugars that served as
fermentable substrates for the KlebsielZu. In turn,
the Kfebsielfu excreted growth factors, identified
as biotin and p-aminobenzoic acid, which were
required by the Clostridium. Furthermore, dem-
Correspondence to: E. Canale-Parola, Department of Microbiology, University of Massachusetts, Amherst, MA 01003
U.S.A.
*
Present address: Laboratory of Microbial Ecology, National
Institute of Dental Research, National Institutes of Health,
Bethesda, MD 20892 U S A ,
onstration of NH -repressible acetylene reduction by co-cultures growing in medium lacking
combined nitrogen showed that the Clostridium
fixed N,, thus allowing growth of the Klebsiellu,
which was not a nitrogen fixer. The mutualistic
relationships observed in the co-cultures may be
representative of interactions that take place in
natural environments in which cellulose-containing plant materials are biodegraded.
2. INTRODUCTION
In anaerobic environments cellulose is converted largely to CH, and CO, by the combined
activities of many diverse microorganisms. Among
these microorganisms there exist mutually beneficial interactions which include interspecies hydrogen transfer [ 1,2], removal of inhibitory substances [3,4], production of growth factors [3-51,
and formation of fermentable soluble sugars
[2,3,5]. Interspecies interactions of this or similar
type have also been observed during microbial
degradation of other biopolymers such as chitin
[6,7], pectin [8], and gelatin [9]. Microbial interac-
238
tions appear to be important processes in the
anaerobic degradation of water-insoluble biopolymers.
In nature, cellulose is present primarily in plant
cell walls, together with other polymers such as
hemicelluloses, pectin, and lignin. Microbial communities that degrade plant material in anaerobic
environments include bacteria that produce extracellular cellulases which hydrolyze plant cell
wall cellulose with formation of soluble sugars
(e.g., cellobiose, glucose, cellodextrins). Inasmuch
as these cellulose-derived sugars are formed extracellularly, they are available as fermentable
substrates not only to the cellulolytic bacteria
that generate them, but also to non-cellulolytic
bacteria.
Plant cell walls, and plant material in general,
usually have a high C : N ratio and, therefore, the
rate of their biodegradation may be limited by N
deficiency [10,11]. In previous work [12] we have
shown that anaerobic cellulolytic bacteria which
occur in environments rich in decaying plant material are able to fix N,. It is believed that the
nitrogenase activity of these cellulolytic bacteria
serves to provide combined nitrogen for the
degradation process [ 121.
To study the physiological interactions between diverse bacteria that utilize cellulose-derived soluble sugars as fermentable substrates, we
established a stable co-culture of a cellulolytic
bacterium capable of N, fixation (Clostridium
strain C7) and a non-cellulolytic bacterium (Kfebsiella strain W1) in a medium containing cellulose
as the carbon and energy source. Both bacterial
strains were isolated from similar aquatic environments (see section 3.1). Clostridium strain C7
has been shown to produce an extracellular multiprotein complex which serves to hydrolyze crystalline cellulose [ 13,141. In the co-culture both
Clostridium strain C7 and Klebsiella strain W1
grew at the expense of soluble sugars produced
from cellulose by the clostridial multiprotein
complex.
In this article we describe cross-feeding interactions that enable N,-fking, cellulolytic bacteria
and non-cellulolytic bacteria to co-exist in anaerobic environments in which cellulose serves as the
carbon and energy source.
3. MATERIALS AND METHODS
3.1. Bacterial strains and culture conditions
The mesophilic cellulolytic Cfostridium species
(strain C7) was routinely cultured in chemically
defined liquid media containing either cellobiose
(medium CB) or cellulose (medium C) as carbon
and energy source. Medium CB was identical to
medium MJ of Johnson et at. [15], except that N a
citrate was omitted and the only vitamins added
were d-biotin and p-aminobenzoic acid (2 X 1 0 - 5
and 4 x
g per 100 ml of medium, respectively). Medium C was identical to medium CB
except that, instead of cellobiose, it Contained
cellulose (0.6 g [dry wtl ball-milled Whatman no.
1 filter paper per 100 ml of medium) [16].
The non-cellulolytic bacterium (strain "1) was
grown in monoculture in media that differed from
medium CB as follows: medium CB-V lacked all
vitamins; medium CB-VU lacked all vitamins and
urea. Strain W1 was grown in co-culture with
strain C7 in medium C and in media that differed
from medium C as follows: medium C-V lacked
all vitamins; medium CT-VU lacked all vitamins,
urea, and cysteine hydrochloride, but contained
titanium (111) nitrilotriacetate (2.5 mM, final concentration) [17] as the reducing agent. The latter
medium did not contain any source of combined
nitrogen utilizable for growth by either strain.
Medium GS2M, a yeast extract-containing
medium used in some experiments, was identical
to GS2 medium of Johnson et al. [151, except that
it contained 0.2 g each of cellobiose and cysteine
hydrochloride per 100 ml, and the pH was adjusted to 7.0. In GS2M agar medium plates the
concentrations of agar (Difco Laboratories, Detroit, MI) and cellobiose were 1.5 and 0.5 g per
100 ml of medium, respectively.
Incubation temperature was 30°C unless specified otherwise. All media for anaerobic cultures
were prereduced [181. For anaerobic growth, liquid cultures were incubated in N,, and agar
medium cultures in an anaerobic chamber (10%
CO,:7% H,:83% N,, v/v/v; Coy Lab. Products, Ann Arbor, MI). Media used to detect
sporulation of strain W1 were the meat broth
described by Holdeman et al. [191 (supplemented
239
with D-glucose, 0.5%, w/v, final concentration)
and Sweet E agar medium plates [191.
Both strains W1 and C7 were isolated from
freshwater environments rich in decaying plant
material. The non-cellulolytic strain W1 was isolated from mud of a shallow freshwater pond by
means of a procedure that involved streaking on
GS2M agar medium plates and anaerobic incubation. Cells from colonies that developed on the
plates were tested for the ability to grow with
cellobiose but not cellulose as carbon and energy
source, and for the ability to grow in stable coculture with Clostridium strain C7 in a chemically
defined medium (medium C>containing cellulose
as the fermentable substrate. Strain W1 used
cellobiose as carbon and energy source, grew in
medium C in co-culture with strain (17, but did
not grow in medium C in pure culture. The
cellulolytic strain C7 was isolated from mud collected several centimeters below the surface of a
freshwater swamp, as described previously [161.
3.2. Determination of cell numbers in co-cultures
Numbers of viable cells of strains C7 and W1
growing in co-culture in chemically defined liquid
medium C-V were determined as follows: a 0.3-ml
volume of a 72-h-old C-V medium co-culture
(both strains in the log phase) was transferred
into each of 32 test tubes each containing 5 ml of
medium C-V. Beginning immediately after inoculation, and at daily intervals, a sample to be used
for serial dilutions was taken from each of four of
the 32 cultures, and the supernatant fluid remaining in the four cultures was recovered by centrifugation and stored at -55°C prior to being assayed for fermentation products. The samples
taken from the four cultures were serially diluted,
under N2 atmosphere, into sterile GS2M broth
from which cellobiose was omitted. To determine
the number of strain W1 cells, 0.1-ml volumes of
each of the four serially diluted samples were
plated in duplicate into GS2M agar medium and
incubated in air. For the determination of strain
C7 cell numbers, 0.1-ml volumes of the four serially diluted samples were plated in duplicate into
GS2M agar medium containing 10 p g of
polymyxin E (Sigma) per ml, and incubated in an
anaerobic chamber. Polymyxin E, at the above-
mentioned concentration, inhibited the growth of
strain W1. Each viable count value was calculated
from the average number of colonies in eight
plate cultures (each containing 100-300 colonies)
prepared with the serially diluted samples from
the four C-V broth cultures.
3.3. Assays
Amounts of d-biotin and p-aminobenzoic acid
produced by strain W1 were determined by assaying the supernatant fluid from log-phase cultures
growing in medium CB-V in N, atmosphere.
Quantitative growth response assays [20] were
used. The assay bacterium was Lactobacillus
plantarum ATCC 8014. The assay media were
Bacto-biotin assay medium (Difco), and a medium
identical to the biotin assay medium, except that
it contained biotin (4 x
g/100 ml of
medium) but lacked p-aminobenzoic acid.
The amount of cellulose degraded by cultures
was determined by measuring the dry weight of
residual cellulose Ill. Acids in culture fluids were
determined by gas-liquid chromatography as described previously [21]. The methods of Sleat and
Mah [22] and of Miller et al. [23] were used to
measure formate and reducing sugars, respectively. Ethanol was determined using Sigma’s alcohol dehydrogenase kit No. 332-A.
Nitrogenase activity was assayed by the acetylene reduction test as described previously [12]
except that monocultures of strain W1 were grown
(to approximately lo8 cells per ml) in medium
CB-VU. In attempts to grow strain W1 anaerobically in the absence of a utilizable source of
combined nitrogen, a reducing agent other than
L-cysteine hydrochloride was added to medium
CB-VU. L-cysteine hydrochloride was replaced by
the following reducing agents (final concentrations): dithiothreitol, 0.2 g/100 ml; Na sulfide,
0.025 g/100 ml; Na thioglycolate, 0.2 g/100 ml;
or titanium (111) nitrilotriacetate [17], 2.5 mM.
Co-cultures of strains C7 and W1 used in nitrogenase assays were grown in medium CT-VU.
3.4. Phenotypic characteristics of strain Wl
Standard procedures [24] were used for the
catalase and oxidase tests. Urea hydrolysis, glucose and citrate fermentation, production of or-
240
nithine decarboxylase and H,S, and the VogesProskauer reaction were determined using the
Enterotube system for Enterobacteriaceae (Roche
Diagnostics, Nutley, NJ).
Capsular material was observed by light microscopy of India ink capsule stain preparations
[25]. Cells were negatively stained and examined
by electron microscopy as previously described
D61.
4. RESULTS
4.1. Characterization of the non-cellulolytic isolate
and establishment of co-culture
Strain W1, the non-cellulolytic isolate, was a
Gram-negative, facultatively anaerobic, rodshaped bacterium that did not form spores in any
of the culture media used (see section 3.1). Strain
W1 cells measured approximately 1 X 2.7 bm,
were surrounded by a capsule, were not motile,
and lacked flagella as shown by electron microscopy of negatively stained preparations. Determination of other phenotypic characteristics
(see section 3.4) indicated that, according to current taxonomic criteria [27], the non-cellulolytic
isolate was a strain of Klebsiella. As mentioned
above (section 3. l), the non-cellulolytic isolate
(Klebsiella strain W1) did not grow in monoculture in medium C, which contained cellulose as
the carbon and energy source, but grew in that
same medium in co-culture with the cellulolytic
Clostridium strain C7. Co-cultures were started
by inoculating medium C or medium C-V with
equal volumes of Clostridium strain C7 and Klebsiella strain W1 cultures (e.g., 0.2 ml of each
culture in log phase per 5 ml of co-culture
medium). The two strains persisted indefinitely in
co-culture through repeated transfers.
4.2. Interactions in co-cultures of the cellulolytic
and non-cellulolytic strains
Klebsiella strain W1 did not utilize, as fermentabIe substrate, any of the end products (e.g.,
acetate, lactate, ethanol, succinate) of cellulose
fermentation formed by strain C7, as indicated by
the failure of strain W1 to grow anaerobically in
media containing any one of these end products
Table 1
Utilization of soluble carbohydrates by strains WI and C7 in
monocultures a
Carbohydrate
No carbohydrate added
Glucose
Cellobiose
Cellotriose
Ce I lot e t raose
Cellopentaose
Cellohexaose
a
Strain W1
Strain C7
-
-
+
+
+
+
+
+
+
+
+
-
The growth media (CB-V for strain WI and GSZM for
strain C7) contained either cellobiose or, in its place, one of
the other soluble carbohydrates listed above. Carbohydrate
concentration was 0.1 g per 100 ml of medium. Cellotriose
and higher molecular mass cellmligosaccharides were purchased from V-Labs, Inc., Covington, LA.
Growth, +; no growth, -.
as carbon and energy source. Furthermore, Klebsiella strain W1 did not utilize amino acids as
fermentable substrates, inasmuch as it did not
grow anaerobically in media lacking carbohydrates but containing either peptone (Difco, 0.2
g/100 ml of medium) or acid-hydrolyzed casein
(Difco Casamino Acids, 0.2 g/100 ml of medium)
as carbon and energy source. Thus, it was inferred that neither fermentation end products
nor amino acids resulting from the hydrolysis of
extracellular cellulase or from cell lysis served as
carbon and energy sources for the Klebsiella
growing anaerobically in co-culture in cellulosecontaining media. Cell lysis was not observed
microscopically in co-cultures during the log phase
of growth of either strain.
In monoculture both strains utilized glucose,
cellobiose, or cellotriose as fermentable substrates for growth, whereas cellotetraose, eellopentaose, or cellohexaose served as fermentable substrate only for Clostridium strain C7
(Table 1). In addition, previous work had shown
that growing cells of Clostridium strain C7 produce an enzyme complex which hydrolyzes cellulose extracellularly [13,141, and that products of
cellulose hydrolysis are present in the culture
medium (281. These results, as well as the substrate utilization pattern of the Klebsiella and
other observations described in section 4.3, in&
24 1
cated that soluble sugars produced by the hydrolysis of cellulose by the clostridium served as fermentable substrates for Klebsiella strain W1
growing in co-culture in chemically defined media.
The vitamins biotin and p-aminobenzoic acid
were included in medium C because they were
required for the growth of Clostridium strain C7
[29]. The clostridium did not grow when either of
the two vitamins (or both) was omitted from
medium C. However, the clostridium grew in
co-culture with strain W1 in medium C-V, which
was identical to medium C except that both vitamins were omitted. Late log-phase cell numbers
of strains C7 and W1 in medium C-V co-cultures
were 5.4 X lo8 and 2.3 X lo8 viable cells per ml,
respectively. Similar cell numbers were observed
in co-cultures in medium C, which contained
biotin and p-aminobenzoic acid. These observations indicated that, in the co-culture, the noncellulolytic Klebsiella strain W1 produced the two
vitamins, and that the clostridium utilized them
for its growth. Support for this conclusion was
provided by quantitative growth response assays,
which showed that the supernatant fluid of CB-V
log-phase monocultures of strain W1 contained
0.21 and 0.19 ng of biotin and p-aminobenzoic
acid, respectively, per ml. Neither of these vitamins was detected in the medium immediately
after inoculation.
The above-mentioned experiments indicated
that, in co-cultures, a mutualistic relationship existed between the cellulolytic clostridium and the
non-cellulolytic Klebsiella strain W1. The clostridium provided strain W1 with growth substrates in
the form of cellulose-derived soluble sugars,
whereas strain W1 produced biotin and p aminobenzoic acid which were required for
growth of the clostridium.
A co-culture of Clostridium strain C7 and
Klebsiella strain W1 was established in medium
CT-VU, in which cellulose was the fermentable
substrate and no utilizable source of combined
nitrogen was present. The co-culture, which was
incubated in N, atmosphere, was stable, i.e. the
two strains persisted through repeated transfers.
As reported previously [12], Clostridium strain C7
is capable of utilizing cellulose as an energy source
for N, fixation. In contrast, we found that Klebsiellu strain W1 grew in monoculture in cellobiose-containing media only in the presence of
a utilizable source of combined nitrogen such as
L-cysteine. For example, in medium CB-VU,
strain W1 failed to grow aerobically in the absence of t-cysteine. It also failed to grow anaerobically in medium CB-VU (N,atmosphere) either
in the absence of L-cysteine or when L-cysteine
was replaced by another reducing agent (see section 3.3). When grown in the presence of L-cysteine, Klebsiella strain W1 lacked nitrogenase
activity, as determined by the acetylene reduction
test. Log-phase co-cultures of strains C7 and W l
in medium CT-VU reduced acetylene (70 nmoles
acetylene/ h/ mg cell dry weight) in the absence
of combined nitrogen, and did not show significant acetylene reduction when ammonium chloride (0.1 g/100 ml medium) was present during
growth. These results indicated that, in co-cultures in medium CT-VU, Clostridium strain C7
fixed N, and provided combined nitrogen for the
growth of Klebsiella strain W1.
4.3. Growth of co-cultures in cellulose-containing
medium
Cell numbers in Clostridium strain C7 monocultures and in co-cultures of the two strains
growing in cellulose-containing medium were
monitored by means of colony counts (Fig. lA, B).
Direct counts by light microscopy were not carried out because cells of the two strains growing
in co-culture had similar morphologies. Numbers
of viable cells of strain C7 either in monoculture
or in co-culture increased during the first 72 h,
then decreased at a relatively fast rate (Fig.
lA, B). At 72 h cell numbers of strain C7 were
higher in the monoculture than in the co-culture
(Fig. lA, B), as would be expected in the presence of competition for soluble sugars between
the two strains growing in co-culture.
During the first 96 h of incubation the concentration of reducing sugars in the monoculture or
co-culture supernatant fluid was relatively low,
but it increased rapidly as the number of viable
cells decreased (Fig. lA, B). Apparently, after
viable cell numbers began to decrease, the rate of
cellulose hydrolysis (to soluble sugars such as
242
]A
C7 Mono-
lB
40r--71
1
Cocullure
Lactate
100
200
100
200
Hour4
Fig. 1. Cell numbers and accumulation of reducing sugars in
Clostridium strain C7 monoculture (A) and in co-culture (B)of
Closrndium strain C7 with Klebsrellu strain W1. The cultures
were in cellulose-containing media (monoculture in medium
C, co-culture in medium C-V). Numbers of strain C7 cells ( 0 )
in the monoculture were determined by plate counts in GS2M
agar medium. Cell numbers of strain C7 ( 0 ) and of strain W1
( 0 ) growing in co-culture were determined as described in
Section 3.2. Reducing sugars ( 0 ) present in culture supernatant fluids are expressed as glucose equivalents. Numbers
on the horizontal axis indicate hours of incubation (in N,
atmosphere).
cellobiose) by cellulose-bound enzyme was greater
than the rate at which these sugars were fermented by the cell population. The onset of the
increase in soluble sugar concentration in the
co-culture fluid occurred 24 h after the cell number of strain C7 began to decrease, but it coincided with the beginning of the decrease in the
number of viable Kfebsieffucells (Fig. 1B). This
observation is in agreement with the conclusion
that the Klebsiellu grew at the expense of soluble
sugars released by the extracellular cellulase.
Accumulation of fermentation end products
formed by the co-culture continued as the number of viable cells decreased after 96 h (Fig. lB,
2). The continued accumulation probably resulted
from sugar metabolism by cells that were still
enzymatically active. Lactate and acetate were
the major acids produced by the co-culture (Fig.
2). Low levels of ethanol were detected (Fig. 2).
Acids produced by Klebsiellu strain W1 monocultures in medium CB-V were primarily acetate
and smaller amounts of succinate (data not
shown). Non-gaseous end products of Clostridium
C7 monocultures were acetate, ethanol and lac-
Hours
Fig. 2. End products of cellulose fermentation by the Clostridium strain C7 and Klebsiellu strain W1 co-culture (in medium
C-V, N, atmosphere). End products are expressed as mmol
per liter of co-culture supernatant fluid. Numbers on the
horizontal axis indicate hours of incubation.
tate [16]. Acetoin and 2,3-butanediol, which USUally are fermentation end products of Klebsielza
species, were not determined.
The rates of cellulose hydrolysis by Closrridium strain C7 growing in medium C, which contained biotin and p-aminobenzoic acid, and i n
co-culture with Klebsielfu strain W1 in medium
C-V, were approximately the same (Fig. 3). The
level of reducing sugars during growth of strain
I
I
I
I
I
I
t
100
150
Hours
Fig. 3. Cellulose utilization during growth of the Closrridium
C7 monoculture ( 0 ) and of the co-culture with KIebsieffu
(o),
in N, atmosphere. Each point on the curves represents
the average of triplicate cultures. Remaining cellulose is expressed in mg per 5-ml culture.
"0
50
w1
243
C7 was somewhat lower in the co-culture than in
the strain C7 monoculture (Fig. lA, B). However,
in either culture the reducing sugar level may
have been too low to affect the rate of cellulose
hydrolysis (e.g., by inhibiting cellulase activity).
The pH of strain C7 monocultures and co-cultures decreased during incubation from 7.2 at the
time of inoculation to approximately 5.3 after 168
h.
5. DISCUSSION
As reported previously [14], the extracellular
multiprotein complex of Clostridium strain C7
hydrolyzes crystalline cellulose forming, as hydrolysis end products, cellobiose and, in smaller
amounts, cellotriose and glucose. In addition, soluble cellooligomersof higher molecular mass (e.g.,
cellotetraose, cellopentaose, cellohexaose) may be
formed as intermediate products during cellulose
hydrolysis by the cellulase complex. Thus, in our
cellulose-utilizing co-cultures of Clostridium
strain C7 and Klebsiellu strain W1, different cellulose-derived soluble sugars apparently were
available to the bacterial strains. We found that
Clostridium strain C7 was capable of utilizing the
entire spectrum of cellulose-derived soluble sugars from glucose to cellohexaose, whereas Ktebsiella strain W1 utilized only glucose, cellobiose,
and cellotriose. The ability of the two bacterial
strains to persist in stable co-culture indicates
that they have different affinities for the soluble
sugars that serve as fermentable substrates for
both of them, and/or that Clostridium strain C7
grows at the expense of soluble sugars not utilized by Klebsiella strain W1.
Neither Clostridium strain C7 nor Klebsiella
strain W1 grew in monoculture in a medium
containing cellulose as fermentable substrate and
lacking biotin and p-aminobenzoic acid. However, when the Clostridium and the Klebsiellu
were inoculated together in this medium, a
cross-feeding relationship developed which allowed both strains to grow. Clostridium cells released into the medium a multiprotein complex
which hydrolyzed cellulose, forming soluble sugars that were used as fermentable substrates by
the non-cellulolytic Klebsiellu. In turn, the Klebsiellu cells secreted biotin and p-aminobenzoic
acid which were required for the growth of the
Clostridium. Results of previous studies of cellulolytic co-cultures indicated that growth factors
required by one bacterial species were produced
by the other bacterial species in the co-culture,
but the growth factors were not identified [3,5,30].
A co-culture of the two strains was established
in a cellulose-containing, vitamin-lacking medium
from which sources of combined nitrogen were
omitted. The co-culture, which was incubated in
N, atmosphere, persisted through repeated transfers and fixed N,, as determined by the acetylene
reduction test. Inasmuch as the Klebsiellu did not
grow in monoculture in media lacking combined
nitrogen, and the Clostridium is known to be a N,
fixer [12], it was concluded that the Clostridium
provided the Klebsiellu with a source (or sources)
of combined nitrogen that allowed the latter organism to grow in the co-culture.
Previous reports have described mixed cultures
consisting of cellulolytic microorganisms and N,fixing bacteria growing in cellulose-containing
media lacking combined nitrogen [10,11,31]. In
these systems, non-cellulolytic, N,-fixing bacteria
provided combined nitrogen for the co-culture
while growing at the expense of soluble sugars
produced by the activity of 0,-respiring, cellulose-hydrolyzing microorganisms. Our Clostridium C7-Klebsiella W1 co-culture differs from previously described N,-fixing, cellulolytic systems
because it is entirely anaerobic and because the
cellulolytic microorganism is the N, fixer. It may
be surmised that, in nature, cellulolytic bacteria
benefit from their ability to fix N, because environments in which cellulose-containing plant material is biodegraded are often deficient in combined nitrogen [10,11]. Furthermore, in these environments, growth of 0,-scavenging facultative
anaerobes, such as Klebsiella W1, may serve to
maintain anoxic conditions favorable to cellulolytic N,-fixing clostridia.
We have not determined the nature of the
nitrogenous compound(s) produced by Clostridium strain C7 and utilized by the Klebsiella as
nitrogen source in the co-culture. One possibility
is that the Klebsiellu may obtain amino acids
244
utilizable as nitrogen sources by producing proteinases that hydrolyze extracellular cellulolytic
proteins released by the clostridium. Lysis of
clostridial cells probably was not a significant
source of combined nitrogen because cell lysis
was not observed microscopically while Klebsiellu
cells increased in number in the co-culture.
In some of the previous studies on cellulose
degradation by mixed cultures it was found that
cellulose hydrolysis was enhanced by growing the
cellulolytic microorganism in co-culture with a
secondary saccharolytic strain (e.g. refs. 3, 28).
The investigators suggested that the secondary
strain, which fermented soluble sugar produced
extracellularly by the cellulose degrader, maintained the soluble sugar in the culture fluid at
levels too low to inhibit cellulase activity. In another study [5], cellulose degradation by Acetiuibrio cellulolyticus was decreased when this bacterium was grown in co-culture with Clostridium
succhurofyticum, the decrease being the result of
substrate competition. Under the growth condition used in our study, the rate of cellulose hydrolysis by Clostridiurn strain C7 was neither enhanced nor diminished in co-cultures with Klebsiellu strain W1 in medium C-V, as compared to
the rate observed when strain C7 was grown in
monoculture in medium C (Fig. 3). Cell numbers
of strain C7 were higher in the monoculture than
in the co-culture (Fig. lA, B) and, therefore, it is
possible that a larger total amount of cellulase
was produced in the monoculture. Cellulase levels during growth of the strain C7 monoculture
and of the co-culture could not be accurately
compared because the enzyme complex was
largely bound to the insoluble cellulose. Thus, it
is not clear why similar rates of cellulose degradation were observed in the co-culture and in the
strain C7 monoculture, even though higher cell
numbers were present in the monoculture.
It is likely that the kinds of cross-feeding relationships observed in the co-cultures of cellulolytic and non-cellulolytic bacteria are representative of interactions that take place in natural
environments in which cellulose is the primary
fermentable substrate. In these environments,
soluble sugars produced by the activity of extracellular cellulases may serve as carbon and energy
sources for non-cellulolytic bacteria that secrete
growth factors required by the cellulose hydrolyzers. Apparently, N,-fixing cellulolytic bacteria are
widespread in anaerobic environments in which
cellulose-rich plant material is decomposed by
microorganisms [121. Inasmuch as in these environments the C:N ratio is high and nitrogen
limitation generally occurs [10,1I], combined nitrogen formed by N,-fixing cellulolytic bacteria
[12] may be utilized for growth by non-N,-fixing
microorganisms which establish cross-feeding interactions with the cellulose degraders. These
mutually advantageous interactions among microorganisms that participate in biopolymer
degradation may occur commonly in natural environments and most likely play an important role
in the turnover of carbon in the biosphere,
ACKNOWLEDGEMENTS
We are grateful to Tom Warnick for perfoming the acetylene reduction experiments. This research was supported by U.S. Department of
Energy Grant DE-FG02-88ER13898 and by U.S.
Department of Agriculture Grant 87-CRCR-12398.
REFERENCES
[l] Weimer, P.J. and Zeikus, J.G. (1977) Fermentation of
cellulose and cellobiose by Clostridium rhermocellum in
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