See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/7422354

Effects of Physicochemical Factors on the Adhesion to Cellulose Avicel of the

Ruminal Bacteria Ruminococcus flavefaciens and Fibrobacter succinogenes
subsp. succinogenes

Article  in  Applied and Environmental Microbiology · November 1990

DOI: 10.1128/AEM.56.10.3081-3087.1990 · Source: PubMed

CITATIONS READS

121 150

4 authors, including:

Gérard Fonty Sylvie Bony

French National Centre for Scientific Research French National Institute for Agriculture, Food, and Environment (INRAE)
41 PUBLICATIONS   2,167 CITATIONS    94 PUBLICATIONS   1,897 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

SEDIGEST : Ecotoxicological risk assessment linked to infilling quarries with treated dredged seaport sediments View project

Transgenerational effects of a parental exposure in the sentinel specie Gammarus fossarum View project

All content following this page was uploaded by Sylvie Bony on 19 May 2014.

The user has requested enhancement of the downloaded file.

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1990, p. 3081-3087 Vol. 56, No. 10
0099-2240/90/103081-07$02.00/0
Copyright C) 1990, American Society for Microbiology

Effects of Physicochemical Factors on the Adhesion to Cellulose

Avicel of the Ruminal Bacteria Ruminococcus flavefaciens and
Fibrobacter succinogenes subsp. succinogenes
VALERIE ROGER,1 GE1RARD FONTY,l.2* SYLVIE KOMISARCZUK-BONY,3t AND PHILIPPE GOUET1
Laboratoire de Microbiologie, INRA, CR de Clermont-Ferrand-Theix, 63122 Ceyrat,1 Laboratoire de Biologie
Compar&e des Protistes, CNRS URA 138, Universite Blaise Pascal, Clermont II, 63170 Aubiere,2
and Station de Recherches de Nutrition, INRA, 78350 Jouy-en-Josas, France
Received 23 March 1990/Accepted 14 July 1990

Ruminococcus flavefaciens adhered instantly to cellulose, while Fibrobacter succinogenes had the highest
percentage of adherent cells after about 25 min of contact between bacteria and cellulose. Adhesion of R.
flavefaciens was unaffected by high concentrations of sugars (5%), temperature, pH, oxygen, metabolic
inhibitors, and lack of Na+. In contrast, the attachment was affected by the removal of divalent cations (Mg2+
and Ca2+), the presence of cellulose derivatives (methylcellulose and hydroxyethylcellulose), and cystine.
Adhesion of F. succinogenes was sensitive to low and high temperatures, high concentrations of glucose and
cellobiose (5%), hydroxyethylcellulose (0.1%), redox potential, pH, lack of monovalent cations, and the
presence of an inhibitor of membrane ATPases or lasalocid and monensin. Cells of F. succinogenes heated at
100°C no longer were adherent. On the other hand, adhesion was insensitive to the lack of divalent cations
(Mg2+ and Ca2+), the presence of 2,4-dinitrophenol, tetrachlorosalicylanilide, or inhibitors of the electron
transfer chains. Adhesion of F. succinogenes seems to be related to the metabolic functions of the cell. External
proteins and/or cellulases themselves might play a part in the attachment process. Several mechanisms are
probably involved in the adhesion of R. flavefaciens, the main one being the interaction between the large
glycocalyx and the divalent cations Ca2' and Mg2+. Hydrophobic bonds and enzymes may also be involved.

In the rumen, fibrolytic microorganisms rapidly colonize
plant particles after their ingestion (2, 19). Adhesion of
cellulolytic bacteria brings the cell into close contact with its
specific substrate and concentrates hydrolytic enzymes on
cellulose. Several qualitative electron microscopic studies
have been done on adhesion of the three dominant ruminal
cellulolytic bacteria to cellulose and plant cell walls (3, 4, 8,
9, 11, 13, 17, 19, 22). The influence of some physicochemical
factors on the adhesion of these bacteria has been investi-
gated (14, 20, 21, 24, 27, 32). The adherence process of these
species, however, is still unknown. Ruminococcus albus and
Ruminococcusflavefaciens possess a large glycocalyx that is
mainly glycoproteic (19). The integrity of the cell coat would
seem to be essential for their adhesion since the action of
periodate or pepsin prevents bacterial adhesion (18). On
contact with the substrate, the coat is often thicker than on
the free surface of the bacterium, as the result of a stretching
of the extracellular material (1). The glycocalyx of Fibrobac-
ter succinogenes, formerly Bacteroides succinogenes (23),
unlike that of R. flavefaciens and R. albus, is very thin (19).
The bacterium seems to make up for the small quantity of
this extracellular material by the flexibility of the cell wall,
which enables it to conform to the topography of the
substrate. F. succinogenes also has fine filaments that link
the cells and might be involved in the adhesion to cellulose
(15). In old cultures, small vesicles deriving from the bacte-
rial outer membrane have been observed in close contact
with cellulose (13). These vesicles have been shown to have
cellulase and xylanase activities, and it has been suggested
*
Corresponding author.
t Present address: Laboratoire de la Chaire d'Alimentation,
Ecole Nationale Vdterinaire de Lyon, 69280 Marcy l'Etoile, France.
3081
but not proved that they are involved in bacterial adhesion
(11).
The aim of this work was to compare the effects of some
abiotic factors on the adhesion of F. succinogenes and R.
flavefaciens to cellulose.
MATERIALS AND METHODS
Bacterial strains used. We studied the effects of all factors
on R. flavefaciens 007 and F. succinogenes subsp. succino-
genes S85 (ATCC 19169), kindly provided by C. S. Stewart
(Rowett Research Institute, Aberdeen, United Kingdom)
and M. P. Bryant (University of Illinois, Urbana-Cham-
paign), respectively.
We also investigated the effects of some of these factors
(see Results) on the adhesion of the following newly isolated
ruminal cellulolytic strains provided by K. J. Cheng (Re-
search Station, Lethbridge, Alberta, Canada): R. flave-
faciens 083 and F. succinogenes LRS095 isolated from a
steer and R. flavefaciens 131 and F. siuccinogenes LRS128
isolated from a buffalo.
Strains S85 and LRS095 were grown on Ml culture
medium derived from that of Bryant et al. (7), modified by S.
Komisarczuk-Bony and G. Fonty in our laboratory (Table 1)
(unpublished data), and strains 007 and 083 were grown on
the medium of Scott and Dehority (31). Since R. flavefaciens
131 and F. succinogenes LRS128 do not grow on synthetic
or semisynthetic medium, they were grown on medium
RGMC (20), modified in our laboratory to contain the
following (per 100 ml): mineral solution 1, 7.5 ml; mineral
solution 2, 7.5 ml; resazurin (0.1%), 0.1 ml; NaHCO3, 0.4 g;
clarified ruminal fluid, 30 ml; cellobiose, 0.2 g; cysteine
hydrochloride, 0.05 g; distilled water, 85 ml. Mineral solu-
tions 1 and 2 were those described previously by Bryant and
Burkey (5).
3082 ROGER ET AL.
TABLE 1. Composition of medium Ml
Substance or parameter Amt
Mineral Ml solution"................ 75 ml
Vitamin solutionb ................ 10 ml
Trace mineral solution ................ 10 ml
Fatty acid solution"................ 3 ml
Hemin solutione................ 1 ml
Resazurin solutiont ................ 1 ml
Cellobiose ................ 3g
NaHCO3 ................ 4g
Cysteine hydrochloride ................ 0.6 g
pHI ................ 7 also prepared.
Gas phase ................ 100% CO2
Mineral Ml solution consists of the following (grams per liter of distilled
water): KH2PO4, 6; (NH4)2S04, 12; NaCI, 12; KCI, 6; MgSO4- 7H20, 1.2;
CaCl,- 2H20, 0.6.
Vitamin solution, consists of the following (milligrams per liter of distilled
water): biotin, 50; para-aminobenzoic acid, 100; pyridoxamine, 1.000.
' Trace mineral solution consists of the following (milligrams per liter of
distilled water): FeSO4 7H20, 1,000; MnSO4* H20, 150; CoCI2 6H20, 150.
d Fatty acid solution consists of the following (milliliters): acetic acid, 17;
propionic acid, 6; butyric acid, 4; isobutyric acid, 1; N-valeric acid, 1;
isovaleric acid, 1; 2-methylbutyric acid, 1.
e Hemin (0.1 g) is dissolved in 10 ml of ethanol and then brought to 1,000 ml
with 0.05 M NaOH.
fResazurin (0.1 g) is dissolved in 100 ml of distilled water.
g Final pH 7 is adjusted with 3 M NaOH.
All strains were cultured under 100% CO2 in Hungate
tubes (16 by 25 mm; Bellco Glass, Inc., Vineland, N.J.)
containing 9 ml of medium and sealed with butyl stoppers.
Cellobiose was the sole energy source in the media.
Determination of percentage of bacteria attached to cellu-
lose. The effects of the different factors were investigated
when bacterial growth was at the end of the exponential
phase. At this time, the optical density was usually between
0.6 and 0.7 for R. flavefaciens and 1.2 and 1.3 for F.
succinogenes. The bacterial cultures were centrifuged at
3,500 x g for 10 min. The supernatant was decanted, and the
pellet was suspended in 9 ml of medium of the same
composition containing the test factor and without cellobiose
and resazurin, which may have modified optical density
readings.
The percentage of bacteria adhering to cellulose was
calculated by the method of Minato and Suto (20), which
consists of measuring the optical density of the culture
before and after the addition of cellulose. Optical density
was read at 600 nm. We studied adhesion to microcrystalline
cellulose Avicel (microcrystalline cellulose for chromatogra-
phy; Macherey Nagel, Duren, Federal Republic of Ger-
many) added at a concentration of 0.2%. At least four
replicates were made for each factor.
Effects of physicochemical factors on adhesion of bacteria to
cellulose Avicel. (i) Contact time between bacteria and cellu-
lose. After the addition of cellulose Avicel, the following
contact times between bacteria and cellulose were studied:
1-min hand shaking (S); S + 4 min of incubation at 38°C; S +
14 min of incubation at 38°C; S + 29 min of incubation at
38°C; S + 59 min of incubation at 38°C. The cultures were
hand shaken every 5 min to resuspend the cellulose.
(ii) Influence of temperature. Adhesion was measured at 4,
13, 20, 30, 38, 42, 43, 44, 46, 48, and 52°C. The cultures of R.
flavefaciens 007 were left for 10 min at the desired temper-
ature before cellulose was added. They were then shaken for
1 min and immediately centrifuged at 500 x g. The cultures
of F. succinogenes S85 were left at the desired temperature
for 24 min after the addition of the cellulose and 1 min of
shaking. A bacterial culture of each species was also heated
APPL. ENVIRON. MICROBIOL.
at 100°C for 10 min. Its adhesion was then compared with
that of an untreated control culture.
(iii) Effects of carbohydrates. The effects of cellobiose,
glucose, mannose, xylose, maltose, and soluble starch were
studied at concentrations of 0.1, 0.2, 0.5, 1, and 5%. To
eliminate any traces of cellobiose, which had been used as
energy source during culture, the bacteria were centrifuged
at 3,500 x g for 10 min, washed in a medium of the same
composition but without cellobiose or resazurin, centrifuged
again at the same speed, and then placed in the same medium
containing the test carbohydrate. A sugar-free control was
The percentage of adherent cells was also determined after
subculturing F. succinogenes S85 one, five, or ten times into
4% glucose or cellobiose. The percentage of adhesion in
these different subcultures was compared with that of a
control containing the same sugar at a concentration of
0.3%.
(iv) Effects of cellulose derivatives. The effects of methyl-
cellulose (MC) (2% viscosity, 400 cP; Sigma Chemical Co.,
St. Louis, Mo.), carboxymethyl cellulose (CMC), and hy-
droxyethylcellulose (E. Merck AG, Darmstadt, Federal Re-
public of Germany) were studied at a concentration of 0.1%.
The bacterial cultures were treated as above; the cells were
suspended after a second centrifugation in a sugar-free
medium of the same composition supplemented with MC,
CMC, or hydroxyethylcellulose at the required concentra-
tion. Controls without cellulose derivatives were prepared at
the same time.
(v) Effect of divalent cations Mg2' and Ca2+. F. succino-
genes S85 and R. flavefaciens 007 were grown on Ml culture
medium (Table 1). For R. flavefaciens, an amino acid
solution containing isoleucine, leucine, and valine (100 mg of
each per 100 ml of distilled water) was added at a concen-
tration of 1% to satisfy its nutritional requirements (6). After
centrifugation, the pellet was resuspended in 9 ml of Ml
medium containing no resazurin or cellobiose and lacking
Ca2+, Mg2+, or both. The mineral composition of the
medium was as follows (grams per 100 ml): KH2PO4, 0.6;
(NH4)2SO4, 1.2; NaCl, 1.2; KCl, 0.6. In addition,
MgSO4- 7H20 was added at a concentration of 0.12 g/100 ml
to the medium lacking Ca2+ and CaCl2 2H20 was added at
a concentration of 0.06 g/100 ml to the medium lacking
Mg2 . The bacterial cells were centrifuged two more times
and resuspended after each centrifugation in their respective
media. At the same time, controls containing Ca2+ or Mg2+
were prepared.
(vi) Effect of pH. Adhesion of R. flavefaciens 007 and F.
succinogenes S85 was studied in a pH range of 3.5 to 9 and
4.5 to 7, respectively, at intervals of 0.5 unit. After centrif-
ugation, R. flavefac iens and F. succinogenes were sus-
pended in the medium of Scott and Dehority (31) and in Ml
medium, respectively, containing no cellobiose, no res-
azurin, and no buffer (NaHCO3). The medium of Scott and
Dehority (31) was adjusted to the required pH with 3 M
NaOH or with 1 M HCI. In Ml medium, a double concen-
tration of NaCl and 8 ml of 3 M NaOH per liter were added
to compensate for the sodium lost by removal of the buffer.
Then the required pH was adjusted with 3 M KOH or with 1
M HCl. This was done since upon total sodium deprivation
F. succinogenes did not adhere at all (see Results). Experi-
ments were done under nitrogen to avoid the variations in
pH caused by the dissolution of CO2 in the medium.
(vii) Effect of oxygen. After the addition of cellulose, the
bacterial culture was left in contact with air for 5, 15, 30, or
60 min.
VOL. 56, 1990 ADHESION OF RUMINAL CELLULOLYTIC BACTERIA TO CELLULOSE 3083

(viii) Effect of redox potential. Adhesion of R. flavefaciens 1,6

007 and F. succinogenes S85 was studied in media contain- 11--k
ing a reducing agent under 100% CO2, without a reducing I
agent under 100% CO2, containing a reducing agent and
8
prepared in aerobiosis, or without a reducing agent and
prepared in aerobiosis. Z.

Three different reducing agents (cysteine hydrochloride, Ea 40

dithiothreitol, and sodium sulfide) were used at a concentra-
tion of 0.1% for R. flavefaciens 007. With F. succinogenes * 20 I
S85, cysteine hydrochloride was the only reducing agent
9
studied. Both species were incubated at 38°C for 25 min after
the addition of cellulose. The redox potential was measured
with an rH meter (CG 819; Schott Gerate, Hofheim, Federal
Republic of Germany); the electrode was immersed in the Time (hours)
bacterial culture for 20 s.
(ix) Effect of sodium and gas phase. R. flavefaciens 007 was
grown on Ml medium containing amino acids as detailed
above. Two media were tested: medium Ml and medium Ml
- Na from which all sodium sources had been removed and go
the pH adjusted to 7 with 3 M KOH. Both were tested under 8

a gas phase of 100% N2 or 100% CO2.

Adhesion of F. succinogenes S85 was studied on the
following media: medium Ml - Na from which all sodium
sources had been removed; medium Ml + NaHCO3 in
which the sodium was added at a concentration of 50 mM in
the. form of NaHCO3; medium Ml + Na2CO3 in which the
sodium was added at a concentration of 50 mM in the form
of Na2CO3; medium Ml + NaCl in which the sodium was 17 22
added at a concentration of 50 mM in the form of NaCl; Time (hours)
medium Ml + KHCO3 in which no sodium ions were added
to the medium (the concentration of KHCO3 was calculated FIG. 1. Effect of the growth phase on the adhesion of F. succi-
so that the supply of HCO3- ions was the same as that in the nogenes S85 (a) and R. flavefaciens 007 (b). Symbols: A, growth
medium Ml + NaHCO3); medium Ml + LiCl in which no (A6w); *, adherent cells (%).
sodium was added to the medium and lithium was added at
a concentration of 50 mM.
All the media were prepared under 100% N2. The medium flavefaciens adhered in the early exponential phase. There-
with LiCl and those without sodium were also prepared after, the adhesion of F. succinogenes fell abruptly, whereas
under 100% CO2. When NaCl was removed from the mineral the high percentage of adherent ruminococci was maintained
solution, it was replaced by the same concentration of KCI. until the late stationary phase (Fig. la and b).
In all media, the pH was adjusted to 7 with 3 M KOH. R. flavefaciens 007 and 083 adhered rapidly to cellulose
We also studied the adhesion of the bacteria on medium Avicel; after only 1 min of contact, 80 to 90% of the bacterial
Ml under 100% N2 or 100% CO2. The latter was considered cells were adherent. For the study of other factors, this
a control. species was maintained in contact with cellulose for 5 min.
(x) Effect of metabolic inhibitors. We tested monensin and In contrast, the greatest number of adhering cells of F.
lasalocid (ionophore antibiotics) at a concentration of 0.02 succinogenes S85 was reached between 15 and 30 min of
mM, 2,4-dinitrophenol and tetrachlorosalicylanilide (proton- contact with cellulose; after 1 min of contact, only 30% of
ophores) at concentrations of 1.6 and 0.02 mM, respectively, the cells adhered (Table 2). The effects of other factors on
N,N'-dicyclohexylcarbodiimide (DCCD) (inhibitor of mem- this species were therefore studied with cultures harvested
brane ATPases) at a concentration of 0.02 mM, antimycin A at the end of the exponential growth phase and after 25 min
and hydroxyquinoline-N-oxide at a concentration of 0.1 of contact with cellulose.
mM, and sodium azide at a concentration of 40 mM (the Adhesion of both species was maximal at 38°C. Adhesion
latter three are all inhibitors of electron transfer chains). of F. succinogenes varied according to the temperature
These inhibitors were added to the bacterial culture and
left in contact with the bacteria for 10 min before the addition
of cellulose. Sodium azide was dissolved in distilled water, TABLE 2. Effect of contact time between cellulose and bacteria
hydroxyquinoline-N-oxide was dissolved in dimethyl sulfox- on percentage of cells adhering to cellulose Avicel
ide, and the other inhibitors were dissolved in ethanol. % of adherent cellsa
Control cultures containing ethanol or dimethyl sulfoxide at Contact time
(min) R. flavefaciens R. flavefaciens F. succinogenes
the same concentrations as in the test tubes were made to 007 083 S85
check the eventual effects of these compounds. The final
concentration of dimethyl sulfoxide or ethanol in the bacte- 1 74 4 71 3 36 1
rial culture was less than 1 or 3%, respectively. 5 81 3 82 1 57 4
15 83 2 75 8 78 5
RESULTS 30 82 7 82 2 83 3
60 80 6 89 2 83 4
F. succinogenes had the highest percentage of adherent
cells at the end of the exponential growth phase, while R. a
Average of four replicates + standard deviation.
3084 ROGER ET AL. APPL. ENVIRON. MICROBIOL.

TABLE 3. Effect of temperature on adhesion of F. succinogenes TABLE 4. Effect of sugars, cellulose derivatives, and lack of
S85 and R. flavefaciens 007 to cellulose Avicel divalent cations on adhesion of F. succinogenes S85 and
R. flavefaciens 007 to cellulose Avicel
Temp % of adherent cellsa % of adherent cellsa
(OC) R. flavefaciens F. succinogenes Factor studied
R. flavefaciens F. succinogenes
4 99±7 71±3
13 98 5 81± 2 Glucose (5%) 82 ± 4 64 ± 2
20 98 7 84 ±+5 Cellobiose (5%) 82 ± 6 54 ± 2
30 100 96 ± 3 Mannose (5%) 89 ± 1 71 ± 8
42 100 98± 3 Xylose (5%) 90 ± 1 77 ± 6
43 NDb 85 ± 6 Maltose (5%) 93 ± 2 100
44 ND 77 3 Soluble starch (5%) 90 ± 5 100
46 ND 83 6
48 ND 39 4 CMC (0.1%) 100 100
52 93 ± 3 ND HEC (0.1%) 21 3 70 ± 3
MC (0. 1%) 46 ± 4 100
'
Percentage of adherent cells compared with a control tube at 38°C
(average of four replicates ± standard deviation). Minus Ca2+ 91 ± 4 100
b ND, Not determined.
Minus Mg2+ 87 ± 4 100
Minus Ca2' and Mg2" 77 ± 5 100
(Table 3), while that of R. flavefaciens was unaffected. The a Percentage of adherent cells compared with a control culture containing
percentage of adhering cells of F. succinogenes decreased Ca2" and Mg2+ and without carbohydrates (average of four replicates +
markedly at 48°C. After exposure of the bacterial cuitures to standard deviation).
100°C, the adhesion of F. succinogenes S85 and LRS095 was
completely suppressed, while there was a decrease of only
15% in attachment of R. flavefaciens. hesion of R. flavefaciens was not greatly affected by redox
At low concentrations (0.1 to 1%), carbohydrates had no potential (Table 5). The presence of cysteine or Na2S under
effect on the adhesion of the two species. At a concentration aerobic conditions almost completely inhibited adhesion of
of 5%, the adhesion of R. flavefaciens was slightly depressed the ruminococci (Table 5).
while that of F. succinogenes was partly affected by xylose Under a nitrogen atmosphere and in the absence of sodium
and mannose and more by cellobiose and glucose (Table 4). ions, F. succinogenes did not adhere to cellulose (20% of
There was a considerable decrease in the adhesion of F. adherent bacteria compared with control values). An atmo-
succinogenes after it had been transferred several times on sphere of CO2 or the presence of Na+ was required for the
cellobiose or glucose at a high concentration (4%). The adhesion of this species. In its effect, Li' could substitute
number of adherent cells of F. succinogenes decreased by for Na+ (Table 6). The adhesion of the bacterium was also
60, 67, and 80% after 1, 5, and 10 transfers, respectively, on strongly inhibited by DCCD and by certain ionophore anti-
a high concentration (4%) of cellobiose. On 4% glucose, biotics (Table 7). At the same concentration, the effect of
whatever the number of transfers, there was only a 35% lasalocid was greater than that of monensin. The other
decrease. Hydroxyethylcellulose partly inhibited the adhe- protonophores and inhibitors of the electron transfer chains
sion of F. succinogenes and greatly inhibited that of R. did not significantly alter the percentage of adherent bacte-
flavefaciens, while MC only inhibited the adhesion of R. ria.
flavefaciens and CMC had no effect on either species (Table Adhesion of R. flavefaciens was little affected by the
4). absence of sodium ions in the medium (90% adherent cells
Only the attachment of R. flavefaciens was affected by a compared with the control) (Table 6) or by the action of the
lack of divalent cations (Table 4). Deprivation of either Ca2" different metabolic inhibitors tested (Table 7).
or Mg2" had little effect, but the lack of both cations
inhibited adhesion to a greater extent (Table 4). The number
of adherent cells of F. succinogenes increased regularly from DISCUSSION
pH 4.5 to 6.0 (30% of adherent cells at pH 4.5, 80% at pH From the various quantitative techniques used for the
6.0), remained stable between pH 6.0 and 7.0, and fell determination of bacterial adhesion, we chose that described
abruptly at pH 7.5 (25% of adherent bacteria). The adhesion by Minato and Suto (20) because it is simple, rapid, repro-
of R. flavefaciens was stable at pHs between 3.5 and 7.5, ducible, and allows the study of many factors. While it is
decreased at pH 8.0 (40% of adherent cells), and remained true that with this method nonadherent bacterial cells may
the same at pH 8.5 and 9.0. After 5 min of exposure to air, be entrapped within cellulose during centrifugation (27), this
80% inhibition of adherent cells of F. succinogenes S85, should not be considered a major drawback. If large num-
LRS095, and LRS128 was observed. After 1 h, adhesion was bers of cells were to be entrapped during centrifugation,
completely inhibited. Adhesion of R. flavefaciens 007 was strong inhibitory factors would never be found; yet we
little affected by the presence of oxygen, but strains 083 and observed that adhesion of R. flavefaciens in the presence of
131 were slightly inhibited: 25% after 5 min and 35% after 1 cysteine in aerobic medium and that of F. succinogenes in
h of exposure. the presence of several factors (Na+ deprivation under an
The percentage of adherent bacteria of F. succinogenes atmosphere of N2, presence of oxygen) were almost com-
decreased with oxidation of the medium compared with the pletely inhibited. Furthermore, in each assay we compared
control (medium with cysteine and under 100% CO2. -250 our results with those of controls lacking the factor the effect
mV). It decreased by 20% in medium without cysteine under of which was to be studied.
100% CO2 (-150 mV), by 80% in medium with cysteine R. flavefaciens adheres in less than 1 min to cellulose,
under aerobic conditions (-50 mV), and by 90% in medium which suggests that the adherence process involves a struc-
without cysteine under aerobic conditions (+100 mV). Ad- ture that is an integral part of the bacterial cell. The adhesion
VOL. 56, 1990 ADHESION OF RUMINAL CELLULOLYTIC BACTERIA TO CELLULOSE 3085

TABLE 5. Effect of redox potential on adhesion of R. flavefaciens 007 to cellulose Avicel

Gas phase Reducing

agent Cysteine hydrochloride
% of adherent cells with Eh (mV)a
Dithiothreitol Na2S

100% C02 Used 90 ± 7 (-210 ± 88) 94 + 4 (-290 + 23) 94 + 2 (-190 ± 24)

Omitted 86 + 11 (-80 ± 38) 88 ± 8 (-180 ± 32) 95 ± 2 (-60 ± 46)
100% air Used 13 ± 10 (+40 ± 78) 60 ± 15 (-220 ± 45) 9 ± 9 (+120 ± 18)
Omitted 58 ± 18 (+140 ± 59) 56 ± 15 (+210 ± 17) 57 ± 7 (+200 ± 24)
a
Average of four replicates ± standard deviation.

of this bacterium, as shown for R. albus (25), occurs within albus (24), soluble starch did not inhibit adhesion to cellu-
S min after the addition of the substrate. R. flavefaciens and lose, even at high concentrations, which suggests the spec-
R. albus appear therefore to be able to colonize plant cell ificity of the binding with the substrate. Moreover, it has
tissues as soon as they arrive in the rumen and possibly been observed elsewhere (G. Fonty, I. Guerry, and P.
colonize plant tissues faster than F. succinogenes. In an Gouet, First Congress of the French Society for Microbiol-
environment in which there is strong competition from other ogy, Toulouse, 1986) that R. flavefaciens adheres weakly or
microorganisms, this may represent an ecological advan- not at all to several noncellulolytic substrates (Sephadex,
tage. Our findings are in line with those of Akin (3), who Amberlite resin, starch granules).
observed by electron microscopy a larger number of F. Unlike Minato and Suto (20), Kudo et al. (16), and
succinogenes adherent to plant cell walls during the later Rasmussen et al. (27), we observed no inhibitory effect of
stages of cellulolysis. CMC. This controversial observation might be explained by
The sudden drop in the percentage of adherent cells of F. the fact that the inhibitory activity of CMC on cellulases was
succinogenes at low and high temperatures is reminiscent of transitory, as this compound was very inhibitory in immedi-
an enzymatic type of bacterium-cellulose linkage. This is ate assays but a portion of this inhibition was relieved by
further suggested by the fact that adhesion reached its preincubating CMC with the enzyme source (26). Under our
maximum at the end of the exponential phase, i.e., when experimental conditions, F. succinogenes was in contact
enzyme production is at its maximum. with cellulose Avicel for 25 min, and this might explain why
The carbohydrates studied, all constitutive elements of the our results were different. They might also be due to the
polyosides of the plant cell walls, had an inhibitory effect on bacterial strains and the cellulose source used. Morris (24)
adhesion only at a high concentration (5%), which indicates recently observed that the adhesion of R. albus differed
that they have a weak specificity for possible receptors of the according to strains, while Wood et al. (35) have shown that
bacterial wall. The inhibitory effect of glucose and cellobiose the cellulases of this bacterium varied in size depending on
on F. succinogenes at high concentrations may be due to a the energy source. However, our results on the effect of MC
retroactive inhibitory effect of these sugars on enzymatic on R. flavefaciens are in agreement with those of Rasmussen
activity and strongly suggests that the cellulases of F. et al. (27). We also observed an eluting effect of MC (V.
succinogenes are involved in the adherence process. After Roger and G. Fonty, unpublished data) against this bacte-
10 transfers on these sugars at 4%, we observed that this rium. Rasmussen et al. (26) and White et al. (34) have
species was still able to degrade filter paper. Furthermore, recently shown that MC at a low concentration has an
Taylor et al. (33) have recently shown that these sugars can
repress certain cellulase genes of the bacterium. As for R.
TABLE 7. Effect of metabolic inhibitors on adhesion of
F. succinogenes S85 and R. flavefaciens 007 to cellulose Avicel
TABLE 6. Effect of sodium and gas phase on adhesion of % of adherent cellsb
F. succinogenes S85 and R. flavefaciens 007 to cellulose Avicel Inhibitor (mM)'
R. flavefaciens F. succinogenes
Gas
a Ion supple- cells'
phase
Gas
mentb Metal ionophores
R. flavefaciens F. succinogenes
Lasalocid (0.02) 82 ± 14 6± 4
100% N2 Minus Na 91 5 19 7 Monensin (0.02) 85 ± 6 70 ± 8
NaHCO3 NDd 84 6
Na2CO3 ND 86 3 Proton ionophores
NaCl ND 57 4 2,4-dinitrophenol (1.6) 100 94 ± 3
KHCO3 ND 10 6 Tetrachlorosalicylanilide (0.02) 100 95 ± 4
Nae 89 5 84 ± 2
LiC1f ND 48 6 Membrane ATPase inhibitor 94 ± 1 10 ± 6
(DCCD [0.2])
100% C02 Minus Na 89 + 4 81 8
LiC1f ND 91 4 Electron transport inhibitors
Antimycin A (0.1) 87 ± 9 88 ± 10
a
Media prepared under either 100% N2 or 100% CO2 gas phase. Hydroxyquinoline-N-oxide (0.1) 90 ± 4 90 ± 4
b
Na+, Li', or HC03- supplied at 50 mM concentration. NaN3 (40) 100 100
' Percentage of adherent cells compared with a control culture (average of

four replicates standard deviation). a

All factors are dissolved in ethanol except NaN3 and hydroxyquinoline-
d ND, Not determined. N-oxide, which were dissolved, respectively, in distilled water and dimethyl
eAll the sodium sources of medium Ml are present (NaCI, NaHCO3, and sulfoxide.
NaOH). b Percentage of adherent cells of a control culture (average of four repli-
f No sodium added. cates ± standard deviation).
3086 ROGER ET AL.
inhibitory effect on an exo-,B-1,4-glucanase (called exo A)
and has surfactant properties at a high concentration. These
observations and our own results suggest that this enzyme
acts in the adherence process and that hydrophobic interac-
tions are involved in the adherence of R. flavefaciens.
However, these statements have to be demonstrated.
Unlike F. succinogenes, R. flavefaciens requires the pres-
ence of Ca2" and Mg2" ions for its adhesion. These cations
might bind the large glycocalyx observed by electron micros-
copy (8, 19) and cellulose. However, the fact that in their
absence adhesion decreased by only 20 to 30% indicates that
ionic interactions are not the sole mechanism of adhesion of
this bacterial species. The greatest number of adherent cells
of F. succinogenes observed at pHs between 5.5 and 7.0 is
consistent with an enzymatic-type adhesion process since at
those pHs the different cellulases of F. succinogenes exhib-
ited the greatest activity. However, pH could have also
affected electrostatic interactions between the cell surface
and cellulose. In agreement with Rasmussen et al. (27), we
observed that the adhesion of R. flavefaciens, like that of R.
albus (24), was not affected by low pHs.
The attachment of R. flavefaciens would seem to be
unaffected by cell metabolism, as evidenced by the high
adhesion percentage obtained in the presence of metabolic
inhibitors and in a culture medium heated at 100°C. On the
contrary, adhesion of F. succinogenes seems to require the
intact metabolic functions of the bacteria. Indeed, the adhe-
sion decreases in the absence of Na, which is necessary for
growth and glucose uptake (12, 29), and is almost completely
inhibited by oxygen. This finding was confirmed by the
observation that heat-treated cultures (100°C for 10 min) do
not adhere and is another argument in favor of the partici-
pation of proteins in adhesion. The effect of redox potential
supports this hypothesis; the more the medium is oxidized,
and thus incompatible with the survival of the bacterium, the
fewer bacteria there are adhering to cellulose. DCCD, which
alkylates the carboxyl groups, might act by inhibiting
ATPases or altering a protein involved in the adhering
process. However, Gong and Forsberg (14) recently demon-
strated that the carboxyl groups of a protein do not play an
important role in adhesion. Thus, DCCD seems to inhibit
adhesion via an inhibition of ATPases. The decrease in the
degree of adhesion of F. succinogenes in the presence of
monensin and lasalocid might be due to modifications of
ionic gradients. The proton motive force of the bacterium,
which might be an energetic source for different cellular
functions, is reduced in the presence of monensin (10, 28,
30). Our results suggest that only live cells of F. succino-
genes are able to adhere to cellulose. However, this hypoth-
esis is not in agreement with results of Gong and Forsberg
(14), who observed that Formalin-treated cells still adhere.
The absence of effects of protonophores and inhibitors of
electron transfer chains, used at concentrations that inhibit
the growth of F. succinogenes (12), remains unexplained.
Contrary to what might be expected for a strictly anaero-
bic bacterium, attachment of R. flavefaciens remains high in
the presence of oxygen or in a medium prepared under
aerobic conditions in the absence of reducing agent. The
almost total inhibition of adhesion in the presence of cyste-
ine in a medium prepared in aerobiosis might be due to the
fact that under these conditions, cysteine is transformed into
cystine, a highly hydrophobic amino acid, which could
therefore compete with the hydrophobic interactions that
possibly exist between the bacterium and its specific sub-
strate. The absence of an effect of dithiothreitol in aerobiosis
can be explained by the low redox potential still reached.
APPL. ENVIRON. MICROBIOL.
The effect of a reducing agent might also be explained by the
presence of the SH groups.
The adherence process seems to be different for R. flave-
faciens and F. succinogenes. The adhesion of the latter is
affected by low temperatures, the presence of oxygen, the
lack of sodium, and the inhibition of its membrane ATPases.
These results suggest that proteins and bacterial cellulases
themselves play a part in the adhesion of F. succinogenes.
The proteins responsible for adhesion should now be iso-
lated and purified. Several mechanisms appear to be in-
volved in the adhesion of R.flavefaciens, the main one being
the interaction between the bacterial cell and the divalent
cations Ca2" and Mg2+. Hydrophobic interactions and en-
zymes may also be involved.
LITERATURE CITED
1. Akin, D. E. 1976. Ultrastructure of rumen bacterial attachment
to forage cell walls. Appl. Environ. Microbiol. 31:562-568.
2. Akin, D. E. 1979. Microscopic evaluation of forage digestion by
rumen microorganisms-a review. J. Anim. Sci. 48:701-710.
3. Akin, D. E. 1980. Evaluation by electron microscopy and
anaerobic culture of types of rumen bacteria associated with
digestion of forage cell walls. Appl. Environ. Microbiol. 39:242-
252.
4. Akin, D. E., and F. E. Barton II. 1983. Rumen microbial
attachment and degradation of plant cell walls. Fed. Proc.
42:114-121.
5. Bryant, M. P., and L. A. Burkey. 1953. Cultural methods and
some characteristics of some of the more numerous groups of
bacteria in the bovine rumen. J. Dairy Sci. 36:205-217.
6. Bryant, M. P., and I. M. Robinson. 1962. Some nutritional
characteristics of predominant cultural ruminal bacteria. J.
Bacteriol. 84:605-614.
7. Bryant, M. P., I. M. Robinson, and H. Chu. 1959. Observations
on the nutrition of Bacteroides succinogenes-a ruminal bacte-
rium. J. Dairy Sci. 42:1831-1847.
8. Cheng, K. J., C. S. Stewart, D. Dinsdale, and J. W. Costerton.
1983/1984. Electron microscopy of bacteria involved in the
digestion of plant cell walls. Anim. Feed Sci. Technol. 10:93-
120.
9. Dinsdale, D., E. J. Morris, and J. S. D. Bacon. 1978. Electron
microscopy of the microbial populations present and their
modes of attack on various cellulosic substrates undergoing
digestion in the sheep rumen. Appl. Environ. Microbiol. 36:160-
168.
10. Forano, E. 1988. Effets de la monensine sur les gradients
transmembranaires de H', Na+ et K' chez Bacteroides succi-
nogenes, bacterie cellulolytique du rumen. Reprod. Nutr. Dev.
28(Suppl. 1):81-82.
11. Forsberg, C. W., T. J. Beveridge, and A. Hellstrom. 1981.
Cellulase and xylanase release from Bacteroides succinogenes
and its importance in the rumen environment. Appl. Environ.
Microbiol. 42:886-896.
12. Franklund, C. V., and T. L. Glass. 1987. Glucose uptake by the
cellulolytic ruminal anaerobe Bacteroides succinogenes. J. Bac-
teriol. 169:500-506.
13. Gaudet, G., and B. Gaillard. 1987. Vesicle formation and
cellulose degradation in Bacteroides succinogenes cultures:
ultrastructural aspects. Arch. Microbiol. 148:150-154.
14. Gong, J., and C. W. Forsberg. 1989. Factors affecting adhesion
of Fibrobacter succinogenes subsp. succinogenes S85 and ad-
herence-defective mutants to cellulose. Appl. Environ. Micro-
biol. 55:3039-3044.
15. Groleau, D., and C. W. Forsberg. 1981. Cellulolytic activity of
the rumen bacterium Bacteroides succinogenes. Can. J. Micro-
biol. 27:517-530.
16. Kudo, H., K. J. Cheng, and J. W. Costerton. 1987. Electron
microscopic study of the methylcellulose-mediated detachment
of cellulolytic rumen bacteria from cellulose fibers. Can. J.
Microbiol. 33:267-272.
17. Lamed, R., J. Naimark, E. Morgenstern, and E. A. Bayer. 1987.
 VOL. 56, 1990 ADHESION OF RUMINAL CELLULOLYTIC BACTERIA TO CELLULOSE
Specialized cell surface structures in cellulolytic bacteria. J.
Bacteriol. 169:3792-3800.
18. Latham, M. J. 1980. Adhesion of rumen bacteria to plant cell
walls, p. 339-350. In R. C. W. Berkeley, J. M. Lynch, J.
Melling, P. R. Rutter, and B. Vincent (ed.), Microbial adhesion
to surfaces. Ellis Horwood Limited Publishers, Chichester,
United Kingdom.
19. Latham, M. J., B. E. Brooker, G. L. Pettipher, and P. J. Harris.
1978. Ruminococcus flavefaciens cell coat and adhesion to
cotton cellulose and to cell walls in leaves of perennial ryegrass
(Lolium perenne). AppI. Environ. Microbiol. 35:156-165.
20. Minato, H., and T. Suto. 1978. Technique for fractionation of
bacteria in rumen microbial ecosystem. It. Attachment of
bacteria isolated from bovine rumen to cellulose powder in vitro
and elution of bacteria attached to it. J. Gen. Appl. Microbiol.
16:1-16.
21. Minato, H., and T. Suto. 1981. Technique for fractionation of
bacteria in rumen microbial ecosystem. IV. Attachment of
rumen bacteria to cellulose powder and elution of bacteria
attached to it. J. Gen. Appl. Microbiol. 27:21-31.
22. Miron, J., M. T. Yokoyama, and R. Lamed. 1989. Bacterial cell
surface structures involved in lucerne cell wall degradation by
pure cultures of cellulolytic rumen bacteria. Appl. Microbiol.
Biotechnol. 32:218-222.
23. Montgomery, L., B. Flesher, and D. Stahl. 1988. Transfer of
Bacteroides suc cinogenes (Hungate) to Fibrobaccter gen. nov.
as Fibrobacter succinogenes comb. nov. and description of
Fibrobacter intestinalis sp. nov. Int. J. Syst. Bacteriol. 38:430-
435.
24. Morris, E. J. 1988. Characteristics of the adhesion of R. albuis to
cellulose. FEMS Microbiol. Lett. 51:113-118.
25. Morris, E. J., and 0. J. Cole. 1987. Relationship between
cellulolytic activity and adhesion to cellulose in R. albus. J.
View publication stats
3087
Gen. Microbiol. 133:1023-1032.
26. Rasmussen, M. A., R. B. Hespell, B. A. White, and R. J.
Bathast. 1988. Inhibitory effects of methylcellulose on cellulose
degradation by Ruminococcus flav'efaciens. Appl. Environ.
Microbiol. 54:890-897.
27. Rasmussen, M. A., B. A. White, and R. B. Hespell. 1989.
Improved assay for quantitating adherence of ruminal bacteria
to cellulose. Appl. Environ. Microbiol. 55:2089-2091.
28. Russell, J. B. 1987. A proposed mechanism of monensin action
in inhibiting ruminal bacterial growth: effects on ion flux and
protonmotive force. J. Anim. Sci. 64:1519-1525.
29. Russell, J. B. 1987. Effect of extracellular pH on growth and
proton motive force of Bacteroides suc cinogenes, a cellulolytic
ruminal bacterium. Appl. Environ. Microbiol. 53:2379-2383.
30. Russell, J. B., and H. J. Strobel. 1989. Effect of ionophores on
ruminal fermentation. Appl. Environ. Microbiol. 55:1-6.
31. Scott, H. W., and B. A. Dehority. 1965. Vitamin requirements of
several cellulolytic bacteria. J. Bacteriol. 89:1169-1175.
32. Stack, R. J., and R. E. Hungate. 1984. Effect of 3-phenyl
propanoic acid on capsule and cellulases of Rumninococcus
albus 8. Appl. Environ. Microbiol. 48:218-223.
33. Taylor, K. A., B. Crosby, M. McGavin, C. W. Forsberg, and
D. Y. Thomas. 1987. Characteristics of the endoglucanase en-
coded by a cel gene from Bacteroides succinogenes expressed
in Escheric/hia coli. Appl. Environ. Microbiol. 53:41-46.
34. White, B. A., M. A. Rasmussen, and R. M. Gardner. 1988.
Methylcellulose inhibition of exo-,-1,4-glucanase A from Rumi-
nococcusflavefaciens FD1. Appl. Environ. Microbiol. 54:1634-
1636.
35. Wood, T. M., C. A. Wilson, and C. S. Stewart. 1982. Prepara-
tion of the cellulase from the cellulolytic anaerobic rumen
bacterium Rumninococcus albus and its release from the bacte-
rial cell wall. Biochem. J. 205:129-137.