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Keywords:

  • rumen bacteria;
  • fiber digestion;
  • coculture;
  • Fibrobacter succinogenes

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

In a previous study, we reported the ecological significance of uncultured bacterial group U2 in the rumen. In this study, the involvement of a recently cultured group U2 bacterium, strain R-25, in fiber digestion was tested in coculture with the fibrolytic bacterium Fibrobacter succinogenes S85. Dry matter (DM) digestion, growth and metabolites were examined in culture using rice straw as the carbon source. Although strain R-25 did not digest rice straw in monoculture, coculture of strain R-25 and F. succinogenes S85 showed enhanced DM digestion compared with that for F. succinogenes S85 monoculture (36.9 ± 0.6% vs. 32.8 ± 1.3%, < 0.05). Growth of strain R-25 and production of the main metabolites, d-lactate (strain R-25) and succinate (F. succinogenes S85), were enhanced in the coculture. Enzyme assay showed increased activities of carboxymethylcellulase and xylanase in coculture of strain R-25 and F. succinogenes S85. Triculture including strain R-25, F. succinogenes S85 and Selenomonas ruminantium S137 showed a further increase in DM digestion (41.8 ± 0.8%, < 0.05) with a concomitant increase in propionate, produced from the conversion of d-lactate and succinate. These results suggest that the positive interaction between strains R-25 and F. succinogenes S85 causes increased rice straw digestion.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Ruminant animals utilize plant fiber as an energy source by converting cellulose and hemicellulose to short-chain fatty acids by ruminal fermentation. The microbial ecosystem in the rumen is comprised of bacteria, protozoa, anaerobic fungi, methanogenic archaea, and bacteriophages (Klieve & Bauchop, 1988; Morvan et al., 1996; Flint, 1997). Of the rumen microorganisms, bacteria possess high fibrolytic activities and comprise a significant biomass. Brulc et al. (2009) reported that more than 90% of coding sequences in the rumen metagenome was derived from bacteria. Therefore, bacteria play a key role in the biological fiber degradation in the rumen.

Comprehensive analysis of 16S rRNA genes from rumen samples revealed that 300–400 different bacterial species are present in the rumen (Edwards et al., 2004; Yu et al., 2006). Among these, only 2–31% showed high similarity (≥ 97%) with cultivated species (Kobayashi, 2006). Koike et al. (2003) reported that the majority (77%) of fiber-associated bacterial community in the rumen had < 97% similarity with 16S rRNA gene sequences of known bacteria. These results indicate that there is limited knowledge about ruminal fibrolytic species and the possible involvement of uncultured bacteria in ruminal fiber digestion.

Through phylogenetic analysis of the fiber-associated community in the rumen, several bacterial groups consisting only of uncultured bacteria have been detected (Koike et al., 2003; Shinkai et al., 2010). Among these uncultured groups, our research group has been focusing on unknown group 2 (U2) that belongs to the phylum Firmicutes (Koike et al., 2003, 2010; Koike & Kobayashi, 2009). Group U2 has been detected as a large phylogenetic group with > 200 clones showing more than 97% similarity to the 16S rRNA gene sequence. The population size of U2 in the rumen was significantly higher in the solid fraction compared with liquid fraction. Strong fluorescent signals from U2 cells attached to plant fibers were observed by fluorescence in situ hybridization in the rumen (Koike et al., 2010). Therefore, U2 seems to occupy a significant metabolically active niche in the fiber-associated bacterial community in the rumen. In a previous study, we successfully isolated two strains belonging to U2 (strains R-25 and B76) and found that several of their hemicellulolytic enzyme activities were higher than those of xylanolytic Butyrivibrio fibrisolvens H17c (Koike et al., 2010). Group U2 was phylogenetically distant from representative rumen isolates and formed a cluster with nonruminal, fibrolytic strains (Fig. 1). However, U2 strains could not utilize insoluble substrates, such as cellulose or xylan, and grew only on soluble sugars (Koike & Kobayashi, 2009). On the basis of these ecological and physiological findings, U2 members are expected to play a supporting role in the rumen plant fiber digestion.

image

Figure 1. Phylogenetic placement of 16S rRNA gene sequences of recently cultured group U2 bacterium within the representative ruminal species. Nonruminal species (*) showing the similarity with group U2 were included in the tree. The accession numbers in GenBank are shown in parentheses. Bootstrap values are shown as the percentage of 1000 replicates. The horizontal bar represents nucleotide substitutions per sequence position.

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The involvement of nonfibrolytic bacteria in rumen fiber digestion has been observed in coculture studies (Dehority & Scott, 1967; Kudo et al., 1987; Osborne & Dehority, 1989; Fondevila & Dehority, 1996; Sawanon & Kobayashi, 2006; Sawanon et al., 2011). In these trials, digestion was enhanced by coexistence of fibrolytics and nonfibrolytics. Contribution of nonfibrolytics to fiber digestion is likely to be in an indirect manner, such as by hydrogen transfer or by cross-feeding of degradation and/or fermentation products derived from plant fiber (Flint, 1997).

In this study, we investigated the role of a recently cultured bacterium belonging to group U2 in ruminal fiber digestion. Of the two strains from group U2, we used strain R-25 for coculture experiments with a representative ruminal fibrolytic bacterium, Fibrobacter succinogenes S85. A triculture test with a lactate and succinate-utilizing bacterium, Selenomonas ruminantium S137, was also carried out.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Bacteria and medium

Rumen bacterium R-25, which was isolated from the rumen of sheep and classified in the group U2 (Koike et al., 2010), was used in this study. Fibrobacter succinogenes S85, which is a fibrolytic bacterium, was purchased from American Type Culture Collection. Selenomonas ruminantium S137, which was isolated from sheep rumen (Sawanon et al., 2011), was used as a metabolite (lactate and succinate) utilizer.

Basal medium was prepared anaerobically according to the method of Bryant (1972) to have the following composition (100 mL−1): 7.5 mL of mineral solutions I and II (Bryant & Burkey, 1953), 0.1 mL of 0.1% resazurin, 40 mL of clarified rumen fluid, 39 mL of distilled water, 1 mL of 5% l-cysteine-HCl·H2O, and 5 mL of 8% Na2CO3.

Substrate, inocula, and incubations

Rice straw was air-dried in an oven at 60 °C, ground to pass through a 1-mm sieve, and used as a substrate to measure fiber digestion. Strain R-25 and S. ruminantium S137 were grown to the end of log phase at 39 °C in basal medium containing cellobiose and glucose [0.5% (w/v) each] as carbon sources. After three passages, the cultures were centrifuged (2300 g, 4 °C, 10 min) to pellet the bacteria. Anaerobic dilution solution (Bryant & Burkey, 1953) was added to the pellet to resuspend the bacteria to an optical density at 660 nm (OD660 nm) of 0.2. Fibrobacter succinogenes S85 was grown for 48 h in basal medium containing 1.0% (w/v) rice straw as the sole carbon source. After three passages, the culture was centrifuged (377 g, 4 °C, 1 min) to separate the rice straw particles and supernatant containing bacterial cells (Minato & Suto, 1978). The supernatant was collected in another sterile tube and centrifuged (2300 g, 4 °C, 10 min) to pellet the bacteria. The pellet was resuspended as above, and these OD-adjusted cell suspensions were used as inocula.

Each inoculum was added at 0.1 mL to 10 mL of basal medium containing 0.1 g of rice straw as the sole carbon source. Six test tubes were prepared for respective mono- and cocultures and incubated at 39 °C under anaerobic condition. On the basis of a previous study (Shinkai et al., 2009), samples were collected at three time points after incubation; 0 h (corresponding to inocula), 48 h (middle of digestion) and 96 h (endpoint of digestion). Samples were collected from three of six test tubes at 48 h, and the rest of three test tubes were incubated until 96 h. Tubes with no inocula were prepared as a blank and treated in the same manner.

Measurement of DM digestion and metabolites

After 96 h incubation, the cultures were cooled on ice for 30 min to detach bacterial cells from fiber particles (Minato & Suto, 1978) and centrifuged (377 g, 4 °C, 10 min), and the supernatant containing bacterial cells was collected. The residue was washed with 10 mL of 0.1 M potassium phosphate buffer and re-centrifuged (2300 g, 4 °C, 10 min). The washed residue was dried at 105 °C for 48 h and weighed to calculate dry matter (DM) digestion. Collected supernatant containing bacterial cells was centrifuged (16 000 g, 4 °C, 10 min) to obtain clear supernatant that was used for measurement of metabolites. Short-chain fatty acids (SCFAs) were determined by gas chromatography (GC-14B; Shimadzu, Kyoto, Japan). Succinate and d-/l-lactate were measured by commercial assay kits (Megazyme, Wicklow, Ireland).

Real-time PCR

To monitor the growth of each bacterial strain in culture, copy number of 16S rRNA gene was quantified by real-time PCR. Repeated bead beating plus column method (Yu & Morrison, 2004) was employed for DNA extraction and purification from 1 mL of inocula and cultures at 48 and 96 h. PCR targeting the 16S rRNA gene was performed with a LightCycler 480 system (Roche Applied Science, Mannheim, Germany) and a KAPA SYBR FAST qPCR kit (KAPA Biosystems, Woburn, MA). Primer sets specific to each bacterial strain were used as follows: U2_Fw (5′-CTAGGTGTAGGGGGTATC-3′) and U2_Rv (5′-GCTGCCCTCTGTCGTTG-3′) for strain R-25 (Koike et al., 2010), 193f (5′-GGTATGGGATGAGCTTGC-3′) and 654r (5′-GCCTGCCCCTGAACTATC-3′) for F. succinogenes S85 (Tajima et al., 2001) and Sele.rumi_Fw (5′-TGCTAATACCGAATGTTG-3′) and Sele.rumi_Rv (5′-TCCTGCACTCAAGAAAGA-3′) for S. ruminantium S137 (Tajima et al., 2001). All other quantification procedures, including the standard plasmids, PCR conditions, and calculations, were according to Koike et al. (2007, 2010).

Enzyme assays

To measure the fibrolytic activity in culture, fibrolytic enzyme assays were carried out for extracellular and intracellular fractions. Culture supernatant and bacterial cells from strains R-25 and F. succinogenes S85 monocultures and their coculture were separated by centrifugation (16 000 g, 4 °C, 10 min). The supernatant was placed in dialysis tubing (12 000- to 14 000-Da cut-off, Seamless Cellulose Tubing, Sanko-junyaku, Tokyo, Japan) in potassium phosphate buffer (50 mM, pH 6.8) overnight. The dialyzed fraction was condensed with polyethylene glycol (MW 20,000) and used in extracellular enzyme assays. Cell-free extract was obtained by ultrasonic disruption of the cell pellet (10 × 1 min on ice, 20 kHz, 25 watts) using a VC-70 Ultrasonic Processor (Sonics and Materials, Newton, CT) followed by centrifugation (16 000 g, 4 °C, 20 min) and was used in intracellular enzyme assay.

The carboxymethylcellulase (CMCase) and xylanase activities were determined by monitoring the increase in reducing sugar formation from the substrates using dinitrosalicylic acid reagents, as described by Cotta (1988). Carboxymethylcellulose and oat spelt xylan were dissolved in 50 mM potassium phosphate buffer (pH 6.8) at 1% (w/v) and used as the substrates. The protein concentration was determined using Bio-Rad Protein Assay kit (Bio-Rad, Hercules, CA) with bovine gamma globulin as a standard. Enzyme activity was expressed as specific activity (formation of 1 nmol of sugar min−1 mg of protein−1) or total activity mL−1 culture (formation of 1 nmol of sugar min−1 mL−1 of original culture).

Utilization of fiber digestion products by strain R-25

To confirm the utilization of fiber digestion product by strain R-25, the following experiment was carried out. Fibrobacter succinogenes S85 was incubated in medium containing rice straw as the sole carbon source for 48 h and centrifuged (2300 g, 4 °C, 10 min), and the supernatant was filtered through a sterile filter (0.22 μm; Millipore, Billerica, MA) in the anaerobic chamber (Coy, Grass Lake, MI) maintained at the atmosphere of 95% CO2 and 5% H2. A cell suspension of strain R-25 with OD660 nm = 0.2 was inoculated to the obtained culture supernatant of F. succinogenes S85 and grown to mid-log phase. The control (rice straw medium without inoculation of F. succinogenes S85) was processed as above. In addition, cultures of strain R-25 incubated in basal medium containing 0.5% (w/v) cellooligosaccharides (SEIKAGAKU BIOBUSINESS, Tokyo, Japan) or xylooligosaccharides (Wako, Osaka, Japan) as the sole carbon source was used for comparative study. Extracellular and intracellular enzyme assays were performed following the protocol described above.

Scanning electron microscopy observations

Rice straw particles in the monoculture and coculture were sampled at 48 h. The samples were washed three times with 50 mM potassium phosphate buffer (pH 6.8) and fixed with 3% glutaraldehyde in the same buffer for 1 h at room temperature. After fixation, the samples were washed four times with 50 mM potassium phosphate buffer and postfixed for 30 min in 1% osmic acid (OsO4) in the same buffer. After washing four times, the samples were dehydrated by graded ethanol solution series [50, 70, 90, 99.5% (v/v), 10 min at each concentration] and exposed to isoamyl acetate for 20 min twice. Isoamyl acetate was removed with a critical point dryer using liquid CO2 (HCP-2; Hitachi, Tokyo, Japan) in eight 15-min treatments. The samples were coated with gold in an ion sputter (E101; Hitachi) and observed in a JSM-6301 low vacuum scanning electron microscope (JEOL, Tokyo, Japan) at an accelerating voltage of 5 kV.

Statistical analysis

The means of DM digestion, metabolites, 16S rRNA gene copy number, and enzyme activity for each treatment were analyzed by one-way analysis of variance of spss ver. 16.0 J (IBM, Tokyo, Japan). < 0.05 was regarded as significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Two-member coculture experiment

DM digestion of rice straw by F. succinogenes S85 was 32.8%, while strain R-25 did not digest rice straw (Table 1). DM digestion with coculture of strains R-25 and F. succinogenes S85 was 1.13-fold higher (< 0.05) than that of monoculture of F. succinogenes S85 (36.9% and 32.8%, respectively). The extracellular CMCase and xylanase activities in monoculture of strain R-25 or F. succinogenes S85 and their coculture are shown in Table 1. For both CMCase and xylanase, the activities in coculture were higher than those of the F. succinogenes S85 monoculture (< 0.05).

Table 1. Dry matter digestibilities and extracellular enzyme activities by monocultures of strain R-25 or F. succinogenes S85 and by their coculture after 96-h incubation
StrainDM digestibility (%)Enzyme activity (nmol min−1 mg−1 protein)
CMCaseXylanase
  1. Values are shown as mean ± SD (= 3).

  2. Within a column, means followed by different letters are significantly different (< 0.05).

Strain R-25 0 5.2± 3.2c 3.9± 3.4c
F. succinogenes S8532.8 ± 1.3b55.7± 0.8b98.7± 13.1b
R-25 + S8536.9 ± 0.6a81.8± 6.2a142.8± 7.5a

Changes in 16S rRNA gene copy number for strains R-25 and F. succinogenes S85 in monoculture and in their coculture are presented in Table 2. At the beginning of incubation, the copy numbers of 16S rRNA gene for strain R-25 and F. succinogenes S85 were 8.1 × 106 mL−1 and 9.0 × 106 mL−1, respectively. After 48 h of incubation, strain R-25 increased more than ten times greater (< 0.05) in coculture with F. succinogenes S85 than in monoculture. Significantly higher growth (< 0.05) of strain R-25 in coculture was also observed at end point. Although the growth of F. succinogenes S85 in coculture with strain R-25 was lower (< 0.05) than that for F. succinogenes S85 monoculture after 48 h of incubation, higher copy number (< 0.05) was observed in coculture with strain R-25 than in monoculture after 96 h of incubation.

Table 2. Changes in 16S rRNA gene copy numbers and main metabolite production in monoculture of strain R-25 or F. succinogenes S85 and their coculture
 Incubation time (h)
04896
  1. Values are shown as mean ± SD (= 3).

  2. Statistical analyses were performed between mono- and coculture at each time point within the respective items. Within the different set of letters (ab, xy, ABC, or XYZ), means followed by different letters are significantly different.

  3. nd, not detected.

16S rRNA gene copy numbers(Log copy number mL−1 of culture)
Strain R-25
Monoculture6.89 ± 0.177.83 ± 0.05b8.09 ± 0.03b
Coculture with S856.89 ± 0.178.98 ± 0.11a8.79 ± 0.08a
F. succinogenes S85
Monoculture6.95 ± 0.028.93 ± 0.05x7.38 ± 0.04y
Coculture with R-256.95 ± 0.028.67 ± 0.03y7.83 ± 0.05x
Metabolites(μmol mL−1 of culture)
d-Lactate
R-25 monoculturend2.00 ± 0.21A2.01 ± 0.35B
S85 monoculturend0.12 ± 0.01B0.08 ± 0.13C
Coculturend1.50 ± 0.06A6.04 ± 0.63A
Succinate
R-25 monoculturend0.01 ± 0.03Z0.20 ± 0.06Y
S85 monoculturend3.81 ± 0.27Y6.62 ± 0.81X
Coculturend5.68 ± 0.88X6.56 ± 0.57X

In monoculture containing rice straw as a carbon source, strain R-25 produced d-lactate, acetate, l-lactate, and succinate, meanwhile F. succinogenes S85 released succinate, acetate, propionate, and d-lactate (Supporting information, Table S1). Among these organic acids, d-lactate and succinate were the main metabolites produced by strains R-25 and F. succinogenes S85, respectively; therefore, only d-lactate and succinate production are shown (Table 2). Lactate production in monoculture of strain R-25 was 2.0 μmol mL−1 of culture at 48 h and did not increase over the period of 48–96 h. In contrast, lactate production in coculture of strain R-25 with F. succinogenes S85 increased continuously up to 96 h. In particular, there was a marked increase from 48 to 96 h. Although succinate concentration at 96 h was similar between monoculture and coculture, the rate of production until 48 h was greater in coculture, producing significantly higher concentration at 48 h (< 0.05).

Utilization of fiber digestion product by strain R-25

Growth of strain R-25 in the supernatant of F. succinogenes S85 culture (OD660 nm = 0.10) was comparable with that in cello- or xylo-oligosaccharide medium (OD660 nm = 0.12). Intracellular and extracellular enzyme activities of strain R-25 on various media are shown in Table 3. CMCase activity of strain R-25 was lower than 1 nmol min−1 mL−1 culture, irrespective of the media and enzyme fractions. On the other hand, intracellular xylanase activity was significantly higher (< 0.05) in the supernatant of F. succinogenes S85 culture (6.8 nmol min−1 mL−1 culture) and xylooligosaccharide medium (2.7 nmol min−1 mL−1 culture). However, xylanase activity was low or negligible in the extracellular fraction.

Table 3. CMCase and xylanase activities of strain R-25 grown on the supernatant of rice straw medium, the supernatant of F. succinogenes S85 culture, cellooligosaccharide medium, and xylooligosaccharide medium
Culture substrateEnzyme activity (nmol min−1 mL−1 of original culture)
CMCaseXylanase
IntracellularExtracellularIntracellularExtracellular
  1. Values are shown as mean ± SD (= 3).

  2. Within a column, means followed by different letters are significantly different (< 0.05).

  3. nd, not detected.

Rice straw medium supernatant0.11 ± 0.470.35 ± 0.210.28 ± 0.66c0.26 ± 0.22
S85 culture supernatantnd0.19 ± 0.466.80 ± 0.42and
Cellooligosaccharide medium0.02 ± 0.060.28 ± 0.43ndnd
Xylooligosaccharide medium0.73 ± 0.720.14 ± 0.292.73 ± 0.31bnd

Three-member coculture experiment

DM digestion of rice straw and concentration of major organic acids in the culture of strain R-25, F. succinogenes S85, S. ruminantium S137, and in combination are shown in Table 4. DM digestion was significantly higher in coculture than in monoculture of F. succinogenes S85, and the highest digestion was observed in triculture (< 0.05). The major organic acids in monocultures of strains R-25, F. succinogenes S85, and S. ruminantium S137 were d-lactate, succinate, and propionate, respectively. In coculture of strains R-25 and F. succinogenes S85, succinate, d-lactate, and acetate were detected. The main acids in coculture of F. succinogenes S85 and S. ruminantium S137 were propionate and acetate. The main products in the triculture were also propionate and acetate. These acids were significantly increased in triculture, meanwhile d-lactate and succinate were lower in the triculture than in the coculture of strain R-25 and F. succinogenes S85.

Table 4. Dry matter digestibility and concentration of major organic acids in the culture of strain R-25, F. succinogenes S85, S. ruminantium S137, and their combinations after 96-h incubation
Bacterial strainDM digestibility (%)Acid production (μmol mL−1 of culture)
AcetatePropionated-LactateSuccinateSuma
  1. Values are shown as mean ± SD (= 3).

  2. Within a column, means followed by different letters are significantly different (< 0.05).

  3. a

    Sum of acetate, propionate, d-lactate, and succinate.

  4. nd, not detected.

Monocultures
R-250ndnd2.2 ± 0.5b0.2 ± 0.1b2.4 ± 0.4e
F. succinogenes S8532.4 ± 1.3c2.1 ± 0.1cndnd6.6 ± 0.8a8.7 ± 0.7c
S. ruminantium S13702.2 ± 0.1c4.2 ± 0.2cnd0.2 ± 0.2b6.6 ± 0.4d
Cocultures
R-25 + S8536.6 ± 0.8b2.9 ± 0.6cnd6.0 ± 0.6a6.6 ± 0.3a15.5 ± 0.8b
S85 + S13736.1 ± 0.5b5.5 ± 0.5b9.4 ± 0.6bnd0.3 ± 0.1b15.2 ± 0.8b
R-25 + S13701.8 ± 0.7c4.2 ± 0.4c0.3 ± 0.6cnd6.3 ± 1.5d
Triculture
R-25 + S85 + S13741.8 ± 0.8a7.3 ± 0.7a12.9 ± 0.8a0.5 ± 0.4c0.5 ± 0.1b21.2 ± 1.1a

The 16S rRNA gene copy numbers for these strains at 96 h of incubation were significantly higher (< 0.05) in triculture than in monocultures and two-member coculture (Fig. 2a). Scanning electron microscopy (SEM) observations showed that all three strains attached to rice straw in monoculture (Fig. 2b, i–iii). In the triculture, the three strains were shown to be closely located on the rice straw (Fig. 2b, iv).

image

Figure 2. Copy number of 16S rRNA gene (a) and scanning electron micrographs (b) in the mono-, coculture or triculture of strain R-25, F. succinogenes S85 and S. ruminantium S137. (a) Quantification was performed on the samples after 96-h incubation. Error bars show the standard deviation in triplicate tests. Different superscript letters indicate significant difference within the strain (< 0.05). ND, not determined. (b) Images show monoculture of strain R-25 (i), F. succinogenes S85 (ii) or S. ruminantium S137 (iii), and their triculture (iv) grown on rice straw for 48 h. Arrows indicate each bacterium (R-25, strain R-25; Fs, F. succinogenes S85; Sr, S. ruminantium S137). Magnifications, ×10 000. Bars, 1 μm.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Although the positive interaction between rumen bacteria has been reported in the previous studies, the present result is the first demonstration of synergism between the newly cultured group U2 bacterium R-25 and F. succinogenes. The extent of increase in DM digestion by coculture of strain R-25 and F. succinogenes S85 was comparable with the previous coculture studies using the combinations of F. succinogenes and several nonfibrolytic species, where DM digestion was enhanced in coculture at 1.05–1.18-fold (Dehority & Scott, 1967; Kudo et al., 1987; Osborne & Dehority, 1989; Fondevila & Dehority, 1996; Sawanon et al., 2011). Growth and fermentation patterns of F. succinogenes S85 were altered in coculture with strain R-25. Higher level of 16S rRNA gene copy number (at 96 h) and succinate production (at 48 h) of F. succinogenes S85 suggest that strain R-25 had a positive effect on fermentation activity of F. succinogenes S85. Enzyme activity in coculture of strain R-25 with F. succinogenes S85 partly supports this suggestion. Although extracellular activity of CMCase and xylanase was significantly higher in coculture of strains R-25 and F. succinogenes S85, activity of extracellular CMCase and xylanase from strain R-25 alone was almost negligible. Therefore, elevated extracellular activity of fibrolytic enzyme in the coculture is likely to be solely attributable to F. succinogenes S85. Possible explanations of this positive alteration of F. succinogenes S85 activity by strain R-25 include the consumption of oligosaccharides and hydrogen, which can accumulate in the monoculture. Previous research has shown that endoglucanase activity of F. succinogenes S85 is repressed by cellobiose (McGavin et al., 1990). Furthermore, the consumption of hydrogen by methanogenic archaea leading to increased ATP production and/or organic acid concentration of fibrolytic strains has been reported as interspecies hydrogen transfer (Latham & Wolin, 1977; Williams et al., 1994; Rychlik & May, 2000). Consumption of oligosaccharides and hydrogen to produce lactate by strain R-25 could lead to the maintenance of the fibrolytic activity of F. succinogenes S85, resulting in enhanced DM digestion in coculture.

Judging from higher growth and lactate production of strain R-25, benefit for this strain in coculture seemed to be availability of xylooligosaccharides that are released along with rice straw degradation by F. succinogenes S85. In fact, intracellular xylanase activity of strain R-25 was induced by the supernatant of F. succinogenes S85 culture and xylooligosaccharides medium. Induction of xylanolytic enzyme by xylooligosaccharides was reported on known rumen bacterium S. ruminantium and Prevotella bryantii (Cotta & Whitehead, 1998; Miyazaki et al., 2005). Fibrobacter succinogenes S85 can degrade the xylan chain of hemicellulose by its own xylanolytic enzymes (Matte & Forsberg, 1992; Matte et al., 1992). However, recent genomic study indicates that F. succinogenes S85 lacks many of the genes necessary to transport and metabolize the hydrolytic products of noncellulose polysaccharides such as xylan (Suen et al., 2011). Therefore, strain R-25 might be able to utilize xylooligosaccharides produced by F. succinogenes S85 in the coculture without competition.

Although the DM digestion was improved in coculture of strains R-25 and F. succinogenes S85, the fermentation products of these two strains accumulated. As d-lactate and succinate are rarely accumulated in the rumen, these organic acids should be removed to maintain the function of F. succinogenes S85 and strain R-25. Selenomonas ruminantium is known as a succinate-utilizing and propionate-producing bacterium in the rumen (Strobel & Russell, 1991) and is classified into two subspecies, lactate nonutilizing subsp. ruminantium and lactate utilizing subsp. lactilytica (Flint & Bisset, 1990). Our previous studies showed that S. ruminantium S137, which was a lactate–succinate-utilizing strain, enhanced fibrolytic activity of F. succinogenes (Sawanon et al., 2011) and Ruminococcus flavefaciens (Sawanon & Kobayashi, 2006). Therefore, S. ruminantium S137 was used in this study as a lactate–succinate-utilizing bacterium to determine whether this strain is helpful for metabolizing organic acids that accumulate in coculture of strains R-25 and F. succinogenes S85. Rice straw digestion and bacterial population were highest in triculture. As predicted, lactate/succinate consumption and propionate production was observed when S. ruminantium S137 was included to form a triculture. These observations strongly suggest that the consumption of d-lactate and succinate by S. ruminantium S137 could improve the growth of strains R-25 and F. succinogenes S85, resulting in increased digestion in the triculture. Other than S. ruminantium, there are many kinds of rumen bacteria that can metabolize lactate and/or succinate, such as Megasphaera elsdenii, Schwartzia succinivorans, Succiniclasticum ruminis, and Veillonella parvula. These metabolite utilizers may play a similar role to S. ruminantium S137 in ruminal fiber digestion. Although rice straw digestion was not observed in mono- and coculture of strain R-25 and S. ruminantium S137, metabolites were detected in these cultures. Probably, these strains utilized soluble sugars derived from rice straw for their growth in the culture without F. succinogenes S85. SEM observation indicates that three strains form aggregates on rice straw in the triculture. This result suggests the occurrence of interspecies cross-feeding and hydrogen transfer. Our research group has proposed that rumen bacterial group U2, including strains R-25, F. succinogenes, and S. ruminantium, can be a core member of the fibrolytic community in the rumen (Koike et al., 2003, 2007, 2010). The findings in this study support this proposition.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This study was supported in part by a Grant-in Aid for Scientific Research (No. 22780238 to S.K. and No. 17380157 to Y.K.) from the Japanese Ministry of Education, Culture, Sports, Science and Technology.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
fml2649-sup-0001-TableS1.docWord document35KTable S1. Metabolite production in monoculture of strain R-25 and F. succinogenes S85 after 96 h incubation in rice straw medium.

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