Improved culturability of cellulolytic rumen bacteria and phylogenetic diversity of culturable cellulolytic and xylanolytic bacteria newly isolated from the bovine rumen.

RESEARCH ARTICLE

Improved culturability of cellulolytic rumen bacteria and phylogenetic diversity of culturable cellulolytic and xylanolytic bacteria newly isolated from the bovine rumen Thet Nyonyo1, Takumi Shinkai2 & Makoto Mitsumori1,2 1

Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Japan; and 2National Institute of Livestock and Grassland Science, Tsukuba, Japan

Correspondence: Makoto Mitsumori, National Institute of Livestock and Grassland Science, Tsukuba, Ibaraki 305-0901, Japan. Tel.: +81 29 836 8660; fax: +81 29 838 8606; e-mail: [email protected] Received 31 October 2013; revised 2 March 2014; accepted 4 March 2014. DOI: 10.1111/1574-6941.12318

MICROBIOLOGY ECOLOGY

Editor: Julian Marchesi Keywords fibre digestion; cellulose-degrading bacteria; xylan-degrading bacteria; isolation; culture media.

Abstract The phylotypes of rumen bacteria have increased by the accumulation of 16S rRNA gene sequences, and they show a complex microbial community structure in the rumen. However, most of the biochemical properties of rumen bacteria defined by phylotypes are still unknown. We attempted to improve the culturability of cellulolytic bacteria from the rumen using an agar medium (CA) and a gellan gum medium (CG) containing azo-carboxymethylcellulose as a carbon source. We isolated 129 strains from these media, and the numbers of isolates that showed filter paperase, carboxymethylcellulase and xylanase activity were 51, 117 and 105, respectively. The isolates were classified into six phyla by 16S rRNA gene sequences. In accordance with other studies, fibreadherent rumen bacteria from the phylum Firmicutes were the most abundant cultured isolates obtained (82.2%). Isolates that were unclassified (< 97% similarity) totalled 19.4%, indicating that the media used in this study was successfully able to improve the culturability of rumen cellulolytic bacteria. Moreover, as the Chao1 richness of CG was higher than that of CA, we estimated that, compared with CA, CG supports the growth of a wide variety of rumen bacteria. These results demonstrate that culturable species of ruminal cellulolytic bacteria can be increased using improved culture media.

Introduction The degradation of cellulosic materials such as cellulose and hemicellulose by members of the rumen microbial community is an important process in which plant fibres are converted into short-chain fatty acids (SCFAs), which are energy sources for ruminants (Cheng et al., 1984). Therefore, cellulose- and hemicellulose-degrading bacteria and their roles in rumen fermentation have been intensively investigated using both culture-dependent and culture-independent methods (Stewart et al., 1997; Brulc et al., 2009; Koike & Kobayashi, 2009; Morrison et al., 2009). It was confirmed by both types of method that Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens are the predominant cellulolytic bacteria in the rumen (Stewart et al., 1997; Russell et al., 2009). Moreover, some rumen bacteria including Clostridium longisporum, Clostridium lochheadii, Butyrivibrio fibrosolvens, Prevotella ruminicola, Eubacterium ruminantium FEMS Microbiol Ecol && (2014) 1–10

and Eubacterium cellulosolvens are known to be fibrolytic bacterial species (Stewart et al., 1997). However, it was indicated that the majority (77%) of clones detected from the fibre-associated rumen bacterial community consisting of both cellulolytic and noncellulolytic bacteria had low similarity (< 97%) with the 16S rRNA gene sequences of known bacteria (Koike et al., 2003). Approximately one-half of the bacteria showing carboxymethylcellulase (CMCase) in the rumen are uncharacterized and/or unclassified bacteria (Kong et al., 2012), and thus, many uncultured/unknown cellulolytic bacteria are presumed to be involved in cellulose degradation in the rumen. Additionally, most of the biochemical characteristics of phylotypes detected from the rumen culture-independent methods are fully defined by pure-culture studies (Koike et al., 2010). The isolation of previously uncultured rumen bacteria has been attempted by modifying culture methods, and these modifications have enabled the cultivation of many ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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novel rumen bacteria (Kenters et al., 2011; Nyonyo et al., 2013). However, the sugar availability of bacterial strains cultured in those studies was not examined, and the roles of these strains in rumen fermentation were unknown. Thus, the main aim of the present study was to isolate cellulolytic bacteria with new culture media devised in our previous study (Nyonyo et al., 2013). As a soluble substrate within the media, we used azo-carboxymethylcellulose (azo-CMC). Azo-CMC is derived from cellulose and as an azo-dye can be used for detecting endoglucanase activity associated with bacteria isolated from the rumen (Ko et al., 2011; Seo et al., 2013). Moreover, the ability of isolates to degrade insoluble cellulose, CMC and xylan was tested using filter paper, azo-CMC and azoxylan. These substrates have been used previously for measuring xylanase activity in feed (Cosson et al., 1999) and in ruminal microorganisms (Wallace et al., 2001). We also characterized the diversity and phylogeny of the bacteria using 16S rRNA gene sequence analysis.

Materials and methods Media preparation

We used an agar-modified basal medium (A-MBM) and a gelrite-modified basal medium (G-MBM) described in our previous work (Nyonyo et al., 2013) to isolate cellulolytic bacteria from the rumen, with some modifications. Briefly, to prepare clarified rumen fluid (Bryant & Robinson, 1961), rumen contents collected from ruminally fistulated cows were sterilized by autoclaving at 121 °C for 20 min, then squeezed through two layers of cheese cloth and clarified by centrifugation at 30 000 g for 15 min. The modified basal medium (MBM) was formulated with 0.9 g L1 Na Cl, 0.9 g L1 (NH4)2SO4, 0.16 g L1 MnCl2, 0.066 g L1 CaCl2, 0.1 g L1 MgSO4, 0.02 g L1 FeSO4, 0.02 g L1 ZnSO4, 0.002 g L1 CoCl2, 0.001 g L1 resazurin, 2 g L1 casamino acid, 0.0005 g L1 hemin, 1 g L1 MgCl2, 0.5 g L1 cellobiose, 2 g L1 azo-CMC (Megazyme International Co., Wicklow, Ireland), 4 g L1 Na2CO3, 0.5 g L1 L-cysteine∙hydrochloride and 400 mL L1 clarified rumen fluid, 66.7 mL L1 volatile fatty acid (VFA) complex solution, which was composed of 20 mL L1 acetic acid, 1 mL L1 iso-butyric acid, 1.2 mL L1 each of isovaleric acid, valeric acid, iso-valeric acid and 2-methylbutyric acid and 10 mL L1 vitamin complex solution, which was composed of 0.2 g L1 each of pyridoxal hydrochloride, riboflavin, thiamine hydrochloride, nicotinamide, Dpantothenic acid calcium, 0.01 g L1 P-amino benzonic acid, 0.005 g L1 each of folic acid and D-biotin, and 0.0005 g L1 cyanocobalamin. The MBM was mixed with 12 g L1 agar (01162-15; Nacalai Tesque, Kyoto, Japan) or 2.5 g L1 Gelrite (G1910; Sigma-Aldrich, St. Louis, MO) ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

T. Nyonyo et al.

for the preparation of A-MBM or G-MBM, respectively. For subcultures, the slant media were prepared with the same compositions, except that 0.2% CMC (C4888; SigmaAldrich) was used instead of azo-CMC. Animals and sampling

The use of animals in this study was according to the guidelines of the Animal Care Committee of the National Institute of Livestock and Grassland Science (NILGS), Tsukuba, Japan. Three fistulated dry Holstein cows (average body weight, 663 kg) kept at the NILGS were used. The cows were fed a diet consisting of Timothy hay, steam-rolled corn grain and soybean meal (67%, 19% and 14% dry matter, respectively) twice a day (3.6 kg dry matter per feed). Rumen contents collected from the three cows through the rumen fistula before a morning feeding were separated into the liquid and solid phases by squeezing the contents through two layers of cheesecloth. The liquid (600 mL: mixture consisting of 200 mL each) and solid (150 g: mixture consisting of 50 g each) phases were combined and blended in a Waring blender for 30 s under a stream of O2-free CO2 to make blended rumen content. Cultivation of rumen bacteria

Inocula were prepared by serial 10-fold dilutions of the blended rumen content with anaerobic dilution fluid (Bryant & Burkey, 1953) as described previously (Nyonyo et al., 2013). A tube containing 10 mL of azoCMC A-MBM (CA) or azo-CMC G-MBM (CG) preheated at 60 °C was inoculated with 0.2 mL anaerobic dilution fluid and was rolled in an ice bath to make a roll tube. The roll tubes were then incubated at 38 °C. For subcultures, colonies were randomly picked at 3, 8 and 9 days of incubation and transferred into a slant medium of CA or CG containing preadded liquid medium (0.2 mL), which had the same composition as the slant medium except that no agar or no gelrite was included. The tubes were subcultured at 38 °C for 1– 2 days. Degradation of filter paper, CMC and xylan

For the detection of the filter paperase (FPase) activity of each isolate, we transferred an isolate from a subculture into a 12 9 100 mm glass test tube containing MBM (3 mL) with a piece of filter paper (FP, 27 mg, No. 5A; Tokyo Roshi, Kaisha, Tokyo). For the detection of CMCase and xylanase activities, the isolate was stab-inoculated into a 12 9 100 mm glass test tube containing slant medium (3 mL), which was composed of MBM FEMS Microbiol Ecol && (2014) 1–10

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Isolation of novel cellulolytic bacteria from the rumen

without sugars but contained 0.2% azo-CMC or 0.2% azo-xylan and was solidified with agar or gelrite. The tubes were incubated at 38 °C. The degrees of filter paper degradation were classified into four levels by the change of fitter paper on day 14 after incubation, that is, level 0, no change (no degradation); level 1, disturbance on the surface or fraying of the filter paper; level 2, deformed filter paper or open holes on the filter paper; level 3, collapse of the filter paper. The degradation of CMC and xylan by each isolate was estimated by the colour change of the azo (Remazol Brilliant Blue) dye in the media. Namely, transmitted light images of slant media on a white background were digitally taken on days 0, 3, 7 and 14 after incubation. We analysed the colour images using IMAGEJ software (version 1.46r; National Institutes of Health, Bethesda, MD) according to its user guide (http://rsbweb.nih.gov/ij/docs/ user-guide.pdf) and the protocol described by Hartig (2013). To obtain brightness values, each captured image, a red-green-blue (RGB) image, was treated with the auto brightness/contrast command and then split into three greyscale image layers, that is, greyscale images of the red, green and blue components of the original using the RGB split command. The blue image layer was used for further measurement. From the images of a test tube, the brightest field (B), the darkest field (D) and a medium region (M), that is, B, a region without medium on a test tube (background); D, a part of a butyl-rubber cap; M, a part of the butt, were selected. Three areas of each target region (B, D or M) were sampled. The average of brightness was calculated using the brightness values of each target region to obtain the averages of B, D and M (B-ave, D-ave and M-ave). The proportion of the brightness of M-ave to that of B-ave, that is, M-bri, was calculated by the following equation: M-bri = (M-ave – Dave)/(B-ave – D-ave) 9 100 (%). The degradation values of CMC (DEGcmc) and xylan (DEGxyl) were estimated as: DEGcmc (%) = (1 M-bri of day 14/M-bri of day 0) 9 100 and DEGxyl (%) = (1 M-bri of day 7/M-bri of day 0) 9 100, respectively. PCR amplification of 16S rRNA genes and sequencing

The culture fluid (0.1 mL) from each subculture was collected and centrifuged at 12 000 g for 10 min. After removal of the supernatant, the cell pellet was resuspended in distilled water and heated at 100 °C for 5 min, followed by centrifugation at 12 000 g for 10 min (Moore et al., 2008). The supernatant (50 lL) was collected and used as a template for polymerase chain reaction (PCR). FEMS Microbiol Ecol && (2014) 1–10

The 16S rRNA genes were amplified by PCR using the forward primer 27F (50 -GAGTTTGATCMTGGCTCAG-30 ) and the reverse primer 1492r (50 -GGYTACCTTGTTACGACTT-30 ; Lane, 1991). The PCR was performed at a volume of 50 lL, which contained 0.25 lL of ExTaqs polymerase (TaKaRa Bio Inc., Japan), 5 lL of PCR buffer, 1 lL of each primer (10 pmol lL1), 4 lL of dNTPs mix (2.5 mM each, TaKaRa Bio Inc.) and 1 lL template DNA. The PCR program consisted of 2 min at 94 °C followed by 35 cycles of 35 s at 94 °C, 45 s at 55 °C and 2 min at 72 °C with a final 2 min extension at 72 °C. PCR was conducted with Platinum Taq (Invitrogen, Carlsbad, CA) and an iCycler thermal cycler (Bio-Rad, Hercules, CA). The amplified 16S rRNA gene products were sequenced using the primers 16SIMP-F (50 -CTACCAGGGTATCTAATCCTG-30 ), 16SIMP-R(50 -CAGGATT AGATACCCTGGTAG-30 ; Delvasto et al., 2006), 704F (50 GTAGCGGTGAAATGCGTAGA-30 ), 357F (50 -CTCCT ACGGGAGGCAGCAG-30 ), 787R (50 -CTACCAGGGTATCTAAT-30 ), 1492r (50 -GGYTACCTTGTTACGACTT-30 ), 704R (50 -TCTACGCATTTCACCGCTAC-30 ; Lane, 1991) and 357R (50 -CTGCTGCCTCCCGTA-30 ; Stackebrandt & Charfreitag, 1990). Sequencing was performed by FASMAC Co. (Atsugi, Japan). Sequence analysis

We identified putative chimeric sequences using a decipher chimera detection tool (http://decipher.cee.wisc.edu/ FindChimeras.html). The remaining sequences were compared with database sequences using the NCBI BLAST program (Altschul et al., 1990) and classified by the Ribosomal Database Project (RDP) sequence match tool (http://rdp.cme.msu.edu/; Cole et al., 2009, 2014). The operational taxonomic units (OTU), based on sequence similarity of at least 97%, the Chao1 richness and the Shannon–Wiener index were calculated using the FASTGROUP II Web-based bioinformatics platform (http://fast group.sdsu.edu/fg_tools.htm). All sequences were aligned with MEGA5 version 5.2.2 (Tamura et al., 2011). The phylogenetic tree was constructed by the neighbour-joining method (Saitou & Nei, 1987) using CLUSTALX version 2.0.11 (Larkin et al., 2007) and was drawn by MEGA5.

Results Identification and diversity indices of isolates

We classified the phylotypes of the 129 isolates showing FPase, CMCase or xylanase in this study into six phyla: Firmicutes, Proteobacteria, Spirochaetes, Bacteroidetes, Fibrobacteres and Actinobacteria, and into known and unclassified genera (Table 1). The unclassified taxa at the ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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T. Nyonyo et al.

genus level accounted for 19.4% of the 129 isolates (Table 1). Pseudobutyrivibrio was the most abundant genus in the total isolates and the isolates from the medium CA, whereas unclassified Lachnospiraceae was the most abundant in the isolates from CG (Table 1). The numbers of Streptococcus were significantly different (P < 0.01) between CA and CG (Table 1). The isolates were divided into 66 OTUs (Table 2), and those 16S rRNA gene sequences showed 83.2–100% similarity to valid taxon (Table 3). The Chao1 index and the Shannon–Wiener diversity index of the CG isolates were higher than those of the CA isolates (Table 2). The 16S rRNA gene sequences of the isolates obtained in this

study were deposited in the DDBJ nucleotide sequence databases under accession numbers AB849327–AB849455. FPase, CMCase and xylanase activities of isolates

The ability of the isolates to degrade FP, CMC and xylan is illustrated in Fig. 1 and Supporting Information, Fig. S1. The number of isolates with FPase activities (level of FP degradation > 1) was 51 (39.5% of the total; Fig. 2). Isolates with middle FPase activity (level of FP degradation = 2) were found in the genera Ruminococcus, Fibrobacter, Pseudobutyrivibrio, unclassified Lachnospira-

Table 1. Phylotype of isolates Phylum

Order, family or genus

Firmicutes

Pseudobutyrivibrio

34 (26.4)

U-Lachnospiraceae

22 (17.1)

Butyrivibrio

22 (17.1)

Streptococcus

12 (9.3)

Enterococcus

1 (0.8)

Anaerovibrio

1 (0.8)

Selenomonas

5 (3.9)

U-Clostridiales

1 (0.8)

Saccharofermentans

2 (1.6)

Ruminococcus

5 (3.9)

Clostridium cluster IV

1 (0.8)

Proteobacteria

Succinivibrio

5 (3.9)

Spirochaetes

Treponema

2 (1.6)

Bacteroidetes

U-Bacteroidales

2 (1.6)

Prevotella

Total isolates (% of isolates)

10 (7.8)

Fibrobacteres

Fibrobacter

3 (2.3)

Actinobacteria

Actinomyces

1 (0.8)

Total (all taxa)

129 (100) U-taxa

25 (19.4)

Medium

No. of isolates (% of isolates)*

CA CG CA CG CA CG CA CG CA CG CA CG CA CG CA CG CA CG CA CG CA CG CA CG CA CG CA CG CA CG CA CG CA CG CA CG CA CG

19 15 6 16 15 7 0 12 0 1 1 0 1 4 1 0 1 1 5 0 0 1 3 2 1 1 2 0 7 3 2 1 0 1 64 65 9 17

(29.7) (23.1) (9.4) (24.6) (23.4) (10.8) (0.0)† (18.5)† (0.0) (1.5) (1.6) (0.0) (1.6) (6.2) (1.6) (0.0) (1.6) (1.5) (7.8) (0.0) (0.0) (1.5) (4.7) (3.1) (1.6) (1.5) (3.1) (0.0) (10.9) (4.6) (3.1) (1.5) (0.0) (1.5) (100) (100) (14.1) (26.2)

*% of isolates in each medium is shown. †Significantly different at 0.01 by RDP analysis. U-, unclassified.

ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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Table 2. Diversity indices of isolates Media

Isolates

OTUs

Chad

H’

CA CG Total

64 65 129

34 40 66

122.2 221.5 316.0

3.24 3.31 3.70

H’ means the Shannon–Wiener diversity index.

ceae, Butyrivibrio, Streptococcus, Enterococcus, Saccharofermentans and Prevotella (Figs 1 and S1). Isolates showing high FPase activity (level of FP degradation = 3) were assigned to only Ruminococcus. The number of isolates showing the ability to degrade two or three substrates was 110 (85.3% of the total; Fig. 2).

Discussion Fibre-degrading bacteria such as cellulose-degrading bacteria and hemicellulose-degrading bacteria are thought to be pivotal in the maintenance of rumen function, because they can convert plant fibre, major components of plant biomass, to low-molecular-weight carbohydrates and SCFAs, which are used by other rumen bacteria and ruminants as energy sources (Flint et al., 2008). Fibredegrading bacteria have been identified mostly by culturedependent methods, because their fibre-degrading ability can be detected by pure cultures with cellulosic materials. However, culture-independent methods have detected many fibre-degrading enzymes produced by known/ unknown microorganisms and have indicated that fibreadherent rumen microorganisms, which are thought to be composed of a combination of fibre-degrading bacteria and related bacteria, have a complex microbial ecosystem (Flint et al., 2008; Toyoda et al., 2009). In the present study, new media were devised based on the media described in our previous study (Nyonyo et al., 2013) using azo-CMC as a substrate and an indicator for detecting cellulolytic bacteria to improve the culturability of cellulolytic rumen bacteria. Additionally, FPase, CMCase or xylanase activities of the isolates were measured by the medium containing filter paper, azo-CMC or azo-xylan, respectively, to define these enzymatic activities promptly. The media enabled the collection of 129 isolates that consisted of 66 OTUs and were classified into six phyla and into known and unclassified genera. The fibre-adherent microbial fraction is thought to be responsible for much of the degradation of plant fibre, and earlier studies have demonstrated that Firmicutes was the dominant phylum associated with the plant-adherent fraction by metagenomic or 16S rRNA gene sequence analysis (Koike et al., 2003; Larue et al., 2005; Brulc et al., 2009). As the number of members of Firmicutes in the present study was 82.2% of the total (106 isolates), FirmiFEMS Microbiol Ecol && (2014) 1–10

cutes was the most dominant of this culturable rumen microbiota, similar to other fibre-adherent microbiota. The media used in this study successfully improved the culturability of rumen cellulolytic bacteria; isolates with < 97% 16S rRNA gene sequence similarity to known taxa (Table 1) represented 19.4% of the total isolates obtained and branched separately from other groups, suggesting that these unassigned isolates represent novel species. Among the 129 isolates, the 16S rRNA gene sequence of only eight shared > 97% similarity to Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens, which are considered the major cellulolytic species in the rumen (Koike & Kobayashi, 2001; Krause et al., 2003; Russell et al., 2009). Moreover, one strain of Clostridium cluster IV, which includes Ruminococcus albus and Ruminococcus flavefaciens (Krause et al., 1999), was isolated and showed FPase activity. Thus, all of Clostridium cluster IV isolates cultured in the present study were FP-degrading bacteria. Butyrivibrio and Pseudobutyrivibrio, which were created by the reclassification of Butyrivibrio (Kopecny et al., 2001), are recognized as the major xylanolytic bacteria in the rumen (Krause et al., 2003). Some Butyrivibrio strains from the rumen were reported to possess FPase activity (Bryant & Small, 1956; Das & Qin, 2012). In this study, most of the isolates of Butyrivibrio and Pseudobutyrivibrio also degraded xylan and CMC; isolates of showing CMCase and xylanase activity accounted for 95.5% and 90.9% of the total isolates of Butyrivibrio, respectively, and 91.2% and 94.1% of those of Pseudobutyrivibrio, respectively. Regarding FPase, nine strains (40.9%) of Butyrivibrio isolates and six strains (17.6%) of Pseudobutyrivibrio isolates showed FPase activity. Moreover, 86.4% and 86.4% of the total isolates of unclassified Lachnospiraceae, which are located close to clades of Butyrivibrio and Pseudobutyrivibrio (Table 1), showed xylanase and CMCase activities, respectively. The isolates of Butyrivibrio, Pseudobutyrivibrio and unclassified Lachnospiraceae cultured in this study therefore presumably functioned as xylan- and solubilized polysaccharide-degrading bacteria, which are closely associated with cellulose-degrading bacteria (Flint et al., 2008). Twelve isolates of Streptococcus, thought to be members of the Streptococcus bovis/Streptococcus equinus complex (SBSEC; Jans et al., 2012), showed xylanase and CMCase activity except for two isolates. The finding that strains of Streptococcus were cultured by only CG suggests that CG should be more selective for growth of Streptococcus compared with CA. Several members of the SBSEC are known to be cellulolytic bacteria in the foregut of the dromedary camel (Samsudin et al., 2012) and in the cattle rumen (Das & Qin, 2012). The phylogenetic analysis from this study identified an Enterococcus isolate obtained from CG media, with significant FPase activity that grouped with ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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Table 3. Bacterial 16S rRNA gene sequences from isolates cultured from two different media Phylogenetic classification† Representatives of clades*

Phylum, order or family

Genus

Nearest valid taxon (GenBank accession)

Similarity (%)‡

CA37 CA16 CG33 CG64 CG63 CA29 CG29 CG30 CA38 CG53 CA34 CG58 CG54 CG55 CA60 CG22 CG2 CA63 CA19 CA58 CA4 CA43 CG18 CA9 CA75 CA23 CG37 CG26 CA71 CG48 CA20 CG50 CG47 CG51 CG10 CA72 CA84 CG86 CG36 CG74 CA24 CA7 CA22 CA10 CG7 CA81 CG46 CA45 CA17 CG11 CA25

Lachnospiraceae Lachnospiraceae Lachnospiraceae Lachnospiraceae Lachnospiraceae Lachnospiraceae Lachnospiraceae Lachnospiraceae Lachnospiraceae Lachnospiraceae Lachnospiraceae U-Lachnospiraceae U-Lachnospiraceae U-Lachnospiraceae U-Lachnospiraceae U-Lachnospiraceae U-Lachnospiraceae U-Lachnospiraceae U-Lachnospiraceae U-Lachnospiraceae U-Lachnospiraceae U-Lachnospiraceae U-Lachnospiraceae Lachnospiraceae Lachnospiraceae Lachnospiraceae Lachnospiraceae Lachnospiraceae Lachnospiraceae Lachnospiraceae Lachnospiraceae Streptococcaceae Streptococcaceae Streptococcaceae Enterococcaceae Selenomonadales Selenomonadales Selenomonadales Selenomonadales Selenomonadales Ruminococcaceae U-Clostridiales Ruminococcaceae Ruminococcaceae Ruminococcaceae Proteobacteria Spirochaetes U-Bacteroidales Bacteroidales Bacteroidales Bacteroidales

Pseudobutyrivibrio Pseudobutyrivibrio Pseudobutyrivibrio Pseudobutyrivibrio Pseudobutyrivibrio Pseudobutyrivibrio Pseudobutyrivibrio Pseudobutyrivibrio Pseudobutyrivibrio Pseudobutyrivibrio Pseudobutyrivibrio NA NA NA NA NA NA NA NA NA NA NA NA Butyrivibrio Butyrivibrio Butyrivibrio Butyrivibrio Butyrivibrio Butyrivibrio Butyrivibrio Butyrivibrio Streptococcus Streptococcus Streptococcus Enterococcus Anaerovibrio Selenomonas Selenomonas Selenomonas Selenomonas Saccharofermentans NA Ruminococcus Ruminococcus Clostridium cluster IV Succinivibrio Treponema NA Prevotella Prevotella Prevotella

Pseudobutyrivibrio ruminis Ce4 (AY178843) Pseudobutyrivibrio ruminis Ce1 (AY178841) Strain NCDO 2397 (X89979) Pseudobutyrivibrio ruminis pC-XS7 (AF202262) Pseudobutyrivibrio ruminis 153 (JN619348) Pseudobutyrivibrio ruminis pC-XS7 (AF202262) Pseudobutyrivibrio ruminis pC-XS7 (AF202262) Pseudobutyrivibrio ruminis pC-XS7 (AF202262) Pseudobutyrivibrio ruminis INIIa3 (JN662465) Pseudobutyrivibrio ruminis pC-XS7 (AF202262) Butyrivibrio fibrisolvens AR27 (BFU77337) Lachnobacterium sp. wal 14165 (AJ518873) Butyrivibrio sp. 3 (EU714406) Roseburia faecis M6/1 (AY804149) Strain WG-1 (HQ404371) Eubacterium rectale M104/1 (FP929043) Pseudobutyrivibrio ruminis INIIa3 (JN662465) Roseburia faecis JCM17581 (AB661433) Eubacterium ruminantium GA195 (AB008552) Eubacterium ruminantium GA195 (AB008552) Eubacterium ruminantium GA195 (AB008552) Butyrivibrio hungatei Su6 (AY178635) Butyrivibrio hungatei Su6 (AY178635) Butyrivibrio fibrisolvens OB251 (BFU77341) Butyrivibrio hungatei Su6 (AY178635) Butyrivibrio fibrisolvens Bu 43 (X89976) Butyrivibrio fibrisolvens M55 (AY699273) Butyrivibrio fibrisolvens INIIa16 (KC904269) Butyrivibrio fibrisolvens NCDO 2221 (X89970) Butyrivibrio fibrisolvens NCDO 2221 (X89970) Butyrivibrio hungatei NK4A153 (GU324403) Streptococcus equinus W1 (AB563225) Streptococcus lutetiensis 033 (CP003025) Streptococcus bovis B315 (AF396920) Enterococcus asini NBRC 100681 (AB681224) Anaerovibrio lipolyticus DSM 3074 (AB034191) Selenomonas ruminantium TAM6421 (NR_075026) Selenomonas sp. MCB2 (EF195237) Selenomonas ruminantium TAM6421 (NR_075026) Selenomonas ruminantium S109 (AB198432) Clostridium alkalicellum Z-7026 (AY959944) Dehalobacter restrictus TEA (Y10164) Ruminococcus flavefaciens 17 (AM748742) Ruminococcus albus KF1 (AY445596) Ruminococcus bromii YE282 (DQ882649) Succinivibrio dextrinosolvens 0554 (Y17600) Treponema bryantii NK4A124 (GU324416) Tannerella forsythia HG3 (AB053941) Prevotella brevis AC15-1 (AB501175) Prevotella ruminicola BP1-162 (AB501167) Prevotella ruminicola BP5-11 (AB501168)

98.3 94.0 91.8 97.0 95.1 100.0 98.1 98.3 96.0 96.6 96.9 94.9 94.3 92.4 99.5 96.4 91.5 93.1 91.4 91.8 94.3 90.6 90.0 98.4 99.9 97.5 99.9 98.1 99.8 99.1 99.7 100.0 99.7 95.8 99.0 98.0 97.7 96.8 98.3 99.0 89.3 87.6 97.8 98.5 94.5 99.8 97.0 83.2 93.8 96.8 97.9

(continued)



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Table 3. Continued Phylogenetic classification† Representatives of clades*

Phylum, order or family

Genus

Nearest valid taxon (GenBank accession)

CA40 CA8 CG73 CG67

Bacteroidales Bacteroidales Fibrobacteres Actinobacteria

Prevotella Prevotella Fibrobacter Actinomyces

Prevotella ruminicola AC5-13 (AB501173) Prevotella ruminicola 23 (CP002006) Fibrobacter succinogenes S85 (CP001792) Actinomyces nasicola R2014 (AJ508455)

Similarity (%)‡ 96.5 97.1 98.5 99.0

*

Representatives were selected from each clade shown in Fig. 1. Taxonomic affiliations were determined using the RDP Classifier with an 80% bootstrap cut-off. ‡ Sequence similarity between a representative of OTU and its nearest valid taxon was calculated. U-, unclassified ; NA, not applicable. †

Enterococcus faecalis. It was reported that cellulolytic bacterial strains assigned to Enterococcus species were isolated from the human colon (Robert & Bernalier-Donadille, 2003). Although Anaerovibrio lipolyticus is known to be a lipolytic bacterium in the rumen (Prins et al., 1975), the Anaerovibrio isolate CA72 showed CMCase and xylanase activities. Because another Anaerovibrio strain isolated in our previous study (Nyonyo et al., 2013) also showed both enzymatic activities (data not shown), further study is needed to re-evaluate the substrate degradability of Anaerovibrio species in the rumen. Because the Selenomonas isolates had relatively high CMC activity (48– 100% degradation of CMC), those isolates would be involved in fibre digestion as reported previously (Koike et al., 2003; Sawanon et al., 2011). Two isolates of Saccharofermentans, which is a phylogenetic relative of some Clostridium species (Chen et al., 2010), were cultured and one of them had intermediate-level cellulose-degrading ability, whereas the other showed no cellulose degradation. Five Succinovibrio isolates, closely related to Succinovibrio dextrinosolvens, were obtained in the present study. A cellulolytic Succinovibrio strain, presumed to be Succinovibrio dextrinosolvens, was also isolated from the foregut of dromedary camel (Samsudin et al., 2012). Treponema-related sequences similar to Treponema bryantii were detected as a member of a fibre-associated community in the sheep rumen (Koike et al., 2003). The present study identified two Treponema isolates possessing low-cellulose degradation ability from the rumen. A wide variety of Prevotella species showing CMCase and xylanase activities have been isolated from the rumen (Avgustin et al., 1997; Stewart et al., 1997). Moreover, Prevotella ruminicola has cellulase genes on its genome (Purushe et al., 2010), and cellulolytic Prevotella strains were isolated from the dromedary camel (Samsudin et al., 2012). In the present study, 10 Prevotella strains showing CMCase activity were isolated, and 60% of them degraded FP. One Actinomyces isolate with high sequence similarity (99.9%) to Actinomyces nasicola, which FEMS Microbiol Ecol && (2014) 1–10

possesses beta-glucosidase (Hall et al., 2003), showed low FPase activity. Twenty-five strains affiliated with unclassified Lachnospiraceae, Clostridiales and Bacteroidales and having varying levels of cellulolytic activity could not be identified to the species level. While the contribution of these unclassified isolates to cellulose degradation in the rumen is still unclear, further studies to understand their metabolic characteristics and potential role in plant fibre breakdown are required. No significant difference in the degree of substrate degradation or in the Shannon–Wiener diversity indexes was observed between the isolates obtained with CA and CG. As the Chao1 of CG was higher than that of CA, CG has probably supported the growth of a wide range of rumen bacteria compared with CA. Indeed, Streptococcus strains were only isolated by CG. The 51 isolates (40.0% of the total) showed FPase activity. This result was presumably caused by the use of CMC, a soluble cellulose derivative, as a substrate in the media. Because many unclassified strains and strains possessing 16S rRNA gene sequences with low similarity (< 97%) to a valid taxon were isolated, it appears that the media used in this study improved the culturability of cellulolytic rumen bacteria. Kong et al. (2012) reported that CMC-degrading bacteria contributed between 8.2% and 10.1% of the total bacterial cell numbers in the rumen, and populations of the major cellulolytic bacteria species, R. flavefaciens, R. albus and F. succinogenes, constituted 44.5–53.1% of the total CMC-degrading bacteria. Findings from this study suggest that bacteria with some CMC-degrading activity consist not only the major cellulolytic bacteria species but also other bacterial species affiliated with the Phyla Firmicutes, Proteobacteria, Spirochaetes, Bacteroidetes and Actinobacteria. In conclusion, the present study highlights the bacterial diversity of culturable cellulolytic bacteria in the bovine rumen and rapidly isolated a wide range of cellulolytic rumen bacteria, including unclassified species, using the media developed in this study. These results provide a better understanding of the mechanisms of cellulose ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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T. Nyonyo et al.

Fig. 1. Simplified neighbour-joining phylogenetic tree of 16S rRNA gene sequences derived from the isolates obtained in this study shown in boldface, and those of reference sequences with GenBank accession numbers provided in parentheses. Aquifex pyrophilus is used as an outgroup sequence. The frequency with which nodes were recovered in a bootstrap analysis is indicated by symbols: □, at nodes recovered in ≥ 85% of trees generated from bootstrapped data sets; ■, at nodes recovered in ≥ 99% of trees. Nodes recovered in < 85% of trees have no symbol. The labels on the righthand side of the figure indicate the representing family or genus. The number of isolates, medium (media) that supported their growth and average enzymatic activities of isolates were shown outside of triangles. The horizontal bar represents nucleotide substitutions per sequence position. F, Filter paperase; C, CMCase; X, xylanase.




FPase 2 (1.6%)

10 (7.8%)

5 (3.9%) 34 (26.4%)

12 (9.3%)

61 (47.3%)

CMCase

5 (3.9%) Xylanase

Fig. 2. Venn diagram indicating the shared isolates’ numbers among the groups showing FPase, CMCase and Xylanase activities. Isolates showing FPase activity (the level of FP degradation > 1), CMCase activity (the degradation value > 0%) or xylanase activity (the degradation value > 0%) were counted.

degradation in the rumen for improving feed efficiency, although further studies for characterizing their biochemical properties, especially cellulase and hemicellulase activities against various insoluble substrates, and sequencing their genomes need to be performed.

Acknowledgement This work was supported by Grants-in-Aid for Scientific Research (JSPS Grant No. 24580402).

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Supporting Information Additional Supporting Information may be found in the online version of this article: Fig. S1. Neighbour-joining phylogenetic tree of 16S RNA gene sequences derived from the isolates obtained in this study shown in boldface, and those of reference sequences with GenBank accession numbers provided in parentheses.


Magnesium requirement of some of the principal rumen cellulolytic bacteria.

Effects of glycerol on the growth, adhesion, and cellulolytic activity of rumen cellulolytic bacteria and anaerobic fungi.

An isolated cellulolytic Escherichia coli from bovine rumen produces ethanol and hydrogen from corn straw.

Diversity of rumen bacteria in canadian cervids.

Effects of Methylcellulose on Cellulolytic Bacteria Attachment and Rice Straw Degradation in the In vitro Rumen Fermentation.

Ultrastructure of cell envelopes of bacteria of the bovine rumen.

Cellulolytic and proteolytic ability of bacteria isolated from gastrointestinal tract and composting of a hippopotamus.

Cellulolytic and xylanolytic enzymes from thermophilic Aspergillus terreus RWY.

Inclusion of xylan in a medium for the enumeration of total culturable rumen bacteria.

Draft Genome Sequence of Cellulolytic and Xylanolytic Cellulomonas sp. Strain B6 Isolated from Subtropical Forest Soil.

Deoxyribonuclease activity in rumen bacteria.

Recovering glycoside hydrolase genes from active tundra cellulolytic bacteria.

Interactions among cellulolytic bacteria from an anaerobic digester.

Production and assay of cellulolytic enzyme activity of Enterobacter cloacae WPL 214 isolated from bovine rumen fluid waste of Surabaya abbatoir, Indonesia.

Diversity structure of culturable bacteria isolated from the Fildes Peninsula (King George Island, Antarctica): A phylogenetic analysis perspective.

Production of alkaline phosphatase by epithelial cells and adherent bacteria of the bovine rumen and abomasum.

Alkaline phosphatase activity of rumen bacteria.

Production of cellulolytic and xylanolytic enzymes by an Arthrographis species.

Comparison of rumen bacteria distribution in original rumen digesta, rumen liquid and solid fractions in lactating Holstein cows.

Isolation and characterization of cellulolytic anaerobic fungi and associated mycoplasmas from the rumen of a steer fed a roughage diet.

Effect of fumarate reducing bacteria on in vitro rumen fermentation, methane mitigation and microbial diversity.

Degradation of maize stem by two rumen fungal species, Piromyces communis and Caecomyces communis, in pure cultures or in association with cellulolytic bacteria.

Draft Genome Sequence of Cellulolytic and Xylanolytic Paenibacillus sp. A59, Isolated from Decaying Forest Soil from Patagonia, Argentina.

Comparative growth rates of various rumen bacteria in clarified rumen fluid from cows and sheep fed different diets.