Microb Ecol (2013) 66:448–461 DOI 10.1007/s00248-013-0185-4

HOST MICROBE INTERACTIONS

Broad Diversity and Newly Cultured Bacterial Isolates from Enrichment of Pig Feces on Complex Polysaccharides

Cherie J. Ziemer

Received: 26 September 2012 /Accepted: 9 January 2013 /Published online: 29 January 2013 # Springer Science+Business Media New York (outside the USA) 2013

Abstract One of the fascinating functions of mammalian cells between 1010 and 1012 cells/mL [24, 34, 47]. This high intestinal microbiota is fermentation of plant cell wall com- bacterial density is coupled with broad diversity and a ponents. Eight-week continuous culture enrichments of pig multitude of complex interactions. A fascinating function feces with cellulose and xylan/pectin were used to isolate of the gut microbiota in mammalian herbivores and omni- bacteria from this community. A total of 575 bacterial iso- vores is fermentation of plant cell wall components by the lates were classified phylogenetically using 16S rRNA gene microbiota; mammals lack the enzymes required to break- sequencing. Six phyla were represented in the bacterial down β-(1,4) and other linkages between the monosacchar- isolates: Firmicutes (242), Bacteroidetes (185), Proteobac- ides that make up plant cell walls [23]. Without the intestinal teria (65), Fusobacteria (55), Actinobacteria (23), and Syn- microbiota the host is unable to tap into energy stored in ergistetes (5). The majority of the bacterial isolates had plants; plant biomass is the main storage of the sun's energy ≥97 % similarity to cultured bacteria with sequences in the on earth, equivalent to ~1020 J/year [35]. The plant cell wall RDP, but 179 isolates represent new species and/or genera. is made up of cellulose and hemicelluloses, the primary Within the Firmicutes isolates, most were classified in the structural components of plants, with smaller and variable families of Lachnospiraceae, Enterococcaceae, Staphylo- amounts of pectins, β-glucans, oligosaccharides, lignins, coccaceae, and Clostridiaceae I. The majority of the Bacter- and glycoproteins [25]. oidetes were most closely related to Bacteroides A number of intestinal microbial metagenomes have dem- thetaiotaomicron, Bacteroides ovatus, and B. xylanisolvens. onstrated enriched carbohydrate transport and metabolism Many of the Firmicutes and Bacteroidetes isolates were genes, including the rumen [2, 10], human [12, 17], pig identified as species that possess enzymes that ferment plant [18], and chicken [34]. Ley et al. [23] found that the type of cell wall components, and the rest likely support these diet (herbivorous, omnivorous, or carnivorous) strongly pre- bacteria. The microbial communities that arose in these dicted the fecal microbial composition across a broad sam- enrichment cultures had broad bacterial diversity. With over pling of mammals. The capacity of human and pig gut bacteria 30 % of the isolates not represented in culture, there are new to utilize plant cell wall components has been long recognized opportunities to study genomic and metabolic capacities of [32, 38]. Depending upon fiber type, age, and feeding regime, these members of the complex intestinal microbiota. between 7 and 30 % of a pig's maintenance energy require- ment was supplied by absorbed short chain fatty acid [15, 36]. While a great deal of information is currently gathered Introduction using metagenomic techniques, it is still useful to have isolated microorganisms available for study. In order to Large intestines of mammals are among the most densely determine the bacteria associated with cellulose and hemi- populated environments for microorganisms with bacterial cellulose fermentation in the large intestine of the pig, continuous culture fermentors were used to enrich for those microorganisms. The aim was to isolate a broad range of * C. J. Ziemer ( ) bacteria from these enrichments and characterize them by USDA, Agricultural Research Service, National Laboratory for Agriculture and the Environment, Ames, IA 50010, USA 16S rRNA gene phylogeny. This work allows for a large e-mail: [email protected] collection of bacterial isolates available for further study. Bacterial Isolates from Plant Cell Wall Enrichments of Pig Feces 449

Table 2 Fermentor nu- Materials and Methods a trient medium Ingredient g/L

Animals and Fermentor Operation Peptone water 2.0 Yeast extract 2.0 All animal procedures were approved by the Iowa State NaCl 0.1

University Institutional Animal Care and Utilization Com- K2HPO4 0.04 mittee (log # 3-05-5834-S). Six growing pigs (45+ kg body KH2PO4 0.04 weight) were adapted to high fiber diets (Table 1) for at least MgSO4-7H2O 0.01 6 weeks prior to fresh fecal collection. Freshly voided feces CaCl2-6H2O 0.01 (at least 500 g) were collected from individual pigs and NaHCO3 2.0 returned to the lab for processing within 30 min. Fecal slurry Cysteine–HCl 0.5 (1 in 10 w/v) was made using anaerobic phosphate buffered Bile salts 0.5 saline (PBS) and mixed for 3 min in a lab blender (Seward Heminb 0.05 Stomacher Model 400, Fisher Scientific) in an anaerobic aAll chemicals obtained mL/L chamber (Coy, Grass Lake, MI, USA). Two 300 mL inocu- from Sigma-Aldrich Tween 80 2.0 lum slurries from each fecal sample were prepared for bHemin solution: 0.5 g Vitamin K1 0.01 inoculation into fermentors, with one fermentor enriched in 25 mL 1 M NaOH with cellulose (CAS# 9004-34-6, Alphacel, ICN Biomedi- cals, Aurora, OH, USA) and the other with xylan/pectin (2:1 the fecal slurry. Each fermentor was fed 3 g of polysaccha- xylan/pectin; oat spelt xylan CAS# 9014-63-5 Sigma- ride and allowed to ferment without nutrient medium inflow Aldrich, St. Louis, MO, USA and pectin CAS# 9000-69-5, until the next day (~18 h). Fermentors were then allowed to ICN Biomedicals) fed 3 g carbohydrate twice daily at 0600 reach the working volume of 700 mL by addition of nutrient − and 1700 h. medium at a dilution rate of 0.03 h 1. The operating con- BioFlo 110 fermentors (New Brunswick Scientific, Edi- ditions of the fermentors were set to model conditions in the son, NJ, USA), with 350 mL anaerobic nutrient medium large intestine (cecum and colon) of the pig. Contents of the with no carbohydrate (Table 2) previously added, received vessels were continuously mixed and sparged with nitrogen to maintain anaerobic conditions. Working volume was maintained by removal of contents with an outflow pump. Table 1 Composition of diets fed to pigs prior to fecal collection Throughout the 8 weeks of operation, a temperature of Ingredient % of diet 38 °C and pH of 6.7 were maintained.

Corn 44.3 Bacterial Isolations Soybean meal 21.3 Distillers dried grains with solubles 20.0 After 8 weeks, 1 mL of fermentor contents was anaerobically Soybean hulls 10.0 transferred into 9 mL anaerobic 1/2 strength peptone water − Vegetable oil 1.9 (Fisher Scientific) and then serially diluted through 10 10 − − Limestone 0.8 dilution. Dilutions 10 5 through 10 10 were plated onto Dicalcium phosphate 0.7 Wilkens-Chalgren agar plates. Carbohydrate-specific agar Sodium chloride 0.4 plates [21](Table3) were replicate plated [20]; carbohydrates Vitamin mixa 0.3 used were cellulose, carboxymethyl cellulose (CAS#9000-11- Trace mineral mixb 0.3 7, Sigma-Aldrich), oat spelt xylan, beechwood xylan (CAS# Lysine 0.2 9014-63-5, Sigma-Aldrich), and pectin. Plates were incubated Calculated composition anaerobically at 38 °C for 3 to 5 days. In order to get the Metabolizable energy (kcal/kg) 3,300 greatest range of bacteria associated with cellulose and xylan/- Crude protein 20.6 pectin fermentation, the plates with the lowest dilutions with Neutral detergent fiber 20.3 distinct colonies were chosen for bacterial isolations. Acid detergent fiber 10.0 Colonies were selected based on differential colony mor- phology from the carbohydrate-specific agar plates and plated a Provided the following per kilogram of complete diet: 6,600 IU onto anaerobic Wilkens-Chalgren agar to obtain individual μ vitamin A, 1,650 IU vitamin D3, 33 IU vitamin E, 33 g vitamin isolates. Purity of bacterial cultures was determined using B12, 9.9 mg riboflavin, 49.5 mg niacin, and 26.4 mg pantothenic acid Gram stain and microscopic observations. Isolated bacteria b Provided the following per kilogram of complete diet: 17.5 mg Cu (oxide), 175 mg Fe (sulfate), 2 mg I (CaI), 60 mg Mn (oxide), 150 mg were cryopreserved using Microbank® Bacterial Preservation Zn (oxide), and 0.3 mg Se System (Pro-Lab Diagnostics, Austin, TX, USA). DNA was 450 C. J. Ziemer

Table 3 Composition of carbohydrate specific agars were included. Closest related sequences were identified Ingredienta g/L using Ribosomal Database Project (RDP) [4] 16S rRNA gene database for the nearly full-length sequences. Se- Carbohydrate 5.0 quence similarity was analyzed using BioNumerics (v. 6.5 Trypticase 4.5 software, Applied Maths, Austin, TX, USA) and an Yeast extract 0.5 unrooted dendogram was created using standard pairwise Agar (gL−1) 20.0 alignment and unweighted pair group method with arithme- mL/L tic mean (UPGMA) clustering. Bacteria isolated from the Mineral 1b 40.0 same fermentor with ≥99 % similarity were removed from Mineral 2b 40.0 the final analysis in order to reduce redundancy. Bacterial Heminc 10.0 isolate data is presented by phylum, except for Firmicutes VFA solutiond 10.0 and Bacteroidetes that are divided into class and family Resazerin (0.1 % solution) 1.0 groupings. The sequences have been deposited in GenBank e with the accession numbers JQ606836–JQ607746. Na2S/L-cysteine HCL solution 10.0 Incubated clarified rumen fluidf 0.0 Distilled water 870.0 Results a All chemicals were purchased from Sigma-Aldrich b −1 −1 Mineral 1=6 g L K2HPO4 and mineral 2=6 g L KH2PO4,6g A total of 902 bacteria were isolated from the cellulose and −1 −1 −1 −1 L (NH4)2SO4,12gL NaCl, 2.45 g L MgSO4, and 1.69 g L xylan/pectin enrichments with pig (n=6) fecal inoculum. CaCl ·2H O 2 2 Exclusion of 327 bacteria, due to ≥99 % similarity to bac- c Added as solution from 0.5 g hemin in 25 mL 1 M NaOH teria isolated from the same fermentor and fecal donor, d VFA solution contained the following acids (mL/L): acetic 6.85, propionic 3.00, butyric 1.85, isobutyric 0.50, 2-methyl butyric 0.55, resulted in a collection of 575 bacteria. Six phyla were N-valeric 0.55, and isovaleric 0.55 represented in the bacterial isolates: Firmicutes (242), Bac- e Anaerobic solution (200 mL) at pH10 with 2.5 gL-cysteine HCL· teroidetes (185), Proteobacteria (65), Fusobacteria (55), H2O and 2.5 g Na2S·9H2O Actinobacteria (23), and Synergistetes (5). The majority of f Incubated clarified rumen fluid modified from Allison et al. [1]as bacterial isolates had ≥97 % similarity to cultured bacteria follows 400 mL rumen fluid with addition of 300 mL mineral 1, with sequences in the RDP. The distribution of 69 bacterial 300 mL mineral 2, and 4 g Na2CO3, addition of second centrifugation isolates with <95 % similarity to previously cultured bacte- after 4 °C overnight storage and pH7.0 adjustment prior to autoclaving. Stored frozen at −20 °C until used ria was 28 Firmucutes, 31 Bacteroidetes, two Proteobacte- ria, four Fusobacteria, two Actinobacteria, and two Synergistetes. A total of 110 isolates had similarities be- isolated using DNeasy Blood and Tissue Kit (Qiagen, Valen- tween 95 and 97 % to previously cultured bacteria and were cia, CA, USA) according to the manufacturer's instructions for distributed across phyla as follows: 49 Firmucutes, 81 Bac- bacterial cells. PCR amplification of 16S rRNA genes was teroidetes, 12 Proteobacteria, 26 Fusobacteria, three Actino- done with 27 F (5′-AGAGGTTTGATCMTGGCTCAG-3′) bacteria, and two Synergistetes. and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′) pri- The number of isolates obtained from enrichments with mers [19] (all primers from Invitrogen, Carlsbad, CA, USA) each carbohydrate was similar: 274 bacteria from the cellulose and a Dyad DNA Engine thermocycler (MJ Research, Water- and 301 from xylan/pectin enrichments. However, distribution town, MA, USA). The PCR reaction mixture (50 μL) of isolates recovered from each polysaccharide differed across contained 1× Qiagen PCR buffer, 1.25 U of Taq polymerase phyla and genera within a phylum (Table 4). The most striking (Qiagen), 0.25 mM of each dNTP (AMRESCO, Solon, OH, differences in isolate numbers between cellulose and xylan/- USA), 25 pmol of each primer and 80 ng of template DNA. pectin enrichments by phyla were Bacteroidetes (110 versus Amplified products were cleaned using QIAquick 96 PCR 74, respectively), Proteobacteria (10 versus 55, respectively) Purification (Qiagen) and sequenced by the Iowa State Uni- and Fusobacteria (15 versus 40, respectively). While the total versity DNA Sequencing and Synthesis Facility (Ames, IA, number of Firmicutes isolates was similar, the distribution USA) using ABI Prism 377 sequencer (Applied Biosystems, among genera differed. Cellulose enrichments yielded greater Foster City, CA, USA). numbers of Bacillus, Enterococcus, Eubacterium, Staphylo- coccus,andStreptococcus while Clostridium and Tissierella Sequence Analysis were more frequently isolated from xylan/pectin enrichments. The numbers of bacteria isolated from each fecal donor were Consensus sequences were made using VNTI 11.1 software comparable (73 from pig A, 86 from pig B, 134 from pig C, (Invitrogen), and isolates with ≥1,200-bp length sequences 118 from pig D, 78 from pig E, and 86 from pig F). For the Bacterial Isolates from Plant Cell Wall Enrichments of Pig Feces 451

Table 4 Distribution of bacteria isolated from cellulose or xylan/ other Clostridia were from the families Peptostreptococcus pectin enrichments of pig feces by phylum and genus (isolates 11), Clostridiaceae I (30 isolates), Clostridiales_In- Phylum Genus Cellulose Xylan/ certae sedis XI (13 isolates), and Eubacteriaceae (20 isolates) pectin (Fig. 1c). Two isolates were classified as Negativicutes family Number of isolates Veillonellaceae. The bacterial isolates identified as Bacteroidetes are in- Firmicutes 125 118 cluded in Fig. 2 as A: Bacteroides thetaiotaomicron–Bac- Anaerosporobacter 04 teroides ovatus–B. xylanisolvens grouping (63.8 % of Bacillus 11 5 Bacteroidetes) and B: Bacteroidaceae (other than those in Clostridium 41 63 A, 29.7 % of Bacteroidetes), Porphyromonadaceae (5.4 % Enterococcus 21 14 of Bacteroidetes), and Rikenellaceae (0.5 % of Bacteroi- Eubacterium 17 2 detes) family groupings. All of the 118 bacterial isolates in Staphylococcus 21 3 the B. thetaiotaomicron–B. ovatus–B. xylanisolvens group- Streptococcus 13 5 ing (Fig. 2a) were within 96 % similarity. The two largest Tissierella 013 similarity groupings, at ≥99 % similarity, were for bacterial Veillonella 11isolates identified as B. thetaiotaomicron (Bacteroidetes Bacteroidetes 110 74 group B) and B. xylanisolvens (Bacteroidetes group C). Alistipes 01The other Bacteroidaceae isolates (Fig. 2b) were primarily Bacteroides 110 63 grouped with B. uniformis and B. vulgatus, and only 11 Dysgonomonas 03Bacteroidaceae isolates in this grouping were identified as Parabacteroides 07other Bacteroides species. Proteobacteria 10 55 Bacteria in the Proteobacteria phylum were Gammapro- Desulfovibrio 02teobacteria from the Escherichia/Shigella genera, only two Escherichia 10 53 Deltaproteobacteria were isolated (Fig. 3). Forty-six of these Synergistetes Cloacibacillus 50isolates clustered with ≥99 % similarity to Escherichia coli Fusobacteria Fusobacteria 15 40 and E. fergusonii strains. Figure 4 contains bacterial isolates Actinobacteria 9 14 from the remaining three phyla. Most of the Fusobacteria Actinomyces 01were closely related (46 of the 55 isolates). Within the Bifidobacterium 31Actinobacteria phylum, four families were identified in the Eggerthella 24bacterial isolates: Propionibacteriaceae (52 % of Actinobac- Propionibacterium 48teria), Bifidobacteriaceae (17 %), Coribacteriaceae (23 %), and Dermatophiliaceae (4 %). The Synergistetes isolates were all closely related (>96 % similarity). most part, bacterial isolates from the different fecal donors were evenly distributed within the phyla; however, no Syner- gistetes were isolated from fecal enrichments from pigs A and Discussion D and no Proteobacteria were isolated from pigs E and F fecal enrichments. A wide range of bacteria were successfully isolated from Firmicutes were the largest proportion of the bacterial these enrichments and while not strictly quantitative, a isolates (42.1 %). For ease of discussion, the Firmicutes rough estimation of phyla abundance can be obtained. As were divided into the classes Bacilli (101 isolates) and expected, the Firmicutes and Bacteroidetes isolates predo- Clostridia (139 isolates), with two Negativicutes isolates minated at 74.2 % of isolates, followed by Proteobacteria, included with the Clostridia, with further division based on Fusobacteria, Actinobacteria, and Syngistetes,(11.3%, family classification (Fig. 1). The Bacilli (Fig. 1a)were 9.6 %, 4.0 %, and 0.9 %, respectively). Results reported from four families: the Enterococcaceae (32 isolates), Staph- ylococcaceae (31 isolates), Streptococcaceae (14 isolates), „ and Bacillaceae I (12 isolates). The largest family grouping Fig. 1 Cluster analysis with unrooted dendogram of bacterial isolates from cellulose and xylan/pectin enrichments of pig feces in the phylum within the isolates was Lachnospiraceae (65 isolates) in the Firmicutes: a isolates in the Bacilli class, b isolates in the Clostridia Clostridia class of the Firmicutes (Fig. 1b). Within the class and Lachnospiraceae family, and c isolates in the Clostridia class Lachnospiraceae, the majority of the bacteria fell into the Peptostreptococcaceae, Clostridiaceae I, Clostridiales_Incertae sedis RDP genus Clostridium XIVa, and only four isolates were XI, and Eubacteriaceae families and Negativicutes class Veillonella- ceae family. Reference strains have genus, species, and NCBI acces- identified in the Coprococcus genus (NLAE-zl-P520, sion number included. Bacteria with ≥99 % similarity are grouped in NLAE-zl-P521, NLAE-zl-P506, and NLAE-zl-P802). The order to simplify the dendogram. Line at the top represents % similarity 452 C. J. Ziemer a all sequences Key Family 90 91 93 95 98 100 92 94 96 97 99

99.7 98.1 Streptococcaceae group A: NLAE-zl-P496, NLAE-zl-P582 NLAE-zl-P819 Streptococcaceae 97.1 Streptococcaceae group B: NLAE-zl-P807, NLAE-zl-P888, NLAE- 99.6 96.6 zl-P758, NLAE-zl-P891, Streptococcus parasanguinis GJ561390, NLAE-zl-P889 96.1 NLAE-zl-P580 Streptococcaceae

99.7 97.1 Streptococcaceae group C: NLAE-zl-P281, NLAE-zl-P879 95.7 NLAE-zl-P186 Streptococcaceae

99.6 Streptococcaceae group D: NLAE-zl-P567, NLAE-zl-P581, NLAE- zl-P662, NLAE-zl-P68, NLAE-zl-P460,

Enterococcaceae group A: NLAE-zl-P274, NLAE-zl-P382, NLAE- zl-P847, NLAE-zl-P164, NLAE-zl-P114, NLAE-zl-P116, NLAE-zl- P158, NLAE-zl-P664, NLAE-zl-P775, NLAE-zl-P89, NLAE-zl-P43, 99.1 NLAE-zl-P867, NLAE-zl-P583, NLAE-zl-P849, Enterococcus

90.7 faecium DQ403193, NLAE-zl-P185, NLAE-zl-P857, NLAE-zl- P425, NLAE-zl-P148, NLAE-zl-P97 98.5

98.3

99.4 98.3 Enterococcaceae group B: NLAE-zl-P435, NLAE-zl-P795 NLAE-zl-P722 Enterococcaceae NLAE-zl-P435 Enterococcaceae 97.5

Enterococcaceae group C: NLAE-zl-P650, NLAE-zl-P779, NLAE- 99.4 zl-P449, NLAE-zl-P715, NLAE-zl-P765, NLAE-zl-P401, NLAE-zl- 96.7 P487, NLAE-zl-P831, NLAE-zl-P851, NLAE-zl-P735, NLAE-zl- P824

Enterococcus faecalis EU168400 Enterococcaceae

89.3 Bacillaceae 1 group A: NLAE-zl-P643, NLAE-zl-P15, NLAE-zl- 99.1 P423, NLAE-zl-P424, NLAE-zl-P170, NLAE-zl-P429, NLAE-zl-P1, NLAE-zl-P805, NLAE-zl-P459, NLAE-zl-P57 98.9

93.7 NLAE-zl-P331 Bacillaceae 1 93.3 99.7 Bacillaceae 1 group B: NLAE-zl-P442, NLAE-zl-P443 NLAE-zl-P8 Bacillaceae 1

Staphylococcaceae group A: NLAE-zl-P596, NLAE-zl-P783,

99.0 NLAE-zl-P44, NLAE-zl-P781, NLAE-zl-P654, NLAE-zl-P467, Staphylococcus epidermidis EU071611, NLAE-zl-P533, NLAE-zl- P29, NLAE-zl-P54, NLAE-zl-P323, NLAE-zl-P811, NLAE-zl-P865,

98.4 NLAE-zl-P723, NLAE-zl-P9, NLAE-zl-P465, NLAE-zl-P30 92.4

98.1 99.9 Staphylococcaceae group B: NLAE-zl-P19, NLAE-zl-P426, NLAE- zl-P769 NLAE-zl-P150 Staphylococcaceae

98.0 Staphylococcaceae group C: NLAE-zl-P471, NLAE-zl-P6, NLAE- 99.0 zl-P728, Staphylococcus hominis L37601, NLAE-zl-P656, NLAE-

98.4 zl-P821, NLAE-zl-P655, NLAE-zl-P472

97.5 98.2 NLAE-zl-P566 Staphylococcaceae

99.1 Staphylococcaceae group D: NLAE-zl-P20, NLAE-zl-P26, NLAE- 98.5 zl-P836 NLAE-zl-P63 Staphylococcaceae

99.5 Staphylococcaceae group E: NLAE-zl-P2, NLAE-zl-P657 Bacterial Isolates from Plant Cell Wall Enrichments of Pig Feces 453

b all sequences 88 98 90 92 94 96 Key Family 100

Lachnospiraceae group A: NLAE-zl-P606, NLAE-zl-P611, NLAE-zl- P625, NLAE-zl-P607, NLAE-zl-P598, NLAE-zl-P620, NLAE-zl-P605, NLAE-zl-P666, NLAE-zl-P746, NLAE-zl-P701, NLAE-zl-P705, NLAE- 99.0 zl-P144, NLAE-zl-P684, NLAE-zl-P541, NLAE-zl-P76, Clostridium sp. ArC5 AF443594, NLAE-zl-P638, NLAE-zl-P641, NLAE-zl-P645, NLAE-zl-P763, NLAE-zl-P106, NLAE-zl-P571, NLAE-zl-P40, NLAE-zl- P18, NLAE-zl-P698

98.2

97.9

94.4 NLAE-zl-P28 Lachnospiraceae NLAE-zl-P48 Lachnospiraceae NLAE-zl-P45 Lachnospiraceae

Lachnospiraceae group B: Clostridium clostridioforme HM008264, 99.0 NLAE-zl-P777, NLAE-zl-P100, NLAE-zl-P109, NLAE-zl-P120, NLAE- zl-P691, NLAE-zl-P130, NLAE-zl-P726, NLAE-zl-P309, NLAE-zl-P99, NLAE-zl-P124, NLAE-zl-P123 98.5 93.2

98.4 NLAE-zl-P55 Lachnospiraceae 98.0 NLAE-zl-P47 Lachnospiraceae 96.6 NLAE-zl-P31 Lachnospiraceae NLAE-zl-P751 Lachnospiraceae

99.1 Lachnospiraceae group C: NLAE-zl-P415, NLAE-zl-P766, NLAE- 92.9 98.8 zl-P406, NLAE-zl-P212

95.3 98.8 NLAE-zl-P883 Lachnospiraceae 98.7 NLAE-zl-P855 Lachnospiraceae 98.4 NLAE-zl-P390 Lachnospiraceae 98.3 NLAE-zl-P835 Lachnospiraceae 92.4 98.1 NLAE-zl-P885 Lachnospiraceae NLAE-zl-P762 Lachnospiraceae 99.3 Lachnospiraceae group D: NLAE-zl-P154, NLAE-zl-P753 Clostridium leptum AF262239 Lachnospiraceae 91.5 94.8 NLAE-zl-P484 Lachnospiraceae 93.8 99.3 Lachnospiraceae group E: NLAE-zl-P93, NLAE-zl-P94 95.0 91.2 Butyrate-producing bacterium A2-231 AJ270484 Lachnospiraceae Lachnospira pectinoschiza AY169414 Lachnospiraceae

86.3 99.4 Lachnospiraceae group F: NLAE-zl-P520, NLAE-zl-P521, NLAE- 98.1 zl-P506 NLAE-zl-P802 Lachnospiraceae

99.4 Lachnospiraceae group C: NLAE-zl-P594, NLAE-zl-P630

Fig. 1 (continued) 454 C. J. Ziemer c all sequences 92 86 90 84 88 94 98 96 Key Family 100 99.5 Peptostreptococcaceae group B: NLAE-zl-P12, NLAE-zl-P83 92.0 NLAE-zl-P536 Peptostreptococcaceae 91.1 Peptostreptococcaceae group B: NLAE-zl-P112, NLAE-zl-P69, NLAE-zl- 99.4 P513, Clostridium bifermentans DQ978211, NLAE-zl-P749, NLAE-zl-P516, 89.0 NLAE-zl-P446

NLAE-zl-P34 Peptostreptococcaceae 97.6 NLAE-zl-P65 Peptostreptococcaceae

99.3 Clostridiaceae group A: NLAE-zl-P748, NLAE-zl-P856, NLAE-zl-P428, NLAE- zl-P287, NLAE-zl-P808 96.3

99.8 Clostridiaceae group B: NLAE-zl-P818, Clostridium sp. BG-C36 FJ384373, 95.1 NLAE-zl-P822 86.0 99.5 Clostridiaceae group C: NLAE-zl-P61, NLAE-zl-P81, NLAE-zl-P719 94.5 98.1 NLAE-zl-P49 Clostridiaceae 1 94.2 Sarcina ventriculi AF110272 Clostridiaceae 1

93.3 99.9 Clostridiaceae group D: NLAE-zl-P10, NLAE-zl-P115, NLAE-zl-P142

NLAE-zl-P153 Clostridiaceae 1

91.2 Clostridiaceae 1 group E: NLAE-zl-P628, NLAE-zl-P856, NLAE-zl-P468, NLAE-

99.0 zl-P902, NLAE-zl-P612, Clostridium sporogenes DQ278865, NLAE-zl-P601, NLAE-zl-P816, NLAE-zl-P602, NLAE-zl-P600, NLAE-zl-P903, NLAE-zl-P812, NLAE-zl-P813, NLAE-zl-P814

98.8

85.5 NLAE-zl-P815 Clostridiaceae 1

Clostridiales_Incertae Sedis XI group A: NLAE-zl-P628, NLAE-zl-P856, NLAE-zl- 99.1 P468, NLAE-zl-P902, NLAE-zl-P612, Clostridium sporogenes DQ278865, NLAE- zl-P601, NLAE-zl-P816, NLAE-zl-P602, NLAE-zl-P600, NLAE-zl-P903, NLAE-zl- 98.9 P812, NLAE-zl-P813, NLAE-zl-P814 97.3 NLAE-zl-P603 Clostridiales_Incertae sedis XI 96.3 99.5 Clostridiales_Incertae Sedis XI group B: NLAE-zl-P155, NLAE-zl-P160 92.2 99.4 Clostridiales_Incertae Sedis XI group C: NLAE-zl-P152, NLAE-zl-P162 NLAE-zl-P544 Clostridiales_Incertae Sedis XI NLAE-zl-P46 Eubacteriaceae 98.8 NLAE-zl-P56 Eubacteriaceae 82.7

87.6 98.2 Eubacteriaceae group: NLAE-zl-P307, NLAE-zl-P320, NLAE-zl- P473, NLAE-zl-P439, NLAE-zl-P482, Eubacterium limosum AB298909, NLAE-zl-P475, NLAE-zl-P80, NLAE-zl-P143, 99.0 Eubacterium callanderi X96961, NLAE-zl-P59, NLAE-zl-P67,

97.9 NLAE-zl-P479, NLAE-zl-P558, NLAE-zl-P169, NLAE-zl-P432, NLAE-zl-P27, NLAE-zl-P62, NLAE-zl-P172

NLAE-zl-P58 Eubacteriaceae 99.9 Veillonellaceae group: NLAE-zl-P568, Veillonella sp. ACP1 HQ616359 97.7

91.4 NLAE-zl-P799 Veillonellaceae Megasphaera elsdenii U95027 Veillonellaceae

Fig. 1 (continued) Bacterial Isolates from Plant Cell Wall Enrichments of Pig Feces 455 a all sequences 96 97 99 98

100 Key Family

99.1 Bacteroidetes group A: NLAE-zl-P375, NLAE-zl-P379, NLAE-zl-P358 98.8 NLAE-zl-P850 Bacteroidaceae

Bacteroidetes group B: NLAE-zl-P32, NLAE-zl-P35, NLAE-zl-P188, NLAE-zl-P279, NLAE-zl-P171, NLAE-zl-P182, NLAE-zl-P146, NLAE-zl-P265, NLAE-zl-P370, NLAE-zl-P437, NLAE-zl-P736, NLAE-zl-P223, NLAE-zl-P163, NLAE-zl-P213, NLAE-zl-P299, NLAE-zl-P277, NLAE-zl-P211, NLAE-zl-P262, NLAE-zl-P873, NLAE-zl-P858, NLAE-zl-P268, NLAE-zl-P273,

98.8 NLAE-zl-P147, NLAE-zl-P744, NLAE-zl-P882, NLAE-zl-P175, NLAE-zl-P393, NLAE-zl-P830, NLAE-zl-P846, NLAE-zl-P827, NLAE-zl-P874, NLAE-zl-P693, NLAE-zl-P745, NLAE-zl-P228, 99.0 NLAE-zl-P362, NLAE-zl-P412, NLAE-zl-P917, NLAE-zl-P920, NLAE-zl-P923, NLAE-zl-P737, NLAE-zl-P174, NLAE-zl-P757, NLAE-zl-P750, NLAE-zl-P176, NLAE-zl-P187, NLAE-zl-P195, NLAE-zl-P351, NLAE-zl-P224, NLAE-zl-P353, NLAE-zl-P373, NLAE-zl-P324, NLAE-zl-P346, NLAE-zl-P369, NLAE-zl-P350, NLAE-zl-P298, NLAE-zl-P270, NLAE-zl-P407, NLAE-zl-P852, NLAE-zl-P699, NLAE-zl-P88, Bacteroides thetaiotaomicron AB510710, NLAE-zl-P700, NLAE-zl-P219, NLAE-zl-P853, NLAE- zl-P269, NLAE-zl-P354, NLAE-zl-P754, NLAE-zl-P780, NLAE-zl- P366, NLAE-zl-P861, NLAE-zl-P436, NLAE-zl-P271, NLAE-zl-P64, 98.5 98.9 NLAE-zl-P391

98.8

98.4

97.1 NLAE-zl-P32 Bacteroidaceae NLAE-zl-P348 Bacteroidaceae NLAE-zl-P378 Bacteroidaceae NLAE-zl-P372 Bacteroidaceae NLAE-zl-P190 Bacteroidaceae

Bacteroidetes group C: NLAE-zl-P7, NLAE-zl-P402, NLAE- zl-P900, NLAE-zl-P887, NLAE-zl-P834, NLAE-zl-P854, NLAE-zl-P828, NLAE-zl-P282, NLAE-zl-P349, NLAE-zl- 97.0 P747, NLAE-zl-P295, NLAE-zl-P732, NLAE-zl-P225, NLAE- 99.0 zl-P909, NLAE-zl-P913, Bacteroides xylanisolvens AM230650, NLAE-zl-P139, NLAE-zl-P218, NLAE-zl-P125, NLAE-zl-P371, NLAE-zl-P229, NLAE-zl-P233, NLAE-zl-

98.8 P833, NLAE-zl-P352, NLAE-zl-P377, NLAE-zl-P764, NLAE- zl-P318, NLAE-zl-P733 96.0

98.8

98.6 NLAE-zl-P833 Bacteroidaceae NLAE-zl-P718 Bacteroidaceae 98.3 NLAE-zl-P409 Bacteroidaceae

95.7 NLAE-zl-P388 Bacteroidaceae NLAE-zl-P360 Bacteroidaceae

99.0 Bacteroidetes group D: NLAE-zl-P105, NLAE-zl-P714, NLAE-zl-P60, NLAE- 98.9 zl-P358 98.7 NLAE-zl-P60 Bacteroidaceae 98.1 NLAE-zl-P141 Bacteroidaceae 96.3 Bacteroides ovatus X83952 Bacteroidaceae NLAE-zl-P41 Bacteroidaceae.

Fig. 2 Cluster analysis with unrooted dendogram of bacterial isolates Porophyromonadaceae, and Dysgonomonas families. Reference strains from cellulose and xylan/pectin enrichments of pig feces in the phylum have genus, species, and NCBI accession number included. Bacteria Bacteroidetes: a isolates in the B. thetaiotoaomicron–B. xylanisolvens– with ≥99 % similarity are grouped in order to simplify the dendogram. B. ovatus assemblage of the family Bacteroideaceae and b isolates in Line at the top represents % similarity the Bacteroidaceae (other than those in group A), Rikenellaceae, 456 C. J. Ziemer

b all sequences Key Family 86 94 84 88 90 92 96 98 100 Alistipes finegoldii AY643083 Rikenellaceae 95.7 NLAE-zl-P117 Rikenellaceae

99.0 Porphyromonaceae group A: NLAE-zl-255, NLAE-zl-636, NLAE-zl-743, NLAE-zl-340, NLAE-zl-344 98.7

98.1 NLAE-zl-P599 Porphyromonadaceae 93.3 NLAE-zl-P241 Porphyromonadaceae

87.8 Parabacteroides merdae EU722738 Porphyromonadaceae

99.3 Porphyromonaceae group B: NLAE-zl-545, Dysgonomonas mossii AJ319867, NLAE-zl-794, NLAE-zl-800

82.8

Bacteroidaceae Group E: NLAE-zl-P167, NLAE-zl-P633, NLAE-zl- P447, NLAE-zl-P108, NLAE-zl-P592, NLAE-zl-P151, NLAE-zl- 99.0 P338, NLAE-zl-P96, NLAE-zl-P168, NLAE-zl-P332, NLAE-zl- P631, NLAE-zl-P918, Bacteroides uniformis EU722741, NLAE-zl- P448, NLAE-zl-P559, NLAE-zl-P591, NLAE-zl-P157

98.9

98.6 NLAE-zl-P191 Bacteroidaceae 98.0 NLAE-zl-P286 Bacteroidaceae 86.8 92.6 NLAE-zl-P79 Bacteroidaceae 99.5 Bacteroidaceae Group F: Bacteroides fragilis AB54764, NLAE-zl- P343

Bacteroidaceae Group G: NLAE-zl-P569, NLAE-zl-P356, NLAE-zl- P301, NLAE-zl-P302, NLAE-zl-P317, NLAE-zl-P572, NLAE-zl- 99.0 P497, NLAE-zl-P278, NLAE-zl-P430, NLAE-zl-P440, Bacteroides 91.6 vulgates EF608209, NLAE-zl-P803, NLAE-zl-P56, NLAE-zl- P575, NLAE-zl-P559, NLAE-zl-P478, NLAE-zl-P564, NLAE-zl- P493, NLAE-zl-P776,N LAE-zl-P488, NLAE-zl-P445, NLAE-zl- P489

98.8

98.4

90.5 NLAE-zl-P897 Bacteroidaceae 97.8 NLAE-zl-P557 Bacteroidaceae 93.9 NLAE-zl-P560 Bacteroidaceae

99.6 Bacteroidaceae Group H: NLAE-zl-P103, NLAE-zl-177

99.5 Bacteroidaceae Group I: NLAE-zl-P551, NLAE-zl-P514, NLAE-zl-

98.9 P524, NLAE-zl-P512,

98.6 NLAE-zl-P624 Bacteroidaceae 99.2 98.9 Bacteroidaceae Group J: NLAE-zl-P236, NLAE-zl-P159 NLAE-zl-P341 .Bacteroidaceae

Fig. 2 (continued) Bacterial Isolates from Plant Cell Wall Enrichments of Pig Feces 457

all sequences Key Family

Enterobacteriaceae group A: NLAE-zl-P121, NLAE-zl-P868, NLAE-zl- P235, NLAE-zl-P884, NLAE-zl-P651, Escherichia fergusonii HQ259942, NLAE-zl-P844, NLAE-zl-P248, NLAE-zl-P245, NLAE-zl-P876, NLAE-zl- P841, NLAE-zl-P119, NLAE-zl-P676, NLAE-zl-P670, NLAE-zl-P673, NLAE-zl-P672, NLAE-zl-P669, NLAE-zl-P685, NLAE-zl-P17, NLAE-zl- P122, NLAE-zl-P869, NLAE-zl-P127, NLAE-zl-P25, NLAE-zl-P679, NLAE-zl-P21, NLAE-zl-P118, NLAE-zl-P242, NLAE-zl-P280, NLAE-zl- P275, NLAE-zl-P846, NLAE-zl-P729, NLAE-zl-P731, NLAE-zl-P875, NLAE-zl-P877, NLAE-zl-P826, NLAE-zl-P194, NLAE-zl-P135, NLAE-zl- 99.0 P660, NLAE-zl-P738, NLAE-zl-P256, NLAE-zl-P51, NLAE-zl-P253, NLAE-zl-P52, NLAE-zl-P839, Escherichia coli X80725, NLAE-zl-P276, NLAE-zl-P272, NLAE-zl-P717

98.9

98.9

98.9 NLAE-zl-P717 Enterobacteriaceae

98.8 NLAE-zl-P823 Enterobacteriaceae NLAE-zl-P678 Enterobacteriaceae 98.7 NLAE-zl-P829 Enterobacteriaceae

99.1 98.798.9 Enterobacteriaceae group B: NLAE-zl-P198, NLAE-zl-P201 NLAE-zl-P832 Enterobacteriaceae

98.7 NLAE-zl-P671 Enterobacteriaceae 99.1 Enterobacteriaceae group C: NLAE-zl-P237, NLAE-zl-P838 98.9

98.698.8 NLAE-zl-P250 Enterobacteriaceae NLAE-zl-P126 Enterobacteriaceae 98.5 NLAE-zl-P203 Enterobacteriaceae 98.3 NLAE-zl-P716 Enterobacteriaceae NLAE-zl-P840 Enterobacteriaceae 97.7 98.8 NLAE-zl-P842 Enterobacteriaceae 83.0 NLAE-zl-P23 Enterobacteriaceae 99.8 Desulfovibrionaceae group: NLAE-zl-P434, NLAE-zl-P72 98.0 Desulfovibrio intestinalis Y12254 Desulfovibrionaceae.

Fig. 3 Cluster analysis with unrooted dendogram of bacterial isolates genus, species, and NCBI accession number included. Bacteria with from cellulose and xylan/pectin enrichments of pig feces in the phylum ≥99 % similarity are grouped in order to simplify the dendogram. Line Proteobacteria, γ-proteobacteria class Enterobacteriaceae family and δ- at the top represents % similarity proteobacteria class Desulfovibrionaceae family. Reference strains have 458 C. J. Ziemer by Ley et al. [23] support the phyla abundance from these similar to Clostridium bifermentans, a xylanolytic Peptos- enrichments, with similar results across feces from 60 dif- treptococcaceae bacterium [40]. For a number of the species ferent mammalian species for Firmicutes and Bacteroidetes related to the isolates, especially those in the Eubacteriaceae (82 % of nearly 20,000 classified sequences) and the other and Bacillaceae, information on plant cell wall utilization phyla (14.2 % Proteobacteria, Fusobacteria, and Actinobac- was not readily available and will need to be explored in teria). Pig feces was used as the source of bacteria for the greater detail. An additional niche that some Firmicutes may enrichments because the majority of data available for mam- fill in these enriched communities is fermentation of pep- mals is from microbial analysis of feces and the enrichments tides and amino acids. Cotta et al. [6] identified a number of could be readily replicated with other mammals, in particular, Clostridium, Enterococcus, and Staphylococcus species iso- food production animals. Plant cell wall-utilizing microbial lated from pig feces and stored manure with this capacity. communities could be very different in other parts of the Members of the Bacteroidaceae family have demonstrat- intestine; the microbial community obtained from these cul- ed utilization of a wide range of carbohydrate, including ture results may differ from that in the pig's cecum and colon. plant cell wall (cellulose, xylan, pectins, and β-glucans and Nearly one-third of the isolates (n=179) were less than glactans) and host mucopolysaccharides and glycoproteins 97 % similar to cultured bacteria and represent new species [38]. Genomic analysis of Bacteroides species confirms the and/or genera, using 97 % similarity as species and 95 % as enrichment of genes for enzymes targeting carbohydrates genera cutoffs [41]. While use of selective media introduces and that the range of these enzymes is quite broad [14]. bias, in general, easy to grow bacteria are isolated while many Active enzymes targeting plant cell wall carbohydrates have others will not be detected; the fact that so many of these been demonstrated in B. ovatus, B. thetaiotaomicron, B. isolates represent previously uncultured bacteria indicates that xylanisolvens, B. uniformis, and B. vulgatus [29, 31, 39]. many uncultured intestinal bacteria could probably be recov- Among the isolates, the majority of Bacteroidetes were ered by using a broader range of differential nutrient media. A closely related to B. ovatus, B. thetaiotaomicron,andB. large number of isolates were ≥99 % similar, demonstrating a xylanisolvens; both B. ovatus and B. thetaiotaomicron have great overlap in phylogenetic similarity in multiple phyla and multiple polysaccharide utilization loci that encode for a families across the different fecal donors. broad range of enzymes targeting various plant cell wall Differences in the type of bacteria that were isolated from components [29]. Many of these Bacteroides species are the different carbohydrate enrichments were evident across able to ferment multiple carbohydrates so it is unclear why all phyla. Changing the level and type of nonstarch poly- more isolates were obtained from the cellulose enrichments saccharides, using bran or oligosaccharides, resulted in compared to xylan/pectin enrichments. While the Bacter- changes in fecal microbial community profiles [13, 16]. oides are some of the most studied intestinal bacteria, Inclusion of resistant starch, insoluble or soluble nonstarch 60.5 % of Bacteroidetes isolates had less than 97 % simi- polysaccharides in pig diets altered the number and types of larity to previously cultured strains. bacteria isolated from cecal contents, with Gram-positive Most of the Proteobacteria isolated were Gammaproteo- bacteria predominating [9]. Isolates from cellulose enrich- bacteria in the Enterobacteriaceae family, primarily from the ments were evenly distributed between Gram-positive and Escherichia/Shigella genera. Enterobacteriaceae bacteria are Gram-negative bacteria (134 and 140, respectively); how- members of the mammalian intestinal microbiota, at 7–8% ever, Gram-negative bacteria were more frequent among of the microbiota [23], perhaps up to 10 % in pig feces [18]. isolates from xylan/pectin enrichments (169 and 132 Among these isolates, Proteobacteria were 3.6 % of total Gram-positive). The differences in the distribution of iso- bacteria from cellulose and 18.3 % from xylan/pectin lates among the various genera warrant further exploration, enrichments. In contrast with the current results, pigs fed especially with regards to isolates from Clostridium, Entero- cellulose and carboxymethylcellulose had more Enterobacter- coccus, and Bacteroides. iaceae in their feces than pigs fed oat β-glucan diets [30]. In Members of the Lachnospiraceae family were the most addition to their ability to utilize a wide range of less complex frequently isolated bacteria in the Firmicutes phyla. This substrates (sugars, yeast extract, peptones, etc.), members of family includes the clostridal groups XIVa and XIVb [5], a the Enterobacteraecia with the ability to utilize plant cell wall predominate group in pig and other mammal's intestinal substrates have been previously isolated [7, 45, 46]. Addition- tract [3, 11, 22]. Many of the Firmicutes in the intestinal ally, Escherichia and Klebsiella species isolated from the pig tract have cellulose and hemicellulose degradation enzymes intestine were able utilize a number of amino acid with 40 to including members of the Lachnospiraceae, Clostridiaceae I 60 % of those utilized for protein synthesis [8]. Two Desulfo- families, with a few in Eubacteriaceae and Bacillaceae I vibrionaceae (Deltaproteobacteria) were isolated even though families [27, 42, 43]. Two examples among the isolates were the media were not designed to grow sulfate-reducing bacte- 12 bacteria closely related to Clostridium celerescens,a ria. However, many of the enrichment cultures appeared to cellulolytic Lachnospiraceae bacterium [33]andthree have a sulfate-reducing bacterial population (significant Bacterial Isolates from Plant Cell Wall Enrichments of Pig Feces 459

all sequences Key Family

99.0 Synergistaceae group: NLAE-zl-P578, NLAE-zl-P579, NLAE-zl- 98.4 P452, NLAE-zl-P702, NLAE-zl-P261 95.2 NLAE-zl-P261 Synergistaceae Cloacibacillus evryensis CU463952 Synergistaceae

Fusobacteriaceae group A: NLAE-zl-321, NLAE-zl-386, NLAE-zl- 417, NLAE-zl-404, NLAE-zl-392, NLAE-zl-617, NLAE-zl-528, NLAE-zl-78, NLAE-zl-590, Fusobacterium varium AB640694, NLAE-zl-518, NLAE-zl-553, NLAE-zl-173, NLAE-zl-312, NLAE-zl- 99.1 197, NLAE-zl-548, NLAE-zl-181, NLAE-zl-156, NLAE-zl-312, NLAE-zl-197, NLAE-zl-389, NLAE-zl-134, NLAE-zl-184, NLAE-zl- 79.5 498, NLAE-zl-13, NLAE-zl-210, NLAE-zl-217, NLAE-zl-183, NLAE- zl-418, NLAE-zl-305, NLAE-zl-316, NLAE-zl-179, NLAE-zl-397, NLAE-zl-499, NLAE-zl-129, NLAE-zl-501, NLAE-zl-207, NLAE-zl- 383, NLAE-zl-322, NLAE-zl-539, NLAE-zl-588, NLAE-zl-335, NLAE-zl-258, NLAE-zl-532, NLAE-zl-635, NLAE-zl-38, NLAE-zl-87

98.8

98.7

98.7 99.8 Fusobacteriaceae group B: NLAE-zl-193, NLAE-zl-249 NLAE-zl-P311 Fusobacteriaceae

98.6 NLAE-zl-P202 Fusobacteriaceae 98.9

78.5 NLAE-zl-P329 Fusobacteriaceae

98.2 99.1 98.8 Fusobacteriaceae group C: NLAE-zl-37, NLAE-zl-36 NLAE-zl-P330 Fusobacteriaceae NLAE-zl-P214 Fusobacteriaceae Actinomyces odontolyticus X53227 Actinomycetaceae 89.7 NLAE-zl-P862 Dermatophilaceae

88.1

Propionibacteriaceae group: NLAE-zl-P71, NLAE-zl-P755, 99.1 NLAE-zl-P677, NLAE-zl-P586, NLAE-zl-P70, NLAE-zl-P503, Propionibacterium acnes AY642051, NLAE-zl-P809, NLAE-zl- 86.1 P113, NLAE-zl-P825, NLAE-zl-P66, NLAE-zl-P50, NLAE-zl- P688

99.8 Bifidobacteriaceae group: NLAE-zl-P863, NLAE-zl-P898, NLAE- 96.3 zl-P804, NLAE-zl-784 82.7 Bifidobacterium choerinum D86186 Bifidobaceriaceae

99.6 Coriobacteriaceae group: NLAE-zl-P537, NLAE-zl-P806, NLAE- 98.6 zl-P768, NLAE-zl-892, NLAE-zl-893 93.8 NLAE-zl-P665 Coriobacteriaceae Eggerthella lenta AB558167 Coriobacteriaceae. 460 C. J. Ziemer

ƒFig. 4 Cluster analysis with unrooted dendogram of bacterial isolates from cellulose and xylan/pectin enrichments of pig feces in the phyla insight and comprehension of total plant cell wall- Synergistetes, Fusobacteria, and Actinobacteria. Reference strains have fermenting microbial communities. Over 30 % of the isolates genus, species, and NCBI accession number included. Bacteria with were not included as cultured bacteria in the RDP. These ≥ 99 % similarity are grouped in order to simplify the dendogram. Line bacteria represent new opportunities to study the genomic at the top represents % similarity and metabolic capacities of these members of the complex intestinal microbiota.

amounts of black precipitate were visible in those fermentors), Acknowledgments This research was supported by a grant from and investigation of this group using nonculture methods Defense Advanced Research Projects Agency as part of its Intestinal could determine the predominance of these bacteria. Fortitude Program to C.J. Ziemer. The author would like to thank Todd Atherly, Kerrie Franzen, and John Tenhundfeld for technical analyses. Isolates identified in the remaining three phyla (Fusobac- teria, Actinobacteria, and Synergistetes) have been detected in mammalian intestinal tracts [23, 44]. While it is not clear what function isolates from these phyla carry out in the References microbial communities in cellulose and xylan/pectin enrich- ments, they have a wide range of fermentative capacities. 1. Allison MJ, Robinson IM, Bucklin JA, Booth GD (1979) Com- They may support the plant cell wall-utilizing bacteria by parison of bacterial populations of the pig cecum and colon based actively fermenting nitrogen compounds, and bacteria in upon enumeration with specific energy sources. Appl Environ Microbiol 37:1142–1151 both the Actinobacteria and Fusobacteria phyla have those 2. Brulc JM, Antonopoulos DA, Miller ME, Wilson MK, Yannarell properties [44]. Of the Fusobacteria isolated, 54.5 % had AC, Dinsdale EA, Edwards RE, Frank ED, Emerson JB, Wacklin less than 97 % similarity to previously cultured strains. The P, Coutinho PM, Henrissat B, Nelson KE, White BA (2009) Gene- specific role of the Fusobacteria in these enrichment cultures centric metagemomics of the fiber-adherent bovine rumen micro- biome reveals forage specific glycoside hydrolases. Proc Natl is unclear as they use a limited number of carbohydrates; Acad Sci USA 106:1948–1953 however, their main fermentation product is butyrate, an 3. Castillo M, Skene G, Roca M, Anguita M, Badiola I, Duncan SH, important energy source for host intestinal cells [37, 44]. Flint HJ, Martín-Orúe SM (2007) Application of 16S rRNA gene- Although only 23 isolates identified as Actinobacteria, they targeted fluorescence in situ hybridization and restriction fragment length polymorphism to study porcine microbiota along the gas- had broad phylogenetic diversity. Proprionibacterium acnes trointestinal tract in response to different sources of dietary fiber. and other species contribute to the proteolysis and peptidase FEMS Microbiol Ecol 59:138–146 activity in the large intestine [26, 47]. A large amount of 4. 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Only five Synergistetes were isolated and acterization and comparison of bacteria from swine faeces and while their presence in the intestinal tract of mammals has manure storage pits. Environ Microbiol 5:737–745 been documented, their function within the intestinal micro- 7. Crittenden R, Karppinen S, Ojanen S, Tenkanen M, Fagerström R, Mättö J, Saarele M, Mattila-Sandholm T, Poutanen K (2002) In biota is unknown [28, 44]. vitro fermentation of cereal dietary fiber carbohydrates by pro- The microbial communities that arose in these enrichment biotic and intestinal bacteria. J Sci Food Agric 82:781–789 cultures had broad bacterial diversity. Many of the bacteria 8. Dai Z-L, Zhang J, Wu G, Zhu W-Y (2010) Utilization of amino isolated are closely related to species demonstrated to produce acids by bacteria from the pig small intestine. Amino Acids 39:1201–1215 enzymes that ferment cellulose and hemicellulose fractions of 9. 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