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Microbial Ecology

Newly Cultured Bacteria with Broad Diversity Isolated from Eight-Week Continuous Culture Enrichments of Cow Feces on Complex Polysaccharides

Cherie J. Ziemer
Cherie J. Ziemer
USDA, Agricultural Research Service, National Laboratory for Agriculture and the Environment, Ames, Iowa, USA
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DOI: 10.1128/AEM.03016-13
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    FIG 1

    Cluster analysis with an unrooted dendrogram of bacterial isolates from cellulose and xylan-pectin enrichments of cow feces in the phylum Firmicutes. (A) Isolates in the Bacilli, Erysipelotrichia (family Erysipelotrichaceae), and Negativicutes (family Veillonellaceae) classes; (B) isolates in the Clostridia class and Lachnospiraceae family; (C) isolates in the Clostridia class and Peptostreptococcaceae, Clostridiaceae 1, Clostridiales incertae sedis XI, and Eubacteriaceae families. The genus and species names and GenBank (NCBI) accession number are included for the reference strains. Bacteria with ≥99% similarity are grouped in order to simplify the dendrogram. The line at the top represents percent similarity across isolates; Schlesneria paludicola (Plantomycetes) is used as the outgroup.

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    FIG 2

    Cluster analysis with an unrooted dendrogram of bacterial isolates from cellulose and xylan-pectin enrichments of cow feces in the phylum Bacteroidetes. (A) Isolates in the B. thetaiotaomicron- B. xylanisolvens-B. ovatus assemblage of the family Bacteroidaceae; (B) isolates in the Bacteroidaceae (other than those in the group shown in panel A) and Porphyromonadaceae families. The genus and species names and GenBank (NCBI) accession number are included for the reference strains. Bacteria with ≥99% similarity are grouped in order to simplify the dendrogram. The line at the top represents percent similarity across isolates; Schlesneria paludicola (Plantomycetes) is used as the outgroup.

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    FIG 3

    Cluster analysis with an unrooted dendrogram of bacterial isolates from cellulose and xylan-pectin enrichments of cow feces in the phyla Proteobacteria; Gammaproteobacteria class, Enterobacteriaceae family; Deltaproteobacteria class, Desulfovibrionaceae family; Synergistetes; Fusobacteria; and Actinobacteria. The genus and species names and GenBank (NCBI) accession number are included for the reference strains. Bacteria with ≥99% similarity are grouped in order to simplify the dendrogram. The line at the top represents percent similarity across isolates; Schlesneria paludicola (Plantomycetes) is used as the outgroup.

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  • TABLE 1

    Fermentor nutrient medium

    IngredientaConcn
    Peptone water2.0 g/liter
    Yeast extract2.0 g/liter
    NaCl0.1 g/liter
    K2HPO40.04 g/liter
    KH2PO40.04 g/liter
    MgSO4 · 7H2O0.01 g/liter
    CaCl2 · 6H2O0.01 g/liter
    NaHCO32.0 g/liter
    Cysteine-HCl0.5 g/liter
    Bile salts0.5 g/liter
    Heminb0.05 g/liter
    Tween 802.0 ml/liter
    Vitamin K10.01 ml/liter
    • ↵a All chemicals were obtained from Sigma.

    • ↵b Hemin solution consisted of 0.5 g hemin in 25 ml 1 M NaOH.

  • TABLE 2

    Composition of carbohydrate-specific agars

    IngredientaConcn
    Carbohydrate5.0 g/liter
    Trypticase4.5 g/liter
    Yeast extract0.5 g/liter
    Agar20.0 g/liter
    Mineral 1b40.0 ml/liter
    Mineral 2c40.0 ml/liter
    Hemind10.0 ml/liter
    VFA solutione10.0 ml/liter
    Resazurin (0.1% solution)1.0 ml/liter
    Na2S–l-cysteine HCl solutionf10.0 ml/liter
    Incubated clarified rumen fluidg0.0 ml/liter
    Distilled water870.0 ml/liter
    • ↵a All chemicals were purchased from Sigma.

    • ↵b Mineral 1 is K2HPO4, 6 g liter−1.

    • ↵c Mineral 2 is KH2PO4, 6 g liter−1; (NH4)2SO4, 6 g liter−1; NaCl, 12 g liter−1; MgSO4, 2.45 g liter−1; and CaCl2 · 2H2O, 1.69 g liter−1.

    • ↵d Added as a solution of 0.5 g hemin in 25 ml 1 M NaOH.

    • ↵e Short chain fatty acid solution contained the following acids, in ml/liter: acetic, 6.85; propionic, 3.00; butyric, 1.85; isobutyric, 0.50; 2-methyl butyric, 0.55; N-valeric, 0.55; and isovaleric, 0.55.

    • ↵f Two hundred milliliters anaerobic solution at pH 10 with 2.5 g l-cysteine HCl · H2O and 2.5 g Na2S · 9H2O.

    • ↵g Incubated clarified rumen fluid modified from the work of Allison et al. (50) as follows: 400 ml rumen fluid with addition of 300 ml mineral 1, 300 ml mineral 2, and 4 g Na2CO3; addition of a second centrifugation after overnight storage at 4°C; and adjustment to pH 7.0 prior to autoclaving. The fluid was stored frozen at −20°C until it was used.

  • TABLE 3

    Distribution of bacteria isolated from cellulose or xylan-pectin enrichments of cow feces by phylum and genus

    PhylumGenusNo. of isolates
    CelluloseXylan-pectin
    FirmicutesAnaerofilum11
    Anaerosporobacter180
    Anaerostipes01
    Anaerovorax20
    Blautia811
    Butyricicoccus03
    Clostridium214
    Clostridium IV34
    Clostridium XI01
    Clostridium XIVa2137
    Clostridium XIVb20
    Clostridium XVIII314
    Enterococcus51
    Erysipelotrichaceae incertae sedis315
    Eubacterium20
    Flavonifractor01
    Lachnospiraceae incertae sedis30
    Lachnospiraceae, unclassified51
    Lactonifactor11
    Megasphaera02
    Paenibacillus10
    Peptostreptococcus01
    Oscillibacter105
    Ruminococcus, unclassified51
    Sedimentibacter10
    Sporanaerobacter20
    Staphylococcus10
    Streptococcus11
    Tissierella140
    BacteroidetesBacteroides3955
    Dysgonomonas1910
    Parabacteroides92
    Proteiniphilum80
    ProteobacteriaAeromonas61
    Citrobacter10
    Desulfovibrio151
    Escherichia01
    Morganella10
    Proteus214
    SynergistetesCloacibacillus61
    FusobacteriaFusobacterium41
    ActinobacteriaBifidobacterium04
    Olsenella01
    Paraeggerthella10
    Propionibacterium04
    Micrococcineae, unclassified10
    Actinomycetales, unclassified50
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Newly Cultured Bacteria with Broad Diversity Isolated from Eight-Week Continuous Culture Enrichments of Cow Feces on Complex Polysaccharides
Cherie J. Ziemer
Applied and Environmental Microbiology Jan 2014, 80 (2) 574-585; DOI: 10.1128/AEM.03016-13

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Newly Cultured Bacteria with Broad Diversity Isolated from Eight-Week Continuous Culture Enrichments of Cow Feces on Complex Polysaccharides
Cherie J. Ziemer
Applied and Environmental Microbiology Jan 2014, 80 (2) 574-585; DOI: 10.1128/AEM.03016-13
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