Fütterungsbedingte mikrobielle Zusammensetzung von Rinderkot ...

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5. Microbial biomass in faeces of dairy cows affected by a nitrogen deficient diet 41 determined by reversed-phase HPLC with 100% methanol as the mobile phase and detected at a wavelength of 282 nm (Jost et al., 2011). 5.2.5. Amino sugars The amino sugars muramic acid, mannosamine, glucosamine and galactosamine were determined according to Indorf et al. (2011). Moist samples of 2 g fresh faeces were weighed into 20 ml test tubes, mixed with 10 ml 6 M HCl, and heated for 2 h at 105 °C. After HCl removal from the filtered hydrolysates in a vacuum rotary evaporator at 40 °C and centrifugation, the samples were transferred to vials and stored at -18 °C until the HPLC measurements. Chromatographic separations were performed on a Phenomenex (Aschaffenburg, Germany) Hyperclone C18 column (125 mm length × 4 mm diameter), protected by a Phenomenex C18 security guard cartridge (4 mm length × 2 mm diameter) at 35 °C. The HPLC system consisted of a Dionex (Germering, Germany) P 580 gradient pump, a Dionex Ultimate WPS – 3000TSL analytical autosampler, with in-line split-loop injection and thermostat, and a Dionex RF 2000 fluorescence detector set at 445 nm emission and 330 nm excitation wavelengths, with medium sensitivity. OPA reagent (o-Phthalaldehyde), sample and preparation vials were stored in the autosampler at 15 °C. For the automated pre-column derivatisation, 50 µl OPA and 30 µl sample were mixed in the preparation vial, and 15 µl of the indole derivates were injected after 120 sec reaction time. The mobile phase consisted of two eluents and was delivered at a flow rate of 1.5 ml min -1 . Eluent A was a 97.8/0.7/1.5 (v/v/v) mixture of an aqueous phase, methanol and tetrahydrofuran (THF). The aqueous phase contained 52 mmol sodium citrate and 4 mmol sodium acetate, adjusted to pH 5.3 with HCl. Methanol and THF were then added. Eluent B consisted of 50% water and 50% methanol (v/v). Fungal C (mg g -1 dry weight) was calculated by subtracting bacterial glucosamine from total glucosamine as an index for fungal residues, assuming that muramic acid and glucosamine occur at a 1 to 2 molar ratio in bacterial cells (Engelking et al., 2007): mg fungal C g -1 dry weight = (mmol glucosamine − mmol muramic acid) × 179.2 g mol -1 × 9, where 179.2 is the molecular weight of glucosamine and 9 the conversion value of fungal glucosamine to fungal C (Appuhn and Joergensen, 2006). Bacterial C (µg g -1 dry weight) was calculated as an index for bacterial residues by multiplying the
5. Microbial biomass in faeces of dairy cows affected by a nitrogen deficient diet 42 concentration of muramic acid in µg g -1 dry weight by 45 (Appuhn and Joergensen, 2006). 5.2.6. Statistical analysis The results presented in the tables are arithmetic means and expressed on an oven- dry basis (about 72 h at 60°C). The significance of difference was tested by one-way analysis of variance for repeated measures. All statistical analyses were performed using JMP 7.0 (SAS Inst. Inc.). 5.3 Results The dry matter percentage of faeces samples ranged between 13 and 17 % (data not shown). Mean organic C (data not shown) and N content averaged 604 and 29 mg g -1 DM, respectively, without significant differences between treatments (Table 2). The mean faecal pH-value was significantly higher in NB (6.7, P
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Fütterungsbedingte mikrobielle Zus
Page 5 and 6: Mis queridos
Page 7: Vorwort Die vorliegende Dissertatio
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Page 12 and 13: Abbildungsverzeichnis Abb. 1a, b Pi
Page 14 and 15: Abkürzungsverzeichnis ADF Säure-D
Page 16 and 17: 1. Einleitung 1 1. Einleitung In de
Page 18 and 19: 1. Einleitung 3 Stickstoff aus mikr
Page 20 and 21: 2. Ziele der Arbeit 5 auf die Kotqu
Page 22 and 23: 3. Methodik und Vorversuche 7 Der E
Page 24 and 25: 3. Methodik und Vorversuche 9 (Tabe
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Page 98 and 99: 7. Zusammenfassung 83 signifikant n
Page 100 and 101: 8. Summary 85 ratio, which differed
Page 102 and 103: 9. Schlussfolgerung und Ausblick 87
Page 104 and 105: 10. Literatur 89 Engelking, B., Fle
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10. Literatur 91 Vandermeer, J., 19
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11. Danksagung 93 Meiner Bürokolle
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Fütterungsbedingte mikrobielle Zusammensetzung von Rinderkot ...

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