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Appendix 119 ___________________________________________________________________________ 7 APPENDIX Appendix 120 ___________________________________________________________________________ Appendix 3.1. HPLC method. Appendix 121 ___________________________________________________________________________ Appendix 3.2. Optical density versus dry weight calibration curves. 1.6y = 1.164x1.4 2R = 0.99781.210.80.60.40.200 0.2 0.4 0.6 0.8 1 1.2 1.4Optical Density at 650 nm Figure 7.1. Calibration curve for Clostridium thermolacticum DSM 2910. 0.16y = 0.1451x - 0.00080.142R = 0.99490.120.10.080.060.040.0200 0.2 0.4 0.6 0.8 1 1.2Optical Density at 650 nmFigure 7.2. Calibration curve for Clostridium thermoautotrophicum DSM 7417. Biomass [g/L]Biomass [g/L]Appendix 122 ___________________________________________________________________________ Appendix 3.3. Screening results. 1. Thermoanaerobacter brockii ssp brockii DSM 1457 ...
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Appendix 119 ___________________________________________________________________________
7
APPENDIX
Appendix 120 ___________________________________________________________________________
A
endix 3.1.
HPLC emthod
.
y = 1.164x R 2 = 0.9978
Appendix 121 ___________________________________________________________________________ Appendix 3.2. Optical density versus dry weight calibration curves. 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Optical De ns ity at 650 nm Figure 7.1. Calibration curve for Clostridium thermolacticum DSM 2910. 0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0 0 0.2 0.4 0.6 0.8 1 1.2 Optical Density at 650 nm Figure 7.2. Calibration curve for Clostridium thermoautotrophicum DSM 7417.
y = 0.1451x - 0.0008 R 2 = 0.9949
Appendix 122 ___________________________________________________________________________ Appendix 3.3. Screening results. 1. Thermoanaerobacter brockii ssp brockii DSM 1457 Table 7.1. Composition of the growth medium of Thermoanaerobacter brockii
Basal medium NH 4 Cl NaCl MgCl 2 × 6H 2 O KH 2 PO 4 K 2 HPO 4 Trace elt sol. FeSO 4 × 7H 2 O Resazurin Vitamin sol. Na 2 S × 9H 2 O Yeast extract Trypticase Lactose Distilled water
Quantity Trace element Quantity Vitamin solution Quantity solution 0.9 g Nitrilotriacetic 12.8 g Biotin 2 mg acid 0.9 g FeCl 3 × 4H 2 O 0.2 g Folic acid 2 mg 0.4 g MnCl 2 × 4H 2 O 0.1 g Pyridoxin-HCl 10 mg 0.750 g CoCl 2 × 6H 2 O 0.17 g Thiamin-HCL 5 mg 1.5 g CaCl 2 × 2H 2 O 0.1 g Riboflavin 5 mg 9 ml ZnCl 2 0.1 g Nicotinic acid 5 mg 3 mg CuCl 2 0.02 g D-Ca- 5 mg pantothenate 1 mg H 3 BO 3 0.01 g Vitamin B 12 0.1 mg 5 ml Na 2 MoO 4 × 2H 2 O 0.01 g p-Aminobenzoic 5 mg acid 1 g NiCl 2 × 6H 2 O 0.026 g Lipoic acid 5 mg 3 g NaCl 1 g Distilled water 1000 mL 10 g Na 2 SeO 3 × 5H 2 O 0.02 g ~ 20 g Dist ter 1000 mL illed wa 1000 mL
Appendix 123 ___________________________________________________________________________
H 2
100 90 80 70 60 50 40 30 20 10 0 0 10 20 30 40 50 60 70 80 90 100 Time (h)
CO 2
Ethanol Galactose Lactate
14 12 10 8 6 4 2 0
70 Lactose 60 50 40 30 20 Glucose 10 Acetate 0 0 10 20 30 40 50 60 70 80 90 100 Time (h)
Figure 7.3. Fermentation of Thermoanaerobacter brockii DSM 1457 grown on milk permeate at 65°C, initial pH at 7.4, final pH of 4.76, final biomass concentration of 0.98 g/L.
Appendix 124 ___________________________________________________________________________ 2. Thermoanaerobacter ethanolicus DSM 2246
Table 7.2. Composition of the growth medium of Thermoanaerobacter ethanolicus
Basal medium Tryptone Yeast extract FeSO 4 × 7H 2 O Na 2 SO 3 Na 2 S 2 O 3 × 5H 2 O Resazurin Lactose Distilled water
Quantity 10 g 2 g 0.2 g 0.2 g 0.8 g 1 mg ~ 20 g 1000 mL
Appendix 125 ___________________________________________________________________________
H 2
CO 2
160 9 140 8 120 7 100 6 5 80 4 603 402 201 0 0 0 10 20 30 40 50 60 70 80 90 Time (h)
Cells
Ethanol
70 1.6 Lactose 60 1.4 1.2 50 1 40 Lactate 0.8 30 Galactose 0.6 20 Glucos e 0.4 Acetate 100.2 0 0 0 10 20 30 40 50 60 70 80 90 Time (h) Figure 7.4. Fermentation of Thermoanaerobacter ethanolicus DSM 2246 grown on milk permeate at 65°C, initial pH at 7.57, final pH of 4.71, final biomass concentration of 0.88 g/L.
Appendix 126 ___________________________________________________________________________ 3. Thermoanaerobacter ethanolicus DSM 2355 Composition of the growth medium (see DSM 1457 medium)
CO 2
8 7 6 5 4 3 2 1 0 70
160 140 120 100 H 2 80 60 40 20 0 0 10 20 30 40 50 60 Time (h) 80 4 Cells 70 Ethanol 3.5 Lactose 60 3 50 Galactos e 2.5 40 2 Lactate 30 1.5 20 Glucose 1 Acetate 10 0.5 0 0 0 10 20 30 40 50 60 70 Time (h) Figure 7.5. Fermentation of Thermoanaerobacter ethanolicus DSM 2355 grown on milk permeate at 65°C, initial pH at 7.28, final pH of 4.80, final biomass concentration of 1.31 g/L.
Appendix 127 ___________________________________________________________________________ 4. Thermoanaerobacter finnii DSM 3389 Composition of the growth medium (see DSM 1457 medium)
CO 2
H 2
50 45 40 35 30 25 20 15 10 5 0 0 10 20 30 40 50 60 70 80 90 100 Time (h) 60 Lactose Galactose 50
40 30 Lactate 20 10
Glucose Ethanol
Acetate
12 10 8 6 4 2 0
0 0 10 20 30 40 50 60 70 80 90 100 Time (h) Figure 7.6. Fermentation of Thermoanaerobacter finnii DSM 3389 grown on milk permeate at 65°C, initial pH at 7.41, final pH of 4.76, final biomass concentration of 0.61 g/L.
Appendix 128 ___________________________________________________________________________ 5. Thermoanaerobacter thermohydrosulfuricus DSM 2247 Composition of the growth medium (see DSM 2246 medium)
CO 2
H 2
16 14 12 10 8 6 4 2 0
400 350 300 250 200 150 100 50 0 0 10 20 30 40 50 60 70 80 90 Time (h) 140 120 100 80 Lactose 60 40 Lactate 20 Galactos e Acetate 0 0 10 20 30 40 50 60 70 80 90 Time (h)
Ethanol
Figure 7.7. Fermentation of Thermoanaerobacter thermohydrosulfuricus DSM 2247 grown on milk permeate at 65°C, initial pH at 7.52, final pH of 4.70, final biomass concentration of 1.87 g/L.
400 350 300 250 200 150 100 50 0
Appendix 129 ___________________________________________________________________________ 6. Thermoanaerobacter thermohydrosulfuricus DSM 567 Composition of the growth medium (see DSM 2246 medium) The fermentation is not shown due to the unidentification of certain by-products. 7. Thermoanaerobacterium thermosaccharolyticum DSM 571 Composition of the growth medium (see DSM 2246 medium) 600 H 2 500 400 CO 2 300 200 100 0 0 10 20 30 40 50 60 70 80 90 100 80 70 60 Lactose 50 40 30 20 10 0 0 10 20 30 40 50 60 70 80 90 100 Time (h) Figure 7.8. Fermentation of Thermoanaerobacterium thermosaccharolyticum DSM 571 grown on milk permeate at 60°C, initial pH at 7.70, final pH of 4.82, final biomass concentration of 0.98 g/L
Ethanol
Acetate
