Experimental Study of Biodegradation of Ethanol and Toluene Vapors

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CHAPTER THREE – EXPERIMENTAL STUDIES This chapter describes the materials and methods used in the experimental studies of this research. It covers the microorganisms, the growth nutrients and culture, and the organic chemicals used in this project. The analytical methods and the experimental procedures applied in this study will also be discussed in this section. 3.1 Microorganisms and Chemicals 3.1.1 Microorganism and Medium Strains of Pseudomonas are well known for their ability to degrade aromatic hydrocarbons (Stanier et al., 1966; Reardon et al., 1994, 2000; Brown et al., 2000). Pseudomonas putida (ATCC 23973), which was used in this study, was kindly provided by Dr. W. A. Brown at McGill University (Montreal, Canada). Pictures of these bacteria were taken using a 505 Philips (Holland) scanning electron microscope (SEM) in the Department of Biology, University of Saskatchewan. The preparation of slides for SEM were based on the procedure reported by Klainer and Betsch (1970) and Hill (1974). The unicellular rod shape of P. putida is easily observed with the SEM pictures (See Figures B-1 and B-2). The nutrient medium contained (grams in 1 litre of deionized, reverse osmosis water): KH 2 PO 4 , 1.5; K 2 HPO 4 , 5.0; (NH 4 ) 2 SO 4 , 2.0; NaCl, 5.0; CaCl 2 , 0.06; MgSO 4 •7H 2 O, 0.2; 0.1mL trace element solution (Dr. Brown, McGill University). The trace 32
element solution consisted of (grams in 100 mL water): Na 2 MoO 4 • 2H 2 O, 0.5; MnSO 4 , 0.5; CuSO 4 , 0.5; FeCl 3 , 0.5. The medium was prepared by mixing the appropriate inorganic chemicals with one liter of distilled water that resulted in a buffered medium with a starting pH of 6.7-6.9. The media, culture flasks and bioreactor were sterilized at 121 o C for at least 30 minutes and then the organic compounds were added in a biofilter cabinet to reach the desired initial concentrations. 3.1.2 Organic Chemicals For all bioremediation experiments, only benzyl alcohol, ethanol and/or toluene were used as carbon sources. Ethanol (100%, Commercial Alcohols Inc., Brampton, Canada), toluene (99.5%, BDH, Toronto, Canada) and benzyl alcohol (99%, Aldrich Chemical Co., Milwaukee, USA) were used as the growth carbon sources. 1-butanol (99.4%, BDH, Inc., Toronto, Canada) was used as internal standard to prepare liquid samples for gas chromatography (GC) analyses. Antifoam “B” (BDH, Toronto, Canada) was added to the bioreactor at a concentration of approximately 1.5 g/L to prevent foam formation. Experimental observations in this study confirmed that the cells do not grow on Antifoam “B”. The effect of Antifoam “B” on mass transfer was not studied. 3.2 Growth of Microorganisms 3.2.1 Shake Flask The microorganisms used in the experiments were Pseudomonas putida (ATCC 23973). To propagate bacteria from the agar plates, a 250-mL Erlenmeyer flask with 100-150 mL sterile medium containing a certain amount of substrate (ethanol or benzyl 33
Page 1 and 2: BIOREMEDIATION OF VOLATILE ORGANIC
Page 3 and 4: ABSTRACT The mass transfer of ethan
Page 5 and 6: effect on the growth of P. putida.
Page 7 and 8: ACKNOWLEDGMENT Of the countless peo
Page 9 and 10: TABLE OF CONTENTS PERMISSION TO USE
Page 11 and 12: 4.3 Batch Growth on Ethanol and Ben
Page 13 and 14: LIST OF FIGURES Figure 3-1. Schemat
Page 15 and 16: Figure 5.3 (b) Simulation of ethano
Page 17 and 18: Figure 5-16. Continuous removal of
Page 19 and 20: NOMENCLATURE a - Interfacial area,
Page 21 and 22: CHAPTER ONE - INTRODUCTION 1.1 Air
Page 23 and 24: esults (van Groenestijn and Hesseli
Page 25 and 26: experimental results are presented
Page 27 and 28: Table 2.2 VOC Emissions in the U.S.
Page 29 and 30: (paint shop effluents, for instance
Page 31 and 32: combustion provides 70 to 90% air p
Page 33 and 34: used to degrade ethanol vapours and
Page 35 and 36: g m -3 . This would suggest that th
Page 37 and 38: treatment, This excessive biomass f
Page 39 and 40: The simultaneous degradation of tol
Page 41 and 42: emoves only water-soluble gases. Wa
Page 43 and 44: 2.2.6 Pseudomonas It is well known
Page 45 and 46: 2.3.2 Growth Models with Inhibition
Page 47 and 48: where µ is the total specific grow
Page 49 and 50: 2.4.1 Batch Reactor In a batch reac
Page 51: discharged from the other end. The
Page 55 and 56: growth on ethanol, 20-48 hours for
Page 57 and 58: Gas phase samples of toluene were c
Page 59 and 60: Inlet sampler Exit sampler To fume
Page 61 and 62: Once the oxygen meter indicated a s
Page 63 and 64: centrifuged and the supernatant was
Page 65 and 66: where k L a is the volumetric mass
Page 67 and 68: Figure 4-2a shows ethanol liquid co
Page 69 and 70: coefficient (k L a) in the well-mix
Page 71 and 72: calculated by dividing the gas volu
Page 73 and 74: Average bubble diameter, mm 8 7 6 5
Page 75 and 76: Bioflow III bioreactor. Equation (4
Page 77 and 78: 3.5 y=Ln(S0/S) 3 2.5 2 1.5 1 y = 12
Page 79 and 80: Ethanol and biomass concentration,
Page 81 and 82: 4.3.2 Batch Growth on Benzyl Alcoho
Page 83 and 84: Biomass and benzyl alcohol concentr
Page 85 and 86: ethanol loading was beyond the maxi
Page 87 and 88: Table 4-2. Comparison of bioremedia
Page 89 and 90: Biomass, benzyl alcohol and toluene
Page 91 and 92: 4.4.3 Toluene-ethanol Bioremediatio
Page 93 and 94: Substrate and biomass concentration
Page 95 and 96: Biomass and benzyl alcohol concentr
Page 97 and 98: Biomass and benzoic acid concentrat
Page 99 and 100: ethanol. These results clearly indi
Page 101 and 102: CHAPTER FIVE - MATHEMATICAL MODELIN
Page 103 and 104:
The stoichiometry of the biomass sy
Page 105 and 106:
synthesis and in the ethanol catabo
Page 107 and 108:
1 max Y ex = − 0.635 + 2δ + K 3
Page 109 and 110:
Biomass Eq.5-18 Benzyl Alcohol Eq.5
Page 111 and 112:
r x r N -1.27 -1 0 1 0 0 -0.2 0 0 2
Page 113 and 114:
φ = −θ b ⋅ν (5-31) φ b = 1.
Page 115 and 116:
The data sets used to evaluate the
Page 117 and 118:
Figures 5.4a and 5.4b present 95% c
Page 119 and 120:
Ethanol and biomass concentration (
Page 121 and 122:
Ethanol measured (C-mol/L) 0.3 0.25
Page 123 and 124:
Figures 5.5 (a-d) demonstrate the s
Page 125 and 126:
Benzyl alcohol and biomass concentr
Page 127 and 128:
Benzyl alcohol measured (C-mol/L) 0
Page 129 and 130:
x = 5 ∑ i= 1 x N i (5-40) where x
Page 131 and 132:
ethanol in a steady state mode is l
Page 133 and 134:
where ν 1 , ν 2 , ν 3, ν 4 are
Page 135 and 136:
Biomass concentration, g/L 1.5 1 0.
Page 137 and 138:
φ T =ν 4 (5-53b) φx = −ν 1 (5
Page 139 and 140:
In this study, a metabolic model ha
Page 141 and 142:
Equations (5-34) and (5-35) and the
Page 143 and 144:
ATP 1 = Y max ATP r x + m ATP ⋅ C
Page 145 and 146:
Ethanol, biomass and ammonium conce
Page 147 and 148:
Figure 5-13a shows that the lower t
Page 149 and 150:
Biomass, substrate concentrations,
Page 151 and 152:
C e DKe = (5-63) μ − D m Equatio
Page 153 and 154:
corresponds with the value associat
Page 155 and 156:
Biomass and ethanol concentrations
Page 157 and 158:
than 4.0 mg/L at all dilution rates
Page 159 and 160:
Biomass, benzyl alcohol and ammonia
Page 161 and 162:
negligible ( y out , e = 0) , and e
Page 163 and 164:
Figure 5-17 shows the results at a
Page 165 and 166:
Biomass Concentrations, g/L 3.5 3 2
Page 167 and 168:
C x ( 1 max Q max = ⋅{ D( Cb0 −
Page 169 and 170:
1.2 g/L, the system could not be op
Page 171 and 172:
The continuous models can capture a
Page 173 and 174:
• Air stripping coefficients obta
Page 175 and 176:
Toluene acts as an inhibitor on the
Page 177 and 178:
Bruining, W. J., G. E. H. Joosten,
Page 179 and 180:
Falatko, D. M., and J. T. Novak, 19
Page 181 and 182:
Keuning, S., and D. Jager, 1994.
Page 183 and 184:
Orshansky, F., and N. Narkis, 1997.
Page 185 and 186:
Togna, A. P, M. Singh, 1994. “Bio
Page 187 and 188:
APPENDIX A. Important Metabolic Pat
Page 189 and 190:
III. Toluene Pathway (Adapted from
Page 191 and 192:
Appendix B. Calibration Procedure a
Page 193 and 194:
Figure B-2. Pseudomonas putida (ATC
Page 195 and 196:
Appendix B-3. Calibration of Chemic
Page 197 and 198:
Appendix B-4. Calibration of Toluen
Page 199 and 200:
Appendix C. Experimental Results: A
Page 201 and 202:
Probe response time, s 12 10 8 6 4
Page 203 and 204:
Appendix C-2. Fed-batch Operation B
Page 205 and 206:
Appendix D: Mathematical Solutions
Page 207 and 208:
The microscopic model allows the fo
Page 209 and 210:
13δ Y o , = 0.585 2 x = (D-24) (8K
Page 211 and 212:
2 σ = −1.3E − 06 (E-11) Y xe Y
Page 213:
Biomass, ethanol, and acetic acid c
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Experimental Study of Biodegradation of Ethanol and Toluene Vapors

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