BIOTECHNOLOGY A N D B I O E N G I N E E R I N G
VOL. X V I I (1975)
Production of Alcohol by Bacillus stearothermophilus T h e thermophilic bacterium Bacillus slearolhermophilus (ATCC 7954, NCA 1503, NCIB 8924) has been shown to produce high levels of the enzyme alcohol dehydrogenase,l.z amounting to 1-254 of the soluble cell protein when grown in complex media3 containing sucrose. This enzyme is also produced in continuous culture.' Alcohol dehydrogenase (AIlH) is known to be responsible for the t,erminal step in the formation of alcohol by yeasts, and, in the course of investigat,ions of H . stearothermophilus as a source of thermostable proteins,5 we briefly examined it.s ability t o produce alcohol under anaerobic conditions at high temperatures and the possibility of removing any alcohol so produced by stripping with an inert gas. This study reports alcohol production by h'. stearothermophilus grown in a chemostat' on complex media3 with 3% glucose as the carbon source. The gr0wt.h temperature was 65°C and nitrogen, 0.2 to 2.0 liter/min, was dispersed in the culture (stirrer speed 250 rpm) to establish anaerobic conditions for growth at dilution rates (D)of 0.1 and 0.2 hr-1.
ALCOHOL ESTIMATION The effluent gases from the culture vessel were passed through a reflux condenser and the condensate collected in a chilled container. Alcohol concentration in both the culture supernatant and the condensate was measured by gas-liquid chromatography using a Pye GCV fitted with dual flame ionization detectors. Samples (1 pl) of distillate or culture supernatant were injected onto a column (1.5 m X 4 mm) packed with 10% carbowax 2011 on Iliatomite C HMIlS 60-80 mesh (J.J's. Chromatography Ltd., Kings Lynn, England). Helium (40 ml/min) was used as the carrier gas; the chromatograph was run isothermally (75°C) ; injector and detector temperatures were 100°C and l;iO"C, respectively. T h e alcohol present was measured by comparison of peak heights with authentic alcohol standards of appropriate concentration. T h e attenuation was set to give approximat,ely 709; full scale deflection for each standard used. The results are shown in Table I. Although the t,otal alcohol produced was significantly higher at the higher dilution rate, the amount of alcohol distilled depended almost entirely upon t.he rate of gas flow through the culture (see Table I ) ; doubling the stirrer speed made no significant difference. It is significant that, in contrast to growth under aerobic conditions,' the culture supernatants from anaerobic cultures consistently contained less than O.Ol(% acetate. Itamalingham and Finn6 have described a "vacuferm process" for the recovery of fermentation alcohol from yeast cultures. Our experiments, although not directed to this end, suggest t h a t alcohol production by thermophilic organisms may present a simpler alternative for concentrating the product while simultaneously reducing its concentration within t,he culture, thus permitting the fermentation of normally unacceptably high levels of carbohydrate. The level of alcohol in the distillate was only 5.3% (max 7G/,), but t.his represents a 76-fold concentrat.ion of the alcohol produced by the culture and a consequent, considerable 1375 @ 1975 by John Wiley & Sons, Inc.
.o
18.3 21.6 27.3 17.1 23.0
0.13 0.11 0.02 0.03 0.05
12 hr vol culture from chemostat X EtOH concn
5.3 1.48 4.78 3.07 0.88 0.07 0.07 0.10 0.06 0.10
Glucose used ADH Ethanol (ygv/v) (mg/ml) (p/mg dry wt) condensate supernatant
12 hr vol condensate X EtOH concentration X 100
2.0
1
1.8 1.8 2.6 2.3 2.0
0.2 1.0 0.2
0.1
0.2
Cell dry wt (mg/ml)
Nz flow (liter/min)
~
D (hr-1)
*Recovery ratio (To) =
~~
76 21 48 31 15
Concentration factor
~
3.78 33.1 1.35 26.1 37.6
Recovery ratio (%)a
~~~~
TABLE I Effect of Dilution Rate and Gas Flow Rate Upon the Evolution and Recovery of Alcohol in Anaerobic Cultures of B. stearothermophilus
U
s
r x
c 0
z
9
COMMUNICATIONS TO T H E EDITOR
1377
reduction of the concentration in the culture vessel. This particular organism is obviously not suitable for producing alcohol, but we feel that the discovery of a thermophilic organism capable of an economic conversion of sugar to alcohol could now be very useful. References 1. E. Kobl and J. I. Harris, Biochem. J . , 124,76P (1971). 2. A. Atkinson, B. W. Phillips, p. S. Callow, W. It. Stones, and P. A. Bradford, Riochem. J., 127, 63P (1972). 3. K. Sargeant, D. N. East, A. R. Whitaker, and It. Elsworth, J . Gen. Microbiol., 65, 3 (1971). 4. A. Atkinson, C. G. T. Evans, and R. G. Yeo, J . A p p l . Bacteriol., 38, 301 (1975). 5. A. Atkinson, Process Riochem., 8, (8) 9 (1973). 6. A. Ramalingham and R. K. Finn, “The vacuferm process,” Proc. Amer. Chem. Soc., 168th Meeting, Atlantic City, New Jersey, Sept. 1974.
A. ATKINSON D. C. ELLWOOD C. G. T. EVANS R. G. YEO Microbiological Research Establishment Porton, Salisbury, Wiltshire England Accepted for Publication May 6, 1975
VOL. X V I I (1975)
Production of Alcohol by Bacillus stearothermophilus T h e thermophilic bacterium Bacillus slearolhermophilus (ATCC 7954, NCA 1503, NCIB 8924) has been shown to produce high levels of the enzyme alcohol dehydrogenase,l.z amounting to 1-254 of the soluble cell protein when grown in complex media3 containing sucrose. This enzyme is also produced in continuous culture.' Alcohol dehydrogenase (AIlH) is known to be responsible for the t,erminal step in the formation of alcohol by yeasts, and, in the course of investigat,ions of H . stearothermophilus as a source of thermostable proteins,5 we briefly examined it.s ability t o produce alcohol under anaerobic conditions at high temperatures and the possibility of removing any alcohol so produced by stripping with an inert gas. This study reports alcohol production by h'. stearothermophilus grown in a chemostat' on complex media3 with 3% glucose as the carbon source. The gr0wt.h temperature was 65°C and nitrogen, 0.2 to 2.0 liter/min, was dispersed in the culture (stirrer speed 250 rpm) to establish anaerobic conditions for growth at dilution rates (D)of 0.1 and 0.2 hr-1.
ALCOHOL ESTIMATION The effluent gases from the culture vessel were passed through a reflux condenser and the condensate collected in a chilled container. Alcohol concentration in both the culture supernatant and the condensate was measured by gas-liquid chromatography using a Pye GCV fitted with dual flame ionization detectors. Samples (1 pl) of distillate or culture supernatant were injected onto a column (1.5 m X 4 mm) packed with 10% carbowax 2011 on Iliatomite C HMIlS 60-80 mesh (J.J's. Chromatography Ltd., Kings Lynn, England). Helium (40 ml/min) was used as the carrier gas; the chromatograph was run isothermally (75°C) ; injector and detector temperatures were 100°C and l;iO"C, respectively. T h e alcohol present was measured by comparison of peak heights with authentic alcohol standards of appropriate concentration. T h e attenuation was set to give approximat,ely 709; full scale deflection for each standard used. The results are shown in Table I. Although the t,otal alcohol produced was significantly higher at the higher dilution rate, the amount of alcohol distilled depended almost entirely upon t.he rate of gas flow through the culture (see Table I ) ; doubling the stirrer speed made no significant difference. It is significant that, in contrast to growth under aerobic conditions,' the culture supernatants from anaerobic cultures consistently contained less than O.Ol(% acetate. Itamalingham and Finn6 have described a "vacuferm process" for the recovery of fermentation alcohol from yeast cultures. Our experiments, although not directed to this end, suggest t h a t alcohol production by thermophilic organisms may present a simpler alternative for concentrating the product while simultaneously reducing its concentration within t,he culture, thus permitting the fermentation of normally unacceptably high levels of carbohydrate. The level of alcohol in the distillate was only 5.3% (max 7G/,), but t.his represents a 76-fold concentrat.ion of the alcohol produced by the culture and a consequent, considerable 1375 @ 1975 by John Wiley & Sons, Inc.
.o
18.3 21.6 27.3 17.1 23.0
0.13 0.11 0.02 0.03 0.05
12 hr vol culture from chemostat X EtOH concn
5.3 1.48 4.78 3.07 0.88 0.07 0.07 0.10 0.06 0.10
Glucose used ADH Ethanol (ygv/v) (mg/ml) (p/mg dry wt) condensate supernatant
12 hr vol condensate X EtOH concentration X 100
2.0
1
1.8 1.8 2.6 2.3 2.0
0.2 1.0 0.2
0.1
0.2
Cell dry wt (mg/ml)
Nz flow (liter/min)
~
D (hr-1)
*Recovery ratio (To) =
~~
76 21 48 31 15
Concentration factor
~
3.78 33.1 1.35 26.1 37.6
Recovery ratio (%)a
~~~~
TABLE I Effect of Dilution Rate and Gas Flow Rate Upon the Evolution and Recovery of Alcohol in Anaerobic Cultures of B. stearothermophilus
U
s
r x
c 0
z
9
COMMUNICATIONS TO T H E EDITOR
1377
reduction of the concentration in the culture vessel. This particular organism is obviously not suitable for producing alcohol, but we feel that the discovery of a thermophilic organism capable of an economic conversion of sugar to alcohol could now be very useful. References 1. E. Kobl and J. I. Harris, Biochem. J . , 124,76P (1971). 2. A. Atkinson, B. W. Phillips, p. S. Callow, W. It. Stones, and P. A. Bradford, Riochem. J., 127, 63P (1972). 3. K. Sargeant, D. N. East, A. R. Whitaker, and It. Elsworth, J . Gen. Microbiol., 65, 3 (1971). 4. A. Atkinson, C. G. T. Evans, and R. G. Yeo, J . A p p l . Bacteriol., 38, 301 (1975). 5. A. Atkinson, Process Riochem., 8, (8) 9 (1973). 6. A. Ramalingham and R. K. Finn, “The vacuferm process,” Proc. Amer. Chem. Soc., 168th Meeting, Atlantic City, New Jersey, Sept. 1974.
A. ATKINSON D. C. ELLWOOD C. G. T. EVANS R. G. YEO Microbiological Research Establishment Porton, Salisbury, Wiltshire England Accepted for Publication May 6, 1975
