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Continuous solvent production from whey permeate using cells of Clostridium acetobutylicum immobilized by adsorption onto bonechar
Continuous solvent production from whey permeate using cells of Clostridium acetobutylicum immobilized by adsorption onto bonechar N. Qureshi and I. S. Maddox Biotechnology Department, Massey University, Palmerston North, New Zealand
(Received 11 December 1986; revised 15 June 1987)
Cells of Clostridium acetobutylicum were immobilized by adsorption onto bonechar and used in a packed bed reactor for the continuous production of solvents from whey permeate. A maximum solvent productivity of 4.1 g l-1 h-1, representing a yield of 0.23 g solvent/g lactose utilized, was observed at a dilution rate of 1.0 h- 1. The reactor was operated under stable conditions for 61 days. High concentrations of lactose in the whey permeate favored solventogenesis, while low concentrations favored acidogenesis.
Keywords:solvent production;wheypermeate; bonechar;cell immobilization Introduction
Materials
There is continuing interest in the production of acetonebutanol-ethanol from various raw materials using newer fermentation technologies such as immobilized cell and cell recycle systems. These techniques result in high biomass concentrations, and thus allow high reactor productivities to be achieved. Various authors have reported the use of calcium alginate-immobilized cells of Clostridium acetobutylicum or C. be(jerinckii for solvent production from glucose, 1- 5 while cell recycle techniques have also been described using this substrate. 6'7 A potential commercial substrate for solvent production is whey permeate, a byproduct of the dairy industry, and several reports are available describing production from this raw material using calcium alginate-immobilized cells.S- lo The economic feasibility of immobilized cell fermentation systems can be realized only when the process is of low capital and operating cost, relatively simple to perform, stable and readily scaled-up. In this context, an immobilization technique has been reported whereby C. acetobutylicum was adsorbed on beechwood shavings and used for solvent production from glucose. 1x The purpose of the present work was to apply this technique to solvent production from whey permeate using the material bonechar, as used in sugar refining, on which to adsorb the bacterial cells.
Cheese whey permeate (lactose concentration 45-50 g 1-1) was obtained from the New Zealand Dairy Research Institute (Palmerston North, New Zealand), while yeast extract was from Difco Laboratories (Detroit, MI, USA). Bonechar, of average particle size 600#m (range 210 /zm-850 #m, nonporous), as supplied by the New Zealand Sugar Co. Ltd. (Auckland, New Zealand).
Materials and methods Organism Clostridium acetobutylicum P262 was obtained from D.
R. Woods (University of Cape Town, South Africa), and was maintained as spores in distilled water at 4°C.
668
Enzyme Microb. Technol., 1987, vol. 9, November
Cell cultivation
Spore suspension (0.1 ml) was inoculated into Cooked Meat Medium (20 ml, Difco Laboratories) supplemented with lactose (10 g 1- t) and heat-shocked at 75°C for 2 min. The culture was incubated at 34°C, anaerobically, until highly motile cells were observed (18-20 h), and was then used to inoculate (0.14).5% inoculum) whey permeate (80 ml) supplemented with yeast extract (5 g 1-1). Upon appearance of highly motile cells (16-18 h at 34°C), this culture was used to inoculate 1.2 liters of the same medium contained in a 2-liter glass vessel installed on a Microferm fermenter unit (New Brunswick Scientific Co., New Brunswick, N J, USA). Fermentation was allowed to proceed for 16-20 h at 34°C. Anaerobic conditions were maintained by sweeping oxygen-free nitrogen gas across the surface of the culture. Cell immobilization
A glass column reactor of 110 × 25mm was packed with bonechar and sterilized in an autoclave at 121°C for 15 min. The initial void volume of the reactor was 15.5 ml. The culture described above was circulated through the reactor, followed by commencement of feed medium (whey permeate supplemented with yeast extract, 5 g 1-1, pH 5.0-5.2) at a dilution rate of 0.25 h - 1 and at 34°C. The 0141 0229/87/110668 04 $03.00 © 1987 Butterworth Publishers
Solvent production from whey permeate: N. Oureshi and I. S. Maddox feed medium was maintained anaerobic by continuously surface-flushing with oxygen-free nitrogen gas. After two days of operation the surface of the bonechar was observed to be covered with a thin layer of cell growth. The dilution rate was then increased to 0.41 h - t and maintained for a further 3-5 days, after which time the bonechar was fully covered with biomass. The dilution rate was then reduced to 0.31 h - t , and the reactor was found to be stable with regard to lactose utilization and solvent production after a further two days of operation. All medium flow was in the upward direction and the reactor was maintained in the vertical position. All results described below were obtained under steady-state conditions, and the reactor productivity was calculated as the effluent solvent concentration x dilution rate (based on total reactor volume).
Analyses Solvents and acids were determined by gas chromatography (Model GC-8A, Shimadzu Corporation, Kyoto, Japan) using a flame ionization detector and a column of Porapak Q. The carrier gas (N2) flowrate was 30ml m i n - t and the column temperature was 200°C. Prior to analysis samples were acidified using orthophosphoric acid, and sec-butanol was added as an internal standard. Lactose was determined by high performance liquid chromatography. ~2
Results Experiments were performed to investigate the effect of dilution rate on the performance of the column reactor. The results are summarized in Figure 1. An increase in dilution rate over the range 0.3-1.0h - t resulted in increased solvent and acid (acetic + butyric) productivities. The maximum solvent productivity observed was 4.1 g 1- t h - 1 at a dilution rate of 1.0 h - 1 (solvent yield 0.23 g/g lactose utilized; lactose utilization 30%). This corresponded to concentrations in the effluent of acetone, 1.3 g 1- t, butanol, 2.7 g 1- ~, and ethanol, 0.1 g 1- t. Solvent yield and lactose utilization, however, showed maxima at a dilution rate of 0.4 h - t , corresponding to a yield of 0.32 g solvents/g lactose utilized, and 44% lactose utilization. At dilution rates higher than 0.4 h-1, therefore, increased solvent productivity was obtained at the expense of lactose utilization and solvent yield.
Since one of the advantages of using immobilized cells in a continuous flow fermentation system is the avoidance of cell washout, experiments were performed to determine the requirement for yeast extract supplementation of the whey permeate feed medium. Initially, the reactor was started with a yeast extract concentration of 5 g l-1 of whey permeate. After the biomass had accumulated, a steady-state condition was attained at a dilution rate of 0.3 h - t. The yeast extract concentration was then reduced in increments, allowing a steady-state to be achieved at each stage. The feed medium pH value was maintained at pH 5.25 and the temperature at 34°C. Figure 2 shows the effect of yeast extract concentration on the solvent and acid productivities and the solvent yield. At a concentration of 1 g 1-t or above, these parameters showed little variation. However, below a yeast extract concentration of 1 g 1-1, a reduction in solvent productivity was observed, with a simultaneous increase in the ratio of total acids: total solvents from 0.6 to 0.8. The results in Figure I had shown that operation of the reactor at high dilution rates resulted in high solvent productivity, but was accompanied by increased concentrations of residual lactose in the effluent stream. One possible technique to overcome this problem is to reduce the lactose concentration in the feed medium, i.e., dilute the whey permeate. Thus, experiments were performed at a dilution rate of 0.6 h - t where the lactose concentration was reduced incrementally by diluting the whey permeate but maintaining the yeast extract concentration at 5 g 1- t A steady-state was achieved at each stage, and the effects on acid and solvent productivities are shown in Figure 3.
1.6
A
A
•
Ti T
1.2
0.6 -o
•~
0.8
0.4
0.4
0.2 co
>
O
o
O
tt
0 0 Y e a s t e x t r a c t , gl - t
F i g u r e 2 Effect of yeast extract concentration on reactor performance Total solvent productivity, A ; total acid productivity, • ; solvent yield, A
5 T
"4
7
/
~3
0.3
--~
z= >
1
• 0.2
7
0.4
0.6
0.8
4 Acldogenlc
3
I Solventogenic IA~ A-
a~
>;
40
2
20 o ,-J
0.27
7e=
1.0
Dilution rate. h - 1 F i g u r e 1 Effect of dilution rate on reactor performance. Butanol productivity, (3; total solvent productivity, A ; total acid productivity, • ; solvent yield, A ; lactose utilization, [ ]
O :D "0
o
1
13.
I !
0
I
:
|
20
40
i
60
Lactose, gl-i
F i g u r e 3 Effect of lactose concentration on reactor performance Total solvent productivity, A ; total acid productivity, •
Enzyme M i c r o b Technol, 1987, vol. 9, November
669
Papers 1.0
0.8 .o tr.
0.8
0.4 0.2
0
~
0
20
,
40 Lactose, gl -~
|
60
Figure 4 Effect of lactose concentration on the ratio of acetic acid to butyric acid ( A ) , and of acetone to butanol ( © )
At lactose concentrations greater than 40 g 1-1, the fermentation was solventogenic, whereas at lower lactose concentrations it became acidogenic. Interestingly, the ratio of acetone:butanol in the effluent decreased with lower lactose concentration, as did the ratio of acetic acid:butyric acid (Figure 4). In terms of operational stability the reactor was used in continuous fermentation for a period of 1472 h. During this period, blockage due to excess biomass occurred at the base (840 h) but the problem was solved by inverting the reactor. Otherwise, performance was unaffected. The system was finally stopped because of blockage.
Discussion Immobilization by adsorption is a simple technique whereby cells stick to the immobilization support either by ionic/hydrogen bonding or by an adhesive polymer produced by the cell itself. In adsorbed cell packed bed reactors the flowrate of medium, the medium ingredients (particularly for technical substrates such as cheese whey permeate) and the various conditions of product concentration affect immobilization. Various theories regarding adsorption and desorption have been described. 11 In the present work, cells of C. acetobutylicum were immobilized by adsorption onto bonechar. This support was chosen because of its low cost and availability, and the immobilization technique has proved to be simple and
Table 1
mild. In addition, there were few problems of gas hold-up within the reactor. The accumulation of biomass within the reactor, and the attainment of steady state conditions, took approximately seven days. Biomass accumulation is favored by operating at dilution rates high enough so that growth-inhibitory concentrations of solvent are not formed. Once biomass is accumulated, however, dilution rates may be decreased so that excessive biomass growth is prevented by inhibitory solvent concentration. The maximum solvent productivity attained in the reactor was 4.1 g 1-1 h-1 at a dilution rate of 1.0 h -1. This value compares favorably with those obtained using other immobilization techniques (Table 1). When using whey permeate as a substrate for solvent production, most workers have added supplementary yeast extract, presumably as a nutrient source. Batch fermentations without supplementary yeast extract perform less well.1a In the present study it has been shown that a minimum yeast extract supplementation of 1 g 1-1 must be used when using cheese whey permeate in order to maintain a higher solvent productivity. Free cells were continuously released from the reactor, showing that continual cell growth occurs. The high solvent productivity attained in the reactor was at the cost of incomplete lactose utilization. Attempts to avoid this problem by dilution of the lactose in the whey permeate led to the fermentation changing from being solventogenic to acidogenic. Similar observations have been made by other workers when using whey permeate or lactose as a substrate? 4'15 Simultaneously, there was a change in the ratio of acetone:butanol in the effluent stream, and similar changes have also been observed previously.16 However, the precise reasons for these changes are not clear. In conclusion, the technique of immobilizing cells of C. acetobutylicum on bonechar for solvent production appears to have several advantages over other techniques. Bonechar is a readily available and cheap support material. No other material or chemical is required for cell immobilization and the process is simple and mild. Finally, operation of the reactor is simple, high productivities are attained and the process is stable for long periods.
Acknowledgement N.Q. thanks the New Zealand University Grants Committee for a post-doctoral fellowship to support this work.
Solvent production by Clostridium acetobutylicum in various types of reactors
Fermenter mode
Substrate
Productivity gl 1 h 1
Yield gg-1
Batch Continuous/alginate entrapment Continuous/alginate entrapment Continuous/bonechar adsorption Continuous/beechwood shavings adsorption Continuous/cell recycle Continuous/cell recycle
whey permeate whey permeate
0.29 1.0
0.28
whey permeate
1.79
0.27
650
10
whey permeate
4.1
0.23
1472
this work
glucose
1.53
0.26
864
11
glucose
6.5
0.37
200
6
glucose
5.4
0.3
670
Enzyme Microb. Technol., 1987, vol. 9, November
Working period, h
Reference
13 8
7
Solvent production from whey permeate. N. Oureshi and I. S. Maddox
References 1 Haggstrom, L. and Molin, N. BiotechnoL Lett. 1980, 2, 241-246 2 Krouwel, P. G., van der Laan, W. F. M. and Kossen, N. W. F. Biotechnol. Lett. 1980, 2, 253-258 3 Krouwel, P. G. et al. Enzyme Microb. Technol. 1983, 5, 46-54. 4 Forberg, C., Enfors, S.-O. and Haggstrom, L. Eur. J. ,4ppl. Microbiol. Biotechnol. 1983, 17, 143-147 5 Largier, S. T. et al. Appl. Environ. Microbiol. 1985, 50, 477-481 6 Pierrot, P., Fick, M. and Engasser, J. M. Biotechnol. Lett. 1986, 8, 253-256 7 Afschar, A. S. et al. Appl. Microbiol. Biotechnol. 1985, 22, 394-398. 8 Schoutens, G. H., Nieuwenhuizen, M. C. H. and Kossen, N. W. F. Appl. Microbiol. Biotechnol. 1985, 21, 282-286
9 Schoutens, G. H. and Kossen, N. W. F. Chem. Eng. J. (Lausanne) 1986, 32, B51-56 10 Ennis, B. M., Maddox, I. S. and Schoutens, G. H. New Zealand J. Dairy Sci. Technol. 1986, 21, 99-109 11 Forberg, C. and Haggstrom, L. Enzyme Microb. Technol. 1985, 7, 230-234 12 Ennis, B. M. and Maddox, I. S. Biotechnol. Lett. 1985, 7, 601-606 13 Maddox, I. S. Biotechnol. Lett. 1980, 2, 493-498 14 Ennis, B. M. and Maddox, I. S. BiotechnoL Bioeng. 1987, 29, 329-334 15 Welsh, F. W. and Veliky, I. A. Biotechnol. Lett. 1986, 8, 43-46 16 Bahl, H. et al. Appl. Environ. Microbiol. 1986, 52, 169-172
Enzyme Microb. Technol., 1987, vol. 9, November
671
(Received 11 December 1986; revised 15 June 1987)
Cells of Clostridium acetobutylicum were immobilized by adsorption onto bonechar and used in a packed bed reactor for the continuous production of solvents from whey permeate. A maximum solvent productivity of 4.1 g l-1 h-1, representing a yield of 0.23 g solvent/g lactose utilized, was observed at a dilution rate of 1.0 h- 1. The reactor was operated under stable conditions for 61 days. High concentrations of lactose in the whey permeate favored solventogenesis, while low concentrations favored acidogenesis.
Keywords:solvent production;wheypermeate; bonechar;cell immobilization Introduction
Materials
There is continuing interest in the production of acetonebutanol-ethanol from various raw materials using newer fermentation technologies such as immobilized cell and cell recycle systems. These techniques result in high biomass concentrations, and thus allow high reactor productivities to be achieved. Various authors have reported the use of calcium alginate-immobilized cells of Clostridium acetobutylicum or C. be(jerinckii for solvent production from glucose, 1- 5 while cell recycle techniques have also been described using this substrate. 6'7 A potential commercial substrate for solvent production is whey permeate, a byproduct of the dairy industry, and several reports are available describing production from this raw material using calcium alginate-immobilized cells.S- lo The economic feasibility of immobilized cell fermentation systems can be realized only when the process is of low capital and operating cost, relatively simple to perform, stable and readily scaled-up. In this context, an immobilization technique has been reported whereby C. acetobutylicum was adsorbed on beechwood shavings and used for solvent production from glucose. 1x The purpose of the present work was to apply this technique to solvent production from whey permeate using the material bonechar, as used in sugar refining, on which to adsorb the bacterial cells.
Cheese whey permeate (lactose concentration 45-50 g 1-1) was obtained from the New Zealand Dairy Research Institute (Palmerston North, New Zealand), while yeast extract was from Difco Laboratories (Detroit, MI, USA). Bonechar, of average particle size 600#m (range 210 /zm-850 #m, nonporous), as supplied by the New Zealand Sugar Co. Ltd. (Auckland, New Zealand).
Materials and methods Organism Clostridium acetobutylicum P262 was obtained from D.
R. Woods (University of Cape Town, South Africa), and was maintained as spores in distilled water at 4°C.
668
Enzyme Microb. Technol., 1987, vol. 9, November
Cell cultivation
Spore suspension (0.1 ml) was inoculated into Cooked Meat Medium (20 ml, Difco Laboratories) supplemented with lactose (10 g 1- t) and heat-shocked at 75°C for 2 min. The culture was incubated at 34°C, anaerobically, until highly motile cells were observed (18-20 h), and was then used to inoculate (0.14).5% inoculum) whey permeate (80 ml) supplemented with yeast extract (5 g 1-1). Upon appearance of highly motile cells (16-18 h at 34°C), this culture was used to inoculate 1.2 liters of the same medium contained in a 2-liter glass vessel installed on a Microferm fermenter unit (New Brunswick Scientific Co., New Brunswick, N J, USA). Fermentation was allowed to proceed for 16-20 h at 34°C. Anaerobic conditions were maintained by sweeping oxygen-free nitrogen gas across the surface of the culture. Cell immobilization
A glass column reactor of 110 × 25mm was packed with bonechar and sterilized in an autoclave at 121°C for 15 min. The initial void volume of the reactor was 15.5 ml. The culture described above was circulated through the reactor, followed by commencement of feed medium (whey permeate supplemented with yeast extract, 5 g 1-1, pH 5.0-5.2) at a dilution rate of 0.25 h - 1 and at 34°C. The 0141 0229/87/110668 04 $03.00 © 1987 Butterworth Publishers
Solvent production from whey permeate: N. Oureshi and I. S. Maddox feed medium was maintained anaerobic by continuously surface-flushing with oxygen-free nitrogen gas. After two days of operation the surface of the bonechar was observed to be covered with a thin layer of cell growth. The dilution rate was then increased to 0.41 h - t and maintained for a further 3-5 days, after which time the bonechar was fully covered with biomass. The dilution rate was then reduced to 0.31 h - t , and the reactor was found to be stable with regard to lactose utilization and solvent production after a further two days of operation. All medium flow was in the upward direction and the reactor was maintained in the vertical position. All results described below were obtained under steady-state conditions, and the reactor productivity was calculated as the effluent solvent concentration x dilution rate (based on total reactor volume).
Analyses Solvents and acids were determined by gas chromatography (Model GC-8A, Shimadzu Corporation, Kyoto, Japan) using a flame ionization detector and a column of Porapak Q. The carrier gas (N2) flowrate was 30ml m i n - t and the column temperature was 200°C. Prior to analysis samples were acidified using orthophosphoric acid, and sec-butanol was added as an internal standard. Lactose was determined by high performance liquid chromatography. ~2
Results Experiments were performed to investigate the effect of dilution rate on the performance of the column reactor. The results are summarized in Figure 1. An increase in dilution rate over the range 0.3-1.0h - t resulted in increased solvent and acid (acetic + butyric) productivities. The maximum solvent productivity observed was 4.1 g 1- t h - 1 at a dilution rate of 1.0 h - 1 (solvent yield 0.23 g/g lactose utilized; lactose utilization 30%). This corresponded to concentrations in the effluent of acetone, 1.3 g 1- t, butanol, 2.7 g 1- ~, and ethanol, 0.1 g 1- t. Solvent yield and lactose utilization, however, showed maxima at a dilution rate of 0.4 h - t , corresponding to a yield of 0.32 g solvents/g lactose utilized, and 44% lactose utilization. At dilution rates higher than 0.4 h-1, therefore, increased solvent productivity was obtained at the expense of lactose utilization and solvent yield.
Since one of the advantages of using immobilized cells in a continuous flow fermentation system is the avoidance of cell washout, experiments were performed to determine the requirement for yeast extract supplementation of the whey permeate feed medium. Initially, the reactor was started with a yeast extract concentration of 5 g l-1 of whey permeate. After the biomass had accumulated, a steady-state condition was attained at a dilution rate of 0.3 h - t. The yeast extract concentration was then reduced in increments, allowing a steady-state to be achieved at each stage. The feed medium pH value was maintained at pH 5.25 and the temperature at 34°C. Figure 2 shows the effect of yeast extract concentration on the solvent and acid productivities and the solvent yield. At a concentration of 1 g 1-t or above, these parameters showed little variation. However, below a yeast extract concentration of 1 g 1-1, a reduction in solvent productivity was observed, with a simultaneous increase in the ratio of total acids: total solvents from 0.6 to 0.8. The results in Figure I had shown that operation of the reactor at high dilution rates resulted in high solvent productivity, but was accompanied by increased concentrations of residual lactose in the effluent stream. One possible technique to overcome this problem is to reduce the lactose concentration in the feed medium, i.e., dilute the whey permeate. Thus, experiments were performed at a dilution rate of 0.6 h - t where the lactose concentration was reduced incrementally by diluting the whey permeate but maintaining the yeast extract concentration at 5 g 1- t A steady-state was achieved at each stage, and the effects on acid and solvent productivities are shown in Figure 3.
1.6
A
A
•
Ti T
1.2
0.6 -o
•~
0.8
0.4
0.4
0.2 co
>
O
o
O
tt
0 0 Y e a s t e x t r a c t , gl - t
F i g u r e 2 Effect of yeast extract concentration on reactor performance Total solvent productivity, A ; total acid productivity, • ; solvent yield, A
5 T
"4
7
/
~3
0.3
--~
z= >
1
• 0.2
7
0.4
0.6
0.8
4 Acldogenlc
3
I Solventogenic IA~ A-
a~
>;
40
2
20 o ,-J
0.27
7e=
1.0
Dilution rate. h - 1 F i g u r e 1 Effect of dilution rate on reactor performance. Butanol productivity, (3; total solvent productivity, A ; total acid productivity, • ; solvent yield, A ; lactose utilization, [ ]
O :D "0
o
1
13.
I !
0
I
:
|
20
40
i
60
Lactose, gl-i
F i g u r e 3 Effect of lactose concentration on reactor performance Total solvent productivity, A ; total acid productivity, •
Enzyme M i c r o b Technol, 1987, vol. 9, November
669
Papers 1.0
0.8 .o tr.
0.8
0.4 0.2
0
~
0
20
,
40 Lactose, gl -~
|
60
Figure 4 Effect of lactose concentration on the ratio of acetic acid to butyric acid ( A ) , and of acetone to butanol ( © )
At lactose concentrations greater than 40 g 1-1, the fermentation was solventogenic, whereas at lower lactose concentrations it became acidogenic. Interestingly, the ratio of acetone:butanol in the effluent decreased with lower lactose concentration, as did the ratio of acetic acid:butyric acid (Figure 4). In terms of operational stability the reactor was used in continuous fermentation for a period of 1472 h. During this period, blockage due to excess biomass occurred at the base (840 h) but the problem was solved by inverting the reactor. Otherwise, performance was unaffected. The system was finally stopped because of blockage.
Discussion Immobilization by adsorption is a simple technique whereby cells stick to the immobilization support either by ionic/hydrogen bonding or by an adhesive polymer produced by the cell itself. In adsorbed cell packed bed reactors the flowrate of medium, the medium ingredients (particularly for technical substrates such as cheese whey permeate) and the various conditions of product concentration affect immobilization. Various theories regarding adsorption and desorption have been described. 11 In the present work, cells of C. acetobutylicum were immobilized by adsorption onto bonechar. This support was chosen because of its low cost and availability, and the immobilization technique has proved to be simple and
Table 1
mild. In addition, there were few problems of gas hold-up within the reactor. The accumulation of biomass within the reactor, and the attainment of steady state conditions, took approximately seven days. Biomass accumulation is favored by operating at dilution rates high enough so that growth-inhibitory concentrations of solvent are not formed. Once biomass is accumulated, however, dilution rates may be decreased so that excessive biomass growth is prevented by inhibitory solvent concentration. The maximum solvent productivity attained in the reactor was 4.1 g 1-1 h-1 at a dilution rate of 1.0 h -1. This value compares favorably with those obtained using other immobilization techniques (Table 1). When using whey permeate as a substrate for solvent production, most workers have added supplementary yeast extract, presumably as a nutrient source. Batch fermentations without supplementary yeast extract perform less well.1a In the present study it has been shown that a minimum yeast extract supplementation of 1 g 1-1 must be used when using cheese whey permeate in order to maintain a higher solvent productivity. Free cells were continuously released from the reactor, showing that continual cell growth occurs. The high solvent productivity attained in the reactor was at the cost of incomplete lactose utilization. Attempts to avoid this problem by dilution of the lactose in the whey permeate led to the fermentation changing from being solventogenic to acidogenic. Similar observations have been made by other workers when using whey permeate or lactose as a substrate? 4'15 Simultaneously, there was a change in the ratio of acetone:butanol in the effluent stream, and similar changes have also been observed previously.16 However, the precise reasons for these changes are not clear. In conclusion, the technique of immobilizing cells of C. acetobutylicum on bonechar for solvent production appears to have several advantages over other techniques. Bonechar is a readily available and cheap support material. No other material or chemical is required for cell immobilization and the process is simple and mild. Finally, operation of the reactor is simple, high productivities are attained and the process is stable for long periods.
Acknowledgement N.Q. thanks the New Zealand University Grants Committee for a post-doctoral fellowship to support this work.
Solvent production by Clostridium acetobutylicum in various types of reactors
Fermenter mode
Substrate
Productivity gl 1 h 1
Yield gg-1
Batch Continuous/alginate entrapment Continuous/alginate entrapment Continuous/bonechar adsorption Continuous/beechwood shavings adsorption Continuous/cell recycle Continuous/cell recycle
whey permeate whey permeate
0.29 1.0
0.28
whey permeate
1.79
0.27
650
10
whey permeate
4.1
0.23
1472
this work
glucose
1.53
0.26
864
11
glucose
6.5
0.37
200
6
glucose
5.4
0.3
670
Enzyme Microb. Technol., 1987, vol. 9, November
Working period, h
Reference
13 8
7
Solvent production from whey permeate. N. Oureshi and I. S. Maddox
References 1 Haggstrom, L. and Molin, N. BiotechnoL Lett. 1980, 2, 241-246 2 Krouwel, P. G., van der Laan, W. F. M. and Kossen, N. W. F. Biotechnol. Lett. 1980, 2, 253-258 3 Krouwel, P. G. et al. Enzyme Microb. Technol. 1983, 5, 46-54. 4 Forberg, C., Enfors, S.-O. and Haggstrom, L. Eur. J. ,4ppl. Microbiol. Biotechnol. 1983, 17, 143-147 5 Largier, S. T. et al. Appl. Environ. Microbiol. 1985, 50, 477-481 6 Pierrot, P., Fick, M. and Engasser, J. M. Biotechnol. Lett. 1986, 8, 253-256 7 Afschar, A. S. et al. Appl. Microbiol. Biotechnol. 1985, 22, 394-398. 8 Schoutens, G. H., Nieuwenhuizen, M. C. H. and Kossen, N. W. F. Appl. Microbiol. Biotechnol. 1985, 21, 282-286
9 Schoutens, G. H. and Kossen, N. W. F. Chem. Eng. J. (Lausanne) 1986, 32, B51-56 10 Ennis, B. M., Maddox, I. S. and Schoutens, G. H. New Zealand J. Dairy Sci. Technol. 1986, 21, 99-109 11 Forberg, C. and Haggstrom, L. Enzyme Microb. Technol. 1985, 7, 230-234 12 Ennis, B. M. and Maddox, I. S. Biotechnol. Lett. 1985, 7, 601-606 13 Maddox, I. S. Biotechnol. Lett. 1980, 2, 493-498 14 Ennis, B. M. and Maddox, I. S. BiotechnoL Bioeng. 1987, 29, 329-334 15 Welsh, F. W. and Veliky, I. A. Biotechnol. Lett. 1986, 8, 43-46 16 Bahl, H. et al. Appl. Environ. Microbiol. 1986, 52, 169-172
Enzyme Microb. Technol., 1987, vol. 9, November
671