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Pilot plant production of SCP from sugarcane bagasse
Biological Wastes 20 (1987) 273-280
Pilot Plant Production of SCP from Sugarcane Bagasse Sh. I. EI-Shawarby, E. A. E1-Zanaty, A. H. E1-Refai, F. A. Hamissa & H. Shaker Pilot Plant Laboratory, National Research Dokki, Cairo, Egypt
Centre,
(Received 20 April 1986; accepted 2 October 1986)
ABSTRACT A pilot fermentation unit (about 20 kg scale) was used to produce S C P by microbial conversion of sugarcane bagasse. The bagasse was first treated with 1% N a O H solution for 8 h, then it was dewatered and washed by centrifugation. A rotary drum reactor was used for the fermentation at ambient temperature. Steam was applied to ensure aseptic conditions. A continuous stream of humid air was used to give aerobic conditions and stirring was applied for thorough mixing. The bagasse was inoculated with Trichoderma viride 253-M16 plus a nutrient solution. Samples were withdrawn twice a week, the fermentations lasted for about 28 days and the moisture content was kept constant at about 75-80%. The crude protein content in the product could reach about 28% if the ambient temperature was suitable (i.e. about 30°C).
INTRODUCTION The world protein shortage, together with waste-disposal problems, have greatly stimulated interest and research in the production of unconventional protein-rich food- and feed-stuffs. A range of unconventional potential protein sources (SCP) produced by fermentation has been developing, as mentioned by many investigators such as Mateles & Tannenbaum (1968), Lipinsky & Litchfield (1970), Kihlberg (1972), Gulcho (1973), Davis (1974), Humphery (1974), Tannenbaum & Wang (1975) and Rockwell (1976). As a result of this development, a number 273 Biological Wastes 0269-7483/87/$03.50 © ElsevierApplied SciencePublishers Ltd, England, 1987. Printed in Great Britain
Alkalipreparation . ~ 1"1o- NaOH
~ig. 1.
I .
}~
I
Preparation of nutrients
Fermentation 30°C 21days
]lock flow-sheet for SCP production.
Centrifugation 750 rpm 110kg
,l
Preparation of inoculum
product
SCP
~ater v a p o u r
~rying 80"C
I
,)
Pilot plant production of SCPfrom sugarcane bagasse
275
of large SCP plants are now in operation and several more are under construction (Bunker, 1968; Burrows, 1970; Peppier, 1970 and Solbkowicz, 1976). Underkofler (1969), Wolnak (1972), Dekker & Richards (1973), Cowling (1975), Erikson (1975), Mandels (1975), Berc (1977), Ghose (1977), Fan et al. (1981) and EI-Refai :(1982) have shown that sugar-cane bagasse is one of these wastes which can be fermented to produce a biomass rich in protein. In Egypt, surplus amounts of sugarcane bagasse are produced every year. A programme has been directed by EI-Refai~(1982), in the National Research Centre of Egypt, to increase the protein content and to improve bagasse digestibility for use as an animal fodder. The objective of the present work was to conduct a scaling-up operation to verify the optimum conditions determined in the laboratory, and to obtain representative quantities of SCP for further testing and evaluation. Based on the preliminary laboratory experiments, the scaling-up of the process seemed promising. The production of large amounts of protein on a pilot scale is easily controlled and offers the advantage of optimised process operation and product analyses. The process also allows the production of protein of constant composition which can be tested for safety and quality.
METHODS The process was divided into three main parts--the preparation of the raw material, the fermentation of the bagasse and the drying of the product. Figure 1 shows a schematic of the various steps. Preparation of the raw material Alkali treatment. Bagasse was treated with sodium hydroxide solution [1% (w/w)] for 20 h in plastic barrels, with a solid:liquid ratio of 1:10 (w/v). Each barrel was filled with 5 kg of crude bagasse together with 50 litres of the caustic solution. Centrifugation. To remove the caustic solution, the treated bagasse was centrifuged. About 100 kg of the wet composite was fed to the centrifuge each time and was centrifuged for 15 min. Tap water was then applied for washing until neutrality was reached. The batch was then dewatered by centrifugation for another 15 min. The treated bagasse was then left in the open air at ambient temperature (30°C) for 5 days for further drying. The moisture content of the dried bagasse was about 5%.
H20
;) ~"
l
Nutrients
h (11)
product
SCP
Fig. 2. Equipment flow-sheet for SCP production. (1) Alkali dilution tank; (2) bagasse heap; (3) treatment tank; (4J air compressor; (5) air-filter; (6) centrifuge; (7)inoculum incubator; (8) nutrients tank; (9) fermentor; (10) steam generator; (11) vacuum shelf-dryer.
Air
drain
To ~
,J c6, l
Inoculum
:x
:x
?
t',.,.J O
Pilot plant production of SCP from sugarcane bagasse
277
Fermentation Preparation o f the inoculum The Triehoderma viride 253-M 16 employed was a mutant of the local strain of T. viride that was isolated from crude bagasse by EI-Refai et aL (1981). It was grown in a vegetative medium containing (per litre) 2-6 g (NH4) 2 SO4, 1"0 g calcium citrate, 2-0 g KH2PO4, 0-5 g glucose and 0.1 g peptone. Sufficient medium for inoculation was apportioned into Erlenmeyer flasks (250 ml capacity), each containing 50 ml. The flasks were plugged with cotton wool and sterilized at 121°C for 20 min. When the flasks had cooled to room temperature they were inoculated with a standard spore suspension of the organism. The inoculated flasks were incubated in a shaker incubator (125rpm) at 30°C for 72h. This vegetative medium was used for the inoculation of the fermentation medium. Fermentation medium The medium used as a nutritive solution contained (per litre): 2-4 g (NH4) 2 SO4; 2-0 g KH2PO4, 0.5 g glucose and 1"0 g soybean meal. Fermentation of treated bagasse A horizontal stainless steel drum equipped with an internal stirrer was used as the fermentor (Fig. 2). The drum was insulated and had different inlets for air and steam. There was also a top opening for charging and a side door for discharging. All inlets and outlets were fitted with rubber gaskets for sealing. The fermentor was connected to a steam generator, an air filter and a compressor. The fermentor was charged with 15, 20 or 25 kg, in the three test runs, of the dry, treated bagasse and each batch was mixed thoroughly with the mixer. The nutrient solution (about 2 litres per kilogram of dry bagasse) was then added slowly from the top opening to ensure its homogeneous spreading over the bagasse. Direct steam at about 202 kPa and 121°C was then admitted to the fermentor to produce aseptic conditions for the growth of the fungus. Sterilization of the whole system, including the pipes and air filter, lasted for about 1 h. The fermentor and its contents were then left to cool to room temperature. The inoculum (about 0"7 litres per kilogram of dry bagasse) was then poured over the bagasse while mixing to ensure uniform distribution. Sterile air from the compressor via the air filter was passed through sterile water and fed into the fermentor to keep the previously determined moisture level and aeration for the process. Each batch fermentation continued for about 28 days, during which two samples were withdrawn every week for the determination of crude protein and moisture content. Water saturated air was fed continuously for 6 h day- 1 at a rate of about 2 m 3 m - 3 h - ~, while mixing was applied twice a day for 0"5 h. The moisture content of the fermenting bagasse was 75-80%.
278
Sh. L EI-Shawarby, E. A. El-Zanaty, A. H. EI-Refai, F. A. Hamissa, H. Shaker
Drying the product After completion of the fermentation, which was indicated by a more or less constant protein content, the whole batch was manually transferred into a shelf dryer. Drying was accomplished at a constant temperature of 80°C under vacuum. Figure 2 is a schematic of the equipment flow sheet of the pilot-scale unit.
RESULTS A N D DISCUSSION The results given in Table 1 show an improvement in the nutritional value as judged by the increase in protein content of the crude bagasse. A consistent relationship was recorded between the percentage loss of the hollocellulose (cellulose + hemicellulose) fraction, and the increase of the culture protein with time. A loss of about 8% of the hollocellulose fraction TABLE 1 Changes in N Content and Protein Content of Bagasse During Fermentation Time (days)
N content
Protein content
(%)
(%)
Batch 1 (30°C)
5 10 14 17 21 25 28
2.10 3.02 3.48 2.98 3-64 3.76 4.57
13.12 18.87 21.73 18.61 22.75 23-50 28.60
Batch 2 (15°C)
5 8 12 15 19 25 30
2.11 2.66 2.68 2-87 2.57 2.50 2.59
13-20 16.60 16-75 17-94 16.10 15.60 16.25
Batch 3 (20°C)
3 7 11 17 23 27
2-85 3.33 3-17 2.92 3.75 3.27
17.80 20-80 19-80 18-30 23.44 20.44
N = Kjeldahi N. Protein = N × 6'25.
Pilot plant production of SCP from sugarcane bagasse
279
was attained after 5 days of fermentation, while about a fourfold decrease ( = 32%) of the same fraction was achieved at the end of the process period (28 days). This was accompanied by a parallel increase in the protein content of the biomass, which reached a maximum of 28.5 % at the end of the process for the first batch. The second batch was carried out in December, when the ambient temperature was about 15°C on average as compared with 30°C during the first run. Hence, the maximum protein content was lower at about 16% at the end of the process. The third batch was more successful as the ambient temperature reached 20-22°C and the maximum protein content was 23.4%. These data showed that the highest protein content resulted from fermenting at the highest temperature. This emphasises the importance of temperature for this type of fermentation and shows that, for a continuous process, all the year round, the fermentor would have to be heated and its temperature controlled. The nutritional improvement of the alkali-treated bagasse obtained after 28 days of fermentation was also determined by Farid et al. (1984). This work showed that the nutritive value, expressed as IVDMD, increased from 21.5% to 52.9% as given by Hamessa et al. (1984).
REFERENCES Berc, H. T. (1977). International Symposium on Bioconversions of Cellulosic Substances into Energy; Chemical and Microbial Proteins, Delhi, India. Bunker, H. J. (1968). Single cell protein, MIT Press, Cambridge, Massachusetts, USA, 67. Burrows, S. (1970). The yeasts, Academic Press, London, 349. Cowling, E. B. (1975). Bioteeh. Bioeng. Symp. No. 5 (Wike, C. R. (Ed.)), New York, USA, 165. Davis, P. (1974). Single cell protein, Academic Press, New York, USA. Dekker, R. F. H. & Richards, G. N. (1973). J. Sci. Food Agric., 24, 375. El-Refai, A. H. (1982). Utilization of Cane Sugar Bagasses, Grant No. FG.EG-180 USA, Fifth Annual Research Report, NRC, Cairo, Egypt. Erikson, K. E. (1975). In: Symposium on Enzymatic Hydrolysis of Cellulose, Aulanko, Finland (M. Bailey, T. M. Erani & M. Linko (Eds)), 263. Fan, L. T., Lee, Y. H. & Gharpuray, M. M. (1981). Adv. Biochem. Bioeng., 23, 158. Farid, M., Shaker, H. & E1-Refai, A. H. (1984). Productivity of T. viride 253, Annual Report, NRC, Cairo, Egypt. Ghose, T. K. (1977). Adv. Biochem. Eng., 6, 39. Gulcho, S. (1973). Proteins from hydrocarbons, Noyes Data Corp., Park Ridge, New Jersey, USA. Hamessa, F. A., EI-Dewany, A. I. & E1-Refai, A. H. (1984). Microbios Letters, 26, 129. Humphery, A. E. (1974). Chem. Eng. (N. Y.), 81(26), 98.
280
Sh. I. EI-Shawarby, E. A. EI-Zanaty, A. H. EI-Refai, F. A. Hamissa, H. Shaker
Kihlberg, R. (1972). Ann. Rev., Microbiol., 26, 427. Lipinsky, E. S. & Litchfield, O. H. (1970). Critical Rev. Food TechnoL, 1, 580. Mandels, M. (1975). Biotech. Bioeng. Syrup. No. 5. (Wilke, C. R. (Ed)), New York, USA, 81. Mateles, R. I. & Tannenbaum, S. R. (1968). SCP II, MIT Press, Cambridge, Massachusetts, USA. Peppier, H. J. (1970). The.yeasts, Academic Press, London, 421. Rockwell, P. J. (1976). Single cell protein from cellulose and hydrocarbons, Noyes Data Corp., Park Ridge, New York, USA. Solbkowicz, G. (1976). Food from wastes. (Birch, G. G., Parker, K. J. & Worgan, J. T. (Eds)), Applied Science Publishers, London, 42. Tannenbaum, S. R. & Wang, D. I. C. (1975). Single cell protein II., MIT Press, Cambridge, Massachusetts, USA. Underkofler, L. A. (1969). Adv. Chem. Ser., 95, 343. Wolnak, B. (1972). Present and future technological and commercial status of enzymes. National Foundation Rep. No. NSF/RAX-73-002, USA Research and Development Association, Natick, Massachusetts.
Pilot Plant Production of SCP from Sugarcane Bagasse Sh. I. EI-Shawarby, E. A. E1-Zanaty, A. H. E1-Refai, F. A. Hamissa & H. Shaker Pilot Plant Laboratory, National Research Dokki, Cairo, Egypt
Centre,
(Received 20 April 1986; accepted 2 October 1986)
ABSTRACT A pilot fermentation unit (about 20 kg scale) was used to produce S C P by microbial conversion of sugarcane bagasse. The bagasse was first treated with 1% N a O H solution for 8 h, then it was dewatered and washed by centrifugation. A rotary drum reactor was used for the fermentation at ambient temperature. Steam was applied to ensure aseptic conditions. A continuous stream of humid air was used to give aerobic conditions and stirring was applied for thorough mixing. The bagasse was inoculated with Trichoderma viride 253-M16 plus a nutrient solution. Samples were withdrawn twice a week, the fermentations lasted for about 28 days and the moisture content was kept constant at about 75-80%. The crude protein content in the product could reach about 28% if the ambient temperature was suitable (i.e. about 30°C).
INTRODUCTION The world protein shortage, together with waste-disposal problems, have greatly stimulated interest and research in the production of unconventional protein-rich food- and feed-stuffs. A range of unconventional potential protein sources (SCP) produced by fermentation has been developing, as mentioned by many investigators such as Mateles & Tannenbaum (1968), Lipinsky & Litchfield (1970), Kihlberg (1972), Gulcho (1973), Davis (1974), Humphery (1974), Tannenbaum & Wang (1975) and Rockwell (1976). As a result of this development, a number 273 Biological Wastes 0269-7483/87/$03.50 © ElsevierApplied SciencePublishers Ltd, England, 1987. Printed in Great Britain
Alkalipreparation . ~ 1"1o- NaOH
~ig. 1.
I .
}~
I
Preparation of nutrients
Fermentation 30°C 21days
]lock flow-sheet for SCP production.
Centrifugation 750 rpm 110kg
,l
Preparation of inoculum
product
SCP
~ater v a p o u r
~rying 80"C
I
,)
Pilot plant production of SCPfrom sugarcane bagasse
275
of large SCP plants are now in operation and several more are under construction (Bunker, 1968; Burrows, 1970; Peppier, 1970 and Solbkowicz, 1976). Underkofler (1969), Wolnak (1972), Dekker & Richards (1973), Cowling (1975), Erikson (1975), Mandels (1975), Berc (1977), Ghose (1977), Fan et al. (1981) and EI-Refai :(1982) have shown that sugar-cane bagasse is one of these wastes which can be fermented to produce a biomass rich in protein. In Egypt, surplus amounts of sugarcane bagasse are produced every year. A programme has been directed by EI-Refai~(1982), in the National Research Centre of Egypt, to increase the protein content and to improve bagasse digestibility for use as an animal fodder. The objective of the present work was to conduct a scaling-up operation to verify the optimum conditions determined in the laboratory, and to obtain representative quantities of SCP for further testing and evaluation. Based on the preliminary laboratory experiments, the scaling-up of the process seemed promising. The production of large amounts of protein on a pilot scale is easily controlled and offers the advantage of optimised process operation and product analyses. The process also allows the production of protein of constant composition which can be tested for safety and quality.
METHODS The process was divided into three main parts--the preparation of the raw material, the fermentation of the bagasse and the drying of the product. Figure 1 shows a schematic of the various steps. Preparation of the raw material Alkali treatment. Bagasse was treated with sodium hydroxide solution [1% (w/w)] for 20 h in plastic barrels, with a solid:liquid ratio of 1:10 (w/v). Each barrel was filled with 5 kg of crude bagasse together with 50 litres of the caustic solution. Centrifugation. To remove the caustic solution, the treated bagasse was centrifuged. About 100 kg of the wet composite was fed to the centrifuge each time and was centrifuged for 15 min. Tap water was then applied for washing until neutrality was reached. The batch was then dewatered by centrifugation for another 15 min. The treated bagasse was then left in the open air at ambient temperature (30°C) for 5 days for further drying. The moisture content of the dried bagasse was about 5%.
H20
;) ~"
l
Nutrients
h (11)
product
SCP
Fig. 2. Equipment flow-sheet for SCP production. (1) Alkali dilution tank; (2) bagasse heap; (3) treatment tank; (4J air compressor; (5) air-filter; (6) centrifuge; (7)inoculum incubator; (8) nutrients tank; (9) fermentor; (10) steam generator; (11) vacuum shelf-dryer.
Air
drain
To ~
,J c6, l
Inoculum
:x
:x
?
t',.,.J O
Pilot plant production of SCP from sugarcane bagasse
277
Fermentation Preparation o f the inoculum The Triehoderma viride 253-M 16 employed was a mutant of the local strain of T. viride that was isolated from crude bagasse by EI-Refai et aL (1981). It was grown in a vegetative medium containing (per litre) 2-6 g (NH4) 2 SO4, 1"0 g calcium citrate, 2-0 g KH2PO4, 0-5 g glucose and 0.1 g peptone. Sufficient medium for inoculation was apportioned into Erlenmeyer flasks (250 ml capacity), each containing 50 ml. The flasks were plugged with cotton wool and sterilized at 121°C for 20 min. When the flasks had cooled to room temperature they were inoculated with a standard spore suspension of the organism. The inoculated flasks were incubated in a shaker incubator (125rpm) at 30°C for 72h. This vegetative medium was used for the inoculation of the fermentation medium. Fermentation medium The medium used as a nutritive solution contained (per litre): 2-4 g (NH4) 2 SO4; 2-0 g KH2PO4, 0.5 g glucose and 1"0 g soybean meal. Fermentation of treated bagasse A horizontal stainless steel drum equipped with an internal stirrer was used as the fermentor (Fig. 2). The drum was insulated and had different inlets for air and steam. There was also a top opening for charging and a side door for discharging. All inlets and outlets were fitted with rubber gaskets for sealing. The fermentor was connected to a steam generator, an air filter and a compressor. The fermentor was charged with 15, 20 or 25 kg, in the three test runs, of the dry, treated bagasse and each batch was mixed thoroughly with the mixer. The nutrient solution (about 2 litres per kilogram of dry bagasse) was then added slowly from the top opening to ensure its homogeneous spreading over the bagasse. Direct steam at about 202 kPa and 121°C was then admitted to the fermentor to produce aseptic conditions for the growth of the fungus. Sterilization of the whole system, including the pipes and air filter, lasted for about 1 h. The fermentor and its contents were then left to cool to room temperature. The inoculum (about 0"7 litres per kilogram of dry bagasse) was then poured over the bagasse while mixing to ensure uniform distribution. Sterile air from the compressor via the air filter was passed through sterile water and fed into the fermentor to keep the previously determined moisture level and aeration for the process. Each batch fermentation continued for about 28 days, during which two samples were withdrawn every week for the determination of crude protein and moisture content. Water saturated air was fed continuously for 6 h day- 1 at a rate of about 2 m 3 m - 3 h - ~, while mixing was applied twice a day for 0"5 h. The moisture content of the fermenting bagasse was 75-80%.
278
Sh. L EI-Shawarby, E. A. El-Zanaty, A. H. EI-Refai, F. A. Hamissa, H. Shaker
Drying the product After completion of the fermentation, which was indicated by a more or less constant protein content, the whole batch was manually transferred into a shelf dryer. Drying was accomplished at a constant temperature of 80°C under vacuum. Figure 2 is a schematic of the equipment flow sheet of the pilot-scale unit.
RESULTS A N D DISCUSSION The results given in Table 1 show an improvement in the nutritional value as judged by the increase in protein content of the crude bagasse. A consistent relationship was recorded between the percentage loss of the hollocellulose (cellulose + hemicellulose) fraction, and the increase of the culture protein with time. A loss of about 8% of the hollocellulose fraction TABLE 1 Changes in N Content and Protein Content of Bagasse During Fermentation Time (days)
N content
Protein content
(%)
(%)
Batch 1 (30°C)
5 10 14 17 21 25 28
2.10 3.02 3.48 2.98 3-64 3.76 4.57
13.12 18.87 21.73 18.61 22.75 23-50 28.60
Batch 2 (15°C)
5 8 12 15 19 25 30
2.11 2.66 2.68 2-87 2.57 2.50 2.59
13-20 16.60 16-75 17-94 16.10 15.60 16.25
Batch 3 (20°C)
3 7 11 17 23 27
2-85 3.33 3-17 2.92 3.75 3.27
17.80 20-80 19-80 18-30 23.44 20.44
N = Kjeldahi N. Protein = N × 6'25.
Pilot plant production of SCP from sugarcane bagasse
279
was attained after 5 days of fermentation, while about a fourfold decrease ( = 32%) of the same fraction was achieved at the end of the process period (28 days). This was accompanied by a parallel increase in the protein content of the biomass, which reached a maximum of 28.5 % at the end of the process for the first batch. The second batch was carried out in December, when the ambient temperature was about 15°C on average as compared with 30°C during the first run. Hence, the maximum protein content was lower at about 16% at the end of the process. The third batch was more successful as the ambient temperature reached 20-22°C and the maximum protein content was 23.4%. These data showed that the highest protein content resulted from fermenting at the highest temperature. This emphasises the importance of temperature for this type of fermentation and shows that, for a continuous process, all the year round, the fermentor would have to be heated and its temperature controlled. The nutritional improvement of the alkali-treated bagasse obtained after 28 days of fermentation was also determined by Farid et al. (1984). This work showed that the nutritive value, expressed as IVDMD, increased from 21.5% to 52.9% as given by Hamessa et al. (1984).
REFERENCES Berc, H. T. (1977). International Symposium on Bioconversions of Cellulosic Substances into Energy; Chemical and Microbial Proteins, Delhi, India. Bunker, H. J. (1968). Single cell protein, MIT Press, Cambridge, Massachusetts, USA, 67. Burrows, S. (1970). The yeasts, Academic Press, London, 349. Cowling, E. B. (1975). Bioteeh. Bioeng. Symp. No. 5 (Wike, C. R. (Ed.)), New York, USA, 165. Davis, P. (1974). Single cell protein, Academic Press, New York, USA. Dekker, R. F. H. & Richards, G. N. (1973). J. Sci. Food Agric., 24, 375. El-Refai, A. H. (1982). Utilization of Cane Sugar Bagasses, Grant No. FG.EG-180 USA, Fifth Annual Research Report, NRC, Cairo, Egypt. Erikson, K. E. (1975). In: Symposium on Enzymatic Hydrolysis of Cellulose, Aulanko, Finland (M. Bailey, T. M. Erani & M. Linko (Eds)), 263. Fan, L. T., Lee, Y. H. & Gharpuray, M. M. (1981). Adv. Biochem. Bioeng., 23, 158. Farid, M., Shaker, H. & E1-Refai, A. H. (1984). Productivity of T. viride 253, Annual Report, NRC, Cairo, Egypt. Ghose, T. K. (1977). Adv. Biochem. Eng., 6, 39. Gulcho, S. (1973). Proteins from hydrocarbons, Noyes Data Corp., Park Ridge, New Jersey, USA. Hamessa, F. A., EI-Dewany, A. I. & E1-Refai, A. H. (1984). Microbios Letters, 26, 129. Humphery, A. E. (1974). Chem. Eng. (N. Y.), 81(26), 98.
280
Sh. I. EI-Shawarby, E. A. EI-Zanaty, A. H. EI-Refai, F. A. Hamissa, H. Shaker
Kihlberg, R. (1972). Ann. Rev., Microbiol., 26, 427. Lipinsky, E. S. & Litchfield, O. H. (1970). Critical Rev. Food TechnoL, 1, 580. Mandels, M. (1975). Biotech. Bioeng. Syrup. No. 5. (Wilke, C. R. (Ed)), New York, USA, 81. Mateles, R. I. & Tannenbaum, S. R. (1968). SCP II, MIT Press, Cambridge, Massachusetts, USA. Peppier, H. J. (1970). The.yeasts, Academic Press, London, 421. Rockwell, P. J. (1976). Single cell protein from cellulose and hydrocarbons, Noyes Data Corp., Park Ridge, New York, USA. Solbkowicz, G. (1976). Food from wastes. (Birch, G. G., Parker, K. J. & Worgan, J. T. (Eds)), Applied Science Publishers, London, 42. Tannenbaum, S. R. & Wang, D. I. C. (1975). Single cell protein II., MIT Press, Cambridge, Massachusetts, USA. Underkofler, L. A. (1969). Adv. Chem. Ser., 95, 343. Wolnak, B. (1972). Present and future technological and commercial status of enzymes. National Foundation Rep. No. NSF/RAX-73-002, USA Research and Development Association, Natick, Massachusetts.