- Email: [email protected]
Protein enrichment of banana plant wastes by yeast cultivation
Biological Wastes 24 (1988) 127-136
Protein Enrichment of Banana Plant Wastes by Yeast Cultivation C. H. Horn, J. C. du Preez* & P. M. Lategan Department of Microbiology, University of the Orange Free State, PO Box 339, 9300 Bloemfontein,South Africa (Received 28 August 1987; accepted 23 September 1987)
A BSTRA CT Banana plant wastes, which are generated in copious amounts during normal agricultural practice, contain between 35% and 60% starch on a moisturefree basis. A dextrin-containing liquid extract, obtained after liquefaction of the plant starch with a commercial thermostable ~-amylase, was used as substrate for the cultivation of Candida utilis. Saccharification of the dextrins, to enable growth of C. utilis, was effected with either a commercial glucoamylase or by the co-cultivation of Lipomyces kononenkoae with C. utilis. The saccharification efficiency obtained with this amylolytic yeast proved comparable with that of the commercial glucoamylase, and a dry biomass concentration of 14.86g litre-l, with a crude protein content of 47.4%, was reached in the hydrolysate. Supplementation of the hydrolysate with banana peels resulted in a dry biomass concentration of 37'7 g litre -1 (31"1% crude protein) and increased the yield to 0.23g crude, protein/g glucose equivalents. By mixing in this biomass with the original plant solids, a biomass product with a crude protein content of 17.85% (dry weight) was obtained. Undesired ethanol production could be minimized by controlling the rate of starch saccharification by using an appropriate glucoamylase concentration or, in the case of co-cultivation, a suitable C. utilis to L. kononenkoae ratio.
INTRODUCTION In b a n a n a plantations the plants are routinely thinned out by removing those which have borne fruit. In South African plantations in the Transvaal * To whom correspondence should be addressed. 127 Biological Wastes 0269-7483/88/$03.50 © 1988 Elsevier Applied Science Publishers Ltd, England. Printed in Great Britain
128
C. H. Horn, J. C. du Preez, P. M. Lategan
province this practice produces about 100 tons of waste material per hectare annually, with a 70:30 ratio of plant stem and conn. Limited amounts of banana peels are available during a 4-month peak season as a fruitprocessing waste. Although the utilization of waste bananas and banana peels for animal feed has been investigated (Neto & Panek, 1975; Chung & Meyers, 1979; Pujol & Bahar, 1983), to our knowledge no literature on utilization of the plant wastes, which contain 35% to 60% starch on a moisture-free basis, is available. Enzymatic hydrolysis of starch has several advantages over acid hydrolysis; acid-resistant equipment is not required, higher sugar yields are obtained, and lower concentrations of inhibitory degradation products, such as furfurals, are formed (Moreton, 1978). The enzymatic hydrolysis of starch to glucose requires two steps, liquefaction and saccharification (Aschengreen et al., 1979). These procedures can efficiently be carried out using a thermostable or-amylase (Termamyl) produced by Bacillus licheniformis (Rosendal et al., 1979; Aschengreen et aL, 1979; NiElson & Rosendal, 1980) and Aspergillus niger glucoamylase (AMG) (Rosendal et al., 1979; Aschengreen et al., 1979). As an alternative to the use of commercial amylases, amylolytic yeasts have been investigated for the direct utilization of starch or for the hydrolysis of starch to glucose, which could then be utilized in co-culture by fast-growing yeasts for single-cell-protein production (Spencer-Martins & Van Uden, 1979; Oteng-Gyang et al., 1980; Van Uden et al., 1980; SfiCorreia & Van Uden, 1981; Sills & Stewart, 1982; Estrela et al., 1982; Dhawale & Ingledew, 1983). A recent comparative study showed that Schwanniomyces occidentalis, and especially Lipomyces kononenkoae, exhibited a high amylolytic activity, possessing s-amylase and glucoamylase as well as debranching activity (Horn et al., 1987). The aim of this study was to cultivate yeasts on the starch component of banana-plant wastes to increase the protein content, thereby enhancing the value of the wastes as an animal fodder. The semi-solid nature of the banana plant wastes precluded the direct application of conventional submerged yeast cultivation, and necessitated a pretreatment step to obtain a more liquid substrate.
METHODS
Enzymatic liquefaction and pretreatment A banana plant mixture consisting of 1750g stem and 750g corm (wet weight) was shredded and diluted with 1000 ml distilled water to facilitate
Yeast culture on banana wastes
129
stirring. Liquefaction was effected with 0.36ml Termamyl 120L (Novo Industries) in a Magnaferm fermentor (New Brunswick Scientific Co.), equipped with a 3 litre glass vessel, after the addition of 0.63 g CaC12 . 2H2 O to stabilize the enzyme. The temperature was maintained at 95°C for 30 min by passing steam through the baffles while stirring the mixture at 400 rpm. A cheese press was used to separate the liquid hydrolysate from the banana plant solids, yielding 3 litres of hydrolysate which contained 41 g litre-1 total sugars as glucose. Additional nutrients for yeast growth (per litre hydrolysate); 3.0g urea, 5.0g KH2PO 4 and 0.21g MgSO4.7H20, were added to the hydrolysate and the pH adjusted to 4.5 with 5N H 2 S O 4 prior to autoclaving at 121°C for 15 min. When banana peels were included, the urea concentration was increased to 5.0 g litre-1 Yeast strains Candida utilis ATCC 9256 was used as the primary SCP producer, and Lipomyces kononenkoae IGC 4052B, obtained from Professor N. van Uden, Gulbenkian Institute of Science, Portugal, as the amylase producer.
Shake-flask cultivation C. utilis was inoculated from a fresh slant into a 1000-ml Erlenmeyer shake flask with 100 ml medium, containing (per litre distilled water); 30 g maltose, 6.7 g yeast nitrogen base (Difco) and 4 g KH2PO 4, adjusted to pH 4.5, which was incubated for 18 h at 30°C at 180 rpm. A 20 ml volume of this culture served as inoculum for each of 1000 ml Erlenmeyer flasks containing 200 ml banana plant hydrolysate, which were incubated as above. Immediately prior to inoculation, A M G 200L (Novo Industries) was added to the flasks at concentrations of 100, 50, 15 and 2.5/A litre- i. The inoculum medium for L. kononenkoae contained (per litre distilled water); 10g soluble starch (Merck), 6-7 g yeast nitrogen base (Difco) and 10 g NaH2PO 4 . H20. OtengGyang et al. (1980) showed that sodium ions enhanced the excretion of amylases. The cultivation conditions were the same as above, but with a 52 h incubation period. L. kononenkoae reached a biomass of 6 g litre- 1 and C. utilis 12"2 glitre-1 at the time of inoculation. A 10ml inoculum of each yeast suspension was used in the co-cultivation studies, with the incubation conditions as described above but with no A M G added.
Fermentor cultivations The media for the C. utilis and L. kononenkoae inocula were as described in the foregoing section, except that the starch content was increased to 15 g
130
C. H. Horn, J. C. du Preez, P. M. Lategan
litre-1 for L. kononenkoae. These yeasts were inoculated from fresh slants into 500-ml Erlenmeyer shake flasks containing 80 ml of each medium, and L. kononenkoae was incubated for 56 h and C. utilis for 24 h (180 rpm; 30°C). At the time of fermentor inoculation C. utilis and L. kononenkoae had reached biomass concentrations of 13.2 and 6.6g litre -1 (dry mass), respectively, so that a 50ml inoculum of each yeast suspension gave an initial ratio of 2:1 of the co-culture in the fermentor. A 1:1 inoculum ratio was obtained by increasing the starch content of the medium for the L. kononenkoae inoculum to 30 g litre-1, which resulted in a cell density of 13.5 g litre- 1 (dry mass). A New Brunswick Multigen F-2000 bioreactor was used for batch cultivations at 30°C under aseptic conditions. The 2 litre glass vessel, fitted with a reflux cooler to minimize evaporation, contained 1 litre sterile hydrolysate which was supplemented with 200 g litre- 1 banana peels where stated. The stirring speed was 800 rpm and the aeration rate 1.0 litre min- 1. The pH was automatically controlled at 4.5 with 5~q H2SO 4. The dissolved oxygen tension (DOT) was monitored with a polarographic oxygen electrode (WTW, Weilheim, West Germany).
Analytical procedures Weighed plant samples were dried at 105°C to determine the moisture content. The starch content of the banana plant waste (100g of each component) was determined by liquefying the starch with 0.5 ml Termamyl 120L at 95°C for 30 min followed by saccharification with 0"5 ml A M G 200L at 65°C for 12h. The total free sugars of the banana plant wastes were extracted according to Friedmann et al. (1967). Growth was monitored by biuret protein determinations'(Herbert et al., 1971). The Kjeldahl procedure, as outlined by Du Preez et al. (1985), was used to determine the protein content of the banana plant wastes as well as the final product after cultivation. The supernatants of samples were analysed for free sugars by HPLC and for ethanol by gas-liquid chromatography (Du Preez et al., 1985). Oxygen in the fermentor exhaust was measured with a Beckman OM14 polarographic oxygen analyser, while the CO2 content was measured with a Lira Model 303 infrared CO2 analyser (Mine Safety Appliances Co.). RESULTS A N D DISCUSSION
Composition of banana plant wastes The banana plant wastes had a very high moisture content (Table 1). Expressed on a moisture-free basis, the starch content of the stem, and
Yeast culture on banana wastes
131
TABLE 1 C o m p o s i t i o n o f Banana Plant Wastes
Plant constituents
Percentage content a Corms
Moisture Free sugars ~ Starch b Protein b
92.63 9.72 60.17 4.56
__.0-21 +__0.81 ___3.10 _ 0.03
Stems
Peels
84-86 ___0.49 4-57 + 0-46 35-80 _ 2.16 4-25 + 0-07
89.36 ___0.09 41.54 + 2.23 0 10.90 + 0.14
Leaves 66.19 9-79 11.40 4-59
_ 0.53 ___0-37 _ 0"86 + 0-15
M e a n value and s t a n d a r d deviation o f triplicates. b Expressed on a moisture-free basis. a
especially the corm, was appreciable. The starch content of the leaves, together with their very high fibre content, made them a poor and difficult substrate for protein enrichment. The free-sugar content of the peels was high with no detectable starch because the fruits were fully ripe when peeled. A mixture of the corm and stem in a 30:70 ratio gave an average starch content of 43 % on a moisture-free basis. The protein content of these wastes was low, the peels having the highest value of 10.9% protein (moisture free). Shake-flask cultivation
Cultivating C. utilis in the hydrolysate saccharified with 15/~1 litre- 1 A M G produced the highest final protein-content, although undesired ethanol production occurred (Table 2). Higher concentrations of A M G had the effect that ethanol production by C. utilis increased, which decreased protein TABLE 2 Yeast G r o w t h in Banana Plant Wastes (Corm and Stem) Following Starch Saccharification with Commercial Glucoamylase ( A M G ) or by L. kononenkoae
Yeasts
C. C. C. C. L.
utilis utilis utilis utilis kononenkoae and C. utilis
AMG (#l litre- 1)
i~x (h- l)
Protein production (g litre- 1)
Ethanol production (g litre- 1)
Cultivation period a (h)
100.0 50"0 15"0 2'5 0
0"325 0.32 0" 132 0-127 0" 12
4'73 5.02 6' 19 3.75 5'44
5"25 4-70 1"60 0-50 1"94
16 16 24 30 24
° Time required to reach stationary phase.
132
C. H. Horn, J. C. du Preez, P. M. Lategan
production. Insufficient saccharification of starch was obtained with 2.5/A litre -1 AMG, which resulted in a low protein production. The joint maximum specific growth rate of the co-culture was 0.12 h - 1, indicating that the growth rate was limited by the rate of starch hydrolysis by the L. kononenkoae amylases. The final protein content of the co-culture was 5"44 g litre- 1, which was lower than the best value (6.19 g litre- 1) obtained with the C. utilis mono-culture. This was in part due to the slightly higher ethanol production in this instance, presumably by C. utilis, since L. kononenkoae is a non-fermentative yeast (Wilson & Ingledew, 1982), and also because of the low protein content of L. kononenkoae (Ogden & Tubb, 1984). Ethanol production during these experiments might have been due to insufficient aeration in the shake flasks. Fermentor co-cultivations In order to investigate the influence of the initial C. utilis to L. kononenkoae ratio on the co-cultivation, two different inoculum ratios for the co-culture were used. Initial co-culture ratio o f 2: l (w/w) No initial lag phase was evident and the stationary phase was reached after 22h (Fig. 1A). The maximum specific growth-rate, calculated by linear regression from the increase in biuret protein concentration, was 0.14h-1 which was slightly higher than that obtained in shake flasks. For the first 12 h glucose was not detected and only maltose accumulated briefly, indicating that the growth-rate of the co-culture was dependent upon the rate of glucose liberation from the dextrins by L. kononenkoae. Microscopy indicated that C. utilis dominated over L. kononenkoae during the cultivation and was therefore the major protein source. Ethanol production was prevented by the very low residual glucose concentration, plus the fact that the DOT was maintained above 20% of air saturation. The respiration quotient (RQ) at no stage exceeded a value of 1.0 (Fig. 1A). After cessation of growth the total dry solids in the hydrolysate amounted to 10.42 g litre- 1 containing 48"4% Kjeldahl protein, corresponding to a crude protein yield of 0.12g per g glucose equivalents initially present in the hydrolysate. Remixing of the culture solids with the original plant solids residue, which contained 6% Kjeldahl protein after starch hydrolysis, resulted in a final product containing 12% crude protein on a moisture-free basis. Initial co-culture ratio o f 1:1 (w/w) With this inoculum the maximum specific growth-rate increased to 0.23 h - 1 The maximum specific growth-rate of the co-culture was still limited by the
133
Yeast culture on banana wastes
A 6 t. ~2
2
I
I:zO
1 z ~o 3
I
0 0
6
12
TIME
- 18 ,
2t,
125
h
Fig. 1. The respiratory quotient (O) and residual maltose (O) and glucose(A) levelsduring growth, monitored by biuret protein (/X) production, of the yeast co-culture in the banana plant hydrolysate. The inoculum ratio of C. utilis to L. kononenkoae was 2:1 (A) and 1: 1 (B), respectively, on a dry mass basis. rate o f dextrin saccharification by L. kononenkoae, however, because this growth rate was far below the value o f 0.53 h - 1 for C. utilis on glucose reported by M o r e t o n (1978). The stationary phase was reached earlier at 20 h, and except for maltose no free sugar was observed at any time (Fig. 1B). C. utilis dominated over L. kononenkoae throughout the cultivation. N o ethanol was produced and the RQ values remained below 1 (Fig. 1B). The total dry solids in the hydrolysate after cultivation amounted to 14-86 g litre- 1 with a crude protein content o f 47-4%, representing a yield o f 0.17 g crude protein per g glucose equivalents. By altering the inoculum ratio the a m o u n t o f protein produced was therefore increased by 40%. This was probably due to the more efficient hydrolysis o f the starch as a result of the m o r e intensive amylase production by the larger L. kononenkoae inoculum. Mixing this hydrolysate biomass with the original plant solids from which the hydrolysate had been prepared yielded a final product containing 13-1% crude protein on a moisture-free basis. Supplementation o f hydrolysate with banana peels After inoculating the hydrolysate supplemented with 20% (w/v) banana peels with a 1:1 ratio o f the co-culture, no lag-phase occurred and the stationary-phase was reached after 15 h (Fig. 2B). The m a x i m u m specific growth rate of 0-22h -1 was slightly lower than that obtained in the
C. H. Horn, J. C. du Preez, P. M. Lategan
134
30
i
~-~ -,6 20
1.2 O'
O.8 0A
8 I
7
I
I
I
C12
B
r
t~
"
10
3
s~ t~ t~ O
!Oo
3
6
9 TIU.E, h
12
15
Fig. 2. The respiratory quotient (0), specificrates of 0 2 uptake (rq) and CO2 production (A), ethanol concentration (ll), and residualmaltose (0), glucose(A) and fructose(I-q)levels during yeast growth, monitored by biuret protein (A) production, in banana plant hydrolysate supplementedwith 20% (w/v)banana peels. The inoculum ratio of C. utilis to L. kononenkoae was 1:1 on a dry mass basis. hydrolysate only. Because the peels contained the free sugars glucose and fructose, their initial presence resulted in the production of a small amount of ethanol (0-9 glitre-~) concomitant with an increase in the respiratory quotient to a value of 1-4 (Fig. 2A), which is indicative of a fermentative metabolism. The production of ethanol was not due to oxygen limitation, however, as the DOT never decreased to below 20% of air saturation. The ethanol production was probably due to the glucose-effect (Moss et al., 1969). Even though C. utilis is regarded as a 'glucose-insensitive' yeast (De Deken, 1966), Moss et al. (1969) found that high glucose concentrations (> 19 g litre-~) could elicit ethanol production under aerobic conditions. The gas-exchange data gave a reliable indication of the time of carbohydrate exhaustion (Fig. 2A), which was useful when working with turbid culture media. Due to the inclusion of the peels the dry solids content after cultivation was increased to 37.7g litre -1, of which 31.1% was crude protein, representing a yield of 0.23 g crude protein per g glucose equivalents. Mixing this product with the original plant solids resulted in a final biomass product containing 17.85% Kjeldahl protein on a moisture-free basis. By using the amylolytic L. kononenkoae the commercial glucoamylase could be dispensed with, while maintaining a high efficiency of starch
Yeast culture on banana wastes
135
saccharification. Undesired ethanol production could be minimized by controlling the rate of starch saccharification, either by using an appropriate glucoamylase concentration or a suitable C. utilis to L. kononenkoae ratio, together with a simultaneous saccharification and yeast-cultivation procedure. Obviously, the economics of the process would be enhanced by the addition of a carbohydrate-rich adjunct, such as banana peels, to the stem-corm mixture. A n unfortunate consequence of the physical nature of the banana plant material, and contributing to the cost and complexity of the process, is that an initial starch liquefaction with 0~-amylase is required to obtain a liquid medium necessary for conventional aerobic submerged cultivation. Another approach, i.e. a semi-solid type of cultivation procedure using an amylolytic microorganism, might obviate the need for commercial enzymes altogether.
ACKNOWLEDGEMENTS We thank 'Landbou-Energie' Ltd and the Central Research Fund of the U.O.F.S. for their financial support, and P. J. Botes for his competent technical assistance with the chromatographic analyses.
REFERENCES Aschengreen, N. H., Ni~lson, B. H., Rosendal, P. & Ostergaard, J. (1979). Liquefaction, saccharification and isomerization of starches from sources other than maize. Starch/Stiirke, 31, 64-6. Chung, S. L. & Meyers, S. P. (1979). Bioprotein from banana wastes. Dev. Ind. MicrobioL, 20, 723-32. De Deken, R. H. (1966). The crabtree effect: A regulatory system in yeast. J. Gen Microbiol., 44, 149-56. Dhawale, M. R. & Ingledew, W. M. (1983). Starch hydrolysis by derepressed mutants of Schwanniomyces castellii. Biotechnol. Lett., 5, 185-90. Du Preez, J. C., De Jong, F., Botes, P. J. & Lategan, P. M. (1985). Fermentation alcohol from grain sorghum starch. Biomass, 8, 101-17. Estrela, A. I., Lemos, M. & Spencer-Martins, I. (1982). A note on the effect of growth temperature on the production of amylases by the yeast Lipomyces kononenkoae. J. Appl. Bacteriol., 52, 465-7. Friedmann, T. E., Witt, N. F., Neighbors, B. W. & Weber, C. W. (1967). Determination of available carbohydrates in plant and animal foods. J. Nutr., 91, 9-10. Herbert, D., Phipps, P. J. & Strange, R. E. (1971). Chemical analysis of microbial cells. In: Methods in microbiology, Vol. 5B (Norris, J. R. & Ribbons, D. W. (Eds)), Academic Press, London, 210-65.
136
C. H. Horn, J. C. du Preez, P. M. Lategan
Horn, C. H., Du Preez, J. C. & Lategan, P. M.'(1988). A comparative study of the amylolytic ability of Lipomyces and Schwanniomyces yeast species. System. Appl. MicrobioL I, 10, 106-10. Moreton, R. S. (1978). Growth of Candida utilis on enzymatically hydrolysed potato waste. J. Appl. BacterioL, 44, 373-82. Moss, F. J., Rickard, P. A. D., Beech, G. A. & Bush, F. E. (1969). The response by microorganisms to steady state growth in controlled concentrations of oxygen and glucose. I. Candida utilis. Biotechnol. Bioeng., 11, 561-80. Neto, J. S. A. & Panek, A. D. (1975). Growth ofCandida utilis on extracts of banana leaves as a cellulosic waste. Rev. Microbiol. (S. Paulo), 6, 59-62. Ni~lson, B. H. & Rosendal, P. (1980). Application of low-temperature liquefaction on production of ethanol from starch. In: Prov. IVth Int. Symp. on AlcoholFuels Technology, Vol. 1, Brazil, 51-5. Ogden, K. & Tubb, R. S. (1984). A strain of Saccharomyces cereoisiae which grows efficiently on starch. Enzyme Microb. Technol., 7, 2204. Oteng-Gyang, K., Moulin, G. & Galzy, P. (1980). Effect of medium composition on excretion and biosynthesis of the amylases of Schwanniomyces castellii. Eur. J. Appl. Microbiol. Biotechnol., 9, 129-32. Pujol, F. & Bahar, S. (1983). Production of single cell protein from green plantain skin. Eur. J. Microbiol. Biotechnol., 18, 361-8. Rosendal, P., Ni~lson, B. H. & Lange, N. K. (1979). Stability of bacterial alphaamylase in the starch liquefaction process. Starch/Stiirke, 31, 368-72. S~-Correia, I. & Van Uden, N. (1981). Production of biomass and amylases by the yeast Lipomyces kononenkoae in starch-limited continuous culture. Eur. J. Appl. Microbiol. Biotechnol., 13, 24-8. Sills, A. M. & Stewart, G. G. (1982). Production of amylolytic enzymes by several yeast species. J. Inst. Brew., 88, 313-16. Spencer-Martins, I. & Van Uden, N. (1979). Extracellular amylolytic system of the yeast Lipomyces kononenkoae. Eur. J. Appl. Microbiol. BiotechnoL, 6, 241-50. Southgate, D. A. T. (1976). Determination of food carbohydrates. Applied Science Publishers Ltd, London, 34, 108-9. Van Uden, N., Cabeca-Silva, C., Madeira-Lopez, A. & Spencer-Martins, I (1980). Selective isolation of derepressed mutants of an a-amylase yeast by the use of 2deoxyglucose. Biotechnol. Bioeng., 12, 651-4. Wilson, J. J. & Ingledew, W. M. (1982). Isolation and characterization of Schwanniomyces alluvius amylolytic enzymes. AppL Environ. MicrobioL, 44, 301-7.
Protein Enrichment of Banana Plant Wastes by Yeast Cultivation C. H. Horn, J. C. du Preez* & P. M. Lategan Department of Microbiology, University of the Orange Free State, PO Box 339, 9300 Bloemfontein,South Africa (Received 28 August 1987; accepted 23 September 1987)
A BSTRA CT Banana plant wastes, which are generated in copious amounts during normal agricultural practice, contain between 35% and 60% starch on a moisturefree basis. A dextrin-containing liquid extract, obtained after liquefaction of the plant starch with a commercial thermostable ~-amylase, was used as substrate for the cultivation of Candida utilis. Saccharification of the dextrins, to enable growth of C. utilis, was effected with either a commercial glucoamylase or by the co-cultivation of Lipomyces kononenkoae with C. utilis. The saccharification efficiency obtained with this amylolytic yeast proved comparable with that of the commercial glucoamylase, and a dry biomass concentration of 14.86g litre-l, with a crude protein content of 47.4%, was reached in the hydrolysate. Supplementation of the hydrolysate with banana peels resulted in a dry biomass concentration of 37'7 g litre -1 (31"1% crude protein) and increased the yield to 0.23g crude, protein/g glucose equivalents. By mixing in this biomass with the original plant solids, a biomass product with a crude protein content of 17.85% (dry weight) was obtained. Undesired ethanol production could be minimized by controlling the rate of starch saccharification by using an appropriate glucoamylase concentration or, in the case of co-cultivation, a suitable C. utilis to L. kononenkoae ratio.
INTRODUCTION In b a n a n a plantations the plants are routinely thinned out by removing those which have borne fruit. In South African plantations in the Transvaal * To whom correspondence should be addressed. 127 Biological Wastes 0269-7483/88/$03.50 © 1988 Elsevier Applied Science Publishers Ltd, England. Printed in Great Britain
128
C. H. Horn, J. C. du Preez, P. M. Lategan
province this practice produces about 100 tons of waste material per hectare annually, with a 70:30 ratio of plant stem and conn. Limited amounts of banana peels are available during a 4-month peak season as a fruitprocessing waste. Although the utilization of waste bananas and banana peels for animal feed has been investigated (Neto & Panek, 1975; Chung & Meyers, 1979; Pujol & Bahar, 1983), to our knowledge no literature on utilization of the plant wastes, which contain 35% to 60% starch on a moisture-free basis, is available. Enzymatic hydrolysis of starch has several advantages over acid hydrolysis; acid-resistant equipment is not required, higher sugar yields are obtained, and lower concentrations of inhibitory degradation products, such as furfurals, are formed (Moreton, 1978). The enzymatic hydrolysis of starch to glucose requires two steps, liquefaction and saccharification (Aschengreen et al., 1979). These procedures can efficiently be carried out using a thermostable or-amylase (Termamyl) produced by Bacillus licheniformis (Rosendal et al., 1979; Aschengreen et aL, 1979; NiElson & Rosendal, 1980) and Aspergillus niger glucoamylase (AMG) (Rosendal et al., 1979; Aschengreen et al., 1979). As an alternative to the use of commercial amylases, amylolytic yeasts have been investigated for the direct utilization of starch or for the hydrolysis of starch to glucose, which could then be utilized in co-culture by fast-growing yeasts for single-cell-protein production (Spencer-Martins & Van Uden, 1979; Oteng-Gyang et al., 1980; Van Uden et al., 1980; SfiCorreia & Van Uden, 1981; Sills & Stewart, 1982; Estrela et al., 1982; Dhawale & Ingledew, 1983). A recent comparative study showed that Schwanniomyces occidentalis, and especially Lipomyces kononenkoae, exhibited a high amylolytic activity, possessing s-amylase and glucoamylase as well as debranching activity (Horn et al., 1987). The aim of this study was to cultivate yeasts on the starch component of banana-plant wastes to increase the protein content, thereby enhancing the value of the wastes as an animal fodder. The semi-solid nature of the banana plant wastes precluded the direct application of conventional submerged yeast cultivation, and necessitated a pretreatment step to obtain a more liquid substrate.
METHODS
Enzymatic liquefaction and pretreatment A banana plant mixture consisting of 1750g stem and 750g corm (wet weight) was shredded and diluted with 1000 ml distilled water to facilitate
Yeast culture on banana wastes
129
stirring. Liquefaction was effected with 0.36ml Termamyl 120L (Novo Industries) in a Magnaferm fermentor (New Brunswick Scientific Co.), equipped with a 3 litre glass vessel, after the addition of 0.63 g CaC12 . 2H2 O to stabilize the enzyme. The temperature was maintained at 95°C for 30 min by passing steam through the baffles while stirring the mixture at 400 rpm. A cheese press was used to separate the liquid hydrolysate from the banana plant solids, yielding 3 litres of hydrolysate which contained 41 g litre-1 total sugars as glucose. Additional nutrients for yeast growth (per litre hydrolysate); 3.0g urea, 5.0g KH2PO 4 and 0.21g MgSO4.7H20, were added to the hydrolysate and the pH adjusted to 4.5 with 5N H 2 S O 4 prior to autoclaving at 121°C for 15 min. When banana peels were included, the urea concentration was increased to 5.0 g litre-1 Yeast strains Candida utilis ATCC 9256 was used as the primary SCP producer, and Lipomyces kononenkoae IGC 4052B, obtained from Professor N. van Uden, Gulbenkian Institute of Science, Portugal, as the amylase producer.
Shake-flask cultivation C. utilis was inoculated from a fresh slant into a 1000-ml Erlenmeyer shake flask with 100 ml medium, containing (per litre distilled water); 30 g maltose, 6.7 g yeast nitrogen base (Difco) and 4 g KH2PO 4, adjusted to pH 4.5, which was incubated for 18 h at 30°C at 180 rpm. A 20 ml volume of this culture served as inoculum for each of 1000 ml Erlenmeyer flasks containing 200 ml banana plant hydrolysate, which were incubated as above. Immediately prior to inoculation, A M G 200L (Novo Industries) was added to the flasks at concentrations of 100, 50, 15 and 2.5/A litre- i. The inoculum medium for L. kononenkoae contained (per litre distilled water); 10g soluble starch (Merck), 6-7 g yeast nitrogen base (Difco) and 10 g NaH2PO 4 . H20. OtengGyang et al. (1980) showed that sodium ions enhanced the excretion of amylases. The cultivation conditions were the same as above, but with a 52 h incubation period. L. kononenkoae reached a biomass of 6 g litre- 1 and C. utilis 12"2 glitre-1 at the time of inoculation. A 10ml inoculum of each yeast suspension was used in the co-cultivation studies, with the incubation conditions as described above but with no A M G added.
Fermentor cultivations The media for the C. utilis and L. kononenkoae inocula were as described in the foregoing section, except that the starch content was increased to 15 g
130
C. H. Horn, J. C. du Preez, P. M. Lategan
litre-1 for L. kononenkoae. These yeasts were inoculated from fresh slants into 500-ml Erlenmeyer shake flasks containing 80 ml of each medium, and L. kononenkoae was incubated for 56 h and C. utilis for 24 h (180 rpm; 30°C). At the time of fermentor inoculation C. utilis and L. kononenkoae had reached biomass concentrations of 13.2 and 6.6g litre -1 (dry mass), respectively, so that a 50ml inoculum of each yeast suspension gave an initial ratio of 2:1 of the co-culture in the fermentor. A 1:1 inoculum ratio was obtained by increasing the starch content of the medium for the L. kononenkoae inoculum to 30 g litre-1, which resulted in a cell density of 13.5 g litre- 1 (dry mass). A New Brunswick Multigen F-2000 bioreactor was used for batch cultivations at 30°C under aseptic conditions. The 2 litre glass vessel, fitted with a reflux cooler to minimize evaporation, contained 1 litre sterile hydrolysate which was supplemented with 200 g litre- 1 banana peels where stated. The stirring speed was 800 rpm and the aeration rate 1.0 litre min- 1. The pH was automatically controlled at 4.5 with 5~q H2SO 4. The dissolved oxygen tension (DOT) was monitored with a polarographic oxygen electrode (WTW, Weilheim, West Germany).
Analytical procedures Weighed plant samples were dried at 105°C to determine the moisture content. The starch content of the banana plant waste (100g of each component) was determined by liquefying the starch with 0.5 ml Termamyl 120L at 95°C for 30 min followed by saccharification with 0"5 ml A M G 200L at 65°C for 12h. The total free sugars of the banana plant wastes were extracted according to Friedmann et al. (1967). Growth was monitored by biuret protein determinations'(Herbert et al., 1971). The Kjeldahl procedure, as outlined by Du Preez et al. (1985), was used to determine the protein content of the banana plant wastes as well as the final product after cultivation. The supernatants of samples were analysed for free sugars by HPLC and for ethanol by gas-liquid chromatography (Du Preez et al., 1985). Oxygen in the fermentor exhaust was measured with a Beckman OM14 polarographic oxygen analyser, while the CO2 content was measured with a Lira Model 303 infrared CO2 analyser (Mine Safety Appliances Co.). RESULTS A N D DISCUSSION
Composition of banana plant wastes The banana plant wastes had a very high moisture content (Table 1). Expressed on a moisture-free basis, the starch content of the stem, and
Yeast culture on banana wastes
131
TABLE 1 C o m p o s i t i o n o f Banana Plant Wastes
Plant constituents
Percentage content a Corms
Moisture Free sugars ~ Starch b Protein b
92.63 9.72 60.17 4.56
__.0-21 +__0.81 ___3.10 _ 0.03
Stems
Peels
84-86 ___0.49 4-57 + 0-46 35-80 _ 2.16 4-25 + 0-07
89.36 ___0.09 41.54 + 2.23 0 10.90 + 0.14
Leaves 66.19 9-79 11.40 4-59
_ 0.53 ___0-37 _ 0"86 + 0-15
M e a n value and s t a n d a r d deviation o f triplicates. b Expressed on a moisture-free basis. a
especially the corm, was appreciable. The starch content of the leaves, together with their very high fibre content, made them a poor and difficult substrate for protein enrichment. The free-sugar content of the peels was high with no detectable starch because the fruits were fully ripe when peeled. A mixture of the corm and stem in a 30:70 ratio gave an average starch content of 43 % on a moisture-free basis. The protein content of these wastes was low, the peels having the highest value of 10.9% protein (moisture free). Shake-flask cultivation
Cultivating C. utilis in the hydrolysate saccharified with 15/~1 litre- 1 A M G produced the highest final protein-content, although undesired ethanol production occurred (Table 2). Higher concentrations of A M G had the effect that ethanol production by C. utilis increased, which decreased protein TABLE 2 Yeast G r o w t h in Banana Plant Wastes (Corm and Stem) Following Starch Saccharification with Commercial Glucoamylase ( A M G ) or by L. kononenkoae
Yeasts
C. C. C. C. L.
utilis utilis utilis utilis kononenkoae and C. utilis
AMG (#l litre- 1)
i~x (h- l)
Protein production (g litre- 1)
Ethanol production (g litre- 1)
Cultivation period a (h)
100.0 50"0 15"0 2'5 0
0"325 0.32 0" 132 0-127 0" 12
4'73 5.02 6' 19 3.75 5'44
5"25 4-70 1"60 0-50 1"94
16 16 24 30 24
° Time required to reach stationary phase.
132
C. H. Horn, J. C. du Preez, P. M. Lategan
production. Insufficient saccharification of starch was obtained with 2.5/A litre -1 AMG, which resulted in a low protein production. The joint maximum specific growth rate of the co-culture was 0.12 h - 1, indicating that the growth rate was limited by the rate of starch hydrolysis by the L. kononenkoae amylases. The final protein content of the co-culture was 5"44 g litre- 1, which was lower than the best value (6.19 g litre- 1) obtained with the C. utilis mono-culture. This was in part due to the slightly higher ethanol production in this instance, presumably by C. utilis, since L. kononenkoae is a non-fermentative yeast (Wilson & Ingledew, 1982), and also because of the low protein content of L. kononenkoae (Ogden & Tubb, 1984). Ethanol production during these experiments might have been due to insufficient aeration in the shake flasks. Fermentor co-cultivations In order to investigate the influence of the initial C. utilis to L. kononenkoae ratio on the co-cultivation, two different inoculum ratios for the co-culture were used. Initial co-culture ratio o f 2: l (w/w) No initial lag phase was evident and the stationary phase was reached after 22h (Fig. 1A). The maximum specific growth-rate, calculated by linear regression from the increase in biuret protein concentration, was 0.14h-1 which was slightly higher than that obtained in shake flasks. For the first 12 h glucose was not detected and only maltose accumulated briefly, indicating that the growth-rate of the co-culture was dependent upon the rate of glucose liberation from the dextrins by L. kononenkoae. Microscopy indicated that C. utilis dominated over L. kononenkoae during the cultivation and was therefore the major protein source. Ethanol production was prevented by the very low residual glucose concentration, plus the fact that the DOT was maintained above 20% of air saturation. The respiration quotient (RQ) at no stage exceeded a value of 1.0 (Fig. 1A). After cessation of growth the total dry solids in the hydrolysate amounted to 10.42 g litre- 1 containing 48"4% Kjeldahl protein, corresponding to a crude protein yield of 0.12g per g glucose equivalents initially present in the hydrolysate. Remixing of the culture solids with the original plant solids residue, which contained 6% Kjeldahl protein after starch hydrolysis, resulted in a final product containing 12% crude protein on a moisture-free basis. Initial co-culture ratio o f 1:1 (w/w) With this inoculum the maximum specific growth-rate increased to 0.23 h - 1 The maximum specific growth-rate of the co-culture was still limited by the
133
Yeast culture on banana wastes
A 6 t. ~2
2
I
I:zO
1 z ~o 3
I
0 0
6
12
TIME
- 18 ,
2t,
125
h
Fig. 1. The respiratory quotient (O) and residual maltose (O) and glucose(A) levelsduring growth, monitored by biuret protein (/X) production, of the yeast co-culture in the banana plant hydrolysate. The inoculum ratio of C. utilis to L. kononenkoae was 2:1 (A) and 1: 1 (B), respectively, on a dry mass basis. rate o f dextrin saccharification by L. kononenkoae, however, because this growth rate was far below the value o f 0.53 h - 1 for C. utilis on glucose reported by M o r e t o n (1978). The stationary phase was reached earlier at 20 h, and except for maltose no free sugar was observed at any time (Fig. 1B). C. utilis dominated over L. kononenkoae throughout the cultivation. N o ethanol was produced and the RQ values remained below 1 (Fig. 1B). The total dry solids in the hydrolysate after cultivation amounted to 14-86 g litre- 1 with a crude protein content o f 47-4%, representing a yield o f 0.17 g crude protein per g glucose equivalents. By altering the inoculum ratio the a m o u n t o f protein produced was therefore increased by 40%. This was probably due to the more efficient hydrolysis o f the starch as a result of the m o r e intensive amylase production by the larger L. kononenkoae inoculum. Mixing this hydrolysate biomass with the original plant solids from which the hydrolysate had been prepared yielded a final product containing 13-1% crude protein on a moisture-free basis. Supplementation o f hydrolysate with banana peels After inoculating the hydrolysate supplemented with 20% (w/v) banana peels with a 1:1 ratio o f the co-culture, no lag-phase occurred and the stationary-phase was reached after 15 h (Fig. 2B). The m a x i m u m specific growth rate of 0-22h -1 was slightly lower than that obtained in the
C. H. Horn, J. C. du Preez, P. M. Lategan
134
30
i
~-~ -,6 20
1.2 O'
O.8 0A
8 I
7
I
I
I
C12
B
r
t~
"
10
3
s~ t~ t~ O
!Oo
3
6
9 TIU.E, h
12
15
Fig. 2. The respiratory quotient (0), specificrates of 0 2 uptake (rq) and CO2 production (A), ethanol concentration (ll), and residualmaltose (0), glucose(A) and fructose(I-q)levels during yeast growth, monitored by biuret protein (A) production, in banana plant hydrolysate supplementedwith 20% (w/v)banana peels. The inoculum ratio of C. utilis to L. kononenkoae was 1:1 on a dry mass basis. hydrolysate only. Because the peels contained the free sugars glucose and fructose, their initial presence resulted in the production of a small amount of ethanol (0-9 glitre-~) concomitant with an increase in the respiratory quotient to a value of 1-4 (Fig. 2A), which is indicative of a fermentative metabolism. The production of ethanol was not due to oxygen limitation, however, as the DOT never decreased to below 20% of air saturation. The ethanol production was probably due to the glucose-effect (Moss et al., 1969). Even though C. utilis is regarded as a 'glucose-insensitive' yeast (De Deken, 1966), Moss et al. (1969) found that high glucose concentrations (> 19 g litre-~) could elicit ethanol production under aerobic conditions. The gas-exchange data gave a reliable indication of the time of carbohydrate exhaustion (Fig. 2A), which was useful when working with turbid culture media. Due to the inclusion of the peels the dry solids content after cultivation was increased to 37.7g litre -1, of which 31.1% was crude protein, representing a yield of 0.23 g crude protein per g glucose equivalents. Mixing this product with the original plant solids resulted in a final biomass product containing 17.85% Kjeldahl protein on a moisture-free basis. By using the amylolytic L. kononenkoae the commercial glucoamylase could be dispensed with, while maintaining a high efficiency of starch
Yeast culture on banana wastes
135
saccharification. Undesired ethanol production could be minimized by controlling the rate of starch saccharification, either by using an appropriate glucoamylase concentration or a suitable C. utilis to L. kononenkoae ratio, together with a simultaneous saccharification and yeast-cultivation procedure. Obviously, the economics of the process would be enhanced by the addition of a carbohydrate-rich adjunct, such as banana peels, to the stem-corm mixture. A n unfortunate consequence of the physical nature of the banana plant material, and contributing to the cost and complexity of the process, is that an initial starch liquefaction with 0~-amylase is required to obtain a liquid medium necessary for conventional aerobic submerged cultivation. Another approach, i.e. a semi-solid type of cultivation procedure using an amylolytic microorganism, might obviate the need for commercial enzymes altogether.
ACKNOWLEDGEMENTS We thank 'Landbou-Energie' Ltd and the Central Research Fund of the U.O.F.S. for their financial support, and P. J. Botes for his competent technical assistance with the chromatographic analyses.
REFERENCES Aschengreen, N. H., Ni~lson, B. H., Rosendal, P. & Ostergaard, J. (1979). Liquefaction, saccharification and isomerization of starches from sources other than maize. Starch/Stiirke, 31, 64-6. Chung, S. L. & Meyers, S. P. (1979). Bioprotein from banana wastes. Dev. Ind. MicrobioL, 20, 723-32. De Deken, R. H. (1966). The crabtree effect: A regulatory system in yeast. J. Gen Microbiol., 44, 149-56. Dhawale, M. R. & Ingledew, W. M. (1983). Starch hydrolysis by derepressed mutants of Schwanniomyces castellii. Biotechnol. Lett., 5, 185-90. Du Preez, J. C., De Jong, F., Botes, P. J. & Lategan, P. M. (1985). Fermentation alcohol from grain sorghum starch. Biomass, 8, 101-17. Estrela, A. I., Lemos, M. & Spencer-Martins, I. (1982). A note on the effect of growth temperature on the production of amylases by the yeast Lipomyces kononenkoae. J. Appl. Bacteriol., 52, 465-7. Friedmann, T. E., Witt, N. F., Neighbors, B. W. & Weber, C. W. (1967). Determination of available carbohydrates in plant and animal foods. J. Nutr., 91, 9-10. Herbert, D., Phipps, P. J. & Strange, R. E. (1971). Chemical analysis of microbial cells. In: Methods in microbiology, Vol. 5B (Norris, J. R. & Ribbons, D. W. (Eds)), Academic Press, London, 210-65.
136
C. H. Horn, J. C. du Preez, P. M. Lategan
Horn, C. H., Du Preez, J. C. & Lategan, P. M.'(1988). A comparative study of the amylolytic ability of Lipomyces and Schwanniomyces yeast species. System. Appl. MicrobioL I, 10, 106-10. Moreton, R. S. (1978). Growth of Candida utilis on enzymatically hydrolysed potato waste. J. Appl. BacterioL, 44, 373-82. Moss, F. J., Rickard, P. A. D., Beech, G. A. & Bush, F. E. (1969). The response by microorganisms to steady state growth in controlled concentrations of oxygen and glucose. I. Candida utilis. Biotechnol. Bioeng., 11, 561-80. Neto, J. S. A. & Panek, A. D. (1975). Growth ofCandida utilis on extracts of banana leaves as a cellulosic waste. Rev. Microbiol. (S. Paulo), 6, 59-62. Ni~lson, B. H. & Rosendal, P. (1980). Application of low-temperature liquefaction on production of ethanol from starch. In: Prov. IVth Int. Symp. on AlcoholFuels Technology, Vol. 1, Brazil, 51-5. Ogden, K. & Tubb, R. S. (1984). A strain of Saccharomyces cereoisiae which grows efficiently on starch. Enzyme Microb. Technol., 7, 2204. Oteng-Gyang, K., Moulin, G. & Galzy, P. (1980). Effect of medium composition on excretion and biosynthesis of the amylases of Schwanniomyces castellii. Eur. J. Appl. Microbiol. Biotechnol., 9, 129-32. Pujol, F. & Bahar, S. (1983). Production of single cell protein from green plantain skin. Eur. J. Microbiol. Biotechnol., 18, 361-8. Rosendal, P., Ni~lson, B. H. & Lange, N. K. (1979). Stability of bacterial alphaamylase in the starch liquefaction process. Starch/Stiirke, 31, 368-72. S~-Correia, I. & Van Uden, N. (1981). Production of biomass and amylases by the yeast Lipomyces kononenkoae in starch-limited continuous culture. Eur. J. Appl. Microbiol. Biotechnol., 13, 24-8. Sills, A. M. & Stewart, G. G. (1982). Production of amylolytic enzymes by several yeast species. J. Inst. Brew., 88, 313-16. Spencer-Martins, I. & Van Uden, N. (1979). Extracellular amylolytic system of the yeast Lipomyces kononenkoae. Eur. J. Appl. Microbiol. BiotechnoL, 6, 241-50. Southgate, D. A. T. (1976). Determination of food carbohydrates. Applied Science Publishers Ltd, London, 34, 108-9. Van Uden, N., Cabeca-Silva, C., Madeira-Lopez, A. & Spencer-Martins, I (1980). Selective isolation of derepressed mutants of an a-amylase yeast by the use of 2deoxyglucose. Biotechnol. Bioeng., 12, 651-4. Wilson, J. J. & Ingledew, W. M. (1982). Isolation and characterization of Schwanniomyces alluvius amylolytic enzymes. AppL Environ. MicrobioL, 44, 301-7.