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Utilization of cattle manure for Torula yeast production from straw hydrolysates
Animal Feed Science and Technology, 5 (1980) 175--182 Elsevier Scientific Publishing Company, Amsterdam --Printed in The Netherlands
175
UTILIZATION OF CATTLE MANURE FOR TORULA YEAST PRODUCTION FROM STRAW HYDROLYSATES
M. MOO-YOUNG, D.S. CHAHAL and D. VLACH, Biochemical Engineering Group, Chemical Engineering Department, University of Waterloo, Waterloo, Ontario (Canada) (Received 27 August 1979; accepted for publication 18 Febuary 1980)
ABSTRACT Moo-Young, M., Chahal, D.S. and Vlach, D., 1980. Utilization of cattle manure for Torula yeast production from straw hydrolysates. Anita. Feed Sci. Technol., 5: 175--182. Good growth of Torula (Candida utilis) was obtained from mixtures of anaerobically fermented cattle manure liquor and barley straw acid hydrolysate. Relatively good growth of this yeast was also obtained from mixtures of acid- or alkali-pretreated cattle manure and barley straw acid hydrolysate. However, a significant amount of reducing sugars (30--40%) was usually left unutilized, indicating the need for a better strain of C. utilis capable of utilizing such sugars as galactose and arabinose which are released (in additon to glucose and xylose) from the hemicellulose during the acid hydrolysis of barley straw. These experiments demonstrate the practicability of using these nutrient sources, obtainable from abundant waste materials, to produce useful single cell protein (SCP) products. In particular, an integrated two stage anaerobic--aerobic fermentation process for the co-production of methane fuel gas and SCP offers an economically attractive option.
INTRODUCTION Vast q u a n t i t i e s o f animal m a n u r e s o c c u r universally as waste residues. In t h e past, these residues have f r e q u e n t l y b e e n used as soil fertilizer. However, increasingly stringent g o v e r n m e n t regulations on e n v i r o n m e n t a l p o l l u t i o n , especially n e a r u r b a n areas, n o w restrict this a p p l i c a t i o n o f manures. A n i m a l m a n u r e s r e p r e s e n t a p o t e n t i a l l y valuable s o u r c e o f n u t r i e n t s for f e r m e n t a t i o n processes. If i m p l e m e n t e d , this a p p l i c a t i o n w o u l d be an environm e n t a l l y - a c c e p t a b l e and r e s o u r c e - c o n s e r v i n g m e t h o d o f utilizing manures. R h o d e s a n d O r t o n ( 1 9 7 5 ) have tried t o utilize f e e d l o t waste filtrate with the i n d i g e n o u s m i c r o f l o r a for f e r m e n t a t i o n o f c r a c k e d maize as animal feed. H o w e v e r , the end p r o d u c t o f f e r m e n t a t i o n s h o w e d v e r y little increase in t h e p r o t e i n c o n t e n t o f t h e processed materials. In a d d i t i o n , feeding trials with mice given this f e r m e n t e d m a i z e s h o w e d very p o o r g r o w t h responses c o m p a r e d t o t h e c o n t r o l diet (a c o m m e r c i a l feed). The a u t h o r s r e p o r t e d no 0377-8401/80/0000--0000/$02.25 © 1980 Elsevier Scientific Publishing Company
176 toxicity of the fermented maize product! However, there was no check on the real possibility of multiplication of toxin-producing organism(s) in such an uncontrolled system of fermentation. Moo-Young and Chahal (1979) have recently shown that acid- or alkalipretreated cattle manure can be a good supplementary nutrient source for single cell protein (SCP) production from crop residues such as cornstover, using the cellulolytic organism, Chaetomium cellulolyticum. They also found that anaerobically-prefermented cattle manure liquor from a methane-generating digestor provided an even better source of supplementary nutrients. Thomas and Evison (1978) have studied the possibility of inhibiting the methanogenesis phase in an anaerobic digestion of pig manure so as to maximize volatile f a t t y acids concentration in the effluent (through the acidogenesis phase), which can then be used as a carbon source for SCP production by growing Candida utilis. Unfortunately, this approach minimizes the yield of methane. In addition, more than 50% of the volatile solids are discarded in the solid residue, which would pose a pollution problem in practical applications. In the present study, we have avoided the above disadvantages by using crop residues as the main carbon source for a biomass-producing aerobic fermentation, and the liquor from the stabilized anaerobic digestion of cattle manure is used primarily for non-carbon nutrient supplements (N,P,K) for yeast SCP production. Since anaerobic digestors often fail in practice, the relative effectiveness of alkali- and acid-pretreated manure as the source of nutrient supplements was also investigated. MATERIALS AND METHODS
Organism Candida utilis (ATCC 9226) was maintained on yeast--malt agar (yeast extract, 3 g; malt extract, 3 g, peptone, 5 g; glucose, 10 g; agar, 15 g; in one litre of solution). The inoculum was pre-adapted on the respective fermentation media by two serial transfers before use. A 5 or 10% v/v inoculum was used as indicated in the respective experiments. ChemicaUy-pretreated cattle manure liquor (CCML ) In attemping to release any recalcitrant inherent nutrients, a sample of wet manure was boiled in a 0.5 N H2 SO4 or 0.25 N NaOH solution in a 5% w/v slurry for 1 h. The liquor was obtained by filtering the pretreatment slurry through muslin cloth.
Anaerobically-fermented cattle manure liquor (ACML) In this case, the cattle manure was anaerobically fermented (for methane co-production) for 15 days. The anaerobic sludge was stored in a cold room
177 overnight to allow solids to settle out. The decanted supernatant contained 1.2 g N 1-1 and a small q u a n t i t y of volatile f a t t y acids (Scharer and MooYoung, 1979).
Barley straw acid hydrolysate (BSAH) Chopped barley straw was boiled with 0.5 N H2 SO4 solution in a 5% w/v slurry for 1 h to release the hemicellulose sugars. The BSAH was obtained by filtering the slurry through muslin cloth. The solid residue can be used as ruminant-grade forage (Moo-Young et al., 1979). In some cases, more chopped barley straw was boiled in the BSAH for an extra hour to increase the concentration of the reducing sugars (RS) in the acid hydrolysate solution.
Fementation media A. Anaerobically fermented cattle manure liquor (ACML) (i) ACML and B S A H mixture. Double-treated BSAH was mixed with ACML in a 1:2 v/v ratio giving a final mixture containing 11.5 g reducing sugars 1-1 with a C:N ratio of 5.4. A 10% v/v inoculum was used and fermentation was carried out under non-aseptic conditions since low pH 4.5 operations were used.
(ii) ACML, xylose and glucose mixture. The ACML was diluted with water in a 2:1 v/v ratio and xylose (8.33 g 1-1) and glucose (1.67 g 1-1) were added giving a final mixture containing 8.5 g RS 1-1 and 0.5 g N 1-1 with a C:N ratio of 6.8. A 10% v/v inoculum was used, and fermentation was carried out in a 10-1 fermentor (Microferm Fermentor, New Brunswick Scientific Co., NJ) at 29°C with 1 VVM aeration and 400 r.p.m, propeller speed. The medium along with the fermentor was sterilized at 121°C for 1 h. Xylose and glucose were mixed in a ratio of 5:1 w/w as this is the usual ratio f o u n d in the hemicellulose c o m p o n e n t of barley straw.
B. Chemically-pretreated cattle manure liquor (CCML ) The CCML was used primarily for the N source and the BSAH for the C source. Both solutions were mixed in approximately a 1:1 v/v ratio so that the C:N ratio was about 7.0. The pH of the mixture was adjusted with Ca(OH)2 to 4.5. Most of the precipitated CaSO4 was removed by sedimentation. Fermentation was carried out in Erlenmeyer flasks (50 ml quantities of the medium were sterilized at 121°C for 20 min). The inoculated (5% v/v) flasks were incubated on a gyrotory shaker at 250 r.p.m, and 29°C. Samples (washed twice) were analysed for protein by the Kjeldahl m e t h o d and for reducing sugars by the dinitrosalicyclic acid method.
178 RESULTS AND DISCUSSION
1. Anaerobically-fermented cattle manure liquor (ACML) Typical results for a sample of anaerobically fermented cattle manure liquor (ACML) mixed with barley straw acid hydrolysate (BSAH) are shown in Fig. 1. Good growth was obtained, giving a production of 2.4 g 1-1 of PX (PX = concentration of microbial protein in the medium) with a protein yield of 22 g g- 1 RS after 32 h of fermentation. However, a b o u t 26% of the RS were unutilized. The pH of the medium remained fairly constant at 4.5. Diauxic growth was observed with a short period of high growth rate (0.12 h -1 ) in the initial stage followed by a longer period of slower growth rate (0.03 h -1 ) in the later stage of fermentation. It is believed that the relatively short period of fast growth was due to the utilization of glucose and the longer period of slower growth rate was due to the utilization of xylose. When ACML and BSAH were mixed in 2:3 v/v ratio, a clearer diauxic growth pattern was observed. To check the above inference, C. utilis was grown in a mixture of diluted ACML, xylose and glucose (Fig. 2). The same pattern of diauxic growth was 16 r
3.;
r
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:
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....... t3]t5 -
4-0 -
"
00
10
20
30
40
50
TIME ( h )
Fig. 1. T y p i c a l t i m e courses for T o r u l a y e a s t c u l t i v a t e d o n a m i x t u r e o f a n a e r o b i c a l l y f e r m e n t e d c a t t l e m a n u r e a n d b a r l e y - s t r a w acid h y d r o l y s a t e . RS = R e d u c i n g sugars. P X = Microbial p r o t e i n . 16
3.2
2.8 ~12.
4
,~2A
0.8
OA o-
o(
~
J 10
20 TIME
30
40
50
( h )
Fig. 2. Typical t i m e c o u r s e for f e r m e n t a t i o n o f a m i x t u r e o f x y l o s e ( 8 . 3 3 g 1-1 ) a n d glucose (1.67 g 1-1 ) w i t h a n a e r o b i c a l l y f e r m e n t e d c a t t l e m a n u r e (pH 4.5). RS = R e d u c i n g sugars. P X = Microbial p r o t e i n .
179
observed (i.e. a short period of fast growth rate (0.15 h -1 ) on glucose followed b y a longer period of slower growth rate (0.08 h -1 ) on xylose). In another experiment, when the content of RS was increased by a factor of 1.5 in the mixture of ACML and BSAH, the same protein yield (0.22 g g-1 ES) and the same proportion of unutilized residual RS were obtained. The unutilized sugars are probably galactose, arabinose and oligosaccharides, having reducing properties which are released in the acid hydrolysate of the barley straw. 2. Chemically pretreated cattle manure liquor (CCML) Figure 3 shows typical results for the growth of C. utilis in a mixture of acid-treated CCML and BSAH using a 5% v/v inoculum. Similar results were obtained for b o t h aseptic (this case) and non-aseptic fermentation conditions, indicating as before the practical utility of the low pH conditions. Moreover the acid-treatment of b o t h substrates (manure and barley straw) provided presterilization. As before, a maximum protein yield of 0.22 g g-1 RS was obtained after a 30-h fermentation, during which time the pH changes were slight. However, a large proportion of unutilized RS (usually 30--40%) remained unutilized, indicating that some of the RS (galactose, arabinose, etc.) found in the acid hydrolysate of straw cannot be metabolized by this strain of C. utilis, as is possible with a new strain which is n o w available in our laboratory (Gonzalez, 1979). Diauxic growth was also apparent in this case, b u t the first period of higher rate (0.17 h -i ) was longer than the second period of slow growth rate (0.017 h -t ). It was assumed that more glucose might have been released from the manure with the acid treatment. 20
2C
16
\.1/-.
v~ 12
1E
4.o I 3.21 2.81
~ 2.4 2.c
z:: 4
°o
1'o
3'0 TIME
( h )
4fO
Q8, 0
do TIME
( h )
Fig. 3. T y p i c a l r e s u l t s f o r fermentation of acid-treated cattle manure liquor and b a r l e y straw acid h y d r o l y s a t e . R S = Reducing sugars. P X = Microbial protein. Fig. 4. R e s u l t s f o r f e r m e n t a t i o n o f alkali-treated cattle manure liquor and barley straw acid hydrolysate. R S = Reducing sugars. P X = Microbial protein.
I
19.0 18.0
Alkali t r e a t e d m a n u r e l i q u o r + acid t r e a t e d s t r a w liquor
8.5
Anaerobically fermented manure liquor + xylose/glucose mixture
Acid t r e a t e d m a n u r e l i q u o r + acid t r e a t e d s t r a w l i q u o r
11.5
7.0
8.2
0.5
3.1
Final
(g I-I)
Initial
(g 1"I)
R e d u c i n g sugars
Anaerobically fermented m a n u r e l i q u o r + acid t r e a t e d s t r a w liquor
Fermentation medium
2.8
2.7
2.1
2.4
Microbial protein ( g 1-1)
28
30
36
32
Growth time (h)
0.14
0.17
0.15
0.12
u~
0.05
0.02
0.08
0.03
~2
Apparant g r o w t h r a t e ( h "1 )
S u m m a r y o f results for u t i l i z a t i o n o f c a t t l e m a n u r e a n d b a r l e y s t r a w f o r T o r u l a y e a s t SCP p r o d u c t i o n
TABLE I
100.0
90.0
58.3
76.5
Protein productivity (rag 1-1 h -1)
(3O
181 Figure 4 shows results for the yeast growth on a mixture (1:1 v/v ratio) of the alkali-treated manure and BSL having a C:N ratio of 8. (Alkali-treated manure has an advantage over the acid-pretreatment in that smaller quantaties of Ca(OH)2 are required for pH adjustment.) As before, the protein yield was the same (0.22 g g-1 RS) and about 38% RS were left unutilized after 30 h of fermentation. A similar pattern of diauxic growth was observed as in Fig. 2, that is, a short period of fast_growth rate (0.14 h -1) followed by a longer period of slower growth rate (0.05 h-' ). Results for the utilization of cattle manure (Table I) indicated that the apparent growth rate of C. utilis was slightly lower with ACML than with acid- and alkali-treated manure, but protein productivity was high when compared on the basis of initial RS supplied in the medium. Higher protein productivity might be due to the fact that ACML contained some volatile fatty acids which act as additional C sources. In the present study, a protein yield of 0.22 g g-1 of RS utilized was obtained, which is very close to the theoretical value (0.25 g g-1 RS utilized). However, a possible deficiency of this particular system was that this strain of C. utilis was unable to utilize about 30--40% of RS, which remained in the fermented broth. Similarly, Han et al. (1976) have found that only 0.598 g of protein per litre with C. utilis (ATCC 9256) was produced from acid hydrolysate of ryegrass (Lolium multiform lam.) which contained 6 g of RS. Thus, their particular strain of yeast also failed to utilize all the RS from the straw hydrolysate, in spite of their addition of a substantial quantity of yeast extract (1.25 g 1-1 ); only about 0.1 g of protein was produced per g of RS supplied. Recently, we have selected a new strain of C. utilis (isolated from peat moss) which is capable of utilizing more than 95% of the sugars in our crop-residue hydrolysates (including galactose and arabinose). At present, this capability is being exploited by Gonzalez (1979) in this laboratory for the utilization of maize stover and sugar cane bagasse to produce yeast SCP. It is felt that unless a higher conversion of the reducing sugars in the fermentation media is possible, the economic feasibility of the process is low, despite the potential credit of methane co-production with the SCP (Moo-Young et al., 1979). In independent studies, preliminary feeding trials on rats with the SCP product obtained from these types of culture indicate good response when compared to casein as the dietary protein source. CONCLUSION Candida utilis was capable of utilizing most of the soluble carbohydrates from the mixture of anaerobically fermented cattle manure liquor and acid hydrolysate of barley straw for SCP production. Both acid- and alkali-pretreated cattle manure liquors also proved to be good sources of nutients for this yeast to produce SCP from the acid hydrolysate of barley straw. About 30--40% of RS was left unutilized by the strain of C. utilis. In order more fully to utilize the hydrolysate sugars such as galactose and arabinose, a new
182 strain o f C. utilis is being investigated. Utilization o f a n a e r o b i c a l l y f e r m e n t e d m a n u r e has t h e a d d e d a t t r a c t i o n t h a t useful m e t h a n e fuel gas can be co-prod u c e d b y t h e i n t e g r a t e d two-stage f e r m e n t a t i o n process. ACKNOWLEDGEMENTS Financial assistance f r o m t h e National Research Council and the Departent o f Agriculture, C a n a d a is a c k n o w l e d g e d . We are t h a n k f u l f o r some o f the e x p e r i m e n t a l d a t a p r o v i d e d b y Peter H r y b . REFERENCES Gonzalez, A., 1979. Ph.D. Thesis, University of Waterloo, in preparation. Han, Y.W., Cheeke, P.R., Anderson, A.W. and Lekprayon, C., 1976. Growth of Aurobasidium pullulans on straw hydrolysate. Appl. Environ. Microbiol., 32: 799--802. Moo-Young, M. and Chahal, D.S., 1979. Utilization of cattle manure for single-cell protein production with Chaetomium cellulolyticum. Anim. Feed Sci. Technol., 4: 199--208. Moo-Young, M., Daugulis, A.J. and Moreira, A., 1979. Economic evaluation on a farm co-operative plant for producing animal feed from agricultural wastes. In: M. MooYoung and G.J. Farquhar (Editors), Waste Treatment and Utilization. Pergamon, Oxford. Rhodes, R.A. and Orton, W.L., 1975. Solid substrate fermentation of feedlot waste combined with feed grains. Trans. Am. Soc. Agric. Eng., 18: 728--733. Scharer, J.M. and Moo-Young, M., 1979. Methane generation by anaerobic digestion of cellulose-containing wastes. Adv. Biochem. Eng., 11 : 85--101. Thomas, K. and Evison, L.M., 1978. A new approach to anaerobic digestion: Process control to produce effluents suitable for SCP recovery. Proc. Int. Res. Syrup. SCI on New Processes of Water Treatment and Recovery, London, September 1977.
175
UTILIZATION OF CATTLE MANURE FOR TORULA YEAST PRODUCTION FROM STRAW HYDROLYSATES
M. MOO-YOUNG, D.S. CHAHAL and D. VLACH, Biochemical Engineering Group, Chemical Engineering Department, University of Waterloo, Waterloo, Ontario (Canada) (Received 27 August 1979; accepted for publication 18 Febuary 1980)
ABSTRACT Moo-Young, M., Chahal, D.S. and Vlach, D., 1980. Utilization of cattle manure for Torula yeast production from straw hydrolysates. Anita. Feed Sci. Technol., 5: 175--182. Good growth of Torula (Candida utilis) was obtained from mixtures of anaerobically fermented cattle manure liquor and barley straw acid hydrolysate. Relatively good growth of this yeast was also obtained from mixtures of acid- or alkali-pretreated cattle manure and barley straw acid hydrolysate. However, a significant amount of reducing sugars (30--40%) was usually left unutilized, indicating the need for a better strain of C. utilis capable of utilizing such sugars as galactose and arabinose which are released (in additon to glucose and xylose) from the hemicellulose during the acid hydrolysis of barley straw. These experiments demonstrate the practicability of using these nutrient sources, obtainable from abundant waste materials, to produce useful single cell protein (SCP) products. In particular, an integrated two stage anaerobic--aerobic fermentation process for the co-production of methane fuel gas and SCP offers an economically attractive option.
INTRODUCTION Vast q u a n t i t i e s o f animal m a n u r e s o c c u r universally as waste residues. In t h e past, these residues have f r e q u e n t l y b e e n used as soil fertilizer. However, increasingly stringent g o v e r n m e n t regulations on e n v i r o n m e n t a l p o l l u t i o n , especially n e a r u r b a n areas, n o w restrict this a p p l i c a t i o n o f manures. A n i m a l m a n u r e s r e p r e s e n t a p o t e n t i a l l y valuable s o u r c e o f n u t r i e n t s for f e r m e n t a t i o n processes. If i m p l e m e n t e d , this a p p l i c a t i o n w o u l d be an environm e n t a l l y - a c c e p t a b l e and r e s o u r c e - c o n s e r v i n g m e t h o d o f utilizing manures. R h o d e s a n d O r t o n ( 1 9 7 5 ) have tried t o utilize f e e d l o t waste filtrate with the i n d i g e n o u s m i c r o f l o r a for f e r m e n t a t i o n o f c r a c k e d maize as animal feed. H o w e v e r , the end p r o d u c t o f f e r m e n t a t i o n s h o w e d v e r y little increase in t h e p r o t e i n c o n t e n t o f t h e processed materials. In a d d i t i o n , feeding trials with mice given this f e r m e n t e d m a i z e s h o w e d very p o o r g r o w t h responses c o m p a r e d t o t h e c o n t r o l diet (a c o m m e r c i a l feed). The a u t h o r s r e p o r t e d no 0377-8401/80/0000--0000/$02.25 © 1980 Elsevier Scientific Publishing Company
176 toxicity of the fermented maize product! However, there was no check on the real possibility of multiplication of toxin-producing organism(s) in such an uncontrolled system of fermentation. Moo-Young and Chahal (1979) have recently shown that acid- or alkalipretreated cattle manure can be a good supplementary nutrient source for single cell protein (SCP) production from crop residues such as cornstover, using the cellulolytic organism, Chaetomium cellulolyticum. They also found that anaerobically-prefermented cattle manure liquor from a methane-generating digestor provided an even better source of supplementary nutrients. Thomas and Evison (1978) have studied the possibility of inhibiting the methanogenesis phase in an anaerobic digestion of pig manure so as to maximize volatile f a t t y acids concentration in the effluent (through the acidogenesis phase), which can then be used as a carbon source for SCP production by growing Candida utilis. Unfortunately, this approach minimizes the yield of methane. In addition, more than 50% of the volatile solids are discarded in the solid residue, which would pose a pollution problem in practical applications. In the present study, we have avoided the above disadvantages by using crop residues as the main carbon source for a biomass-producing aerobic fermentation, and the liquor from the stabilized anaerobic digestion of cattle manure is used primarily for non-carbon nutrient supplements (N,P,K) for yeast SCP production. Since anaerobic digestors often fail in practice, the relative effectiveness of alkali- and acid-pretreated manure as the source of nutrient supplements was also investigated. MATERIALS AND METHODS
Organism Candida utilis (ATCC 9226) was maintained on yeast--malt agar (yeast extract, 3 g; malt extract, 3 g, peptone, 5 g; glucose, 10 g; agar, 15 g; in one litre of solution). The inoculum was pre-adapted on the respective fermentation media by two serial transfers before use. A 5 or 10% v/v inoculum was used as indicated in the respective experiments. ChemicaUy-pretreated cattle manure liquor (CCML ) In attemping to release any recalcitrant inherent nutrients, a sample of wet manure was boiled in a 0.5 N H2 SO4 or 0.25 N NaOH solution in a 5% w/v slurry for 1 h. The liquor was obtained by filtering the pretreatment slurry through muslin cloth.
Anaerobically-fermented cattle manure liquor (ACML) In this case, the cattle manure was anaerobically fermented (for methane co-production) for 15 days. The anaerobic sludge was stored in a cold room
177 overnight to allow solids to settle out. The decanted supernatant contained 1.2 g N 1-1 and a small q u a n t i t y of volatile f a t t y acids (Scharer and MooYoung, 1979).
Barley straw acid hydrolysate (BSAH) Chopped barley straw was boiled with 0.5 N H2 SO4 solution in a 5% w/v slurry for 1 h to release the hemicellulose sugars. The BSAH was obtained by filtering the slurry through muslin cloth. The solid residue can be used as ruminant-grade forage (Moo-Young et al., 1979). In some cases, more chopped barley straw was boiled in the BSAH for an extra hour to increase the concentration of the reducing sugars (RS) in the acid hydrolysate solution.
Fementation media A. Anaerobically fermented cattle manure liquor (ACML) (i) ACML and B S A H mixture. Double-treated BSAH was mixed with ACML in a 1:2 v/v ratio giving a final mixture containing 11.5 g reducing sugars 1-1 with a C:N ratio of 5.4. A 10% v/v inoculum was used and fermentation was carried out under non-aseptic conditions since low pH 4.5 operations were used.
(ii) ACML, xylose and glucose mixture. The ACML was diluted with water in a 2:1 v/v ratio and xylose (8.33 g 1-1) and glucose (1.67 g 1-1) were added giving a final mixture containing 8.5 g RS 1-1 and 0.5 g N 1-1 with a C:N ratio of 6.8. A 10% v/v inoculum was used, and fermentation was carried out in a 10-1 fermentor (Microferm Fermentor, New Brunswick Scientific Co., NJ) at 29°C with 1 VVM aeration and 400 r.p.m, propeller speed. The medium along with the fermentor was sterilized at 121°C for 1 h. Xylose and glucose were mixed in a ratio of 5:1 w/w as this is the usual ratio f o u n d in the hemicellulose c o m p o n e n t of barley straw.
B. Chemically-pretreated cattle manure liquor (CCML ) The CCML was used primarily for the N source and the BSAH for the C source. Both solutions were mixed in approximately a 1:1 v/v ratio so that the C:N ratio was about 7.0. The pH of the mixture was adjusted with Ca(OH)2 to 4.5. Most of the precipitated CaSO4 was removed by sedimentation. Fermentation was carried out in Erlenmeyer flasks (50 ml quantities of the medium were sterilized at 121°C for 20 min). The inoculated (5% v/v) flasks were incubated on a gyrotory shaker at 250 r.p.m, and 29°C. Samples (washed twice) were analysed for protein by the Kjeldahl m e t h o d and for reducing sugars by the dinitrosalicyclic acid method.
178 RESULTS AND DISCUSSION
1. Anaerobically-fermented cattle manure liquor (ACML) Typical results for a sample of anaerobically fermented cattle manure liquor (ACML) mixed with barley straw acid hydrolysate (BSAH) are shown in Fig. 1. Good growth was obtained, giving a production of 2.4 g 1-1 of PX (PX = concentration of microbial protein in the medium) with a protein yield of 22 g g- 1 RS after 32 h of fermentation. However, a b o u t 26% of the RS were unutilized. The pH of the medium remained fairly constant at 4.5. Diauxic growth was observed with a short period of high growth rate (0.12 h -1 ) in the initial stage followed by a longer period of slower growth rate (0.03 h -1 ) in the later stage of fermentation. It is believed that the relatively short period of fast growth was due to the utilization of glucose and the longer period of slower growth rate was due to the utilization of xylose. When ACML and BSAH were mixed in 2:3 v/v ratio, a clearer diauxic growth pattern was observed. To check the above inference, C. utilis was grown in a mixture of diluted ACML, xylose and glucose (Fig. 2). The same pattern of diauxic growth was 16 r
3.;
r
2.6 2A x
:
1.e
•
....... t3]t5 -
4-0 -
"
00
10
20
30
40
50
TIME ( h )
Fig. 1. T y p i c a l t i m e courses for T o r u l a y e a s t c u l t i v a t e d o n a m i x t u r e o f a n a e r o b i c a l l y f e r m e n t e d c a t t l e m a n u r e a n d b a r l e y - s t r a w acid h y d r o l y s a t e . RS = R e d u c i n g sugars. P X = Microbial p r o t e i n . 16
3.2
2.8 ~12.
4
,~2A
0.8
OA o-
o(
~
J 10
20 TIME
30
40
50
( h )
Fig. 2. Typical t i m e c o u r s e for f e r m e n t a t i o n o f a m i x t u r e o f x y l o s e ( 8 . 3 3 g 1-1 ) a n d glucose (1.67 g 1-1 ) w i t h a n a e r o b i c a l l y f e r m e n t e d c a t t l e m a n u r e (pH 4.5). RS = R e d u c i n g sugars. P X = Microbial p r o t e i n .
179
observed (i.e. a short period of fast growth rate (0.15 h -1 ) on glucose followed b y a longer period of slower growth rate (0.08 h -1 ) on xylose). In another experiment, when the content of RS was increased by a factor of 1.5 in the mixture of ACML and BSAH, the same protein yield (0.22 g g-1 ES) and the same proportion of unutilized residual RS were obtained. The unutilized sugars are probably galactose, arabinose and oligosaccharides, having reducing properties which are released in the acid hydrolysate of the barley straw. 2. Chemically pretreated cattle manure liquor (CCML) Figure 3 shows typical results for the growth of C. utilis in a mixture of acid-treated CCML and BSAH using a 5% v/v inoculum. Similar results were obtained for b o t h aseptic (this case) and non-aseptic fermentation conditions, indicating as before the practical utility of the low pH conditions. Moreover the acid-treatment of b o t h substrates (manure and barley straw) provided presterilization. As before, a maximum protein yield of 0.22 g g-1 RS was obtained after a 30-h fermentation, during which time the pH changes were slight. However, a large proportion of unutilized RS (usually 30--40%) remained unutilized, indicating that some of the RS (galactose, arabinose, etc.) found in the acid hydrolysate of straw cannot be metabolized by this strain of C. utilis, as is possible with a new strain which is n o w available in our laboratory (Gonzalez, 1979). Diauxic growth was also apparent in this case, b u t the first period of higher rate (0.17 h -i ) was longer than the second period of slow growth rate (0.017 h -t ). It was assumed that more glucose might have been released from the manure with the acid treatment. 20
2C
16
\.1/-.
v~ 12
1E
4.o I 3.21 2.81
~ 2.4 2.c
z:: 4
°o
1'o
3'0 TIME
( h )
4fO
Q8, 0
do TIME
( h )
Fig. 3. T y p i c a l r e s u l t s f o r fermentation of acid-treated cattle manure liquor and b a r l e y straw acid h y d r o l y s a t e . R S = Reducing sugars. P X = Microbial protein. Fig. 4. R e s u l t s f o r f e r m e n t a t i o n o f alkali-treated cattle manure liquor and barley straw acid hydrolysate. R S = Reducing sugars. P X = Microbial protein.
I
19.0 18.0
Alkali t r e a t e d m a n u r e l i q u o r + acid t r e a t e d s t r a w liquor
8.5
Anaerobically fermented manure liquor + xylose/glucose mixture
Acid t r e a t e d m a n u r e l i q u o r + acid t r e a t e d s t r a w l i q u o r
11.5
7.0
8.2
0.5
3.1
Final
(g I-I)
Initial
(g 1"I)
R e d u c i n g sugars
Anaerobically fermented m a n u r e l i q u o r + acid t r e a t e d s t r a w liquor
Fermentation medium
2.8
2.7
2.1
2.4
Microbial protein ( g 1-1)
28
30
36
32
Growth time (h)
0.14
0.17
0.15
0.12
u~
0.05
0.02
0.08
0.03
~2
Apparant g r o w t h r a t e ( h "1 )
S u m m a r y o f results for u t i l i z a t i o n o f c a t t l e m a n u r e a n d b a r l e y s t r a w f o r T o r u l a y e a s t SCP p r o d u c t i o n
TABLE I
100.0
90.0
58.3
76.5
Protein productivity (rag 1-1 h -1)
(3O
181 Figure 4 shows results for the yeast growth on a mixture (1:1 v/v ratio) of the alkali-treated manure and BSL having a C:N ratio of 8. (Alkali-treated manure has an advantage over the acid-pretreatment in that smaller quantaties of Ca(OH)2 are required for pH adjustment.) As before, the protein yield was the same (0.22 g g-1 RS) and about 38% RS were left unutilized after 30 h of fermentation. A similar pattern of diauxic growth was observed as in Fig. 2, that is, a short period of fast_growth rate (0.14 h -1) followed by a longer period of slower growth rate (0.05 h-' ). Results for the utilization of cattle manure (Table I) indicated that the apparent growth rate of C. utilis was slightly lower with ACML than with acid- and alkali-treated manure, but protein productivity was high when compared on the basis of initial RS supplied in the medium. Higher protein productivity might be due to the fact that ACML contained some volatile fatty acids which act as additional C sources. In the present study, a protein yield of 0.22 g g-1 of RS utilized was obtained, which is very close to the theoretical value (0.25 g g-1 RS utilized). However, a possible deficiency of this particular system was that this strain of C. utilis was unable to utilize about 30--40% of RS, which remained in the fermented broth. Similarly, Han et al. (1976) have found that only 0.598 g of protein per litre with C. utilis (ATCC 9256) was produced from acid hydrolysate of ryegrass (Lolium multiform lam.) which contained 6 g of RS. Thus, their particular strain of yeast also failed to utilize all the RS from the straw hydrolysate, in spite of their addition of a substantial quantity of yeast extract (1.25 g 1-1 ); only about 0.1 g of protein was produced per g of RS supplied. Recently, we have selected a new strain of C. utilis (isolated from peat moss) which is capable of utilizing more than 95% of the sugars in our crop-residue hydrolysates (including galactose and arabinose). At present, this capability is being exploited by Gonzalez (1979) in this laboratory for the utilization of maize stover and sugar cane bagasse to produce yeast SCP. It is felt that unless a higher conversion of the reducing sugars in the fermentation media is possible, the economic feasibility of the process is low, despite the potential credit of methane co-production with the SCP (Moo-Young et al., 1979). In independent studies, preliminary feeding trials on rats with the SCP product obtained from these types of culture indicate good response when compared to casein as the dietary protein source. CONCLUSION Candida utilis was capable of utilizing most of the soluble carbohydrates from the mixture of anaerobically fermented cattle manure liquor and acid hydrolysate of barley straw for SCP production. Both acid- and alkali-pretreated cattle manure liquors also proved to be good sources of nutients for this yeast to produce SCP from the acid hydrolysate of barley straw. About 30--40% of RS was left unutilized by the strain of C. utilis. In order more fully to utilize the hydrolysate sugars such as galactose and arabinose, a new
182 strain o f C. utilis is being investigated. Utilization o f a n a e r o b i c a l l y f e r m e n t e d m a n u r e has t h e a d d e d a t t r a c t i o n t h a t useful m e t h a n e fuel gas can be co-prod u c e d b y t h e i n t e g r a t e d two-stage f e r m e n t a t i o n process. ACKNOWLEDGEMENTS Financial assistance f r o m t h e National Research Council and the Departent o f Agriculture, C a n a d a is a c k n o w l e d g e d . We are t h a n k f u l f o r some o f the e x p e r i m e n t a l d a t a p r o v i d e d b y Peter H r y b . REFERENCES Gonzalez, A., 1979. Ph.D. Thesis, University of Waterloo, in preparation. Han, Y.W., Cheeke, P.R., Anderson, A.W. and Lekprayon, C., 1976. Growth of Aurobasidium pullulans on straw hydrolysate. Appl. Environ. Microbiol., 32: 799--802. Moo-Young, M. and Chahal, D.S., 1979. Utilization of cattle manure for single-cell protein production with Chaetomium cellulolyticum. Anim. Feed Sci. Technol., 4: 199--208. Moo-Young, M., Daugulis, A.J. and Moreira, A., 1979. Economic evaluation on a farm co-operative plant for producing animal feed from agricultural wastes. In: M. MooYoung and G.J. Farquhar (Editors), Waste Treatment and Utilization. Pergamon, Oxford. Rhodes, R.A. and Orton, W.L., 1975. Solid substrate fermentation of feedlot waste combined with feed grains. Trans. Am. Soc. Agric. Eng., 18: 728--733. Scharer, J.M. and Moo-Young, M., 1979. Methane generation by anaerobic digestion of cellulose-containing wastes. Adv. Biochem. Eng., 11 : 85--101. Thomas, K. and Evison, L.M., 1978. A new approach to anaerobic digestion: Process control to produce effluents suitable for SCP recovery. Proc. Int. Res. Syrup. SCI on New Processes of Water Treatment and Recovery, London, September 1977.