Deschamps, F.

Garcia, Jean-Louis

Labat, Marc

Meyer, F.

Horizon / Pleins textes

I 
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Biotechnology Letters V o l  6 No 6 379-384 (1984) 
I 
ANAEROBIC DIGESTION 
OF SUGAR BEET PULPS 
M.  LA BAT^, J.L. GAR CIA^*, F.  MEYER^ 
and F. DESCHAMPSb 
, a. Laboratoire de Microbiologie ORSTOM, Université de 
Provence, 13331 Marseille cedex 3, France. 
b. IRCHA, B.P. nol, 91710 Vert le Petit, France. 
SUMMARY 
Anaerobic digestion of sugar bee; pulps was studied in a 
70 1 digestor. with sequential feeding, after enzymatic hydrolysis 
by Trichoderma harzianum cellulases. buring the 130 days feeding, 
3.6 m of biogas were produced with an' average content of 5 8 ' %  
CH4 from 270 1 of hydrolysed pulps at 20 g VS/1. Average yield 
3 
and production rate were respectively 0.67 m 3 /kg VS and 0.4 m 3 / 
' kg VS and 0.4  m3/m3 of digestor'per day. 
INTRODUCTION' 
Bioconversion of agro-industry cellulosic by-products is 
promising for proteins, enzymes, alcohol or biogas production. 
Sugar-beet by-products are diversely used: yeasts and various 
metabolites are obtained from molasses, pulps are used f o r  
complementation in animal feeding after ensilage. 
Several assays have been made to produce cellulolytic 
enzymes from pulps by culture of moulds in liquid (CONTRERAS 
et al- , 1982; ROUSSOS et al., 1983) or solid media (ROUSSOS 
et al., 1983). 
0. 'k. 8. T. 8. M. fonds ~~~~~~~~~~~~~ 
IVO: A-I3113,QAJL. 
379 Cote I p 
We used the culture method of fungal growth in solid state 
fermentation as described by RAIMBAULT- and ALAZARD (1980) to 
produce cellulases by a strain of Trichoderma harzianum. The 
enzymes obtained were used to hydrolyse pulps before anaerobic 
digestion. 
MATERIALS AND METHODS 
1. Microorganism : the organism used for the production 
of cellulo1,ytic enzymes-was T.harzianum CCM F-470 (Czechoslovack 
Collection of Microorganisms, Br'nÖ , Czechoslovackia) . Stock 
cultures were maintained on Malt-Agar slants (Difco) at 29OC. 
6 
2. Aerobic fermentation medium : 100 g of dry pulps were 
ground ( 0.5 mm) and moistened with a solution containing : 
(NH4)2S04 , 9.75 g ; urea , 2.38 g ; KH2P04 , 5 g ; distilled 
water , 100 ml ; pH 4.2 , The mixture was autoclaved at llO°C 
for 1 h . 
water containing Tween 80 (1% v/v) at the rat.e of 3,107 spores/ 
g dry material. The moisture content was adjusted to 70%.Then, 
the final mixture was transferred into incubators according to 
the method of. RAIMBAULT and ALAZARD (1980). These columns 
incubators were placed in a 29°C regulated water bath and 
aerated with air saturated with water to maintain aerobic 
conditions, 
Inoculation was made with a spore suspension in distilled 
According to ROUSSOS et al. (1983), the production of 
cellulases was 2,000 IU FPA (Filter Paper Activity) and 22,000 
IU CMCA (Carboxy Methyl Cellulose Activity) from 100 g o f  
initial dry material. This product, enriched.in cellulases, 
was stored at -25OC before utilization. 
3 .  Pretreatment for p u l p  hydrolysis : 10 kg o f  wet pulps 
(20% dry matter) were mixed with 200 g of cellulases enriched 
.pulps and introduced in a reactor in 100 1 of water, Pretreatment 
380 
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was p e r f o r m e d  a t  50°C f o r  6 0 . h  w i t h  c o n t i n u o u s  m i x i n g .  A t  t h e  
e n d ,  t h e  w h o l e  p u l p s  were l i q u e f ' i e d  a n d  s t o r e d  i n  a d e e p  
f r e e z e r  b e f o r e  u t i l i z a t i o n .  
4. M e t h a n o g e n i c  f e r m e n t a t i o n  o f  s o l u b i l i z e d  p u l p s  : , 
A n a e r o b i c  d i g e s t i o n  of h y d r o l y s e d  p u l p s  was c o n d u c t e d  i n  a 70 1 
d i g e s t o ' r .  T h e  35'C t e m p e r a t u r e  c o n t r o l  was o b t a i n e d  by  c i r c u l a -  
t i o n  o f  r e g u 1 a t e . d  water i n  a n  o u t s i d e  j a c k e t .  I n t e r m i t t e n t  
r e c y c l i n g  o f  t h e  d i g e s t o r  c o n t e n t s  was made b y  a pump. ' 
T h e  d i g e s t o r  'was i n o c u l a t e d  w i t h  a m i x t u r e  c o n t a i n i n g :  . 
s w i n e  was te  30 1, f e r m e n t e d  m a n u r e  30 1, f e r m e n t e d  s w i n e  waste 
5 1 a n d  water  5 1. T h e  a d d i t i o n  o f  h y d r o l y s e d  p u l p s  s t a r t e d  
o n l y  a t  t h e  e n d  o f  g a s  p r o d u c t l o n  by t h i s  i n i t i a l  m i x t u r e .  
I n t r o d u c t i o n  of s o l u b i l i z e d  p u l p s  was made  o n c e  o r  twice 
a d a y  by  r e p l a c i n g  e q u a l ' v o l u m e  o f  d i g e s t o r  j u i c e  c o n t e n t . T h e  
V.S. c o n t e n t  h a s  b e e n  i n c r e a s e d  i n  a r a n g e  of o n e  t o  f o u r  
d u r i n g  t h e  18 w e e k s  o f  f e r m e n t a t i o n .  F j o m  t h r e e f o l d  i n c r e a s e ,  
l o a d i n g  was p e r f o r m e d  twice  a d a y .  
5 .  M e t h o d s  o f  a n a l y s i s  : g a s  p r o d u c t i o n  was m e a s u r e d  w i t h  
a g a s m e t e r  a n d  a u t o m a t i c a l l y  a n a l y s e d  by  t h e r m a l  c o n d u c t i v i t y  
w i t h  a g a s  c h r o m a t o g r a p h  (GIRDEL 30) a c c o r d i n g  t o  t h e  c a l i b r a -  
t i o n  d e s c r i b e d  by G A R C I A  e t  a 1 . ( 1 9 8 2 ) .  V o l a t i l e s  f a t t y  , a c i d s  
(VFA) were a n a l y s e d  a s  p r e v i o u s l y  d e s c r i b e d  ( G A R C I A  e t  a 1 . , 1 9 8 2 ) .  
T h e  pH v a l u e  o f  t h e  d i g e s t o r  c o n t e n t s  was f o l l o w e d  a l l  a l o n g  
t h e  a n a e r o b i c  d i g e s t i o n .  - -  
RESULTS 
D u r i n g  t h e  e a r l y  13 w e e k s ,  s e q u e n t i a l  f e e d i n g  was made  
w i t h  2 1 a d a y  o f  s o l u b i l i z e d  p u l p s  ( 2 0  g V S / l ) .  T h e n  a n  
i n c r e a s i n g  d a y l y  l o a d  was a p p l i e d  e v e r y  2 w e e k s  u n t i l  i t  
r e a c h e d  f o u r  t imes  t h e  . i n i t i a l  l o a d .  A t  t h e ,  e n d  o f  t h e  e x p e r i -  
m e n t s ,  f e e d i n g  was p e r f o r m e d  w i t h  f e r m e n t e d  s o l u b i l i z e d  p u l p s  
i n  o r d e r  t o  o b s e r v e  e f f e c t s  o n  m e t h a n o g e n e s i s .  
# 
38 1 
During the 18 weeks of fermentation, the digestor produced 
3 3,617 m 
of hydrolysed pulps. Total gas yield was 0.67 m /kg VS with an 
average production rate of 0.4 m 3 / m 3  of digestor per day. 
of biogas with an average content of 58% CH4 from 270 1 
3 
As shown in Fig.1, the weekly production of biogas reached 
a constant value after 1.5 weeks loading time and maintained 
its rate over the 13 weeks at the same daily load. When double 
I feeding was applied, the gas production increased three times 
and maintained its rate despite of the next increase of loading, 
When fermented pulps were added after 18 weeks of fermentation, 
the gas production decrkased dramatically. 
As shown in Fig.2, the gas yield was affected by load 
'increase. The best results appeared for a 4 1 of splubilized 
pulps per day load ( 8 0  VS) i.e. 5.7 kg of fresh pulps at 80% 
moisture per cubic meter per day. The decrease of gas produc- 
tion observed between the 6th and loth week of fermentation was 
probably due to the introduction of air during feeding as 
confirmed by bacterial counts (unpublished results). 
Fig.3 shows the relation between pH and CH4 production. 
Introduction of fermented pulps brought about an important drop 
of pH from 7 . 0  to 4.0 in 2 weeks. Evolution of VFA concentra- 
tion followed.*this drop of pH as shown in Fig.4. Propionic acid 
was the product mainly accumulated. During steady state of  
anaerobic digestion, VFA concentration was very low. It raised 
. since loading increased threefold. 
DISCUSSION 
Current results observed in industrial methanization with 
solid substrates are : average loading 2 to 6 Kg VS/m 3 o f  
production 0 . 5  to 1 m 3 3  /m per day, gas yield 0.2 to 0.3 m 3 /kg VS 
digestor per day, hydraulic retention time 10 to 25 days, gas * 
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- - - I (  ~ - .--......,, "..I 
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k t  
s D T F  
Fig.1- Weekly average production Fig.2- Weekly average yield 
of biogas 0 and CH4,.A o f  biogas.. and CH4 A 
S = simple charge loading 
D = double charge loading 
T = threefold charg!:, 
F = fourfold charge 
t 
 CH^ 
s 6 T F  
Fig.3- Weekly variations of pH0 Fig.4- Weekly average pro- 
and CH4 evolution A during anaero- 
bic digestion of prehydrolysed acids: acetic 0 ;propionic A ; 
sugar beet pulps. butyric . 
duction of volatile fatty 
383 
. .  
(NYNS, 1979). Other results obtained with sugar beet pulps by 
LESCURE (1982), without pretreatment, showed that 88% of the 
carbon content were digested over 96 days in a 25 m3 digestor 
by a two stages digestion. 
. In our experiments, the best conditions to methanize 
3 
* day, hydraulic retention time 17 days, gas production 1 m /m 
solubilized pulps seem t o  be : average 1oadin.g 1 kg VS/m per 
3 3  
per day, gas yield 0.74 m /kg VS. Enzymatic hydrolysis of pulps 
releases a lot of  metabolizable sugars f rom cellulose and 
probably from hemicelluloses and pectines, Given these consi- 
derations, the suitable COD loading is not allowed to be as 
3 
high a8 for crude substrates. However, biological pretreatment 
widely increases the susceptibility of carbon, which explains 
the high yield obtained. 
Acknowledgement : this work was supported by a grant 11'82.21. 
091.3261 from the Agence Française pour la MaPtrise de 1' 
Energie (AFME). 
.L 
REFERENCES 
CONTRERAS, I., GONZALEZ, R., RONCO, A., and ASENJO, J ,A. (1982). 
Biotechnol.lett. 4 , 51-56 . 
GARCIA,J.L., GUYOT,J.P., OLLIVIER,B., TRAD,M., and PAYCHENG,C. 
(1982). Cah.ORSTOM,SCr.Biol, 45 , 3-15 . 
LESCURE,J.P. (1982). Rapport COMES n'80.75.0168, AFME, PARIS. 
NYNS,E.S, (1979). XXe Cycle Perfect.Gknie Chim.Bel'g. , Louvain, 
47 pp , Univ.Cathol.Louvain. 
RAIMBAULT,M. and ALAZARD,D. (1980). Eur.J.Appl.Microbiol.Biotechno1. 
9 I 199-209 
ROUSSOS,S., GARCIA,J.L.,and RAIMBAULT,M. (1983). 1nd.Alim.Agri.c. 
100 , 449-452 . 
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Anaerobic digestion of sugar beet pulps

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