Hissette M.
1-*, Destain J.
2,Théwis A.
1, Thonart P.
2and Beckers Y.
1INTRODUCTION :The valorisation of by-products from biofuel industry will promote the application of the 2010 (2003/30/EC) and 2020 European Directives, stipulating the inclusion of
biofuels in transport sector. Use of exogenous enzymes to valorize these by-products in monogastric animals opens very wide and interesting opportunities toward Sustainable Development.
The aim of the study :Utilization of fibrolytic ruminal enzymes to valorize by-products in the digestive tract of the poultry.
Potential of ruminal cellulosome to valorise
biofuel by-products
Animal Science
1and Bio-industry
2Units, Gembloux Agro-Bio Tech /University of Liège
,
Belgium
(
*mhissette@ulg.ac.be)
Ruminal fibrolytic enzymes Biofuel
industries
By-products
Metabolisable monomers
Broiler
RUMINAL FIBROLYTIC BACTERIA
Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens => Fiber adherent bacteria (cellulosome) = 80-90 % of ruminal cellulolytic
activities
Figure 2 : General methodology to produce crude extract from ruminal solid content (A). Influence of pH in the extractive buffer (B) and influence of surfactant solution (C) at the first phase of crude extract obtention. The solution obtained from the first phase is secondary blended (Ultrathurax homogenizer) with triton X-100 to destabilize the bacterial outer membrane.
I
PRODUCTION OF RUMINAL CRUDE EXTRACT FROM RUMINAL SOLID CONTENT
The efficiency of the ruminal crude extract was tested on commercial cellulosic substrates. Each condition of production was evaluated on the aptitude of the enzymatic solution to degrade
different cellulosic substrates in vitro (in ruminal conditions).
Step 1: Separation bacteria (with enzymes)
fibers Step 2 : Extraction enzymes bacteria Bacteria Enzymes Bacteria Dosage Bacteria Enzymes Enzymes Cellulose Cellulose A
Conclusion :The extraction protocol used to obtain a crude extract rich in fibrolytic rumen enzymes was defined on specific conditions of production and
extraction. This extract hydrolyses in vitro by-products of the biofuel industry by producing quantities of reduciong sugars varying from 30 to 50 mg per g of by-products (wheat distiller grain, wheat bran and rapeseed meal). In these conditions, ruminal cellulosome can hydrolyse half-part of cellulose content in the by-products.
Figure 1 : Cellulosome structure (A to H ; from Bayer et al., 2004), specific activities of cellulolytic enzymes (I) and hydrolysis mecanism of cellulolytic bacteria (J; from Mouriño et al., 2001)
Bibliography:Bayer et al., 2004. Annual Review of Microbiology. Vol 58. 521-554. 2004. Mouriño et
al., 2001. J. Dairy Sci. 84:848-859.
J
Figure 4 : Hydrolysis of cellulose content (9-10% in these products) in different types of by-products (A) and effect of evolution of crude extract activity (Fpase activity) on the quantity of reducing sugars produced (B) and digestion rate (amount of solubilised matter) of wheat bran and wheat distiller grains (C). Kinetic of by-products cellulolysis in a 1,5 liter reactor (scale up =100x ; D).
Figure 3 : Influence of the diets fed by the cow (A), extraction site (rumen vs duodenum ; B) and extraction time (preprandial vs postprandial ; C) on ruminal crude extract activities.
CONDITIONS OF EXTRACTION OF CRUDE EXTRACT CELLULOLYTIC POTENTIAL OF RUMINAL CRUDE EXTRACT ON BY-PRODUCTS
This work was supported by the DGARNE of the Walloon Region
Study of the cellulolytic activities in different conditions of extraction to optimise the crude extract potential. 0 1 2 3 4 5 6 7 8 9 10
Endoglucanase Exoglucanase Cellobiase Crude cellulase
E n zy m a ti c a ct iv it ie s ( U I/ g D M ) p H 4 p H 6 p H 4 p H 4 p H 4 p H 8 p H 6 p H 8 p H 6 p H 8 p H 6 pH 8 B 0 1 2 3 4 5 6 7 8 9 Endoglucanase Cellobiase E n zy m a ti c a ct iv it ie s (U I/ g D M ) M e th y lc e ll u lo se T w e e n 8 0 T w e e n 8 0 M e th y lc e ll u lo se B la n co C 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85
Endoglucanase Exoglucanase Cellobiase Cellulase brute
E n zy m a ti c a ct iv it ie s ( U I/ g D M ) P re p ra n d ia l P o st p ra n d ia l P re p ra n d ia l P re p ra n d ia l P re p ra n d ia l P o st p ra n d ia l P o st p ra n d ia l P o st p ra n d ia l C 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85
Endoglucanase Exoglucanase Cellobiase Cellulase brute
R u m e n E n zy m a ti c a ct iv it ie s ( U I/ g D M ) D u o d e n u m D u o d e n u m D u o d e n u m D u o d e n u m R u m e n R u m e n R u m e n B 0 0,5 1 1,5 2 2,5
Grass hay Grass sillage Fresh grass
C ru d e c e ll u la se a ct iv it ie s (U I/ g r D M ) A 0 10 20 30 40 50 60
Wheat distiller grains Wheat bran Rapeseed meal
W h e ig h t o f re d u ci n g s u g a rs p ro d u ce d b y c e ll u lo ly si s (m g /g o f b y -p ro d u ct s) A 0 5 10 15 20 25 30 35 40 45 0 50 100 150 200 250 300 350 400 450 W e ig h t o f re d u ci n g s u g a rs p ro d u ct e d (m g r/ g o f co p ro d u ct )
Crude cellulase activity - FPase activities (UI/15ml)
Wheat bran
Wheat distiller grain B 40% cellulolysis 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 D ig e st io n r a te ( % )
Crude cellulase activities of ruminal crude extract (UI/15ml)
Wheat bran
Wheat distiller grain C 0 0,5 1 1,5 2 2,5 3 3,5 4 4,5 5 0 50 100 150 200 250 300 350 400 450 500 W e ig h t o f re d u ci n g s u g a rs p ro d u ce b y ce ll u lo ly si s (g r/ 1 ,5 l)
Incubation time (min)
