Caribbean Experiences: The Role of
Bacteria in the Hydrolysis of Sugarcane
Bagasse
Winston F. Tinto
Laboratory of Bioorganic Chemistry
University of the West Indies,
Cave Hill Campus
What are Biofuels?

Biofuels are derived from biomass
What are Biofuels?
 Biofuel products derived from a renewable source.
Biogas
Biodiesels
Bioalcohols
Renewable Energy Sources
Electricity, water heating  Stand Alone Energy Sources
Environmental Benefits
1. Lower emissions
1.1 Carbon neutral
Biomass as a Renewable Energy Source
2. Source of carbon
Ethanol: C2H2OH
Butanol: C4H9OH
Fuel
Acetic acid: C2H4O2
Organic solvent
Glycerol: C3H8O3
Pharmaceutical ,
cosmetic products
Lactic acid: C3H6O3
Pharmaceutical
Succinic acid: C4H6O4
Food and beverages
Aromatics: C6H6
Range of products
Fuel
Biochemical Composition of Biomass
References: Rubin, E. Genomics of cellulosic biofuels. Nature 454, 841–845 (2008)
Biochemical Composition of Biomass
Biomass Composition
20%
Lignin
Hemicellulose
50%
Cellulose
30%
Non-selective
depolymerization
Laccases
Manganese peroxidases
Lignin peroxidases
Biodegradation of Hemicelluloses
Glucuronic acid
Biomass Composition
Xylose backbone
Lignin
20%
Hemicellulose
50%
Cellulose
30%
Arabinose
Ferulic acid
Ethanol
Fermentation
Hydrolysis
Xylose monomers
ß-Xylosidases
Acetate
Biodegradation of Hemicelluloses
Glucuronic acid
Xylose backbone
50%
Arabinose
Ferulic acid
Hydrolysi
Hydrolysis
ß-Xylosidases
Glucuronidase
Feruloyl esterases
Arabinofuranosidases
Acetyl xylan esterases
Acetate
Biodegradation of Cellulose
Biomass Composition
20%
Lignin
Hemicellulose
50%
Cellulose
30%
Ethanol
Hydrolysis
Fermentation
Glucose
Monomers
Cellulase:
Exoglucananses
Endoglucanases
ß-Glucosidases
Classification of Biofuels
 Classified by type of feedstock used.
– First generation
– Second generation
– Third generation
First Generation Biofuels
Cereals
 Obtained directly from food sources
 Seed oils: Europe
 Sugars: Brazil
Corn
 Corn: USA
 Cereals: Canada
First
Generation
Biofuels
Sugar
Seed oils
Second Generation Biofuels
Agricultural waste
 Obtained from non-food sources
 Municipal solid waste/animal waste: USA
 Energy crops:- switch grass, miscanthus: USA
 Agricultural waste: bagasse : Brazil
Energy
crops
Focus of our research
Second
Generation
Biofuels
Municipal
waste
Third Generation Biofuels
 Algae and genetic modification
 Algae: USA, Europe
 Genetic modification: plant or
microorganisms
Genetic
engineering
Third
Generation
Biofuels
Algae
Sugarcane as a Feedstock
 Barbados’s main export
crop.
 Consists of 3 parts:
– Stem: milled to produce
sugar
– Bagasse
– Tops: fresh green leaves
– Dry leaves
Reference: Canilha L et. Al. 2012
Deriving Biofuels from Biomass
 Biotechnology
 Biological
conversion
Microorganisms
 Most environmentally
friendly
White and brown rot fungi
P. chrysosporium
(Lignin removal)
Fungi
Bacteria
Enzyme
P. putida
S. vividosporus
Aims of Research
 To create a bacterial culture collection.
 To identify isolates able to hydrolyze hemicelluloses
and cellulose to fermentable sugars.
 To investigate production of enzymes for
biodegradation of lignin and cellulose.
To produce biofuels from fermentation of sugars.
 Characterize fuel product
Characterize the bi-products of fermentation
Culturing and Isolation of Bacteria
 Culturing of samples:
– General purpose media: Tryptic soy broth and agar (TSB,
TSA)
– Serial dilutions
– Spread plating of dilutions
– Various colonies selected
– Streak plating until purity was achieved
Culturing and Isolation of Bacteria
 Spread plating
Bagasse
Dry leaves
Green leaves
Soil
Culturing and Isolation of Bacteria

Streak plating
Bacterial Culture Collection
 Total number of isolates: 104
–
–
–
–
Bagasse: 44
Soil: 27
Dry leaves: 20
Green leaves: 13
 Isolates randomly selected for initial work
– Hydrolysis of hemicellulose
– Hydrolysis of cellulose
Hydrolysis of Hemicellulose During 12 Hours
1 .4
X y lo s e ( m g /m L )
S3
1 .2
S6
1 .0
S10
S12
0 .8
0 .6
0 .4
0 .2
0 .0
0
2
4
6
8
10
12
14
T im e ( h r s )
Figure 1. Hydrolysis of hemicellulose during 12 hours
Production of Glucose During 168 Hours
G lu c o s e P r o d u c t io n 1 6 8 h r s
Glucose (mg/mL)
1 .4
S3
1 .2
S6
S 10
1 .0
S 12
0 .8
0 .6
0 .4
0
24
48
72
96
120
144
168
192
T im e ( h r s )
Figure7. Hydrolysis of cellulose during 168 hours
Conversion of Biomass to Fermentable Sugars
 Conversion of biomass to fermentable sugars an
important aspect.
 An understanding of the ability of bacteria to perform
hydrolysis is necessary.
 This will be used for second generation biofuels
production.
Production of MnP Enzymes for Lignin Biodegradation
 Dye decolorization by lignin enzymes
– Methylene blue dye substrate
– 96 well plate screen
 Degradation lignin to expose hemicelluloses and
cellulose
P>0.05
150
C o n tro l
S3
S6
100
S 10
S 12
50
Control vs. each
isolate
P<0.05
Is o la te
2
S
1
0
S
1
6
S
S
o
C
o
n
tr
3
0
l
D e g re e o f D e c o lo r is a tio n (% )
Decolorization of Methylene Blue
P<0.05
S6 vs. S3
S10 vs. S12
S6 vs. S10
S3 vs. S12
Figure 9. Degree of decolorization of methylene blue
Production of Exoglucanase Enzymes
S3
S6
1% cellulose agar
S10
S12
 Exoglucanases are active against crystalline cellulose.
 Able to cleave externally to produce glucose.
 Hydrolysis by use of Grams Iodine Kasana (2010).
Production of Endoglucanase Enzymes
 CMC: sodium methyl carboxy groups (-CH2-COO-Na+)
attached to ends.
– Makes cellulose soluble
– Endoglucanases hydrolyze internal bond and are active
against CMC.
S3
S10
1% CMC agar
S12
S6
Future Prospects
Conversion of Biomass to Fermentable Sugars
 Selection of isolates efficient at hydrolysis.
 Effects of physiological conditions on hydrolysis:
– pH
– Temperature
– Nutrient availability
 Optimization of hydrolysis process to obtain
maximum fermentable sugars.
Biofuels Production
NMR
analysis
GC-MS
analysis
Aromatics
Bacterial
hydrolysis
Other
products
Bacterial
Fermentation
Biofuels end product
Acknowledgements
 SIRDC (Sugarcane Industry Research and Development
Committee)
 WICSCBS (Sugarcane Breeding Station)
 BAMC (Barbados Agricultural Management
Corporation)
 Thank you
For more information
email: [email protected]
visit:
www.alcue-net.eu
ALCUE-NET is funded by the European Commission under the 7th
Framework Programme addressing “Mobilising Environmental
Knowledge for Policy, Industry and Society” supporting “Strategic
International R&I Cooperation”.
02/06/2015
Presentation Title
35