Bioethanol Production

Mohamed M. Abo-Elgheit
Mohamed M. Abo-ElgheitQC Microbiologist, MSc. Student à Egypt Otsuka Pharmaceutical Co.
Mohamed Mosaad Abo El-Gheit
MSc. Student, Applied Microbiology, SCU,
Ismailia, Egypt
mhmd.aboelgheit@gmail.com
Contents:
 Biofuel
 Types of Biofuel
 1st and 2nd generation of Bioethanol
 Lignocellulosic Biomass in Egypt
 Composition of Lignocellulose
 Pretreatment of Lignocellulose
 Microbial Enzymatic Hydrolysis
 Bioprocessing of Biomass
 Cellulosic Activities in Actinomycetes
 Microbial Consortia
Biofuel:
Energy from newly-growing plant
sources
CO2-neutral
alternative source of energy to the
current traditional sources e.g. gasoline
CO2 –Neutral?
1st Generation of Bioethanol
Sugars
extract ferment
ethanol
sugarcane
BRAZIL
(sucrose)
Sugars
Hydrolyze
(enzymes)
ferment
ethanol
USA
(starch)
Cosgrove; 2005
Types of Biofuel
 Solid  animal wastes and agricultural residues can be
used as sources of energy by direct burning (primitive way)
 Liquid  Bioethanol C2H5OH ( fermentation of
sugar)
 Biodiesel (by saturation of vegetable
oils)
 Gas  e.g. methane and biogas derived from organic
wastes by anaerobic digestion
Organic wastes Heat energy
Direct burning
2nd Generation of Bioethanol
Cosgrove; 2006
Lignocellulosic Biomass
 Agricultural Residues:
Source: Quantitative appraisal of biomass resource and their
energy potential in Egypt; 2013
Lignocellulosic Biomass:
 Energy crops: plants which grow at low cost, to make
biofuel.

Composition of Lignocellulose
 Cellulose
 Hemicelluloses
 Lignin
Ash
Extractives
Cellulose
Hemicellulose
(both 5 and 6 carbon sugars)
(need modified microbe to
convert to ethanol)
Ash
Extractives
Lignin (phenols)
(6 carbon sugars)
Chapple, 2006; Ladisch, 1979, 2006
Pretreatment
 break down the
shield formed by
lignin and
hemicellulose
 Open the fiber
structure
 reduce the degree
of polymerization
of cellulose.
Source: Overview of biomass pretreatment
for cellulosic ethanol production; 2009
 Pretreatment has been viewed as one of the most
expensive processing steps within the conversion of
biomass to fermentable sugar
 Pretreatment methods maybe: physical, chemical or
biological
 Biological:
 Adv. : no chemicals, no energy requirements, mild
environmental conditions
 Disadv.: slow, the activity of the microorganisms maybe
not specific to lignin only!
Pretreated Lignocellulose
 What is “Pretreated Biomass”?
increased surface area,
solubilization of cellulose,
redistribution of cellulose and lignin
 Cellulose 35-50%
 Hemicellulose 20- 35%
 Lignin 5-30%
Microbial cellulose utilization fundamental and biotechnology; 2002
Enzymatic Treatment
Pretreated
Lignocellulose
Pentoses and
hexoses + lignin and
lignin degradation
Enzymatic
Hydrolysis
cellulose glucose
hemicellulose glucose + xylose+ other
C5 and C6 sugars
Microbial cellulose utilization fundamental and biotechnology; 2002
Microbial Enzyme system:
 Substrate  cellulose + hemicellulose
 Enzymes:
 endoglucanases: cut at random internal sites
along the cellulose/hemicellulose chain
 exoglucanases: act at reducing and nonreducing
ends
 beta-glucosidase: break betaglucoside bond to
form glucose
Enzyme system
Cellulose Oligosaccharides (<10)
Endogluconase
Cellobiose
+ glucose
glucose
Exoglucanase
Beta-glucosidase
Microbial cellulose utilization fundamental and biotechnology; 2002
Lignocellulosic Activities of
Actinomycetes
 According to Lynd et al (2002) there is a considerable
concentration of cellulytic capabilities among
Actinomyceltales.
 Actinomycetes are well known for their ability to
decompose complex molecules, particularly
lignocellulose components
 Micromonospora spp and Strptomyces spp are well
known for their decomposition ability on Biomass
Actinomycetes and cellulytic
activities
Growth TempSpeices
mesophilicM. chalcea
mesophilicS. roseflavus
MesophilicS. reticuli
ThermophilicThermobifidia fusca
mesophilicKibdelosporanguim Philippinenses
Most of actinomycete species can be isolated from both soil and
water.
Bioprocessing of cellulosic Biomass
 Steps (mediated events):
1) Cellulase production
2) Hydrolysis of
cellulose/hemicellulose
3) Fermentation of cellulose
hydrolysis products e.g.
glucose
4) Fermentation of
hemicellulose hydrolysis
products other than
glucose e.g. xylose
biomass
fuel
Microbial cellulose utilization fundamental and biotechnology; 2002
Bioprocessing of cellulosic Biomass
This diagram shows the capability of consolidation or separation of
mediate events (steps) of bioprocessing of Biomass
Source: Microbial cellulose utilization fundamental and biotechnology; 2002
• SHF: Separated
Hydrolysis and
Fermentation
• SSF: Simultaneous
Saccharification and
Fermentation
• SSCF: Simultaneous
Saccharification and
Cofementation
• CBP: Consolidate
Bioprocessing
Consolidated Bioprocessing CBP
 In which all bioprocessing steps are combined together
as one process
 Biomass processing technology has exhibited a trend
toward increasing consolidation over time
 Advantages  Efficiency + Economically effective
 CBP organisms:
 Single organism
 Community of organisms( symbiotic consortium)
(which is more efficient???)
Symbiotic Consortium
 A community of organisms
 i.e 2 or more organisms living in association
 Symbiosis may be : mutualism, commensalism, o
antagonism
 Types:
 Natural consortuim
 Engineered consortuim
Genetically
Recombined natural capabilities
i.e. ecological approaches
Natural Consortium
 The main problem  doesn’t accumulate high levels
of biofuel why?
 Biofuel molecules are molecules of energy
 Biofuels represents an a pportunity for a new consortia
member (organism) to exploit
 Natural consortia tend to thermodynamically free
energy of molecules till the lowest level
 Be overcome by  engineering consortia
Models of microbial interactions in
a consortuim (dual culture)
Sequential utilization
 2 oranisms M1 and M2
 The fuel molecule (F1) is
considered a waste product of
M1. However, it is degraded by
M2 as source of energy e.g.
commensalism
 No accumlation of fuel
molecules
Co-utilization
 M1 & M2 are competing to
utilize the substrate , producing
fuel molecules
 Competitive symbiosis i.e.
controlled by inhibitors
/activatiors
 Fuel considered waste product
of both organsims
 There is accumulation of fuel
Substrate transformation
 M1 acts on substrate converting
it to a form that can be utilized
by M2
 e.g. pretreatment of
lignocellulosic material
 mutualsim
Product transformation
 M1 produces fuel products as
waste product
 M2 act on fuel to convert it into
an alternative fuel
 Look like sequential utilization.
However, the fuel molecules are
converted to alternative fuel ,
not completely utilized
Bioethanol Production
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Bioethanol Production

  • 1. Mohamed Mosaad Abo El-Gheit MSc. Student, Applied Microbiology, SCU, Ismailia, Egypt mhmd.aboelgheit@gmail.com
  • 2. Contents:  Biofuel  Types of Biofuel  1st and 2nd generation of Bioethanol  Lignocellulosic Biomass in Egypt  Composition of Lignocellulose  Pretreatment of Lignocellulose  Microbial Enzymatic Hydrolysis  Bioprocessing of Biomass  Cellulosic Activities in Actinomycetes  Microbial Consortia
  • 3. Biofuel: Energy from newly-growing plant sources CO2-neutral alternative source of energy to the current traditional sources e.g. gasoline
  • 5. 1st Generation of Bioethanol Sugars extract ferment ethanol sugarcane BRAZIL (sucrose) Sugars Hydrolyze (enzymes) ferment ethanol USA (starch) Cosgrove; 2005
  • 6. Types of Biofuel  Solid  animal wastes and agricultural residues can be used as sources of energy by direct burning (primitive way)  Liquid  Bioethanol C2H5OH ( fermentation of sugar)  Biodiesel (by saturation of vegetable oils)  Gas  e.g. methane and biogas derived from organic wastes by anaerobic digestion Organic wastes Heat energy Direct burning
  • 7. 2nd Generation of Bioethanol Cosgrove; 2006
  • 8. Lignocellulosic Biomass  Agricultural Residues: Source: Quantitative appraisal of biomass resource and their energy potential in Egypt; 2013
  • 9. Lignocellulosic Biomass:  Energy crops: plants which grow at low cost, to make biofuel. 
  • 10. Composition of Lignocellulose  Cellulose  Hemicelluloses  Lignin Ash Extractives Cellulose Hemicellulose (both 5 and 6 carbon sugars) (need modified microbe to convert to ethanol) Ash Extractives Lignin (phenols) (6 carbon sugars) Chapple, 2006; Ladisch, 1979, 2006
  • 11. Pretreatment  break down the shield formed by lignin and hemicellulose  Open the fiber structure  reduce the degree of polymerization of cellulose. Source: Overview of biomass pretreatment for cellulosic ethanol production; 2009
  • 12.  Pretreatment has been viewed as one of the most expensive processing steps within the conversion of biomass to fermentable sugar  Pretreatment methods maybe: physical, chemical or biological  Biological:  Adv. : no chemicals, no energy requirements, mild environmental conditions  Disadv.: slow, the activity of the microorganisms maybe not specific to lignin only!
  • 13. Pretreated Lignocellulose  What is “Pretreated Biomass”? increased surface area, solubilization of cellulose, redistribution of cellulose and lignin  Cellulose 35-50%  Hemicellulose 20- 35%  Lignin 5-30% Microbial cellulose utilization fundamental and biotechnology; 2002
  • 14. Enzymatic Treatment Pretreated Lignocellulose Pentoses and hexoses + lignin and lignin degradation Enzymatic Hydrolysis cellulose glucose hemicellulose glucose + xylose+ other C5 and C6 sugars Microbial cellulose utilization fundamental and biotechnology; 2002
  • 15. Microbial Enzyme system:  Substrate  cellulose + hemicellulose  Enzymes:  endoglucanases: cut at random internal sites along the cellulose/hemicellulose chain  exoglucanases: act at reducing and nonreducing ends  beta-glucosidase: break betaglucoside bond to form glucose
  • 16. Enzyme system Cellulose Oligosaccharides (<10) Endogluconase Cellobiose + glucose glucose Exoglucanase Beta-glucosidase Microbial cellulose utilization fundamental and biotechnology; 2002
  • 17. Lignocellulosic Activities of Actinomycetes  According to Lynd et al (2002) there is a considerable concentration of cellulytic capabilities among Actinomyceltales.  Actinomycetes are well known for their ability to decompose complex molecules, particularly lignocellulose components  Micromonospora spp and Strptomyces spp are well known for their decomposition ability on Biomass
  • 18. Actinomycetes and cellulytic activities Growth TempSpeices mesophilicM. chalcea mesophilicS. roseflavus MesophilicS. reticuli ThermophilicThermobifidia fusca mesophilicKibdelosporanguim Philippinenses Most of actinomycete species can be isolated from both soil and water.
  • 19. Bioprocessing of cellulosic Biomass  Steps (mediated events): 1) Cellulase production 2) Hydrolysis of cellulose/hemicellulose 3) Fermentation of cellulose hydrolysis products e.g. glucose 4) Fermentation of hemicellulose hydrolysis products other than glucose e.g. xylose biomass fuel Microbial cellulose utilization fundamental and biotechnology; 2002
  • 20. Bioprocessing of cellulosic Biomass This diagram shows the capability of consolidation or separation of mediate events (steps) of bioprocessing of Biomass Source: Microbial cellulose utilization fundamental and biotechnology; 2002 • SHF: Separated Hydrolysis and Fermentation • SSF: Simultaneous Saccharification and Fermentation • SSCF: Simultaneous Saccharification and Cofementation • CBP: Consolidate Bioprocessing
  • 21. Consolidated Bioprocessing CBP  In which all bioprocessing steps are combined together as one process  Biomass processing technology has exhibited a trend toward increasing consolidation over time  Advantages  Efficiency + Economically effective  CBP organisms:  Single organism  Community of organisms( symbiotic consortium) (which is more efficient???)
  • 22. Symbiotic Consortium  A community of organisms  i.e 2 or more organisms living in association  Symbiosis may be : mutualism, commensalism, o antagonism  Types:  Natural consortuim  Engineered consortuim Genetically Recombined natural capabilities i.e. ecological approaches
  • 23. Natural Consortium  The main problem  doesn’t accumulate high levels of biofuel why?  Biofuel molecules are molecules of energy  Biofuels represents an a pportunity for a new consortia member (organism) to exploit  Natural consortia tend to thermodynamically free energy of molecules till the lowest level  Be overcome by  engineering consortia
  • 24. Models of microbial interactions in a consortuim (dual culture)
  • 25. Sequential utilization  2 oranisms M1 and M2  The fuel molecule (F1) is considered a waste product of M1. However, it is degraded by M2 as source of energy e.g. commensalism  No accumlation of fuel molecules
  • 26. Co-utilization  M1 & M2 are competing to utilize the substrate , producing fuel molecules  Competitive symbiosis i.e. controlled by inhibitors /activatiors  Fuel considered waste product of both organsims  There is accumulation of fuel
  • 27. Substrate transformation  M1 acts on substrate converting it to a form that can be utilized by M2  e.g. pretreatment of lignocellulosic material  mutualsim
  • 28. Product transformation  M1 produces fuel products as waste product  M2 act on fuel to convert it into an alternative fuel  Look like sequential utilization. However, the fuel molecules are converted to alternative fuel , not completely utilized