Production of Bioethanol from Lignocellulosic Agricultural Wastes.ppt

Kishore

1. Production of Bioethanol from
Lignocellulosic Agricultural Wastes
Procedure, Strains, and Recent
Advancements (Second-Generation
Biofuels)

2. Introduction: The Need for 2G
Bioethanol
•First Generation (1G) Biofuel: Produced from food
crops (e.g., corn, sugarcane juice).
•Major Drawback: The "Food vs. Fuel" ethical dilemma.
•Second Generation (2G) Biofuel: Produced from non-
food lignocellulosic biomass (agricultural residues like
straw, stover, bagasse, cobs).
•Advantages: Utilizes abundant, cheap waste; reduces
environmental pollution; no competition with food
supply.
•The Challenge: The complex, recalcitrant structure of
lignocellulose requires intensive pretreatment.

3. Lignocellulosic Biomass: Structure &
Components
•Lignocellulose is a composite material:
–Cellulose (40-50%): Crystalline polymer of Glucose
(C6
​ sugar). The primary target for ethanol.
–Hemicellulose (25-35%): Amorphous polymer of
Pentose (C5
​) sugars like
Xylose and C6
​ sugars like
Mannose.
–Lignin (15-20%): A complex, amorphous phenolic
polymer. Acts as a physical barrier (the "glue")
shielding cellulose from enzymes.
•Goal: Efficiently separate and hydrolyze these
components into simple, fermentable sugars.

4. Overall Procedure: The Bioconversion
Pathway
•Biomass Preparation (Milling): Reduce particle size.
•Pretreatment: Break down the Lignin-Carbohydrate
Complex (LCC).
•Hydrolysis (Saccharification): Convert
Cellulose/Hemicellulose to simple sugars.
•Fermentation: Convert simple sugars to ethanol.
•Recovery & Purification: Distillation and dehydration.
•[Flowchart: Biomass → Pretreatment → Hydrolysis →
Fermentation → Ethanol]

5. Step 1: Biomass Preparation & Size
Reduction
•Goal: Increase the surface area-to-volume ratio for
greater chemical/enzymatic access.
•Process: Mechanical methods (chipping, grinding,
milling).
•Dry Milling: Grinding the biomass in its dry state.
•Wet Milling: Grinding after soaking (sometimes
preferred).
•Optimal Size: Usually 1
 mm to 10 mm depending on
the feedstock and subsequent pretreatment method.
•Result: Homogenized feedstock ready for
pretreatment.

6. Step 2: Pretreatment Methods (I)
•Chemical Methods: Primarily used to dissolve or
modify lignin and hemicellulose.
•Dilute Acid Hydrolysis: Uses H2
​SO4​ or HCl at high
temp. 120−160 C.
∼ ∘
–Pro: Excellent C5
​ (hemicellulose) sugar release.
–Con: Generates fermentation inhibitors (e.g., furfural,
HMF, acetic acid).
•Alkaline Pretreatment: Uses NaOH or Ca(OH)2
​ (Lime).
–Pro: Highly effective at lignin removal (delignification);
few inhibitors.
–Con: Does not significantly degrade hemicellulose or
increase surface area as much as acid.

7. Step 2: Pretreatment Methods (II)
•Physicochemical Methods: Combine physical disruption with
chemical effects.
•Steam Explosion (SE): Biomass is treated with high-pressure steam
( 160−260 C) and then rapidly depressurized.
∼ ∘
–Pro: Low cost, effective for many feedstocks.
–Con: Incomplete lignin removal; generates inhibitors.
•Ammonia Fiber Explosion (AFEX): Uses liquid ammonia at
moderate temperature and pressure, followed by rapid
decompression.
–Pro: High sugar recovery; preserves almost all hemicellulose; minimal
inhibitor formation.
–Con: High cost of ammonia and recovery.
•Final Step: All pretreated solids must be washed and detoxified to
remove inhibitors before fermentation.

8. • Methods for Removing Inhibitors from Lignocellulosic Hydrolysate
• Detoxification methods are broadly categorized into physical, chemical, and biological approaches, often used in combination.
• 1. Chemical Methods ??????
• These methods involve adding chemical agents to neutralize or precipitate inhibitory compounds.
• Overliming (Most Common):
–Process: The pH of the hydrolysate is raised to pH 10–12 using calcium hydroxide (Ca(OH)2
​)
or lime. It is held there for 30–60 minutes, then lowered back to
the fermentation range (pH 4.5–5.5) using sulfuric acid (H2
​SO4​).
–Mechanism: The high pH promotes the polymerization and precipitation of various phenolics (lignin derivatives) and aldehydes (like furfural and HMF) into
insoluble forms, which are then removed by filtration.
–Drawback: It leads to significant sugar loss and the formation of a large gypsum sludge (CaSO4
​) waste stream.
• Sulfite Addition (Sodium Sulfite, Na2
​SO3​):
–Process: Sulfite is added to the hydrolysate at the fermentation pH.
–Mechanism: Sulfite forms adducts (non-toxic complexes) with aldehyde inhibitors, such as furfural and HMF, effectively rendering them harmless to the
microbes.
–Advantage: Minimal sugar loss and simpler process compared to overliming.
• 2. Physical Methods
??????
• These methods rely on separating inhibitors based on physical properties like volatility or adsorption.
• Activated Carbon Adsorption:
–Process: The hydrolysate is passed through a column packed with activated carbon or synthetic resins.
–Mechanism: Inhibitors, especially the hydrophobic phenolic compounds and HMF/furfural, adsorb onto the high surface area of the carbon.
–Drawback: Activated carbon is expensive, requires periodic regeneration (which adds cost), and may also adsorb some valuable sugars.
• Evaporation/Stripping:
–Process: Steam or inert gas is bubbled through the hydrolysate, or the hydrolysate is simply heated.
–Mechanism: Highly volatile inhibitors, primarily acetic acid and furfural, are removed from the liquid phase.
–Advantage: Effective for volatile components but less effective for HMF or phenolics.
• 3. Biological Methods (Biotreatment)
??????
• These methods use microorganisms or enzymes to break down toxic compounds before introducing the primary fermenting organism.
• Microbial Treatment:
–Process: Treating the hydrolysate with a detoxification-only microbe, often a fungus like Coniothyrium species or a yeast like Candida species, prior to the
ethanol fermentation.
–Mechanism: These organisms possess specific enzymes (e.g., aldehyde reductases) that convert toxic aldehydes (HMF and furfural) into their less-toxic
alcohol forms (HMF→HMF alcohol; furfural → furfuryl alcohol).
–Advantage: Environmentally friendly and avoids the use of harsh chemicals.
–Note: The ethanol-producing strain (S.
 cerevisiae) can often perform this conversion during the initial lag phase of fermentation, a process called
in situ
detoxification.
• The choice of method depends heavily on the specific pretreatment method used and the resulting inhibitor profile of the hydrolysate. Often, a combination (e.g.,
overliming followed by activated carbon or biotreatment) is required for high-efficiency 2G ethanol production.

9. Step 3: Hydrolysis (Saccharification)
•Goal: Convert the liberated polymers into monomers
(simple sugars).
•Method: Enzymatic Hydrolysis (EH) is the industry
standard.
–Uses a cocktail of commercial enzymes: Cellulases (Endo-,
Exo-glucanases) and β-glucosidase.
–Also requires Hemicellulases (e.g., Xylanase) for C5
​ sugars.
•Conditions: Typically 50 C, pH 4.8, for 48−72 hours.
∘ ∼
•Output: A sugar-rich broth (hydrolysate) containing
Glucose (C6
​) and
Xylose (C5
​).

10. Step 4: Fermentation
•Process: Microorganisms convert the sugars to ethanol under
anaerobic conditions.
•C6
​H12​O6​→2C2​H5​OH+2CO2​ (Glucose)
•C5
​H10​O5​→ 2.5C2​H5​OH+ 2.5CO2​ (Xylose/Pentoses - less ∼ ∼
efficient)
•Fermentation Modes:
•Separate Hydrolysis and Fermentation (SHF): Hydrolysis and
Fermentation occur in two separate reactors (easier temp. control).
•Simultaneous Saccharification and Fermentation (SSF): Hydrolysis
and Fermentation occur in the same reactor (favored in industry).
•Simultaneous Saccharification and Co-fermentation (SSCF): Same
as SSF, but the strain can utilize both C6
​ and C5​ sugars.

11. Step 5: Ethanol Recovery and
Purification
•The "Wash": The fermented broth contains
8−12%
 v/v ethanol, water, yeast, and residual ∼
solids.
•Distillation: Used to separate ethanol from the
broth due to the difference in boiling points.
•Fractional Distillation yields a maximum of
95.6% ethanol (the azeotrope).
∼
•Dehydration: Needed to produce Anhydrous
Ethanol ( 99.8%) for fuel blending (E10, E85).
∼
•Methods: Molecular sieves (adsorption),
Azeotropic distillation, or Extractive distillation.

12. •Methods for Producing Anhydrous Ethanol
•Since distillation alone cannot break the 95.6% water/ethanol azeotrope, special dehydration techniques
are required to produce the final anhydrous product:
•1. Molecular Sieve Dehydration (Adsorption)
•Mechanism: This is the most common industrial method today. The 95.6% ethanol vapor is passed
through beds packed with a zeolite (molecular sieve) material. These sieves have pores sized precisely to
adsorb (trap) the smaller water molecules while allowing the larger ethanol molecules to pass through.
•Process: The beds are cycled; when one bed is saturated with water, the process stream is diverted to a
fresh bed, and the saturated bed is regenerated by heating or depressurization.
•2. Extractive Distillation
•Mechanism: An azeotrope-breaking solvent (called an entrainer), typically glycol or glycerol, is added to
the ethanol/water mixture. The entrainer changes the volatility of the water relative to the ethanol.
•Process: The entrainer remains in the liquid phase, increasing the relative volatility of water, allowing the
ethanol to be distilled overhead as a nearly pure vapor. The entrainer is later recovered and recycled.
•3. Pressure-Swing Distillation
•Mechanism: This method exploits the fact that the composition of the ethanol-water azeotrope changes
with pressure.
•Process: Distillation is carried out in a sequence of columns operating at different pressures. The
azeotrope produced in the first column (e.g., at low pressure) is fed to a second column operating at a
different pressure (e.g., high pressure), where the azeotrope shifts composition, allowing the desired pure
component (ethanol) to be drawn off.
•All these processes result in the final, high-purity anhydrous ethanol ( 99.8%) necessary for blending into
∼
fuel.

13. Key Strain: Saccharomyces
 cerevisiae
(Yeast)
•The Workhorse of Industry (1G & 2G): High
industrial robustness and ethanol tolerance
( 15−20%
 v/v).∼
•Metabolic Strength: Highly efficient conversion
of Glucose (C6
​)
to ethanol (high yield).
•Major Drawback: Cannot naturally ferment
Pentose Sugars (C5
​)
like Xylose, which accounts
for up to 30% of the sugars in lignocellulose.
•Strategy: Requires Genetic Engineering (GE) to
utilize C5
​ sugars for 2G applications.

14. Alternative Strain: Zymomonas
 mobilis
(Bacteria)
•Gram-negative Bacterium: Used in some
industrial processes.
•Metabolic Strength: High specific rates of
glucose uptake and ethanol production via the
Entner-Doudoroff (ED) pathway (higher
yield/productivity than S.
 cerevisiae).
•Drawback: Naturally limited to C6
​ sugars;
generally lower ethanol tolerance and is
sensitive to low pH and high temperatures.
•Strategy: Also requires GE to ferment C5
​ sugars
and enhance robustness.

15. Engineered Strains: The 2G Solution
•C5
​ Utilization:
The primary focus of strain engineering.
•Method 1 (XR-XDH Pathway): Introducing genes for Xylose
Reductase (XR) and Xylitol Dehydrogenase (XDH) from
organisms like Pichia
 stipitis into S. cerevisiae.
•Method 2 (XI Pathway): Introducing the gene for Xylose
Isomerase (XI) from bacteria like Thermus
 thermophilus.
•Inhibitor Tolerance: Strains are engineered to better resist
toxic compounds (furfural, HMF) generated during
pretreatment.
•Consolidated Bioprocessing (CBP) Strains: Engineered to
produce cellulase enzymes and ferment the resulting
sugars (e.g., cellulolytic S.
 cerevisiae).

16. •Detailed Explanation of the XR-XDH Pathway
•The XR-XDH pathway is naturally found in many xylose-utilizing yeasts (like Pichia
 stipitis) but not in
Saccharomyces
 cerevisiae. To make S. cerevisiae a second-generation biofuel producer, the genes
for these two enzymes are introduced.
•The Two Enzymes
•Xylose Reductase (XR): This enzyme catalyzes the first step.
•Xylitol Dehydrogenase (XDH): This enzyme catalyzes the second step.
•The Biochemical Process
•The conversion proceeds in two steps, using the cofactors NAD(P)H and NAD+:
•Reduction (by XR): Xylose is reduced to an intermediate sugar alcohol, xylitol.
•Xylose+NAD(P)HXylose
 Reductase (XR)​Xylitol+NAD(P)+
•Oxidation (by XDH): Xylitol is then oxidized to xylulose.
•Xylitol+NAD+Xylitol
 Dehydrogenase (XDH)​Xylulose+NADH
•Final Step: Xylulose is phosphorylated by the native yeast enzyme, xylulokinase, to enter the
pentose phosphate pathway and eventually join glycolysis for conversion into ethanol.
•The Major Drawback: Cofactor Imbalance
•The reason this pathway is less efficient than the XI pathway is a fundamental issue with the
required cofactors:
•XR typically requires NADPH (or both NADH and NADPH) for the first step.
•XDH strictly requires NAD+ for the second step.
•In the cytoplasm of S.
 cerevisiae, there is an unequal availability and regeneration rate for these
cofactors. This mismatch leads to an intermediate build-up of xylitol inside the cell. Xylitol
accumulation is toxic and slows down the entire metabolic process, resulting in lower ethanol yield
and slower productivity—a major economic hurdle in commercial 2G production.
•The alternative Xylose Isomerase (XI) pathway directly converts xylose to xylulose in a single step,
without the need for cofactors, thus avoiding the xylitol bottleneck.

17. Summary & Future Outlook
•Procedure: Lignocellulosic bioethanol production
is a multi-step, complex process whose efficiency
hinges on Pretreatment and C5
​ Fermentation
.
•Strains: S.
 cerevisiae remains dominant, but its
future lies in Genetic Engineering for robustness
and C5
​ utilization.
•Advancements: The shift to SSF/SSCF, CBP, and
Lignin Valorization is transforming 2G technology
from a research concept to industrial reality.
•Future Goal: Achieving an economically viable,
zero-waste integrated biorefinery model.

18. Production of Ethanol from
Sugarcane Molasses and Sugar Beet

19. •Production of Ethanol from Sugarcane
Molasses and Sugar Beet
•Both molasses (a byproduct of sugarcane
refining) and sugar beet are rich in readily
fermentable sugars, making their conversion
to ethanol one of the oldest and most efficient
processes, classified as First-Generation (1G)
Bioethanol.

20. Feedstock Primary Sugar Status Feedstock
Sugarcane
Molasses
Sucrose (40%
−60%), Glucose,
Fructose
Byproduct/Waste
of sugar mills.
Sugarcane
Molasses
Sugar Beet
Beta
 vulgaris
Sucrose (15%−20%)
Dedicated
crop/Primary raw
material.
Sugar Beet

21. •That's an excellent clarification! While the previous slides focused
on Second-Generation (2G) Bioethanol from lignocellulosic wastes
(like bagasse or straw), sugarcane molasses and sugar beet are
sources for First-Generation (1G) Bioethanol.
•Here is a detailed explanation of the process for alcohol (ethanol)
production using these sugar-rich agricultural byproducts.
•Production of Ethanol from Sugarcane Molasses and Sugar Beet
•Both molasses (a byproduct of sugarcane refining) and sugar beet
are rich in readily fermentable sugars, making their conversion to
ethanol one of the oldest and most efficient processes, classified as
First-Generation (1G) Bioethanol.

22. •1. Feedstock Composition
•FeedstockPrimary SugarStatusSugarcane MolassesSucrose (40%−60%), Glucose,
FructoseByproduct/Waste of sugar mills.Sugar BeetSucrose (15%−20%)Dedicated
crop/Primary raw material.
•Export to Sheets
•2. The Conversion Process (A Simplified 1G Pathway)
•The 1G pathway is significantly simpler than the 2G pathway because it skips the
complex, expensive, and inhibitory pretreatment and hydrolysis steps.
•A. Pre-Fermentation Preparation (Hydrolysis)
•Sugarcane Molasses: Molasses contains sucrose, a disaccharide. To be fermented by
yeast, sucrose must first be broken down into monosaccharides (glucose and fructose).
–Inversion: This is done by adding sulfuric acid and/or the enzyme invertase (often
secreted by the yeast itself) to convert the sucrose into its fermentable
monomers. Sucrose + H2
​O Invertase/Acid​
Glucose+Fructose
•Sugar Beet: Sugar beets are mashed and heated to extract the sucrose-rich juice. This
juice is then treated (often with lime and carbon dioxide) to remove non-sugar
impurities before proceeding to fermentation. Like molasses, the sucrose is usually
inverted.

23. •B. Fermentation
•Dilution and Nutrient Addition: The concentrated sugar solution
(molasses or beet juice) is diluted with water to an optimal sugar
concentration (15%−20%) to prevent osmotic stress on the yeast.
Essential nutrients like nitrogen and phosphorus are added.
•Inoculation: The mixture is inoculated with the robust fermenting
organism, typically Saccharomyces
 cerevisiae
(Brewer's Yeast).
•Bioreaction: The yeast consumes the readily available hexose
sugars (glucose and fructose) via glycolysis and the subsequent
ethanol fermentation pathway under anaerobic conditions.
•Glucose/Fructose→2Ethanol+2CO2
​+Heat
•Result: A fermented wash (or "beer") with an ethanol
concentration typically ranging from 8% to 15%.

24. •C. Purification and Recovery
•Distillation: The ethanol wash is subjected to
fractional distillation to separate the ethanol
(B.P. 78.37 C) from the water (B.P. 100 C). This
∘ ∘
achieves a maximum concentration of 95.6%
∼
ethanol (the azeotrope).
•Dehydration: If anhydrous ethanol ( 99.8%) is
∼
required for fuel use, the azeotrope is broken
using advanced methods like molecular sieves.

25. •Advantages of 1G Production
•High Efficiency & Yield: Sugars are immediately
fermentable, resulting in very high conversion efficiencies
( 90−95% of theoretical yield) and rapid fermentation
∼
times.
•Established Technology: The process is fully mature,
standardized, and highly reliable worldwide.
•Low Inhibitor Concentration: Since no harsh pretreatment
is needed, inhibitor formation (like HMF and furfural) is
minimal or non-existent.
•The main drawback of 1G production is the "Food vs. Fuel"
competition, which is why research is heavily focused on
the 2G process using true agricultural wastes.

27. Production of Bioethanol Using
Zymomonas
 mobilis and Clostridium

28. •1. Zymomonas
 mobilis (A Highly Efficient Alternative to Yeast)
•Zymomonas
 mobilis is a gram-negative bacterium that is being extensively
researched and used as an alternative to the traditional yeast
(Saccharomyces
 cerevisiae) for industrial ethanol fermentation.
Feature
Zymomonas
 mobilis
(Bacteria)
Saccharomyces
 cerevisiae
(Yeast)
Metabolic Pathway
Entner-Doudoroff (ED)
Pathway
Embden-Meyerhof-Parnas
(EMP) Pathway (Glycolysis)
Theoretical Ethanol Yield
∼98% (Closer to
theoretical maximum)
∼90−93%
Ethanol Productivity
Higher (Faster specific
glucose uptake rate)
Lower
Byproducts
Minimal (produces less
biomass and CO2
​)
More CO2
​ and cell mass
(biomass)
Osmotic Tolerance
Lower (sensitive to high
sugar concentration)
Higher (more robust)
Ethanol Tolerance
High (can tolerate up to
13%v/v ethanol)
High

29. •Application in Ethanol Production
•Z.
 mobilis is particularly efficient when using
pure glucose or sucrose as a feedstock (First-
Generation or 1G).
•Genetic Engineering: Strains are often
engineered to utilize a broader range of
sugars found in agricultural wastes (like xylose
and arabinose from lignocellulose) to compete
with 2G engineered yeast.

30. •Clostridium is a genus of anaerobic, spore-forming bacteria known for its
diverse metabolic capabilities, particularly its role in ABE fermentation
(Acetone-Butanol-Ethanol) and its ability to break down complex
carbohydrates.
Species Primary ProductSubstrates UsedSignificance
C.
 acetobutylicum
Butanol, Acetone,
Ethanol
Starch, Sugars,
Hemicellulose
Traditional ABE
fermentation (ABE
process is
historically
significant for bio-
butanol, with
ethanol as a co-
product).
C.
 thermocellum
Ethanol, Acetate,
Lactate
Cellulose (direct
breakdown)
Capable of
Consolidated
Bioprocessing
(CBP)—combining
enzyme production,
cellulose hydrolysis,
and fermentation in
one step.

31. Application in Ethanol Production
•Lignocellulosic Degradation: Certain Clostridium
strains are vital for 2G and 3G ethanol production due
to their ability to naturally secrete powerful cellulase
enzymes.
•Consolidated Bioprocessing (CBP): C.
 thermocellum
can directly convert cellulose-rich agricultural wastes
(e.g., switchgrass, corn stover) into ethanol, drastically
reducing the cost and complexity associated with
adding commercial enzymes.
•Tolerance to Inhibitors: Clostridium strains often show
higher tolerance to common fermentation inhibitors
found in pretreated waste biomass compared to yeast.

32. Summary of Microbial Roles in
Bioethanol
•The use of these microorganisms is driving innovation
in biofuel production:
•Z.
 mobilis
offers high yield and productivity for sugar-
rich feedstocks.
•Clostridium offers cost reduction and simplification by
enabling direct utilization of cellulose-rich agricultural
wastes through CBP.
•Both organisms are crucial for developing efficient,
economically viable, and sustainable ethanol
production processes that move beyond traditional
food crops.