1. Development of efficient methods for the
production of bioethanol from macroalgae
Dr Indra Neel Pulidindi
Professor (Em.) Aharon Gedanken
Department of Chemistry, Bar-Ilan University, Ramat-Gan
Israel

2. Alternate Energy Sources
A trade off between new energy sources and clean environment is the problem facing mankind
Why alternate fuels?
Depleting fossil fuels
Non-uniform global distribution of fossil fuels
Growing population
Increasing demand for transportation fuel
CO2 emissions
Why bioethanol?
Tested transportation fuel
Carbon neutral
Renewable and Sustainable
Terrestrial and marine biomass as feedstock 2

3. Drive for research in Bioethanol – Cost reduction
World wide focus - bioethanol as a substitute to gasoline
Feedstocks for ethanol - Brazil (sugar cane molasses), United States (corn),
India (sugar cane), Thailand (cassava),
France (sugar beet), China (corn), Canada (wheat)
Brazil in billion liters of bioethanol produced from sugar cane juice or molasses
US in produced 50 billion liters of bioethanol from corn
Bioethanol Industry
In Brazil bioethanol processing plants and nearly 33, 620 ethanol service stations
In US ethanol biorefineries in operation with 1073 ethanol stations
Possible ways of reducing bioethanol production costs
Identifying appropriate feedstock
Improving/eliminating the feed stock pretreatment stage
Shortening the fermentation time

4. reactions in the conversion of biomass to fuel ethanol
Advantages of Bioethanol
Renewable and environmentally friendly and carbon neutral fuel
Blended with petrol or used as neat alcohol
Higher octane no. and higher heat of vapourisation
Scheme 1. Schematic representation of the formation of ethanol from biomass
Hydrolysis of biomass to sugars and fermentation of sugars to ethanol are the two important
reactions in the conversion of biomass to fuel ethanol

5. (D-glucose, D-sucrose)
How to make the fermentation faster?
Use of bathsonicator accelerated the fermentation rate
Glucose fermentation to ethanol
Sugars
(D-glucose, D-sucrose)
Ethanol + CO2
Yeast (Saccharomyces cerevisiae)
Theoretical yield of ethanol:
1 g glucose yields 0.51 g ethanol
1 g sucrose yields 0.54 g ethanol
Yeast strain employed: Bakers yeast (Bravo brand)

6. Acceleration of Fermentation of Glucose at 30 ºC
Glucose conc. = 20 %
sonication: 38% conversion in 5 h
Stirring: only 14.5 % conversion
in 5 h
ksonication = x 10-6 sec-1
kstirring = 6.67 x 10-6 sec-1
Kinetics from 13C NMR and from wt. loss 6

7. Complete glucose fermentation under sonication at 30 °C
Glucose conc. – 20 %
13C NMR spectra (from 0 to 11 h)‏
With time intensity of the peaks corresponding to ethanol increased and
the intensity of peaks corresponding to glucose decreased
Complete conversion of glucose to ethanol in 11 h 7

8. Acceleration of Fermentation of Glucose at 20 ºC
Fermentation is 2.6 times faster using sonication
Glucose conc. = 20 %
Sonication: 100%
conversion in 19 h
Stirring: 36 h is required for
the complete conversion
ksonication: 6.31 x 10-6 sec-1
kstirirng: x 10-6 sec-1
Kinetics from wt. loss of the broth 8

9. Glucose conversion with sonication Vs stirring
Acceleration of fermentation even at 40 % glucose concentration
Glucose conversion with sonication Vs stirring
Sonication: 19 % conversion in 18 h
Stirring: 11 % conversion in 18 h 9

10. How does yeast cells appear? – Sonication Vs Stirring
Aggregates of yeast cells
Stirring
Dispersed yeast cells
Sonication
Sonication facilitated dispersion of yeast aggregates
The yeast is reusable even after sonication
Extrapolating the sonication methodology to other feedstocks

11. One step conversion of a macroalgae to ethanol using sonication
Algea – Ulva rigida
Process – Simultaneous saccharification and fermentation (SSF)
Proximate composition
Relative % on dry weight basis
Carbohydrate
Cellulose
37±3.9
23.8 ±1.2
Starch
7.6±1.1
Protein
6.2±0.9
Carbon
28.1±1.2
Nitrogen
4.5±0.7
Hydrogen
5.5±1.3
Sulphur
2.3±0.4
Cellulose & Starch constitute the
major fraction (31.4 wt.%) of carbohydrates that could yield glucose
Glucose is the most easily fermentable sugar for ethanol production
The objective is to selectively produce
glucose from the cellulose and starch components of algae and subsequently
convert the same to ethanol under sonication

12. Enzymatic saccharification of Ulva rigida
Before carrying the SSF process, the saccharification process is evaluated in isolation
Sonication Vs incubation at 37 °C
1.68 g of dried U. rigida 40 mL of distilled water
40 mL of 200 μM sodium acetate (buffer)
(2.1 % w/v)
100 µL amyloglucosidase 300units/mL,
40 µL α-amylase 250units/mL,
0.1 g cellulase, 0.3 units/mg
All the contents were taken in a 100 mL glass
media bottles with cap
3.6 times higher yield of glucose is obtainable employing sonication during the hydrolysis stage relative to incubation
The enhancement in the release of glucose from the algae upon sonication is attributed to mechanical and thermal effects
Ultrasound improved the hydrolysis process by the reduction of the structural rigidity of the cellulose and starch components in the biomass
Ultrasound-assisted process reduced the hydrolysis reaction time by improving mixing and phase transfer, and by enhancing the diffusion of enzymes across cell membranes (algae), so that enzymes can easily reach the bulk of the substrate

13. Selective production of glucose from Ulva rigida
Sonication
120 min
Incubation
Sodium acetate
buffer
glucose
13C NMR spectra of hydrolyzate of ulva rigida produced under sonication and incubation
at 37 °C at 120 min.
Exclusive production of glucose
No other sugars produced upon hydrolysis
Use of cellulose and amylase selectively hydrolyzed cellulose and starch fractions respectively

14. Sonication based SSF process for bioethanol production
SSF batch:
Sonication Vs Incubation at 37 °C
1.68 g of dried U. rigida in 40 mL of distilled water
40 mL of 200 μM sodium acetate (buffer)
(2.1 % w/v)
100 µL amyloglucosidase 300 units/mL
40 µL α-amylase 250 units/mL,
0.1 g cellulase (0.3 units/mg)
0.5 g of Baker’s yeast
100 mL glass media bottles with cap were used
Analogous to the saccharification process of algae with enzymes, the SSF process was also found to be faster under sonication relative to incubation .
In 30 min. the yield of ethanol under sonication is significantly high (4.3±0.26 wt.% Vs 1.0±0.13 wt.% under incubation)
Even after incubation for 48 h, the ethanol yield under incubation is only 6.1±0.13 wt.% which could be achieved in a short duration of 120 min. with the use of mild sonication.
Acceleration in the SSF process by the action of sonication could be due to the possibility of generation of fresh surface on the yeast cells by the faster removal of ethanol and CO2 formed as the metabolites during fermentation

15. Quantification of ethanol produced from Ulva regida
The appearance of 3H (t, 1.18 ppm) and
2H (q, 3.64 ppm) are typical of the
presence of ethanol in the analyte.
The peak , 3H, s, 1.9 ppm,
is characateristic of sodium acetate
employed as buffer.
The peak, 1H, s, 8.5 ppm, is typical
of the internal standared, HCOONa.
Based on the relative integral values of
the internal standard and the ethanol
peaks, the amount of ethanol was
found to be 6.0±0.16 wt
(Vs 4.0±0.07 wt.% using incubation).
1H NMR spectrum of the aliquot of sample collected from the fermentation (SHF)
broth under mild sonication at 120 min.

16. Process efficiency (%)
Efficiency of SSF process : sonication Vs incubation
SSF process
Glucose yield
(mg)
Ethanol yield
Theoretical
ethanol yield (mg)
Process efficiency (%)
Sonication
(t=180 min)
330 ± 17
110 ± 13
170
65.5
Incubation
(t=48 h)
290 ± 15
100 ± 11
150
67.9
Reaction conditions:
biomass (DW) = 1.68 g;
distilled water = 40 mL;
cellulase = 0.1 g (0.3 units/mg):
α-amylase = 40 µL (250 units/mL);
amyloglucosidase = 100 µL, (300 units/mL);
Sodium acetate buffer = 40 mL
The lower process efficiency could be attributed to the formation of glycerol as the secondary metabolite
during the fermentation.
Use of improved quality of yeast strain may lead to the selective production of ethanol from the fermentable sugars.

17. Salient features of the sonication based SSF process
(i) no requirement of pretreatment of the biomass
(ii) only one stage of operation
(iii) exclusive production of glucose as the sole sugar as a result of enzymatic saccharification
(iv) faster production of glucose from algae and simultaneous conversion of glucose to ethanol
What are the other avenues to improve the process efficiency and reduce the process cost?
Lowering of enzyme dosage or developing a robust chemical hydrolysis process
Improving over all starch and cellulose hydrolysis
Use of improved yeast strains as substitute to Baker’s yeast
Screening & culturing the marine algea with high carbohydrate content

18. Microwave assisted production of glucose from cellulose
Hydrolysis conditions:
Cellulose (Avicel® PH-101, 1.0 g),
Catalyst - 1 M HCl, 20 mL
Microwave irradiation for 7 min.
under stirring in a 100 mL RB flask
Glucose obtained as the only hydrolysis product
13C NMR spectrum of the hydrolysate from cellulose hydrolysis
Peaks typical of D-glucose (60.2 (C6), 69.0 (C4), 70.7, 72.2 (C2), 73.6 (C3), 75.1 (C5) and 95.1 (C1, β), 91.4 (C1α) ppm)
only were observed
Fast and Selective production of glucose from cellulose possible using microwave irradiation
No other products such as hydroxyl methyl furfural (HMF) or levulinic acid or formic acid were observed

19. Effect of HCl concentration on the amount of glucose produced
Effect of dilute acid concentration on glucose amount
Effect of HCl concentration on the amount of glucose produced
The highest glucose yield (0.67 g/g of cellulose) is obtained for the 7.5 wt. % HCl.
The cellulose conversion was found to be 67 %.
The unreacted residue (33 wt.%) was found to be cellulose.

20. Optimization of irradiation time and microwave power
Effect of irradiation time on the glucose amount produced
Effect of microwave power on the yield of glucose
Cellulose hydrolysis carried out at
600, 840 and 1200 W for 7 min
Energy consumption for 600, 800 and 1200 W
Power is KWh, KWh and 0.09 KWh
repectively 1200, 840 and 600 W
70 % (840 W) is the optimum power
The yield of glucose increased as a function of time for irradiation from 2 to 7 min.
After 7 min. no further increase was noted
The optimal irradiation time is 7 min.
High lights:
Fast and selective hydrolysis of Cellulose (Avicel® PH-101)
Glucose is the sole hydrolysis product
7 min. of microwave irradiation with 2.38 M HCl as optimum reaction conditions

21. Fermentation of the hydrolyzate to ethanol
Glucose amount was decreased from
0.67 to g in a duration of 12 h of
fermentation indicating 98.3 wt.%
glucose conversion
Variation of glucose amount in the fermentation broth as a function of time
Glucose fermentation with Saccharomyces cerevisiae
Fermentation was carried out in a 100 mL Erlenmeyer flask.
The fermentation medium: 20 mL of neutralized hydrolysate
To this medium, 0.2 g of yeast is added.
Fermentation time - 12 h incubation
Fermentation temperature - 30 °C
Peaks characteristic of ethanol (3H, t at 1.1 ppm and 2H, q at 3.6 ppm) are observed
1H NMR spectrum of ethanol obtained from
the fermentation of cellulose hydrolysate at 12 h

22. 20 wt.% HSiW/activated carbon
Starch hydrolysis using solid acid catalyst
Starch (potato)
(0.2 g) +
20 wt.% HSiW/activated carbon
20 mL H2O
Glucose
Hydrothermal
process
Optimization of reaction temperature: 100, 120, 150 °C
Reaction time – 4 h
150 °C was found to be the
optimum reaction temperature
for the complete conversion
of starch
Starch is hydrolyzed selectively to
Glucose
No side products like HMF,
levulinic acid and formic acid
were formed.
13C NMR spectra of the hydrolyzate from starch as a function of temperature

23. Optimization of reaction time for starch hydrolysis
With out catalyst there is
No conversion of starch
After 2 h of reaction time,
Traces of unreacted starch
Still present
4 h hydrothermal treat at
150 °C is required for
The complete conversion of
Starch
Exclusive formation of
glucose is observed
13C NMR spectra of the hydrolyzate from starch as a function of time

24. Optimization of catalyst amount for starch hydrolysis
Reaction conditions:
Reaction time – 4 h
Reaction temp. – 150 C
Starch – 0.2 g
Water – 20 mL
Catalyst – g
13C NMR spectra of the hydrolyzate from starch as a function of time
Complete and selective conversion of starch to glucose is achieved with a low amount of
catalyst
A ratio of substrate to catalyst of 1:1 is optimum

25. Upscaling studies of glucose production from starch
Reaction conditions:
Reaction time – 4 h
Reaction temp. – 150 C
Starch – 0.2 – 1.0 g
Water – 20 mL
Catalyst –
20 wt.% HSiW/activated carbon g
13C NMR spectra of the hydrolyzate from starch as a function of amount of substrate

26. Reusability of the HSiW/activated carbon catalyst
An active, selective and reusable solid acid catalyst is designed for glucose
production from starch
HSiW/activated carbon could be a possible substitute to amylase for starch hydrolysis

27. summary
Industrial scale conversion of biomass to ethanol is much awaited.
Sonication based SSF process need to be developed further to improve the process efficiency
Microwave irradiation is a potential tool to accelerate the cellulose hydrolysis
HSiW/C is a promising catalyst for starch hydrolysis that could be substitute to enzyme catalysts