1 Development of efficient methods for the - PowerPoint Presentation

1. 1Development of efficient methods for the production of bioethanol from macroalgaeProfessor (Em.) Aharon GedankenDepartment of Chemistry, Bar-Ilan University, Ramat-Gan Israel

2. 2Alternate Energy SourcesWhy alternate fuels? Depleting fossil fuels Non-uniform global distribution of fossil fuels Growing population Increasing demand for transportation fuel CO2 emissionsWhy bioethanol? Tested transportation fuel Carbon neutral Renewable and Sustainable Terrestrial and marine biomass as feedstock2A trade off between new energy sources and clean environment is the problem facing mankind

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 2010 - 27 billion liters of bioethanol produced from sugar cane juice or molasses US in 2010 - produced 50 billion liters of bioethanol from cornBioethanol Industry In Brazil - 243 bioethanol processing plants and nearly 33, 620 ethanol service stations In US - 113 ethanol biorefineries in operation with 1073 ethanol stationsPossible ways of reducing bioethanol production costs Identifying appropriate feedstock Improving/eliminating the feed stock pretreatment stage Shortening the fermentation time3

4. 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 vapourisationScheme 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 ethanol4

5. 5Glucose fermentation to ethanolSugars(D-glucose, D-sucrose)Ethanol + CO2Yeast (Saccharomyces cerevisiae)Yeast strain employed: Bakers yeast (Bravo brand)Theoretical yield of ethanol: 1 g glucose yields 0.51 g ethanol1 g sucrose yields 0.54 g ethanol How to make the fermentation faster?Use of bathsonicator accelerated the fermentation rate

6. 6Acceleration of Fermentation of Glucose at 30 ºCKinetics from 13C NMR and from wt. loss Glucose conc. = 20 % sonication: 38% conversion in 5 hStirring: only 14.5 % conversionin 5 hksonication = 15.35 x 10-6 sec-1 kstirring = 6.67 x 10-6 sec-16

7. 713C NMR spectra (from 0 to 11 h)‏Complete glucose fermentation under sonication at 30 °C 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 hGlucose conc. – 20 %7

8. 8Acceleration of Fermentation of Glucose at 20 ºC Glucose conc. = 20 % Sonication: 100% conversion in 19 hStirring: 36 h is required forthe complete conversion ksonication: 6.31 x 10-6 sec-1kstirirng: 2.465 x 10-6 sec-1Fermentation is 2.6 times faster using sonicationKinetics from wt. loss of the broth8

9. 9Acceleration of fermentation even at 40 % glucose concentrationGlucose conversion with sonication Vs stirring Sonication: 19 % conversion in 18 h Stirring: 11 % conversion in 18 h9

10. 10How does yeast cells appear? – Sonication Vs StirringAggregates of yeast cellsStirringDispersed yeast cellsSonication Sonication facilitated dispersion of yeast aggregatesThe yeast is reusable even after sonicationExtrapolating the sonication methodology to other feedstocks

11. Proximate compositionRelative % on dry weight basisCarbohydrateCellulose37±3.923.8 ±1.2Starch7.6±1.1Protein6.2±0.9Carbon28.1±1.2Nitrogen4.5±0.7Hydrogen5.5±1.3Sulphur2.3±0.4One step conversion of a macroalgae to ethanol using sonicationAlgea – Ulva rigidaProcess – Simultaneous saccharification and fermentation (SSF)Cellulose & Starch constitute the major fraction (31.4 wt.%) of carbohydrates that could yield glucoseGlucose is the most easily fermentable sugar for ethanol productionThe objective is to selectively produce glucose from the cellulose and starch components of algae and subsequently convert the same to ethanol under sonication 11

12. Enzymatic saccharification of Ulva rigidaBefore carrying the SSF process, the saccharification process is evaluated in isolation3.6 times higher yield of glucose is obtainable employing sonication during the hydrolysis stage relative to incubationThe enhancement in the release of glucose from the algae upon sonication is attributed to mechanical and thermal effectsUltrasound improved the hydrolysis process by the reduction of the structural rigidity of the cellulose and starch components in the biomassUltrasound-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 substrateSonication Vs incubation at 37 °C1.68 g of dried U. rigida 40 mL of distilled water40 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/mgAll the contents were taken in a 100 mL glass media bottles with cap12

13. Sonication120 minIncubation120 minSodium acetatebufferglucoseExclusive production of glucoseNo other sugars produced upon hydrolysisUse of cellulose and amylase selectively hydrolyzed cellulose and starch fractions respectively Selective production of glucose from Ulva rigida 13C NMR spectra of hydrolyzate of ulva rigida produced under sonication and incubation at 37 °C at 120 min.13

14. Sonication based SSF process for bioethanol productionSSF batch: Sonication Vs Incubation at 37 °C1.68 g of dried U. rigida in 40 mL of distilled water40 mL of 200 μM sodium acetate (buffer) (2.1 % w/v) 100 µL amyloglucosidase 300 units/mL40 µL α-amylase 250 units/mL,0.1 g cellulase (0.3 units/mg)0.5 g of Baker’s yeast100 mL glass media bottles with cap were usedAnalogous 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 fermentation14

15. Quantification of ethanol produced from Ulva regida1H NMR spectrum of the aliquot of sample collected from the fermentation (SHF) broth under mild sonication at 120 min.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).15

16. SSF processGlucose yield(mg)Ethanol yield(mg)Theoreticalethanol yield (mg)Process efficiency (%)Sonication(t=180 min)330 ± 17110 ± 1317065.5Incubation(t=48 h)290 ± 15100 ± 1115067.9Efficiency of SSF process : sonication Vs incubationReaction 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 mLThe 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. 16

17. (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 Salient features of the sonication based SSF process17What 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 algae with high carbohydrate content

18. Microwave assisted production of glucose from cellulose13C NMR spectrum of the hydrolysate from cellulose hydrolysisHydrolysis conditions:Cellulose (Avicel® PH-101, 1.0 g), Catalyst - 1 M HCl, 20 mLMicrowave irradiation for 7 min. under stirring in a 100 mL RB flaskPeaks 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 observedFast and Selective production of glucose from cellulose possible using microwave irradiationNo other products such as hydroxyl methyl furfural (HMF) or levulinic acid or formic acid were observed Glucose obtained as the only hydrolysis product

19. Effect of dilute acid concentration on glucose amountEffect of HCl concentration on the amount of glucose producedThe 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. 20Optimization of irradiation time and microwave powerEffect of irradiation time on the glucose amount producedEffect of microwave power on the yield of glucose High lights:Fast and selective hydrolysis of Cellulose (Avicel® PH-101)Glucose is the sole hydrolysis product7 min. of microwave irradiation with 2.38 M HCl as optimum reaction conditionsThe 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. Cellulose hydrolysis carried out at 600, 840 and 1200 W for 7 minEnergy consumption for 600, 800 and 1200 WPower is 0.183 KWh, 0.121 KWh and 0.09 KWhrepectively 1200, 840 and 600 W70 % (840 W) is the optimum power

21. Fermentation of the hydrolyzate to ethanolVariation of glucose amount in the fermentation broth as a function of timeGlucose fermentation with Saccharomyces cerevisiae Fermentation was carried out in a 100 mL Erlenmeyer flask. The fermentation medium: 20 mL of neutralized hydrolysateTo this medium, 0.2 g of yeast is added. Fermentation time - 12 h incubationFermentation temperature - 30 °CGlucose amount was decreased from 0.67 to 0.011 g in a duration of 12 h of fermentation indicating 98.3 wt.% glucose conversionPeaks characteristic of ethanol (3H, t at 1.1 ppm and 2H, q at 3.6 ppm) are observed1H NMR spectrum of ethanol obtained from the fermentation of cellulose hydrolysate at 12 h

22. 22Starch hydrolysis using solid acid catalystStarch (potato)(0.2 g)+20 wt.% HSiW/activated carbon(0.2 g)+20 mL H2OGlucoseHydrothermalprocessOptimization of reaction temperature: 100, 120, 150 °CReaction time – 4 h150 °C was found to be the optimum reaction temperaturefor the complete conversion of starchStarch is hydrolyzed selectively toGlucoseNo side products like HMF,levulinic acid and formic acidwere formed.13C NMR spectra of the hydrolyzate from starch as a function of temperature

23. 23Optimization of reaction time for starch hydrolysiso13C NMR spectra of the hydrolyzate from starch as a function of time With out catalyst there isNo conversion of starchAfter 2 h of reaction time,Traces of unreacted starchStill present4 h hydrothermal treat at 150 °C is required forThe complete conversion ofStarchExclusive formation ofglucose is observed

24. 24Optimization of catalyst amount for starch hydrolysis13C NMR spectra of the hydrolyzate from starch as a function of time Reaction conditions:Reaction time – 4 hReaction temp. – 150 CStarch – 0.2 gWater – 20 mLCatalyst – 0.2-1 gComplete 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. 25Upscaling studies of glucose production from starch13C NMR spectra of the hydrolyzate from starch as a function of amount of substrateReaction conditions:Reaction time – 4 hReaction temp. – 150 CStarch – 0.2 – 1.0 gWater – 20 mLCatalyst – 20 wt.% HSiW/activated carbon 0.2-1.0 g

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

27. summary27Industrial scale conversion of biomass to ethanol is much awaited. Sonication based SSF process need to be developed further to improve the process efficiencyMicrowave irradiation is a potential tool to accelerate the cellulose hydrolysisHSiW/C is a promising catalyst for starch hydrolysis that could be substitute to enzyme catalysts