Bioethanol production from lignocellulosic biomass

1. Bioethanol production from lignocellulosic biomass Sachin Kumar: SNIRE, Kapurthala S. P. Singh: DPT, IIT Roorkee, Saharanpur Campus I. M. Mishra: DChE, IIT Roorkee A. D. Adhikari: IIP, Dehradun

2. Energy from biomass is sustainable • Fuels from biomass • Clean, carbon-neutral, and sustainable energy • Ethanol has potential of an alternative automotive fuel, preferably in a blend with gasoline • Use of ethanol in transport sector important for countries like India that depend heavily on import of crude oil • Use of ethanol as fuel reduces CO2 emission • Fossil fuels • Reserves depleting fast • Rising price and uncertainty in availability • Environmentally unfriendly - blamed for the global climate change to a large extent

3. The present study • Screening of a thermophilic strain that can ferment glucose and xylose to ethanol. Strain has been characterized as yeast Kluyveromyces sp. IIPE453. • Sugars for fermentation obtained from acid hydrolysis of sugarcane bagasse. • Two-stage acid hydrolysis – dilute acid treatment followed by strong acid treatment – enabled about 92% recovery of the sugars present in biomass • Optimum growth and fermentation were observed at 50 C and pH of 5

4. Feed-stocks for bioethanol • Lignocellulosic biomass: available more abundantly - forest residues, agricultural residues, industrial residues, energy crops such as switch grass • Traditional: such as corn, food grains, sugarcane juice, and cane molasses face social and economic barriers as these materials are used substantially for human and animal consumption.

5. Composition of lignocellulosic biomass • Cellulose (20-50 %): Linear polymer of glucose units linked by β-(1–4)-glycosidic bonds. • Hemicellulose (20-40 %): Highly branched and complex heteropolymer that contains hexoses, pentoses, and uronic acids. Hemicellulose is more easily hydrolyzed to its constituent monosaccharides than cellulose. • Lignin (15-25 %): Aromatic polymer containing phenolic residues. • Other components: Small quantity

6. Composition of sugars in various lignocellulosic biomass

7. Conversion of biomass to biofuels • There are several biochemical and chemical routes to produce biofuels from biomass • The main processes are fermentation of sugars to alcohol, gasification and chemical synthesis, and direct liquefaction. • Many different fuels such methanol, ethanol, hydrogen, synthetic diesel, biodiesel, and bio-oil can be produced from biomass.

8. Hydrolysis Fermentable sugar Fermentation Ethanol, Butanol, Hydrogen Biochemical process Residue Bio-oil Anaerobic digestion SNG CH4 Biogas Purification FT Diesel Biomass DME Catalytic synthesis Gasification Syn gas Methanol Hydro treating and refining Pyrolysis Bio-oil Hydrocarbon Chemical process Hydrothermal liquefaction Power generation Residue Extraction of oil Oil plant Trans-esterification Biodiesel Oil Conversion of biomass to biofuels

9. Biochemical processes for converting biomass to ethanol • Hydrolysis of hemicellulose/ cellulose to monomer sugars • Separation of sugars • Fermentation of sugars • Product recovery and concentration by distillation

10. Major differences in biochemical processes • Hydrolysis of biomass By acid or by enzymes • Selection of microbes For fermentation of sugars to ethanol (ethanologens) • For economical production of ethanol from lignocellulosic biomass, the microorganisms must be capable of fermenting both glucose and xylose. • Both anaerobic and aerobic ethanologens available • Facultative aerobes preferred for industrial applications due to difficulties in maintaining strict anaerobic conditions in large scale fermentations using thermophilic anaerobes

11. By-products in bioethanol production • In spite of several breakthroughs, the cost of bioethanol produced from lignocellulosic feed-stocks remains high. • Processes having potential of high ethanol recovery with some value-added by-products may improve the economy. • Some examples of value-added by-products from bioethanol production follow:

12. By-products during ethanol production from biomass • Xylitol: conversion of xylose to xylitol during hydrolysis of biomass. Useful in prevention of tooth decay and ear infection in children, sugar substitute for diabetic patients • Furfural: Is a valuable chemical formed by conversion of xylose to furfural • Single cell protein: Useful for animal feed and can be produced by utilization of xylose solution for growing Candida utilis. • Lignin: it is the residue after extraction of sugars. Useful as additive, adhesives, adsorptive materials

13. EXPERIMENTAL WORK • Isolation of thermophilic and thermotolerant strains • Screening of isolates for ethanol production • Growth of screened microbe • Hydrolysis of sugarcane bagasse • Fermentation conditions • Analytical methods

14. Isolation of Thermophilic and Thermotolerant Strains • Thermophiles were isolated from soil samples in nutrient broth (NB) and yeast extract peptone and dextrose (YPD) media • Soil samples were collected from dumping sites of crushed bagasse in a sugar mill. • Pure colonies were isolated by using 2% agar and 1% gelrite as solidifying agent at 45 oC and 60 oC, respectively.

15. Composition of NB & YPD media NB: Nutrient Broth YPD: Yeast extract Peptone and Dextrose

16. Screening of Isolates for Ethanol Production • Isolated ethanologens screened using different sugars: glucose, mannose, galactose, xylose, arabinose, sucrose, cellobiose and lactose. • New isolate inoculated into phenol red broth medium and incubated overnight at 45-60 oC • Change in pH due to acid production by ethanologens indicated by color change from red to yellow • One potential yeast Kluyveromycessp.IIPE453 (KS) was selected based on faster growth and color change in the phenol red medium

17. Growth of screened microbe • The screened strain KS was grown in salt medium (SM) • The cells were grown in 250 ml flasks in shaker at 50 oC and 150 rpm on glucose, mannose, galactose, xylose, arabinose, sucrose, cellobiose and lactose, 10 g/l each separately. • The cells were also produced in large quantity by growing in a Bioflow-110 bioreactor (ca. 5 liters) on glucose and xylose. The temperature, pH and dissolved oxygen were controlled at 50 oC, 5.0 and 40 % saturation, respectively, during the growth phase.

18. Composition of salt medium The pH was adjusted 5.0 by 1N hydrochloric acid

19. Hydrolysis of bagasse • First stage hydrolysis (acid conc 2-10 %) 2 kg crushed bagasse with acid solution charged to a 30-L digester fitted with an agitator (200 rpm), solid to liquid ratio 1:8 to 1:4, maintained at100 oC for 90 min. Samples withdrawn at 15 min interval. After 90 min the digested biomass washed to recover the sugars. • Second stage hydrolysis (acid conc 18-40 %) Residual bagasse of first stage treated with stronger acid in the same digester for 90 min at 80 oC and 200 rpm. Samples withdrawn at 15 min interval. The digested biomass wasahed to recover the sugars

20. Fermentation Conditions • Batch fermentation of the hydrolysate in a Bioflow-110 bioreactor (ca. 2 liters) by free cells of Kluyveromyces sp.IIPE453. • Temperature: 50 oC, pH: 5.0 •  Fermentation medium (g/l): di-sodium hydrogen ortho phosphate, 0.15; potassium di-hydrogen ortho phosphate, 0.15; ammonium sulphate, 1.0; yeast extract, 1.0.

21. Analytical Methods • HPLC used for analysis of sugars (glucose, fructose, sucrose and xylose) and xylitol: High Performance Carbohydrate Column (Waters) at 30 oC; Acetonitrile and water mixture (75:25) at a flow rate of 1.4 ml/min as mobile carrier; Refractive index detector (Waters 2414). • Gas chromatography used for analysis of ethanol: Ashco Neon II Gas Analyzer; 2 m long and 1/8" diaPorapak-QS column with 80/100 mesh packing; Sample injection at 220oC; Oven temperature 150 oC; Flame ionization detector at 250 oC; Nitrogen gas as carrier.

22. RESULTS AND DISCUSSION • Isolation and characterization of microbe • Products of 1st stage hydrolysis • Products of 2nd stage hydrolysis • Sugar recovery from the hydrolyzate • Fermentation of 1st stage hydrolysate to ethanol • Fermentation of 2nd stage hydrolysate to ethanol

23. Isolation and characterization of microbe • From several thermophilic strains isolated from soil samples collected from dumping site of sugarcane bagasse where the temperature was usually high, only one microorganism showed growth and fermentation on glucose, mannose, galactose, xylose, sucrose, cellobiose and lactose. • This strain was selected for further fermentation studies and characterized as yeast Kluyveromyces sp. IIPE453 (deposited in ‘Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh (India)’ with deposition no. MTCC 5314). • The optimum temperature and pH for growth and fermentation were found to be 50 C and 5.0, respectively.

24. 1st stage hydrolysis: Xylose concentrationEffect of acid concentration Temp: 100 °C

25. 1st stage hydrolysis: Glucose concentration Effect of acid concentration Temp: 100 °C

26. 1st stage hydrolysis: Furfural concentration Effect of acid concentration Temp: 100 °C

27. 1st stage hydrolysis: Xylose concentrationEffect of solid/liquor ratio Temp: 100 °C Acid conc: 8%

28. 1st stage hydrolysis: Glucose concentration Effect of solid/liquor ratio Temp: 100 °C Acid conc: 8%

29. 1st stage hydrolysis: Furfural concentration Effect of solid/liquor ratio Temp: 100 °C Acid conc: 8%

30. 2nd stage hydrolysis: Xylose concentrationEffect of acid concentration Temp: 80 °C

31. 2nd stage hydrolysis: Glucose concentration Effect of acid concentration Temp: 80 °C

32. 2nd stage hydrolysis: Furfural concentration Effect of acid concentration Temp: 80 °C

33. Hydrolysis of Sugarcane Bagasse • Week acid predominantly hydrolyzes hemicelluloses to xylose and strong acid cellulose to glucose. • Hydrolysis to furfural is low for both week as well as strong acids. • Maximum recovery of xylose was obtained at acid concentration of 8 % and solid to liquid ratio of 1:4. • Maximum recovery of glucose was obtained at acid concentration of 40 %. • About 92 % of total sugars present in bagasse could be recovered in the two-stage acid hydrolysis.

34. Sugar recovery from the hydrolyzate • Sugars and the acid from hydrolysis mixture could be separated by ion-exchange technique. • About 95 % acid free sugars were recovered with strong anion and weak anion mixture in the ratio of 5:2 and residence time 44 minutes, and • About 95 % acid was recovered in the regeneration step.

35. Fermentation of 1st stage hydrolysate by yeast KS 7 g/l glucose and 18 g/l xylose Temp: 50 °C Concentration Sugar present in hydrolysate was consumed within 8 h

36. Batch fermentation of dilute acid bagasse hydrolysate Ethanol yield was low due to the low percentage of glucose in hydrolysate. No inhibition was observed during fermentation by the inhibitors such as furfural.

37. Fermentation of 2nd stage glucose rich hydrolysate by yeast KS Temp: 50 °C Concentration

38. Batch fermentation of 2nd stage hydrolysate The results are better than reported in literature: Ballesteros et al. (44) and Tomás-Pejó et al. (25) with different strain and biomass

39. Batch fermentation of 2nd stage hydrolysate • The dry cell weight was almost constant throughout the process. • The final ethanol concentration in broth was low due to low initial sugar concentration in the hydrolysate, which could be overcome either by improvement of saccharification of sugarcane bagasse or by mixing other high sugar containing feed-stocks.

40. CONCLUSIONS • A thermophilic strain was isolated from the soil samples collected from the dumping site of sugarcane bagasse. • The strain was characterized as yeast Kluyveromyces sp. IIPE453 (deposited in ‘Microbial Type Culture Collection, Institute of Microbial Technology, Chandigarh (India)’ with deposition no. MTCC 5314).

41. CONCLUSIONS • The yeast strain showed growth and fermentation on glucose, mannose, galactose, xylose, sucrose, cellobiose and lactose. • The optimum temperature and pH for growth and fermentation were observed to be 50 C and 5.0 respectively.

42. CONCLUSIONS • About 92 % of the sugars present in the bagasse biomass could be recovered by acid hydrolysis in two steps, first with dilute acid to hydrolyze hemicelluloses and then with concentrated acid to hydrolyze cellulose. • Bagasse hydrolysate could be fermented to ethanol using the yeast Kluyveromyces sp. IIPE453 either in batch process or in continuous process.

43. CONCLUSIONS • No inhibition during fermentation by the presence of inhibitors like furfural in the hydrolysate was observed. • The ethanol yield was, however, low due to low fermentation of xylose to ethanol and low percentage of glucose present in the bagasse hydrolysate. • The yield could be increases by increasing the glucose concentration in the hydrolysate through addition of molasses, sugarcane juice or some similar material.

44. Thank youfor your kind attention