Ethanosolv Pretreatment of Bamboo with Dilute Acid for Efficient Enzymatic Saccharification

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1 Ethanosolv Pretreatment of Bamboo with Dilute Acid for Efficient Enzymatic Saccharification Zhiqiang LI Ph.D. 55th International Convention of Society of Wood Science and Technology

2 Main content 1. Introduction 2. Pretreatments of bamboo 3. Results and discussions 4. Conclusions

3 1 Introduction Energy crisis Global warming Renewable biofuel

4 Bioethanol production technical route Source: Pan X. Wisconsin bioenergy summit. 2009

5 Pretreatment of biomass The solubilization and separation of one or more components of biomass Reduce the degree of crystallinity of the cellulose and increase the fraction of amorphous cellulose, the most suitable form for enzymatic attack. Bring about a change in the macroscopic and microscopic size and structure of biomass as well as submicroscopic structure and chemical composition.

6 Goals of an effective pretreatment process (i) To form sugars directly or subsequently by hydrolysis (ii) To avoid loss and/or degradation of sugars formed (iii) To limit formation of inhibitory products (iv) To reduce energy demands (v) To minimize costs

7 Ethanosolv pretreatment platform Source: Pan X. Wisconsin bioenergy summit. 2009

8 Bamboo bioethanol production Bamboo Bamboo, with its advantages of fast growth, short renovation, easy propagation and rich in cellulose and hemicellulose(>2/3 w/w), is a potential material source of bioethanol feedstock. Cellulose Glucose Hemicellulose Arabinose Galactose Xylose Lignin Other chemical products Mannose World bamboo production and consumption map Bioethanol Physical pretreatment Physicochemical pretreatment Chemical pretreatment Biological pretreatment

9 2 Pretreatment of bamboo Chemical Pretreatment

10 Pretreatment method Ethanosolv: 2% H 2 SO 4, 75% (v/v) ethanol DA: Dilute acid (2%, H 2 SO 4 ), water

11 Analytical methods According to National Energy Laboratory (NREL) Analytical Procedure: Determination of Structural Carbohydrates and Lignin in Biomass (with modifications) UV-Visible spectrophotometer : Acid-soluble lignin (205 nm) Improved high-performance anion exchange chromatography (Dionex HPLC system ICS-3000) : Carbohydrate and fermentation inhibitors.

12 3 Results and Discussions Table 1. Chemical analyses of untreated bamboo and pretreated bamboo substrates after 30 min at 180 Acid charge on OD bamboo (%) Pretreated solutions Component weight (%) Arabinose Galactose Glucose Xylose Mannos e Acidinsoluble lignin Acidsoluble lignin Untreated bamboo 1.1± ± ± ± ± ± ±0.0 0 Ethanosolv 1.0± ± ± ±0.9 ND 24.9± ±0.0 2 Ethanosolv 0.1±0.0 ND 84.5± ± ± ± ±0.1 2 Water 0.1±0.0 ND 52.0± ±0.2 ND 35.0± ±0.4 The ethanosolv pretreatment with sulfuric acid could minimize the loss of cellulose, which served the main resource in bioethanol production. DA substrate The addition of acid to the liquid mixture played a very important role in catalyzing the removal of hemicellulose and lignin.

13 Table 2. Chemical analyses of pretreated spent liquors Acid charge on OD bamboo (%) Pretreated solutions Component in spent liquors (g/l) Arabinose Galactose Glucose Xylose Mannos e Acidsoluble lignin 0 Ethanosolv 0.0± ± ± ± ± ±0.9 2 Ethanosolv 1.6± ± ± ± ± ±0.3 2 Water 0.04± ± ± ±0.6 ND 5.7±0.3 More glucose and xylose were detected in ethanosolv pretreatment spent liquors than that in DA pretreated spent liquors.

14 Table 3. Mass balance of 100 g oven-dry bamboo powder during pretreatments Acid charge on OD bamboo (%) Pretreated solutions Component recovery, g Arabinose Galactos e Glucose Xylose Mannose Lignin Sum Recovery 0 Ethanosolv 2 Ethanosolv 2 Water Sub. 0.9± ± ± ±0.9 ND 23.1± Liquor 0.0± ± ± ± ± ± Sub. 0.0± ± ± ± ± ± Liquor 1.0± ± ± ± ± ± Sub. 0.1± ± ± ± ± ± Liquor 0.0± ± ± ± ± ± Table. Sugar recovery % Glucose Xylose Total Ethanosolv DA The result indicated xylose was subject to greater degradation than glucose in acid pretreatment, especially in water pretreated solution.

15 Glucose yield of bamboo and wood CGCY (%) Ethanosolv with acid Microcrystalline cellulose Water with acid Ethanosolv without acid Untreated bamboo Enzymatic hydrolysis time (h) Figure 1. Comparison of time-dependent enzymatic hydrolysability of different pretreated bamboo substrates with a enzyme loading of 15 FPU cellulase and 30 IU β-glucosidase per gram of cellulose, 50 C, ph 4.8 and on a 220 rpm shaker. CGCY: cellulose-to-glucose yield. Source: Pan et al.: Organosolv Fractionation for Bioconversion Biotechnology and Bioengineering. DOI /bit The cellulose-to-glucose conversion yield of ethanosolv pretreatment (77.1%) was higher than that of water pretreatment (57.3%). Which were even higher than microcrystalline cellulose to glucose conversion yield (76.2%)? But lower than hybrid poplar wood.

16 Inhibitors formed in the pretreatment Inhibitors for fermentation, but useful chemicals for others!

17 Table 4. Fermentation inhibitors in pretreated spent liquors after 30 min at 180 Acid charge on OD bamboo (%) Pretreated solutions Inhibitors (g/l) Acid-soluble lignin Formic acid Acetic acid Furfural HMF Levulinic acid 0 Ethanosolv 5.5± ± ± ± ± ± Ethanosolv 5.0± ± ± ± ± ± Water 5.7± ± ± ± ± ± Total DA Pretreatment HMF Furfural Ethanosolv Pretreatment HMF Furfural DA and ethanosolv pretreatment can be used for HMF and furfural production!

18 Sugar yields of DA pretreated biomass Lignocellulosic biomass Temperature ( ) Pretreatment conditions Acid concentration (% w/w) Residence time (min) Sugar yield (g/100g a ) Rye straw Bermuda grass Olive tree Saline biomass Bamboo Bamboo Note: a-basis on 100g raw material

19 Substrate s available surface area (pore volume) Table. Density and hardness of biomass Material Desity (g/cm 3 ) Hardness (Mpa) Moso bamboo Paulownia Populus ussuriensis Picea jezoensis Birch Other factors: Degree of polymerization Crystallinity The microscopic structure Bamboo species Table. Density of moso bamboo in different ages Growth ages (a) Thickness of culm wall (mm) Basic density (g/cm 3 ) oven-dried volume shrinkage(%) air-dried volume shrinkage(%)

20 Cell wall thickness (coarseness) The waxy barrier comprising grass cuticle and tree bark impedes penetration of enzymes; Even milled, plant stems and woody tissues limit liquid penetration by their nature. Debarked Fat and wax in bamboo is %

21 4 Conclusions Bamboo is more difficult than other biomass on efficient pretreatment; The glucose yield of ethanosolv is 77.1% and 57.3% for DA pretreatment, still lower than % of wood; Untreated bamboo is just about 2.4%; DA and ethanosolv pretreatment can be used for HMF and furfural production with some modification.

22 Acknowledgement Director Zehui Jiang, Dr. Benhua Fei, ICBR Very appreciate FPL and UW for providing me opportunity to study and work there in the U.S.A. Dr. Zhiyong Cai, FPL and other staff Dr. Xunjun Pan, UW Funding sources: International Centre for Bamboo and Rattan (ICBR), Grant. No

23 Thank you for your attention!