1. High solid loading enzymatic hydrolysis of various paper wastes Methods and Kinetic model Lei Wang *, Richard Templer ‡ & Richard J. Murphy * * Division of Biology, Imperial College London, London, SW7 2AZ, UK ‡ Department of Chemistry, Imperial College London, London, SW7 2AZ, UK Introduction & Motivation As a potential feedstock for producing bioethanol, paper wastes have the following advantages: Abundance in the UK. The collected paper from waste stream reached 8.1 million tons in 2009 [1]. Prices in the range of £30/ton to £45/ton [2]. GHG emission savings. 82.9 billion litres of waste paper-derived cellulosic ethanol can be produced globally, replacing 5.36% of gasoline consumption with GHG emissions savings up to 86.1% [3]. This study of the technological feasibility of producing fermentable sugars from various paper wastes investigated: The detailed compositional analysis High solids loading of enzymatic hydrolysis Understanding kinetics of enzymatic hydrolysis Comparison between two enzyme systems Composition profile a Effect of blending on glucose yields Glucan and xylan hydrolysis model Relationship between glucose yield and lignin content Comparison of two different enzyme systems References [1,2] WRAP, (2010), Market situation report. [3] Shi et al., (2009), GCB Bioenerg. 1, 317-320. [4] Shen and Agblevor, (2008), Chem. Eng. Commun.195 (1), 107-112. [5] Zhang et al. (2009), Biotechnol. Bioeng. 104 (5), 920-931. Acknowledgements The authors wish to thank the Porter Institute (Imperial College) for financial support and Novozymes A/S, Demark for donation of Cellic Ctec 1 and Lars Saaby Pedersen from Nozoymes A/S for useful discussions on the results. Paper wastes were collected locally. Celluclast 1.5 L and Novozymes 188 were purchased (overall activity of the mixture in an activity ratio of 1:1 was 95FPU/ml); Cellic Ctec 1 (120 FPU/ml) was donated by Novozymes A/S Denmark. Composition of paper materials and enzyme activities were determined following NREL protocols. Blending was performed at 15% (w/w) solids loading in a bench-top blender. Its effect was evaluated by enzymatic hydrolysis at 5% (w/w). The high solids loading 15% (w/w) enzymatic hydrolysis was carried out in a overhead stirred reactor. A three parameters model involving Langmuir adsorption and second order enzyme deactivation was adopted [4]. The data fit was modified, using binary regression (two dimensions). A simple correlation between glucan and xylan hydrolysis rate was adopted and modified to develop xylan hydrolysis model [5]: NewspaperUsed office paperMagazineCardboard Glucan47.20 (0.05) b 58.64 (0.05)35.91 (0.08)52.61 (0.06) Xylan7.11 (0.02)14.65 (0.08)4.72 (0.06)8.23 (0.06) Galactan1.90 (0.02)0.001.98 (0.02)1.89 (0.01) Mannan7.31 (0.01)0.005.67 (0.04)5.07 (0.01) Arabinan1.86 (0.07)0.001.82 (0.03)1.55 (0.03) ASL c 1.06 (0.01)1.41 (0.01)0.98 (0.03)1.59 (0.02) AIL d 17.08 (0.43)4.68 (0.29)13.85 (0.29)14.18 (0.19) Extractives3.93 (0.05)1.97 (0.05)3.45 (0.06)2.55 (0.02) CaO2.13 (0.04)8.12 (0.02)2.63 (0.04)4.20 (0.01) Ash10.51 (0.11)7.97 (0.08)30.14 (0.16)9.89 (0.07) Total100.0997.44101.15101.76 a All results are presented as percentages of oven dry weight. b Three replications were performed. Standard errors are in brackets. c ASL presented acid soluble lignin which was measured using UV-vis at 330 nm determined in-house. d AIL is acid insoluble lignin. The glucose yield was significantly increased at 10 and 15 FPU/ g glucan enzyme loading potentially because: The enzyme absorption is sensitive to particle size in this range of enzyme loading. For higher enzyme loading (>20 FPU), the particle size appears to be non-limiting for hydrolysis efficiency because of the excess of free enzyme. For lower enzyme loading (5 FPU), the increase of accessible surface area is not an advantage due to the limited amount of enzyme applied. Newspaper Used office paper Magazine Cardboard Experimental results; Glucan hydrolysis model prediction; Xylan hydrolysis prediction Celluclast 1.5L+Novozyme 188Cellic Ctec 1 Two enzyme systems were at the same weight concentration loading. Glucose yield was decreased linearly with increasing lignin content for both enzyme systems. Cellic Ctec 1 resulted in higher glucose yield in the range of 5% to 15%. Cellic Ctec 1 could reduce enzyme dose to obtain the same glucose yield because of its higher activity (120 FPU/ml) than the other system (95 FPU/ml). This saves 30% enzyme loading for office paper. Waste paper, as a cellulosic fraction of municipal solid waste, with varied sugar content from 50% in magazine to 74% in used office paper has great potential as a raw material to produce fermentable sugars and ethanol. A wet blending step prior to enzymatic hydrolysis can increase glucose conversion efficiency by 5 to 10 percentage points for newspaper e.g. from 48% to 58%. Experimental data of high solids loading hydrolysis using two different enzyme systems was fitted well to glucan and xylan hydrolysis models providing scientific insight and inputs for industrial process design. Glucose yields of papers were found to be inversely proportional to their lignin contents over the range of 6 to 18% lignin. Cellic Ctec 1 had potential for saving total enzyme loadings. Results Conclusions Fig. 1. Enzymatic hydrolysis of newspaper after blending for different periods of time at different enzyme loadings (Celluclast 1.5L + Novozymes 188 system. Three replications were performed and standard error was shown) Fig. 2. Kinetics of glucan and xylan hydrolysis for four types of paper using Celluclast 1.5L + Novozymes 188 Fig. 3. The correlation between glucose yield and lignin content for four types of paper at three different enzyme concentrations for two enzyme systems Fig. 4. Glucose yield at 72h for four types of paper using two different enzyme systems