Effect of process conditions on high solid enzymatic hydrolysis of pre-treated pine

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1 Effect of process conditions on high solid enzymatic hydrolysis of pre-treated pine Abstract Anders Josefsson Department of Chemical Engineering, Lund University, Sweden In this study a relatively uncharacterized lignocellulosic feedstock was examined during enzymatic hydrolysis for bioethanol production. The study evaluated the difference of two enzyme generations from Novozymes at different temperatures as well as the effect of varying enzyme dose and stirrer intensity. Furthermore, the characteristics of pine were compared to those of other lignocellulosic feedstocks. A comparison between two different impeller systems was also conducted where a system with only an anchor impeller was compared to a system with both an anchor and a central impeller. The results show that temperature has a clear impact on the hydrolysis and that the newer Cellic Ctec3 enzyme mixture increases the effectiveness of the hydrolysis by around 1% compared to the older Cellic Ctec2. The comparison between enzyme load and impeller intensity show that increased impeller intensity and/or increased enzyme load increase the productivity of the hydrolysis. The stirrer intensity was shown to have a larger effect on high enzyme loads. Pine was shown to behave similar to spruce in an enzymatic hydrolysis when comparing torque profiles and power consumption under similar process conditions. It is indicated that an impeller set-up with both a central impeller and an anchor may be more efficient than a set-up with only an anchor. Keywords: Enzymatic hydrolysis, bioethanol, lignocellulosic biomass, pretreated pine, high solids. Introduction Using lignocellulosic feedstocks for bioethanol production is being investigated in order to decrease the dependency of oil and the carbon dioxide emissions this dependency causes (Hahn-hägerdal, et al., 26). Since lignocellulosic feedstocks vary in composition (Wiselogel, 1997) (Hayn, et al., 1993) they behave quite differently during the production process. This means that the effects of varying process conditions of different feedstocks are important to determine in the pursuit of large scale production of biofuels from lignocellulosic sources. An important aspect of achieving an economically feasible ethanol production from lignocellulosic materials is to achieve a high ethanol concentration in the process by increasing the WIS content (Galbe & Zacchi, 22). Higher ethanol content will decrease the cost waste water handling as well as distillation costs in the. Increased WIS content has negative effects as well such as decreased yields (Kristensen, et al., 29) (Humbird, et al., 21) and increased viscosity of the biomass (Viamajala, et al., 29) (Wiman, et al., 212) which in turn increases the power input required for mixing (Palmqvist & Lidén, 212) (Zhang, et al., 21).

2 This study investigates the effect of process parameters such as stirring, enzyme dose and temperature on enzymatic hydrolysis of pretreated pine at high WIS content. It also compares the effectiveness of the newest cellulase mixture from Novozymes with an older one. The material used is the relatively uncharacterized pine tree. Materials and methods Raw material and pretreatment The material used in this study is pretreated pine. The pre-treatment was carried out by SO 2 steam explosion. The material was impregnated in 2.% SO 2 for 2 minutes prior to being steam pretreated at 21 C for minutes in a reactor previously described by Palmqvist, et al., The material was then pressed to achieve the desired WIS concentration of 16.4%. The contents of the starting material are shown in tables 1 & 2. Table 1, WIS content and lignin and glucan fraction of WIS in starting material. WIS content Lignin Glucan 16.4% 49.7% 46.% Table 2, sugar concentrations in liquid fraction of starting material. Glucose Mannose Xylose [g/l] [g/l] [g/l] Hydrolysis experiments The hydrolysis experiments were carried out in duplicates in two reactors, Hanna and Memmalys (Belach Bioteknik). The Memmalys reactor was used for the enzyme generation and temperature experiments. The enzymes Cellic Ctec3 and Cellic Ctec2 (kindly provided by Novozymes A/S) were evaluated at both 34 C and C with constant impeller power input. The reactor was equipped with an anchor impeller as well as a central impeller which could be controlled separately. The Hanna reactor was used for the impeller intensity and enzyme dose experiments. Cellic Ctec3 was used and the temperature was C. The impeller speed was set to either 1 or rpm. The enzyme doses tested were 3% or 3% of the total WIS-content in the load, i.e. 7.g or 7g (g total load and 16.4% WIS). Samples from all the experiments were taken at, 3, 6, 9, 24 and 48 hours after the start of the hydrolysis. The h sample was taker prior to the addition of enzymes. Analysis The analysis of sugar content was carried out by centrifuging sampled hydrolysis liquid in 2mL Eppendorf tubes for minutes at 13 rpm. The supernatant was filtered through 2 µm filters. These samples were then stored at -2 C prior to being analyzed. A HPLC system was used to measure sugar concentrations. The system was fitted with a polymer column (Aminex HPX-87P, Bio-Rad Laboratories) at 8 C. The eluent used was deionized water at.6 ml per minute and detection was done with a refractive index detector (WATERS 241, Milford). Results Enzyme and temperature comparison The results from the comparison between the two enzyme generations are shown in figure 1. They show an increase in glucose production at higher temperature for both enzyme mixtures with the biggest difference being the initial rate. The newer product proved more efficient giving around 1% higher glucose production compared to the older enzyme generation.

3 2 1 Ctec2 34 C Ctec2 C Ctec3 34 C Ctec3 C 2 4 Figure 1, Glucose produced in the experiments using the two enzyme generations Ctec2 and Ctec3 at 34 C and C Stirring and enzyme dose evaluation Stirring intensity and enzyme load comparison results are shown in figure 2. They show that the glucose produced is increased with increasing stirrer intensity as well as with increasing enzyme dose. The impeller speed, however, has a larger relative effect on the glucose productivity using a high enzyme load compared to a lower. When increasing the rmp from 1 to on 3% of WIS as enzyme load the glucose produced increases by.6% while using 3% of WIS as the enzyme load the hydrolysis yields 22.% more glucose. Material comparison When comparing pine to other materials, yield, torque profile and power consumption during the hydrolysis was compared to similar experiments on other lignocellulosic materials (Palmqvist & Lidén, 212). The torque profile during hydrolysis of pine can be seen in figure 3 and relevant data in table 3. Table 3, relative costs of glucose in experiments in terms of enzyme and power consumption. Parameters Average power [W] Total power [kj] kj / g gluc g enz / g gluc Yield % 1rpm 3% enz rpm 3% enz rpm 3% enz rpm 3% enz

4 1 rpm 3% enzyme load rpm 3% enzyme load rpm 3% enzyme load 2 4 rpm 3% enzyme load 2 4 Figure 2, glucose produced in the stirrer intensities 1rpm and rpm with 3% and 3% of WIS enzyme load. Impeller system evaluation Results from the comparison between the reactors (i.e. the impeller set-up comparison) can be seen in figure 4. The two Hanna reactor runs at 1 and rpm respectively both produce less glucose than the run in Memmalys reactor with the dual impeller setup. The Memmalys reactor had some problems with resistances in the impeller mount system which makes discussing exact power consumptions difficult. It is however known that the Memmalys reactor did not consume more than 1W for the duration of the hydrolysis while the single impeller runs consumed.17w and 1.2W respectively, showing that the dual impeller system may very well be more efficient than the single anchor system. Discussion Enzyme and temperature comparison The results clearly showed that the temperature closest to the considered optimal for enzymatic hydrolysis, C, was the most effective. The newer enzyme batch (Ctec3) proved 1% more efficient than the older (Ctec2). This is somewhat less than expected based on the experience from other materials. Enzyme load and stirrer intensity evaluation Both the enzyme load and stirrer intensity have a clear impact on the effectiveness of the hydrolysis. This has been shown to be true for other materials as well (Palmqvist, et al., 211) (Samaniuk, et al., 211). The fact that an Torque [Nm] rpm 3% enzyme load Figure 3, torque profile of hydrolysis of pine.

5 2 1 1 rpm 2 1 rpm rpm Figure 4, comparison between the two reactors, the left and the middle showing Hanna results and the right Memmalys results. increased impeller intensity has an increased effect on the hydrolysis has not previously been seen although it may be related to the milling effect mentioned in previous studies (Palmqvist, et al., 211). If related to the milling effect, this could indicate that at low rpm, the material contains enough free seats to support most of the enzymes in the low dose while the higher enzyme load reactor will contain a higher amount of inactive enzymes in the bulk of the reactor. As the rpm is increased, the milling effect is increased and the higher enzyme load runs benefit more as there are more free enzymes to start hydrolyzing the new available fibers in the solution. The yield is, however, increased in both the high and low enzyme load experiments. There are several other factors this may be attributed to. The mass transport of sugars from the fibers is one example. As the hydrolysis progress, the areas in close proximity to the fibers will accumulate cellobiose due to stagnant layers around the fibers caused by fluid dynamics. Increasing the fluid velocity in the reactor will decrease the size of these layers. This increases the mass transport to and from the fibers and lessens the impact of product inhibition on the hydrolysis. The size decrease of the stagnant layers surrounding the fibers could also increase the transportation of enzymes to and from the fiber surface enabling more of the enzymes to be active at a time by assisting the adsorption of the enzymes which has been shown to be an issue using high WIS lignocellulose (Kristensen, et al., 29). Comparison with other materials The effect of stirring on the hydrolysis has been evaluated on other lignocellulosic materials prior to this study (Samaniuk, et al., 211) (Palmqvist, et al., 211). However, to our knowledge, pine is a relatively uncharacterized material for biofuel production. Experiments on spruce, also softwood like pine, have shown a large effect of mixing on the hydrolysis with increasing yield with increased mixing intensity. This is only true until a certain point where additional mixing has no further effect on the hydrolysis. (Tengborg, et al., 21). The torque profiles from the 1 rpm experiments with 3% enzyme load can be compared to similar experiments run on % WIS pre-treated spruce and arundo (Palmqvist & Lidén, 212). For arundo the torque and power input is lowered very quickly during the start of the hydrolysis, taking only 1-2 hours to reach its lowest level. For spruce this decrease takes a longer time, being visible for at least the first 1 hours, very much like the results acquired for pine. This indicates that pine could be assumed, as expected, to have more similarities to spruce than other materials, at least rheology-wise. Comparison between impeller systems A comparison between the reactors was made at 3% enzyme dose, C and Ctec3. The highest yield was achieved in the Memmalys reactor although due to the power

6 losses in the impeller system of the Memmalys reactor no exact comparison of the energy consumption of the experiments can be made. The total power used in the Memmalys reactor, however, was limited to 1W for the duration of the experiment, giving it lower average power consumption than the rpm experiment in the Hanna reactor (table 3). Previous comparisons between impeller types indicate that anchor impellers are more effective compared to other types during enzymatic hydrolysis (Kinnarinen, et al., 212). The anchor impellers, however, risk losing pumping power due to the viscosity decrease during the hydrolysis and thus the mixing becomes worse as the hydrolysis progresses. As the Memmalys reactor has two central impellers this could mean that the mixing in this reactor is increased later in the hydrolysis due to the central propellers, even at lower power inputs. Conclusions The results show a difference in hydrolysis rate between the evaluated temperatures and also between the two enzyme batches. It is shown that higher stirrer intensity gives a higher glucose production. The effect is, however, larger at high enzyme concentrations than at low. As expected, torque measurements show more similarities to spruce than other lignocellulosic materials. Along with similar yields and power consumptions that indicate that spruce and pine are relatively similar. Furthermore, it is indicated that the impeller system in the Memmalys reactor can acquire a higher efficiency over time than the Hanna reactors impeller system. References Hayn, M., Steiner, W., Klinger, R. & Steinmüller, H., Hayn, M., Steiner, W., Klinger, R., & Steinmüller, H. (1993). Basic research and pilot studies on the enzymatic conversion of lignocellulosics. BIOTECHNOLOGY IN AGRICULTURE, pp Humbird, D., Mohagheghi, A., Dowe, N. & Schell, D. J., 21. Economic impact of total solids loading on enzymatic hydrolysis of dilute acid pretreated corn stover. Biotechnology Progress, 26(), pp Kinnarinen, T. et al., 212. Effect of mixing on enzymatic hydrolysis of cardboard waste: Saccharification yield and subsequent separation of the solid residue using a pressure filter. Bioresource Technology, Volume 11, p Kristensen, J. B., Felby, C. & Jørgensen, H., 29. Yield-determining factors in high-solids enzymatic hydrolysis of lignocellulose. Biotechnology for Biofuels, 2(11). Palmqvist, B. & Lidén, G., 212. Torque measurements reveal large process differences between materials during high solid enzymatic hydrolysis of pretreated lignocellulose. Biotechnology for biofuels. Palmqvist, B., Wiman, M. & Lidén, G., 211. Effect of mixing on enzymatic hydrolysis of steam-pretreated spruce: a quantative analysis of conversion and power consumption. Biotechnology for biofuels. Palmqvist, E. et al., Design And Operation Of A Bench-Scale Process Development Unit For The Production Of Ethanol From Lignocellulosics. Bioresource Technolog, Volume 8, pp Samaniuk, J. R., Scott, T., Root, T. W. & Klingenberg, D. J., 211. The effect of high intensity mixing on the enzymatic hydrolysis of concentrated cellulose fiber suspensions. Bioresource Technology, 12(6), pp Tengborg, C., Galbe, M. & Zacchi, G., 21. Influence of Enzyme Loading and Physical Parameters on the Enzymatic Hydrolysis of Steam-Pretreated Softwood. Biotechnology Progress, 17(1), pp Viamajala, S., McMillan, J., Schell, D. & Elander, R., 29. Rheology of corn stover slurries at high solids concentrations - Effects of saccharification and particle size. Bioresour Technol, 1(2), pp Wiman, M., Palmqvist, B., Tornberg, E. & Lidén, G., 212. Rheological Characterization of Dilute Acid Pretreated Softwood. Biotechnology Bioengineering, pp Wiselogel, A., Biomass feedstock resources and composition. Fuel and Energy Abstracts, 38(2), pp (1). Zhang, J. et al., 21. Simultaneous saccharification and ethanol fermentation at high corn stover solids loading in a helical stirring bioreactor. Biotechnology and Bioengineering, (4), pp Galbe, M. & Zacchi, G., 22. A review of the production of ethanol from softwood. Applied Microbiolology Biotechnology, Volume 9, pp Hahn-hägerdal, B. et al., 26. Bio-ethanol the fuel of tomorrow from the residues of today. Trends in Biotechnology, 24(12), pp

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