Simultaneous saccharification and fermentation of Arundo donax - Comparison of feeding strategies

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1 Simultaneous saccharification and fermentation of Arundo donax - Comparison of feeding strategies Bhargav Prasad Kodaganti Abstract Department of Chemical Engineering, Lund University, Sweden September, 211 Due to the current economic growth witnessed worldwide and to meet the demands of the increasing population, an increased usage of non-renewable fuels has resulted. This is not sustainable and alternative fuels, which should be renewable, environmentally friendly and also economically feasible are much needed. Bioethanol from lignocellulosic substrate is considered to be one of the important alternatives which can provide a suitable solution and Arundo donax is considered to be a promising bioenergy plant to be used as the substrate for the bioethanol production. In this study, two differently pretreated Arundo Donax materials were investigated for the production of ethanol. The two materials were evaluated in a simultaneous saccharification and fermentation (SSF) process and tested with standard enzyme (2 FPU/g glucan) and high enzyme dosage (1 FPU/g glucan) at 1% water insoluble solids (WIS). The slightly more severely pretreated material (PDU) showed a higher ethanol yield compared to other material (CTX). The milder pretreated material was further investigated in fed-batch SSF. Fed-batch SSF strategies (enzyme and substrate) were designed tested and compared with the batch SSF mode. Results showed that adding enzymes at an early stage gave higher ethanol yields than when enzymes were added later. In fact, batch SSF gave higher ethanol yields than any enzyme fed batch SSF tested. Also in substrate feed SSF experiment, the highest ethanol yield was obtained for batch processes. Substrate feed, however, improved the ease of stirring. Keywords: Arundo donax, Lignocellulose, Batch and, feeding strategies Introduction Increasing usage and continuous depletion of non-renewable resource has become a major area of concern for developed and developing countries. Today s world economy and development is totally determined by the nonrenewable fuels. Renewable fuels on the other hand are safe, environmentally friendly and reduce the carbon dioxide emission to the atmosphere. Due to this alarming situation, a worldwide search is on to produce renewable fuels which should be viable, sustainable and economically feasible [1, 2]. Bioethanol produced from Arundo donax has the potential to become a promising option [3]. However there are some hurdles to overcome this. Lignocellulosic biomass is the most complex polymer which is made up of cellulose, hemicellulose and lignin. Before the conversion from biomass to ethanol, biomass must have a steam pretreatment step required to break down the hemicellulose part in order to access the cellulose. Cellulose fibres are now exposed to cellulase enzyme which converts them to the glucose monomers. Enzymatic hydrolysis is considered to be most important step in the ethanol conversion [4]. Yeast (Saccharomyces cerevisiae) is the most common organism used for the fermentation of glucose to ethanol. By considering the above factors, Simultaneous saccharification and fermentation (SSF) provides a suitable process configuration which can provide with better ethanol productivity and economy [5].

2 Materials and methods Raw Material Two materials were used for the experimental work. Arundo donax material was provided by the Chemtex industry, Italy (CTX) and process development unit (PDU), Department of Chemical engineering, Lund University, Sweden. The materials were received steam pre-treated (solid matter) and stored at 4 C. These materials used different pre-treatment conditions. Yeast cultivation process Pre-culture Preparation Yeast (Ethanol Red) inoculum was prepared in a 25 ml conical flask containing the medium composition of 16.5 g/l Glucose, 7.5 g/l (NH 4 ) 2 SO 4, 3.5 g/l KH 2 PO 4,.744 g/l MgSO 4.7H 2 O, 1 ml/l Trace metal solution and 1 ml/l Vitamin solution. The trace metal and vitamin solution was prepared according to Taherzadeh et.al [2]. The salt and trace metal solutions were mixed together and ph was adjusted to 5.5 (.25 M NaOH). The glucose solution was prepared separately. The solutions were autoclaved at 121 C for 2 min. The autoclaved salt and glucose solution was mixed together and the vitamin solutions were added through sterile filter. The medium was inoculated by using a sterilized loop and the culture was incubated at 3 C at 18 rpm in rotary shaker for 24 hours. Batch cultivation At the end of the pre-culture phase, batch cultivation was started. The reactor working volume was 7 ml. The medium contains 2g/l Glucose, 2 g/l (NH 4 ) 2 SO 4, 1 g/l KH 2 PO 4, 2 g/l MgSO 4, 27 ml/l Trace metal solution and 2.7 ml/l Vitamin solution. The batch cultivation was started exactly after the end of 24 hours pre-culture. The batch cultivation was run at 3 C with a stirrer speed of 8 rpm and aeration rate of 12 ml/min, the ph was maintained at 5 by automatic addition of 3 M NaOH. contained soluble sugars and fermentation inhibitors. The 1 litre feed was fed at an initial rate of.4 L/h and increasing it linear to.1 L/h during the 16 hour cultivation phase. The same batch cultivation conditions are used in the fed-batch phase with one slight change of increased aeration supply to 15 ml/min. A few drops of antifoam were added during the addition of feed to avoid foaming. After the end of 16 hours of feeding, the produced yeast was centrifuged for 8 min at 3 rpm (HERMLE Z 513 K centrifuge, HERMLE Labortechnik, Germany). The supernatant were discarded and the yeast pellets were suspended with.9% NaCl. The suspended yeast pellets were used for further SSF experiments. SSF process All batch and fed batch SSF experiments were carried out in duplicates under anaerobic conditions using 2.5 L bioreactor (Biostat AB, Braun Biotech International, Melsungen, Germany). The following experimental condition were used for the all the SSF investigation carried out. A standard 1% WIS was used with 1.2 L working volume and a stirring speed of 3 rpm. The ph electrodes were always calibrated before start up. The ph were always maintained at 5 and 3 M NaOH were added automatically to maintain the ph. The temperature was set at 34 C. Addition of enzymes and yeast suspension leads to start of the experiment. Samples were taken at different time intervals according to the experiments for the HPLC analysis. Total run time for the SSF process was 96 hours. Table 1 SSF recipe mixture WIS 1% Working volume Yeast suspension Yeast extract (NH 4 ) 2 HPO 4 MgSO 4.7H 2 O 1.2 L 4 g/l 1 g/l.5 g/l.25 g/l Fed-Batch cultivation The full utilization of glucose and ethanol in the batch reactor marks the end of the batch phase. At this point, 1 litre of PDU hydrolysate was fed into the reactor. This marks the start of the fed-batch phase. The PDU hydrolysate

3 Enzyme used and strategies performed Cellic CTEC 2 (Novozymes, Denmark), a class of cellulase enzymes, was used for the complete study. The enzyme filter paper activity was found to be FPU/ml according to NREL procedure [6]. For the SSF experiments, enzymes were added directly without sterilization or through sterile filter. Enzyme addition volumes were calculated based on the glucan content of the material. Batch SSF Batch SSF process is similar to the normal SSF process (i.e. addition of materials as a whole). The two different materials (CTX and PDU) were used in batch SSF process with various enzyme loadings. 2, 6, or 1 FPU/g glucan Cellic CTEC2 were used for the CTX material, whereas enzyme loadings of 2 or 1 FPU/g glucan Cellic CTEC 2 were used for PDU material. All the fed batch SSF experiments used CTX material as the substrate with 1% WIS loading. The standard enzymes loading (2 FPU/g glucan Cellic CTEC 2) were used in all the fed batch SSF strategies. Enzyme feeding strategy Table 2 shows the enzyme fed batch SSF methods with their respective enzyme addition volume pattern followed in different time intervals. Time (hour) Case 1 Case 2 Case 3 Case 4 25% 25% 25% 25% 12 25% 24 25% 11% 43% 25% 36 25% 48 25% 21% 21% 72 25% 43% 11% Substrate feeding strategy In this feeding strategy, substrates are added in portions. 2 FPU/g glucan Cellic CTEC 2 enzymes were used (based on the final material addition). The enzyme activity on a glucan basis varies from the actual values, as there are variations in the WIS % present in the reactor (Table 3). Table 3 explains the substrate addition pattern involved in the substrate feeding strategy Time (h) WIS % Enzyme activity FPU/g glucan Material analysis Material analysis was performed for the two steam pre-treated materials (CTX and PDU) by following the National Renewable Energy Laboratory (NREL) standard procedure [7]. And the following results were obtained for the CTX and PDU material. The dry weight was found by washing the fibres with deionized water over filter paper and the values are shown in Table 4. Table 4 shows the material analysis results for the PDU and CTX material Material Type Glucan Xylan Lignin PDU 52.3% 6,1% 33.2% CTX 5.2% 6.4% 36.8% Dry weight analysis (of yeast cells) DM content was determined by drying the yeast samples in an oven at 15 C until a constant weight was obtained. HPLC analysis All samples taken from the SSF process were analysed using HPLC. To be used in the HPLC for analysis, the following sample preparation protocol was followed. The samples were centrifuged at 14 rpm for 5 minutes. Then the supernatant was filtered through a.2 µm sterile filter and the samples were stored at 2ºC. The stored samples were injected in the column for quantification of sugars, ethanol and other metabolites.

4 Aminex HPX-87H column, Bio-Rad Laboratories were used for the analysis of ethanol, acetate, lactate, furfural and HMF at 6 ºC with the flow rate of.6 ml/min. The eluent used was 5 mm H2SO4. To quantify the sugars and glycerol, Aminex HPX-87P column, Bio-Rad Laboratories, was used at 85 ºC and.6 ml/min. The eluent used was MilliQwater. Yield calculation Ethanol yield was calculated based on the glucan content present in the material. The theoretical mass of glucose released during hydrolysis is 1.11 times the mass of glucan (due to the addition of water). The following formula shows how to calculate the ethanol yield. Yse = (Concentration of Ethanol (g/l)) (Total amount of added glucose (g/l)) The maximum theoretical ethanol yield from glucose is.51 g/g. The fraction of theoretical ethanol yield, Yse* was obtained by, Yse*=Yse.51 Resultss and discussion SSF batch experiments A standard experiment was carried out to show how a normal SSF process will work. In the SSF experiments, the ethanol profile seems to be the most interesting one to t follow (for obvious reason since it s the main product of interest) ). The glucose which was continuously released from the fibres due to the action of the enzymes was readily consumed by the yeast. Therefore the accumulation of glucose cannot be seen with the SSF experiments (Figure 1). Xylose released from fibres were partly converted xylitol, ethanol. but not metabolized to Figure 1 showss the how a normal SSF experiment would run. CTXX material with 1% WIS is used as substrate with standard s enzyme load (2 FPU cellic CTEC 2/g glucan). Each data point indicates the sampling done at a that time. Material comparison A comparisonn study wass made between the two materials (CTX and PDU) with different Cellic CTEC 2 enzyme loadings. 2 and 1 FPU/g glucann Cellic CTEC 2 enzyme load correspond to standard and high enzyme loading respectively. With the high enzyme load (1 FPU/g glucan) ), ethanol yield was higher than standard enzyme load (2 FPU/g glucan enzyme), but not close to the theoretical ethanol yield (.51 g/g). With the standard enzyme load (2 FPU/g glucan Cellic CTEC 2), ethanol yields were.27 and.29 g/ /g in the CTX and PDU materials respectively (refer Table 5 and Figure 2). Concentration of Ethanol (g/l) Comparison of CTX and PDU in normal and high enzyme Time (h) CTX material 2 FPU/g glucan CTX material 1 FPU/g glucan PDU material 2 FPU/g glucan Figure 2 explains the ethanol curve for the CTX and PDU material with w standard (2 FPU/g glucan) and high (1 FPU/gg glucan) enzyme load.

5 Table 5 shows the yield comparison between the CTX and PDU material with two different enzyme loadings of 2 and 1 FPU/g glucan Cellic CTEC 2 Enzyme load. The ethanol concentration and yield values correspond to the last hour sample i.e. 96 hour sample. Material Concentration of ethanol (g/l) Yield of ethanol (g/g) 2 FPU/g glucan Cellic CTEC 2 Enzyme load 1 FPU/g glucan Cellic CTEC 2 Enzyme load 2 FPU/g glucan Cellic CTEC 2 Enzyme load CTX FPU/g glucan Cellic CTEC 2 Enzyme load PDU The high enzyme load for the CTX and PDU material reached a yield of 73.9% and 78.5% of the theoretical value and the standard enzyme load reached 54.5% and 58.6% of the theoretical ethanol yield for the CTX and PDU material respectively (refer materials and methods section for calculation). Even though the PDU material had produced higher ethanol yields, Arundo donax CTX material was further selected for the fed-batch SSF strategies. The main motives for comparing the CTX and PDU material was to show, how much difference it could make with ethanol yields by using a different pretreatment conditions on the same material. Since a stronger pretreatment technique was used for the PDU when compared to the CTX material, the PDU produced a higher ethanol yield but at the same time we have to consider the fact that the high inhibitors will be present in the PDU material. Therefore further fed batch SSF studies were performed on the CTX material. In the figure 3, different enzyme loadings (2, 6 and 1 FPU/g glucan) were tested with CTX material. 1 FPU/ g glucan enzyme seems to produce the better ethanol concentration than the other two enzyme loadings. This is due to the fact that the larger enzyme volumes are added to the fibres, which in turn leads to the high release of glucose from the fibres and higher ethanol concentration. Concentration of ethanol (g/l) Ethanol profile for CTX material in batch SSF mode 2 FPU/g glucan Cellic CTEC2 enzyme Time (h) Figure 3 explains the different enzyme dosages used for CTX material (1% WIS) with batch SSF mode. Fed-batch SSF feeding strategy Enzyme feeding strategy Concentration of Ethanol (g/l) batch SSF feeding strategies Time (h) Figure 4 explains the results obtained for the ethanol profile with four different cases of enzyme feeding strategies performed. All the four cases and batch SSF process used 2 FPU/g glucan Cellic CTEC 2 enzymes. 6 FPU/g glucan Cellic CTEC2 enzyme 1 FPU/g glucan Cellic CTEC2 enzyme Batch SSF batch case 1 batch case 2 batch case 3 batch case 4

6 Table 6 shows the concentration and yield of ethanol from the enzyme fed batch SSF cases Concentration of ethanol (g/l) Yield of ethanol (g/g) Case 1 Case 2 Case 3 Case 4 Case 1 Case 2 Case 3 Case The batch SSF produced a higher ethanol concentration compared to all the 4 fed-batch cases performed (Figure 4). The case 4 seems to be the best fed batch SSF strategy with respect to ethanol yield and concentration. From Table 6, it is clearly seen that the case 4 strategy is different from other 3 cases with respect to enzyme volume and timeline of addition. But it is similar with case 1 in the enzyme volumes added (i.e. 25% addition at each time). In spite of this, fed batch case 4 did not produce higher ethanol yields than the batch SSF method. In Table 3, case 3 is the one which adds high amount of enzyme during the initial addition when compared to case 1 and 2 (i.e. more than 5% of enzyme volumes are added within two additions in the case 3). Therefore better initial ethanol productivity was produced during the second and third additions. But still after the fourth addition case 3 almost reaches a similar level as the other two cases (case 1 and 2). From the above experiments, we can gather a few important findings for the enzyme feeding. The previous studies suggest that the fed batch enzyme strategy produced better ethanol yields than the batch SSF [8, 9]. This is not the case in my work. Of course the conditions used are different. Enzymes should be added as quickly as possible, so they have more time to react with the fibres to release glucose and perform fermentation to ethanol. And the majority of enzymes should be added during the first initial addition, which gives a better yield. Substrate feeding strategy The substrate feeding was not successful when compared to the batch SSF mode, i.e. the yields are low compared to the batch. The substrate feeding greatly improved the mixing Concentration of ethanol (g/l) Comparison between batch and fed batch SSF Time (hour) Figure 5 show the comparison of the ethanol concentration between the batch and fed batch SSF strategy. of the substrate and stirring problems were solved easily, but the ethanol yield of the batch SSF was better when compared with fed batch SSF (Refer figure 6). Even though previous studies showed better ethanol yields with fed batch mode, the material and the conditions (steam pretreatment, fermentation parameters etc.) used are quite different when compared to the current studies [9]. case 4 provided better ethanol yields compared to other cases followed (shown in Figure 6). The substrate feeding strategy did not work out very well, the reasons can be due to the slow hydrolysis rate of the material or the CTX material requires better steam pretreatment conditions than the one which used in the current method. Perhaps if the PDU material was tried out for the fed-batch strategies, a better understanding of the fibre conversion to higher ethanol yield could have been accomplished. Other interesting aspects to look out in the Figure 6 is during the first two additions of substrate feeding strategy, initial ethanol productivity was better than the enzyme Batch SSF case 1 Case 4 substrate feeding strategy

7 feeding strategy. But after the third addition the productivity considerably drops behind the enzyme feeding strategy. Conclusion Two differently steam pretreated materials were compared in batch SSF. The ethanol yields were higher for the PDU material, which was pretreated using SO2, than the second material (CTX) tested. The PDU material had lower xylan content and higher glucan content than the CTX material. Previous work on pretreated wheat straw has shown promising results with higher ethanol yields with fed-batch compared to batch SSF. Therefore in the case of Arundo donax (CTX) material, a number of fed-batch SSF methods were tried out in order to improve the ethanol yields compared to the batch SSF process. Running the fed batch SSF mode in Arundo donax (CTX) greatly increased the mixing of the substrate especially in the high dry matter content. From the current study, it can be seen that batch SSF process works better than the fed batch SSF methods. Perhaps the condition and feedstock s composition used in other studies were different. On the overall I can conclude that, Arundo material seems to be quite different from the other feedstock available; therefore a careful and exclusive fed-batch SSF strategy has to be designed in order to get the better ethanol yield improvement. References [1] Bessou C, Ferchaud F and Gabrielle B, Mary B, (211). Biofuels, greenhouse gases and climate change. A review, 31, [2] Sun Y and Cheng J, (22). Hydrolysis of lignocellulosic materials for ethanol production: A review, Bioresour Technol 83, [3] Williams CMJ, Biswas TK, Black I, Harris PI, Heading S, Marton L, Czako M, Pollock R and Virtue JG (submitted). Use of poor quality water to produce high biomass yields of giant reed (Arundo donax L.) on marginal lands for biofuel or pulp/paper. Proceedings of International Symposium on Underutilised plants. Tanzania, March 2-7th 28. (Acta Horticulturae, submitted) [4] Kumar P, Barrett DM, Delwiche MJ and Stroeve P, (29). Methods for Pretreatment of Lignocellulosic Biomass for Efficient Hydrolysis and Biofuel Production, Ind. Eng. Chem. Res., 48, [5] Wingren A, Galbe M and Zacchi G, (23). Techno-Economic Evaluation of Producing Ethanol from Softwood: Comparison of SSF and SHF and Identification of Bottlenecks, Biotechnol. Prog., 19, [6] Adney B and Baker J, (1996), Measurement of Cellulase Activities (LAP) NREL, Golden, CO [7] Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D, Crocker D, (28), Determination of structural carbohydrates and lignin in biomass (LAP). NREL, Golden, CO [8] Hoyer K, Galbe M and Zacchi G, (21). Effects of enzyme feeding strategy on ethanol yield in fed-batch simultaneous saccharification and fermentation of spruce at high dry matter, Biotechnology for Biofuels, 3, 14 [9] Olofsson K, Palmqvist B and Liden G, (21). Improving simultaneous saccharification and co-fermentation of pretreated wheat straw using both enzyme and substrate feeding, Biotechnology for biofuels, 3,117