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Feasibility of a facile butanol bioproduction using planetary mill pretreatment
Bioresource Technology 199 (2016) 283–287
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Feasibility of a facile butanol bioproduction using planetary mill pretreatment Jeong Heo Kwon a,b,1, Hyunsoo Kang c,1, Byoung-In Sang b, Yunje Kim d, Jiho Min e, Robert J. Mitchell c,2, Jin Hyung Lee a,⇑,2 a
Korea Institute of Ceramic Engineering and Technology (KICET), 101, Soho-ro, Jinju-si, Gyeongsangnam-do 52851, Republic of Korea Division of Chemical Engineering & Bio Engineering, Hanyang University, Seoul, Republic of Korea School of Nano-Bioscience and Chemical Engineering, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea d Clean Energy Research Center, Korea Institute of Science and Technology, Seoul, Republic of Korea e Division of Chemical Engineering, Chonbuk National University, Jeonju, Jeonbuk, Republic of Korea b c
h i g h l i g h t s Convenient butanol production method proposed using planetary mill pretreatment. Planetary mill pretreatment eliminates need for biomass washing and buffer changes. Planetary milled Pinus rigida hydrolysates utilized directly without detoxication. Improved biobutanol yields with sugars from the hydrolysates than purified glucose.
a r t i c l e
i n f o
Article history: Received 30 June 2015 Received in revised form 14 August 2015 Accepted 17 August 2015 Available online 28 August 2015 Keywords: Planetary mill Pretreatment Enhanced bioconversion Biobutanol Biofuel
a b s t r a c t A facile butanol bioproduction process was developed using planetary milling, and Pinus rigida wood waste as a model substrate for fermentable sugars. The use of planetary milling as the pretreatment eliminates the need for washing and transfer of the biomass prior to enzymatic hydrolysis. Moreover, using this pretreatment process resulted in the production of only 0.072 ± 0.003 g/L soluble phenolic compounds, a concentration that was not inhibitory towards Clostridium beijerinckii NCIMB 8052. As the milling was performed in a compatible buffer (50 mM acetate, pH 4.8), the enzymatic hydrolysis step was initiated by simply adding the cellulase cocktail powder directly to pretreated biomass without washing the biomass or exchanging the buffer, resulting in a glucose yield of 31 g/L (84.02%). Fermentation of the hydrolysate samples by C. beijerinckii NCIMB 8052 gave slightly better butanol yields than cultures grown in a typical lab media (P2), with final concentrations of 6.91and 6.66 g/L, respectively. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Recently, the production of biofuels and their commercialization has garnered a significant amount of interest globally due to both fossil fuel depletion and the need to reduce net CO2 emissions (Kim et al., 2010). The use of biofuels is an answer to these problems since their use reduces global greenhouse gas (GHG) emissions while also improving both national energy security and rural development (van Eijck et al., 2014). As such, biofuels offer
⇑ Corresponding author. Tel.: +82 55 792 2752; fax: +82 55 792 2740. E-mail address: [email protected] (J.H. Lee). These authors contributed equally to this work.
1,2
http://dx.doi.org/10.1016/j.biortech.2015.08.074 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
a promising and potential substitute for petroleum usage, supporting the argument for the adequacy of biofuels as an alternative energy source (Fung et al., 2014). One of the more current trends in biofuel production is the use of lignocellulosic biomasses as they are readily available, generally have a low cost and are non-food feedstocks (Chandra et al., 2012). The production of biofuels from a lignocellulosic biomass requires several steps, including biomass pretreatment, hydrolysis of the cellulose and hemicellulose to sugar and the subsequent fermentation of this sugar (Balat, 2011). The complexity of the process required for lignocellulosic biofuels is mainly due to the recalcitrance of the biomass and, hence, the pretreatment step is key in determining the efficacy of the downstream steps. Typically, the pretreatment step involves an exposure of the biomass to some
284
J.H. Kwon et al. / Bioresource Technology 199 (2016) 283–287
harsh chemical, which may include a dilute acid, sodium hydroxide or ammonia. The use of such steps necessitates additional processes and steps, such as filtration of the treated solids, washing of the biomass with deionized water, pH neutralization and drying of the solids prior to the downstream enzymatic hydrolysis step (Kim et al., 2008; Li et al., 2014; Pappas et al., 2014). Moreover, these chemical-based pretreatments produce toxic byproducts that are known to undergo irreversible interactions with cellulose hydrolyzing enzymes, as well as inhibit the fermentative activity of the microorganisms. As such, the buffers used during the pretreatment step must be replaced and the biomass washed prior to the subsequent enzymatic hydrolysis and fermentation steps, both of which generate wastewater that must be treated. The capital costs associated with this treatment are significant, encompassing 21% of the total biofuel process cost (NREL, 2011). Though chemical pretreatments usually requires a lower energy consumption than physical pretreatments (Hendriks and Zeeman, 2009), they also have limitations that should be considered in industrial applications. To side-step this limitation, Li et al. (2014) recently reported a novel configuration for cellulosic ethanol production; a one-pot simultaneous saccharification and fermentation (One-pot SSF) that did not require rinsing and detoxification of the treated biomass. To achieve this, they used a combination of size reduction and alkali pretreatment in their system. However, the highest glucose concentration obtained was only 6.75 g/L, a value that is not feasible for commercial applications. Moreover, as their system used an alkali pretreatment, it is very likely that the inhibitors were also generated. To address both the costs associated with wastewater treatment and the generation of fermentative inhibitors, a facile butanol bioproduction process is proposed in this study using a planetary milling pretreatment step. The potential use of planetary milling processes for the pretreatment of lignocellulosic biomass and to improve fermentable sugar production was reported previously (Kim et al., 2013a,b). This process does not require any extraneous steps, such as rinsing of the biomass or changing of the buffer, since planetary milling neither uses chemicals nor does it produce significant quantities of enzyme/microbe inhibitors (Kim et al., 2013b). Furthermore, to demonstrate the feasibility of this facile butanol bioproduction system, pitch pine wood waste was planetary milled and hydrolyzed to fermentable sugars and this was successfully converted to butanol using Clostridium beijerinckii NCIMB 8052.
2. Methods 2.1. Materials Pitch pine (Pinus rigida) wood waste was kindly provided by Suncheon Forestry Cooperative, Republic Korea. Sugar standards (i.e., glucose, cellobiose, xylose, galactose and mannose), 3,5dinitrosalicylic acid, sodium acetate trihydrate and sodium citrate were all purchased from Sigma–Aldrich (USA). A cellulose cocktail was purchased from Worthington Biochemical Co. (USA).
2.2. Planetary mill pretreatment of the P. rigida biomass Initially, 7.2 g of pitch pine waste was added to a zirconia jar container, followed by 180 ml of 50 mM sodium acetate (pH 4.8). For milling, 3 mm diameter zirconia balls were added. Planetary milling was carried out using a Pulverisette 5 (Fritsch, Germany) at room temperature for 4 h. The jar was rotated at a velocity of 300 rpm.
2.3. Enzymatic hydrolysis of the biomass Before starting the enzyme hydrolysis, half of supernatant was removed to obtain a biomass solid loading of 8.0%. Powdered cellulase (Worthington Biochemical Co., USA) was added directly to the supernatant and biomass slurry to a final concentration of 15 FPU/ g-biomass. This mixture was then incubated at 50 °C for 96 h with agitation using a rotator (JEIO Tech, Rep. Korea) set to 200 rpm. The glucose yield after treatment was calculated using the following equation:
Glucose yield ð%Þ ¼
Glucose produced by enzyme hydrolysis Glucan in biomass 1:11
2.4. Fermentation by C. beijerinckii NCIMB 8052 A 1 ml suspension of C. beijerinckii NCIMB 8052 spores, which were stored at 4 °C in sterile distilled water, were germinated by heat shocking them at 75 °C for 10 min. They were then inoculated into 25 mL of anaerobic Reinforced Clostridial Medium (RCM; Difco Laboratories, USA) in a serum bottle and grown for 12 h at 37 °C and 250 rpm. Afterwards, an inoculum (5%) was transferred to 25 mL of fresh RCM medium and grown for 6 h to ensure that the culture was in the exponential growth phase. From this culture, 2.5 mL was inoculated into 50 mL of modified P2 medium as defined below and grown as previously described (Dwidar et al., 2012). The P2 medium used in this study was prepared by adding the following amounts of compounds into 50 mL of enzymatic hydrolysate: 1 g/L yeast extract, 2 g/L (NH4)2SO4, 1% P2 salt solution A, 1% P2 salt solution B, and 0.1% vitamin solution. For the control cultures, the same components were added to 50 ml of 50 mM sodium acetate containing the same concentration of glucose (Sigma, USA) as found in the biomass hydrolysate. All the media were prepared aseptically within a UV-sterilized laminar flow hood. The pH of the media was adjusted to pH 6.0 by adding 10 M NaOH. The media was converted to an anaerobic state by purging the fermentation broth with sterile filtered Ar gas. After 30 min of purging, the unit was sealed using sterilized caps and this was used directly for the fermentation, which was performed in a shaking incubator (Vision Science, ROK) set to 250 rpm and 37 °C. Each experiment was performed in triplicate. 2.5. Analytical methods The size of pretreated pitch pine wood waste was investigated using a Scanning Electron Microscope (JSM-6700F, JEOL, Tokyo, Japan). The crystallinity of the biomass was measured using Xray diffraction (XRD, Rigaku D/max-RB powder diffractometer, Tokyo, Japan) with Cu ja radiation (k = 1.542 Å). The crystallinity index was calculated using the follow equation:
Crystallinity Index ð%Þ ¼ ½ðI2h¼22:5 I2h¼18:7 Þ=ðI2h¼22:5 Þ 100 The phenolic content of the milled hydrolysate was measured using Folin–Ciocalteau colorimetry as described previously (Kim et al., 2013b). A standard GC (HP-6890, Agilent, USA), equipped with a time-of-flight mass spectrometer (TOFMS) and a column (HP-U2, 20 m 0.20 mm, 0.11 lm), was used to identify the phenolic compounds produced during the pretreatment steps. The ion source temperature was 220 °C and the resulting solution (2 lL) was injected in split mode (2:1) at an injector temperature of 250 °C and separated through an Ultra-2 capillary column (17 m 0.2 mm i.d., 0.11 lm film thickness; Agilent Technologies). The initial oven temperature was 60 °C for 1.00 min, ramp to 255 °C at 5 °C/min, 1 min hold. Helium (99.9999%) was used as the carrier gas (1.0 mL/min. constant flow at an oven temperature of 150 °C). The mass spectrometer was operated at 70 eV with
J.H. Kwon et al. / Bioresource Technology 199 (2016) 283–287
selected ion monitoring (SIM) and mass range was 35–650 m/z. The sugar content was measured using an Agilent 1200 Series HPLC fitted with a refractive index detector (Agilent Technologies, USA). The column used was an Aminex HPX-87P column (BioRad, USA) and deionized water was used as the solvent. Likewise, the acetate, butanol, acetic acid and butyric acid concentrations were all determined using an Agilent 7890A FID-GC system (Agilent Technologies, USA) equipped with an Agilent HP-INNOWAX GC column and a flame ionization detector. The optical densities of the cultures were measured using a spectrophotometer (BioPhotometer, Eppendorf, Germany). 3. Results and discussion 3.1. Concept of the facile butanol bioproduction process using planetary mill pretreatment The conventional process involves a washing step, recycling of the catalyst and detoxification of the sample due to the chemical catalyst use. Together these contribute to both increasing the process complexity as well as the costs associated with the production of the biofuel. One example of an extraneous cost associated with conventional processes is the use of water that should be treated or recycled before it can be released into the environment. In contrast, the facile process proposed in this study does not require any buffer exchange or washing steps and, thus, significantly reduces the wastewater output and the associated costs required for its subsequent treatment (Fig. S1). Moreover, as the pretreatment, hydrolysis and fermentation steps are carried out using the same aqueous solution during the process, no biomass is lost. 3.2. Effects of planetary mill pretreatment on P. rigida Planetary mill treatment of plant biomass increases the surface area by reducing the size of the biomass to the level of fibers and fiber bundles, which ultimately increases the enzyme accessibility to the cellulose. This is illustrated in Fig. S2, where pitch pine (P. rigida) wood waste was significantly reduced in size after a planetary mill pretreatment. This treatment regime also changes the surface properties by disrupting the typically rigid surface and its intact phytoliths (Fig. S3). Before planetary milling, the crystallinity index (CI) of the pitch pine was 52.5 ± 0.9%, but this was reduced to 29.9 ± 3.7% after milling. This reduction indicates that planetary milling weakened the hydrogen bonding network within the crystalline cellulose while also potentially increasing access to the cellulose available for saccharification. One major benefit of planetary mill pretreatments is its disruption of the strong physical interactions within the biomass to reduce its recalcitrance to enzymatic and microbial hydrolysis while not changing the chemical composition of the biomass (Data not shown). However, as the soluble phenolic compounds formed from soluble hemicelluloses and lignin can inhibit microbial activities (Alvira et al., 2010; Lee et al., 2012, 2015), an analysis of the phenolic compounds produced during the pretreatment step was performed before conducting the downstream fermentative processes. Planetary milling of the pitch pine produced only 0.072 ± 3 g/L of soluble phenolic compounds. This concentration was smaller than found in previous study (Kim et al., 2013b), even though a higher solid-to-liquid ratio was used here. This difference is likely due to the properties of the biomass used, which were pitch pine wood waste in the current study and rice straw previously. Using GC/TOF-MS, the pretreatment products were identified (Fig. S4). A total of fourteen phenolic compounds such as benzaldehyde and 2-methoxyphenol, were identified (Table S1), with each existing at very low concentrations.
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3.3. Enzymatic hydrolysis of the planetary milled P. rigida After enzymatic treatment of the milled biomass with an enzyme load of 15 FPU/g-biomass, it was found that three sugars were primarily present in the hydrolysate, including cellobiose (6.5 ± 0.5 g/L), xylose (2.0 ± 0.9 g/L) and glucose (31.0 ± 0.2 g/L). No galactose, arabinose or mannose was detected in any of the samples because the cellulase selected for this study lacks hemicellulase activity. However, the hemicellulose is an important source of pentoses that can also be utilized by fermentative microbes and improve the solvent yields. As such, the use of another enzyme preparation, such as Fusarium oxysporum enzyme extract, could be employed to hydrolyze both cellulose and hemicellulose for industry applications (Xiros et al., 2011). As the P. rigida wood waste used in this study consists of 41.5% cellulose, the glucose yield is 84.02%. Attempts to enhance the glucose concentration by increasing the amount of biomass treated in each batch were unsuccessful and the highest glucose concentration achieved did not significantly exceed 31.0 g/L (Data not shown). As reported previously, this limitation likely stems from both end-product accumulation and inhibition of the cellulase activity, as well as a low free-water availability (Hsieh et al., 2014; Jorgensen et al., 2007). Several studies stated that these limitations can be overcome by enhancing the cellulase activity (Sun and Cheng, 2002) or by employing a simultaneous saccharification and fermentation (SSF) process, ideas which will be considered in a future study. Moreover, the residues remaining after hydrolysis of the sugars would be enriched for lignin and, as such, could be used for other biochemical processes and productions.
3.4. Evaluation of fermentable sugar produced from planetary milled P. rigida Since the addition of sodium acetate to fermentation broths is beneficial as it increases both the solvent production and glucose utilization by C. beijerinckii NCIMB 8052 (Chen and Blaschek, 1999), 50 mM sodium acetate was selected as the buffering
Fig. 1. Time course characteristics of the cultures grown either on (A) glucose or (B) the pretreated P. rigida hydrolysate, showing the optical densities, glucose consumption and butanol concentrations. Each of the cultures initially had 31 g/L glucose within them either from its addition (A) or from the hydrolysate (B).
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Table 1 Comparison of the solvent and acid production levels in cultures grown for 72 h with either the milled hydrolysate prepared according to this study or glucose (control) added.
Milled hydrolysate Control *
Butanol yield =
Acetone (g/L)
Butanol (g/L)
Acetate (g/L)
Butyrate (g/L)
Butanol yield* (g/g)
3.42 ± 0.11 3.05 ± 0.09
6.91 ± 0.64 6.66 ± 0.21
1.49 ± 0.10 1.09 ± 0.18
1.81 ± 0.35 1.02 ± 0.02
0.25 ± 0.01 0.23 ± 0.02
Weight of butanol produced . Weight of glucose used
solution for the enzymatic hydrolysis. An additional benefit of this buffer is that it can be used for the fermentation steps after simply adjusting the pH and adding the essential vitamins and nutrients. However, based upon the above findings where 31 g/L of glucose was generated from the biomass, no further glucose addition is necessary. Consequently, the efficacy of using these hydrolysate samples directly for fermentative processes was next tested within C. beijerinckii NCIMB 8052 cultures. For this, only the remaining biomass was removed and so the bacterium was exposed to all of the phenolics and sugars produced during the pretreatment stages. Fermentations performed with these hydrolysate samples show a delayed cell growth and an overall lower biomass when compared to the control cultures (Fig. 1). The maximum average cell density, i.e., the optical density at 600 nm (OD600), for the biomass hydrolysate cultures was 5.29 while that of the control was 8.12, both after 48 h. It is thought that this longer lag period and lowered overall bacterial density is due to impurities present in the milled hydrolysate that cause C. beijerinckii NCIMB 8052 stress, and may even be due to the compounds listed in Table S1. For instance, ferulic acid, which has a structure similar with one of the compounds identified, namely, 4-hydroxy-3-methoxy benzeneacetic acid, is known to cause an analogous loss in the bacterial growth (Lee et al., 2015). Besides the cell densities, the only other clear difference between the two cultures was the increased solvent and acid production by the biomass hydrolysate cultures (Table 1). This was particularly evident with the acids, which were produced at about 1.4 to 1.8-fold higher level. The acetone production was also enhanced by about 10% while butanol increased only slightly over the control. However, the butanol/acetone ratio in the biomass hydrolysate was slightly smaller than that seen with the control culture; 2.02 and 2.18 g/g, respectively (Table 1). Considering all of these, the hydrolysate culture produced more than 1.8 g/L more products than the control cultures, which were grown on typical lab media containing glucose. It would also seem that the loss in microbial biomass corresponds with the increased production, a connection that will be studied further. However, the above results clearly demonstrate that the milled hydrolysate sample can be used for fermentations and actually leads to slightly better yields than conventional lab P2 media. This study illustrated the feasibility of a facile butanol bioproduction through the use of a planetary mill pretreatment. Such a facile butanol bioproduction has several advantages. Firstly, there is no need for washing and buffer exchange, which would be beneficial both in terms of saving time and labor and, most importantly, avoiding the loss of biomass and the generation of wastewater. Moreover, only one-vessel is required for the entire process, reducing the need for downstream sample transfer, which is also an important factor for industrial feasibility.
4. Conclusions This study demonstrates that a facile butanol bioproduction is possible when using planetary milling. Fermentations performed using P. rigida biomass treated using this facile process showed slightly improved solvent and acid yields when compared to
cultures grown on a glucose-based media, illustrating that the hydrolysate is readily fermentable and that this facile process doesn’t generate a significant amount of lignin-related inhibitory compounds. As the whole bioconversion process as presented in this study, i.e., from biomass to biofuel, can be achieved within a single buffered media, it could also potentially reduce production costs associated with both wastewater treatment and biomass loss. Acknowledgements This work was supported by the New & Renewable Energy Core Technology Program of the Korea Institute of Energy Technology Evaluation and Planning (KETEP) (Grant # 20153010091990) and partially by the National Research Foundation of Rep. Korea (Grant # NRF-2009-C1AAA001-2009-0093499). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2015.08. 074. References Alvira, P., Tomas-Pejo, E., Ballesteros, M., Negro, M.J., 2010. Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: a review. Bioresour. Technol. 101 (13), 4851–4861. Balat, M., 2011. Production of bioethanol from lignocellulosic materials via the biochemical pathway: a review. Energy Convers. Manage. 52 (2), 858–875. Chandra, R., Takeuchi, H., Hasegawa, T., 2012. Hydrothermal pretreatment of rice straw biomass: a potential and promising method for enhanced methane production. Appl. Energy 94, 129–140. Chen, C.K., Blaschek, H.P., 1999. Effect of acetate on molecular and physiological aspects of Clostridium beijerinckii NCIMB 8052 solvent production and strain degeneration. Appl. Environ. Microbiol. 65 (2), 499–505. Dwidar, M., Lee, S., Mitchell, R.J., 2012. The production of biofuels from carbonated beverages. Appl. Energy 100, 47–51. Fung, T.K.F., Choi, D.H., Scheufele, D.A., Shaw, B.R., 2014. Public opinion about biofuels: the interplay between party identification and risk/benefit perception. Energy Policy 73, 344–355. Hendriks, A.T.W.M., Zeeman, G., 2009. Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresour. Technol. 10, 10–18. Hsieh, C.W.C., Cannella, D., Jorgensen, H., Felby, C., Thygesen, L.G., 2014. Cellulase inhibition by high concentrations of monosaccharides. J. Agric. Food Chem. 62 (17), 3800–3805. Jorgensen, H., Vibe-Pedersen, J., Larsen, J., Felby, C., 2007. Liquefaction of lignocellulose at high-solids concentrations. Biotechnol. Bioeng. 96 (5), 862– 870. Kim, T.H., Taylor, F., Hicks, K.B., 2008. Bioethanol production from barley hull using SAA (soaking in aqueous ammonia) pretreatment. Bioresour. Technol. 99 (13), 5694–5702. Kim, J.S., Park, S.C., Kim, J.W., Park, J.C., Park, S.M., Lee, J.S., 2010. Production of bioethanol from lignocellulose: status and perspectives in Korea. Bioresour. Technol. 101 (13), 4801–4805. Kim, H.J., Chang, J.H., Jeong, B., Lee, J.H., 2013a. Comparison of milling modes as a pretreatment method for cellulosic biofuel production. J. Clean Energy Technol. 1 (1), 45–48. Kim, H.J., Lee, S., Kim, J., Mitchell, R.J., Lee, J.H., 2013b. Environmentally friendly pretreatment of plant biomass by planetary and attrition milling. Bioresour. Technol. 144, 50–56. Lee, S., Nam, D., Jung, J.Y., Oh, M.-K., Sang, B.-I., Mitchell, R.J., 2012. Identification of Escherichia coli biomarkers responsive to various lignin-hydrolysate compounds. Bioresour. Technol. 114, 450–456. Lee, S., Lee, J.H., Mitchell, R.J., 2015. Analysis of Clostridium beijerinckii NCIMB 8052’s transcriptional response to ferulic acid and its application to enhance the strain tolerance. Biotechnol. Biofuels 2015 (8), 68.
J.H. Kwon et al. / Bioresource Technology 199 (2016) 283–287 Li, J.B., Lin, J.H., Zhou, P.F., Wu, K.J., Liu, H.M., Xiong, C.J., Gong, Y.X., Xiao, W.J., Liu, Z. H., 2014. One-pot simultaneous saccharification and fermentation: a preliminary study of a novel configuration for cellulosic ethanol production. Bioresour. Technol. 161, 171–178. NREL (National Renewable Energy Laboratory), 2011. Process Design and Economics for Biochemical Conversion of Lignocellulosic Biomass to Ethanol. Technical Report NREL/TP-5100-47764. Pappas, I.A., Koukoura, Z., Tananaki, C., Goulas, C., 2014. Effect of dilute acid pretreatment severity on the bioconversion efficiency of Phalaris aquatica L. lignocellulosic biomass into fermentable sugars. Bioresour. Technol. 166, 395– 402.
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Sun, Y., Cheng, J., 2002. Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresour. Technol. 83 (1), 1–11. van Eijck, J., Batidzirai, B., Faaij, A., 2014. Current and future economic performance of first and second generation biofuels in developing countries. Appl. Energy 135, 115–141. Xiros, C., Katapodis, P., Christakopoulos, P., 2011. Factors affecting cellulose and hemicellulose hydrolysis of alkali treated brewers spent grain by Fusarium oxysporum enzyme extract. Bioresour. Technol. 102, 1688–1696.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Feasibility of a facile butanol bioproduction using planetary mill pretreatment Jeong Heo Kwon a,b,1, Hyunsoo Kang c,1, Byoung-In Sang b, Yunje Kim d, Jiho Min e, Robert J. Mitchell c,2, Jin Hyung Lee a,⇑,2 a
Korea Institute of Ceramic Engineering and Technology (KICET), 101, Soho-ro, Jinju-si, Gyeongsangnam-do 52851, Republic of Korea Division of Chemical Engineering & Bio Engineering, Hanyang University, Seoul, Republic of Korea School of Nano-Bioscience and Chemical Engineering, Ulsan National Institute of Science and Technology, Ulsan, Republic of Korea d Clean Energy Research Center, Korea Institute of Science and Technology, Seoul, Republic of Korea e Division of Chemical Engineering, Chonbuk National University, Jeonju, Jeonbuk, Republic of Korea b c
h i g h l i g h t s Convenient butanol production method proposed using planetary mill pretreatment. Planetary mill pretreatment eliminates need for biomass washing and buffer changes. Planetary milled Pinus rigida hydrolysates utilized directly without detoxication. Improved biobutanol yields with sugars from the hydrolysates than purified glucose.
a r t i c l e
i n f o
Article history: Received 30 June 2015 Received in revised form 14 August 2015 Accepted 17 August 2015 Available online 28 August 2015 Keywords: Planetary mill Pretreatment Enhanced bioconversion Biobutanol Biofuel
a b s t r a c t A facile butanol bioproduction process was developed using planetary milling, and Pinus rigida wood waste as a model substrate for fermentable sugars. The use of planetary milling as the pretreatment eliminates the need for washing and transfer of the biomass prior to enzymatic hydrolysis. Moreover, using this pretreatment process resulted in the production of only 0.072 ± 0.003 g/L soluble phenolic compounds, a concentration that was not inhibitory towards Clostridium beijerinckii NCIMB 8052. As the milling was performed in a compatible buffer (50 mM acetate, pH 4.8), the enzymatic hydrolysis step was initiated by simply adding the cellulase cocktail powder directly to pretreated biomass without washing the biomass or exchanging the buffer, resulting in a glucose yield of 31 g/L (84.02%). Fermentation of the hydrolysate samples by C. beijerinckii NCIMB 8052 gave slightly better butanol yields than cultures grown in a typical lab media (P2), with final concentrations of 6.91and 6.66 g/L, respectively. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Recently, the production of biofuels and their commercialization has garnered a significant amount of interest globally due to both fossil fuel depletion and the need to reduce net CO2 emissions (Kim et al., 2010). The use of biofuels is an answer to these problems since their use reduces global greenhouse gas (GHG) emissions while also improving both national energy security and rural development (van Eijck et al., 2014). As such, biofuels offer
⇑ Corresponding author. Tel.: +82 55 792 2752; fax: +82 55 792 2740. E-mail address: [email protected] (J.H. Lee). These authors contributed equally to this work.
1,2
http://dx.doi.org/10.1016/j.biortech.2015.08.074 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
a promising and potential substitute for petroleum usage, supporting the argument for the adequacy of biofuels as an alternative energy source (Fung et al., 2014). One of the more current trends in biofuel production is the use of lignocellulosic biomasses as they are readily available, generally have a low cost and are non-food feedstocks (Chandra et al., 2012). The production of biofuels from a lignocellulosic biomass requires several steps, including biomass pretreatment, hydrolysis of the cellulose and hemicellulose to sugar and the subsequent fermentation of this sugar (Balat, 2011). The complexity of the process required for lignocellulosic biofuels is mainly due to the recalcitrance of the biomass and, hence, the pretreatment step is key in determining the efficacy of the downstream steps. Typically, the pretreatment step involves an exposure of the biomass to some
284
J.H. Kwon et al. / Bioresource Technology 199 (2016) 283–287
harsh chemical, which may include a dilute acid, sodium hydroxide or ammonia. The use of such steps necessitates additional processes and steps, such as filtration of the treated solids, washing of the biomass with deionized water, pH neutralization and drying of the solids prior to the downstream enzymatic hydrolysis step (Kim et al., 2008; Li et al., 2014; Pappas et al., 2014). Moreover, these chemical-based pretreatments produce toxic byproducts that are known to undergo irreversible interactions with cellulose hydrolyzing enzymes, as well as inhibit the fermentative activity of the microorganisms. As such, the buffers used during the pretreatment step must be replaced and the biomass washed prior to the subsequent enzymatic hydrolysis and fermentation steps, both of which generate wastewater that must be treated. The capital costs associated with this treatment are significant, encompassing 21% of the total biofuel process cost (NREL, 2011). Though chemical pretreatments usually requires a lower energy consumption than physical pretreatments (Hendriks and Zeeman, 2009), they also have limitations that should be considered in industrial applications. To side-step this limitation, Li et al. (2014) recently reported a novel configuration for cellulosic ethanol production; a one-pot simultaneous saccharification and fermentation (One-pot SSF) that did not require rinsing and detoxification of the treated biomass. To achieve this, they used a combination of size reduction and alkali pretreatment in their system. However, the highest glucose concentration obtained was only 6.75 g/L, a value that is not feasible for commercial applications. Moreover, as their system used an alkali pretreatment, it is very likely that the inhibitors were also generated. To address both the costs associated with wastewater treatment and the generation of fermentative inhibitors, a facile butanol bioproduction process is proposed in this study using a planetary milling pretreatment step. The potential use of planetary milling processes for the pretreatment of lignocellulosic biomass and to improve fermentable sugar production was reported previously (Kim et al., 2013a,b). This process does not require any extraneous steps, such as rinsing of the biomass or changing of the buffer, since planetary milling neither uses chemicals nor does it produce significant quantities of enzyme/microbe inhibitors (Kim et al., 2013b). Furthermore, to demonstrate the feasibility of this facile butanol bioproduction system, pitch pine wood waste was planetary milled and hydrolyzed to fermentable sugars and this was successfully converted to butanol using Clostridium beijerinckii NCIMB 8052.
2. Methods 2.1. Materials Pitch pine (Pinus rigida) wood waste was kindly provided by Suncheon Forestry Cooperative, Republic Korea. Sugar standards (i.e., glucose, cellobiose, xylose, galactose and mannose), 3,5dinitrosalicylic acid, sodium acetate trihydrate and sodium citrate were all purchased from Sigma–Aldrich (USA). A cellulose cocktail was purchased from Worthington Biochemical Co. (USA).
2.2. Planetary mill pretreatment of the P. rigida biomass Initially, 7.2 g of pitch pine waste was added to a zirconia jar container, followed by 180 ml of 50 mM sodium acetate (pH 4.8). For milling, 3 mm diameter zirconia balls were added. Planetary milling was carried out using a Pulverisette 5 (Fritsch, Germany) at room temperature for 4 h. The jar was rotated at a velocity of 300 rpm.
2.3. Enzymatic hydrolysis of the biomass Before starting the enzyme hydrolysis, half of supernatant was removed to obtain a biomass solid loading of 8.0%. Powdered cellulase (Worthington Biochemical Co., USA) was added directly to the supernatant and biomass slurry to a final concentration of 15 FPU/ g-biomass. This mixture was then incubated at 50 °C for 96 h with agitation using a rotator (JEIO Tech, Rep. Korea) set to 200 rpm. The glucose yield after treatment was calculated using the following equation:
Glucose yield ð%Þ ¼
Glucose produced by enzyme hydrolysis Glucan in biomass 1:11
2.4. Fermentation by C. beijerinckii NCIMB 8052 A 1 ml suspension of C. beijerinckii NCIMB 8052 spores, which were stored at 4 °C in sterile distilled water, were germinated by heat shocking them at 75 °C for 10 min. They were then inoculated into 25 mL of anaerobic Reinforced Clostridial Medium (RCM; Difco Laboratories, USA) in a serum bottle and grown for 12 h at 37 °C and 250 rpm. Afterwards, an inoculum (5%) was transferred to 25 mL of fresh RCM medium and grown for 6 h to ensure that the culture was in the exponential growth phase. From this culture, 2.5 mL was inoculated into 50 mL of modified P2 medium as defined below and grown as previously described (Dwidar et al., 2012). The P2 medium used in this study was prepared by adding the following amounts of compounds into 50 mL of enzymatic hydrolysate: 1 g/L yeast extract, 2 g/L (NH4)2SO4, 1% P2 salt solution A, 1% P2 salt solution B, and 0.1% vitamin solution. For the control cultures, the same components were added to 50 ml of 50 mM sodium acetate containing the same concentration of glucose (Sigma, USA) as found in the biomass hydrolysate. All the media were prepared aseptically within a UV-sterilized laminar flow hood. The pH of the media was adjusted to pH 6.0 by adding 10 M NaOH. The media was converted to an anaerobic state by purging the fermentation broth with sterile filtered Ar gas. After 30 min of purging, the unit was sealed using sterilized caps and this was used directly for the fermentation, which was performed in a shaking incubator (Vision Science, ROK) set to 250 rpm and 37 °C. Each experiment was performed in triplicate. 2.5. Analytical methods The size of pretreated pitch pine wood waste was investigated using a Scanning Electron Microscope (JSM-6700F, JEOL, Tokyo, Japan). The crystallinity of the biomass was measured using Xray diffraction (XRD, Rigaku D/max-RB powder diffractometer, Tokyo, Japan) with Cu ja radiation (k = 1.542 Å). The crystallinity index was calculated using the follow equation:
Crystallinity Index ð%Þ ¼ ½ðI2h¼22:5 I2h¼18:7 Þ=ðI2h¼22:5 Þ 100 The phenolic content of the milled hydrolysate was measured using Folin–Ciocalteau colorimetry as described previously (Kim et al., 2013b). A standard GC (HP-6890, Agilent, USA), equipped with a time-of-flight mass spectrometer (TOFMS) and a column (HP-U2, 20 m 0.20 mm, 0.11 lm), was used to identify the phenolic compounds produced during the pretreatment steps. The ion source temperature was 220 °C and the resulting solution (2 lL) was injected in split mode (2:1) at an injector temperature of 250 °C and separated through an Ultra-2 capillary column (17 m 0.2 mm i.d., 0.11 lm film thickness; Agilent Technologies). The initial oven temperature was 60 °C for 1.00 min, ramp to 255 °C at 5 °C/min, 1 min hold. Helium (99.9999%) was used as the carrier gas (1.0 mL/min. constant flow at an oven temperature of 150 °C). The mass spectrometer was operated at 70 eV with
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selected ion monitoring (SIM) and mass range was 35–650 m/z. The sugar content was measured using an Agilent 1200 Series HPLC fitted with a refractive index detector (Agilent Technologies, USA). The column used was an Aminex HPX-87P column (BioRad, USA) and deionized water was used as the solvent. Likewise, the acetate, butanol, acetic acid and butyric acid concentrations were all determined using an Agilent 7890A FID-GC system (Agilent Technologies, USA) equipped with an Agilent HP-INNOWAX GC column and a flame ionization detector. The optical densities of the cultures were measured using a spectrophotometer (BioPhotometer, Eppendorf, Germany). 3. Results and discussion 3.1. Concept of the facile butanol bioproduction process using planetary mill pretreatment The conventional process involves a washing step, recycling of the catalyst and detoxification of the sample due to the chemical catalyst use. Together these contribute to both increasing the process complexity as well as the costs associated with the production of the biofuel. One example of an extraneous cost associated with conventional processes is the use of water that should be treated or recycled before it can be released into the environment. In contrast, the facile process proposed in this study does not require any buffer exchange or washing steps and, thus, significantly reduces the wastewater output and the associated costs required for its subsequent treatment (Fig. S1). Moreover, as the pretreatment, hydrolysis and fermentation steps are carried out using the same aqueous solution during the process, no biomass is lost. 3.2. Effects of planetary mill pretreatment on P. rigida Planetary mill treatment of plant biomass increases the surface area by reducing the size of the biomass to the level of fibers and fiber bundles, which ultimately increases the enzyme accessibility to the cellulose. This is illustrated in Fig. S2, where pitch pine (P. rigida) wood waste was significantly reduced in size after a planetary mill pretreatment. This treatment regime also changes the surface properties by disrupting the typically rigid surface and its intact phytoliths (Fig. S3). Before planetary milling, the crystallinity index (CI) of the pitch pine was 52.5 ± 0.9%, but this was reduced to 29.9 ± 3.7% after milling. This reduction indicates that planetary milling weakened the hydrogen bonding network within the crystalline cellulose while also potentially increasing access to the cellulose available for saccharification. One major benefit of planetary mill pretreatments is its disruption of the strong physical interactions within the biomass to reduce its recalcitrance to enzymatic and microbial hydrolysis while not changing the chemical composition of the biomass (Data not shown). However, as the soluble phenolic compounds formed from soluble hemicelluloses and lignin can inhibit microbial activities (Alvira et al., 2010; Lee et al., 2012, 2015), an analysis of the phenolic compounds produced during the pretreatment step was performed before conducting the downstream fermentative processes. Planetary milling of the pitch pine produced only 0.072 ± 3 g/L of soluble phenolic compounds. This concentration was smaller than found in previous study (Kim et al., 2013b), even though a higher solid-to-liquid ratio was used here. This difference is likely due to the properties of the biomass used, which were pitch pine wood waste in the current study and rice straw previously. Using GC/TOF-MS, the pretreatment products were identified (Fig. S4). A total of fourteen phenolic compounds such as benzaldehyde and 2-methoxyphenol, were identified (Table S1), with each existing at very low concentrations.
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3.3. Enzymatic hydrolysis of the planetary milled P. rigida After enzymatic treatment of the milled biomass with an enzyme load of 15 FPU/g-biomass, it was found that three sugars were primarily present in the hydrolysate, including cellobiose (6.5 ± 0.5 g/L), xylose (2.0 ± 0.9 g/L) and glucose (31.0 ± 0.2 g/L). No galactose, arabinose or mannose was detected in any of the samples because the cellulase selected for this study lacks hemicellulase activity. However, the hemicellulose is an important source of pentoses that can also be utilized by fermentative microbes and improve the solvent yields. As such, the use of another enzyme preparation, such as Fusarium oxysporum enzyme extract, could be employed to hydrolyze both cellulose and hemicellulose for industry applications (Xiros et al., 2011). As the P. rigida wood waste used in this study consists of 41.5% cellulose, the glucose yield is 84.02%. Attempts to enhance the glucose concentration by increasing the amount of biomass treated in each batch were unsuccessful and the highest glucose concentration achieved did not significantly exceed 31.0 g/L (Data not shown). As reported previously, this limitation likely stems from both end-product accumulation and inhibition of the cellulase activity, as well as a low free-water availability (Hsieh et al., 2014; Jorgensen et al., 2007). Several studies stated that these limitations can be overcome by enhancing the cellulase activity (Sun and Cheng, 2002) or by employing a simultaneous saccharification and fermentation (SSF) process, ideas which will be considered in a future study. Moreover, the residues remaining after hydrolysis of the sugars would be enriched for lignin and, as such, could be used for other biochemical processes and productions.
3.4. Evaluation of fermentable sugar produced from planetary milled P. rigida Since the addition of sodium acetate to fermentation broths is beneficial as it increases both the solvent production and glucose utilization by C. beijerinckii NCIMB 8052 (Chen and Blaschek, 1999), 50 mM sodium acetate was selected as the buffering
Fig. 1. Time course characteristics of the cultures grown either on (A) glucose or (B) the pretreated P. rigida hydrolysate, showing the optical densities, glucose consumption and butanol concentrations. Each of the cultures initially had 31 g/L glucose within them either from its addition (A) or from the hydrolysate (B).
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Table 1 Comparison of the solvent and acid production levels in cultures grown for 72 h with either the milled hydrolysate prepared according to this study or glucose (control) added.
Milled hydrolysate Control *
Butanol yield =
Acetone (g/L)
Butanol (g/L)
Acetate (g/L)
Butyrate (g/L)
Butanol yield* (g/g)
3.42 ± 0.11 3.05 ± 0.09
6.91 ± 0.64 6.66 ± 0.21
1.49 ± 0.10 1.09 ± 0.18
1.81 ± 0.35 1.02 ± 0.02
0.25 ± 0.01 0.23 ± 0.02
Weight of butanol produced . Weight of glucose used
solution for the enzymatic hydrolysis. An additional benefit of this buffer is that it can be used for the fermentation steps after simply adjusting the pH and adding the essential vitamins and nutrients. However, based upon the above findings where 31 g/L of glucose was generated from the biomass, no further glucose addition is necessary. Consequently, the efficacy of using these hydrolysate samples directly for fermentative processes was next tested within C. beijerinckii NCIMB 8052 cultures. For this, only the remaining biomass was removed and so the bacterium was exposed to all of the phenolics and sugars produced during the pretreatment stages. Fermentations performed with these hydrolysate samples show a delayed cell growth and an overall lower biomass when compared to the control cultures (Fig. 1). The maximum average cell density, i.e., the optical density at 600 nm (OD600), for the biomass hydrolysate cultures was 5.29 while that of the control was 8.12, both after 48 h. It is thought that this longer lag period and lowered overall bacterial density is due to impurities present in the milled hydrolysate that cause C. beijerinckii NCIMB 8052 stress, and may even be due to the compounds listed in Table S1. For instance, ferulic acid, which has a structure similar with one of the compounds identified, namely, 4-hydroxy-3-methoxy benzeneacetic acid, is known to cause an analogous loss in the bacterial growth (Lee et al., 2015). Besides the cell densities, the only other clear difference between the two cultures was the increased solvent and acid production by the biomass hydrolysate cultures (Table 1). This was particularly evident with the acids, which were produced at about 1.4 to 1.8-fold higher level. The acetone production was also enhanced by about 10% while butanol increased only slightly over the control. However, the butanol/acetone ratio in the biomass hydrolysate was slightly smaller than that seen with the control culture; 2.02 and 2.18 g/g, respectively (Table 1). Considering all of these, the hydrolysate culture produced more than 1.8 g/L more products than the control cultures, which were grown on typical lab media containing glucose. It would also seem that the loss in microbial biomass corresponds with the increased production, a connection that will be studied further. However, the above results clearly demonstrate that the milled hydrolysate sample can be used for fermentations and actually leads to slightly better yields than conventional lab P2 media. This study illustrated the feasibility of a facile butanol bioproduction through the use of a planetary mill pretreatment. Such a facile butanol bioproduction has several advantages. Firstly, there is no need for washing and buffer exchange, which would be beneficial both in terms of saving time and labor and, most importantly, avoiding the loss of biomass and the generation of wastewater. Moreover, only one-vessel is required for the entire process, reducing the need for downstream sample transfer, which is also an important factor for industrial feasibility.
4. Conclusions This study demonstrates that a facile butanol bioproduction is possible when using planetary milling. Fermentations performed using P. rigida biomass treated using this facile process showed slightly improved solvent and acid yields when compared to
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