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Fermentation with continuous ball milling: Effectiveness at enhancing solubilization for several cellulosic feedstocks and comparative tolerance of several microorganisms
Biomass and Bioenergy 134 (2020) 105468
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Research paper
Fermentation with continuous ball milling: Effectiveness at enhancing solubilization for several cellulosic feedstocks and comparative tolerance of several microorganisms Michael L. Balch, Meghan B. Chamberlain, Robert S. Worthen, Evert K. Holwerda *, Lee R. Lynd Thayer School of Engineering, Dartmouth College, Hanover NH.14 Engineering Drive, Hanover, NH, 03755, United States
A R T I C L E I N F O
A B S T R A C T
Keywords: Cotreatment Lignocellulose Fermentation Biofuel Milling
Mechanical disruption of lignocellulose during fermentation, cotreatment, is a nascent approach to increase biologically-mediated carbohydrate solubilization without exposure to high temperatures or chemicals. How ever, ability to tolerate the presence of milling at intensities sufficient to allow high lignocellulose solubilization yields has to date only been evaluated for Clostridium thermocellum and Saccharomyces cerevisiae, and demon stration of high carbohydrate solubilization has only been reported for switchgrass at the time of submission. Continuous ball milling during fermentation by C. thermocellum was found to be sufficient to allow high (>85%) total carbohydrate solubilization of corn stover and poplar as well as switchgrass. Under the conditions tested, ball milling had no apparent effect on soluble sugar fermentation by Escherichia coli and Thermoanaerobacterium saccharolyticum but completely inhibited carbohydrate fermentation by Zymomonas mobilis and Bacillus subtilis. Our results are consistent with the stress of milling being sufficient to overwhelm the inefficient fermentative metabolism of Z. mobilis and B. subtilis, but not to the other microorganisms tested in this work for which fermentation is more efficient and robust.
1. Introduction Cellulosic biofuels will likely be needed to achieve a low-carbon transportation sector [1,2]. In light of the high cost and operational complexity of lignocellulose processing based on thermochemical treatment and fungal cellulase, there is incentive to explore alternatives to this paradigm. One such alternative is to carry out consolidated bio processing (CBP) by thermophilic, saccharolytic microorganisms in conjunction with milling during fermentation, termed cotreatment [3, 4]. CBP combined with cotreatment is referred to herein as C-CBP. Milling as a pretreatment prior to hydrolysis using fungal cellulase has been investigated in several studies [5–7] but energy requirements are widely thought to be too high to be practical [8–10]. However, the physical properties of cellulosic biomass slurries undergo large changes during biologically-mediated solubilization. For example, Ghosh et al. [11] recently observed a 2000-fold reduction in viscosity during fermentation of corn stover by C. thermocellum. Thus it is plausible that energy requirements for milling of partially-fermented material in a cotreatment configuration may be sufficiently low to allow practical application whereas this is not the case for milling as a pretreatment.
Paye et al. [4] compared sequential fermentation of senescent switchgrass by Clostridium thermocellum, a cellulolytic Gram-positive thermophilic bacterium, with ball milling either before fermentation or between the first and the second successive fermentation. Milling partially fermented feedstock was found to result in greater enhance ment of carbohydrate solubilization and particle size reduction as compared to milling prior to fermentation. Upon cultivating C. thermocellum on senescent switchgrass with continuous ball milling, Balch et al. [3] found that 88% of total carbohydrate was solubilized without thermochemical pretreatment or added enzymes. In addition, it was observed that continuous ball milling had little effect on cellobiose fermentation by C. thermocellum but completely halted glucose fermentation by Saccharomyces cerevisiae. Cotreatment is a nascent technology and there are still many important questions to answer including mechanism, energy, and techno-economics. However it is also important to answer foundational questions such as efficacy. Early studies on pretreatment similarly took the approach of demonstrating effect first, later followed by more complex studies which included techno-economic and efficiency considerations.
* Corresponding author. E-mail address: [email protected] (E.K. Holwerda). https://doi.org/10.1016/j.biombioe.2020.105468 Received 18 January 2019; Received in revised form 25 September 2019; Accepted 13 January 2020 Available online 30 January 2020 0961-9534/© 2020 Elsevier Ltd. All rights reserved.
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(JW/SL-YS 485) was cultured on CTFüD with 5 g L 1 cellobiose [35]. Inocula were grown in 125 mL sealed anaerobic culture bottles and incubated for 24 h (at 60 � C for C. thermocellum and T. saccharolyticum and at 37 � C for E. coli, Z. mobilis, and B. subtilis). Inoculum was trans ferred to bioreactors via syringe. The inoculum volume was chosen so that vigorous fermentations ensued within 24 h for unmilled controls: 5% of working volume for lignocellulosic runs with C. thermocellum, 5% for T. saccharolyticum and Z. mobilis, 2.5% for B. subtilis, and 0.2% for E. coli.
For the concept of cotreatment to be a successful approach of reducing lignocellulose recalcitrance, it is important to demonstrate its compatibility with an array of lignocellulosic substrates and biocatalysts (microorganisms). Here we broaden the range of feedstocks and mi croorganisms tested for compatibility with cotreatment using the ball mill bioreactor developed by Balch et al. [3]. Some forms of pretreat ment have been shown to be most effective on a select number of lignocellulosic feedstocks [12]. We believe cotreatment will not be similarly limited, but to date, high carbohydrate solubilization with cotreatment has only been reported for switchgrass. In this study, we tested the effectiveness of cotreatment at enhancing solubilization by C. thermocellum for corn stover, poplar, and late-season harvested switchgrass. These are all of interest as bioenergy feedstocks [13–15], and represent a range of recalcitrance to biological attack in the absence of pretreatment or cotreatment. Mechanical treatments, particularly bead milling, are widely used to disrupt cells [16,17]. It is therefore important to demonstrate the ca pacity of microorganisms to carry out anaerobic fermentation of soluble substrates in the presence of continuous ball milling at an intensity sufficient to enable high total carbohydrate solubilization. Thus far, the impact of cotreatment on microorganisms has only been demonstrated for C. thermocellum and S. cerevisiae. The four microorganisms evaluated in this study - Escherichia coli, Zymomonas mobilis, Bacillus subtilis, and Thermoanaerobacterium saccharolyticum represent a range of physiol ogies and taxonomies and have all been proposed for use in production of cellulosic biofuels (Table 1).
2.2. Bioreactor configuration
2. Materials and methods
All fermentations were carried out anaerobically in custom-made stainless steel bioreactors equipped for continuous ball milling as described by Balch et al. [3]. Anaerobic conditions were ensured by purging the headspace of the reactor overnight with either 100% N2 gas or a 80% N2 – 20% CO2 gas mixture prior to inoculation (as indicated), by the presence of the reducing agent L-cysteine in the media for C. thermocellum and T. saccharolyticum, and by the use of norprene tubing and purged media solutions. For fermentations with milling the bioreactors were filled with 5 mm diameter stainless steel balls numbering a total of around 10,000. The volume of balls was about 600 mL and the volume of cultivation medium was 600 mL making the total final working volume 1200 mL. The height of the bead bed was 80% of the height of the final total working volume. For fermentations without milling, no balls were added and the volume of cultivation medium was doubled such that the total final working volume was constant at 1200 mL.
2.1. Strains, growth medium, and inoculum preparation
2.3. Fermentation of lignocellulosic feedstocks by C. thermocellum
All strains were obtained from Deutsche Sammlung von Mikroorga nismen and Zellkulturen (DSMZ, Braunschweig, Germany) and cultured using growth media shown in previous studies to effectively support the cultivation of each microorganism. Clostridium thermocellum (Rumini clostridium thermocellum) DSM 1313 was cultured in LC medium [31] on 5 g L 1 Avicel PH105 (FMC biopolymers, Philadelphia PA) with 5 g L 1 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (Sigma; St. Louis, MO). Escherichia coli DSM 18039 was cultured in LB medium [32] with 10 g L 1 glucose. Zymomonas mobilis DSM 424 was cultured in DSM medium with 20 g L 1 glucose [33]. Bacillus subtilis DSM 10 was cultured in LB medium [32] with 10 g L 1 glucose and 50 mM NaNO3, which acts as an electron acceptor [34]. Thermoanaerobacterium saccharolyticum
Switchgrass (Panicum virgatum L.), Alamo variety, was harvested in October at the University of Tennessee Knoxville and was kindly pro vided by the Stewart Lab. Corn stover, ground to pass through a 5-mm screen, was received as a gift from the US Department of Energy Great Lakes Bioenergy Research Center and a description of which can be found in Sekhon et al., [36]. Poplar (Populus trichocarpa), was grown at the Oak Ridge National Laboratory, debarked, and milled through a 1 mm screen as described by Trajano et al., [37]. Switchgrass, corn stover, and poplar were pre-rinsed as described in Garlock et al. [38], and then milled with a Retsch mill (Haan, Germany) through a 2-mm screen. For all lignocellulose feedstocks the substrate loading was 5 g L 1 glucan equivalent (14.8 g L 1 dry material for switchgrass, 17.0 g L 1 for corn stover, and 10.0 g L 1 for poplar). The cotreatment bioreactor was set up by layering beads and feed stock in the middle portion of the bed such that all feedstock was exposed to milling. Once loaded the bioreactor was autoclaved at 121 � C for 1 h and then incubated at 30 � C overnight while purging the head space with 30 mL min 1 of 20% CO2 - 80%N2 gas mix (Airgas; White River Junction, VT). Components of LC medium were added via syringe as five separate solutions as described in Holwerda et al. [31]. For corn stover fermentations and milling runs the media was supplemented with 5 g L 1 MOPS buffer. Ball milling was initiated at 100 rpm immediately prior to inoculation except in the case of corn stover, where the culture was grown for 24 h without milling to allow it to initiate growth (and total incubation time was 6 days instead of 5 days). Base addition and gas production were interpreted as signs of metabolic activity and growth. Gas production was measured by using Milligas tip meters (Ritter, Hawthorne, NY). The bioreactors were inoculated with a 5% v/v inoculum, and stirred at 100 rpm independent of the presence of beads. Lignocellulose fermentations were run for 5–7 days (120–168 h) depending on feedstock (see Fig. 1). The pH was maintained at 7.0 [31] with 2 N KOH and 2 N HCl solutions and the temperature was controlled at 60.0 � C [31] by a Sartorius Aplus control tower (Sartorius Stedim, Bohemia NY). 2 mL samples were collected every 24 h for analysis by HPLC (Waters; Milford, MA) on an Aminex HPX-87H column (Bio-Rad,
Table 1 Overview of the microorganisms used in the fermentation of soluble substrates during milling experiments. Microorganism
Selected features
Studies proposing use for cellulosic biofuel production
Escherichia coli
Gram-negative, mesophilic facultative anaerobe, mixedacid fermentation of hexose and pentose sugars [18] Gram-negative, mesophilic obligate anaerobe, homoethanologenic fermentation of glucose [21]
Atsumi and Liao, 2008 [19] Ingram and Doran, 1995 [20] Davis et al., 2005 [22] Rogers et al., 1982 [21] Rogers 1997 [23] Cho et al., 2004 [26] Zhang and Zhang, 2010 [27] Herring et al., 2016 [29] Shaw et al., 2008 [30]
Zymomonas mobilis
Bacillus subtilis
Thermoanaerobacterium saccharolyticum
Gram positive, mesophilic facultative anaerobe [24], mixed acid-butanediol fermentation [25] Gram positive, thermophilic obligate anaerobe, mixed acid fermentation of hemicellulose and derivatives [28]
2
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Fig. 1. Fermentation results for Lignocellulosic substrates: Corn Stover (A,D,G), Switchgrass (B,E,H), and Poplar (C,F,I). Figures show Solubilization (A–C), Gas production (D–F), and Product formation (G–I), with cotreatment (red) and without cotreatment (blue). Values represent the average of duplicate fermentations; error bars represent one standard deviation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 2. Substrate consumption and gas production (red and blue respectively, A-D), and major product formation (green and blue respectively, E-H) for E. coli (A,E), Z. mobilis (B,F), B. subtilis (C,G), and T. saccharolyticum (D,H) with milling (solid lines and closed symbols) and without milling (dashed lines and open symbols). Arrows in B and C indicate times at which reinoculation occurred. Values represent the average of duplicate fermentations; error bars represent one standard de viation. Gas production curves show replicate fermentations. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 3
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Hercules, CA) operated at 50 � C with a 5 mM H2SO4 eluent. All fer mentations were performed in duplicate.
� 5.3% for switchgrass, and 87.8 � 3.3% for poplar. Total gas produc tion with and without cotreatment was highest for corn stover, inter mediate for switchgrass, and lowest for poplar (Fig. 1D 1E, and 1F). Increased gas production was observed with cotreatment relative to without cotreatment, with the magnitude of the increase roughly pro portional to the increase in solubilization. Gas production curves indi cate that fermentation was initiated at about the same time with and without cotreatment for switchgrass and corn stover, but was somewhat delayed for poplar with cotreatment compared to the control without cotreatment. Under all conditions tested, acetate and ethanol were the main fermentation products produced by the wild-type strain of C. thermocellum used in this study. Production of acetate and ethanol was substantially higher with cotreatment than without it for switchgrass and poplar, but not for corn stover for which the difference in solubili zation was not as large. Solubilization data reported here for switchgrass and Populus in the absence of pretreatment or cotreatment are similar to those we have reported previously using C. thermocellum [40], and higher than observed with commercial fungal cellulase [4,40]. Corn stover solubilization by C. thermocellum in the absence of cotreatment or pretreatment is notably higher than the other feedstocks tested as has been reported previously [41]. The extent of solubilization we observed for C. thermocellum in the presence of cotreatment, on the order of 90% of theoretical, is comparable to that observed for thermochemical pre treatment [3,40,42,43].
2.4. Soluble substrate fermentations When testing fermentation of soluble substrate in presence of mill ing, the stainless steel balls were added to the reactor all at once. Growth media components were added to bioreactors prior to autoclaving with the exception of sugars and NaNO3 for B. subtilis. The bioreactors were then autoclaved at 121 � C for 1 h, after which filter-sterilized sugars (glucose or cellobiose), and NaNO3 for B. subtilis, were added. The headspace of bioreactors containing sterilized medium were purged overnight with 30 mL min 1 of filter-sterilized N2 gas (Airgas; White River Junction, VT). Agitation at 100 rpm was initiated prior to inocu lation and continued throughout the duration of the experiment. Fermentations were run for 24 h when metabolic activity, as man ifested by base addition or gas production, was observed, and up to 96 h when metabolic activity was not observed (i.e. a lack of base addition or gas production as measured by gas tip meter). For runs in which fermentation was not initiated, the bioreactors were re-inoculated with a 5% v/v inoculum at time-points indicated in Fig. 2 pH was maintained at 7.0 for E. coli [32] and B. subtilis [34], and 6.0 for Z. mobilis [33] and T. saccharolyticum [35] by addition of 4 N NaOH solutions. Temperature was maintained at 37 � C for E. coli [32], B. subtilis [34], and Z. mobilis [33] and 60 � C for T. saccharolyticum [35]. Fermentation control was performed by Sartorius Aplus control tower. 2 mL samples were collected every 2 h for E. coli, B. subtilis, and T. saccharolyticum, and every 3 h for Z. mobilis, for analysis by HPLC. All fermentations were performed in duplicate.
3.2. Tolerance of various microorganisms to milling Following results in prior work that C. thermocellum readily ferments soluble substrates in the presence of milling whereas S. cerevisiae does not [3], we evaluated the ability of 4 additional microorganisms to ferment soluble substrates appropriate for each organism in the presence of continuous ball milling: E. coli, Z. mobilis, B. subtilis, and T. saccharolyticum. For E. coli (Fig. 2A and E) and T. saccharolyticum (Fig. 2D and H), fermentation proceeded with undiminished rates of substrate con sumption and production of gas and organic fermentation products compared to controls without milling. For Z. mobilis (Fig. 2B and F) and B. subtilis (Fig. 2C and G), there was no evidence of metabolic activity in the presence of milling even after reinoculation although fermentation in controls without milling was readily apparent. Release of gas from liquid medium was lower for the two cultures grown at higher pH, E. coli and B. subtilis. This could be caused by decreased release of CO2 from the culture because of enhanced ionization of CO2 at higher pH [44]. Organic fermentation products in addition to ethanol and acetate were likely formed, particularly for E. coli and B. subtilis, but were not measured since the intended purpose here was to document metabolic activity. The production of fine particulate matter during milling resulted in an increasingly opaque broth in milled fermentations as compared to unmilled fermentations. Because of this it was not possible to accurately measure cell growth by either optical density or dry weight.
2.5. Quantification of fermentation products Samples were centrifuged at 15,000 rpm (21,130�g) for 5 min, and the supernatant analyzed for acetate and ethanol (and glucose or cellobiose for soluble substrate tests) via HPLC. Cumulative gas pro duction was measured via Milligascounter gas tip meters filled with a solution of 0.5 N HCl. 2.6. Calculation of total carbohydrate solubilization The percent total carbohydrate solubilization, TCS, is calculated using equation [1]. ��� � � GS;f þ XS;f þ AS;f TCS ¼ 100%⋅ 1 (1) ðGS;i þ XS;i þ AS;i Þ where G denotes glucan or glucose (mMoles), X denotes xylan or xylose (mMoles), A denotes arabinan or arabinose (mMoles), S denotes solid, f denotes final, and i denotes initial. Initial and final concentrations were determined by multiplying the solids concentration (g L 1 total solids) by the mass fraction of the sugar (e.g. g glucose per g total solid) obtained from quantitative saccharifi cation [39] and dividing by molecular weight.
4. Discussion
3. Results
Recognizing that cotreatment is a new concept with relatively little prior work, the first order of business is to explore the conditions under which it is effective and/or implementable – as was the case for the lignocellulose pretreatment field a quarter century ago. Here we sub stantially extend such exploration with respect to feedstocks and microorganisms. In the presence of cotreatment, but without thermochemical pre treatment other than autoclaving and without added enzymes, final carbohydrate solubilization levels for corn stover senescent switchgrass and poplar are similar to those seen with conventional thermochemical pretreatment [45]. Our switchgrass results are consistent with previous tests on grasses [3,40,46], which show cotreatment roughly doubling
3.1. Solubilization and fermentation of different lignocellulosic feedstocks Fermentation of corn stover, late-season switchgrass, and poplar was carried out by C. thermocellum in the presence and absence of cotreat ment via continuous ball milling. As presented in panels A, B, and C of Fig. 1, total carbohydrate solubilization (TCS) in the absence of cotreatment differed substantially, with 66.0 � 5.8% observed for corn stover, 45.7 � 3.2% for switchgrass, and 17.6 � 2.9% for poplar. Cotreatment substantially increased solubilization for all three feed stocks to rather similar TCS values of 88.3 � 4.7% for corn stover, 87.0 4
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solubilization. Solubilization nearly quadruples for poplar as a result of cotreatment, as also observed by Holwerda et al. [40]. For both C. thermocellum and long-term cultivated cellulolytic mixed enrichments (microbiomes), a fraction of the carbohydrate in lignocel lulosic feedstocks is inaccessible to biological attack even over extensive time periods [47,48]. Clearly, milling decreases this fraction for the three feedstocks examined here. Since the physical impacts of milling are more apparent than chemical impacts, although these are possible [49], it is likely that the primary mode of action of milling is to increase physical accessibility for enzymatic attack. Further elaboration of the mechanistic basis for the effectiveness of cotreatment awaits future study. Final ethanol concentrations with cotreatment are fairly consistent across substrates, between about 0.6 and 0.9 g L 1. Additionally, the ratio of acetate and ethanol produced on poplar is approximately 3:2, which is consistent with results for C. thermocellum on nonlignocellulosic substrates such as Avicel [50]. However, on corn stover and switchgrass, much more acetate is produced both with and without cotreatment. Furthermore, the hexose carbon recovery (80%, see Table S1 in SOM) for poplar is typical for C. thermocellum when not accounting for cells and/or amino acids [50], while the hexose carbon recovery is much higher for corn stover and switchgrass (exceeding 100% for the latter). This is consistent with acetate release from biomass during solubilization. While we note that woody feedstocks tend to have higher acetyl content than grassy feedstocks [51], it may be that the acetyl groups are more readily released from the less recalcitrant grassy feedstocks. Of the six microorganisms evaluated here and by Balch et al. [3] half are tolerant to milling under the conditions tested and half are not. Such tolerance appears to be an ‘all-or-nothing’ phenomenon under the conditions tested, with either little to no effect or complete inhibition of metabolic activity. Among the Gram-positive microorganisms tested here and by Balch et al. [3], T. saccharolyticum and C. thermocellum ferment readily in the presence of milling whereas B. subtilis does not. Among Gram-negative microorganisms, E. coli is tolerant to milling whereas Z. mobilis is not. Milling-tolerant microorganisms include both thermophiles (T. saccharolyticum, C. thermocellum) and mesophiles (E. coli). It is perhaps not surprising that we previously found S. cerevisiae to be sensitive to milling [3] in light of its large size and the absence of a thick cell wall. However, we find here that microorganisms intolerant to milling under the conditions tested include prokaryotes (Z. mobilis and B. subtilis). Additionally, the four organisms tested here are of similar size and shape, so it is not clear that size is a primary factor in sensitivity to milling. Zymomonas mobilis utilizes the Entner-Doudoroff pathway for con version of glucose to pyruvate [21] and thus has half the ATP available per mole glucose compared to micro organisms using the Embden-Meyerhof-Parnas pathway (E. coli [52], T. saccharolyticum [30], or C. thermocellum [41]). B. subtilis was long thought to be an obligate aerobe, and has an inefficient glucose fermentation pathway [24]. We speculate that the stress of milling may be sufficient to overwhelm the inefficient fermentative metabolism of Z. mobilis and B. subtilis, but not the more efficient and robust fermentation of other microorganisms tested in this work.
cotreatment and to evaluate it under industrial conditions, including different milling modalities, energy requirements, and technoecomomic analysis. Declaration of competing interest Lee R. Lynd holds equity in a start-up company that aims to commercialize cellulosic biomass. None of the other authors has a competing interest. Acknowledgements Support for this work was provided by the USDA National Institute for Food and Agriculture (NIFA) under the Biomass Research and Development Initiative (BRDI) [Grant 2016-10008-2531]. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.biombioe.2020.105468. References [1] L.M. Fulton, L.R. Lynd, A. K€ orner, N. Green, L.R. Tonachel, The need for biofuels as part of a low carbon energy future, Biofuels, Bioprod. Biorefining. 9 (2015) 476–483, https://doi.org/10.1002/bbb.1559. [2] OECD/IEA, Fao, How2Guide for Bioenergy Roadmap Development and Implementation, 78, IEA Publ., 2017, ISBN 978-92-5-109586-7. [3] M.L. Balch, E.K. Holwerda, M.F. Davis, R.W. Sykes, R.M. Happs, R. Kumar, E. Wyman, L.R. Lynd, Lignocellulose fermentation and residual solids characterization for senescent switchgrass fermentation by Clostridium thermocellum, Energy Environ. Sci. 10 (2017) 1252–1261, https://doi.org/ 10.1039/C6EE03748H. [4] J.M.D. Paye, A. Guseva, S.K. Hammer, E. Gjersing, M.F. Davis, B.H. Davison, J. Olstad, B.S. Donohoe, T.Y. Nguyen, C.E. Wyman, S. Pattathil, M.G. Hahn, L. R. Lynd, Biological lignocellulose solubilization: comparative evaluation of biocatalysts and enhancement via cotreatment, Biotechnol. Biofuels (2016) 1–13, https://doi.org/10.1186/s13068-015-0412-y. [5] U. Mais, A.R. Esteghlalian, J.N. Saddler, S.D. Mansfield, Enhancing the enzymatic hydrolysis of cellulosic materials using simultaneous ball milling, Appl. Biochem. Biotechnol. 98–100 (2002) 815–832, https://doi.org/10.1385/ABAB:98-100:1-9: 815. [6] M.J. Neilson, R.G. Kelsey, F. Shafizadeh, Enhancement of enzymatic hydrolysis by simultaneous attrition of cellulosic substrates, Biotechnol. Bioeng. 24 (1982) 293–304, https://doi.org/10.1002/bit.260240204. [7] S.K. Ryu, J.M. Lee, Bioconversion of waste cellulose by using an attrition bioreactor, Biotechnol. Bioeng. 25 (1983) 53–65, https://doi.org/10.1002/ bit.260250106. [8] A. Barakat, S. Chuetor, F. Monlau, A. Solhy, X. Rouau, Eco-friendly dry chemomechanical pretreatments of lignocellulosic biomass: impact on energy and yield of the enzymatic hydrolysis, Appl. Energy 113 (2014) 97–105, https://doi.org/ 10.1016/j.apenergy.2013.07.015. [9] A. Hideno, H. Inoue, T. Yanagida, K. Tsukahara, T. Endo, S. Sawayama, Combination of hot compressed water treatment and wet disk milling for high sugar recovery yield in enzymatic hydrolysis of rice straw, Bioresour. Technol. 104 (2012) 743–748, https://doi.org/10.1016/j.biortech.2011.11.014. [10] M.R. Zakaria, M.N.F. Norrrahim, S. Hirata, M.A. Hassan, Hydrothermal and wet disk milling pretreatment for high conversion of biosugars from oil palm mesocarp fiber, Bioresour. Technol. 181 (2015) 263–269, https://doi.org/10.1016/j. biortech.2015.01.072. [11] S. Ghosh, E.K. Holwerda, R.S. Worthen, L.R. Lynd, B.P. Epps, Rheological properties of corn stover slurries during fermentation by Clostridium thermocellum, Biotechnol. Biofuels 11 (2018) 246, https://doi.org/10.1186/ s13068-018-1248-z. [12] B. Yang, C.E. Wyman, Pretreatment: the key to unlocking low-cost cellulosic ethanol, Biofuels, Bioprod. Biorefining. (2008) 26–40, https://doi.org/10.1002/ bbb. [13] S. Kim, X. Zhangm, B. Dale, A.D. Reddy, C.D. Jones, K. Cronin, R.C. Izaurralde, T. Runge, M. Sharara, Corn stover cannot simultaneously meet both the volume and GHG reduction requirements of the renewable fuel standard, Biofuels, Bioprod. Biorefining. 12 (2018) 203–212, https://doi.org/10.1002/bbb. [14] L.R. Lynd, E. Larson, N. Greene, M. Laser, J. Sheehan, B.E. Dale, S. McLaughlin, M. Wang, The role of biomass in America’s energy future: framing the analysis, Biofuels, Bioprod. Biorefining. 3 (2009) 113–123, https://doi.org/10.1002/bbb. [15] P. Sannigrahi, A.J. Ragauskas, G.A. Tuskan, Poplar as a feedstock for biofuels: a review of compositional characteristics, Biofuels, Bioprod. Biorefining. 4 (2010) 209–226, https://doi.org/10.1002/bbb.
5. Conclusion We show that cotreatment implemented in a ball mill bioreactor under the condition tested enables near-complete solubilization of several prominent potential lignocellulosic feedstocks for cellulosic biofuel production. Additionally, we find that some organisms are able to continue fermentation in the presence of ball milling while others are not, but are unable to strongly relate that to physiological features. Our work establishes that cotreatment enables high total carbohydrate sol ubilization to be achieved for a woody feedstock as well as herbaceous feedstocks. More work is needed to understand the mechanistic basis of 5
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Biomass and Bioenergy 134 (2020) 105468 [36] R.S. Sekhon, H. Lin, K.L. Childs, C.N. Hansey, C.R. Buell, N. de Leon, S.M. Kaeppler, Genome-wide atlas of transcription during maize development, Plant J. 66 (2011) 553–563, https://doi.org/10.1111/j.1365-313X.2011.04527.x. [37] H.L. Trajano, N.L. Engle, M. Foston, A.J. Ragauskas, T.J. Tschaplinski, C. E. Wyman, The fate of lignin during hydrothermal pretreatment, Biotechnol. Biofuels 6 (2013) 1, https://doi.org/10.1186/1754-6834-6-110. [38] R.J. Garlock, V. Balan, B.E. Dale, V. Ramesh Pallapolu, Y.Y. Lee, Y. Kim, N. S. Mosier, M.R. Ladisch, M.T. Holtzapple, M. Falls, R. Sierra-Ramirez, J. Shi, M. A. Ebrik, T. Redmond, B. Yang, C.E. Wyman, B.S. Donohoe, T.B. Vinzant, R. T. Elander, B. Hames, S. Thomas, R.E. Warner, Comparative material balances around pretreatment technologies for the conversion of switchgrass to soluble sugars, Bioresour. Technol. 102 (2011) 11063–11071, https://doi.org/10.1016/j. biortech.2011.04.002. [39] A. Sluiter, B. Hames, R. Ruiz, C. Scarlata, J. Sluiter, D. Templeton, D.C. Nrel, Determination of Structural Carbohydrates and Lignin in Biomass Determination of Structural Carbohydrates and Lignin in Biomass, 2012, p. 2011. [40] E.K. Holwerda, R.S. Worthen, N. Kothari, R.C. Lasky, B.H. Davison, C. Fu, Z. Y. Wang, R.A. Dixon, A.K. Biswal, D. Mohnen, R.S. Nelson, H.L. Baxter, M. Mazarei, W. Muchero, G.A. Tuskan, C.M. Cai, E.E. Gjersing, M.F. Davis, M.E. Himmel, C. E. Wyman, P. Gilna, L.R. Lynd, Multiple levers for overcoming the recalcitrance of lignocellulosic biomass, Biotechnol. Biofuels 12 (2019) 1–12, https://doi.org/ 10.1186/s13068-019-1353-7. [41] L.R. Lynd, A.M. Guss, M.E. Himmel, D. Beri, C. Herring, E.K. Holwerda, Advances in consolidated bioprocessing using Clostridium thermocellum and thermoanaerobacter saccharolyticum, in: C. Wittman, J. Liao (Eds.), Ind. Biotechnol. Microorg, Wiley, 2016, pp. 365–394. [42] N. Kothari, E.K. Holwerda, C.M. Cai, R. Kumar, C.E. Wyman, Biomass augmentation through thermochemical pretreatments greatly enhances digestion of switchgrass by Clostridium thermocellum, Biotechnol. Biofuels 11 (2018) 1–14, https://doi.org/10.1186/s13068-018-1216-7. [43] V.A. Thomas, N. Kothari, S. Bhagia, H. Akinosho, M. Li, Y. Pu, C.G. Yoo, S. Pattathil, M.G. Hahn, A.J. Raguaskas, C.E. Wyman, Comparative evaluation of Populus variants total sugar release and structural features following pretreatment and digestion by two distinct biological systems, Biotechnol. Biofuels 10 (2017) 1–16, https://doi.org/10.1186/s13068-017-0975-x. [44] R.F. Weiss, Carbon dioxide in water and seawater: the solubility of a non-ideal gass, Mar. Chem. 2 (1974) 203–215, https://doi.org/10.1016/0304-4203(74)90015-2. [45] C.E. Wyman, Aqueous Pretreatment of Plant Biomass for Biological and Chemical Conversion to Fuels and Chemicals, Wiley, West Sussex, 2013. [46] L.R. Lynd, M. Balch, J.M.D. Paye, USApp20160168596 Publication.PDF, 2016. [47] X. Liang, J.M. Whitham, E.K. Holwerda, X. Shao, L. Tian, Y.W. Wu, V. Lombard, B. Henrissat, D.M. Klingeman, Z.K. Yang, M. Podar, T.L. Richard, J.G. Elkins, S. D. Brown, L.R. Lynd, Development and characterization of stable anaerobic thermophilic methanogenic microbiomes fermenting switchgrass at decreasing residence times, Biotechnol. Biofuels 11 (2018) 1–18, https://doi.org/10.1186/ s13068-018-1238-1. [48] P.J. Van Soest, The uniformity and nutritive availability of cellulose, Fed, SAVE Proc. 32 (1973) 1804–1808. [49] O. Dolotko, J.W. Wiench, K.W. Dennis, V.K. Pecharsky, V.P. Balema, Mechanically induced reactions in organic solids: liquid eutectics or solid-state processes? New J. Chem. 34 (2010) 25, https://doi.org/10.1039/b9nj00588a. [50] L.D. Ellis, E.K. Holwerda, D. Hogsett, S. Rogers, X. Shao, T. Tschaplinski, P. Thorne, L.R. Lynd, Closing the carbon balance for fermentation by Clostridium thermocellum (ATCC 27405), Bioresour. Technol. 103 (2012) 293–299, https:// doi.org/10.1016/j.biortech.2011.09.128. [51] R. Kumar, G. Mago, V. Balan, C.E. Wyman, Physical and chemical characterizations of corn stover and poplar solids resulting from leading pretreatment technologies, Bioresour. Technol. 100 (2009) 3948–3962, https://doi.org/10.1016/j. biortech.2009.01.075. [52] A. Flamholz, E. Noor, A. Bar-even, W. Liebermeister, R. Milo, Glycolytic strategy as a tradeoff between energy yield and protein cost, Proc. Natl. Acad. Sci. U.S.A. (2013), https://doi.org/10.1073/pnas.1215283110.
[16] S.T.L. Harrison, Bacterial cell disruption: a key unit operation in the recovery of intracellular products, Biotechnol. Adv. 9 (1991) 217–240, https://doi.org/ 10.1016/0734-9750(91)90005-G. [17] R. Seetharam, S.K. Sharma, Purification and Analysis of Recombinant Proteins, CRC Press, New York, 1991. [18] L.O. Ingram, P.F. Gomez, X. Lai, M. Moniruzzaman, B.E. Wood, L.P. Yomano, S. W. York, Metabolic engineering of bacteria for ethanol production, Biotechnol. Bioeng. 58 (1998) 204–214, https://doi.org/10.1002/(SICI)1097-0290(19980420) 58:2/3<204::AID-BIT13>3.0.CO;2-C. [19] S. Atsumi, J.C. Liao, Metabolic engineering for advanced biofuels production from Escherichia coli, Curr. Opin. Biotechnol. 19 (2008) 414–419, https://doi.org/ 10.1016/j.copbio.2008.08.008. [20] L.O. Ingram, J.B. Doran, Conversion of cellulosic materials to ethanol, FEMS Microbiol. Rev. 16 (1995) 235–241, https://doi.org/10.1111/j.1574-6976.1995. tb00170.x. [21] P.L. Rogers, K.J. Lee, M.L. Skotnicki, D.E. Tribe, Ethanol production by Zymomonas mobilis, Microb. React. (1982) 37–84, https://doi.org/10.1007/ 3540116982_2. [22] L. Davis, Y.J. Jeon, C. Svenson, P. Rogers, J. Pearce, P. Peiris, Evaluation of wheat stillage for ethanol production by recombinant Zymomonas mobilis, Biomass Bioenergy 29 (2005) 49–59, https://doi.org/10.1016/j.biombioe.2005.02.006. [23] P.L. Rogers, E. Joachimsthal, K. Haggett, Ethanol from lignocellulosics: potential for Zymomonas-based process, AusBiotech 7 (1997) 304–309. [24] M. Nakano, P. Zuber, Anaerobic growth of a “strict aerobe” (Bacillus subtilis), Anu. Rev. Microbiol. 52 (1998) 165–190, https://doi.org/10.1146/annurev. micro.52.1.165. [25] M.M. Nakano, Y.P. Dailly, P. Zuber, D.P. Clark, Characterization of anaerobic fermentative growth of Bacillus subtilis: identification of fermentation end products and genes required for growth, J. Bacteriol. 179 (1997) 6749–6755, https://doi.org/10.1128/jb.179.21.6749-6755.1997. [26] H. Cho, H. Yukawa, M. Inui, R.H. Doi, S. Wong, Production of Minicellulosomes from Clostridium cellulovorans in Bacillus subtilis WB800, Appl. Environ. Microbiol. 70 (2004) 5704–5707, https://doi.org/10.1128/AEM.70.9.5704. [27] X.Z. Zhang, Y.H.P. Zhang, One-step production of biocommodities from lignocellulosic biomass by recombinant cellulolytic Bacillus subtilis: opportunities and challenges, Eng. Life Sci. 10 (2010) 398–406, https://doi.org/10.1002/ elsc.201000011. [28] A.J. Shaw, F.E. Jenney, M.W.W. Adams, L.R. Lynd, End-product pathways in the xylose fermenting bacterium, Thermoanaerobacterium saccharolyticum, Enzym. Microb. Technol. 42 (2008) 453–458, https://doi.org/10.1016/j. enzmictec.2008.01.005. [29] C.D. Herring, W.R. Kenealy, A. Joe Shaw, S.F. Covalla, D.G. Olson, J. Zhang, W. Ryan Sillers, V. Tsakraklides, J.S. Bardsley, S.R. Rogers, P.G. Thorne, J. P. Johnson, A. Foster, I.D. Shikhare, D.M. Klingeman, S.D. Brown, B.H. Davison, L. R. Lynd, D.A. Hogsett, Strain and bioprocess improvement of a thermophilic anaerobe for the production of ethanol from wood, Biotechnol. Biofuels 9 (2016) 1–16, https://doi.org/10.1186/s13068-016-0536-8. [30] A.J. Shaw, K.K. Podkaminer, S.G. Desai, J.S. Bardsley, S.R. Rogers, P.G. Thorne, D. A. Hogsett, L.R. Lynd, Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield, Proc. Natl. Acad. Sci. U.S.A. 105 (2008) 13769–13774, https://doi.org/10.1073/pnas.0801266105. [31] E.K. Holwerda, K.D. Hirst, L.R. Lynd, A defined growth medium with very low background carbon for culturing Clostridium thermocellum, J. Ind. Microbiol. Biotechnol. 39 (2012) 943–947, https://doi.org/10.1007/s10295-012-1091-3. [32] G. Sezonov, D. Joseleau-Petit, R. D’Ari, Escherichia coli physiology in Luria-Bertani broth, J. Bacteriol. 189 (2007) 8746–8749, https://doi.org/10.1128/JB.01368-07. [33] A.E. Goodman, P.L. Rogers, M.L. Skotnicki, Minimal medium for isolation of auxotrophic Zymomonas mutants, Appl. Environ. Microbiol. 44 (1982) 496–498. [34] T. Hoffmann, B. Troup, A. Szabo, C. Hungerer, D. Jahn, The anaerobic life of Bacillus subtilis: cloning of the genes encoding the respiratory nitrate reductase system, FEMS Microbiol. Lett. 131 (1995) 219–225, https://doi.org/10.1016/ 0378-1097(95)00262-4. [35] D.G. Olson, L.R. Lynd, Transformation of clostridium Thermocellum by Electroporation, first ed., Elsevier Inc., 2012 https://doi.org/10.1016/B978-0-12415931-0.00017-3.
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Research paper
Fermentation with continuous ball milling: Effectiveness at enhancing solubilization for several cellulosic feedstocks and comparative tolerance of several microorganisms Michael L. Balch, Meghan B. Chamberlain, Robert S. Worthen, Evert K. Holwerda *, Lee R. Lynd Thayer School of Engineering, Dartmouth College, Hanover NH.14 Engineering Drive, Hanover, NH, 03755, United States
A R T I C L E I N F O
A B S T R A C T
Keywords: Cotreatment Lignocellulose Fermentation Biofuel Milling
Mechanical disruption of lignocellulose during fermentation, cotreatment, is a nascent approach to increase biologically-mediated carbohydrate solubilization without exposure to high temperatures or chemicals. How ever, ability to tolerate the presence of milling at intensities sufficient to allow high lignocellulose solubilization yields has to date only been evaluated for Clostridium thermocellum and Saccharomyces cerevisiae, and demon stration of high carbohydrate solubilization has only been reported for switchgrass at the time of submission. Continuous ball milling during fermentation by C. thermocellum was found to be sufficient to allow high (>85%) total carbohydrate solubilization of corn stover and poplar as well as switchgrass. Under the conditions tested, ball milling had no apparent effect on soluble sugar fermentation by Escherichia coli and Thermoanaerobacterium saccharolyticum but completely inhibited carbohydrate fermentation by Zymomonas mobilis and Bacillus subtilis. Our results are consistent with the stress of milling being sufficient to overwhelm the inefficient fermentative metabolism of Z. mobilis and B. subtilis, but not to the other microorganisms tested in this work for which fermentation is more efficient and robust.
1. Introduction Cellulosic biofuels will likely be needed to achieve a low-carbon transportation sector [1,2]. In light of the high cost and operational complexity of lignocellulose processing based on thermochemical treatment and fungal cellulase, there is incentive to explore alternatives to this paradigm. One such alternative is to carry out consolidated bio processing (CBP) by thermophilic, saccharolytic microorganisms in conjunction with milling during fermentation, termed cotreatment [3, 4]. CBP combined with cotreatment is referred to herein as C-CBP. Milling as a pretreatment prior to hydrolysis using fungal cellulase has been investigated in several studies [5–7] but energy requirements are widely thought to be too high to be practical [8–10]. However, the physical properties of cellulosic biomass slurries undergo large changes during biologically-mediated solubilization. For example, Ghosh et al. [11] recently observed a 2000-fold reduction in viscosity during fermentation of corn stover by C. thermocellum. Thus it is plausible that energy requirements for milling of partially-fermented material in a cotreatment configuration may be sufficiently low to allow practical application whereas this is not the case for milling as a pretreatment.
Paye et al. [4] compared sequential fermentation of senescent switchgrass by Clostridium thermocellum, a cellulolytic Gram-positive thermophilic bacterium, with ball milling either before fermentation or between the first and the second successive fermentation. Milling partially fermented feedstock was found to result in greater enhance ment of carbohydrate solubilization and particle size reduction as compared to milling prior to fermentation. Upon cultivating C. thermocellum on senescent switchgrass with continuous ball milling, Balch et al. [3] found that 88% of total carbohydrate was solubilized without thermochemical pretreatment or added enzymes. In addition, it was observed that continuous ball milling had little effect on cellobiose fermentation by C. thermocellum but completely halted glucose fermentation by Saccharomyces cerevisiae. Cotreatment is a nascent technology and there are still many important questions to answer including mechanism, energy, and techno-economics. However it is also important to answer foundational questions such as efficacy. Early studies on pretreatment similarly took the approach of demonstrating effect first, later followed by more complex studies which included techno-economic and efficiency considerations.
* Corresponding author. E-mail address: [email protected] (E.K. Holwerda). https://doi.org/10.1016/j.biombioe.2020.105468 Received 18 January 2019; Received in revised form 25 September 2019; Accepted 13 January 2020 Available online 30 January 2020 0961-9534/© 2020 Elsevier Ltd. All rights reserved.
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(JW/SL-YS 485) was cultured on CTFüD with 5 g L 1 cellobiose [35]. Inocula were grown in 125 mL sealed anaerobic culture bottles and incubated for 24 h (at 60 � C for C. thermocellum and T. saccharolyticum and at 37 � C for E. coli, Z. mobilis, and B. subtilis). Inoculum was trans ferred to bioreactors via syringe. The inoculum volume was chosen so that vigorous fermentations ensued within 24 h for unmilled controls: 5% of working volume for lignocellulosic runs with C. thermocellum, 5% for T. saccharolyticum and Z. mobilis, 2.5% for B. subtilis, and 0.2% for E. coli.
For the concept of cotreatment to be a successful approach of reducing lignocellulose recalcitrance, it is important to demonstrate its compatibility with an array of lignocellulosic substrates and biocatalysts (microorganisms). Here we broaden the range of feedstocks and mi croorganisms tested for compatibility with cotreatment using the ball mill bioreactor developed by Balch et al. [3]. Some forms of pretreat ment have been shown to be most effective on a select number of lignocellulosic feedstocks [12]. We believe cotreatment will not be similarly limited, but to date, high carbohydrate solubilization with cotreatment has only been reported for switchgrass. In this study, we tested the effectiveness of cotreatment at enhancing solubilization by C. thermocellum for corn stover, poplar, and late-season harvested switchgrass. These are all of interest as bioenergy feedstocks [13–15], and represent a range of recalcitrance to biological attack in the absence of pretreatment or cotreatment. Mechanical treatments, particularly bead milling, are widely used to disrupt cells [16,17]. It is therefore important to demonstrate the ca pacity of microorganisms to carry out anaerobic fermentation of soluble substrates in the presence of continuous ball milling at an intensity sufficient to enable high total carbohydrate solubilization. Thus far, the impact of cotreatment on microorganisms has only been demonstrated for C. thermocellum and S. cerevisiae. The four microorganisms evaluated in this study - Escherichia coli, Zymomonas mobilis, Bacillus subtilis, and Thermoanaerobacterium saccharolyticum represent a range of physiol ogies and taxonomies and have all been proposed for use in production of cellulosic biofuels (Table 1).
2.2. Bioreactor configuration
2. Materials and methods
All fermentations were carried out anaerobically in custom-made stainless steel bioreactors equipped for continuous ball milling as described by Balch et al. [3]. Anaerobic conditions were ensured by purging the headspace of the reactor overnight with either 100% N2 gas or a 80% N2 – 20% CO2 gas mixture prior to inoculation (as indicated), by the presence of the reducing agent L-cysteine in the media for C. thermocellum and T. saccharolyticum, and by the use of norprene tubing and purged media solutions. For fermentations with milling the bioreactors were filled with 5 mm diameter stainless steel balls numbering a total of around 10,000. The volume of balls was about 600 mL and the volume of cultivation medium was 600 mL making the total final working volume 1200 mL. The height of the bead bed was 80% of the height of the final total working volume. For fermentations without milling, no balls were added and the volume of cultivation medium was doubled such that the total final working volume was constant at 1200 mL.
2.1. Strains, growth medium, and inoculum preparation
2.3. Fermentation of lignocellulosic feedstocks by C. thermocellum
All strains were obtained from Deutsche Sammlung von Mikroorga nismen and Zellkulturen (DSMZ, Braunschweig, Germany) and cultured using growth media shown in previous studies to effectively support the cultivation of each microorganism. Clostridium thermocellum (Rumini clostridium thermocellum) DSM 1313 was cultured in LC medium [31] on 5 g L 1 Avicel PH105 (FMC biopolymers, Philadelphia PA) with 5 g L 1 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (Sigma; St. Louis, MO). Escherichia coli DSM 18039 was cultured in LB medium [32] with 10 g L 1 glucose. Zymomonas mobilis DSM 424 was cultured in DSM medium with 20 g L 1 glucose [33]. Bacillus subtilis DSM 10 was cultured in LB medium [32] with 10 g L 1 glucose and 50 mM NaNO3, which acts as an electron acceptor [34]. Thermoanaerobacterium saccharolyticum
Switchgrass (Panicum virgatum L.), Alamo variety, was harvested in October at the University of Tennessee Knoxville and was kindly pro vided by the Stewart Lab. Corn stover, ground to pass through a 5-mm screen, was received as a gift from the US Department of Energy Great Lakes Bioenergy Research Center and a description of which can be found in Sekhon et al., [36]. Poplar (Populus trichocarpa), was grown at the Oak Ridge National Laboratory, debarked, and milled through a 1 mm screen as described by Trajano et al., [37]. Switchgrass, corn stover, and poplar were pre-rinsed as described in Garlock et al. [38], and then milled with a Retsch mill (Haan, Germany) through a 2-mm screen. For all lignocellulose feedstocks the substrate loading was 5 g L 1 glucan equivalent (14.8 g L 1 dry material for switchgrass, 17.0 g L 1 for corn stover, and 10.0 g L 1 for poplar). The cotreatment bioreactor was set up by layering beads and feed stock in the middle portion of the bed such that all feedstock was exposed to milling. Once loaded the bioreactor was autoclaved at 121 � C for 1 h and then incubated at 30 � C overnight while purging the head space with 30 mL min 1 of 20% CO2 - 80%N2 gas mix (Airgas; White River Junction, VT). Components of LC medium were added via syringe as five separate solutions as described in Holwerda et al. [31]. For corn stover fermentations and milling runs the media was supplemented with 5 g L 1 MOPS buffer. Ball milling was initiated at 100 rpm immediately prior to inoculation except in the case of corn stover, where the culture was grown for 24 h without milling to allow it to initiate growth (and total incubation time was 6 days instead of 5 days). Base addition and gas production were interpreted as signs of metabolic activity and growth. Gas production was measured by using Milligas tip meters (Ritter, Hawthorne, NY). The bioreactors were inoculated with a 5% v/v inoculum, and stirred at 100 rpm independent of the presence of beads. Lignocellulose fermentations were run for 5–7 days (120–168 h) depending on feedstock (see Fig. 1). The pH was maintained at 7.0 [31] with 2 N KOH and 2 N HCl solutions and the temperature was controlled at 60.0 � C [31] by a Sartorius Aplus control tower (Sartorius Stedim, Bohemia NY). 2 mL samples were collected every 24 h for analysis by HPLC (Waters; Milford, MA) on an Aminex HPX-87H column (Bio-Rad,
Table 1 Overview of the microorganisms used in the fermentation of soluble substrates during milling experiments. Microorganism
Selected features
Studies proposing use for cellulosic biofuel production
Escherichia coli
Gram-negative, mesophilic facultative anaerobe, mixedacid fermentation of hexose and pentose sugars [18] Gram-negative, mesophilic obligate anaerobe, homoethanologenic fermentation of glucose [21]
Atsumi and Liao, 2008 [19] Ingram and Doran, 1995 [20] Davis et al., 2005 [22] Rogers et al., 1982 [21] Rogers 1997 [23] Cho et al., 2004 [26] Zhang and Zhang, 2010 [27] Herring et al., 2016 [29] Shaw et al., 2008 [30]
Zymomonas mobilis
Bacillus subtilis
Thermoanaerobacterium saccharolyticum
Gram positive, mesophilic facultative anaerobe [24], mixed acid-butanediol fermentation [25] Gram positive, thermophilic obligate anaerobe, mixed acid fermentation of hemicellulose and derivatives [28]
2
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Fig. 1. Fermentation results for Lignocellulosic substrates: Corn Stover (A,D,G), Switchgrass (B,E,H), and Poplar (C,F,I). Figures show Solubilization (A–C), Gas production (D–F), and Product formation (G–I), with cotreatment (red) and without cotreatment (blue). Values represent the average of duplicate fermentations; error bars represent one standard deviation. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 2. Substrate consumption and gas production (red and blue respectively, A-D), and major product formation (green and blue respectively, E-H) for E. coli (A,E), Z. mobilis (B,F), B. subtilis (C,G), and T. saccharolyticum (D,H) with milling (solid lines and closed symbols) and without milling (dashed lines and open symbols). Arrows in B and C indicate times at which reinoculation occurred. Values represent the average of duplicate fermentations; error bars represent one standard de viation. Gas production curves show replicate fermentations. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) 3
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Hercules, CA) operated at 50 � C with a 5 mM H2SO4 eluent. All fer mentations were performed in duplicate.
� 5.3% for switchgrass, and 87.8 � 3.3% for poplar. Total gas produc tion with and without cotreatment was highest for corn stover, inter mediate for switchgrass, and lowest for poplar (Fig. 1D 1E, and 1F). Increased gas production was observed with cotreatment relative to without cotreatment, with the magnitude of the increase roughly pro portional to the increase in solubilization. Gas production curves indi cate that fermentation was initiated at about the same time with and without cotreatment for switchgrass and corn stover, but was somewhat delayed for poplar with cotreatment compared to the control without cotreatment. Under all conditions tested, acetate and ethanol were the main fermentation products produced by the wild-type strain of C. thermocellum used in this study. Production of acetate and ethanol was substantially higher with cotreatment than without it for switchgrass and poplar, but not for corn stover for which the difference in solubili zation was not as large. Solubilization data reported here for switchgrass and Populus in the absence of pretreatment or cotreatment are similar to those we have reported previously using C. thermocellum [40], and higher than observed with commercial fungal cellulase [4,40]. Corn stover solubilization by C. thermocellum in the absence of cotreatment or pretreatment is notably higher than the other feedstocks tested as has been reported previously [41]. The extent of solubilization we observed for C. thermocellum in the presence of cotreatment, on the order of 90% of theoretical, is comparable to that observed for thermochemical pre treatment [3,40,42,43].
2.4. Soluble substrate fermentations When testing fermentation of soluble substrate in presence of mill ing, the stainless steel balls were added to the reactor all at once. Growth media components were added to bioreactors prior to autoclaving with the exception of sugars and NaNO3 for B. subtilis. The bioreactors were then autoclaved at 121 � C for 1 h, after which filter-sterilized sugars (glucose or cellobiose), and NaNO3 for B. subtilis, were added. The headspace of bioreactors containing sterilized medium were purged overnight with 30 mL min 1 of filter-sterilized N2 gas (Airgas; White River Junction, VT). Agitation at 100 rpm was initiated prior to inocu lation and continued throughout the duration of the experiment. Fermentations were run for 24 h when metabolic activity, as man ifested by base addition or gas production, was observed, and up to 96 h when metabolic activity was not observed (i.e. a lack of base addition or gas production as measured by gas tip meter). For runs in which fermentation was not initiated, the bioreactors were re-inoculated with a 5% v/v inoculum at time-points indicated in Fig. 2 pH was maintained at 7.0 for E. coli [32] and B. subtilis [34], and 6.0 for Z. mobilis [33] and T. saccharolyticum [35] by addition of 4 N NaOH solutions. Temperature was maintained at 37 � C for E. coli [32], B. subtilis [34], and Z. mobilis [33] and 60 � C for T. saccharolyticum [35]. Fermentation control was performed by Sartorius Aplus control tower. 2 mL samples were collected every 2 h for E. coli, B. subtilis, and T. saccharolyticum, and every 3 h for Z. mobilis, for analysis by HPLC. All fermentations were performed in duplicate.
3.2. Tolerance of various microorganisms to milling Following results in prior work that C. thermocellum readily ferments soluble substrates in the presence of milling whereas S. cerevisiae does not [3], we evaluated the ability of 4 additional microorganisms to ferment soluble substrates appropriate for each organism in the presence of continuous ball milling: E. coli, Z. mobilis, B. subtilis, and T. saccharolyticum. For E. coli (Fig. 2A and E) and T. saccharolyticum (Fig. 2D and H), fermentation proceeded with undiminished rates of substrate con sumption and production of gas and organic fermentation products compared to controls without milling. For Z. mobilis (Fig. 2B and F) and B. subtilis (Fig. 2C and G), there was no evidence of metabolic activity in the presence of milling even after reinoculation although fermentation in controls without milling was readily apparent. Release of gas from liquid medium was lower for the two cultures grown at higher pH, E. coli and B. subtilis. This could be caused by decreased release of CO2 from the culture because of enhanced ionization of CO2 at higher pH [44]. Organic fermentation products in addition to ethanol and acetate were likely formed, particularly for E. coli and B. subtilis, but were not measured since the intended purpose here was to document metabolic activity. The production of fine particulate matter during milling resulted in an increasingly opaque broth in milled fermentations as compared to unmilled fermentations. Because of this it was not possible to accurately measure cell growth by either optical density or dry weight.
2.5. Quantification of fermentation products Samples were centrifuged at 15,000 rpm (21,130�g) for 5 min, and the supernatant analyzed for acetate and ethanol (and glucose or cellobiose for soluble substrate tests) via HPLC. Cumulative gas pro duction was measured via Milligascounter gas tip meters filled with a solution of 0.5 N HCl. 2.6. Calculation of total carbohydrate solubilization The percent total carbohydrate solubilization, TCS, is calculated using equation [1]. ��� � � GS;f þ XS;f þ AS;f TCS ¼ 100%⋅ 1 (1) ðGS;i þ XS;i þ AS;i Þ where G denotes glucan or glucose (mMoles), X denotes xylan or xylose (mMoles), A denotes arabinan or arabinose (mMoles), S denotes solid, f denotes final, and i denotes initial. Initial and final concentrations were determined by multiplying the solids concentration (g L 1 total solids) by the mass fraction of the sugar (e.g. g glucose per g total solid) obtained from quantitative saccharifi cation [39] and dividing by molecular weight.
4. Discussion
3. Results
Recognizing that cotreatment is a new concept with relatively little prior work, the first order of business is to explore the conditions under which it is effective and/or implementable – as was the case for the lignocellulose pretreatment field a quarter century ago. Here we sub stantially extend such exploration with respect to feedstocks and microorganisms. In the presence of cotreatment, but without thermochemical pre treatment other than autoclaving and without added enzymes, final carbohydrate solubilization levels for corn stover senescent switchgrass and poplar are similar to those seen with conventional thermochemical pretreatment [45]. Our switchgrass results are consistent with previous tests on grasses [3,40,46], which show cotreatment roughly doubling
3.1. Solubilization and fermentation of different lignocellulosic feedstocks Fermentation of corn stover, late-season switchgrass, and poplar was carried out by C. thermocellum in the presence and absence of cotreat ment via continuous ball milling. As presented in panels A, B, and C of Fig. 1, total carbohydrate solubilization (TCS) in the absence of cotreatment differed substantially, with 66.0 � 5.8% observed for corn stover, 45.7 � 3.2% for switchgrass, and 17.6 � 2.9% for poplar. Cotreatment substantially increased solubilization for all three feed stocks to rather similar TCS values of 88.3 � 4.7% for corn stover, 87.0 4
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solubilization. Solubilization nearly quadruples for poplar as a result of cotreatment, as also observed by Holwerda et al. [40]. For both C. thermocellum and long-term cultivated cellulolytic mixed enrichments (microbiomes), a fraction of the carbohydrate in lignocel lulosic feedstocks is inaccessible to biological attack even over extensive time periods [47,48]. Clearly, milling decreases this fraction for the three feedstocks examined here. Since the physical impacts of milling are more apparent than chemical impacts, although these are possible [49], it is likely that the primary mode of action of milling is to increase physical accessibility for enzymatic attack. Further elaboration of the mechanistic basis for the effectiveness of cotreatment awaits future study. Final ethanol concentrations with cotreatment are fairly consistent across substrates, between about 0.6 and 0.9 g L 1. Additionally, the ratio of acetate and ethanol produced on poplar is approximately 3:2, which is consistent with results for C. thermocellum on nonlignocellulosic substrates such as Avicel [50]. However, on corn stover and switchgrass, much more acetate is produced both with and without cotreatment. Furthermore, the hexose carbon recovery (80%, see Table S1 in SOM) for poplar is typical for C. thermocellum when not accounting for cells and/or amino acids [50], while the hexose carbon recovery is much higher for corn stover and switchgrass (exceeding 100% for the latter). This is consistent with acetate release from biomass during solubilization. While we note that woody feedstocks tend to have higher acetyl content than grassy feedstocks [51], it may be that the acetyl groups are more readily released from the less recalcitrant grassy feedstocks. Of the six microorganisms evaluated here and by Balch et al. [3] half are tolerant to milling under the conditions tested and half are not. Such tolerance appears to be an ‘all-or-nothing’ phenomenon under the conditions tested, with either little to no effect or complete inhibition of metabolic activity. Among the Gram-positive microorganisms tested here and by Balch et al. [3], T. saccharolyticum and C. thermocellum ferment readily in the presence of milling whereas B. subtilis does not. Among Gram-negative microorganisms, E. coli is tolerant to milling whereas Z. mobilis is not. Milling-tolerant microorganisms include both thermophiles (T. saccharolyticum, C. thermocellum) and mesophiles (E. coli). It is perhaps not surprising that we previously found S. cerevisiae to be sensitive to milling [3] in light of its large size and the absence of a thick cell wall. However, we find here that microorganisms intolerant to milling under the conditions tested include prokaryotes (Z. mobilis and B. subtilis). Additionally, the four organisms tested here are of similar size and shape, so it is not clear that size is a primary factor in sensitivity to milling. Zymomonas mobilis utilizes the Entner-Doudoroff pathway for con version of glucose to pyruvate [21] and thus has half the ATP available per mole glucose compared to micro organisms using the Embden-Meyerhof-Parnas pathway (E. coli [52], T. saccharolyticum [30], or C. thermocellum [41]). B. subtilis was long thought to be an obligate aerobe, and has an inefficient glucose fermentation pathway [24]. We speculate that the stress of milling may be sufficient to overwhelm the inefficient fermentative metabolism of Z. mobilis and B. subtilis, but not the more efficient and robust fermentation of other microorganisms tested in this work.
cotreatment and to evaluate it under industrial conditions, including different milling modalities, energy requirements, and technoecomomic analysis. Declaration of competing interest Lee R. Lynd holds equity in a start-up company that aims to commercialize cellulosic biomass. None of the other authors has a competing interest. Acknowledgements Support for this work was provided by the USDA National Institute for Food and Agriculture (NIFA) under the Biomass Research and Development Initiative (BRDI) [Grant 2016-10008-2531]. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.biombioe.2020.105468. References [1] L.M. Fulton, L.R. Lynd, A. K€ orner, N. Green, L.R. Tonachel, The need for biofuels as part of a low carbon energy future, Biofuels, Bioprod. Biorefining. 9 (2015) 476–483, https://doi.org/10.1002/bbb.1559. [2] OECD/IEA, Fao, How2Guide for Bioenergy Roadmap Development and Implementation, 78, IEA Publ., 2017, ISBN 978-92-5-109586-7. [3] M.L. Balch, E.K. Holwerda, M.F. Davis, R.W. Sykes, R.M. Happs, R. Kumar, E. Wyman, L.R. Lynd, Lignocellulose fermentation and residual solids characterization for senescent switchgrass fermentation by Clostridium thermocellum, Energy Environ. Sci. 10 (2017) 1252–1261, https://doi.org/ 10.1039/C6EE03748H. [4] J.M.D. Paye, A. Guseva, S.K. Hammer, E. Gjersing, M.F. Davis, B.H. Davison, J. Olstad, B.S. Donohoe, T.Y. Nguyen, C.E. Wyman, S. Pattathil, M.G. Hahn, L. R. Lynd, Biological lignocellulose solubilization: comparative evaluation of biocatalysts and enhancement via cotreatment, Biotechnol. Biofuels (2016) 1–13, https://doi.org/10.1186/s13068-015-0412-y. [5] U. Mais, A.R. Esteghlalian, J.N. Saddler, S.D. Mansfield, Enhancing the enzymatic hydrolysis of cellulosic materials using simultaneous ball milling, Appl. Biochem. Biotechnol. 98–100 (2002) 815–832, https://doi.org/10.1385/ABAB:98-100:1-9: 815. [6] M.J. Neilson, R.G. Kelsey, F. Shafizadeh, Enhancement of enzymatic hydrolysis by simultaneous attrition of cellulosic substrates, Biotechnol. Bioeng. 24 (1982) 293–304, https://doi.org/10.1002/bit.260240204. [7] S.K. Ryu, J.M. Lee, Bioconversion of waste cellulose by using an attrition bioreactor, Biotechnol. Bioeng. 25 (1983) 53–65, https://doi.org/10.1002/ bit.260250106. [8] A. Barakat, S. Chuetor, F. Monlau, A. Solhy, X. Rouau, Eco-friendly dry chemomechanical pretreatments of lignocellulosic biomass: impact on energy and yield of the enzymatic hydrolysis, Appl. Energy 113 (2014) 97–105, https://doi.org/ 10.1016/j.apenergy.2013.07.015. [9] A. Hideno, H. Inoue, T. Yanagida, K. Tsukahara, T. Endo, S. Sawayama, Combination of hot compressed water treatment and wet disk milling for high sugar recovery yield in enzymatic hydrolysis of rice straw, Bioresour. Technol. 104 (2012) 743–748, https://doi.org/10.1016/j.biortech.2011.11.014. [10] M.R. Zakaria, M.N.F. Norrrahim, S. Hirata, M.A. Hassan, Hydrothermal and wet disk milling pretreatment for high conversion of biosugars from oil palm mesocarp fiber, Bioresour. Technol. 181 (2015) 263–269, https://doi.org/10.1016/j. biortech.2015.01.072. [11] S. Ghosh, E.K. Holwerda, R.S. Worthen, L.R. Lynd, B.P. Epps, Rheological properties of corn stover slurries during fermentation by Clostridium thermocellum, Biotechnol. Biofuels 11 (2018) 246, https://doi.org/10.1186/ s13068-018-1248-z. [12] B. Yang, C.E. Wyman, Pretreatment: the key to unlocking low-cost cellulosic ethanol, Biofuels, Bioprod. Biorefining. (2008) 26–40, https://doi.org/10.1002/ bbb. [13] S. Kim, X. Zhangm, B. Dale, A.D. Reddy, C.D. Jones, K. Cronin, R.C. Izaurralde, T. Runge, M. Sharara, Corn stover cannot simultaneously meet both the volume and GHG reduction requirements of the renewable fuel standard, Biofuels, Bioprod. Biorefining. 12 (2018) 203–212, https://doi.org/10.1002/bbb. [14] L.R. Lynd, E. Larson, N. Greene, M. Laser, J. Sheehan, B.E. Dale, S. McLaughlin, M. Wang, The role of biomass in America’s energy future: framing the analysis, Biofuels, Bioprod. Biorefining. 3 (2009) 113–123, https://doi.org/10.1002/bbb. [15] P. Sannigrahi, A.J. Ragauskas, G.A. Tuskan, Poplar as a feedstock for biofuels: a review of compositional characteristics, Biofuels, Bioprod. Biorefining. 4 (2010) 209–226, https://doi.org/10.1002/bbb.
5. Conclusion We show that cotreatment implemented in a ball mill bioreactor under the condition tested enables near-complete solubilization of several prominent potential lignocellulosic feedstocks for cellulosic biofuel production. Additionally, we find that some organisms are able to continue fermentation in the presence of ball milling while others are not, but are unable to strongly relate that to physiological features. Our work establishes that cotreatment enables high total carbohydrate sol ubilization to be achieved for a woody feedstock as well as herbaceous feedstocks. More work is needed to understand the mechanistic basis of 5
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Biomass and Bioenergy 134 (2020) 105468 [36] R.S. Sekhon, H. Lin, K.L. Childs, C.N. Hansey, C.R. Buell, N. de Leon, S.M. Kaeppler, Genome-wide atlas of transcription during maize development, Plant J. 66 (2011) 553–563, https://doi.org/10.1111/j.1365-313X.2011.04527.x. [37] H.L. Trajano, N.L. Engle, M. Foston, A.J. Ragauskas, T.J. Tschaplinski, C. E. Wyman, The fate of lignin during hydrothermal pretreatment, Biotechnol. Biofuels 6 (2013) 1, https://doi.org/10.1186/1754-6834-6-110. [38] R.J. Garlock, V. Balan, B.E. Dale, V. Ramesh Pallapolu, Y.Y. Lee, Y. Kim, N. S. Mosier, M.R. Ladisch, M.T. Holtzapple, M. Falls, R. Sierra-Ramirez, J. Shi, M. A. Ebrik, T. Redmond, B. Yang, C.E. Wyman, B.S. Donohoe, T.B. Vinzant, R. T. Elander, B. Hames, S. Thomas, R.E. Warner, Comparative material balances around pretreatment technologies for the conversion of switchgrass to soluble sugars, Bioresour. Technol. 102 (2011) 11063–11071, https://doi.org/10.1016/j. biortech.2011.04.002. [39] A. Sluiter, B. Hames, R. Ruiz, C. Scarlata, J. Sluiter, D. Templeton, D.C. Nrel, Determination of Structural Carbohydrates and Lignin in Biomass Determination of Structural Carbohydrates and Lignin in Biomass, 2012, p. 2011. [40] E.K. Holwerda, R.S. Worthen, N. Kothari, R.C. Lasky, B.H. Davison, C. Fu, Z. Y. Wang, R.A. Dixon, A.K. Biswal, D. Mohnen, R.S. Nelson, H.L. Baxter, M. Mazarei, W. Muchero, G.A. Tuskan, C.M. Cai, E.E. Gjersing, M.F. Davis, M.E. Himmel, C. E. Wyman, P. Gilna, L.R. Lynd, Multiple levers for overcoming the recalcitrance of lignocellulosic biomass, Biotechnol. Biofuels 12 (2019) 1–12, https://doi.org/ 10.1186/s13068-019-1353-7. [41] L.R. Lynd, A.M. Guss, M.E. Himmel, D. Beri, C. Herring, E.K. Holwerda, Advances in consolidated bioprocessing using Clostridium thermocellum and thermoanaerobacter saccharolyticum, in: C. Wittman, J. Liao (Eds.), Ind. Biotechnol. Microorg, Wiley, 2016, pp. 365–394. [42] N. Kothari, E.K. Holwerda, C.M. Cai, R. Kumar, C.E. Wyman, Biomass augmentation through thermochemical pretreatments greatly enhances digestion of switchgrass by Clostridium thermocellum, Biotechnol. Biofuels 11 (2018) 1–14, https://doi.org/10.1186/s13068-018-1216-7. [43] V.A. Thomas, N. Kothari, S. Bhagia, H. Akinosho, M. Li, Y. Pu, C.G. Yoo, S. Pattathil, M.G. Hahn, A.J. Raguaskas, C.E. Wyman, Comparative evaluation of Populus variants total sugar release and structural features following pretreatment and digestion by two distinct biological systems, Biotechnol. Biofuels 10 (2017) 1–16, https://doi.org/10.1186/s13068-017-0975-x. [44] R.F. Weiss, Carbon dioxide in water and seawater: the solubility of a non-ideal gass, Mar. Chem. 2 (1974) 203–215, https://doi.org/10.1016/0304-4203(74)90015-2. [45] C.E. Wyman, Aqueous Pretreatment of Plant Biomass for Biological and Chemical Conversion to Fuels and Chemicals, Wiley, West Sussex, 2013. [46] L.R. Lynd, M. Balch, J.M.D. Paye, USApp20160168596 Publication.PDF, 2016. [47] X. Liang, J.M. Whitham, E.K. Holwerda, X. Shao, L. Tian, Y.W. Wu, V. Lombard, B. Henrissat, D.M. Klingeman, Z.K. Yang, M. Podar, T.L. Richard, J.G. Elkins, S. D. Brown, L.R. Lynd, Development and characterization of stable anaerobic thermophilic methanogenic microbiomes fermenting switchgrass at decreasing residence times, Biotechnol. Biofuels 11 (2018) 1–18, https://doi.org/10.1186/ s13068-018-1238-1. [48] P.J. Van Soest, The uniformity and nutritive availability of cellulose, Fed, SAVE Proc. 32 (1973) 1804–1808. [49] O. Dolotko, J.W. Wiench, K.W. Dennis, V.K. Pecharsky, V.P. Balema, Mechanically induced reactions in organic solids: liquid eutectics or solid-state processes? New J. Chem. 34 (2010) 25, https://doi.org/10.1039/b9nj00588a. [50] L.D. Ellis, E.K. Holwerda, D. Hogsett, S. Rogers, X. Shao, T. Tschaplinski, P. Thorne, L.R. Lynd, Closing the carbon balance for fermentation by Clostridium thermocellum (ATCC 27405), Bioresour. Technol. 103 (2012) 293–299, https:// doi.org/10.1016/j.biortech.2011.09.128. [51] R. Kumar, G. Mago, V. Balan, C.E. Wyman, Physical and chemical characterizations of corn stover and poplar solids resulting from leading pretreatment technologies, Bioresour. Technol. 100 (2009) 3948–3962, https://doi.org/10.1016/j. biortech.2009.01.075. [52] A. Flamholz, E. Noor, A. Bar-even, W. Liebermeister, R. Milo, Glycolytic strategy as a tradeoff between energy yield and protein cost, Proc. Natl. Acad. Sci. U.S.A. (2013), https://doi.org/10.1073/pnas.1215283110.
[16] S.T.L. Harrison, Bacterial cell disruption: a key unit operation in the recovery of intracellular products, Biotechnol. Adv. 9 (1991) 217–240, https://doi.org/ 10.1016/0734-9750(91)90005-G. [17] R. Seetharam, S.K. Sharma, Purification and Analysis of Recombinant Proteins, CRC Press, New York, 1991. [18] L.O. Ingram, P.F. Gomez, X. Lai, M. Moniruzzaman, B.E. Wood, L.P. Yomano, S. W. York, Metabolic engineering of bacteria for ethanol production, Biotechnol. Bioeng. 58 (1998) 204–214, https://doi.org/10.1002/(SICI)1097-0290(19980420) 58:2/3<204::AID-BIT13>3.0.CO;2-C. [19] S. Atsumi, J.C. Liao, Metabolic engineering for advanced biofuels production from Escherichia coli, Curr. Opin. Biotechnol. 19 (2008) 414–419, https://doi.org/ 10.1016/j.copbio.2008.08.008. [20] L.O. Ingram, J.B. Doran, Conversion of cellulosic materials to ethanol, FEMS Microbiol. Rev. 16 (1995) 235–241, https://doi.org/10.1111/j.1574-6976.1995. tb00170.x. [21] P.L. Rogers, K.J. Lee, M.L. Skotnicki, D.E. Tribe, Ethanol production by Zymomonas mobilis, Microb. React. (1982) 37–84, https://doi.org/10.1007/ 3540116982_2. [22] L. Davis, Y.J. Jeon, C. Svenson, P. Rogers, J. Pearce, P. Peiris, Evaluation of wheat stillage for ethanol production by recombinant Zymomonas mobilis, Biomass Bioenergy 29 (2005) 49–59, https://doi.org/10.1016/j.biombioe.2005.02.006. [23] P.L. Rogers, E. Joachimsthal, K. Haggett, Ethanol from lignocellulosics: potential for Zymomonas-based process, AusBiotech 7 (1997) 304–309. [24] M. Nakano, P. Zuber, Anaerobic growth of a “strict aerobe” (Bacillus subtilis), Anu. Rev. Microbiol. 52 (1998) 165–190, https://doi.org/10.1146/annurev. micro.52.1.165. [25] M.M. Nakano, Y.P. Dailly, P. Zuber, D.P. Clark, Characterization of anaerobic fermentative growth of Bacillus subtilis: identification of fermentation end products and genes required for growth, J. Bacteriol. 179 (1997) 6749–6755, https://doi.org/10.1128/jb.179.21.6749-6755.1997. [26] H. Cho, H. Yukawa, M. Inui, R.H. Doi, S. Wong, Production of Minicellulosomes from Clostridium cellulovorans in Bacillus subtilis WB800, Appl. Environ. Microbiol. 70 (2004) 5704–5707, https://doi.org/10.1128/AEM.70.9.5704. [27] X.Z. Zhang, Y.H.P. Zhang, One-step production of biocommodities from lignocellulosic biomass by recombinant cellulolytic Bacillus subtilis: opportunities and challenges, Eng. Life Sci. 10 (2010) 398–406, https://doi.org/10.1002/ elsc.201000011. [28] A.J. Shaw, F.E. Jenney, M.W.W. Adams, L.R. Lynd, End-product pathways in the xylose fermenting bacterium, Thermoanaerobacterium saccharolyticum, Enzym. Microb. Technol. 42 (2008) 453–458, https://doi.org/10.1016/j. enzmictec.2008.01.005. [29] C.D. Herring, W.R. Kenealy, A. Joe Shaw, S.F. Covalla, D.G. Olson, J. Zhang, W. Ryan Sillers, V. Tsakraklides, J.S. Bardsley, S.R. Rogers, P.G. Thorne, J. P. Johnson, A. Foster, I.D. Shikhare, D.M. Klingeman, S.D. Brown, B.H. Davison, L. R. Lynd, D.A. Hogsett, Strain and bioprocess improvement of a thermophilic anaerobe for the production of ethanol from wood, Biotechnol. Biofuels 9 (2016) 1–16, https://doi.org/10.1186/s13068-016-0536-8. [30] A.J. Shaw, K.K. Podkaminer, S.G. Desai, J.S. Bardsley, S.R. Rogers, P.G. Thorne, D. A. Hogsett, L.R. Lynd, Metabolic engineering of a thermophilic bacterium to produce ethanol at high yield, Proc. Natl. Acad. Sci. U.S.A. 105 (2008) 13769–13774, https://doi.org/10.1073/pnas.0801266105. [31] E.K. Holwerda, K.D. Hirst, L.R. Lynd, A defined growth medium with very low background carbon for culturing Clostridium thermocellum, J. Ind. Microbiol. Biotechnol. 39 (2012) 943–947, https://doi.org/10.1007/s10295-012-1091-3. [32] G. Sezonov, D. Joseleau-Petit, R. D’Ari, Escherichia coli physiology in Luria-Bertani broth, J. Bacteriol. 189 (2007) 8746–8749, https://doi.org/10.1128/JB.01368-07. [33] A.E. Goodman, P.L. Rogers, M.L. Skotnicki, Minimal medium for isolation of auxotrophic Zymomonas mutants, Appl. Environ. Microbiol. 44 (1982) 496–498. [34] T. Hoffmann, B. Troup, A. Szabo, C. Hungerer, D. Jahn, The anaerobic life of Bacillus subtilis: cloning of the genes encoding the respiratory nitrate reductase system, FEMS Microbiol. Lett. 131 (1995) 219–225, https://doi.org/10.1016/ 0378-1097(95)00262-4. [35] D.G. Olson, L.R. Lynd, Transformation of clostridium Thermocellum by Electroporation, first ed., Elsevier Inc., 2012 https://doi.org/10.1016/B978-0-12415931-0.00017-3.
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