Mass Spectrometry Exposes Undocumented Lignin-Carbohydrate

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Research Article Cite This: ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

Mass Spectrometry Exposes Undocumented Lignin-Carbohydrate Complexes in Biorefinery Pretreatment Stream Kelsey S. Boes,† Robert H. Narron,‡ Sunkyu Park,‡ and Nelson R. Vinueza*,† †

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North Carolina State University: Department of Textile Engineering, Chemistry, and Science, 1020 Main Campus Drive, Raleigh, North Carolina 27606, United States ‡ North Carolina State University: Department of Forest and Biomaterials, 2820 Faucette Drive, Raleigh, North Carolina 27607, United States ABSTRACT: The presence and effects of lignin−carbohydrate complexes (LCCs) in biorefinery processes are largely unknown. Recently identified in a significant hydrothermal pretreatment process stream (autohydrolyzate), these molecules likely influence not only downstream processing but also product purity and performance. However, without an understanding of their structures, it is impossible to analyze and cope with their effects. To identify and elucidate LCCs in autohydrolyzate, a new method employed chloride doping and tandem mass spectrometry. The results showed complexes ranging in mass from 326−714 Da with evidence of xylose and glucose units. Further analysis revealed lignin-like levels of unsaturation, considering the number of carbons present. These results suggest that, although the complexes contain one or two carbohydrate units, their primary structures are closer to lignin compounds. This method provides a glimpse into the structures of LCCs in a biorefinery process stream, laying the foundation for easier identification and continued structural elucidation of these enigmatic complexes. KEYWORDS: Chloride attachment, Tandem mass spectrometry, Lignin-carbohydrate complex, Electrospray ionization, Autohydrolyzate, Biomass



INTRODUCTION While lignins and carbohydrates are being explored widely for new plant-based materials, their chemical intersectionthe lignin−carbohydrate complex (LCC)is vastly underinvestigated. Being a covalent combination of lignin derivatives and carbohydrate units, LCCs are speculated to form during plant biosynthesis. Their presence in raw biomass is agreed upon, but little is known concerning their fate through biorefinerytype processes.1−5 This knowledge gap can contribute to processing problems for biorefineries, resulting in decreased efficiency.6−8 Not only do LCCs hinder industrial separation of biomass streams but they affect kraft pulping performance and can cause loss of pulp yield.8,9 Various studies over the past decade have successfully employed nuclear magnetic resonance spectroscopy (NMR) to elucidate the chemical connectivity of LCC structures, which were obtained by degrading biomass to the point that LCC molecules could be extracted from the plant cell.1−3,9 In addition, some researchers have synthesized and characterized model LCC compounds.10,11 Unfortunately, all of these studies were instrumentally limited in their ability to suggest structures of LCCs, resulting in a promising yet vague knowledge base concerning the molecular structures of plant-synthesized LCCs. A recent report showed that molecules containing LCCspecific chemical connectivity could be found in a biorefinery © XXXX American Chemical Society

process stream (pretreatment autohydrolyzate). Within this work, a method was developed to segregate the solubilized lignin-derivatives and LCCs from pretreatment autohydrolyzate using a lignin-specific resin.12 This segregation treatment provided an isolated lignin-LCC fraction, opening the door for more specific LCC analysis. Like previous works, the reported findings concerning characterization of these isolated LCCs were also instrumentally limited. The authors quantified the various LCC-specific chemical bonds across the distribution of isolated LCC molecules but did not characterize the individual components.12 We sought to provide a better understanding of the individual LCC molecules comprising the isolate. To achieve this, we leveraged the ionization dopant chloride together with tandem mass spectrometry to propose possible LCC molecular structures from the isolated lignin-LCC fraction and shed some light on these underexplored molecules.



MATERIALS AND METHODS

Raw Materials. Sweetgum (Liquidambar styraciflua, SGUM) chips were supplied by the tree improvement program at North Carolina Received: May 1, 2018 Revised: June 20, 2018

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DOI: 10.1021/acssuschemeng.8b01986 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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ACS Sustainable Chemistry & Engineering

Figure 1. SGUM (left) and SGRASS (right) Cl-adducts observed in (−) ion mode are identified by their 3:1 isotopic ratios and then matched with corresponding Na-adducts (triangle) and Li-adducts (square) observed in (+) ion mode. State University. Chips were screened according to Scandinavian Pulp, Paper, and Board Testing Committee SCAN-CM 40:01, collecting all chips below and including the large accept chip classification for experimentation. Switchgrass (Panicum virgatum, SGRASS) was obtained from a local farm in southern Wake county, North Carolina, and was used as received, including mostly stalk, tops, and smaller particulates. Autohydrolysis Pretreatment. Both the SGUM and SGRASS samples were subjected to extractives removal using a Soxhlet extraction device and a 2:1 benzene−ethanol (v/v) extraction solvent prior to pretreatment in order to remove nonlignin interferences. Samples were allowed to air-dry under a fume hood until constant mass. Autohydrolysis was performed in a 1 L Parr reactor vessel (Parr Instrument Company, USA). A nominal 50 g of air-dried extractive-free biomass was loaded into the vessel followed by water addition to set the liquid-to-solid ratio at 10:1 (accounting appropriately for biomass moisture). The vessel was heated to 180 °C and held at that temperature for 40 min. Average temperature ramp-up time was ∼30 min. Following 40 min of pretreatment, the reaction vessel was submerged into an ice water bath until internal pressure returned to atmosphere. Autohydrolyzate liquor was separated from cooked solids using filter paper and vacuum filtration. A secondary filtration step was then applied to separate autohydrolyzate to ensure total removal of suspended solids, performed using a vacuum and Pyrex gooch crucibles (Fine “F” grade). Free Carbohydrates Removed by Resin. Amberlight XAD16N resin was used to adsorb lignin from autohydrolyzate as described by Narron et al.12 The resin was mixed with autohydrolyzate liquor (AH-L) for 30 min with mild magnetic stir bar agitation. Following adsorption, the resin was separated from solution by vacuum filtration. The captured lignin-bearing resin was washed with deionized water to remove all unadsorbed AH-L compounds. Methanol was then used to desorb lignin-containing compounds from the resin. The methanol solution, termed extracted AH-L, was then prepared for MS analysis. Sample Prepared for Mass Spectrometry. The extracted AH-L was filtered with Whatman 0.2 μm pore size filters. The sample solution was prepared as follows: 3 mL of methanol/water (50:50, v/v), 300 μL of undiluted filtered extracted AH-L, 600 μL of lithium chloride (1 mM), 100 μL of cellobiose (1 mM), 50 μL of ß-guaiacylglycerol-ß-guaiacyl ether (98%), and 50 μL of 2-methoxy-4methyl phenol (>98.0%). Lithium chloride (LiCl) was added to provide chloride ions for Cl-adduct formation. The latter three chemicals were added as references to correct any mass shifts that might be occurring in the mass spectrometer. Samples for sweetgum and switchgrass were prepared similarly.

Reference chemicals were purchased from SigmaAldrich and used without further purification. LC−MS grade methanol was purchased from J.T. Baker with a purity of ≥99.9%. Pure water was generated using an ELGA water system and measured to have a resistivity of 18.2 Ω. Cellobiose and LiCl were diluted to respective concentrations using ultrapure water. Mass Spectrometry. Extracted AH-L samples were analyzed using an Agilent Technologies 6520 Accurate-Mass QTOF LC/MS (Agilent, Santa Barbara, CA) equipped with an ESI source, operated in both positive and negative ion mode. The QTOF was operated in high resolution mode (4 GHz) with a resolving power ranging from 10 700 for 100 m/z to 24 000 for 1600 m/z. The sample solution was injected into the ESI source using a Harvard PhD 2000 Infusion syringe pump at a rate of 6 μL/min. The operating conditions for optimized high molecular weight ion formation consisted of nitrogen drying gas at a temperature of 355 °C and a rate of 8 L/min, 60 psig nebulizer, 110 V fragmentor voltage, 65 V skimmer voltage, 750 V octopole voltage, 4000 V Vcap voltage, and 0.029 μA capillary current. An injection time of 1 min was used. After being tentatively assigned, the Cl-adduct anions in the LiCldoped samples were subjected to collision-activated dissociation (CAD) CAD experiments involved isolation of the suspected Cl-adduct by using a narrow (∼1.3 m/z) window and acceleration of the anion to collide with nitrogen gas with collision energies 18−38 eV as defined by the MassHunter LC/MS Data Acquisition Workstation Software version B.05.01 for 6200 series interface. CAD energies were optimized to achieve stable product ions and a relative abundance of 10% for the target ion, avoiding an overly fragmented spectrum that contained only congested low mass product-ions. Energies chosen erred on the side of low, ensuring the target ion was always visible in the CAD spectrum. Spectra were analyzed in profile mode with MassHunter Qualitative Analysis Workstation Software version B.06.00 interface. Lignin−Carbohydrate Complex Identification and Structural Elucidation. Without an ionization dopant, the mixture of lignin-derivatives and LCCs generates a complicated mass spectrum that is difficult to analyze. There is no way to know which peaks are free lignin molecules and which are LCCs without extensively analyzing each peak through tandem mass spectrometry. Luckily, the addition of the ionization dopant chloride allows us to selectively tag the LCCs with a chloride ion.13 Free lignins cannot be tagged with a chloride ion because their gas phase basicity prohibits the formation of a stable anionic Cl-adduct.14 Carbohydrates, on the other hand, have a gas phase basicity that allows compounds with a carbohydrate unit to form a stable anionic Cl-adduct. Anionic Cl-adducts (M + Cl) are straightforward to spot in a mass spectrum due to their 3:1 isotopic B

DOI: 10.1021/acssuschemeng.8b01986 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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ACS Sustainable Chemistry & Engineering

Chloride attachment can also be definitively confirmed if potential M+35Cl and M+37Cl ions are fragmented and exhibit the product ions 35 − Cl (34.9688 m/z) and 37Cl− (36.9659 m/z), respectively. The final step is to substantiate the presence of lignin structures in the ions. This was done by plotting the level of unsaturation against the number of carbons (CN) for each proposed molecular formula and then comparing the experimental ratios (unsaturation:CN) toratios of carbohydrate and lignin compounds from the literature. Molecular formulas were proposed for the Cl-adduct peaks in using ChemCalc online software and following suggested constraints of elements and numbers of each element for chlorinated anions below 1000 m/z: C (0−78), H (0−126), O (0−20), Cl (0−12), N (0−20).17,18 After confirming the ions to be LCCs, the structures were investigated using tandem MS. All tandem MS spectra were entered into fragmentation software to identify known product ions and neutral fragments from both lignins and carbohydrates. On the basis of identified product ions and fragments, structures were proposed for the LCCs.



RESULTS AND DISCUSSION Potential Chloride Adducts Identified. In sweetgum and switchgrass, we identified 20 potential chloride adducts using visual inspection of the spectrum, 10 potential adducts in SGUM and 10 potential adducts in SGRASS. Cl-Adducts Confirmed by Na- and Li-Adducts. As can be seen in Figure 1, five potential Cl-adducts were observed in negative ion mode and then matched to corresponding Na- and Li-adducts in the positive-ion mode for the SGUM extraction: 401.0957, 533.1434, 575.1492, 707.2006, and 749.2140 m/z. Unfortunately, the positive-ion mode spectrum of the SGRASS extraction was not as helpful; many more lignin distributions were observed that obscured the presence of Na- and Li-adducts. Only one Cl-adduct in SGRASS could be confirmed using this method: 361.0600 m/z. Cl-Adducts Confirmed by Fragmentation. Through CAD, we corroborated our results from the Li- and Na-adduct method and confirmed two additional Cl-adducts in SGRASS: 559.1497 and 601.1585 m/z. Not only did the fragmentation visually match for each set of potential M + 35Cl and M + 37Cl ions (as seen in Figure 2), but 35Cl− and 37Cl− product ions were observed for each corresponding adduct. The complete list of confirmed Cl-adducts can be found in Table 1. Characteristic Carbohydrate Fragments Observed. After confirming the attachment of chloride, the potential LCCs were analyzed for carbohydrate makeup by looking for characteristic carbohydrate product ions in the CAD spectra. Three stable carbohydrate derivative product-ions were observed in each spectrum, as seen in Figure 3. These three product-ions have been previously confirmed to derive from carbohydrate structures.16 The smallest, C4H6O3, has a m/z of 101.03 Da and

Figure 2. M and M + 2 peaks of a suspected Cl-adduct in SGRASS were fragmented. Matching fragmentation confirms that the observed 3:1 ratio is due to Cl-attachment. ratios, resulting from the two isotopes of chloride (35Cl and 37 Cl).13,15,16 It is possible, although less common, for large oligomers to form doubly chlorinated ions which exhibit a 9:6:1 ratio. This means by using chloride dopant we can tag potential LCCs in the mass spectrum with an isotopic marker for simple identification. Once peaks have been identified that have a characteristic Cl-adduct ratio, it becomes necessary to confirm that this ratio is due to chloride attachment. This can be done in several ways. The first, easiest method, is to look for a corresponding cationic Na- or Li-adduct ([M + Na]+ and [M + Li] +, respectively). This is done by collecting a mass spectrum in positive-ion mode and looking for a strong peak that has a mass-to-charge (m/z) ratio equal to that of the potential [M + 35Cl] peak minus the weight of a 35Cl ion plus the weight of a sodium or lithium ion. For sodium, this is the m/z of [M + 35Cl]− − 34.9688 + 22.9898 Da. For lithium, this is the m/z of [M + 35Cl]− − 34.9688 + 7.0160 Da. Although this method does not definitely confirm chloride attachment, it is a quick first pass for sorting through potential Cl-adducts. The second method for confirming chloride attachment uses tandem mass spectrometry. It is a more involved method but also more definitive. The specific type of tandem mass spectrometry used in this paper is collision-activated dissociation (CAD), where a single ion is identified, fragmented, and then reanalyzed. Since an [M + 35Cl]− and an [M + 37Cl]− ion have identical stabilities, they should fragment identically. Fragmentation patterns are considered a match if peaks with the greatest abundance have the same m/z in both spectra.

Table 1. Molecular Formulas Are Proposed for the LCCs and Corresponding Levels of Unsaturation Are Calculateda biomass SGUM

SGRASS

[M + Cl]− (Da)

M (Da)

proposed molecular formula

error (ppm)

level of unsaturation

401.0905 533.1434 575.1403 707.2006 749.2140 361.0600 559.1497 601.1585

366.1305 498.1834 540.1803 672.2406 714.2540 326.1000 524.1897 566.1985

C18H22O8 C34H26O4 C32H28O8 C34H40O14 C36H42O15 C15H18O8 C25H32O12 C27H34O13

2.7 −0.6 −4.3 1.8 −2.3 0.5 −0.6 2.6

8 22 19 15 16 7 10 11

a M was calculated by subtracting the mass of 35Cl from [M + Cl]−. Ppm error was calculated between M and theoretical molecular weight of the proposed molecular formula with less than ±5 ppm considered acceptable.

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DOI: 10.1021/acssuschemeng.8b01986 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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Figure 4. Plot of level of unsaturation versus number of carbons (CN) in proposed molecular formulas shows that both SGUM (blue) and SGRASS (orange) Cl-adduct ions have structures with hydrogen deficiencies more similar to free lignin compounds (gray) than free carbohydrates (yellow).

of 131.03 Da and is derived from water loss from xylose. The presence of these three stable product ions definitively confirms the carbohydrate nature of the LCCs and reveals new information about their structures: xylose and glucose units. Lignin-like Unsaturation Observed. In our final step, we confirmed the ions to be LCCs by substantiating the presence of lignin-like levels of unsaturation in the ions. Free lignins have a higher ratio of unsaturation:CN than free carbohydrates due to their conjugated aromatic backbone (one unsaturation for each double bond and one for each ring). Carbohydrates only have minimal unsaturation resulting from their unconjugated rings. Plotting carbohydrate and lignin structures from literature confirmed this disparity in ratio, as seen in Figure 4.16,19,20 Lignin derivatives maintain an average slope of 0.54, while carbohydrates follow a much lower trajectory with a slope of 0.15. Since the unsaturation:CN ratio of the LCCs closely follows the lignin derivative trajectory and not that of the carbohydrates, this not only confirms the presence of greater unsaturation indicative of lignin structures but infers that the LCC structures likely contain primarily lignin derivative structures with only one or two carbohydrate units per ion. Lignin-Carbohydrate Complex Structures Proposed. With the ions confirmed to be LCCs, we proceeded to analyze the structure of a low molecular weight LCC ion and a high molecular weight LCC ion through tandem mass spectrometry. Analysis of CAD spectra, fragmentation patterns, and product ions allowed us to propose a structure and a partial structure. The proposed structure of the low molecular weight SGRASS ion361.0600 m/zcan be seen in Figure 5. It contains a xylose ring attached to a guaiacyl unit by an ester linkage. This structure was proposed based on exhibited fragmentation patterns: loss of HCl, followed by loss of C5H8O4, followed by loss of CO2. Intermediate ring-opening fragments (loss of C2H4O2 and C3H6O3) support the presence of a five-membered heterocyclic carbohydrate ring, such as a xylose (C5H10O5).15 This structure was previously proposed in a study of TMS-derivatized acid plant hydrolysates.21 Herein we show evidence of the same proposed structure without the necessity of derivatization. The high molecular weight ion proved difficult to fully elucidate with a quadrupole-time-of-flight due to the inability to successively fragment the ion. With our single-collision study, we propose that the structure contains a terminal glucose unit, as seen in Figure 6, attached to the molecule by a conjugated ester linkage, similar to the low molecular weight ion in Figure 5. This is supported by the observed loss of C6H11O5Cl (198.0295 m/z)

Figure 3. Characteristic carbohydrate product ions observed in fragmentation of Cl-adducts confirms carbohydrate structures present in the target ion.

is derived from water loss from a ring-fragmented glucose. The next largest, C5H5O3, has a m/z of 113.02 Da and is derived from two water losses and a formaldehyde loss from deprotonated glucose. The largest, C5H7O4, has a m/z ratio D

DOI: 10.1021/acssuschemeng.8b01986 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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power means that CAD studies not only select the target Cl-adduct but also other ions that are isobaric to the Cl-adduct or are within a 0.7 Da range of the Cl-adduct. When additional ions are selected with the Cl-adducts, the CAD spectra of the [M + 35Cl]− and [M + 37Cl]− will not match identically because they will also include product ions from nearby ions. Lastly, QTOF-MS only allows a single collision event in tandem mass spectrometry, rather than the successive fragmentation that greatly enhances structure elucidation. Future studies employing successive collision events are underway and could greatly advance our understanding of the structures of high molecular weight LCCs.



CONCLUSION Through chloride doping and tandem mass spectrometry, we identified and analyzed eight undegraded LCCs in extracted AH-L, five in hardwood sweetgum and three in nonwood switchgrass. These complexes ranged in mass from 326−714 Da and exhibited both carbohydrate and lignin characteristics. Evidence of glucose and xylose units was revealed by tandem mass spectrometry. Further analysis of unsaturation levels showed high ligninlike unsaturation. These findings shed new light on the structure and mass range of LCCs. With a confirmed method for identifying LCCs, the path has been laid for greater structural elucidation of these enigmatic complexes.

Figure 5. Fragmentation of SGRASS Cl-adduct ion 361.0600 elucidates potential structure of low molecular weight LCC.



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: 919-515-6558. ORCID

Kelsey S. Boes: 0000-0002-4163-5075 Sunkyu Park: 0000-0002-9332-9061 Nelson R. Vinueza: 0000-0002-2608-5431 Author Contributions

Kelsey Boes performed the primary research and wrote the majority of the article. Robert Narron prepared samples and assisted in ideation and writing. Nelson Vinueza secured funding and assisted in data analysis. Sunkyu Park assisted in securing funding.

Figure 6. Fragmentation of SGUM Cl-adduct ion 707.2006 suggests terminal glucose unit on high molecular weight LCC.

Notes

which was previously observed in the fragmentation of chlorideadducted carbohydrates with terminal glucose units.15 Followingthe loss of the terminal glucose, loss of water (H2O, 18.0145 m/z) and then loss of the conjugated ketone linkage (C3H2O, 54.0104 m/z) are potentially observed. Further structural elucidation would require tandem mass spectrometry studies with two or greater CAD events. Limitations of Quadrupole-Time-of-Flight. We have demonstrated that QTOF-MS provides the tools to identify and elucidate the structure of an undegraded low molecular weight LCC and provides evidence toward partial structures of higher weight LCCs in extracted AH-L. Unfortunately, using a QTOF creates limitations and additional challenges to LCC analysis that a different mass spectrometer such as an ion-trap or Orbitrap would eliminate due to the QTOF’s midrange resolving power and the limit of tandem MS experiments to a single collision event. The midrange resolving power of the QTOF and complexity of the mixture mean that not all peaks that appear to have the 3:1 isotopic ratio of a Cl-adduct are in fact Cl-adducts. Unresolved isobaric peaks can sum in abundance to create the effect of a 3:1 isotopic ratio, which is why it is important to confirm chloride adduction. Additionally, the midrange resolving

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This interdepartmental project is generously supported by the North Carolina State University Startup Fund and the Chancellor’s Faculty Excellence Program.



REFERENCES

(1) Balakshin, M.; Capanema, E.; Gracz, H.; Chang, H.; Jameel, H. Planta 2011, 233, 1097−1110. (2) Lawoko, M. Int. J. Biol. Macromol. 2013, 62, 705−713. (3) Giummarella, N.; Zhang, L.; Henriksson, G.; Lawoko, M. RSC Adv. 2016, 6, 42120−42131. (4) Singh, R.; Singh, S.; Trimukhe, K. D.; Pandare, K. V.; Bastawade, K. B.; Gokhale, D. V.; Varma, A. J. Carbohydr. Polym. 2005, 62, 57− 66. (5) Yuan, T.-Q.; Sun, S.-N.; Xu, F.; Sun, R.-C. J. Agric. Food Chem. 2011, 59, 10604−10614. (6) Silva, V. L.; Jameel, H.; Gomes, F. J. B.; Batalha, L. A. R.; Coura, M. R.; Colodette, J. L. J. Wood Chem. Technol. 2017, 37 (1), 52−61. (7) Chen, X.; Lawoko, M.; Heiningen, A. v. Bioresour. Technol. 2010, 101 (20), 7812−7819.

E

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ACS Sustainable Chemistry & Engineering (8) Santos, R. B.; Jameel, H.; Chang, H.-M.; Hart, P. W. TAPPI J. 2013, 12 (7), 23−30. (9) You, T.-T.; Zhang, L.-M.; Zhou, S.-K.; Xu, F. Ind. Crops Prod. 2015, 71, 65−74. (10) Toikka, M.; Brunow, G. J. Chem. Soc., Perkin Trans. 1 1999, 0 (13), 1877−1883. (11) Nylander, F.; Sunner, H.; Olsson, L.; Christakopoulos, P.; Westman, G. Holzforschung 2016, 70 (5), 385−391. (12) Narron, R. H.; Chang, H.; Jameel, H.; Park, S. ACS Sustainable Chem. Eng. 2017, 5, 10763−10771. (13) Boes, K. S.; Narron, R. H.; Chen, Y.; Park, S.; Vinueza, N. R. Fuel 2017, 188, 190−196. (14) Cai, Y.; Cole, R. B. Anal. Chem. 2002, 74, 985−991. (15) Vinueza, N. R.; Gallardo, V. A.; Klimek, J. F.; Carpita, N. C.; Kenttämaa, H. I. Carbohydr. Polym. 2013, 98, 1203−1213. (16) Vinueza, N. R.; Gallardo, V. A.; Klimek, J. F.; Carpita, N. C.; Kenttämaa, H. I. Fuel 2013, 105, 235−246. (17) Kind, T.; Fiehn, O. BMC Bioinf. 2007, 8 (105), 105. (18) Patiny, L.; Borel, A. J. Chem. Inf. Model. 2013, 53, 1223−1228. (19) Banoub, J. H.; Benjelloun-Mlayah, B.; Ziarelli, F.; Joly, N.; Delmas, M. Rapid Commun. Mass Spectrom. 2007, 21, 2867−2888. (20) Banoub, J. H.; Delmas, M. J. Mass Spectrom. 2003, 38, 900− 903. (21) Mitchell, V. D.; Taylor, C. M.; Bauer, S. BioEnergy Res. 2014, 7, 654−669.

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DOI: 10.1021/acssuschemeng.8b01986 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX