Two-Stage Acidic–Alkaline Hydrothermal Pretreatment of Lignocellulose for the High Recovery of Cellulose and Hemicellulose Sugars
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- Guo, B., Zhang, Y., Yu, G. et al. Appl Biochem Biotechnol (2013) 169: 1069. doi:10.1007/s12010-012-0038-5
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The focus of this work was to develop a combined acid and alkaline hydrothermal pretreatment of lignocellulose that ensures high recovery of both hexose and pentose. Dilute sulfuric acid and lime pretreatments were employed sequentially. Process performance was optimized in terms of catalyst concentration, retention time, and temperature using response surface methodology. Medium operational conditions in the acid stage and harsh conditions in the alkaline stage were desirable with optimal performance at 0.73 wt% H2SO4, 150 °C, 6.1 min in the first stage, and 0.024 g lime/g biomass, 202 °C, 30 min in the second stage. In comparison to single-stage pretreatments with high recovery of either glucose or xylose, two-stage process showed great promises with >80 % glucose and >70 % xylose recovery. In addition, the method greatly improved ethanol fermentation with yields up to 0.145 g/g Miscanthus, due to significantly reduced formation of inhibitory by-products such as weak acids, furans, and phenols. Supplementing biomimetic acids would further increase glucose yield by up to 15 % and xylose yield by 25 %.
KeywordsTwo-stage acidic–alkaline pretreatmentMiscanthusCombined acid hydrolysisResponse surface methodologyLignocellulose
Utilizing lignocellulosic biomass as sustainable material has lately become a compelling alternative among conversion technologies in the biofuels and bio-based industry. Widely distributed and largely untapped, lignocellulose can continuously provide low-cost feedstock , which would avoid disturbing the food supply as is the problem with conventional biofuels. On the other hand, lignocellulose derived biofuels are not yet commercially feasible, due to the associated prohibitive conversion and feedstock logistics costs . Recently, it has been noticed that the unfavorable process economics can be improved by means of efficient co-utilization of cellulose and hemicellulose instead of cellulose fraction alone which was focused in the past . However, the stringent requirement of utilizing all lignocellulose components would impose great challenges on the existing conversion processes, especially the initial pretreatment step. Previously, the pretreatment process was designed with the major objective of effective cellulose recovery, and accordingly, a variety of pretreatment methods have been developed including physical, chemical, physicochemical, and biological methods and their combinations . Unfortunately, meanwhile, none of these methods can obtain high sugar recovery extensively from hemicellulose .
To achieve maximum multiple sugar yields simultaneously, pretreatment streamline was suggested to be divided into separate stages [6, 7]. It was well known that the severity of pretreatment conditions greatly affects the hydrolysis of lignocellulose components, especially hemicellulose . A severe condition would cause significant degradation of hemicellulose sugars into inhibitory compounds, while a relatively high degree of severity is still desirable to enhance the enzymatic digestibility of cellulose. Therefore, in the separate pretreatment process, varied severities were applied, where the first stage was conducted at low severity for efficient hemicellulose hydrolysis, and another stage under more severe conditions was followed to treat the remaining residue . In addition to different severities application, distinctive pretreatment methods were conducted in each stage to further improve the overall biomass utilization. This fractionation strategy was based on an essential feature that most pretreatment methods have varied preference to treat certain specific components. As such, acid pretreatment can be used to mainly hydrolyze hemicellulose while alkaline pretreatment to efficiently modify or remove lignin . Up to date, the scheme of sequential acid and alkaline pretreatment was investigated the most. A wide range of promising pretreatment methods have been employed including dilute acid hydrolysis, steam explosion, and hot water treatment in the acid stage succeeded by ammonia, alkaline peroxide treatment, and Organoslov process in the alkaline stage [10–13]. Many of them proved significantly improved yields of both cellulose and hemicellulose sugars and required fewer enzymes for hydrolysis than single-stage pretreatments.
Although the previous studies on two-stage pretreatments have verified the above-shown benefits, the effects of pretreatment conditions on the production of important hydrolysis products and the overall performance were still not well known. Additionally, there was lack of the basic knowledge of the degradation profiles and fates for major lignocellulose components throughout two-stage processes. All these absent information would be necessary for in-depth understanding of pretreatment mechanism and further process improvement of two-stage methods.
To bridge the knowledge gap, in this study, ACidic–ALkaline pretreatments in succession (ACAL pretreatment) were developed. The two-stage process was carried out with acid pretreatment at low severity in the first stage mainly for hemicellulose hydrolysis and then obtained efficient lignin removal and greatly enhanced cellulose digestibility in the second stage via alkaline pretreatment at elevated severity level. To make the process more commercially feasible, commonly applied dilute acid and lime pretreatments were utilized in each stage, respectively. The process was optimized by using response surface methodology (RSM) analysis. Finally, under the optimal conditions, two-stage acidic–alkaline pretreatments were compared with single-stage acid and alkaline pretreatments in terms of pretreatment effectiveness. The objective of this study was to evaluate the influence of major pretreatment conditions on ACAL process, quantitatively characterize the biomass components degradations, and clearly identify the advantages of ACAL process over single-stage pretreatments.
Materials and Methods
Miscanthus was used in this research as the model feedstock. The material was harvested in spring 2008 on the farm in Urbana, IL, and then air-dried below 45 °C to obtain dry matter content between 91 % and 94 %. The dried material was hammermilled, and the fraction passing through ¼-in. (6.35 mm) sieve was collected and analyzed for its contents of major components according to the National Renewable Energy Laboratory (NREL) standard procedures (Technical Report NREL/TP-510-42618). The chemical composition of the dry-based Miscanthus was 39.2 ± 0.3 % glucan, 19.5 ± 0.4 % xylan, 1.2 ± 0.1 % arabinan, and 24.2 ± 1.1 % lignin.
Pretreatment Setup and Operation
In the first stage of acid pretreatment, experiments were carried out in a batch reactor (Model 4534, PARR Instrument Co., Moline, IL) equipped with 2 L cylindrical pressure vessel (9.5 cm i.d.). One hundred twenty grams of dry-based Miscanthus samples were loaded for each batch with various acid solutions to keep a fixed solid loading of 20 % by weight. The pretreatment applied pure sulfuric acid solutions and sulfuric acid solutions mixed with biomimetic acids individually. The biomimetic acids used in this study were trifluoroacetic acid (TFA) and maleic acid (MA). Preceding the reaction in the vessel, the biomass was steeped in the acid solutions for 9 h at ambient temperature. After loaded with the reactants, the vessel was clamped shut and then heated at 6–8 °C/min. Counting of the reactions was started once the vessel reached the desired temperature, and the vessel was controlled at a constant temperature and pressure with agitation at 400 rpm. Once the pretreatment finished, the system was cooled down to 60 °C in about 10 min, and the pressure was released immediately thereafter. After completion of the acid pretreatment, the solids and liquids were separated through Whatman No.1 filter paper. Hydrolysates (liquid fractions) were stored for chemical analysis and further use in the fermentation tests. Solid residues were air-dried at 37 °C till reaching 90–95 % dry matter contents and then used in the second-stage alkaline pretreatment.
A different batch reactor (Model 4593, PARR Instrument Co., Moline, IL) was set up for the second-stage pretreatment with 100 mL cylinder-shaped pressure vessel (3.3 cm i.d.). The operation procedure of the reactor was the same as that of the acid pretreatment reactor. Differently, 6 g of dried solid residues from first-stage pretreatment were loaded with lime solution to bring the solid loading to 20 % by weight. After the second-stage reaction, the reacted biomass was filtered, and the liquid fractions were collected for chemical analysis. Solid residues were tested for enzymatic digestibility and blended with first-stage hydrolysates accordingly for fermentation tests.
Experimental Design and Statistical Analysis
Coded values of the tested variables at various levels
Range and levels
Acid dosage (wt% H2SO4)
Residence time (min)
Range and levels
Lime loading (g Ca(OH)2/g biomass)
Where y is the predicted response, xi and xj are coded values of the independent variables, β0, βi, βij, and βii are the Taylor expansion coefficients, and e is the error of the fitted model.
The regression and statistical analysis were carried out using Microsoft Origin 8.0, and the visualization of response surfaces were displayed by MATLAB 7.13.
Where wi (1 ≤ i ≤ m) is the weight factor for each desirability. In the study, we assumed all related individual by-products (furans and weak acids) contributed equally to the overall adverse effect on fermentation, and their own inhibitory effect was employed to interpret the individual desirability .
In this work, most pretreatment tests and all fermentation experiments were carried out in duplicate, while enzymatic hydrolysis was performed in triplicate. A 95 % confidence level was applied for data analysis.
The pretreated solid materials were enzymatically hydrolyzed following the NREL standard procedure (Technical Report NREL/TP-510-42629). Hydrolysis was conducted in 50 mM sodium citrate buffer (pH 4.8) at the loading of 1.0 wt% glucan content. Applied enzyme loadings were 15 FPU/g glucan of cellulase (Spezyme CP, Genencor), 2 CBU/FPU of β-glucosidase (Novozym 188, Sigma-Aldrich) supplemented with xylase (Multifect Xylanase, Genencor). The test flasks were incubated at 50 °C for 72 h, and hydrolysates were sampled every 24 h.
Simultaneous Saccharification and Co-Fermentation (SSCF)
Saccharomyces cerevisiae DA2416 was used as the host strain for producing ethanol from xylose and glucose in the pretreated hydrolysates. Methods for strain cultivation were described previously . Simultaneous saccharification and co-fermentation (SSCF) was carried out in 250 mL flasks containing 50 mL of YP (1 % w/v yeast extract, 2 % w/v peptone) with pretreated Miscanthus slurry including solid residue and hydrolysate (10 % w/v solid loading) at 30 °C and 100 rpm. The initial pH of medium was adjusted to 5.0 ± 0.1 through overliming (addition of Ca(OH)2 to pH 10–11 first, followed by H2SO4 down to pH 5). Yeast was inoculated with an initial cell concentration of 0.35 g/L. During SSCF, Spezyme cellulose cocktail (30 FPU/g hydrolysate), Novozyme 188 β-glucosidase (60 CBU/g hydrolysate) and Multifect xylanase (0.25 mL/g hydrolysate) were supplemented for saccharification of hydrolysate. After 48 h of SSCF, newly cultured cells (0.35 g/L) were added in order to enhance sugars consumption.
For pretreatment and enzymatic hydrolysis tests, the concentrations of monosaccharides, furans, and weak acids were measured using a high-performance liquid chromatography (HPLC) system (Shimadzu) equipped with a refractive index detector (Waters) as described previously . Oligosaccharides in the hydrolysates were broken down to monosaccharides through 4 % w/w sulfuric acid hydrolysis at 121 °C for 60 min for quantitative analysis by HPLC. Hydrolysates after pretreatment were analyzed for phenolic compounds by gas chromatography-mass spectrometry (GC-MS) system according to previously reported methods . Prior to the analysis, hydrolysate samples were extracted with ether twice at 3:1, and subsequently, the ether phase was concentrated by nitrogen bubbling. In addition, total phenols of the hydrolysates were determined using the Folin–Ciocalteu assay . Samples were diluted by water to adjust absorbance in 0.1–0.5, and total phenols were expressed in gallic acid equivalent.
For fermentation tests, glucose, xylose, xylitol, glycerol, acetate, and ethanol concentrations were determined by HPLC system (Agilent Technologies 1200 Series) equipped with a refractive index detector using a REzex ROA-Organic Acid H+ (8 %) column (Phenomenex Inc., Torrance, CA). The column was eluted with 5 mM sulfuric acid at 0.6 mL/min at 50 °C.
All the chemicals used in the study were purchased from Fisher Scientific (Pittsburgh, PA) and Sigma-Aldrich (St. Louis, MO).
Results and Discussion
The Effect of Pretreatment Conditions on Acid Stage Performance
As can be observed in Fig. 1a, the isocontours of solubilized xylose yield described a score of partial concentric elliptic shells. Under mild conditions, xylose yield increased with all three variable values, but further raising the levels of operational variables into harsher conditions would result in evident drop in xylose yield. General ranges of acid dosage in 0.8–1.0 wt% sulfuric acid, temperature 145–155 °C, and residence time less than 30 min were desirable to obtain the maximized xylose recovery during the pretreatment. The optimal conditions can be achieved at the center of ellipsoids [coded values of (0.58, −0.58, −0.97)] at 0.90 wt%, 151 °C, and 15.3 min with maximal xylose recovery of 13.9 % dry biomass (62.5 % theoretical). These conditions were comparable to the optimal ranges of 0.9–1.8 wt%, 140–153 °C, and 6–40 min by dilute acid pretreatment on various biomass in other reports [14, 22–24], although xylose yield was lower than those reported 76–93 % theoretically, probably in favor of their lower applied solid loading (5–7 %). In addition, the optimal combined severity factor (1.7) was also lower than 2.0–2.3, the only reported value by dilute acid pretreatment on Miscanthus . Besides, Fig. 1a also presented that the ellipsoids were elongated along the axis of residence time, which indicated less influence of time on xylose yield than the other two parameters. Here, glucose yield was not taken into account for the process optimization in the acid stage, since the primary target was hemicellulose hydrolysis to xylose.
Figure 1b showed contour plots of composite response surface through desirability function approach integrating acetic acid, furfural, and HMF yields. All three hydrolysis by-products would exert evident inhibitory effects on the downstream fermentation. However, at the induced concentration in this study (3.8–15.2 g/L acetic acid, 0.9–13.2 g/L furfural, 0–3.0 g/L HMF), acetic acid presented the greatest inhibition. Additionally, the formation of three by-products increased with pretreatment severity, although as for acetic acid it tended to level off at higher severity level (data not shown). Based on the different inhibitory effects of furans and acetic acid, the impact of furans changed remarkably at greater presence, while that of acetate moved faster at low concentration. When taking account of concentration and individual effect, the composite contour plots described steadily decreasing overall desirability as severity level increased, which meant continuously intensifying inhibitory effects. At low severity, acetic acid contributed the most to the overall desirability change whereas furans took over at high severity. In addition, the isoresponse contour surfaces tuned parallel to the axis of residence time while above 25 min. This implied that any extended reaction time would not significantly affect the hydrolysis after 25 min pretreatment.
Yields of primary phenols (concentration > 5 mg/L) in the hydrolysates (in milligrams per liter)
Mild (−1, −1, −1)
Higher lime loading (1.4,0)
Lower lime loading (−1.4,0)
Higher temp. (0,1.4)
Lower temp. (0, −1.4)
2-(4-Hydroxyphenyl) propionic acid
3-(4-Hydroxyphenyl) propionic acid
Up to date, the influence of operational conditions on the performance of dilute acid pretreatment has been intensively studied, but most of them only focused on sugar recovery [14, 24, 28, 29]. Several studies reported on furans and acetate productions, with limited information provided on the effect by single pretreatment parameter [30, 31], while there is no report on phenols yield. Here, the effect of pretreatment conditions on xylose, furans, acetate, and phenols were described, and their interactive tendencies can be observed when all three graphs in Fig. 1 were put together. It clearly indicated the conditions for maximal xylose yield were not the best pretreatment conditions overall due to strong induction of most inhibitory compounds. In fact, the operational severity leveraged the reaction favorability between hemicellulose decomposition and xylose degradation. Employment of concentrated acid and elevated temperature may provide an acidic environment that accelerates formation of furfural from xylose and induces pyrolysis of lignin into phenolic compounds . In this regard, medium severities would be suggested to obtain acceptably high xylose yield as well as reduced by-products formation that facilitates the xylose fermentation as a whole. In this study, the best pretreatment conditions were located at 0.73 wt%, 150 °C, and 6.1 min. Under these conditions, the pretreatment assured 12.5 % of xylose yield (56.3 % theoretical) and achieved by-products formation of 1.95 g/L furfural, 6.02 g/L acetic acid, and negligible HMF. Furthermore, residence time was found to have little effect on all major products production, so it could be consider least in the further process development of acid pretreatment.
All the quadratic models were tested for adequacy by the analysis of variance. They were highly significant, and the coefficients of determination (R2) were all above 0.9. The chosen optimal conditions were confirmed by pretreatment tests with variances of all major product yields less than 5 % compared with the model predicted values.
The Effect of Pretreatment Conditions on Alkaline Stage Performance
As shown in Fig. 2a, glucose release after enzymatic hydrolysis was mainly affected by temperature but lime loading. Along with temperature increase, glucose yield first increased but then declined. On the other hand, at higher temperature, glucose release was facilitated as lime loading increased, while the opposite tendency was observed at lower temperature. High glucose yield of 0.4 g/g residue can be attained at nearly all applied lime loadings if medium temperature range of 185–220 °C was applied. Contrarily, the profile of weak acids in Fig. 2b was simple. The overall desirability reduced continuously with both lime loading and temperature, which meant generally more acetic, formic, and levulinic acids were induced from the release of acetyl group during hemicellulose and furans degradation. Through the hydrolysis, great presences of acetic and formic acids were detected, with concentrations of 4.9–9.4 and 1.6–10.3 g/L, respectively (levulinic acid 0.3–0.6 g/L in contrast). It was important to note that, contrary to the primary trends shown in this figure, formic acid formation decreased to varied extent when temperature was raised up. As for the case of furans shown in Fig. 2c, the overall desirability was strongly affected at low lime loadings. In fact, the inhibitory effect of furans was mainly attributed to HMF due to its high concentration in the hydrolysate (up to 3.1 g/L). HMF formation accelerated at high temperatures, especially with low lime loading. However, interestingly, HMF accumulation reduced with more lime used in the pretreatment but leveled off at high lime loading. Putting three plots together in Fig. 2, we can conclude that similarly as in hemicellulose hydrolysis under acid conditions, during lime pretreatment, raising temperature could facilitate cellulose hydrolysis, but high temperature noticeably further degraded glucose to other by-products. However, lime could slow down the latter unwanted side reaction to certain extent. Besides, the remaining hemicellulose after acid pretreatment would not only be hydrolyzed to xylose but mostly further down to formic acid.
Primary phenolic compounds generated through lime pretreatment were listed in Table 2 along with their concentrations. It has been found that the phenols present in hydrolysates were strongly dependent on the pretreatment type . For that matter, occurrence of different phenols in lime-treated hydrolysates was noted in comparison with previous acidic hydrolysates. As a result, all phenols were produced through acid pretreatment, but ferulic acid was found during lime pretreatment. Furthermore, lime pretreatment generated some unique phenols like syringol and methylhydroquinone. Among the detected phenols, vanillin and syringol were the most abundant. In addition, most phenols through lime pretreatment were lignin blocks with more complicated structure, which suggested that alkaline pretreatment led to incomplete lignin breakdown compared with acid pretreatment. We can also learn from Table 2 that generally higher operational severities could induce more phenols production.
It has been found that high glucose recovery can be obtained under two conditions during alkaline pretreatment, either long pretreatment time and low temperature, or high temperature for a short time . Previously, alkaline pretreatment was commonly employed at lower temperatures (50–130 °C) for extended times on the order of hours, to avoid the great loss of hemicellulose. In this work, most hemicellulose was removed in the prior acid stage, so lime pretreatment can be explored at temperatures above 170 °C with much shortened reaction time, more favorable from an economic perspective. In fact, the applied temperatures were even higher than the previous stage to attain elevated severities for enhanced biomass susceptibility to enzymatic hydrolysis. Similar as in dilute acid pretreatment, little was known about the effects of pretreatment conditions on lime pretreatment performance especially their interactive effects [16, 33]. Other than that, since acid pretreatment was applied ahead, different profiles after lime pretreatment could be expected in this case. Indeed, small amount of lime was necessary, and there was different effect of applied temperature at high levels on glucose recovery. Normally, a lime loading of up to 0.1 g/g of dry biomass was recommended in terms of high sugar recovery [32, 34], but the amount needed was reduced to as low as 0.01 g/g in the current study. Apparently, lime appeared to be more active at elevated temperature to disrupt the cellulose crystallinity and increase the biomass porosity. Meanwhile, on the flip side, enhanced lime activity also meant calcium ions could easily interact with lignin and carbohydrates with high affinity and thus impact glucose release , implying redundant lime addition was of no benefit. It can be demonstrated by the noticeable decline of glucose yield with increased lime loading at low temperatures in Fig. 2a. In addition, at elevated temperatures, significant drop of glucose yield occurred from its own degradation, which was not observed at mild temperatures. Stripping off most hemicellulose and significant alternation of lignocellulose structure prior to alkaline stage would also cause the cellulose more sensitive to the temperature.
For the optimization of lime pretreatment, when taking account of the four major groups of products (sugars as glucose, weak acids, furans, and phenols), a compromise was made, and the best conditions were located at 0.024 g/g biomass of lime loading and 202 °C. Under these conditions, glucose yield was among the highest (78.2 % theoretical) with generally lower acetic acid, furan, and phenol production, as discussed in the following section.
Fates of Lignocellulose Components
Operational conditions of various pretreatment schemes for comparison
1.0 wt% H2SO4, 170 °C, 15 min
0.024 g/g Ca(OH)2, 202 °C, 30 min
0.73 wt% H2SO4, 150 °C, 6 min
0.024 g/g Ca(OH)2, 202 °C, 30 min
0.375 wt% H2SO4 + 4 mg/L TFA, 150 °C, 6 min
0.024 g/g Ca(OH)2, 202 °C, 30 min
0.548 wt% H2SO4 + 15.6 g/L MA, 150 °C, 6 min
0.024 g/g Ca(OH)2, 202 °C, 30 min
For cellulose degradation products, as can be observed from Fig. 3a, single acid pretreatment (P1) left considerably more cellulose intact than the other pretreatment schemes. It verified that acid catalysts were not efficient in glucose recovery. In contrast, ACAL (P3) led to nearly the same profile of cellulose degradation products as single alkaline pretreatment (P2). Recovered glucose mainly came from the treated residue after enzymatic hydrolysis, indicating the second alkaline stage played the key role for glucose recovery. When combined acid catalysts were adopted in ACAL (P4/P5), glucose recovery was further improved by 8–23 %. On the other hand, for hemicellulose degradation products shown in Fig. 3b, the profile of ACAL was similar to single acid pretreatment instead. As was reported previously, lime pretreatment would be ineffective for hemicellulose decomposition . But unexpectedly here, nearly all the degraded hemicellulose went directly down to furfural. It appeared that lime was more efficient in catalyzing xylose degradation than hemicellulose decomposition, although further work was required for verification. On the contrary, single acid pretreatment could convert most hemicellulose into xylose. However, since an elevated severity was applied as not to lose much glucose, a fair amount of xylose was inevitably degraded to furfural at the same time. ACAL could achieve efficient hemicellulose decomposition, primarily in the acid stage. Meanwhile, the separate pretreatments in ACAL allowed a low severity application in the acid stage and ensured higher xylose recovery. Similarly as for cellulose profile, introduction of combined acid catalysts in ACAL could obtain higher xylose yield through thorough conversion of oligomeric xylose.
Major products in the prehydrolysates under various pretreatment schemes
Weak acids (g/L)
5.7 (81 %)
7.3 (80 %)
8.6 (79 %)
1.3 (23 %)
1.2 (20 %)
1.1 (25 %)
0.9 (97 %)
1.1 (96 %)
1.5 (88 %)
Total phenols (g/L)
3.0 (29 %)
3.9 (18 %)
2.9 (24 %)
Overall Pretreatment Effectiveness
The overall performance of two-stage acidic–alkaline pretreatment was scrutinized and compared with other tested pretreatment alternatives in terms of sugar yields and ethanol yields.
Concentrations of major compounds in the SSCF hydrolysates under various pretreatment schemes (in grams per liter)
Glucose at 0 h
Glucose at 48 h
Glucose at 96 h
Xylose at 0 h
Xylose at 48 h
Xylose at 96 h
Ethanol at 48 h
Ethanol at 96 h
Ethanol yield (grams per gram of dry Miscanthus)
Ethanol yield of theoretical maximum
A pretreatment method with successive acidic and alkaline stages (ACAL pretreatment) was developed. In contrast to single-stage pretreatments which efficiently obtain one sugar product alone, two-stage process could achieve high recovery of both glucose (>80 %) and xylose (>70 %). Xylose was mainly recovered from acid stage, while glucose was secured through lime pretreatment. Meanwhile, production of weak acids, furans, and phenols were remarkably reduced. The best performance could be arrived at medium severities in the acid stage and high severities in the alkaline stage. Integration of combined acid catalysts and ACAL could further improve both sugar yields and reduce primary by-products formation, with ethanol yield of up to 0.145 g/g Miscanthus.
This research was partially funded by Illinois Council on Food and Agricultural Research (C-FAR) under Sentinel Program. We are grateful for the courtesy of Department of Crop Science for providing Miscanthus samples. We would like to express our gratitude towards Dr. Alexander Ulanov from Metabolomics Center at the University of Illinois at Urbana-Champaign for phenolic compounds analysis. Won-Heong Lee and Yong-Su Jin are affiliated with the Energy Biosciences Institute.