Guayule as a feedstock for lignocellulosic biorefineries using ammonia fiber expansion (AFEX) pretreatment

Industrial Crops and Products 37 (2012) 486–492

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Guayule as a feedstock for lignocellulosic biorefineries using ammonia fiber expansion (AFEX) pretreatment Shishir P.S. Chundawat a,b,∗ , Linpei Chang a , Christa Gunawan a , Venkatesh Balan a,b , Colleen McMahan c , Bruce E. Dale a,b a

Biomass Conversion Research Lab (BCRL), Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, MI, United States Great Lakes Bioenergy Research Center (GLBRC), East Lansing, MI, United States c United States Department of Agriculture, Agricultural Research Service, Western Regional Research Laboratory, Albany, CA, United States b

a r t i c l e

i n f o

Article history: Received 2 June 2011 Received in revised form 18 July 2011 Accepted 23 July 2011 Available online 19 August 2011 Keywords: Guayule AFEX pretreatment Enzymatic hydrolysis Cellulosic biofuels

a b s t r a c t Natural rubber latex extraction from guayule leaves behind greater than 90% (by weight) of agricultural residue as a feedstock suitable for conversion to biofuels via a thermochemical or biochemical route. Untreated guayule shrub and bagasse (after latex extraction) has shown to be very recalcitrant to enzymatic hydrolysis, necessitating application of a chemical pretreatment to enhance cellulase accessibility. The objective of this work was to carry out detailed compositional analysis, ammonia fiber expansion (AFEX1 ) pretreatment, enzymatic hydrolysis and ethanol fermentation for various guayule-derived biomass fractions. Plant feedstocks tested were derived from two sources; (a) a mature 2007 AZ-2 whole guayule shrub plant obtained from USDA/ARS2 research fields, and (b) the guayule latex-extracted commercial grade bagasse (62505) from Yulex Corporation. Compositional analysis and enzymatic hydrolysis were carried out using standard NREL3 protocols (www.nrel.gov/biomass/analytical procedures.html). AFEX pretreatment was carried out using concentrated ammonium hydroxide at elevated temperatures for desired residence times in a pressurized reactor. Yeast fermentations on biomass hydrolyzates were carried out micro-aerobically using Saccharomyces cerevisiae (424A strain) in shake flasks. AFEX pretreatment was found to substantially improve overall enzymatic digestibility by 4–20 fold for both untreated guayule shrub and latex-extracted bagasse. Maximum glucan and xylan conversion achieved for the latex-extracted bagasse was 40% and 50%, respectively. The yeast was readily able to ferment both glucose and xylose to ethanol from the guayule bagasse hydrolyzate with or without external nutrient supplementation (i.e., yeast extract and tryptone). Our results highlight the possible utilization of guayule as a feedstock for lignocellulosic refineries co-producing natural rubber latex and biofuels. However, further process improvements (e.g., lignin/resin extraction and cellulose decrystallization using a modified AFEX process) are necessary to increase the effectiveness of ammonia-based pretreatments for further enhancing enzymatic digestibility of guayule-derived hardwood biomass. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Guayule (Parthenium argentatum), part of the Asteraceae family, is a perennial shrub native to the North American Chihauhaun desert that synthesizes natural rubber, cis 1,4-polyisoprene, equivalent to that of the Hevea tree (Hevea brasiliensis) (Hammond and Polhamus, 1965). Natural rubber from guayule can be used as an

∗ Corresponding author at: 3900 Collins Rd, Suite 1045, Lansing, MI 48910, United States. Tel.: +1 517 432 0157. E-mail address: [email protected] (S.P.S. Chundawat). 1 AFEX: ammonia fiber expansion. 2 USDA/ARS: United States Department of Agriculture/Agricultural Research Service. 3 NREL: National Renewable Energy Laboratory. 0926-6690/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.indcrop.2011.07.025

alternate source of latex for manufacture of rubber tires, medical devices, and numerous other products. Guayule also produces appreciable quantities of resins, extractable terpenes, fatty acids, and other useful biobased compounds (Chow et al., 2008). Natural rubber and resins comprise 10–20 dry wt% of the plant, leaving 80+% of the biomass as a waste crop residue. One of the methods used to extract rubber is using an environmentally friendly aqueous-based extraction process that grinds the harvested shrub to produce a suspension of rubber particles in buffer known as guayule latex (Cornish, 1996, 1998). Following latex extraction the waste residual lignocellulosic co-product, referred to as bagasse, could be further developed into bioenergy feedstock, composite boards, soil amendments, and construction related materials (Nakayama, 2005). Biofuels can be produced from lignocellulose biomass via biochemical or thermochemical catalysis of cellulose, hemicellulose

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Guayule Plant YULEX® Latex Extraction Process

LATEX

Whole Guayule Shrub (GS)

Guayule Bagasse (GB)

+ Resin Extracted (GSA)

+ Resin Extracted (GBA)

RESIN RUBBER + Rubber Extracted (GSC) Fig. 1. Guayule plant derived laboratory (GS-guayule shrub) and industrial (GBguayule bagasse) grade bagasse. Where GS was extracted with acetone to remove resins to produce GSA, which was further extracted with cyclohexane to remove rubber to produce GSC. GB was extracted with acetone to produce GBA, which contained 1.5% residual rubber.

and lignin (Chundawat et al., 2011a). Cellulose can be converted into alcohols, glycerols, ketones, and acids (via fermentation route); levulinic acid and hydroxymethyl furfural (via acid hydrolysis and dehydration route); and phenolics and aromatics from lignin via acid hydrolysis and hydrogenation, respectively (Chow et al., 2008; Chundawat et al., 2011a). As harvested acreage of guayule expands, the development of guayule bagasse-derived biofuels could positively impact the sustainability of this industrial crop and contribute to future world energy demands. Previous efforts on pretreatment of guayule bagasse with various chemicals/solvents (e.g., NaOH, cadoxen) followed by enzymatic hydrolysis have yielded low glucose yields due to the fact that the bagasse is very resistant to hydrolysis (Chang and Tsao, 1980). Srinivasan and Ju (2010) have shown that supercritical CO2 pretreatment of guayule bagasse (whole guayule shrub was not tested in that study) is effective in giving a readily digestible feedstock, although only at high enzyme loadings (∼75–80 FPU cellulase/g glucan). The ammonia fiber expansion (AFEX) process is used to pretreat lignocellulosic material which results in delocalizing lignin and hemicellulose, decrystallizing cellulose, and increasing polysaccharide accessibility to enzymes (Chundawat, 2009; Chundawat et al., 2010a, 2010b, 2011a, 2011b, 2011c; da Costa Sousa et al., 2009). Previous work on various lignocellulosic biomasses (e.g., corn stover, switchgrass, poplar) using this pretreatment method has given near theoretical glucan and xylan hydrolysis yields (Balan et al., 2009a; Teymouri et al., 2005). The objective of this work was to carry out detailed compositional analysis, AFEX pretreatment, enzymatic hydrolysis (at industrially viable conditions of lower cellulase loadings; ∼10 FPU/g glucan) and ethanol fermentation (with/without external nutrient supplementation at low initial cell density) studies on various guayule-derived biomass fractions isolated from the latex extraction process to examine the feasibility of using guayule as a cellulosic biorefinery feedstock.

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Sunnyvale, CA, USA). Ten extractions were performed at 100 ◦ C with acetone to fully remove the plant resins (about 8 dry wt%) creating sample GSA. The acetone-extracted material (GSA) was then subject to ten additional extractions with cyclohexane at 140 ◦ C to fully remove the rubber (about 5 dry wt%) creating sample GSC. In addition, guayule latex-extracted ‘commercial’ bagasse (62505) was provided by Yulex Corporation (Maricopa, AZ, USA) as sample GB. This material, the leftover bagasse fraction following latex extraction, is mainly a mixture of wood and bark because the shrubs are defoliated prior to latex extraction. Quantitative extraction (ASE 200, Dionex Corporation, Sunnyvale, CA, USA) determined sample GB contained 7.5% acetone (resin) and 1.5% cyclohexane (solvent-extractable rubber) extractable material. Residual rubber is always found in latex-extracted guayule, since some coagulation is inevitable post-harvest and the aqueous process can only capture the water-soluble latex. Sample GB was ASE extracted 10 times with acetone at 100 ◦ C to fully remove all resin, creating sample GBA. 2.2. AFEX pretreatment A detailed methodology for AFEX pretreatment (Balan et al., 2009a) and its underlying physico-chemical mechanism is provided elsewhere (Chundawat et al., 2011c). The AFEX reactor consisted of a 22 mL #316 stainless steel pressure vessel (PARR Instrument Co, Moline, IL, USA). Feedstock with the appropriate moisture content (e.g., 0.6 g moisture/g dry biomass or 60% moisture loading) was loaded into the vessel. The vessel was clamped shut and the required amount of ammonia was injected using a preweighed cylinder. The reactor was placed in a slotted aluminum block attached to a Vela hot plate (Cole Parmer, Vernon Hills, IL, USA). For even heating and good heat transfer, the slots in the heating block were precision-milled to enable a tight fit around the pressure vessel. The reactor was maintained at the desired reaction temperature throughout the experiment. The residence time of the reaction depended on the feedstock treated, usually 30–60 min to complete one AFEX reaction. The pressure was explosively released by opening a 0.5 in. NPT #316 stainless steel ball valve at the end of the pretreatment residence time. The biomass was removed from the reactor and dried overnight in a fume hood to remove residual ammonia. 2.3. Compositional analysis Polysaccharide (e.g., glucan, xylan, arabinan, mannan, galactan), lignin, acetyl and ash content analysis was performed following the NREL LAP protocols (NREL, 2008). Acid soluble lignin was calculated using an absorptivity factor equal to 30.2 that corresponded to corn stover. Mass balance closures ranging between 75 and 95% were obtained in most cases. All chemicals were procured from Sigma (Sigma–Aldrich, St. Louis, MO, USA).

2. Materials and methods 2.4. Enzymatic hydrolysis 2.1. Biomass substrates The guayule ground shrub (GS) used in several experiments (Fig. 1) was a mature AZ-2 plant harvested from research fields at USDA/ARS US- Arid Lands Research Center, Maricopa, AZ, USA, in March 2007. The shrub was coarsely dry chipped immediately following harvest, and then shipped overnight to the USDA/ARS, Albany, CA, where it was processed through a Corenco model M6 grinder at 3500 rpm to produce a finely divided solid ground material. This sample represents a mature, intact guayule plant, with all plant tissues, and all rubber and resin components. Resin and rubber were then sequentially extracted from the GS material using an Accelerated Solvent Extractor (ASE 200, Dionex Corporation,

Enzymatic hydrolysis procedure was based on the NREL LAP009 standard protocol (NREL, 2008). All samples were hydrolyzed in a 0.05 M citrate buffer (pH 4.5) at 1% glucan loading (15 mL reaction volume) with the necessary commercial cellulase (Spezyme CP, Genencor-Danisco, Rochester, NY, USA), ␤-glucosidase (Novo 188, Sigma–Aldrich, St. Louis, MO, USA), and accessory enzymes (e.g., Multifect Xylanase and Pectinase). Multifect Xylanase, and Multifect Pectinase were kindly provided by Genencor-Danisco (Rochester, NY, USA). Spezyme CP, Novo 188, Multifect Xylanase and Multifect Pectinase protein concentrations, as estimated by Kjeldahl Nitrogen analysis (Chundawat, 2009; Chundawat et al., 2011d), were 88, 150, 35 and 90 mg total protein/mL,

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respectively. All hydrolysis experiments were conducted using low or high enzyme loadings. Low enzyme loading experiments were carried out using 4.4 mg Spezyme CP/g dry biomass (DBM) supplemented with Novo 188 (7.5 mg enzyme/g DBM) to prevent cellobiose inhibition. High cellulase loading experiments were carried out using 17.6 mg Spezyme CP/g dry biomass (DBM) supplemented with Novo 188 (15 mg enzyme/g DBM), Multifect Xylanase (3.5 mg enzyme/g DBM), and Multifect Pectinase (9 mg enzyme/g DBM). All samples were incubated at 50 ◦ C at 150 rpm rotation. The hydrolyzed samples were boiled 20 min to denature the enzymes and filtered through a 0.2-micron nylon membrane filter at predetermined time periods (i.e., 24 and 168 h). The samples were then frozen (−20 ◦ C) for subsequent sugar analysis using the HPLC. All experiments were carried out in duplicates. Sugar identification and quantification was performed using a Waters (Waters Corporation, Milford, MA, USA) high performance liquid chromatograph (HPLC) system equipped with a Bio-Rad (Bio-Rad, Hercules, CA, USA) Aminex HPX-87P carbohydrate analysis column. All sugar standards were procured from Sigma (Sigma–Aldrich, St. Louis, MO, USA). Degassed HPLC grade water with a 0.6 mL/min flow rate was used as the mobile phase at a column temperature of 85 ◦ C. The injection volume was 10 ␮L with a run time of 20 min. Different sugar standards were used to quantify cellobiose and other monosaccharides (glucose, xylose, galactose, arabinose, and mannose) present within the samples. Error bars in figures represent standard deviations between duplicate assays. 2.5. Yeast fermentation Yeast seed cultures were prepared by inoculating the frozen glycerol stock of Saccharomyces cerevisiae 424A (LNH-ST) with an initial cell density (OD600 ) of 0.1 in 100 mL of YEP media (10 g/L yeast extract and 20 g/L tryptone) containing 50 g/L glucose in 250 mL shake flask. Pre-cultures were grown for 20 h in microaerobic conditions with 150 rpm agitation at 30 ◦ C. The fermentation was performed microaerobically in 25 mL shake flask containing 9 mL of guayule hydrolysate (Note: AFEX pretreated GB at 150 ◦ C, 1:1 ammonia loading, 100% moisture and 30 min reaction time was hydrolyzed for 168 h using low cellulase loading as described previously) with or without the addition of YEP media at 150 rpm, 30 ◦ C. The pH of hydrolysate was adjusted to 5.5 using 5 M KOH prior to inoculation. Cell density of pre-culture was measured and the value was used to calculate the amount needed for inoculation with initial cell density of 0.5. Cell pellets were harvested by centrifugation at 13,000 rpm for 15 min and then re-suspended into the media. Samples were taken at 3, 6, and 24 h. Cell density was measured by reading the optical density using UV/Vis Spectrophotometer (DU720, Beckman Coulter, Brea, CA, USA) at wavelength 600 nm in 1 cm path length cuvette. Concentrations of residual glucose and xylose, as well as ethanol produced were measured using a HPLC (LC-20AD, Shimadzu, Columbia, MD, USA) equipped with an auto-sampler (SIL-HTc, Shimadzu, Columbia, MD, USA) and refractive index detector (RID-10A) by injecting 10 ␮L of sample into the Biorad Aminex-HPX 87H (Bio-Rad, Hercules, CA, USA) column. The HPLC was operated at 50 ◦ C using 5 mM sulfuric acid as the eluent at 0.6 mL/min. Sugars were quantified by plotting standard curves of the concentrations of standard solutions and corresponding peak heights. 3. Results and discussion 3.1. Compositional analysis Compositional analysis was determined for all substrates prior to pretreatment and enzymatic hydrolysis (Table 1). The glucan and

xylan content for most samples ranged between 16–30% and 9–18%, respectively. There was also a significant amount of arabinan present in these substrates that probably cross-links hemicellulose via ester-linkages potentially impeding enzyme accessibility. Overall compositional analysis results were similar to previous reports for guayule biomass pentose (Srinivasan and Ju, 2010), and hexose (Boateng et al., 2009; Srinivasan and Ju, 2010) content. Guayule bagasse has significantly higher glucan and xylan content, likely due to the removal of leaves (up to 30% DBM) prior to processing. The expected higher hardwood content was not, however, reflected in relative lignin levels (34.7% for shrub, 36.1% for bagasse). Published results (Boateng et al., 2009; Chow et al., 2008; Srinivasan and Ju, 2010) for lignin content of guayule biomass vary widely (20–50%). This is probably due to variation in leaf content, and/or significant contributions from residual latex/resin, protein and/or ash content. Our values indicate about one-third by weight of the biomass is composed of lignin, not unusual for a hardwood plant. Acetone and acetone–cyclohexane extractions on GS and GB reduced the Klason lignin content, but had little effect on sugar composition. Acetone and cyclohexane are known to remove natural plant resins and residual rubber (Chow et al., 2008; Nakayama, 2005). It is possible that guayule-derived resin and latex components contribute to the stability of the insoluble lignin quantified, or that repeated heat/solvent exposure affect lignin stability. Note that ten cycles of hot cyclohexane extraction increased the ash content of guayule shrub four-fold. Enzymatic hydrolysis of untreated guayule resulted in extremely low hydrolysis yields (Fig. 2). Untreated guayule shrub derived (GS, GSA, GSC) substrates gave 4–8% glucan and 1–2% xylan conversions. Glucan and xylan conversion (168 h) after acetone and/or cyclohexane extractions of untreated GB and GS was marginally higher. Interestingly, glucan digestibility for bagasse (GB, GBA; GB is comparable to feedstock used by Srinivasan and Ju, 2010) derived samples was substantially lower than guayule shrub (GS) derived substrates. Also, the digestibility of untreated GB was 5–7 fold lower than what has been reported recently due to significantly lower cellulase loadings employed in this study (Srinivasan and Ju, 2010). There are two main differences between the biomass sources, shrub and bagasse, (1) the aforementioned leaf content, and (2) the heat and solvent history. Comparing shrub GS to bagasse GB, the lower glucan conversions for the bagasse GB sample are likely due to exposure to shear heat and high pH in the latex extraction process. The bagasse was exposed to additional heat during ambient air drying (over 40 ◦ C daily high temp) that may have further resulted in cellulose microfibrils collapse/hornification (Himmel, 2008). In the future, it would be pertinent to minimize extensive heating and drying of the latex-extracted bagasse samples in order to maximize their enzymatic digestibility. Coupling a pretreatment and hydrolysis process along with latex-extraction process on-site would minimize the necessity to dry the bagasse samples and could further increase saccharification yields.

3.2. Effects of AFEX reaction temperature on guayule digestibility AFEX treatment significantly increased the efficiency of enzymatic hydrolysis for guayule. In most cases, biomass conversion increased with increasing temperature. The effect of reactor temperature on the subsequent enzymatic hydrolysis of AFEX-treated guayule samples is shown in Fig. 3 (glucan to glucose conversion) and Fig. 4 (xylan to xylose conversion). Interestingly, increasing the reaction temperature even further (from 130 ◦ C to 150 ◦ C) helped enhance the glucan conversion quite substantially (data for GB and GBA shown in Fig. 5). This suggests that higher reaction temperatures (>130 ◦ C) during AFEX delocalize the lignin, resin and residual

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Table 1 Percent composition (on dry weight basis) for guayule plant and derived fractions. Standard deviation for replicates in all cases was within ±10% of the mean value reported. Where GB, GBA, GS, GSA, GSC and “*” stands for guayule bagasse, guayule bagasse after acetone extraction, whole guayule shrub, whole guayule shrub after acetone extraction, whole guayule shrub after acetone/cyclohexane extraction and “not detected”, respectively. Samples

Glucan

Xylan

Galactan

Arabinan

Mannan

Klason lignin

Acid soluble lignin

Ash

Acetic acid

GB GBA GS GSA GSC

27.1 29.3 19.3 22.1 16.6

16.4 17.7 9.8 11.6 9.0

1.7 2.2 2.4 2.7 1.9

2.6 2.9 4.3 4.7 3.4

* * * * *

36.1 32.1 34.7 29.1 18.9

1.4 1.4 2.1 2.3 1.8

1.8 2.0 5.4 5.4 19.0

4.3 4.5 3.5 4.0 2.7

10

168 h 24 h

% Conversion

8 6 4 2 0

G

X

G

GB

X

G

GBA

X

G

GS

X

G

GSA

X

GSC

Fig. 2. Glucan (G) and xylan (X) enzymatic hydrolysis yields (after 24 and 168 h) for untreated guayule derived biomass at low enzyme loading (11.9 mg/g glucan). Where GB, GBA, GS, GSA, and GSC stand for guayule bagasse, guayule bagasse after acetone extraction, whole guayule shrub, whole guayule shrub after acetone extraction, and whole guayule shrub after acetone/cyclohexane extraction, respectively.

rubber more effectively to further improve glucan accessibility quite significantly. 3.3. Effect of cellulase loading on enzymatic hydrolysis of AFEX-treated guayule Enzymatic hydrolysis at higher cellulase loadings (supplemented along with hemicellulases and other accessory enzymes) was also performed on the AFEX-treated guayule samples (Fig. 6). There was a substantial increase in glucan digestibility (by nearly 2fold) for GB and GBA upon increasing the enzyme loading. However, there were marginal improvements in glucan and xylan digestibility in most other cases suggesting that the glucan and xylan were not yet readily accessible under the selected AFEX pretreatment

conditions (<130 ◦ C). Further optimization of the pretreatment conditions would be required for an industrially feasible process, as excessive enzyme loading is undesirable due to cost concerns (Chundawat et al., 2011a; Gao et al., 2010). Previous work on pretreated GB has reported using 75–80 FPU cellulase/g glucan that resulted in significantly higher enzymatic digestibility; again, the cost of enzymes would make this process economically unfavorable (Srinivasan and Ju, 2010). Presence of high concentrations of lignin, resin and residual latex that may bind unproductively to enzymes would also result in poor hydrolysis yields especially at lower protein loadings. Recent work has shown that synergy between hydrolytic enzymes is sensitive to relative protein ratio especially at lower protein loadings (Gao et al., 2010), which is expected to be further exacerbated due

% Glucan Conversion

35

168 h 24 h

30 25 20 15 10 5 0

70

100 130 GB

70

100 130 GBA

70

100 130 GS

70

100 130 GSA

70

100 130 GSC

Temperature (ºC) Fig. 3. Effect of AFEX pretreatment reaction temperature on glucan enzymatic digestibility for AFEX-treated guayule substrates at 60% (dwb) moisture content, 1:1 kg of NH3 : kg of dry biomass ammonia loading and 10 min reaction time (low enzyme loading; 11.9 mg/g glucan). Where GB, GBA, GS, GSA, and GSC stands for guayule bagasse, guayule bagasse after acetone extraction, whole guayule shrub, whole guayule shrub after acetone extraction, and whole guayule shrub after acetone/cyclohexane extraction, respectively.

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% Xylan Conversion

30

168 h 24 h

25 20 15 10 5 0

70

100 130

70

100 130

GB

70

GBA

100 130

70

GS

100 130

70

GSA

100 130 GSC

Temperature (ºC) Fig. 4. Effect of AFEX reaction temperature on xylan enzymatic digestibility for AFEX-treated guayule substrates at 60% (dwb) moisture content, 1:1 kg of NH3 : kg of dry biomass ammonia loading and 10 min reaction time (low enzyme loading; 11.9 mg/g glucan). Where GB, GBA, GS, GSA, and GSC stand for guayule bagasse, guayule bagasse after acetone extraction, whole guayule shrub, whole guayule shrub after acetone extraction, and whole guayule shrub after acetone/cyclohexane extraction, respectively.

70

168 h 24 h

% Conversion

60 50 40 30 20 10 0

G

X

G

X

1:1

G

2:1

X

G

1:1

X 2:1

GB

GBA

Fig. 5. Effect of ammonia loading (i.e., 1:1 or 2:1 g NH3 /g dry biomass) on enzymatic conversion of glucan (G) and xylan (X) for AFEX-treated guayule substrates at 60% (dwb) moisture content, reaction temperature of 150 ◦ C and 30 min reaction time (low enzyme loading; 11.9 mg/g glucan). Where GB and GBA stand for guayule bagasse, and guayule bagasse after acetone extraction, respectively.

to non-productive binding of enzymes to lignin. Since no lignin is removed from the cell wall during AFEX, a significant proportion of the cellulases may bind to lignin as reported recently by Gao et al. (2011) for AFEX treated corn stover. Since there is nearly

two-fold greater lignin content in guayule compared to corn stover, the problem of non-specific binding of enzymes is expected to be worse. Removal of lignin during pretreatment is one solution to minimizing the deleterious impact of lignin on enzymes.

35

168 h 24 h

% Conversion

30 25 20 15 10 5 0

G

X GB

G

X GBA

G

X GS

G

X GSA

G

X GSC

Fig. 6. Effect of cellulase loading on digestibility. Enzymatic conversion of glucan (G) and xylan (X) for AFEX-treated guayule substrates at 60% (dwb) sample moisture content, 130 ◦ C reaction temperature, 1:1 kg of NH3 : kg of dry biomass ammonia loading and 10 min reaction time for high enzyme loadings (45.1 mg/g glucan). Where GB, GBA, GS, GSA, and GSC stand for guayule bagasse, guayule bagasse after acetone extraction, whole guayule shrub, whole guayule shrub after acetone extraction, and whole guayule shrub after acetone/cyclohexane extraction, respectively.

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Fig. 7. Fermentation profile of AFEX treated guayule bagasse (GB) hydrolyzate using Saccharomyces cerevisiae 424A (LNH-ST) at low initial cell density. AFEX pretreatment was carried out at 150 ◦ C, 30 min reaction time, 1:1 g NH3 /g dry biomass and at 60% moisture (dwb) loading. Either no external nutrients were supplemented (−) or YEP media was added (+) in either case. Glucose (Glc), xylose (Xyl) and ethanol (EtOH) concentrations (g/L) are shown using diamond, triangle or square symbols, respectively.

3.4. Effects of ammonia to biomass loading during AFEX on enzymatic digestibility Additional experiments were performed on guayule bagasse (GB and GBA), since these materials represent commercial agricultural residues and as such are likely to be readily available as bioenergy feedstocks (i.e., compared to GS, GSA and GSC). Fig. 5 shows the effect of ammonia to biomass loading (i.e., 1:1 and 2:1 kg of anhydrous ammonia: kg of dry biomass) on the subsequent enzymatic hydrolysis of AFEX-treated guayule. AFEX pretreatment was performed at a higher temperature (150 ◦ C) and longer reaction time. Higher AFEX pretreatment severities (longer reaction time and higher temperatures) resulted in enhanced glucan and xylan hydrolysis yields (40–50% conversion) compared to all previous cases. These results suggest that AFEX pretreatment at higher reaction temperatures (>150 ◦ C) and longer residence times (30–60 min) would be required to achieve commercially viable sugar hydrolysis yields. The hydrolysis yields achieved with guayule bagasse (∼50%) are marginally lower than those reported for other hardwood species like Poplar and Black Locust (Balan et al., 2009b; Garlock et al., 2011). However, one should keep in mind that the enzyme loadings employed here were significantly lower than what has been reported for AFEX pretreated hardwoods as well. Removal of lignin (resin and other phenolic compounds) during AFEX pretreatment of hardwoods would further increase their enzymatic digestibility. There are also on-going developments to adapt AFEX to produce altered cellulose allomorphs (like cellulose III) that have been reported to be 2–5 times more rapidly digested compared to native cellulose I (Chundawat et al., 2010a, 2011a, 2011b, 2011c). With further improvements to the AFEX process for hardwoods, the overall sugar yields possible during enzymatic hydrolysis would likely also improve. 3.5. Effect of AFEX pretreatment on ethanol fermentation from guayule bagasse AFEX pretreatment has been reported to be very effective compared to other pretreatments in generating a highly fermentable substrate hydrolyzate (Lau and Dale, 2009; Lau et al., 2010). AFEX is known to produce very few biological small-molecule inhibitors compared to dilute-acid pretreatment for most feedstocks through a unique physicochemical mechanism (Chundawat et al., 2010b, 2011c). We were interested in testing the fermentability of AFEX pretreated guayule bagasse hydrolyzate under industrially relevant fermentation conditions. The hydrolyzate was fermented (Fig. 7) using an engineered S. cerevisiae strain capable of fermenting

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glucose and xylose at low initial cell density and with/without external nutrient supplementation (i.e., YEP media). We found that the glucose was readily consumed within 24 h with or without YEP media supplementation, however, presence of YEP aided glucose uptake. Xylose fermentation was found to be more sluggish with nearly 60% xylose left behind after 24 h fermentation. YEP addition was beneficial to glucose fermentation, unlike previous reports on AFEX treated grass hydrolyzates that had no retardation of glucose fermentation rate (Lau and Dale, 2009; Lau et al., 2010), suggesting that there were significant microbial inhibitors present in the hydrolyzate. Removal of resins, residual rubber and lignin-derived products from the biomass during extractive based AFEX pretreatment would further aid the fermentability of the guayule biomass hydrolyzate. 4. Conclusions Natural rubber latex extraction from guayule leaves behind more than 90% of the crop biomass as a finely divided, free flowing feedstock (guayule bagasse) potentially suitable for conversion to biofuels. Advantages of guayule bagasse include that the agricultural and harvest costs are borne by the primary product (natural rubber) production, it is relatively high in density, high in energy content (21,000 kJ/kg) (Boateng et al., 2009, 2010), can be used for both biochemical and thermochemical processes, and is harvested 12 months/year. Untreated guayule shrub and bagasse biomass has very poor digestibility of polysaccharides during enzymatic hydrolysis, to some extent due to the harsh chemical and/or heat history of the biomass during natural rubber extraction. AFEX pretreatment of the biomass substantially improves enzymatic digestibility and fermentability, especially at high pretreatment temperatures. Residual rubber or resin may have some negative effect on biological processing (i.e., fermentation in particular) of guayule biomass to biofuels. These studies suggest that an extractive based AFEX process that can remove residual resin, latex and lignin during pretreatment can substantially improve both the saccharification and fermentation kinetics for guayule-derived biomass. Acknowledgements We thank Rajesh Gupta (Auburn University) for carrying out compositional analyses, and Alex Arceneaux (USDA-ARS) for performing the solvent extractions of guayule samples. The commercial bagasse used in this study was provided by Yulex Corporation, Maricopa, AZ. Dr. Terry Coffelt, USDA-ARS, USALARC, Maricopa, AZ generously provided guayule plants and a review of the manuscript. This work was funded in part by the MSU SPG grant and the DOE Great Lakes Bioenergy Research Center (DOE Office of Science BER DE-FC02-07ER64494). References Balan, V., Bals, B., Chundawat, S.P., Marshall, D., Dale, B.E., 2009a. Lignocellulosic biomass pretreatment using AFEX biofuels. Meth. Protoc., 61–77. Balan, V., Sousa, L.D.C., Chundawat, S.P.S., Marshall, D., Sharma, L.N., Chambliss, C.K., Dale, B.E., 2009b. Enzymatic digestibility and pretreatment degradation products of AFEX-treated hardwoods (Populus nigra). Biotechnol. Prog. 25, 365–375. Boateng, A.A., Mullen, C.A., Goldberg, N., Hicks, K.B., McMahan, C., Cornish, K., Whalen, M., 2009. Energy-dense liquid fuel intermediates by pyrolysis of guayule (Parthenium argentatum) shrub and bagasse. Fuel 88, 2207–2215. Boateng, A.A., Mullin, C.A., McMahan, C.M., Whalen, M.C., Cornish, K., 2010. Guayule (Parthenium argentatum) pyrolysis and analysis by PY-GC/MS. J. Anal. Appl. Pyrolysis 87, 14–23. Chang, M., Tsao, G.T., 1980. Hydrolysis of guayule cellulose for alcohol production. In: Gregg, E.C., Tipton, J.L., Huang, H.T. (Eds.), Proceedings of the Third International Guayule Conference Guayule Rubber Society. Pasadena, CA, April 27–May 1, 1980, pp. 211–224. Chow, P., Nakayama, F.S., Blahnik, B., Youngquist, J.A., Coffelt, T.A., 2008. Chemical constituents and physical properties of guayule wood and bark. Ind. Crop. Prod. 28, 303–308.

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Chundawat, S., 2009. Ultrastructural and physicochemical modifications within ammonia treated lignocellulosic cell walls and their influence on enzymatic digestibility. Chemical Engineering & Materials Science. Ph.D. Dissertation. Michigan State University (Ph.D. Dissertation, AAT 3417644), East Lansing. Chundawat, S.P.S., Sousa, L., Cheh, A., Balan, V., Dale, B.E., 2011. Digestible lignocellulosic biomass and extractives and methods for producing same U.S. patent application serial number PCT/US2011/033079 filed on 04/19/2011, USA. Chundawat, S.P.S., Vismeh, R., Sharma, L., Humpula, J., Sousa, L., Chambliss, C.K., Jones, A.D., Balan, V., Dale, B.E., 2010b. Multifaceted characterization of cell wall decomposition products formed during ammonia fiber expansion (AFEX) and dilute-acid based pretreatments. Bioresour. Technol. 101, 8429–8438. Chundawat, S.P.S., Beckham, G.T., Himmel, M., Dale, B.E., 2011a. Deconstruction of lignocellulosic biomass to fuels and chemicals. Annu. Rev. Chem. Biomol. Eng. 2, 121–145. Chundawat, S.P.S., Bellesia, G., Uppugundla, N., Sousa, L., Gao, D., Cheh, A., Agarwal, U., Bianchetti, C., Phillips, G., Langan, P., Balan, V., Gnanakaran, S., Dale, B.E., 2011b. Restructuring the crystalline cellulose hydrogen bond network enhances its depolymerization rate. J. Am. Chem. Soc. 133, 11163–11174. Chundawat, S.P.S., Donohoe, B.S., Sousa, L., Elder, T., Agarwal, U.P., Lu, F., Ralph, J., Himmel, M.E., Balan, V., Dale, B.E., 2011c. Multi-scale visualization and characterization of plant cell wall deconstruction during thermochemical pretreatment. Energy Environ. Sci. 4, 973–984. Chundawat, S.P.S., Lipton, M.S., Purvine, S.O., Uppugundla, N., Gao, D., Balan, V., Dale, B.E., 2011d. Proteomics based compositional analysis of complex cellulase–hemicellulase mixtures. J. Proteome Res., doi:10.1021/pr101234z, ASAP Available online: http://pubs.acs.org/doi/abs/10.1021/pr101234z. Cornish, K., 1996. Hypoallergenic natural rubber products from Parthenium argentatum (Gray) and other non-Hevea brasiliensis species. US Patent 5,580,942. Cornish, K., 1998. Hypoallergenic natural rubber products from Parthenium argentatum (Gray) and other non-Hevea brasiliensis species. US Patent 5,717,050. da Costa Sousa, L., Chundawat, S.P.S., Balan, V., Dale, B.E., 2009. ‘Cradle-to-grave’ assessment of existing lignocellulose pretreatment technologies. Curr. Opin. Biotechnol. 20, 339–347.

Gao, D., Chundawat, S.P.S., Krishnan, C., Balan, V., Dale, B.E., 2010. Mixture optimization of six core glycosyl hydrolases for maximizing saccharification of ammonia fiber expansion (AFEX) pretreated corn stover. Bioresour. Technol. 101, 2770–2781. Gao, D., Chundawat, S.P.S., Uppugundla, N., Balan, V., Dale, B.E., 2011. Binding characteristics of Trichoderma reesei cellulases on untreated, ammonia fiber expansion and dilute-acid pretreated lignocellulosic biomass. Biotech. Bioeng. 108 (8), 1788–1800. Garlock, R.J., Wong, Y.C., Balan, V., Dale, B.E., 2011. AFEX pretreatment and enzymatic conversion of black locust (Robinia pseudoacacia L.) to soluble sugars. Bioenergy Res., doi:10.1007/s12155-011-9134-6, Available online: http://www.springerlink.com/content/wx14g58268k66254/. Hammond, B.L., Polhamus, L.G., 1965. In: U.T.B. 1327 (Ed.), Research in Guayule (Parthenium argentatum): 1942–1959. USDA, Washington, D.C., pp. 157. Himmel, M., 2008. Biomass Recalcitrance: Deconstructing the Plant Cell Wall for Bioenergy. Blackwell, Oxford, UK, pp. 188. Lau, M.W., Dale, B.E., 2009. Cellulosic ethanol production from AFEX-treated corn stover using Saccharomyces cerevisiae 424A(LNH-ST). Proc. Natl. Acad. Sci. U.S.A. 106, 1368–1373. Lau, M.W., Gunawan, C., Balan, V., Dale, B.E., 2010. Comparing the fermentation performance of Escherichia coli KO11, Saccharomyces cerevisiae 424A(LNH-ST) and Zymomonas mobilis AX101 for cellulosic ethanol production. Biotechnol. Biofuels 3, 11. Nakayama, F.S., 2005. Guayule future development. Ind. Crop. Prod. 22, 3–13. NREL, 2008. Standard Biomass Analytical Procedures Technical Reports NREL/TP510-42618 and NREL/TP-510-42629 (http://www.nrel.gov/biomass/analytical procedures.html). National Renewable Energy Laboratory Lab (NREL), U.S. Department of Energy. Srinivasan, N., Ju, L.-K., 2010. Pretreatment of guayule biomass using supercritical carbon dioxide-based method. Bioresour. Technol. 101, 9785–9791. Teymouri, F., Laureano-Perez, L., Alizadeh, H., Dale, B.E., 2005. Optimization of the ammonia fiber explosion (AFEX) treatment parameters for enzymatic hydrolysis of corn stover. Bioresour. Technol. 96, 2014–2018.