Development of a Highly Efficient Pretreatment Sequence for the

Subscriber access provided by University of Cincinnati Libraries

Article

Development of a highly efficient pretreatment sequence for the enzymatic saccharification of loblolly pine wood Xueyu DU, Lucian A. Lucia, and Reza A. Ghiladi ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b00198 • Publication Date (Web): 24 May 2016 Downloaded from http://pubs.acs.org on May 24, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Sustainable Chemistry & Engineering is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sustainable Chemistry & Engineering

Development of a highly efficient pretreatment sequence for the enzymatic saccharification of loblolly pine wood Xueyu Du,†‡ Lucian A. Lucia,†‡‡ and Reza A. Ghiladi*† †

Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh,

North Carolina 27695-8204, United States ‡

Department of Forest Biomaterials, North Carolina State University, 2820 Faucette Drive,

Raleigh, North Carolina 27695-8005, United States ‡

Qilu University of Technology, State Key Laboratory of Pulp & Paper Science and Technology,

Jinan, PR China 250353

* Corresponding Author: Tel: (919) 513-0680, Fax: (919) 515-5079, E-mail: [email protected]

1

ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: The efficient pretreatment of lignocellulosic materials for bioenergy production is a critical step upon which efficient saccharification is highly dependent, particularly in softwoods due to both their high content and condensed lignin structures. In the present study, preliminary pretreatment steps (e.g., Wiley milling, acetone extraction, autohydrolysis, and disc refining) and economical subsequent/corepretreatment steps (e.g., reagents immersion, hydrothermolysis, dilute acid hydrolysis, and ionic liquids treatment) were systematically investigated to identify which combinations led to efficient enzymatic saccharification of loblolly pine wood, the dominant softwood resource in the US. The results demonstrated that 85% phosphoric acid-based immersions were highly efficient for both cellulose crystallinity degradation and enzymatic hydrolysis, and thus can be included as core pretreatment steps. The highest glucan recovery yield obtained was 93.0% after enzymatic hydrolysis when a pretreatment sequence consisting of autohydrolysis, disc refining, acetone drying, and 85% phosphoric acid immersion (50 °C, 1 h) was employed.

KEYWORDS: Pretreatment; Softwood; Delamination; XRD; Phosphoric acid; Saccharification efficiency

2


Page 2 of 33

Page 3 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Lignocellulosic materials are considered the most sustainable feedstocks for biomassbased energy production relative to fossil resources. Via enzymatic hydrolysis, polysaccharides indigenous to wood (the dominant lignocellulosic biomass) can be depolymerized into their corresponding monosaccharides (such as glucose, xylose, mannose, etc.) more mildly than acid hydrolysis, and further fermented to bio-ethanol.1-2 However, many available species of lignocellulosics cannot be directly/readily saccharified without effective pretreatments due to their compact and recalcitrant structures, and represents a bottleneck to their more widespread use. While a number of different approaches3-4 have been applied to this problem, each with its own distinct (dis)advantages, significant efforts are still needed to overcome the challenges of a lignocellulosic feedstock from both process and cost/benefit standpoints (e.g., high production costs, environmental pollution, low efficiency). There are currently three main paradigms for improving and optimizing the enzymatic hydrolysis of lignocellulosic biomass. Among the three, the likely most popular is the search for ideal polysaccharide-rich feedstocks that have a low cost, low lignin content, a fast growth cycle, are easy to collect, and display a high conversion rate to fermentable sugars. Without question, agricultural residues (e.g., wheat straw, sugarcane bagasse, and corn stover), dedicated crops (e.g., switchgrass and salix), and fast-growing hardwoods (e.g., eucalyptus) have emerged to the fore as prime candidates according to the above criteria, and significant advances for this approach have already been achieved.5-11 The second paradigm is to explore and develop more effective pretreatment methods that involve various physical, chemical, physicochemical, and

3



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

biological treatments, either separately or in combination.3-4 Notably, the selection of pretreatments should not only emphasize efficiency, but should also consider overall operational cost. Typically, a higher pretreatment efficiency is often commensurate with higher energy consumption. Therefore, the exploration of multi-step economical pretreatments may be more attractive than pursuing a single high cost treatment and reagents. The third paradigm for enabling lignocellulosic feedstocks is to develop a new generation of cellulase and hemicellulase enzymes with higher activity and adaptability under variable treatment environments.12-13 Softwood, although it has received less attention within the construct of enzymatic hydrolysis due to its relatively high content and high condensation degree of lignin,14-16 maintains its status as the significant lignocellulosic material especially for softwood-rich countries, such as the United States, Canada, and Sweden.17-18 Moreover, its industrial waste (e.g., sawdust, shavings, waste chips, etc.) are an economical and abundant source of polysaccharides. To date, substantial efforts have been devoted for pursuing a complete/enhanced enzymatic digestibility of softwood for ethanol production, by means of technical development16, 19-23 and also economic assessment/evaluation.24-26 However, there still remains a tremendous opportunity to achieve a high sugar conversion rate of softwood by a highly efficient pretreatment sequence. Thus, the focus of the present study is the systematic investigation for more efficient approaches to softwood pretreatment by screening and combining traditional and newlyemerging pretreatment methods, such as size reduction, ionic liquid dissolution, reagents immersion (including acids, urea aqueous solution, and microemulsions), autohydrolysis, and mechanical refining. Although concentrated inorganic acids are corrosive and

4


Page 4 of 33

Page 5 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


hazardous to process equipment, some possess superior abilities for the interruption of inter/intra-hydrogen bonds of cellulose and can further swell and dissolve it, e.g., concentrated phosphoric acid.27-31 The treatment of urea, a non-oxidative chemical reagent, could also open up the fiber structure by disruption of its hydrogen bonds. Its applications have been widely used on, for example, dissolution of wood components and isolation of lignin-carbohydrate complexes (LCCs).32 Microemulsions, commonly composed of surfactant(s), oil (non-polar phase), and water, is a new-emerging technique for fast penetration of porous wood structures.33-35 Although microemulsions themselves do not exhibit remarkable abilities for reducing cellulose crystallinity,33 they are still a good reagent carrier for other treatments due to similarity and intermiscibility theory. The amphiphile surfactant molecules and hydrophobic components present in microemulsions can be absorbed onto the lignin domain of the treated samples which could decrease the subsequent adsorption of cellulase onto lignin structures during enzymatic hydrolysis and thus enhance the efficiency of enzymatic hydrolysis. To target the fermentable sugar conversion rate of softwood, loblolly pine wood was selected as the raw material, the most common softwood species in the US. The initial pretreatment screening efforts focused on cellulose crystallinity as the basis for wood fiber delamination,36 because a well delaminated wood sample is often represented by a lower cellulose crystallinity, a key prerequisite for enzymatic hydrolysis. Verification of the efficacy of the pretreatment methods was demonstrated by their corresponding glucan recovery yields post enzymatic hydrolysis. On the basis of the results obtained from this study, a more optimized multi-step pretreatment sequence for the enzymatic hydrolysis of

5



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

loblolly pine wood is proposed that consists of preliminary steps and subsequent highperformance core steps. EXPERIMENTAL SECTION Materials and reagents. Air-dried loblolly pine wood chips were obtained in-house. Commercial cellulose powder, urea, 1-ethyl-3-methylimidazolium chloride (EmimCl), and 1-butyl-3-methylimidazolium chloride (BmimCl) were each purchased from SigmaAldrich. Cellulase (Cellic CTec2) and hemicellulase (Cellic HTec2) were generously supplied by Novozymes North American (Franklinton, NC, USA). 85 wt-% phosphoric acid, 72 wt-% sulfuric acid and acetic acid were purchased from Fisher Scientific. Deionized (D.I.) water was used for all the experiments. Preliminary pretreatment steps. The originally air dried loblolly pine wood chips were processed into two different starting batches for this study as follows: (A) loblolly pine wood particles, 20-40 mesh (LPWP): Air dried loblolly pine wood chips were Wiley-milled to 20-40 mesh (0.42-0.84 mm) and extracted with acetone in a Soxhlet extractor for 24 h to remove all extractives. The LPWP starting materials were kept in sealed glass bottles after 80 °C oven drying overnight. (B) Autohydrolyzed and refined loblolly pine wood pulp (AHR-LPW): 600 g air-dried loblolly pine wood chips were immersed in 3.5 L D.I. water in a 6.9 L laboratory digester (M/K Systems, Inc). It was heated to 180 °C at 2.5 °C/min and held for 40 min. During the heating, the inner pressure in the digester was relieved once to expel volatile substances when the temperature reached 100 °C. After heating, the treated chips were removed when the temperature was cooled below 60 °C, and further rinsed with cold D.I. water. All the treated wood chips were subsequently mechanically refined by a disc refiner (disc size

6


Page 6 of 33

Page 7 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


8’’; 3600 RPM; Bauer Bros. Co., Springfield, Ohio-Brantford, Ontario) for two times with gap sizes of 0.762 mm and 0.254 mm, respectively. Afterwards, the pulp was dewatered by centrifugation and stored at 4 °C (moisture content = 65.0% after centrifugation). Pretreatment steps. (A) Immersion by acids, urea, and microemulsions. 1.00 g extractive-free LPWP (80 °C oven dried) was fully soaked with 10 mL of different reagents (namely 40 wt-% phosphoric acid, 85% phosphoric acid, 72% sulfuric acid, 100% acetic acid, and microemulsion-1, -2, -3 and -4) in 20 mL glass vials at room temperature. The vials were shaken occasionally, and after a total immersion time of 24 h the residues were recovered from the treated reagents and rinsed thoroughly with D.I. water by vacuum filtration. The purified samples were collected and kept wet. Four O/W type microemulsions were formulated by varying the concentration of major components to investigate their potential effects as pretreatment towards enzymatic hydrolysis efficiency. The detailed composition of these four microemulsions were as follows: Microemulsion1 (H2O: 90.7 wt-%; sodium dodecylsulfate: 1.8 wt-%; dodecane: 2.7 wt-%; 1-pentanol: 3.0 wt-%; NaCl: 1.8 wt-%); microemulsion-2 (H2O: 91.6 wt-%; sodium dodecylsulfate: 3.7 wt-%; dodecane: 1.4 wt-%; 1-pentanol: 1.5 wt-%; NaCl: 1.8 wt-%); microemulsion-3 (H2O: 89.1 wt-%; sodium dodecylsulfate: 3.6 wt-%; dodecane: 2.7 wt-%; 1-pentanol: 2.9 wt-%; NaCl: 1.8 wt-%); microemulsion-4 (H2O: 83.4 wt-%; sodium dodecylsulfate: 3.3 wt-%; dodecane: 7.5 wt-%; 1-pentanol: 4.1 wt-%; NaCl: 1.7 wt-%). (B) Hydrothermolysis. 1.00 g extractive-free LPWP (80 °C oven dried) was pre-soaked with 10 mL D.I. water for 1 h, and the suspension was then placed in a 150 mL capped and pressure-tolerant glass bottle in an autoclave (Market Forge STM-EL autoclave sterilizer).

7



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The temperature was raised to 120 °C and held for 1 h. The chamber was opened when the temperature was < 60 °C. The sample was vacuum filtered and thoroughly rinsed with D.I. water. The purified sample was collected and kept wet. (C) Dilute acid hydrolysis. 1.00 g extractive-free LPWP (80 °C oven dried) was pre-soaked with 10 mL 3% sulfuric acid aqueous solution for 1 h, and then placed in a 150 mL capped and pressure-tolerant glass bottle in an autoclave (Market Forge STM-EL autoclave sterilizer). All following steps were the same as in pretreatment step (B). (D) Dissolution by ionic liquids. 0.50 g and 1.00 g extractive-free LPWP (80 °C oven dried) were charged into 20 mL glass vials containing either 10.00 g pre-heated 1-ethyl-3-methylimidazolium chloride or 10.00 g pre-heated 1-butyl-3-methylimidazolium chloride, respectively. The sealed vials were heated in a 110 °C oil bath with magnetic stirring for 8 h. Afterwards, the mixture was transferred to a beaker containing 100 mL acetone-water solution (v/v=1:1). The suspension was magnetically stirred for 1 h at room temperature and then allowed to sit overnight. All of the insoluble residue was recovered and thoroughly rinsed by vacuum filtration with a fine filtering crucible. The purified sample was collected and kept in the wet state for further analyses. Enzymatic hydrolysis of different pretreated wood samples. A combination of cellulase (Cellic CTec2) and hemicellulase (Cellic HTec2) (VCTec2 : VHTec2 = 9 : 1 (v/v)) was prepared for the enzymatic hydrolysis of different pretreated wood samples. The general parameters of the enzymatic hydrolysis were as follows: consistency (concentration of dry wood sample), 5%; buffer, 0.1 M, HAc/NaAc, pH=4.8; cellulase amount, 10 FPU/g oven-dried sample; time, 72 h; incubation temperature, 50 °C. In addition, lignin-rich LPWP and lignin-rich AHR-LPW were prepared by thorough

8


Page 8 of 33

Page 9 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


enzymatic hydrolyses of 6 h vibratory ball milled LPWP and 6 h vibratory ball milled AHR-LPW according to the above hydrolysis conditions after two rounds (72 h per round with a purification step and addition of fresh enzymes for each round, the lignin recovery yield ≥ 90%). Analytical methods. XRD analyses of the different wood samples were performed on a Rigaku Smartlab X-ray Diffractometer (Cu K-alpha source with a wavelength of 1.5418 Å, Si or LaB6 was used for calibration of the system) to characterize the crystallinity change. Scans were obtained from 10 to 40 degrees (2θ in 0.1 degree steps for 15 seconds per step). The Klason lignin content, acid soluble lignin content, and carbohydrate composition of treated wood samples were determined according to TAPPI Standards T 222 om-6 and T 222 om-02.37 For gel-state NMR experiments, 30 mg ligninrich LPWP and 30 mg lignin-rich AHR-LPW were mixed with 600 µL DMSO-d6, respectively into 5 mm NMR tubes and then swelled to form gel-like states. Two dimensional Heteronuclear Single Quantum Coherence (HSQC) NMR spectra were recorded on a Bruker Avance 500 MHz instrument with a 5 mm double resonance broadband BBI inverse probe using a standard Bruker pulse program at room temperature.

RESULTS AND DISCUSSION Comparison of different preliminary pretreatment steps. To preliminarily open up the wood structure and remove interfering substances (e.g., extractives), the loblolly pine wood chips were treated by two different ways: i) Wiley milling (mechanical disintegration) followed by acetone Soxhlet extraction (thermal and organic solvent treatment), or ii) autohydrolysis (acidic, thermal, and pressurized treatment) followed by

9



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 33

disc refining (mechanical disintegration). Each involved mechanical disintegration and thermal treatment. The yield of wood sample after autohydrolysis and refining was 69.2% based on dry loblolly pine wood. The compositional analyses of the original loblolly pine wood, extractive-free loblolly pine wood particles (LPWP), and its autohydrolyzed and refined product (AHR-LPW) are listed in Table 1. Between the two preliminarily treated materials, extractive-free LPWP retained most of the non-extractive wood components and provided the highest theoretical amount of glucan (42.0%) based on original dry wood. With respect to AHR-LPW, though it suffered a weight loss of 30.8% as a consequence of the removal of a majority of both its extractives (0.3% vs. 9.3% in original wood) and hemicellulose (mannan: 1.8% vs. 10.2% in original wood; xylan: 2.4% vs. 4.5% in original wood), it still contained a high content of glucan, 38.9% (slightly lower than the 42.0% in original wood). A small portion of lignin was removed after autohydrolysis and refining (24.9% in AHR-LPW vs. 26.3% in original wood, both based on original dry wood).

Table 1. Lignin and sugar analyses of original loblolly pine wood, extractive-free LPWP, and AHR-LPW. Samples

Extractive s (%)

Lignin (%)

Main polysaccharides

Glucan Mannan Xylan (%) (%) (%) 9.3 26.3 42.0 10.2 4.5 Loblolly pine wood (105 °C O.D) 0 29.0 46.3 11.2 5.0 Extractive-free LPWP (105 °C O.D) (0) (26.3) (42.0) (10.2) (4.5) 0.5 35.7 55.8 2.8 3.4 AHR-LPW (105 °C O.D) (0.3) (24.9) (38.9) (1.8) (2.4) All results were calculated based on respective dry starting materials. Main polysaccharide content was based upon the amount of the corresponding monosaccharides using a multiplication factor of 0.9 for six carbon sugars, and a factor of 0.88 for five carbon sugars. Data shown in brackets are the amounts based on the original dry loblolly pine wood.

10


Page 11 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


More information related to lignin structural changes can be revealed by interpretation of 2D HSQC NMR spectra for their corresponding products after 6 h vibratory ball milling and thorough enzymatic hydrolysis, viz., lignin-rich LPWP (Figure 1) and lignin-rich AHR-LPW (Figure 2). The dominant lignin signals originated from methoxy groups, β-O-4’ linkages (A), phenylcoumaran (B) and resinol (C) subunits. Signals from residual xylan were also clearly observed. To compare the amounts of major lignin subunits (e.g. β-O-4’ linkages and phenylcoumaran) in these two samples, the C2H2 cross signal (G2) from the guaiacyl (G) units was taken as an internal standard (integration set to 1.00), because this position is never substituted and readily assignable in typical 2D HSQC spectrum.38 The relative amounts of the β-O-4’ linkages and phenylcoumaran subunits in these two lignin-rich samples were semi-quantified by measuring the integrations of their respective Cα-Hα cross signals from lignin terminal functional units. The results showed that the number of β-O-4’ linkages was 0.26/G2 in lignin-rich AHR-LPW, lower than the 0.39 β-O-4’ linkages/G2 in lignin-rich LPWP (Figures 1 and 2). On the other hand, the amount of phenylcoumaran in lignin-rich AHRLPW (0.17/G2) is higher than in lignin-rich LPWP (0.13/G2). These variations in major lignin substructures probably resulted from the partial cleavage of the β-O-4’ linkages during autohydrolysis. Moreover, the amounts of some typical end groups of lignin subunits, e.g., coniferyl alcohol end groups (D) and cinnamyl aldehyde end groups (E), were reduced after autohydrolysis, as shown by 0.03 Dγ/G2 (lignin-rich AHR-LPW) vs. 0.08 Dγ/G2 (lignin-rich LPWP), and 0.02 Eα/G2 (lignin-rich AHR-LPW) vs. 0.05 Eα/G2 (lignin-rich LPWP). Therefore, the lignin structure became slightly more condensed (e.g., a decrease in β-O-4’ linkages), and its original relatively reactive lignin functional units

11



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(e.g., coniferyl alcohol end groups and cinnamyl aldehyde end groups) were readily removed after autohydrolysis, although the total content of lignin after autohydrolysis and refining did not decrease to any large extent.

Figure 1. 2D HSQC NMR spectrum of a lignin-rich sample prepared from the enzymatic hydrolysis of 6 h vibratory ball milled LPWP.

12


Page 12 of 33

Page 13 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2. 2D HSQC NMR spectrum of a lignin-rich sample prepared from the enzymatic hydrolysis of 6 h vibratory ball milled AHR-LPW.

Taken together, the results shown in Table 1 and Figures 1 and 2 suggest that the combination of Wiley milling and acetone extraction increased the sample surface area by mechanically reducing sample size, but microscopically, the original wood structure had not been altered. The extractive-free LPWP was featured as rigid short-fiber wood particles with the presence of almost intact hemicellulose and lignin. However, the preliminary pretreatment combined with autohydrolysis and disc refining resulted in flexible and separated fiber clusters with the loss of most of extractives, substantial loss of hemicellulose, and only a small portion of lignin lost. Screening of subsequent pretreatment steps for cellulose crystallinity degradation. In lignocellulosic structures, cellulose is a primary contributor to the overall crystallinity as evidenced from X-ray diffraction analysis. However, highly ordered regions (long range crystallinity) do not fully dominate the whole structure of cellulose; rather, they often co-exist with generalized amorphous regions including pure amorphous regions and less ordered regions. Within highly ordered and less ordered crystalline regions, the former one contributes most of the cellulose crystallinity and is extremely insusceptible and recalcitrant, while the latter possesses only a very limited crystallinity from short range crystallinity. Therefore, the short crystalline regions are more easily accessed, disrupted, and even removed. An efficient decrystallization method should not only remove the sporadically distributed short crystalline fragments in the generalized amorphous regions, but also be more targeted toward disruption and reduction of the long range crystallinity that is manifested through the high level of inter-

13



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

and intramolecular hydrogen bonds. Traditionally, one popular and empirical way for the determination of crystallinity is by calculation of the height ratio between a major crystalline peak (I002-Iamorphous) and 002 plane peak after deduction of the background absorbance.39 Although the results obtained by this XRD height method are higher than other methods (e.g., the XRD amorphous subtraction, the XRD peak deconvolution, and the NMR C4 peak separation methods40), it is still the easiest and time-saving method for empirical determination of relative crystallinity,39 and therefore the XRD height method was adopted here for the characterization of the cellulose crystallinity changes before and after pretreatment. According to the calculation, if the pretreatment is only effective in removing the crystallinity of less ordered regions (contributing to the intensity of the amorphous signal in the XRD spectrum), the overall crystallinity of the treated sample will slightly increase compared to the starting raw material. On the other hand, if the pretreatment step is sufficiently strong as to disrupt highly ordered crystalline regions into less ordered crystalline domains, the overall crystallinity will be decreased. As is shown in Table 2, the original crystallinity of LPWP (20-40 mesh) ~ 55.4%, far less than commercial cellulose powder (81.0%) because of the coexistence of hemicellulose and lignin. After undergoing 6 h of vibratory ball milling, LPWP is in an almost fully amorphous state with ~ 0% crystallinity. Upon pretreatment, it was noted that mild crystallinity removal occurred only when the less ordered region was decrystallized: immersion (8 M urea, 40% H3PO4, acetic acid, and microemulsions 1-4), hydrothermolysis (120 °C, 1 h), and 3% H2SO4 hydrolysis (120 °C, 1 h) can be considered mild pretreatment steps as their observed crystallinity values (59.7-68.2%) were above that of the original LPWP (55.4%).

14


Page 14 of 33

Page 15 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


On the other hand, concentrated inorganic acids (e.g., 85 wt-% phosphoric acid and 72 wt-% sulfuric acid) and ionic liquids (especially for 1-butyl-3-methylimidazolium chloride) were noted to disrupt the crystallinity of highly ordered crystalline regions, as noted by both a lowering of the crystallinity below 55.4% (Table 2), as well as their subsequent effect on the XRD data (vide infra).

Table 2. Cellulose crystallinity changes before and after various pretreatment steps. Samples

Pretreatments

Crystallinity

LPWP (80 °C oven dried)

No treatment 6 h vibratory ball milling 8 M Urea immersion (24 h, rt) 40% H3PO4 immersion (1 h, rt) 40% H3PO4 immersion (24 h, rt) 85% H3PO4 immersion (1 h, rt) 85% H3PO4 immersion (2 h, rt) 85% H3PO4 immersion (3 h, rt) 85% H3PO4 immersion (24 h, rt) 72% H2SO4 immersion (1 h, rt) 72% H2SO4 immersion (24 h, rt) 100% Acetic acid immersion (24 h, rt) Microemulsion-#1 immersion (24 h, rt) Microemulsion-#2 immersion (24 h, rt) Microemulsion-#3 immersion (24 h, rt) Microemulsion-#4 immersion (24 h, rt) Hydrothermolysis (120 °C, 1 h) 3% H2SO4 hydrolysis (120 °C, 1 h) 1-ethyl-3-methylimidazolium chloride (110 °C, 8 h) 1-butyl-3-methylimidazolium chloride (110 °C, 8 h) No treatment Microemulsion-#1 (24 h, rt) 85% H3PO4 immersion (24 h, rt)

55.4% ̴0% 59.9% 59.7% 60.0% 50.4% 48.3% 48.1% 41.7% 46.2% 46.2% 60.1% 61.4% 60.9% 61.9% 57.9% 61.3% 68.2% 52.9%

Commercial cellulose Powder

Removal of cellulose crystallinity Less ordered Highly ordered region region --Yes Yes Yes No Yes No Yes No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes Yes

47.0%

Yes

Yes

81.0% 82.7% 41.5%

-Yes Yes

-No Yes

Analysis of the XRD data (Figure 3) indicated that for LPWP, after a 1 h immersion in 85% phosphoric acid, the intensities of the 101 plane, 10ī plane, 002 plane, and 040 plane peaks decreased to a large extent, the sample crystallinity dropped to 50.4 %, and a new broad peak ~ 28 (°) emerged that likely originated from the newly formed short

15



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ranges of crystalline fragments. This trend of decrystallization was further extended to 41.7% after 24 h of immersion. At this point, all the original characteristic plane peaks disappeared and were fully replaced by a single and much broader peak ~ 28 (°), which indicates that all the long range ordered crystalline regions have been completely converted into shorter /less ordered regions. In other words, the originally compact loblolly pine wood structure was more closely resembled an amorphous and well delaminated state, which coexisted with sporadically distributed small crystalline regions that remained after concentrated phosphoric acid treatment to give signals ~ 28 ° (2θ).

Figure 3. XRD spectra of LPWP before and after treatment by immersion in concentrated acid (85 wt-% phosphoric acid or 72 wt-% sulfuric acid) or ionic liquids EmimCl and BmimCl.

16


Page 16 of 33

Page 17 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


An even lower crystallinity (46.2%) was obtained after 1 h immersion in 72% sulfuric acid when compared with 1 h immersion in 85% phosphoric acid, which is consistent with the stronger protonation ability of 72% sulfuric acid. Nevertheless, the superiority of 72% sulfuric acid did not maintain after extending the immersion time up to 24 h because the crystallinity of this sample was unexpectedly the same value as the 1 h treated sample. Notably, the color of 72% sulfuric acid immersed LPWP was much darker than 85% phosphoric acid treated LPWP within the same immersion time, (Figure 4), which might have originated from the chromophores formed from lignin.

Figure 4. Color differences of LPWP after room temperature pretreatment with either 85% phosphoric acid or 72% sulfuric acid immersion.

When compared with concentrated acid immersion at room temperature (RT), ionic liquids were not able to achieve the same levels of cellulose decrystallization. For instance, the crystallinity of 1-ethyl-3-mehylimidazolium chloride (EmimCl) treated LPWP (110 °C, 8 h) was only 52.9%, higher than the value of either the 1 h 85%

17



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

phosphoric acid immersed sample (50.4%) or the 1 h 72% sulfuric acid immersed sample (46.2%). The crystallinity of 1-butyl-3-methylimidazolium chloride (BmimCl) treated LPWP (110 °C, 8 h) was 47.0%; while lower than the value of the 1 h 85% phosphoric acid treated LPWP (50.4%), it was still higher than the values for either 24 h 85% phosphoric acid treated or 1 h 72% sulfuric acid treated LPWP samples. Thus, despite attracting tremendous attention as a potential avenue for the full dissolution of lignocellulosics and an effective pretreatment strategy for enzymatic hydrolysis, and when considering the harsh reaction conditions (e.g., high temperature and strict control of moisture content) and toxicity of some ionic liquids, these findings suggest that they are not an effective and viable pretreatment step for loblolly pine wood. Based on the above considerations, a longer time immersion with 85% phosphoric acid and shorter time immersion with 72% sulfuric acid were considered the two most promising means for loblolly pine wood fiber decrystallization, and were thus explored further for their potential positive influence on enzymatic hydrolysis. Comparison of enzymatic hydrolysis efficiency of 72% sulfuric acid-immersed and 85% phosphoric acid-immersed samples. To verify if either of the two acid immersion pretreatment strategies (72% sulfuric acid or 85% phosphoric acid) can benefit subsequent enzymatic hydrolysis, their corresponding 1 h and 24 h treated samples were selected and enzymatically hydrolyzed. The glucan recovery yields (based on theoretical glucan content in dry LPWP) of 1 h and 24 h phosphoric acid-immersed samples were 51.0% and 76.7% respectively, much higher than the direct enzymatic hydrolysis of LPWP (13.1%) (Table 3). However, while 72% sulfuric acid pretreatment led to a higher level of cellulose crystallinity removal within a short time period of

18


Page 18 of 33

Page 19 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


immersion when compared to phosphoric acid, suggesting the potential for a greater susceptibility to enzymatic hydrolysis, it actually exhibited a poorer glucan recovery yield (7%) after 1 h than even the direct enzymatic hydrolysis of LPWP, which worsened to 4.8% after a 24 h immersion. Two possible reasons for the unexpectedly poor performance of the 72% sulfuric acid pretreatment are: i) the loss of water-soluble oligosaccharides (formed during sulfuric acid immersion) during the subsequent workup employing a nylon membrane (150 mesh) filtration step, and/or ii) altered lignin structures in the treated wood residues that inhibited enzymatic hydrolysis.

Table 3. Glucan recovery yields of various pretreated and purified samples after 72 h enzymatic hydrolysis. Sample Separation Method Glucan recovery yield a (%) LPWP -13.1% 1h 72% H2SO4 immersed LPWP Nylon membrane (150 mesh) 7.0% 24h 72% H2SO4 immersed LPWP Nylon membrane (150 mesh) 4.8% 1 h 85% H3PO4 immersed LPWP Nylon membrane (150 mesh) 51.0% 2 h 85% H3PO4 immersed LPWP Nylon membrane (150 mesh) 61.1% 5 h 85% H3PO4 immersed LPWP Nylon membrane (150 mesh) 69.5% Nylon membrane (150 mesh) 73.3% 8 h 85% H3PO4 immersed LPWP 24 h 85% H3PO4 immersed LPWP Nylon membrane (150 mesh) 76.7% 24 h microemulsion-1 immersed LPWP Nylon membrane (150 mesh) 15.6% (24h 85% H3PO4 immersed) followed by (24 h Nylon membrane (150 mesh) 80.4% microemulsion-1 immersed) LPWP 24 h 85% H3PO4 immersed LPWP Pyrex filtering crucible (Porosity: fine) 89.7% Pyrex filtering crucible (Porosity: fine) 91.7% 48 h 85% H3PO4 immersed LPWP 72 h 85% H3PO4 immersed LPWP Pyrex filtering crucible (Porosity: fine) 92.2% Pyrex filtering crucible (Porosity: fine) 77.7% (50 °C, 1h) 85% H3PO4 immersed LPWP a Glucan recovery yield = [(Amount of detected glucose*0.9)/theoretical amount of glucan in the starting raw material]*100%

Further evidence that accounts for the efficiency difference of enzymatic hydrolysis between samples treated by immersion in 72% sulfuric vs 85% phosphoric acid were the topographic features of these two treated and purified wood samples in D.I. water (Figure S1). The 85% phosphoric acid treated wood sample (Figure S1B) was more fibrillated and more delaminated, which is highly favored for enzymatic hydrolysis. On the other hand, the 72% sulfuric acid treated sample (Figure S1A) still showed small black

19



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

compact particles. When taken together, the results demonstrated that although it achieved an obvious cellulose crystallinity removal during a short time period, the 72% sulfuric acid immersion was not an ideal pretreatment for the saccharification of loblolly pine wood. As such, the study focused on the further optimization of the 85% phosphoric acid immersion pretreatment in terms of immersion time, immersion temperature, purification, as well as in combination with other pretreatment steps. Optimization of the 85% phosphoric acid immersion pretreatment step. In the absence of a pretreatment step, the 72 h enzymatic hydrolysis of LPWP resulted in a final glucan yield of 13.1% (Table 3). Pretreatment with 85% phosphoric acid immersion for 1 h at room temperature and atmosphere pressure led to a glucan yield of 51.0% after enzymatic hydrolysis, which increased to 76.7% when pretreated by acid immersion for 24 h. Interestingly, although they did not display as strong an ability for decreasing cellulose crystallinity, microemulsions were able to slightly improve the final efficiency of enzymatic hydrolysis, both alone and in combination with acid immersion. A pretreatment with microemulsion-1 immersion (24 h) resulted in a final glucan yield of 15.6% (vs. 13.1% for enzymatic hydrolysis alone), while the glucan yield for LPWP treated with a 24 h 85% phosphoric acid immersion followed by a 24 h microemulsion-1 immersion was 80.4% (vs. 76.7% for LPWP treated with 24 h 85% phosphoric acid immersion alone). It was rationalized that the slight increase in glucan yield may have arisen from the hydrophobic microemulsion components (e.g., dodecane or the hydrophobic group of SDS) preferentially absorbing onto lignin based on the “similarity and intermiscibility” theory, a result which prevents unwanted absorption of the hydrolysis enzymes onto lignin, thereby leading to the improvements in the glucan yield

20


Page 20 of 33

Page 21 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


observed.41-42 Taken together, these results suggest that a microemulsion immersion is an optional step during a multi-step pretreatment sequence, but that the overall improvements using the microemulsion formulations and conditions studied here were minimal. Additionally, fine wood fibers were observed in the purified acid-treated samples. Such fibers are highly favorable for enzymatic hydrolysis, and their recovery is paramount during the subsequent purification step, otherwise an excess loss of these fine fibers can lead to a drop in glucan yield after enzymatic hydrolysis. When a nylon membrane (150 mesh) was used as a filtration material, a small portion of fine fibers still escaped into the waste liquid, and it was noted that a decrease in the glucan yield occurred. Based on the above considerations, a much finer filtration material, namely a Pyrex filtering crucible (porosity: fine), was employed. As expected, the final glucan yield for 24 h 85% phosphoric acid immersed LPWP increased to 89.7% when employing the fine filtering crucible (Table 3), much higher than 76.7% obtained when using a nylon membrane (150 mesh). Extension of the immersion time to 48 or 72 h did not remarkably increase the final glucan yield after enzymatic hydrolysis (e.g., 91.7% for 48 h immersion and 92.2% for 72 h immersion). Therefore, the 24 h immersion step was selected as the optimal time for 85% phosphoric acid immersion at room temperature. In addition, the effects of 85% phosphoric acid immersion under moderate temperatures (50 °C and 80 °C) were also studied, and a short time period of 1 h was selected from an energy saving point of view. For the 50 °C 1 h immersion of LPWP, the final glucan yield improved to 77.7% over the 1 h room temperature value of 51.0% (Table 3). The sample from the 80 °C 1 h immersion in 85% phosphoric acid was quite

21



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

similar in appearance to the sample from 1 h room temperature immersion in 72% sulfuric acid, likely due to the expedited acidic hydrolysis of the sample using 85% phosphoric acid under higher temperatures, and thus its pretreated residue was not suitable for enzymatic hydrolysis. Therefore, the data suggested that a room temperature 24 h 85% phosphoric acid immersion pretreatment step provides an optimal balance between energy conservation and process time for high glucan yields after enzymatic hydrolysis, with shorter times possible at elevated temperatures, but at a higher energy cost, and slightly higher yields at longer immersion times, but with a higher process time cost. Exploration of an optimized multi-step pretreatment sequence for the enzymatic hydrolysis of loblolly pine wood. An optimized multi-step pretreatment sequence was explored by maintaining the 85% phosphoric acid based immersion as the core step (e.g., 1 h immersion at 50 °C or 24 h at RT) and incorporating other potentially effective pretreatment steps to seek a further enhancement of the whole sequence efficiency. The sequences are noted as entries 1-11 in Table 4. Compared with the low glucan yield (13.1%, sequence 1) of direct enzymatic hydrolysis of extractive-free LPWP, AHR-LWP is more favorable for enzymatic hydrolysis with a glucan yield of 34.8% (sequence 4), likely because the cellulose was more exposed than in LPWP due to barrier removal of most of its hemicellulose and part of the lignin. Therefore, autohydrolysis (180 °C, 40 min) followed by disc refining appeared to be a more efficient combination and was selected as the preliminary step for positing an ideal pretreatment sequence. The next step was to apply 85% phosphoric acid as an immersion agent for the large scale degradation of cellulose crystallinity. However, the following question arose: is it necessary to

22


Page 22 of 33

Page 23 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


regulate the moisture content (65.0%) of AHR-LPW to provide the 85% phosphoric acid immersion the largest degree of cellulose crystallinity removal? The concern here is that by charging 85% phosphoric acid directly into a wet sample, the concentration of phosphoric acid would actually be diluted by the moisture in the sample (~ 76% of actual phosphoric acid concentration for a given sample:phosphoric acid weight ratio of 1:10), which would affect the cellulose crystallinity removal. The relatively low glucan yields of sequences 5 (49.7%) and 6 (57.7%) thus proved the dilution hypothesis. To avoid the acid dilution by the wet sample, oven drying at 105 °C was employed; however, such a step is known to lead to a very solid and compact fiber agglomeration (displaying severe hornification), which is very unfavorable for enzymatic saccharification by reducing enzyme accessibility to cellulose.43-44 The glucan recovery yield of sequence 9 (18.9%) was much lower than sequence 4 (34.8%), demonstrating the negative effects of oven drying and subsequent hornification on enzymatic hydrolysis. In this case, an additional disintegration step, e.g., Wiley milling to ~ 20-40 mesh, was required to increase the sample surface area, and despite fiber hornification being still evident to some degree, the glucan yields of sequence 10 (89.0%) and 11 (90.6%) were improved by the milling and were close to the value from sequence 2 (89.7%). As an alternative to working with either a wet sample (leading to acid dilution) or an oven-dried one (leading to hornification), a mild acetone evaporation drying process was adopted. During this process, the original wet AHR-LPW was rinsed by acetone (multiple low-dose additions) under vacuum filtration to remove the residual wood extractives, the fraction of acetone-soluble lignin, and the majority of the moisture. Then the acetonesaturated sample was continuously ventilated under vacuum to evaporate excess acetone.

23



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

As shown in Table 4, sequence 7 (acetone drying) reached a glucan yield of 81.8% after enzymatic hydrolysis, much higher than the 49.7% from sequence 5. Although sequence 10 reached the highest glucan yield (89.0%) out of these three sequences (sequences 5, 7, and 10), its value was likely improved by the additional Wiley milling process as opposed to oven drying at 105 °C.

24


Page 24 of 33

Page 25 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


Table 4. Different pretreatment sequences for enzymatic hydrolysis of loblolly pine wood. Raw material

Sequence No. #1

Step-1

#2

Wiley milling (20-40 mesh)

#3 #4 #5 Loblolly pine wood chips

#6

Multi-step pretreatment sequence Step-2 Step-3 -85% H3PO4 Acetone extraction (24 h) immersion (rt, 24 h) followed by 80 °C O.D 85% H3PO4 immersion (50 °C, 1 h) -85% H3PO4 immersion (rt, 24 h) 85% H3PO4 immersion (50 °C, 1 h)

#7 #8

Autohydrolysis (180 °C, 40 min)

Disc refining

Acetone wash, filtration & dying under vacuum for 1 h

#9 #10

105 °C O.D & Wiley milling (through 20 mesh)

#11

25


Step-4 --

Glucan recovery yield (%) 13.1

--

89.7

--

77.7

--

34.8

--

49.7

--

57.7

85% H3PO4 immersion (rt, 24 h) 85% H3PO4 immersion (50 °C, 1 h) -85% H3PO4 immersion (rt, 24 h) 85% H3PO4 immersion (50 °C, 1 h)

81.8 93.0 18.9 89.0 90.6


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

As discussed previously, increasing the phosphoric acid immersion temperature to a moderate 50 °C shortens total immersion time that was readily apparent in the multi-step pretreatment sequences 6 (57.7%, vs. 49.7% for 24 h at RT), 8 (93.0%, vs. 81.8% for 24 h at RT) and 11 (90.6%, vs. 89.0% for 24 h at RT), with each exhibiting a higher glucan yield for its respective sequence when compared with the longer immersion time (24 h) at room temperature. Finally, the glucan yield of sequence 11 (90.6%) was a posteriori expected to exceed the yield of sequence 8 (93.0%), based on the comparison result between sequences 7 (81.8%) and 10 (89.0%). However, it could not be neglected that both sequences 10 and 11 included a step of 105 °C oven drying, during which severe fiber hornification occurred, though an additional Wiley milling process was applied to improve enzymatic digestibility. A further increase of the immersion time at 50 °C may help to increase the final glucan yield of sequence 11, but it was not pursued from an energy-saving point of view. Optimized multi-step pretreatment sequence for the enzymatic saccharification of loblolly pine wood. A summary of the observations from sections 3.1-3.5 is as follows: i) a concentration of 85% phosphoric acid needs to be maintained during immersion because even a slight dilution will induce a weaker delamination effect; ii) the room temperature 24 h immersion in 85% phosphoric acid can effectively delaminate shortfiber wood samples compacted with substantial hemicellulose and lignin, but has a considerably lower effect towards crystalline long-fiber wood sample (e.g., AHR-LPW); iii) the 50 °C 1 h immersion in 85% phosphoric acid enables a fast delamination for flexible fiber clusters that contains less hemicellulose and lignin (e.g. AHR-LPW), but is relatively weaker for compact short-fiber wood samples (such as LPWP). iv) high-

26


Page 26 of 33

Page 27 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


temperature drying should be avoided to prevent severe hornification; v) multiple pretreatment sequences were capable of obtaining glucan recovery yields in the ~89-93% range, suggesting that the process economics of each step will be a key consideration in any implementation of the pretreatment strategy. Of all the multi-step pretreatment combinations studied here, sequence 8 may be considered the most effective for loblolly pine wood based solely on glucan recovery yield (93.0%), and a detailed flow chart of that process is delineated in Figure 5. As a function of each of the steps, the originally rigid and compact wood chip becomes considerably more open and softer, beginning first with the loss of a majority of hemicellulose and extractives upon autohydrolysis. After disc refining, the soft wood chips were further converted into coarse lignocellulosic fiber clusters, which greatly increased the surface area of cellulose, although the cellulose chains present in these clusters were still highly crystallized. Via acetone wash and evaporation under room temperature, most of the moisture content in the fiber clusters was removed and, more significantly, there was no fiber hornification during the drying process. After a 1 h 85% phosphoric acid immersion at 50 °C, the residual highly crystalline cellulose structures were extensively disrupted into amorphous and fine wood fibers. These fine fibers were beneficial for the ensuing enzymatic hydrolysis, and thus needed to be extensively recovered by fine porous filtration. In addition, the waste phosphoric acid was partially condensed and recovered for the supplementation of the fresh concentrated phosphoric acid, but a further possibility is that it may also be neutralized for the production of phosphorous based fertilizers.

27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 33

Fresh water Loblolly pine wood chips

Autohydrolysis (180 °C, 40 min)

Wash

Disc refining

Centrifugation

Majority of extractives Majority of hemicellulose Small portion of lignin Small portion of glucan Fresh 85% H3PO4

Fresh acetone

Fresh water Acetone wash & Filtration

85% H3PO4 immersion (50 °C, 1 h)

Evaporation Acetone

Acetone recovery

Purification & concentration Diluted H3PO4

Residue water Waste acetone Residue extractives Acetone dissolved lignin

Enzymatic hydrolysis

Wash & Filtration

Fertilizers (value added by-product)

Fermentable sugars

Figure 5. A suggested economical and effective multi-step pretreatment sequence for the enzymatic saccharification of loblolly pine wood.

CONCLUSIONS 85% phosphoric acid based immersions were screened as core pretreatment steps for both delamination and enzymatic saccharification. From an energy savings point of view, a 24 h immersion at room temperature is preferred, and is more suitable for enzymatic saccharification of short-fiber samples with highly compact hemicellulose and lignin. From a time saving point of view, a shorter 1 h immersion at moderate temperature (50 °C) is more desired, and it can be applied for the effective enzymatic saccharification of

flexible

fiber

clusters

with

28

less

hemicellulose


and

lignin.

Page 29 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ACKNOWLEDGEMENTS This study was generously supported by a grant from the North Carolina Biotechnology Center (2013-MRG-1113).

SUPPORTING INFORMATION The Supporting Information is available free of charge on the ACS Publications website: Extrinsic features of acid treated wood samples (72% sulfuric and 85% phosphoric).

REFERENCES (1) Philippidis, G. P.; Smith, T. K.; Wyman, C. E., Study of the enzymatic hydrolysis of cellulose for production of fuel ethanol by the simultaneous saccharification and fermentation process. Biotechnol. Bioeng. 1993, 41, 846-853. (2) Vallander, L.; Eriksson, K. E. L., Production of ethanol from lignocellulosic materials: State of the art. In Bioprocesses and Applied Enzymology, Springer Berlin Heidelberg: 1990; Chapter 5, pp 63-95. (3) Kumar, P.; Barrett, D. M.; Delwiche, M. J.; Stroeve, P., Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. Ind. Eng. Chem. Res. 2009, 48, 3713-3729. (4) Zheng, Y.; Pan, Z.; Zhang, R., Overview of biomass pretreatment for cellulosic ethanol production. Int. J. Agric. Biol. Eng. 2009, 2, 51-68. (5) Talebnia, F.; Karakashev, D.; Angelidaki, I., Production of bioethanol from wheat straw: An overview on pretreatment, hydrolysis and fermentation. Bioresour. Technol. 2010, 101, 4744-4753. (6) Wanderley, M. C. d. A.; Martín, C.; Rocha, G. J. d. M.; Gouveia, E. R., Increase in ethanol production from sugarcane bagasse based on combined pretreatments and fedbatch enzymatic hydrolysis. Bioresour. Technol. 2013, 128, 448-453. (7) Avci, A.; Saha, B. C.; Kennedy, G. J.; Cotta, M. A., Dilute sulfuric acid pretreatment of corn stover for enzymatic hydrolysis and efficient ethanol production by recombinant Escherichia coli FBR5 without detoxification. Bioresour. Technol. 2013, 142, 312-319. (8) Li, C.; Knierim, B.; Manisseri, C.; Arora, R.; Scheller, H. V.; Auer, M.; Vogel, K. P.; Simmons, B. A.; Singh, S., Comparison of dilute acid and ionic liquid pretreatment of switchgrass: Biomass recalcitrance, delignification and enzymatic saccharification. Bioresour. Technol. 2010, 101, 4900-4906. (9) Lavoie, J.-M.; Capek-Menard, E.; Gauvin, H.; Chornet, E., Production of pulp from Salix viminalis energy crops using the FIRSST process. Bioresour. Technol. 2010, 101, 4940-4946.

29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(10) Romaní, A.; Garrote, G.; Alonso, J. L.; Parajó, J. C., Experimental assessment on the enzymatic hydrolysis of hydrothermally pretreated eucalyptus globulus wood. Ind. Eng. Chem. Res. 2010, 49, 4653-4663. (11) Taherzadeh, M. J.; Karimi, K., Enzymatic-based hydrolysis processes for ethanol. BioResources 2007, 2, 707-738. (12) Van Fossen, A. L.; Ozdemir, I.; Zelin, S. L.; Kelly, R. M., Glycoside hydrolase inventory drives plant polysaccharide deconstruction by the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus. Biotechnol. Bioeng. 2011, 108, 15591569. (13) Blumer-Schuette, S. E.; Lewis, D. L.; Kelly, R. M., Phylogenetic, microbiological, and glycoside hydrolase diversities within the extremely thermophilic, plant biomassdegrading genus Caldicellulosiruptor. Appl. Environ. Microbiol. 2010, 76, 8084-8092. (14) Gregg, D. J.; Saddler, J. N., Factors affecting cellulose hydrolysis and the potential of enzyme recycle to enhance the efficiency of an integrated wood to ethanol process. Biotechnol. Bioeng. 1996, 51, 375-383. (15) Lu, Y.; Yang, B.; Gregg, D.; Saddler, J.; Mansfield, S., Cellulase adsorption and an evaluation of enzyme recycle during hydrolysis of steam-exploded softwood residues. Appl. Biochem. Biotechnol. 2002, 98-100, 641-654. (16) Pan, X.; Xie, D.; Gilkes, N.; Gregg, D.; Saddler, J., Strategies to enhance the enzymatic hydrolysis of pretreated softwood with high residual lignin content. Appl. Biochem. Biotechnol. 2005, 124, 1069-1079. (17) Galbe, M.; Zacchi, G., A review of the production of ethanol from softwood. Appl. Biochem. Biotechnol. 2002, 59, 618-628. (18) Wu, M. M.; Chang, K.; Gregg, D. J.; Boussaid, A.; Beatson, R. P.; Saddler, J. N., Optimization of steam explosion to enhance hemicellulose recovery and enzymatic hydrolysis of cellulose in softwoods. Appl. Biochem. Biotechnol. 1999, 77-79, 47-54. (19) Sannigrahi, P.; Miller, S. J.; Ragauskas, A. J., Effects of organosolv pretreatment and enzymatic hydrolysis on cellulose structure and crystallinity in Loblolly pine. Carbohydr. Res. 2010, 345, 965-970. (20) Wu, S.-f.; Chang, H.-m.; Jameel, H.; Philips, R., Novel Green Liquor Pretreatment of Loblolly Pine Chips to Facilitate Enzymatic Hydrolysis into Fermentable Sugars for Ethanol Production. J. Wood Chem. Technol. 2010, 30, 205-218. (21) Huang, F.; Ragauskas, A. J., Dilute H2SO4 and SO2 pretreatments of Loblolly pine wood residue for bioethanol production. Ind. Biotechnol. 2012, 8, 22-30. (22) Agarwal, U. P.; Zhu, J. Y.; Ralph, S. A., Enzymatic hydrolysis of loblolly pine: effects of cellulose crystallinity and delignification. Holzforschung 2013, 67, 371-377. (23) Li, M.; Tu, M.; Cao, D.; Bass, P.; Adhikari, S., Distinct Roles of Residual Xylan and Lignin in Limiting Enzymatic Hydrolysis of Organosolv Pretreated Loblolly Pine and Sweetgum. J. Agric. Food Chem. 2013, 61, 646-654. (24) Frederick, W. J.; Lien, S. J.; Courchene, C. E.; DeMartini, N. A.; Ragauskas, A. J.; Iisa, K., Production of ethanol from carbohydrates from loblolly pine: A technical and economic assessment. Bioresour. Technol. 2008, 99, 5051-5057. (25) Gonzalez, R.; Treasure, T.; Phillips, R.; Jameel, H.; Saloni, D., Economics of cellulosic ethanol production: green liquor pretreatment for softwood and hardwood, greenfield and repurpose scenarios. BioResources 2011, 6, 2551-2567.

30


Page 30 of 33

Page 31 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(26) Wu, S.; Jameel, H.; Chang, H.-M.; Phillips, R., Techno-economic analysis of the optimum softwood lignin content for the production of bioethanol in a repurposed kraft mill. BioResources 2014, 9, 6817-6830, 14 pp. (27) Zhang, Y. H. P.; Cui, J.; Lynd, L. R.; Kuang, L. R., A transition from cellulose swelling to cellulose dissolution by o-phosphoric acid: Evidence from enzymatic hydrolysis and supramolecular structure. Biomacromolecules 2006, 7, 644-648. (28) Walseth, C. S., Occurrence of cellulases in enzyme preparation from microorganisms. Tappi 1952, 35, 228-232. (29) Ghose, T. K.; Kostick, J. A., Enzymatic saccharification of cellulose in semi- and continously agitated systems. Amer. Chem. Soc.: Washington D.C, 1969; Vol. 95. (30) Stone, J. E.; Scallan, A. M., Digestibility as a simple function of a molecule of similar size to a cellulase enzyme. Amer. Chem. Soc.: Washington D.C., 1969; Vol. 95. (31) Fan, L. T.; Gharpuray, M. M.; Lee, Y.-H., Cellulose hydrolysis. Springer-Verlag: Berlin Heidelberg, 1987; Vol. 3. (32) Henriksson, G.; Lawoko, M.; Martin, M. E. E.; Gellerstedt, G., Lignincarbohydrate network in wood and pulps: A determinant for reactivity. Holzforschung 2007, 61, 668-674. (33) Du, X.; Lucia, L. A.; Ghiladi, R. A., A novel approach for rapid preparation of monophasic microemulsions that facilitates penetration of woody biomass. ACS Sustain. Chem. Eng. 2016, 4, 1665-1672. (34) Carrillo, C. A.; Saloni, D.; Rojas, O. J., Evaluation of O/W microemulsions to penetrate the capillary structure of woody biomass: interplay between composition and formulation in green processing. Green Chem. 2013, 15, 3377-3386. (35) Carrillo, C. A.; Saloni, D.; Lucia, L. A.; Hubbe, M. A.; Rojas, O. J., Capillary flooding of wood with microemulsions from Winsor I systems. J. Colloid Interface Sci. 2012, 381, 171-179. (36) Hall, M.; Bansal, P.; Lee, J. H.; Realff, M. J.; Bommarius, A. S., Cellulose crystallinity – a key predictor of the enzymatic hydrolysis rate. FEBS J. 2010, 277, 15711582. (37) Theander, O.; Westerlund, E. A., Studies on dietary fiber. 3. Improved procedures for analysis of dietary fiber. J. Agric. Food Chem. 1986, 34, 330-336. (38) Sette, M.; Lange, H.; Crestini, C., Quantitative HSQC analyses of lignin: A practical comparison. Comput. Struct. Biotechnol. J. 2013, 6, 1-7. (39) Segal, L.; Creely, J. J.; Martin, A. E.; Conrad, C. M., An empirical method for estimating the degree of crystallinity of native cellulose using the X-ray diffractometer. Text. Res. J. 1959, 29, 786-794. (40) Park, S.; Baker, J.; Himmel, M.; Parilla, P.; Johnson, D., Cellulose crystallinity index: measurement techniques and their impact on interpreting cellulase performance. Biotechnol. Biofuels 2010, 3, 1-10. (41) Fritz, C.; Rojas, O. J. In Microemulsions as a green biomass pretreatment for bioconversion, 247th National Spring Meeting of the American-Chemical-Society (ACS), Dallas, TX, American Chemical Society: Dallas, TX, 2014. (42) Fritz, C.; Ferrer, A.; Salas, C.; Jameel, H.; Rojas, O. J., Interactions between cellulolytic enzymes with native, autohydrolysis, and technical lignins and the effect of a polysorbate amphiphile in reducing nonproductive binding. Biomacromolecules 2015, 16, 3878-3888.

31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(43) Luo, X.; Zhu, J. Y., Effects of drying-induced fiber hornification on enzymatic saccharification of lignocelluloses. Enzyme and Microb. Technol. 2011, 48, 92-99. (44) Nazhad, M. M.; Ramos, L. P.; Paszner, L.; Saddler, J. N., Structural constraints affecting the initial enzymatic hydrolysis of recycled paper. Enzyme and Microb. Technol. 1995, 17, 68-74.

32


Page 32 of 33

Page 33 of 33

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


For Table of Contents Use Only

Development of a highly efficient pretreatment sequence for the enzymatic saccharification of loblolly pine wood Xueyu Du,†‡ Lucian A. Lucia,†‡‡ and Reza A. Ghiladi*† †

Department of Chemistry, North Carolina State University, 2620 Yarbrough Drive, Raleigh,

North Carolina 27695-8204, United States ‡

Department of Forest Biomaterials, North Carolina State University, 2820 Faucette Drive,

Raleigh, North Carolina 27695-8005, United States ‡

Qilu University of Technology, State Key Laboratory of Pulp & Paper Science and Technology,

Jinan, PR China 250353

Synopsis: An economical and sustainable pretreatment sequence for effective enzymatic saccharification of loblolly pine wood.

33


Development of a Highly Efficient Pretreatment Sequence for the

Recommend Documents