Recombinant E. coli cellulases, Î²-glucosidase and polygalacturonase

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Recombinant E. coli cellulases, #-glucosidase and polygalacturonase convert a citrus processing waste into biofuel precursors Eman Ibrahim, Kim Jones, Keith Edward Taylor, Ebtesam N Hosseney, Patrick L. Mills, and Jean Escudero ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b04518 • Publication Date (Web): 03 May 2018 Downloaded from http://pubs.acs.org on May 5, 2018

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ACS Sustainable Chemistry & Engineering

Recombinant E. coli cellulases, β-glucosidase and polygalacturonase convert a citrus processing waste into biofuel precursors Eman Ibrahim1, Kim D. Jones2, Keith E. Taylor3, Ebtesam N. Hosseney4, Patrick L. Mills5 and Jean M. Escudero6,*

1

Department of Environmental Engineering, Texas A&M University-Kingsville, Texas 78363, USA and Department of Botany and Microbiology, Al-Azhar University, Nasr City, Cairo 11884, Egypt; [email protected]

2

Department of Environmental Engineering, Texas A&M University-Kingsville; [email protected]

3

Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada N9B 3P4; [email protected] 4

Department of Botany and Microbiology, Al-Azhar University, Nasr City, Cairo 11884, Egypt; [email protected] 5

Department of Chemical Engineering, Texas A&M University-Kingsville; [email protected]

6,*

Department of Basic Science, St. Louis College of Pharmacy, St. Louis, Missouri 63110-1088, USA; author for correspondence [email protected]

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Abstract Molecular and biochemical characterization of lignocellulohydrolases cel12B, cel8C, β-glucosidase and peh28 from Pectobacterium carotovorum subsp. carotovorum (Pcc) expressed in Escherichia coli (E. coli) was reported in previous work. The current preliminary study investigates the enzymes’ catalytic performance on Rio-Red grapefruit processing waste (GPW) conversion which can lead to the development of low-cost and effective strategies with strain engineering and/or modified catalysts for production of biofuel precursors. The GPW utilized for the study is known for its low ash and lignin contents compared to corn stover, wheat straw and sugarcane bagasse, while yielding soluble sugars and polysaccharide constituents proportionally comparable to wastes from other citrus sources. Pretreatment of GPW at 120°C with 1% w/w NaOH for 15 minutes resulted in significant total solid losses due primarily to conversion of glucans and lignin. Subsequent enzymatic bioconversion using the recombinant E. coli lignocellulolytic system resulted in production of 24, 11 and 14 g/kg solid biomass for the respective glucose, cellobiose and galacturonic acid products from GPW over 24 h at 45°C and pH 5.4. Other sugar products, e.g., xylose, arabinose, galactose, mannose and rhamnose, were also detectable throughout the catalysis but at lower concentrations compared with the main products.

Key words: Escherichia coli, cellulases, β-glucosidase, polygalacturonase, biomass, hydrolysis


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Introduction The world production of citrus was estimated at 109,338 thousand tons for 2009/2010 as reported by Food and Agriculture Organization of the United Nations.1 Twenty-two percent of this production is nonconsumable citrus processing waste. Utilization of citrus waste for biofuel production would have environmental benefits in preventing accumulation of an underutilized by-product that lacks an appropriate disposal strategy, thereby increasing methane toxicity in the aquatic surroundings; citrus processing waste is currently a poorly utilized cattle-feed additive.2 Other valorization strategies have been focused on development to produce essential oils as well other value-added compounds.3 However, the lignocellulosic component of this waste has been largely untapped, hence its choice as the feedstock for the present study. Knowledge of composition is of crucial relevance for processing and/or conversions of biomass. For example, the cellulose to lignin ratio plays a large role in process design for biofuel production. Likewise, the concentration of value-added components will strongly influence recovery strategies and yields from extraction processes, as reviewed by Vassilev et al.,4 based on the work by McKendry et al.5 The usage of inedible agricultural residues, short-rotation energy crops and semi-biomass (biomass contaminated with industrial biomass wastes) have been suggested.4 Lignocellulosic biomass conversion using cellulases is a process that can be adversely affected by several degradation products such as those of lignin-derived and other phenolic compounds,6-7 possibly due to lignin surface properties that increase the amount of enzyme non-productive binding.6 Lignin breakdown provides a complex mixture of p-coumaryl-, coniferyl- and sinapyl alcohols8 in various proportions with p-hydroxyphenyl-, guaiacyl- and syringyl-phenylpropanoid conjugated units from various feedstock species.6 Biomass pretreatment strategies for efficient enzymatic saccharification and/or conversion include steam/steam explosion, grinding/milling and hot water/auto-hydrolysis, acid treatment, and alkali treatment.9 In the latter, NaOH may cause biomass conversion by triggering intramolecular bond cleavages within the lignocellulosic complexes, particularly the ether and ester bonds of the lignin-carbohydrate complexes and/or ester-carbon-to-carbon bonds of ferulic acid in the lignin polymer.9-10 3 ACS Paragon Plus Environment


Due to the complexity of lignocellulosic biomass, details for the mechanism of the cellulose hydrolysis by cellulase catalysis are still underdeveloped.11 Correlation of cellulose accessibility to cellulases with substrate crystalline surfaces and/or morphologies of various biomass sources have been attributed to plant cell wall surface pore sizes.12-13 Synergy between endo- and exo-glucanases as well as between endoglucanases from various sources in the presence or absence of β-glucosidase has been characterized.14-17 Substrate recalcitrance, substrate pretreatment, enzyme biochemical characteristics, as well as the ionic medium composition of the reaction are among the principle factors summarized to influence the degree of cellulase synergy directly.14 Additionally, the role of polygalacturonase in releasing cell wall cellulosic fibrils, which are tightly cemented and embedded into the pectin matrix, facilitating the subsequent biomass enzymatic hydrolysis has been reported by many researchers.18-19 Characterization of three genes from Pectobacterium carotovorum subsp. carotovorum (Pcc) expressed by Escherichia coli (E. coli) and examination of their potential for lignocellulosic biomass conversion and/or biofuel production

was conducted in our earlier study for cellulases, cel8C and cel12B, and

polygalacturonase, peh28.20 The characteristics of the enzymes, cel8C, cel12B and peh28, in terms of protein secondary or tertiary structure as well as their biochemical properties on cellulose, in conjunction with another β-glucosidase from E. coli for cel8C/cel12B, or on galacturonic acid, for peh28, have also been reported.21 Characterization of these enzymes and evaluation of their ability to degrade recalcitrant products found in citrus waste are the main objective of this study. The products of enzymatic conversion of GPW by the recombinant system were determined under industrially relevant conditions after a commonly utilized pretreatment. A gas chromatographic-mass spectrometric (GC-MS) derivatization protocol was used for the assessment of the degree of lignocellulosic biomass conversion of GPW as well as the mono- and di-saccharide products formed due to the biomass alkaline treatment and by enzymatic hydrolysis by a cocktail of the recombinant E. coli enzymes.


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Experimental Section All chemicals in this study were obtained from Sigma-Aldrich (St. Louis, MO), unless otherwise mentioned. Deionized water (DI H2O) used was Nano-purified with a Barnstead DiamondTM Ultrapure water device (cat. no. D11901-7143, Thermo Scientific, Rockford, IL).

Enzyme preparations Recombinants cel12B, cel8C, β-glu and peh28 encoding the respective cellulases, cel12B, cel8C, βglucosidase and polygalacturonase, peh28, expressed in E. coli were used for investigating their combined activities (cocktail formulation given below) on a grapefruit processing waste using their partially purified forms previously described.21

Biomass preparation The study was conducted using Rio Red (Citrus paradisi)-GPW, previously collected as a byproduct biowaste from Rio Grande Juice Company Plant (Mission, TX), as a hammer-mill chopped chemicallyuntreated sample, and stored at -20°C, in which the physical and chemical characterization as well as the biomass crystallinity index were initially determined.22 Freshly collected samples were analyzed in triplicate accordingly, using 20-25 µm particle-size range (GPW was milled for reducing particle size using a Miller stainless-steel knife- grinder (GM-200, cat. no. 024460047, Retsch® (Verder Scientific, Inc.), Newtown, PA)), and their total moisture, solids, pectin (soluble and insoluble), sugars, fats and ash were characterized using published protocols23-25; cellulose, hemicellulose and lignin quantities for this work were determined by Rivas et al., unpublished data, based on the aforementioned protocols. Total sugars were determined through successive analysis of Soxhlet-separated GPW extracts from 18 g moist weight (~72%) of 25-45 µm milled GPW; sequential elutions were carried out for water-soluble, ethanolsoluble and hexane-soluble extractives for 6h per cycle at 100, 60 and 45°C using DI-H2O, ethanol: H2O (80% (v/v)) (ethanol, absolute, 99.9%, cat. no. 64-17-5) and hexane (hexane, 95% anhydrous, Cat. no. 110-54-3), respectively, where ethanol-derived sugar extracts were evaporated and derivatized for gaschromatography and mass-spectrometry analysis, as described below in the biomass hydrolysis section. 5 ACS Paragon Plus Environment


Water-insoluble pectin and hexane-insoluble fats were estimated from dry weights, following overnight oven-drying at 60 and 45±2.0°C, respectively. Total solids and ash were determined in the solid material remaining after water, ethanol and hexane extractions, using the respective dry weights following 24-48 h oven-drying at 105±2.0°C and 4-5h furnace-drying at 550-600°C.

Biomass alkali-pretreatment Samples, 25-45 µm milled GPW, in triplicate were alkali-pretreated using a sodium hydroxide pretreatment protocol described by Wang et al.,26 where 0.5% (w/w) NaOH solution (NaOH, pellets, cat. no. 1310-73-2) was used at 1:10 liquid to biomass solid ratio in autoclaved sealed flasks at 121°C for 15 min. The biomass was subsequently collected on a porcelain Buchner funnel and re-suspended in DI-H2O to an 80% final moist weight and stored at 4°C until enzymatic treatment. Alkali-pretreated GPW was characterized as for chemically-untreated GPW in the preceding section.

Biomass hydrolysis The combined activities of E. coli-expressed cel8C, cel12B, β-glucosidase and peh28 at 9.9 (45.5 U), 12.2 (38.6 U), 16.4 (9.1 U) and 8.1(2.3 U) mg protein per g solid waste (GPW, alkali-pretreated) were evaluated for hydrolytic polymer efficiencies as compared to untreated GPW. The respective activity determinations were carried out using methods described in Ibrahim et al.,21 on authentic pectin/cellulose substrates. GPW was pre-neutralized to pH 5.4 using 0.5 M NaOH and heated to 45±2.0°C before adding the preceding enzyme mixture at 10 g/L solid loading based on the optimum catalytic activities defined previously in Choi et al.27 The hydrolysis at 45°C was followed for up to 24 h; hydrolysate samples were collected every hour for determination of reducing, mono- and disaccharide, sugar products formed due to hydrolysis. Enzyme and substrate blanks were prepared for similar processing at zero-time reaction (t=0; enzyme blank) or with water replacing the enzyme additions (substrate blank) for comparison. A cocktail of growth inhibitors, tetracycline, cycloheximide and chloramphenicol (cat. no. 87128, C104450 and C0378, respectively) was added to all reactions each at 0.1 mg/g solid biomass. Hydrolysate samples (1.0 ml) were diluted with four volumes of ethanol and the filtrates containing mono- and disaccharide hydrolytic products were evaporated for derivatization and GC-MS quantification, as previously 6 ACS Paragon Plus Environment

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described.21 The GC (Model 6890, Agilent Technologies, Inc. Hewlett-Packard, Santa Clara, CA) operating conditions were as follows: 180°C oven temperature for 2 min; increased by 230°C for 15 min at 15°C/min, 250°C injector temperature, 1:10 split ratio, 1.0 µl injection sample/standard and 2°C/min rate flow helium. The MS (Model 5973, Agilent Technologies, Inc.) conditions were however conducted at 1400 electron multiplier voltage, 250°C ion source, 1 s scanning time and 50-550 m/z selected ratios of a mass selective detector (MSD). Retention indices of GC analytes were assigned using derivatives of the respective standards, prepared in the same manner with reference to the corresponding GC-MS spectral data in National Institute of Standards and Technology libraries. The percentages of polymer conversions to the sugars released were estimated following GPW alkali and enzymatic treatments, compared with untreated waste using eq 1.28

( )

% = 100 (1) ℎ

Where is the hydrolysate factor for the respective biomass polysaccharide/sugar combination;

/ is the relative sugar concentration in the hydrolysate following pretreatment/enzymatic hydrolysis as determined by GC-MS, compared with the respective standard; ℎ / is the initial polysaccharide composition in untreated GPW.

Statistical Analysis All tests were performed in triplicate and were analyzed using the GraphPad Prism 6.0 (GraphPad software Inc., La Jolla, CA) statistical program where the Tukey post-test one-way system was utilized for the corresponding data analysis of variance; significance was assigned at P-values less than 0.05.



Results and Discussion Compositional analysis of untreated Rio Red (Citrus paradisi) grapefruit processing waste Knowledge of biomass chemical composition is crucial for its processing to produce biofuels. The significant role of the biomass cellulose to lignin ratio in process design for biofuel production has been reported.29 Additionally, the biomass inorganics (ash) and the technical and environmental problems that may be encountered have also been considered.4 In general, ash content resembles classes of detrital (stable, less reactive and high-melting, e.g., silicates and oxy-hydroxides derivative compounds) and technogenic materials (reactive, less stable and low-melting e.g., opal, oxalates, carbonates, phosphates, sulfates, chlorides and nitrates containing inorganics) which can be used as indicators of biomass contamination by such compounds/elements.4, 30 The composition and the relative proportion of lignin in a given biomass is also detrimental due to influences on enzymatic non-productive binding during biomass hydrolytic biotransformation as reviewed by Saini et al.6 Preliminary analysis was conducted on untreated Rio Red (Citrus paradisi) grapefruit processing waste for total reducing sugars (ethanol-extractives) as well as other ash, fat, pectin, lignin, cellulose and hemicellulose containing derivatives as determined using GC-MS, compared with those for other citrus and non-citrus feedstocks, Table 1. The amount of each constituent of the current GPW was similar to those estimated by Marín et al.31 for grapefruit (Citrus paradisi, whole fruit part, extraction of flavonoids) and for sour orange (Citrus aurantium L., whole fruit part, extraction of flavonoids) wastes. However, variations are seen when GPW is compared to other feedstocks such as sugarcane bagasse, corn stover and wheat straw32 in terms of ash, lignin and cellulose constituents, Table 1. The amount of ash and lignin contents for the GPW are 6-20-fold lower for the sugarcane bagasse and corn stover and 20-40% lower for the wheat straw biomasses indicated (Table 1), suggesting the GPW as an ideal feedstock for subsequent enzymatic hydrolysis. Ash content in a given feedstock significantly influences subsequent biomass industrial processing and/or chemical treatments as reported by Marín et al.31


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Table 1. Chemical composition of untreated GPW as well as other citrus and non-citrus feedstocks, as percent of dry matter Waste by-products

Sugars

Pectin

Fat

Ash

Lignin

GPWa

8.3±0.4

6.5±0.5

0.7±0.1

0.6±0.01

3.5

3.5

1.6

Sour Orangec Grapefruit

c

8.0

13.2

12.5

39.0

8.3

8.1

11.6

23.1

8.1

8.5

0.5

nd

nd

nd

3.7

23.1

16.9

nd

d

nd

nd

nd

10.1

18.6

37.7

nd

d

nd

nd

nd

10.2

16.9

32.6

nd

Sugarcane bagasse

Wheat straw a

Hemicellulose 6.3b

e

d

Corn stover

13.1b

Cellulose 27.2b

GPW is a grapefruit processing citrus waste from Rio-Red (Citrus paradise) collected for the present study.

b

lignin, cellulose and hemicellulose values of GPW were evaluated by Rivas et al. (unpublished data), based on Sluiter et al.23-25

c

values for peel were reported by Marín et al.31

d

values were reported by Carroll and Somerville.32

e

Not determined

Hydrolysis of GPW polysaccharides following alkali-pretreatment and enzymatic conversion using E. coli recombinant cel12B, cel8C, β-glucosidase and peh28 The hydrolytic efficiency of a cocktail of the modified cel12B, cel8C, β-glucosidase and peh28 was primarily investigated in the conversion of polysaccharides of alkali-pretreated GPW at 1% biomass solid loading and approximately 9.9±1.3, 12.2±2.4, 16.4±0.5 and 8.1±1.1 mg protein added for the cel12B, cel8C, β-glucosidase and peh28, respectively, per dry g of biomass. These protein loadings correspond to 45.5, 38.6, 9.1 and 2.1 U of the respective enzyme activities per g of dry GPW. For this initial study, activities of the two cellulases were chosen to be comparable to each other and in total to the activity loading of a successful published study, Wang et al.26 β-Glucosidase activity, since it is ancillary to cellulase action, was chosen to be much lower than the combined cellulases, about 10%, and the pectinase activity was chosen to be 4-fold lower still because the pectin content of GPW is that much lower than the cellulose content of the same waste (Table 1). The enzymatic treatment was carried out at 45°C and the products of hydrolysis were determined in hydrolysates collected over 24 h, using gas chromatography and mass spectrometry (GC-MS) quantitative analysis. An example GC-chromatogram and the MS-profile of 9 ACS Paragon Plus Environment


the hydrolysates of mono- and di-saccharide products of GPW following the 24 h enzymatic hydrolysis are seen in Figure 1, compared with a blank seen in Figure 2, for a similar enzymatic treatment for the GPW at zero-time (t=0). The molecular mass, formula and the structure of mono- and di-saccharide derivatives used in identification are shown in Table 2.

[insert Figure 1] [insert Figure 2]


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Table 2. Fragmentation patterns of selective fragment ions (SFI) for trimethylsilyl (TMS) and trimethylsilyloxime (TMSO) derivatives as analyzed by GC-MS Compounds

a

TMS- and TMSOderivatives

Derivative structure

Molecular weight (g/mol)

Retention time (min)

Selective total fragment ions (SFI) m/za

D-xylose (X)

Xylose, tetrakis (TMS)

438.854

4.282

73, 147, 217, 307

L-arabinose (A)

Arabinose, 2,3,4,5tetrakis-O-(TMS)-

438.854

4.368

73, 147, 217, 307

L-(+)rhamnose (R)

Rhamnose, pyranose, TMS

452.881

5.294

73, 147, 217

D-fructose (syn/antioximes, F 1-2)

Fructose, oxime (TMS)

642.290

F1:5.642 F2:5.716

73, 147, 217, 307

Mannose (M)

Mannose, oxime (TMS)

628.257

5.968

73, 147, 205, 319

D-Galactose (syn/antioximes, Gal 12)

Galactose, oxime hexakis (TMS)

628.257

Gal1:7.540 Gal2:7.631

73, 147, 205, 319

Sucrose (S)

α-DGlucopyranoside, 1,3,4,6-tetrakis-O(trimethylsilyl)-βD-fructofuranosyl 2,3,4,6-tetrakis-O(trimethylsilyl)-

919.745

9.854

73, 147, 217, 361

These mass spectral fragmentation patterns of the derivatives are used for total ion chromatogram (TIC) peak assignments in Figures 1 and 2. The m/z

represents the masses of the fragmentation ions detected for each theoretically derivatized compound relative to the corresponding abundance in integrator units/ng (Iu/ng) shown in the respective GC-chromatograms. The corresponding TIC peak assignments for salicin-internal standard (INSD) α- and β- Dglucopyranose (G1 & G2), Glucose (syn/anti-oximes) (G3 & G4), α- and β- D-cellobiose (C1 & C2), α- and β- D-Galactopyranuronic acid (GA1 & GA2), Galacturonic acid (syn/anti-oximes) (GA3 & GA4), Galacturonic acid (syn/anti-oximes) (GA3 & GA4) shown in Figure 1 and/or Figure 2, were identified earlier in Ibrahim et al.21



Figure 3 shows the progress curves of the products of biomass conversion due to the enzymatic hydrolysis for glucose, cellobiose, galacturonic acid, xylose, arabinose, mannose, fructose, galactose and sucrose, respectively. Figure 4 shows the degree of biomass conversion due to the enzymatic treatment after 24 h, for the respective glucose, galacturonic acid, xylose, arabinose, mannose, fructose and galactose polymers, compared to the biomass following alkali pretreatment alone with NaOH (0.5%, w/v) at 120°C for 15 min prior to the hydrolysis, and compared to the polysaccharide/polymer constituents of untreated GPW (Rivas et al., unpublished data).

[insert Figure 3] [insert Figure 4]

Figure 4 indicates significant glucan transformation/conversion following alkali-pretreatment compared with untreated GPW, unlike that found by Wang et al.26 for a similar pretreatment of coastal Bermuda grass (CBG), but is consistent with that reported by Chen et al.33 in the alkali-pretreatment of corn stover. This extent of conversion is consistent with the loss of biomass total solids detected herein, approximately 28 % (results not shown), in the alkali-pretreatment of GPW, which might also include some alkaliinduced lignin transformation/depolymerization as observed by Wang et al.26 No significant alteration of the galacturonan, rhamnan, arabinan, xylan and galactan constituents of GPW due to alkali pretreatment were found, which is attributed to the lack of pectin as well as hemicellulose transformations by the alkali pretreatment, compared with those of Wang et al.26 The large conversion of the fructan due to GPW alkali-pretreatment could be attributed to the saccharose polymer autohydrolysis at the temperature of treatment (120°C), similar to the finding of Kühnel et al.34 due to a sulfuric acid-thermal pretreatment of a sugar beet-pulp biomass. This investigation provides some useful information on the process of alkali pretreatment of GPW, and suggests additional trials with other pretreatment systems would be warranted in order to compare their influence on the subsequent enzymatic processing of GPW.


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The primary products observed due to the enzymatic treatment of GPW were glucose, cellobiose and galacturonic acid where their ratios increased significantly over 24 h as seen in Figure 3g,j,d, respectively. Increases in the xylose, arabinose, mannose concentrations were also observed over the enzymatic period, but their levels were quite minimal compared with those of glucose, cellobiose and galacturonic acid following the treatment (Figure 3a,b,f). The increases observed with fructose and sucrose over the period of enzymatic treatment were insignificant compared to their initial concentrations in the alkali-pretreated waste (Figure 3e,i). These observations are primarily in agreement with those of Choi et al.27 and Huang et al.,35 for the enzymatic treatment of mandarin (Citrus unshiu) peel and sugarcane bagasse pulp feedstocks, respectively. The xylose concentration increased up to approximately 4.0±0.5 g/kg biomass following the 24 h enzymatic treatment of GPW, accounting for 7.3% total xylan conversion (Figure 3a, and Figure 4). The increase in conversion of the enzymatically treated GPW is attributed to the hemicellulose conversion by analogy with corn stover, poplar, switchgrass and sorghum undergoing similar treatment with Novozymes cellulases, as reported by Wolfrum et al.36 However, the conversion efficiency of the current cellulases for the hemicellulose-xylan polymers of GPW was much lower than the 80-90% yields observed with Novozymes cellulases with the biomasses as reported by Wolfrum et al.36 While the xylan-derived products could be inhibitory to the main hydrolysis of biomass cellulose,36-37 their conversion could improve the process of hydrolysis of corn stover-based biomass as reported by Kumar and Wyman.38 Thus, the process of GPW conversion into biofuel products could be improved if a xylanase were used in the catalysis. Further investigation is warranted into the use and role of xylanase in the catalysis of the present system. Arabinose, galactose, mannose and rhamnose residues were reportedly found among hydrolysates of several enzymatic treatments as co-products of pectinolytic activities, e.g., arabinose, galactose and rhamnose34 and/or hemicellulose breakdown, e.g., galactose and mannose.37 Inhibition by galactose and arabinose co-products of the catalysis and/or main-products generation during the biomass enzymatic 13 ACS Paragon Plus Environment


degradation has been previously reported.34 The inhibitory effects of hemicellulose-derived mannose and galactose on cellulases have also been reported.37,

39

While the inhibitory mechanism of hemi-cellulose

derived sugars has yet to be investigated,37 the inhibition of pectin-derived galactose and arabinose due to the accumulation of attached ferulic acid esters, situated within galactan and arabinan biomass products, was reported.34 Approximately 1.1±0.2 g arabinose and 1.8±0.4 g mannose were produced per kg biomass over the 24 h enzymatic treatment of GPW (Figure 3b,f), accounting for 2.0 and 8.0% arabinan and mannan respective conversions, as seen in Figure 4. The corresponding rhamnose and galactose increases were 49% and 59%, following hydrolysis with the current enzyme system for the GPW (Figure 3c,h), which accounted for 15% and 7% of the respective rhamnan and galactan conversions, as seen in Figure 4. The increases in galactan, arabinan, and rhamnan conversion due to the current enzymatic treatment are, however, lower than those of Kühnel et al.,34 using a cocktail of Chrysosporium lucknowense lignocellulases, which, therefore, could limit their contribution to the process of the biomass degradation by cellulases and/or pectinase, due to accumulation of by-products, based on Xiao et al.37 Approximately 14±0.9 g galacturonic acid per kg biomass was produced over the 24 h enzymatic hydrolysis of GPW (Figure 3d), accounting for 25.0% galacturonan conversion (Figure 4), comparable to the galacturonan conversion biomass level reported by Kühnel et al.,34 following hydrolysis with Chrysosporium lucknowense-lignocellulases and a pectinase/poly-galacturonase in conjunction with soybean peroxidase added to the system. The extent of substrate conversion is, however, lower than that due to peh28 catalysis on a commercial citrus pectin extract by 30.0%, as discussed in Ibrahim et al.,21 which is attributed to increased lignin-derived and other associated inhibitors in the biomass system and/or those arising during pretreatment or saccharification.6 The inhibition observed in the production of galacturonic acid following 2 h of hydrolysis (Figure 3d), supports the hypothesis. The toxicity of weak acid products such as galacturonic acid to subsequent biomass fermentation by yeast would retard their biofuel production.34 The finding that alternative systems, such as E. coli, are able to utilize the galacturonic acid in their hydrolysate products for fermentation,40 would overcome this limitation of yeast


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fermentation. Thus, optimizing the galacturonic acid production from GPW, using the current enzymatic system, could be advantageous using de novo-approaches for biofuel production. Depolymerization of glucan has been observed with the current modified system as demonstrated by the significant increases found in glucose and cellobiose concentrations following the enzymatic treatment compared to raw (untreated) and alkali-pretreated GPW (Figures 3 and 4). The insignificant increases in fructose and other saccharoses following the enzymatic hydrolysis for the GPW (Figure 3), compared to raw (untreated) biomass, suggest that polysaccharide autohydrolysis is not the mechanism for the glucose production. Similar observations have been made with several other lignocellulases and other biomasses by Arantes and Saddler41 and Wolfrum et al.36 The exolytic and endolytic catalysis by the current cellulases (cel12B and cel8C) as well as the major role of β-glucosidase in the conversion of the cellobiose produced has been discussed in detail in Ibrahim et al.21 In general, cellobiose is a product of synergism of cel12B and cel8C, where cel8C solubilizes the cellulose’s amorphous oligomeric residues, liberating them into free oligomers that in turn can be solubilized by cel12B into shorter chains and cellobiose. Due to limitations of the current GC-MS methods to analyze higher oligomers than cellobiose, it is surmised that cellobiose and glucose are the dominant products of catalysis. In this investigation, cellobiose production was not steady throughout the process, from which the role of β-glucosidase in producing the opposite glucose anomer could be inferred. However, glucose production was high in the first 30 min, 12±1.1 g/kg GPW, (Figure 3g); this production only doubled in the remainder of the 24 h incubation. A similar process disparity in the rate of glucose production was observed when Avicel was treated with cellulase and βglucosidase, albeit over a shorter incubation (30 min initial period of 3 h total), as discussed in Ibrahim et al.21 This was suggested to correlate with the enzymes’ consumption of the readily-accessible amorphous regions in the initial period, followed by slower glucose production over the prolonged incubation as enzymes gained access to other amorphous and/or crystalline domains of GPW. Similar explanations have been advanced by Gao et al.,42 for a prolonged incubation of Avicel with the cellulolytic system, Trichoderma cellulase (Novozyme®50013) in combination with a β-glucosidase (Novozyme® 50010). The



utilization of the accessible amorphous glucan system in the early stage of reaction was previously discussed by Arantes and Saddler41 with another biomass. The low rate of enzymatic glucose production after the initial burst on GPW, is consistent with β-glucosidase inhibition by glucose, as discussed in Ibrahim et al.21 The low cellobiose production over that same extended period, might be attributed to putative inhibitory factors from lignin and its attached components that arise during biomass pretreatment and bind non-productively to the enzymes by hydrophobic or electrostatic interaction , as reviewed by Saini et al.6 The overall glucan conversion ratio was 16% of GPW by the current enzyme system, which is lower than what has been observed with other systems where more than 90% was the yield.36, 43 Optimized pretreatment to achieve efficient lignin removal/ separation was not within the scope of the current investigation but it is recognized as a necessary future activity in order to maximize the enzymatic conversion by the current biomass treatment system.26, 43 Additionally, integration with a laccase and/or peroxidase lignin polymerizing system,44 were among several treatments proposed to promote biomass enzymatic processing and/or suppress the non-productive binding by cellulases. This preliminary investigation highlights the hydrolytic properties of the modified system consisting of cel12B, cel8C, βglucosidase and peh28 acting on GPW, with overall glucose production higher than that given by Hamzah et al.45 for a similar preliminary investigation where 4 g/L was the net glucose produced from treated oil palm empty fruit bunch fiber using their cellulase in combination with Novozyme-188. The current system is representative of a new breed in the field of biofuel production, one warranting further optimization and process integration studies.


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Conclusions The Rio Red (Citrus paradisi) GPW utilized for the analysis of enzymatic conversion showed ideal properties due to low levels of ash and lignin which could be vital for the subsequent biotransformation and/or catalysis using the cellulases and polygalacturonase system. The efficacy of the current recombinant system (E. coli cel8C, cel12B, β-glucosidase and peh28) in production of biofuel precursors from a novel biomass, GPW, is demonstrated. Noticeable xylose, arabinose, galactose, mannose and rhamnose in the enzymatic hydrolysate of GPW indicate the relevant hemicellulose and/or pectin transformation with the current recombinants. The low rate of conversion using the current recombinants for the GPW over a 24 h period implies that some inhibition, e.g., due to product accumulation, inhibitors derived from lignin breakdown and/or enzyme inactivation, took place during the catalysis. Integration with other biomass hydrolases. e.g., xylanase and/or other cellulases, having promising synergy with the current recombinants and evaluation of other biomass pretreatments are suggested as a strategy for future process optimization with GPW, in addition to evaluation of product separation and/or process optimization for the catalysis.

Acknowledgments The authors would like to acknowledge both the Institute for Sustainable Energy and the Environment, TAMUK, USA, and Egyptian Ministry of Higher Education, MOHE, Egypt, for funding this research. Our sincere gratitude is extended to Dr. Wafaa M. Abd El-Rahim, Professor of Environmental Microbiology, National Research Centre, Egypt for her invaluable participation in reviewing the study. Gratitude is extended to Prof. Dr. Shad Nelson, Department of Soil and Plant Sciences, Department of Agriculture, TAMUK, Mr. Raul Rivas, Environmental Engineering Department, TAMUK, for stimulating discussion on the topic of the research, and Mr. Jesus R. Hernandez, Environmental Engineering Department, TAMUK, for his technical support during the early analysis with GC-MS.



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Figure Captions Figure 1. GC-MS analysis of GPW enzymatic hydrolysate. GC-MS total ion chromatogram (TIC) of the trimethylsilyl (TMS) and trimethylsilyl-oxime (TMS-oxime) derivatives for compounds of enzymatic treatment of GPW (1% solid biomass loading) using a cocktail of E. coli expressed cel12B, cel8C, βglucosidase and peh28 at 9.9, 12.2, 16.4 and 8.1 mg respective protein loadings per g of solid biomass. As shown, glucose (G), galacturonic acid (GA), fructose (F), galactose (Gal) and cellobiose (C) are characterized as oxime-hexakis-O-TMS (1 and 2 of each sugar representation) and/or O-pentakis-TMS (3 and 4 of each sugar presentation). The two peaks of different retention time and similar mass spectra fragmentation patterns (as in Table 2; mass spectra are not presented) detected for each of these derivative compounds are for the alpha- and beta-stereoisomers, in the case of TMS-sugar derivatives, and/or synand anti-oxime isomers in the case of TMS-oxime comparable derivatives. On the other hand, xylose (X), arabinose (A), rhamnose (R), mannose (M) and sucrose (S) are found in their O-pentakis (TMS)-sugar derivative forms as seen in the single peak configuration and the corresponding mass-ions presented throughout. Salicin internal standard is shown as INSD. Figure 2. GC-MS analysis of GPW enzymatic hydrolysate blank (no enzymes). GC-MS total ion chromatogram (TIC) of the trimethylsilyl (TMS) and trimethylsilyl-oxime (TMS-oxime) derivatives for the blank compounds of GPW without enzymatic hydrolysis. As shown, glucose (G), galacturonic acid (GA), fructose (F) and galactose (Gal) are characterized as oxime-hexakis-O-TMS (1 and 2 of each sugar representation) and/or O-pentakis-TMS (3 and 4 of each sugar presentation). The two peaks of different retention time and similar mass spectra fragmentation patterns (as in Table 2; mass spectra are not presented) detected for each of these derivative compounds are for the alpha- and beta-stereoisomers, in the case of TMS-sugar derivatives, and/or syn- and anti-oxime isomers in the case of TMS-oxime comparable derivatives. On the other hand, xylose (X), arabinose (A), rhamnose (R), mannose (M) and sucrose (S) are found in their O-pentakis (TMS)-sugar derivative forms as seen in the single peak configuration and the corresponding mass-ions presented throughout. Salicin internal standard is shown as



INSD. As seen, the cellobiose corresponding peak(s) are not found while the glucose and galacturonic acid corresponding peak areas are minimal, compared with those identified in Figure 1 above due to the enzymatic hydrolysis.

Figure 3. Time courses for mono- and di-saccharides released during enzymatic hydrolysis. Amounts of xylose (a), arabinose (b), rhamnose (c), galacturonic acid (d), fructose (e), mannose (f), glucose (g), galactose (h), sucrose (i), and cellobiose (j) end-products, in g per kg of GPW, released by enzymatic hydrolysis using a cocktail of E. coli expressed cel12B, cel8C, β-glucosidase and peh28, as determined by GC-MS. The GPW was pretreated with 1% w/w NaOH at 120°C for 15 min, where 1% w/w biomass solid loading was used to initiate the hydrolysis with 9.9±1.3, 12.2±2.4, 16.4±0.5 and 8.1±1.1 mg total protein loading per g dry-equivalent GPW of cel12B, cel8C, β-glucosidase and peh28, respectively, at 45°C and pH 5.4, over the reaction time. Results are given as means of triplicates.

Figure 4. Biomass polymer hydrolysis.

Percent of biomass polysaccharide conversion due to the

enzymatic treatment as compared with alkali-pretreatment of GPW and with baseline levels in the biomass water of untreated GPW, based on the corresponding hydrolysis sugar products from the biomass substrate due to the respective treatments as calculated from GC-MS analysis. The percent of conversion was calculated for each treatment and referred to the initial GPW compositions for the amount of each polymer based on Rivas et al. (unpublished data). Cel12B, cel8C, β-glucosidase and peh28 recombinants from E. coli were used for the enzymatic treatment at 9.9±1.3, 12.2±2.4, 16.4±0.5 and 8.1±1.1 mg total protein loading and at combined 1% w/w loading with respect to the solid biomass, for 24 h at 45°C and pH 5.4, whereas NaOH at 1% w/w and at 121°C was used to conduct the alkaline pretreatment, for the GPW. Xyl, Arab, Rham, Mann, Gluc, Galacturo, Galac and Fruc stand for corresponding xylan, arabinan, rhamnan, galacturonan, fructan, mannan, glucan and galactan polymeric constituents of the GPW. Values presented are means of triplicates ±SE as conducted using multiple range test (Tukey) analysis.


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Figure 1

Figure 2



Figure 3

Figure 4


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Specialized nomenclature (non-standard abbreviations) CBG: coastal Bermuda grass: cel12B: cellulase-12B; cel8C: cellulase-8C; E. coli: Escherichia coli; GC: gas chromatography; GPW: grapefruit processing waste; MS: mass spectrometry; MSD: mass selective detector; peh28: polygalacturonase-28; Pcc: Pectobacterium carotovorum subsp. carotovorum; SFI: selective fragment ion; TIC: total ion current.



TOC/Abstract Graphic

Synopsis Enzymatic hydrolysis liberates biofuel precursors from grapefruit processing waste, a hitherto stranded biomass.


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Recombinant E. coli cellulases, Î²-glucosidase and polygalacturonase

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