Bioprocess Biosyst Eng DOI 10.1007/s00449-016-1575-z
ORIGINAL PAPER
Efficacy of acidic pretreatment for the saccharification and fermentation of alginate from brown macroalgae Damao Wang1 • Eun Ju Yun1 • Sooah Kim1 • Do Hyoung Kim1 • Nari Seo2 Hyun Joo An2 • Jae-Han Kim3 • Nam Yong Cheong4 • Kyoung Heon Kim1
•
Received: 25 November 2015 / Accepted: 16 February 2016 Ó Springer-Verlag Berlin Heidelberg 2016
Abstract This study was performed to evaluate the effectiveness of acidic pretreatment in increasing the enzymatic digestibility of alginate from brown macroalgae. Pretreatment with 1 % (w/v) sulfuric acid at 120 °C for 30 min produced oligosaccharides, mannuronic acid, and guluronic acid. Enzymatic saccharification of pretreated alginate by alginate lyases produced 52.2 % of the theoretical maximal sugar yield, which was only 7.5 % higher than the sugar yield obtained with unpretreated alginate. Mass spectrometric analyses of products of the two reactions revealed that acidic pretreatment and enzymatic saccharification produced saturated monomers (i.e., mannuronic and guluronic acid) with saturated oligosaccharides and unsaturated monomers (i.e., 4-deoxy-L-erythro-5hexoseulose uronic acid; DEH), respectively. While DEH is further metabolized by microorganisms, mannuronic acid and guluronic acid are not metabolizable. Because of the poor efficacy in increasing enzymatic digestibility and owing to the formation of non-fermentable saturated monomers, acidic pretreatment cannot be recommended for enzymatic saccharification and fermentation of alginate.
& Kyoung Heon Kim [email protected] 1
Department of Biotechnology, Graduate School, Korea University, Seoul 02841, Republic of Korea
2
Graduate School of Analytical Science and Technology, Asia-Pacific Glycomics Reference Site, Chungnam National University, Daejeon 34134, Republic of Korea
3
Department of Food and Nutrition, Chungnam National University, Daejeon 34134, Republic of Korea
4
Environmental Analysis Division, Korea Apparel Testing & Research Institute, Seoul 02579, Republic of Korea
Keywords Alginate Brown macroalgae Acidic pretreatment Depolymerization Saccharification
Introduction Strong dependence on fossil fuels has stimulated efforts to produce biofuels and renewable commodity chemical compounds [1]. Ethanol is produced mainly in USA and Brazil (from corn and sugarcane, respectively) [2]. Their ethanol production, however, can be problematic because they rely on the utilization of food resources for production of energy [3, 4]. Therefore, second- and third-generation biomass feedstocks are considered promising because they do not reduce food production. As third-generation biomass, brown macroalgae are now being considered as a renewable resource for production of fuels and chemicals [5]. Because brown macroalgae contain no or much less lignin than plant does, carbohydrates can be fractionated (through a simple downstream process including only milling, leaching, and extraction steps) for subsequent bioconversion processes such as saccharification and fermentation. Among the carbohydrates of brown macroalgae, alginate is the most abundant polysaccharide and constitutes 12–34 % of the total dry weight of these algae [6]. Alginate is a linear polysaccharide composed of b-D-mannuronic acid and its C5 epimer, a-L-guluronic acid [7]; alginate from brown macroalgae is utilized by marine microorganisms. An important prerequisite step for alginate metabolism in marine microorganisms is its enzymatic depolymerization into oligosaccharides and monosaccharides. The final enzymatic saccharification product of alginate is 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH), which can be further converted to 2-keto-3-deoxy-
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Bioprocess Biosyst Eng D-gluconate
(KDG) by a reductase [8]. KDG enters the Entner–Doudoroff pathway and thus generates building blocks and energy for cellular metabolism. This entire pathway for the catabolism of alginate is naturally present in some alginate-metabolizing marine microorganisms such as Sphingomonas sp. A1 [9]. Via introduction of an ethanol-fermenting pathway, this bacterium was engineered to directly produce ethanol from alginate [10]. Industrial microorganisms such as Escherichia coli and Saccharomyces cerevisiae—via introduction of a corresponding catabolic pathway—have also been engineered to produce ethanol by fermenting DEH [11, 12]. Although marine macroalgae contain almost no lignin, they still need to be pretreated to increase their enzymatic digestibility and solubility in water [13, 14]. With regard to alginate from brown macroalgae, acidic [15, 16], alkaline [17, 18], hydrothermal [19], and oxidative pretreatment methods [20] have been used mainly to convert alginate into its oligosaccharides. These pretreatment methods, however, have never been systematically evaluated for their effects in improving the enzymatic digestibility of alginate. The objective of this study was to assess the usefulness of acidic pretreatment of alginate with dilute sulfuric acid; this approach has been successfully applied previously not only to lignocellulose, but also to agarose from red macroalgae [13]. In the present study, pretreated alginate samples that were obtained under various conditions were tested for their enzymatic digestibility by means of depolymerizing enzymes composed of our in-house endotype and exo-type alginate lyases [21, 22]. We also performed rigorous analyses of reaction products of the acidic pretreatment and/or enzymatic saccharification of alginate. This is the first systematic evaluation of acidic pretreatment of alginate in relation to the chemical pretreatment and enzymatic saccharification of brown macroalgae.
Materials and methods Acidic pretreatment of alginate The reaction mixture for the acidic pretreatment of alginate was composed of 2 % (w/v) sodium alginate (SigmaAldrich, St. Louis, MO) and 1 % (w/v) sulfuric acid. The acidic pretreatment was performed in a microwave digester (Milestone, Shelton, CT) at 120, 140, or 160 °C for various periods: 15, 30, or 45 min. After the pretreatment, the reaction products were neutralized (pH 6.0) with 2 M sodium hydroxide for the subsequent saccharification.
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Microbial strains and their cultivation for production of recombinant enzymes Recombinant E. coli BL21 (DE3) harboring alginate lyase genes, alg7D and alg17C, was used for the production of recombinant Alg7D and Alg17C enzymes, respectively. The bacterial cells were grown in Luria–Bertani broth (BD, Sparks, MD) containing 50 mg/L ampicillin in a shaking incubator at 37 °C and 200 rpm until the absorbance of the culture broth at 600 nm reached 0.6. The gene expression was induced by addition of 1 mM isopropyl-b-D-thiogalactopyranoside (Sigma-Aldrich) at 16 °C for 12 h. The cells were collected by centrifugation at 40009g for 15 min and stored at -20 °C until separation of the recombinant proteins. Purification of recombinant alginate lyases Cells were disrupted in lysis buffer (20 mM sodium phosphate, 500 mM sodium chloride, pH 7.4) by sonication, and the disrupted cells were centrifuged at 16,0009g and 4 °C for 1 h. The recombinant protein was purified on a HisTrap affinity column (GE Healthcare, Piscataway, NJ). The purified recombinant protein was concentrated using an Amicon Ultra Centrifugal Filter Unit (UFC903024, MW cutoff of 30 kDa; Millipore, Billerica, MA). The purified protein was quantified by the bicinchoninic acid protein assay (Pierce, Rockford, IL). Enzymatic saccharification of pretreated alginate Activity of the recombinant enzymes that were used in this study was tested by an enzyme assay described in our previous study [23]. One unit (U) of an enzyme was defined as the amount of the enzyme required to release 1 lmol of reducing sugar per minute. Alginate, pretreated alginate, and separated fractions of pretreated alginate after size exclusion chromatography were used as substrates for sequential saccharification with Alg7D and Alg17C. Initially, the enzymatic reaction was performed on 1 mL of a reaction mixture containing 2 % (w/v) of the substrate and 1.16 U of Alg7D at 40 °C for 60 min. Then, 3.68 U of Alg17C was added to the enzymatic reaction mixture, and the latter was incubated at 40 °C for 80 min. The enzymatic reactions were quenched by submerging the reaction tubes in boiling water for 5 min. Reducing sugar released by the enzymatic reaction was quantified by the 3,5-dinitrosalicylic acid assay [24] where glucose was used as a standard. The reducing sugar yield was calculated as the percentage of a theoretical maximum.
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Size exclusion chromatography of pretreated alginate Size exclusion chromatography was performed on an ¨ KTAprime Plus Chromatography system (GE HealthA care) equipped with a Bio-Gel P2 polyacrylamide gel column (Bio-Rad, Hercules, CA) and a UV detector (GE Healthcare). Tris–HCl buffer (20 mM, pH 7.4) was used as the mobile phase at a flow rate of 1.0 mL/min at room temperature. Analyses of enzymatic reaction products by thin layer chromatography These analyses were performed on a silica gel 60 plate (Merck, Darmstadt, Germany). After thin layer chromatography (TLC), we developed the plates with reaction products by placing the plates in a chamber containing the mixture n-butanol–acetic acid–water (3:2:2, v/v/v) and incubating for 1 h, followed by drying of the plates for 2 min. The color of the reaction products was developed by means of a sulfuric acid (95 %, v/v)–ethanol mixture (1:4, v/v). Analyses of enzymatic reaction products by liquid chromatography–mass spectrometry To analyze monosaccharides that were generated during the acidic pretreatment of alginate, chromatography–mass spectrometry (LC–MS) was performed on an Agilent 1200 Series Rapid Resolution Liquid Chromatography (Agilent Technologies, Wilmington, DE) equipped with an electrospray ionization (ESI) source in positive and negative mode and a Q-Exactive orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA). A sample (5 lL) was injected into the LC–MS system and separated on a YMC-Triart C18 column (100 9 3.0 mm, 3 lm particle size; YMC, Kyoto, Japan). Solvent A (0.1 % formic acid in acetonitrile) and Solvent B (0.1 % formic acid in water) were used as the mobile phases at a flow rate of 300 lL/ min. The gradient of the mobile phase was programmed as follows: start at 100 % of Solvent B for 2.5 min and then 70 % of Solvent A and 30 % of Solvent B for 10 min. The temperatures of the autosampler and column were 10 and 20 °C, respectively. The Exactive Orbitrap mass spectra were recorded in the mass range 140–1000 m/z at 1 Hz. The resolution was 70,000, the AGC (automatic gain control) target was set to 3 9 106, and the maximum injection time was 200 ls. The raw data file was analyzed on the Q-Exactive mass spectrometer controlled by the Xcalibur software (version 2.2 SP1.48; Thermo Fisher Scientific).
Matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry of the products of pretreatment and enzymatic processing of alginate These analyses were performed on pretreated alginate and enzymatically processed alginate in positive ion reflectron mode. The purified reaction products were dissolved in water, and 1 lL of the solubilized products was spotted onto a stainless steel target plate, followed by addition of 0.3 lL of 0.01 M NaCl and 0.5 lL of 50 mg/mL 2,5dihydroxybenzoic acid in 50 % (w/w) acetonitrile. The spot was rapidly dried in vacuum for homogenous crystallization. Matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI–TOF/TOF MS) was performed on a Bruker ultrafleXtreme system (Bruker Daltonics, Portland, OR). Each acquired spectrum represented combined signals from 800 laser shots (by means of a 1 kHz laser) at each of the three random locations in the spot, totaling 2400 laser shots. Laser attenuator offset and range were set to 68 and 15 %, respectively, and laser focus was set to 50 %. Mass spectra were recorded in the range 0–5100 m/z. To obtain highresolution data, the detector sampling rate was set to the maximum rate of 4.00 gigasamples/s, and the detector gain was set to 4.09. The mass spectra were externally calibrated using malto-oligosaccharides isolated from commercial beer. A ladder of hexose polymers, spaced at 1 hexose unit (162.053 Da) apart, provided comprehensive coverage of the entire mass acquisition range, enabling accurate mass calibration of the MALDI–TOF/TOF instrument immediately before the sample analysis. Raw MS data were processed using the FlexAnalysis software (version 3.3; Bruker Daltonics). MS peaks were filtered with a signal-to-noise ratio of 3.0 and manually inspected to detect formation of sodium, potassium, or other common adducts. All peaks were then deconvoluted, and a list of all neutral masses in the samples was generated with the abundance values represented by MS peak intensities.
Results and discussion Acidic pretreatment of alginate The use of a high concentration of acid and high temperature [25, 26] for the pretreatment of alginate can result in the formation of degradation products such as glycolic acid and lactic acid and in the production of large amounts of salts after neutralization [19, 27]. Both types of compounds will inhibit the subsequent bioconversion processes such as enzymatic saccharification and fermentation [28].
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Therefore, in this study, we used a low concentration of sulfuric acid (1 %, w/v) and moderate temperatures (120–160 °C) for pretreatment of alginate for 15–45 min. Under these pretreatment conditions, the yield of reducing sugar ranged from 19.8 to 39.6 % of the theoretical maximum (Fig. 1a). At 120 and 140 °C, the reducing sugar yield increased with increasing pretreatment duration. At 160 °C, the highest reducing sugar yield, 39.6 %, was obtained after 15 min of pretreatment. Further increases in pretreatment duration at 160 °C reduced the yield of reducing sugar (Fig. 1a). The TLC of pretreatment products showed that monosaccharides became predominant as temperature increased, especially at 160 °C (Fig. 1b). At lower temperatures, such as 120 and 140 °C, oligosaccharides were formed, but these oligosaccharides seemed to be further converted into monosaccharides and degradation products at increasing pretreatment duration. To minimize the formation of degradation products and to achieve a high
Reducing sugar yield (% of theoretical max.)
A
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40
15 min 30 min 45 min
degree of depolymerization (DP), 120 °C and 30 min were selected as the optimal pretreatment conditions for further experiments. Enzymatic saccharification of pretreated alginate In our previous study, an endo-type lyase, Alg7D, and an exo-type lyase, Alg17C (both derived from Saccharophagus degradans 2-40T), were used in the enzymatic reactions with alginate to produce monosaccharides (i.e., DEH) [21, 22]. In the present study, before the sequential enzymatic reactions with Alg7D and Alg17C, unpretreated alginate and pretreated alginate showed reducing sugar yields of 3.3 and 22.1 % of the theoretical maximum, respectively (Fig. 2). In this study, after the sequential enzymatic reactions with Alg7D and Alg17C, the final reducing sugar yield of unpretreated alginate and pretreated alginate was 44.7 and 52.2 %, respectively (Fig. 2). The difference in the reducing sugar yield between the pretreatment and no-pretreatment experiments was negligible: only 7.5 % of the theoretical maximum. This improvement in enzymatic digestibility after the acidic pretreatment of alginate (i.e., 7.5 %) is significantly smaller than the improvement after acidic pretreatment of agarose (34.4 %) [29].
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Fractionation and analysis of pretreated alginate 20
10
0
120
140
160
Temperature (oC)
To analyze and identify the products of the enzymatic reactions containing pretreated alginate, we fractionated pretreated alginate and then subjected each fraction to the enzymatic reactions. Pretreated alginate was separated into three fractions by size exclusion chromatography (Fig. 3a). These three fractions were analyzed by TLC (Fig. 3b).
B
Reducing sugar yield (% of theroetical max.)
60 50
Before enzyme reaction After enzyme reaction
40 30 20 10 0
Unpretreated alginate
Fig. 1 a The reducing sugar yield (a percentage of the theoretical maximum of reducing sugar) after the acidic pretreatment of alginate with 1 % (w/v) sulfuric acid under various conditions. b TLC of the alginate pretreated with 1 % (w/v) sulfuric acid under various conditions
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Pretreated alginate
Fig. 2 Comparison of enzymatic digestibility by Alg7D and Alg17C (i.e., reducing sugar yields) between unpretreated and pretreated alginate
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Fractions 1 and 2 were combined and used as a substrate for the enzymatic reactions with Alg7D and Alg17C. Analysis of enzymatic reaction products The products of enzymatic saccharification of unpretreated alginate and pretreated alginate were analyzed by TLC (Fig. 4a). Oligosaccharides with DP 2–5 were formed in the enzymatic reaction of unpretreated alginate with Alg7D (Fig. 4a; lane OS), and they were then converted into monosaccharides (DEH) after the reaction with Alg17C (Fig. 4a; lane DEH). When pretreated alginate (Fig. 4a; lane PA) was used as the substrate for the enzymatic reaction with Alg7D, the oligosaccharides with a higher DP and polysaccharides were converted into lower-DP products, judging by disappearance of the starting spot and emergence of new spots on the TLC plate (Fig. 4a; lane PA ? Alg7D). After addition of Alg17C to the Alg7D reaction products, the oligosaccharides were converted into DEH and mannuronic or guluronic acid, according to the spot density of mannuronic acid and guluronic acid (Fig. 4a; lane PA ? Alg7D ? Alg17C). To test our hypothesis about the results in Fig. 4a, Fractions 1 and 2 from pretreated alginate (PA12), which did not contain either mannuronic or guluronic acid (Fig. 3), were combined (Fig. 4b; lane PA12) and used as the substrate in the enzymatic reactions. As in the results shown in Fig. 4b, the enzymatic reaction of PA12 with Alg7D resulted in depolymerization of higher-DP oligosaccharides and polysaccharides (Fig. 4b; lane PA12 ? Alg7D), whereas the further enzymatic reaction (with Alg17C) resulted in the formation of mannuronic and guluronic acid and DEH (Fig. 4b; lane PA12 ? Alg7D ? Alg17C). These results indicated that mannuronic acid, guluronic acid, and DEH are formed by the depolymerizing action of Alg17C.
Fig. 3 a Size exclusion chromatography of pretreated alginate. b TLC of the fractions obtained after the size exclusion chromatography. F1, polysaccharides and oligosaccharides with a high degree of polymerization (DP); F2, low-DP oligosaccharides; F3, monosaccharides. c LC–MS of Fraction 3 from the size exclusion chromatography of pretreated alginate
Among the three fractions, Fraction 1 contained oligosaccharides with the highest MWs and the highest DPs. Oligosaccharides with lower DPs were mostly in Fraction 2. Fraction 3 was identified as either mannuronic acid or guluronic acid (MW of 194.1 for both) based on the m/z ratio 193.03, which was obtained by LC–MS (Fig. 3c). Because Fraction 3 contained only monosaccharides, only
Comparison of reaction products between acidic pretreatment and enzymatic saccharification MALDI–TOF/TOF MS was performed on the reaction products of acidic pretreatment and enzymatic depolymerization of alginate with the endo-type alginate lyase, Alg7D, to compare their mechanisms of action. After the acidic pretreatment, the resulting oligosaccharides with various DPs showed a mass difference of 176 that is identical to the mass of a monomeric unit of alginate, DEH (Fig. 5a). Analysis of these MALDI–TOF/TOF MS results showed that the depolymerization that occurs during acidic pretreatment is the cleavage of b-1,4-glycosidic bonds between unit monomers (not at random sites of alginate). When alginate was depolymerized by Alg7D, oligosaccharides with various DPs were produced with a
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Fig. 4 a TLC of pretreated alginate. b TLC of oligosaccharides from pretreated alginate. OS, oligosaccharides produced by Alg7D; PA, pretreated alginate; M, mannuronic acid; G, guluronic acid; PA12,
Fig. 5 MALDI–TOF/TOF MS of a pretreated alginate and b products of alginate enzymatic depolymerization by Alg7D
mass difference of 176 (Fig. 5b), just as in the acidic pretreatment of alginate shown in Fig. 5a. Nevertheless, comparison of the masses of the corresponding peaks with similar m/z ratios from acid-pretreated alginate (Fig. 5a)
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combined fractions of polysaccharides and oligosaccharides with high DPs from the pretreated alginate
and Alg7D-treated alginate (Fig. 5b)—which appear to have the same DPs—revealed that acid-pretreated alginate had 18 higher m/z ratios than did the alginate processed by Alg7D. These results imply that the acidic pretreatment causes addition of a water molecule to each non-reducing end of monomeric units of alginate. During the acidic pretreatment of alginate, one molecule of water attacks C1 and forms a hydroxyl group at C1 and C4. This reaction leads to the formation of mannuronic acid and guluronic acid directly from alginate, while oligosaccharides with a saturated unit at the non-reducing ends are also formed. To our knowledge, mannuronic or guluronic acid has not been reported to be fermented by any microorganisms. However, the conversion of DEH to KDG is the only reported alginate metabolism pathway [30] and has been used in microorganisms which have been engineered to produce ethanol from alginate [10–12]. This action of acidic pretreatment is different from the mechanism of the enzymatic reaction with Alg7D, an alginate lyase. In addition, monomers that are possibly produced by the acidic pretreatment are mannuronic acid or guluronic acid. Unlike the hydrolysis during the acidic pretreatment, reaction with the alginate lyases depolymerizes alginate through the b-elimination reaction which forms a double bond between C4 and C5 [31]. Figure 6 shows the pathway of depolymerization of alginate. The initial non-reducing end of an alginate polysaccharide is composed of a saturated unit. During the direct enzymatic saccharification of alginate, an endo-type alginate lyase produces
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Fig. 6 A schematic diagram of alginate depolymerization pathways for a acidic pretreatment and b enzymatic saccharification
oligosaccharides containing an unsaturated unit at the nonreducing end, and an exo-type alginate lyase then converts it into an unsaturated monosaccharide, which is then nonenzymatically converted into DEH. In conclusion, acidic pretreatment of alginate improved the enzymatic digestibility of alginate negligibly such as only by 7.5 % in terms of the reducing sugar yield. Furthermore, we found that acidic pretreatment of alginate produces saturated monosaccharides (i.e., mannuronic and guluronic acid) and oligosaccharides with saturated non-reducing ends. Unlike DEH, which results from enzymatic depolymerization of
alginate, mannuronic acid and guluronic acid have not been reported to be fermentable by any microorganisms. Because of the two reasons, namely, the low effectiveness in increasing enzymatic digestibility as well as the low yield of DEH due to the generation of saturated monosaccharides, mannuronic acid and guluronic acid, by acidic pretreatment, the acidic pretreatment cannot be recommended for the saccharification and fermentation of alginate from brown macroalgae. Acknowledgments This work was supported by a grant from the Ministry of Trade, Industry and Energy (10052721). Experiments
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Bioprocess Biosyst Eng were performed at the Korea University Food Safety Hall for the Institute of Biomedical Science and Food Safety.
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ORIGINAL PAPER
Efficacy of acidic pretreatment for the saccharification and fermentation of alginate from brown macroalgae Damao Wang1 • Eun Ju Yun1 • Sooah Kim1 • Do Hyoung Kim1 • Nari Seo2 Hyun Joo An2 • Jae-Han Kim3 • Nam Yong Cheong4 • Kyoung Heon Kim1
•
Received: 25 November 2015 / Accepted: 16 February 2016 Ó Springer-Verlag Berlin Heidelberg 2016
Abstract This study was performed to evaluate the effectiveness of acidic pretreatment in increasing the enzymatic digestibility of alginate from brown macroalgae. Pretreatment with 1 % (w/v) sulfuric acid at 120 °C for 30 min produced oligosaccharides, mannuronic acid, and guluronic acid. Enzymatic saccharification of pretreated alginate by alginate lyases produced 52.2 % of the theoretical maximal sugar yield, which was only 7.5 % higher than the sugar yield obtained with unpretreated alginate. Mass spectrometric analyses of products of the two reactions revealed that acidic pretreatment and enzymatic saccharification produced saturated monomers (i.e., mannuronic and guluronic acid) with saturated oligosaccharides and unsaturated monomers (i.e., 4-deoxy-L-erythro-5hexoseulose uronic acid; DEH), respectively. While DEH is further metabolized by microorganisms, mannuronic acid and guluronic acid are not metabolizable. Because of the poor efficacy in increasing enzymatic digestibility and owing to the formation of non-fermentable saturated monomers, acidic pretreatment cannot be recommended for enzymatic saccharification and fermentation of alginate.
& Kyoung Heon Kim [email protected] 1
Department of Biotechnology, Graduate School, Korea University, Seoul 02841, Republic of Korea
2
Graduate School of Analytical Science and Technology, Asia-Pacific Glycomics Reference Site, Chungnam National University, Daejeon 34134, Republic of Korea
3
Department of Food and Nutrition, Chungnam National University, Daejeon 34134, Republic of Korea
4
Environmental Analysis Division, Korea Apparel Testing & Research Institute, Seoul 02579, Republic of Korea
Keywords Alginate Brown macroalgae Acidic pretreatment Depolymerization Saccharification
Introduction Strong dependence on fossil fuels has stimulated efforts to produce biofuels and renewable commodity chemical compounds [1]. Ethanol is produced mainly in USA and Brazil (from corn and sugarcane, respectively) [2]. Their ethanol production, however, can be problematic because they rely on the utilization of food resources for production of energy [3, 4]. Therefore, second- and third-generation biomass feedstocks are considered promising because they do not reduce food production. As third-generation biomass, brown macroalgae are now being considered as a renewable resource for production of fuels and chemicals [5]. Because brown macroalgae contain no or much less lignin than plant does, carbohydrates can be fractionated (through a simple downstream process including only milling, leaching, and extraction steps) for subsequent bioconversion processes such as saccharification and fermentation. Among the carbohydrates of brown macroalgae, alginate is the most abundant polysaccharide and constitutes 12–34 % of the total dry weight of these algae [6]. Alginate is a linear polysaccharide composed of b-D-mannuronic acid and its C5 epimer, a-L-guluronic acid [7]; alginate from brown macroalgae is utilized by marine microorganisms. An important prerequisite step for alginate metabolism in marine microorganisms is its enzymatic depolymerization into oligosaccharides and monosaccharides. The final enzymatic saccharification product of alginate is 4-deoxy-L-erythro-5-hexoseulose uronic acid (DEH), which can be further converted to 2-keto-3-deoxy-
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(KDG) by a reductase [8]. KDG enters the Entner–Doudoroff pathway and thus generates building blocks and energy for cellular metabolism. This entire pathway for the catabolism of alginate is naturally present in some alginate-metabolizing marine microorganisms such as Sphingomonas sp. A1 [9]. Via introduction of an ethanol-fermenting pathway, this bacterium was engineered to directly produce ethanol from alginate [10]. Industrial microorganisms such as Escherichia coli and Saccharomyces cerevisiae—via introduction of a corresponding catabolic pathway—have also been engineered to produce ethanol by fermenting DEH [11, 12]. Although marine macroalgae contain almost no lignin, they still need to be pretreated to increase their enzymatic digestibility and solubility in water [13, 14]. With regard to alginate from brown macroalgae, acidic [15, 16], alkaline [17, 18], hydrothermal [19], and oxidative pretreatment methods [20] have been used mainly to convert alginate into its oligosaccharides. These pretreatment methods, however, have never been systematically evaluated for their effects in improving the enzymatic digestibility of alginate. The objective of this study was to assess the usefulness of acidic pretreatment of alginate with dilute sulfuric acid; this approach has been successfully applied previously not only to lignocellulose, but also to agarose from red macroalgae [13]. In the present study, pretreated alginate samples that were obtained under various conditions were tested for their enzymatic digestibility by means of depolymerizing enzymes composed of our in-house endotype and exo-type alginate lyases [21, 22]. We also performed rigorous analyses of reaction products of the acidic pretreatment and/or enzymatic saccharification of alginate. This is the first systematic evaluation of acidic pretreatment of alginate in relation to the chemical pretreatment and enzymatic saccharification of brown macroalgae.
Materials and methods Acidic pretreatment of alginate The reaction mixture for the acidic pretreatment of alginate was composed of 2 % (w/v) sodium alginate (SigmaAldrich, St. Louis, MO) and 1 % (w/v) sulfuric acid. The acidic pretreatment was performed in a microwave digester (Milestone, Shelton, CT) at 120, 140, or 160 °C for various periods: 15, 30, or 45 min. After the pretreatment, the reaction products were neutralized (pH 6.0) with 2 M sodium hydroxide for the subsequent saccharification.
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Microbial strains and their cultivation for production of recombinant enzymes Recombinant E. coli BL21 (DE3) harboring alginate lyase genes, alg7D and alg17C, was used for the production of recombinant Alg7D and Alg17C enzymes, respectively. The bacterial cells were grown in Luria–Bertani broth (BD, Sparks, MD) containing 50 mg/L ampicillin in a shaking incubator at 37 °C and 200 rpm until the absorbance of the culture broth at 600 nm reached 0.6. The gene expression was induced by addition of 1 mM isopropyl-b-D-thiogalactopyranoside (Sigma-Aldrich) at 16 °C for 12 h. The cells were collected by centrifugation at 40009g for 15 min and stored at -20 °C until separation of the recombinant proteins. Purification of recombinant alginate lyases Cells were disrupted in lysis buffer (20 mM sodium phosphate, 500 mM sodium chloride, pH 7.4) by sonication, and the disrupted cells were centrifuged at 16,0009g and 4 °C for 1 h. The recombinant protein was purified on a HisTrap affinity column (GE Healthcare, Piscataway, NJ). The purified recombinant protein was concentrated using an Amicon Ultra Centrifugal Filter Unit (UFC903024, MW cutoff of 30 kDa; Millipore, Billerica, MA). The purified protein was quantified by the bicinchoninic acid protein assay (Pierce, Rockford, IL). Enzymatic saccharification of pretreated alginate Activity of the recombinant enzymes that were used in this study was tested by an enzyme assay described in our previous study [23]. One unit (U) of an enzyme was defined as the amount of the enzyme required to release 1 lmol of reducing sugar per minute. Alginate, pretreated alginate, and separated fractions of pretreated alginate after size exclusion chromatography were used as substrates for sequential saccharification with Alg7D and Alg17C. Initially, the enzymatic reaction was performed on 1 mL of a reaction mixture containing 2 % (w/v) of the substrate and 1.16 U of Alg7D at 40 °C for 60 min. Then, 3.68 U of Alg17C was added to the enzymatic reaction mixture, and the latter was incubated at 40 °C for 80 min. The enzymatic reactions were quenched by submerging the reaction tubes in boiling water for 5 min. Reducing sugar released by the enzymatic reaction was quantified by the 3,5-dinitrosalicylic acid assay [24] where glucose was used as a standard. The reducing sugar yield was calculated as the percentage of a theoretical maximum.
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Size exclusion chromatography of pretreated alginate Size exclusion chromatography was performed on an ¨ KTAprime Plus Chromatography system (GE HealthA care) equipped with a Bio-Gel P2 polyacrylamide gel column (Bio-Rad, Hercules, CA) and a UV detector (GE Healthcare). Tris–HCl buffer (20 mM, pH 7.4) was used as the mobile phase at a flow rate of 1.0 mL/min at room temperature. Analyses of enzymatic reaction products by thin layer chromatography These analyses were performed on a silica gel 60 plate (Merck, Darmstadt, Germany). After thin layer chromatography (TLC), we developed the plates with reaction products by placing the plates in a chamber containing the mixture n-butanol–acetic acid–water (3:2:2, v/v/v) and incubating for 1 h, followed by drying of the plates for 2 min. The color of the reaction products was developed by means of a sulfuric acid (95 %, v/v)–ethanol mixture (1:4, v/v). Analyses of enzymatic reaction products by liquid chromatography–mass spectrometry To analyze monosaccharides that were generated during the acidic pretreatment of alginate, chromatography–mass spectrometry (LC–MS) was performed on an Agilent 1200 Series Rapid Resolution Liquid Chromatography (Agilent Technologies, Wilmington, DE) equipped with an electrospray ionization (ESI) source in positive and negative mode and a Q-Exactive orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA). A sample (5 lL) was injected into the LC–MS system and separated on a YMC-Triart C18 column (100 9 3.0 mm, 3 lm particle size; YMC, Kyoto, Japan). Solvent A (0.1 % formic acid in acetonitrile) and Solvent B (0.1 % formic acid in water) were used as the mobile phases at a flow rate of 300 lL/ min. The gradient of the mobile phase was programmed as follows: start at 100 % of Solvent B for 2.5 min and then 70 % of Solvent A and 30 % of Solvent B for 10 min. The temperatures of the autosampler and column were 10 and 20 °C, respectively. The Exactive Orbitrap mass spectra were recorded in the mass range 140–1000 m/z at 1 Hz. The resolution was 70,000, the AGC (automatic gain control) target was set to 3 9 106, and the maximum injection time was 200 ls. The raw data file was analyzed on the Q-Exactive mass spectrometer controlled by the Xcalibur software (version 2.2 SP1.48; Thermo Fisher Scientific).
Matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry of the products of pretreatment and enzymatic processing of alginate These analyses were performed on pretreated alginate and enzymatically processed alginate in positive ion reflectron mode. The purified reaction products were dissolved in water, and 1 lL of the solubilized products was spotted onto a stainless steel target plate, followed by addition of 0.3 lL of 0.01 M NaCl and 0.5 lL of 50 mg/mL 2,5dihydroxybenzoic acid in 50 % (w/w) acetonitrile. The spot was rapidly dried in vacuum for homogenous crystallization. Matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry (MALDI–TOF/TOF MS) was performed on a Bruker ultrafleXtreme system (Bruker Daltonics, Portland, OR). Each acquired spectrum represented combined signals from 800 laser shots (by means of a 1 kHz laser) at each of the three random locations in the spot, totaling 2400 laser shots. Laser attenuator offset and range were set to 68 and 15 %, respectively, and laser focus was set to 50 %. Mass spectra were recorded in the range 0–5100 m/z. To obtain highresolution data, the detector sampling rate was set to the maximum rate of 4.00 gigasamples/s, and the detector gain was set to 4.09. The mass spectra were externally calibrated using malto-oligosaccharides isolated from commercial beer. A ladder of hexose polymers, spaced at 1 hexose unit (162.053 Da) apart, provided comprehensive coverage of the entire mass acquisition range, enabling accurate mass calibration of the MALDI–TOF/TOF instrument immediately before the sample analysis. Raw MS data were processed using the FlexAnalysis software (version 3.3; Bruker Daltonics). MS peaks were filtered with a signal-to-noise ratio of 3.0 and manually inspected to detect formation of sodium, potassium, or other common adducts. All peaks were then deconvoluted, and a list of all neutral masses in the samples was generated with the abundance values represented by MS peak intensities.
Results and discussion Acidic pretreatment of alginate The use of a high concentration of acid and high temperature [25, 26] for the pretreatment of alginate can result in the formation of degradation products such as glycolic acid and lactic acid and in the production of large amounts of salts after neutralization [19, 27]. Both types of compounds will inhibit the subsequent bioconversion processes such as enzymatic saccharification and fermentation [28].
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Therefore, in this study, we used a low concentration of sulfuric acid (1 %, w/v) and moderate temperatures (120–160 °C) for pretreatment of alginate for 15–45 min. Under these pretreatment conditions, the yield of reducing sugar ranged from 19.8 to 39.6 % of the theoretical maximum (Fig. 1a). At 120 and 140 °C, the reducing sugar yield increased with increasing pretreatment duration. At 160 °C, the highest reducing sugar yield, 39.6 %, was obtained after 15 min of pretreatment. Further increases in pretreatment duration at 160 °C reduced the yield of reducing sugar (Fig. 1a). The TLC of pretreatment products showed that monosaccharides became predominant as temperature increased, especially at 160 °C (Fig. 1b). At lower temperatures, such as 120 and 140 °C, oligosaccharides were formed, but these oligosaccharides seemed to be further converted into monosaccharides and degradation products at increasing pretreatment duration. To minimize the formation of degradation products and to achieve a high
Reducing sugar yield (% of theoretical max.)
A
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degree of depolymerization (DP), 120 °C and 30 min were selected as the optimal pretreatment conditions for further experiments. Enzymatic saccharification of pretreated alginate In our previous study, an endo-type lyase, Alg7D, and an exo-type lyase, Alg17C (both derived from Saccharophagus degradans 2-40T), were used in the enzymatic reactions with alginate to produce monosaccharides (i.e., DEH) [21, 22]. In the present study, before the sequential enzymatic reactions with Alg7D and Alg17C, unpretreated alginate and pretreated alginate showed reducing sugar yields of 3.3 and 22.1 % of the theoretical maximum, respectively (Fig. 2). In this study, after the sequential enzymatic reactions with Alg7D and Alg17C, the final reducing sugar yield of unpretreated alginate and pretreated alginate was 44.7 and 52.2 %, respectively (Fig. 2). The difference in the reducing sugar yield between the pretreatment and no-pretreatment experiments was negligible: only 7.5 % of the theoretical maximum. This improvement in enzymatic digestibility after the acidic pretreatment of alginate (i.e., 7.5 %) is significantly smaller than the improvement after acidic pretreatment of agarose (34.4 %) [29].
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Fractionation and analysis of pretreated alginate 20
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0
120
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Temperature (oC)
To analyze and identify the products of the enzymatic reactions containing pretreated alginate, we fractionated pretreated alginate and then subjected each fraction to the enzymatic reactions. Pretreated alginate was separated into three fractions by size exclusion chromatography (Fig. 3a). These three fractions were analyzed by TLC (Fig. 3b).
B
Reducing sugar yield (% of theroetical max.)
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Before enzyme reaction After enzyme reaction
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Unpretreated alginate
Fig. 1 a The reducing sugar yield (a percentage of the theoretical maximum of reducing sugar) after the acidic pretreatment of alginate with 1 % (w/v) sulfuric acid under various conditions. b TLC of the alginate pretreated with 1 % (w/v) sulfuric acid under various conditions
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Pretreated alginate
Fig. 2 Comparison of enzymatic digestibility by Alg7D and Alg17C (i.e., reducing sugar yields) between unpretreated and pretreated alginate
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Fractions 1 and 2 were combined and used as a substrate for the enzymatic reactions with Alg7D and Alg17C. Analysis of enzymatic reaction products The products of enzymatic saccharification of unpretreated alginate and pretreated alginate were analyzed by TLC (Fig. 4a). Oligosaccharides with DP 2–5 were formed in the enzymatic reaction of unpretreated alginate with Alg7D (Fig. 4a; lane OS), and they were then converted into monosaccharides (DEH) after the reaction with Alg17C (Fig. 4a; lane DEH). When pretreated alginate (Fig. 4a; lane PA) was used as the substrate for the enzymatic reaction with Alg7D, the oligosaccharides with a higher DP and polysaccharides were converted into lower-DP products, judging by disappearance of the starting spot and emergence of new spots on the TLC plate (Fig. 4a; lane PA ? Alg7D). After addition of Alg17C to the Alg7D reaction products, the oligosaccharides were converted into DEH and mannuronic or guluronic acid, according to the spot density of mannuronic acid and guluronic acid (Fig. 4a; lane PA ? Alg7D ? Alg17C). To test our hypothesis about the results in Fig. 4a, Fractions 1 and 2 from pretreated alginate (PA12), which did not contain either mannuronic or guluronic acid (Fig. 3), were combined (Fig. 4b; lane PA12) and used as the substrate in the enzymatic reactions. As in the results shown in Fig. 4b, the enzymatic reaction of PA12 with Alg7D resulted in depolymerization of higher-DP oligosaccharides and polysaccharides (Fig. 4b; lane PA12 ? Alg7D), whereas the further enzymatic reaction (with Alg17C) resulted in the formation of mannuronic and guluronic acid and DEH (Fig. 4b; lane PA12 ? Alg7D ? Alg17C). These results indicated that mannuronic acid, guluronic acid, and DEH are formed by the depolymerizing action of Alg17C.
Fig. 3 a Size exclusion chromatography of pretreated alginate. b TLC of the fractions obtained after the size exclusion chromatography. F1, polysaccharides and oligosaccharides with a high degree of polymerization (DP); F2, low-DP oligosaccharides; F3, monosaccharides. c LC–MS of Fraction 3 from the size exclusion chromatography of pretreated alginate
Among the three fractions, Fraction 1 contained oligosaccharides with the highest MWs and the highest DPs. Oligosaccharides with lower DPs were mostly in Fraction 2. Fraction 3 was identified as either mannuronic acid or guluronic acid (MW of 194.1 for both) based on the m/z ratio 193.03, which was obtained by LC–MS (Fig. 3c). Because Fraction 3 contained only monosaccharides, only
Comparison of reaction products between acidic pretreatment and enzymatic saccharification MALDI–TOF/TOF MS was performed on the reaction products of acidic pretreatment and enzymatic depolymerization of alginate with the endo-type alginate lyase, Alg7D, to compare their mechanisms of action. After the acidic pretreatment, the resulting oligosaccharides with various DPs showed a mass difference of 176 that is identical to the mass of a monomeric unit of alginate, DEH (Fig. 5a). Analysis of these MALDI–TOF/TOF MS results showed that the depolymerization that occurs during acidic pretreatment is the cleavage of b-1,4-glycosidic bonds between unit monomers (not at random sites of alginate). When alginate was depolymerized by Alg7D, oligosaccharides with various DPs were produced with a
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Fig. 4 a TLC of pretreated alginate. b TLC of oligosaccharides from pretreated alginate. OS, oligosaccharides produced by Alg7D; PA, pretreated alginate; M, mannuronic acid; G, guluronic acid; PA12,
Fig. 5 MALDI–TOF/TOF MS of a pretreated alginate and b products of alginate enzymatic depolymerization by Alg7D
mass difference of 176 (Fig. 5b), just as in the acidic pretreatment of alginate shown in Fig. 5a. Nevertheless, comparison of the masses of the corresponding peaks with similar m/z ratios from acid-pretreated alginate (Fig. 5a)
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combined fractions of polysaccharides and oligosaccharides with high DPs from the pretreated alginate
and Alg7D-treated alginate (Fig. 5b)—which appear to have the same DPs—revealed that acid-pretreated alginate had 18 higher m/z ratios than did the alginate processed by Alg7D. These results imply that the acidic pretreatment causes addition of a water molecule to each non-reducing end of monomeric units of alginate. During the acidic pretreatment of alginate, one molecule of water attacks C1 and forms a hydroxyl group at C1 and C4. This reaction leads to the formation of mannuronic acid and guluronic acid directly from alginate, while oligosaccharides with a saturated unit at the non-reducing ends are also formed. To our knowledge, mannuronic or guluronic acid has not been reported to be fermented by any microorganisms. However, the conversion of DEH to KDG is the only reported alginate metabolism pathway [30] and has been used in microorganisms which have been engineered to produce ethanol from alginate [10–12]. This action of acidic pretreatment is different from the mechanism of the enzymatic reaction with Alg7D, an alginate lyase. In addition, monomers that are possibly produced by the acidic pretreatment are mannuronic acid or guluronic acid. Unlike the hydrolysis during the acidic pretreatment, reaction with the alginate lyases depolymerizes alginate through the b-elimination reaction which forms a double bond between C4 and C5 [31]. Figure 6 shows the pathway of depolymerization of alginate. The initial non-reducing end of an alginate polysaccharide is composed of a saturated unit. During the direct enzymatic saccharification of alginate, an endo-type alginate lyase produces
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Fig. 6 A schematic diagram of alginate depolymerization pathways for a acidic pretreatment and b enzymatic saccharification
oligosaccharides containing an unsaturated unit at the nonreducing end, and an exo-type alginate lyase then converts it into an unsaturated monosaccharide, which is then nonenzymatically converted into DEH. In conclusion, acidic pretreatment of alginate improved the enzymatic digestibility of alginate negligibly such as only by 7.5 % in terms of the reducing sugar yield. Furthermore, we found that acidic pretreatment of alginate produces saturated monosaccharides (i.e., mannuronic and guluronic acid) and oligosaccharides with saturated non-reducing ends. Unlike DEH, which results from enzymatic depolymerization of
alginate, mannuronic acid and guluronic acid have not been reported to be fermentable by any microorganisms. Because of the two reasons, namely, the low effectiveness in increasing enzymatic digestibility as well as the low yield of DEH due to the generation of saturated monosaccharides, mannuronic acid and guluronic acid, by acidic pretreatment, the acidic pretreatment cannot be recommended for the saccharification and fermentation of alginate from brown macroalgae. Acknowledgments This work was supported by a grant from the Ministry of Trade, Industry and Energy (10052721). Experiments
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Bioprocess Biosyst Eng were performed at the Korea University Food Safety Hall for the Institute of Biomedical Science and Food Safety.
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