Utilization of Seawater for the Biorefinery of Lignocellulosic Biomass

Research Article pubs.acs.org/journal/ascecg

Utilization of Seawater for the Biorefinery of Lignocellulosic Biomass: Ionic Liquid Pretreatment, Enzymatic Hydrolysis, and Microbial Lipid Production Huan Ren, Min-Hua Zong, Hong Wu, and Ning Li* State Key Laboratory of Pulp and Paper Engineering, School of Food Science and Engineering, South China University of Technology, 381 Wushan Road, Guangzhou 510640, China S Supporting Information *

ABSTRACT: The biorefineries of lignocellulosic biomass have attracted increasing interest recently. However, large water consumption in the large-scale biorefineries remains a major problem. In this work, utilization of abundant seawater as an alternative to freshwater for ionic liquid (IL) pretreatment of lignocellulosic biomass was reported for the first time. In addition, enzymatic hydrolysis of IL-pretreated biomass and microbial lipid production from wheat straw hydrolysate were conducted in seawater. It was found that seawater had no significantly negative effect on enzymatic hydrolysis as well as IL pretreatment. After grass lignocelluloses were pretreated by 50% cholinium IL−seawater mixtures at 90 °C for 6 h and washed by seawater, the residues became highly susceptible to enzymatic hydrolysis; the reducing sugar yields of 54−72% were obtained in pH 4.8 seawater in the subsequent enzymatic hydrolysis of the residues. The lipid yield of 4.5 g/L and lipid coefficient of 0.21 g/g of sugar were achieved after cultivation of Trichosporon fermentans on wheat straw hydrolysate with the sugar concentration of approximately 30 g/L for 3 days. KEYWORDS: Biorefinery, Ionic liquids, Enzymatic saccharification, Pretreatment, Lignocelluloses, Wheat straw hydrolysate

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INTRODUCTION In recent years, many efforts have been devoted for the production of biobased fuels and platform chemicals via the biorefineries of renewable and abundant lignocellulosic biomass.1 For example, polysaccharides present in lignocellulosic biomass could be hydrolyzed to the reducing sugars through both chemical and enzymatic approaches, and these sugars could be further upgraded into biofuels and bioactive chemicals via fermentation, or into platform chemicals such as 5-hydroxymethylfurfural and furfural via chemical methods.2 Lignin could be valorized to valuable aromatic chemicals via depolymerization.3,4 However, the biorefineries of lignocellulosic biomass remain challenging, because of its inherent recalcitrance to chemical and biological degradation. As a result, pretreatment and fractionation of lignocellulosic biomass are necessary generally prior to its degradation. Over the past decades, chemists have developed physical, physicochemical, chemical, and biological methods for pretreatment of lignocellulosic biomass.5 Ionic liquid (IL) pretreatment has emerged as a promising technology for deconstruction of lignocellulosic biomass.6 1-Ethyl-3-methylimidazolium acetate ([Emim][OAc]) is one of the most commonly used ILs for efficient pretreatment of lignocelluloses.7 Recently, our group has reported a series of renewable cholinium IL, which have proven to be effective pretreatment solvents.8−10 More © XXXX American Chemical Society

importantly, this type of biobased ILs has low toxicity and is readily biodegradable.11−13 Although significant advances in the development of effective solvents and strategies for pretreatment of lignocellulosic biomass have been achieved, the large-scale biorefineries of lignocelluloses have not been realized yet.14 One of the challenges for successful biorefineries is the large water consumption. It was reported that water of 1.9−5.9 m3 was required for the production of 1 m3 biofuels.15 It is well-known that freshwater shortage has become a severe problem in some countries and regions, likely due to overpopulation and environmental pollution, which may further hinder the successful applications of the biorefineries. Fortunately, there is a vast quantity of seawater (approximately 96.5%) on earth, compared to the small amount of freshwater (2.5%) and brackish water (1%). Therefore, seawater may be a promising option for the biorefineries. Recently, using seawater as an alternative to freshwater has attracted growing interest in the biorefineries such as for enzymatic and fermentative applications.14,16−18 Nevertheless, to our knowledge, there is only one report on the utilization of seawater for pretreatment of Received: July 6, 2016 Revised: August 15, 2016

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DOI: 10.1021/acssuschemeng.6b01562 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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ACS Sustainable Chemistry & Engineering

NaN3 (to inhibit the microbial growth), cellulase with 11 FPU/g of biomass, and β-glucosidase with 25 CBU/g of biomass in citrate buffer (50 mM, pH 4.8). Aliquots (300 μL) were withdrawn at specified time intervals, and boiled for 5 min to quench the enzymatic reaction. After centrifugation (18 000 g, 10 min), the reducing sugar concentrations were measured by the DNS method. Polysaccharide digestibility and reducing sugar yield were calculated according to eqs 2 and 3, respectively:

lignocellulosic biomass in the literature, where seawater-based hydrothermal pretreatment of date palm residues was conducted.15 In this work, we studied the feasibility of the use of seawater to replace freshwater for IL pretreatment of lignocellulosic biomass. In addition, the commercially available enzyme cocktail was demonstrated to be highly active in seawater for the enzymatic hydrolysis of the pretreated lignocellulosic biomass. Also, microbial lipid production from wheat straw hydrolysate by oleaginous yeast, Trichosporon fermentans CICC 1368, was conducted.

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polysaccharide digestibility (%) = amount of polysaccharide hydrolyzed amount of polysaccharide in the biomass used for enzymatic hydrolysis

EXPERIMENTAL SECTION

× 100

Materials. Cellulase (Celluclast 1.5L) with 49 FPU/mL and βglucosidase (Novozymes 188) with 463 CBU/mL were donated kindly by Novozymes (China), and the enzyme activities were determined according to a previous method.19 A choline hydroxide solution (45 wt %) in methanol was bought from Sigma-Aldrich. Rice straw, sugar cane bagasse, eucalyptus, and pine were harvested in Guangzhou, Guangdong (China) in 2013. Wheat straw was harvested in Houma, Shanxi (China) in 2014. Corncob was harvested in Zaozhuang, Shandong (China), in 2012. All lignocelluloses were mechanically powdered to a particle size of 250−420 μm, and stored in a sealed bag at −20 °C. Seawater was obtained from South China Sea (Shenzhen, China), which contains approximately 4.0% solid. Seawater was subjected to filtration using a microporous membrane with a pore diameter of 0.45 μm prior to use. L-Amino acids were obtained from Yuanju Biotechnol Co. (Shanghai, China). Levulinic acid (99%) was purchased from Aladdin Industrial Inc. (Shanghai, China). Taurine (99%) was from Macklin Biochemical Co. (Shanghai, China). 3,5-Dinitrosalicylic acid (99%, DNS) was from Sinopharm Chemical Reagent Co. (China). Citric acid (99%), sodium citrate (99%), potassium sodium tartrate (99%), sodium metabisulfite (99%), and sodium azide (99%, NaN3) were from Fuchen Chemical Reagent Co., Ltd. (Tianjin, China). Cholinium ILs were synthesized according to our previous methods.9,10 [Emim][OAc] was bought from Zhongke Kaidi Chemical Co. (Lanzhou, China). T. fermentans CICC 1368 was supplied by China Center of Industrial Culture Collection and kept on wort agar at 4 °C. Compositional Analysis of Lignocellulosic Biomass. Polysaccharide and lignin contents of biomass samples were determined according to the standard NREL analytical procedure,20 with a minor modification. Briefly, the samples were hydrolyzed with 72% sulfuric acid at 30 °C for 1 h, and then incubated with an acid content of 4% at 121 °C for 1 h. The resulting hydrolysates were neutralized with calcium carbonate, and then the reducing sugar contents were determined by the DNS method.19 The contents of acid-insoluble lignin remaining after acid hydrolysis were determined gravimetrically by using filtering crucibles. The contents of acid-soluble lignin were measured spectrophotometrically at 320 nm using the extinction coefficient of 30 L/g cm. IL Pretreatment of Lignocellulosic Biomass. IL pretreatment was conducted as described recently,9 with slight modifications. Typically, 150 mg biomass samples were incubated under N2 in 3 g of 80% IL−seawater mixtures while being stirred at 90 °C and 500 rpm for 6 h. Then, the suspension was diluted with an equal volume of deionized water or seawater and centrifuged (18 000 g, 15 min). The residues were washed with deionized water or seawater (approximately 350 mL) until the supernatant was colorless. Then, the residues were lyophilized and stored in a sealed bag at −20 °C prior to use. Lignin extractability was calculated according to eq 1:

lignin extractability (%) ⎛ lignin amount in the recovered residues ⎞ = ⎜1 − ⎟ × 100 lignin amount in the untreated biomass ⎠ ⎝

(2)

reducing sugar yield (%) released reducing sugar amount = × 100 theoretic reducing sugar amount in native biomass (3) Fourier Transform Infrared (FTIR) Analysis. Samples were pressed uniformly with KBr into pellets. FTIR analysis was conducted on a Bruker VERTEX 70 spectrometer (Bruker, Germany) in transmission mode. Spectra were recorded over the range 400−4000 cm−1 with spectral resolution of 0.3 cm−1 with air and IL solutions as backgrounds for untreated and pretreated samples, respectively. Microbial Lipid Production. Microbial lipid production was carried out according to a previous report.21 The precultivation was performed in the preculture medium (glucose 20 g/L, xylose 10 g/L, peptone 10 g/L, yeast extract 10 g/L, pH 6.0) at 28 °C and 160 rpm for 24 h. The culture media for lipid production included freshwaterand seawater- and wheat-straw-hydrolysate-based culture media. The composition of freshwater-based culture medium is as follows: glucose 24 g/L, xylose 8 g/L, peptone 0.56 g/L, yeast extract 0.20 g/L, MgSO4·7H2O 0.4 g/L, KH2PO4 2.0 g/L, MnSO4·H2O 0.003 g/L, CuSO4·5H2O 0.0001 g/L, and deionized water. The composition of seawater-based culture medium is the same as that of freshwater-based culture medium, except that the solvent is seawater. Similarly, the composition of hydrolysate-based culture medium is the same as that of freshwater-based culture medium, except that the sugars are replaced with wheat straw hydrolysate containing 22.5 g/L of glucose and 7.8 g/L of xylose. Then, 5% seed culture was inoculated to the culture media (200 mL, pH 6.5), and incubated at 25 °C and 160 rpm for 3 days. All the experiments were conducted in triplicate, and the results were expressed as the means ± standard deviation. Biomass was harvested by centrifugation and its weight was determined in its lyophilized form. The lyophilized biomass was added into 4 M HCl with a loading of 20 g/L, and stood for 2 h. After being boiled for 15 min, the mixture was frozen at −18 °C for 10 min. Then, lipid extraction was conducted with chloroform/methanol (2/1, v/v) for 30 min, followed by centrifugation. The chloroform phase (down phase) was subjected to vacuum evaporation for removing the solvent, thus affording the lipid. The sugar concentrations of the hydrolysate samples were analyzed by HPLC (Waters) equipped with a Bio-Rad Aminex HPX-87H column and a refractive index detector (Waters 2410). The mobile phase consisted of 5 mM sulfuric acid aqueous solution with a flow rate of 0.5 mL/min. The column temperature was 65 °C. The lipid coefficient was defined as gram lipid produced per gram sugar consumed.

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RESULTS AND DISCUSSION Effect of IL Structures on Pretreatment and Enzymatic Hydrolysis. Previously, it was demonstrated that IL−water mixtures were promising alternatives to pure ILs for biomass pretreatment,22−26 because of many advantages such as the reduced viscosity (easy handling), lower cost, and facile recycling of the mixtures (avoiding energy-intensive evaporation for completely removing water). Therefore, pretreatment of wheat straw with 80% IL−seawater mixtures was conducted,

(1)

Enzymatic Hydrolysis of Lignocellulosic Biomass. Enzymatic hydrolysis was carried out at 50 °C, and 200 rpm in a 50 mL vial containing approximately 2.9 mg/mL of biomass, 0.02 mg/mL of B


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ACS Sustainable Chemistry & Engineering Table 1. Effect of ILs on Pretreatment and Enzymatic Hydrolysis of Wheat Straw pretreatmenta

composition of residues (%)d

entry

ILb

lignin extracted (%)

residue recovery (%)

polysaccharides

AIL

ASL

enzymatic hydrolysis of residuese reducing sugar yields (%)

1 2 3 4 5 6 7 8

untreated [Ch][Arg] [Ch][Gly] [Ch][Tau] [Ch][Lev] [Ch][OAc] [Emim][OAc]-90 [Emim][OAc]-150c

0 71.3 61.6 57.0 13.5 27.3 25.2 52.0

100 52.9 61.3 63.6 71.9 68.0 67.6 50.6

60.4 77.9 66.0 69.1 65.0 66.5 67.8 80.2

15.5 8.5 10.2 10.5 19.7 17.5 18.0 15.3

1.9 0.9 1.0 1.2 1.2 1.1 1.2 1.1

15.3 71.0 66.1 70.8 18.0 47.3 42.8 55.4

Samples of wheat straw (150 mg) were incubated in 80% IL−seawater mixtures (3 g) under N2 while being stirred at 90 °C and 500 rpm for 6 h. Then the residues were washed with deionized water prior to compositional analysis and enzymatic hydrolysis. bIL abbreviations: [Ch][Arg], cholinium argininate; [Ch][Gly], cholinium glycinate; [Ch][Tau], cholinium taurinate; [Ch][Lev], cholinium levulinate; [Ch][OAc], cholinium acetate; [Emim][OAc], 1-ethyl-3-methylimidazolium acetate. cSamples of wheat straw (150 mg) were incubated in 50% IL−seawater mixture (3 g) under N2 while being stirred at 150 °C and 500 rpm for 1.5 h. Then the residues were washed with deionized water prior to compositional analysis and enzymatic hydrolysis. dDetermined via the NREL protocol with a minor modification. Polysaccharide content was determined by the DNS method. Results are expressed as a percentage of the residues. AIL, acid-insoluble lignin; ASL, acid-soluble lignin. eReaction conditions: 20 mg of recovered wheat straw, 7 mL of citrate buffer (50 mM, pH 4.8), 11 FPU/g cellulase, 25 CBU/g β-glucosidase, 0.02 mg/mL NaN3, 50 °C, 200 rpm. Values calculated from the time courses shown in Figure 2. a

and the changes in the composition and enzymatic digestibility of the biomass before and after pretreatment were recorded (Table 1). As shown in Table 1, some cholinium ILs including [Ch][Arg], [Ch][Gly], and [Ch][Tau] remained highly effective for wheat straw pretreatment after adding 20% seawater; after pretreatment with these IL−seawater mixtures, 57−71% lignin was removed from wheat straw (entries 2−4). However, for the cases of [Ch][Lev] and [Ch][OAc], low lignin extractabilities (14−27%) were observed (Table 1, entries 5 and 6), possibly due to poor delignification abilities of these ILs.10,24 [Emim][OAc], one of the widely used ILs, was mixed with seawater, and the mixture was used to pretreat wheat straw. Unexpectedly, it was found that only 25% lignin was extracted from wheat straw after pretreatment with an 80% [Emim][OAc]−seawater mixture at 90 °C for 6 h (Table 1, entry 7). Previously, Mazza and co-workers reported that [Emim][OAc]−water mixtures were effective solvents for pretreatment of straw,22,27 in which high pretreatment severity (e.g., 150 °C, 1.5 h) was used. Therefore, similar conditions were applied for wheat straw pretreatment in this work (Table 1, entry 8). Indeed, the delignification degree (52%) increased significantly at higher pretreatment severity with [Emim][OAc]−seawater mixture. As shown in Table 1, the biomass composition changed significantly upon pretreatment. In the cases of [Ch][Arg]-, [Ch][Gly]-, and [Ch][Tau]-pretreated straws, much lower lignin contents (9−12%) were observed (Table 1, entries 2−4), compared to that of the native biomass (entry 1, 17%), due to the strong delignification abilities of these ILs. Consequently, the polysaccharide contents increased to 66−78% in these cases (Table 1, entries 2−4). However, for the cases with poor lignin extractabilities (Table 1, entries 5−7), the polysaccharide contents as well as the lignin contents increased slightly, compared to those of the native biomass. As shown in Table 1, entry 8, the polysaccharide content increased to approximately 80% upon pretreatment with 50% [Emim][OAc]−seawater at 150 °C, while the lignin content changed slightly. The changes in the biomass composition were also verified by comparing the FTIR spectra of wheat straw before and after pretreatment (Figure 1). The characteristic peaks were assigned according to a recent publication.10 As shown in Figure 1, the

Figure 1. FTIR spectra of wheat straw pretreated with various IL− seawater mixtures. Regions of the spectra mentioned in the text are annotated as follows: (a) 1732 cm−1, (b) 1512 cm−1, (c) 898 cm−1.

band at 1732 cm−1 (ester-linked acetyl, feruloyl, and pcoumaroyl groups between hemicellulose and lignin) almost disappeared after pretreatment in most cases, indicating extensive breakage of the ester linkages between hemicellulose and lignin. The peak at 1512 cm−1 derived from CC stretching vibration in phenol rings is one of the fingerprint signals of lignin. This signal decreased significantly and even disappeared upon pretreatment with [Ch][Arg]−, [Ch][Gly]−, and [Ch][Tau]−seawater mixtures. However, this peak intensity became stronger for other biomass materials, compared to the untreated biomass. The peak at 898 cm−1 is the characteristic “anomeric region” absorption band for βlinkage of carbohydrates.28 It was found that the peak intensity enhanced after pretreatment in all cases, suggesting the C


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(polysaccharide digestibility of 23%), likely due to its high lignin content. The polysaccharide digestibility of [Emim][OAc]-pretreated wheat straw showed a clear dependence on the pretreatment severity (Figure 2). The higher the pretreatment severity, the higher the polysaccharide digestibility was in the subsequent enzymatic hydrolysis. It was reported that pretreatment of lignocellulose biomass with pure [Emim][OAc] led to significant decreases in cellulose crystallinity index (CrI) as well as extensive lignin removal.29,30 Therefore, wheat straw was subjected to X-ray diffraction analysis before and after pretreatment with [Emim][OAc]−seawater mixtures (Figure S1, available as Supporting Information). It was found that cellulose CrI almost did not change and even increased slightly after pretreatment, which is opposite to the previous results.29,30 In this work, aqueous [Emim][OAc], instead of pure [Emim][OAc], was used for lignocellulosic biomass pretreatment. It suggests that cellulose dissolution will not occur, because of the fact that the presence of water can significantly inhibit the dissolution of cellulose in imidazolium ILs.31 It was reported that the reduction level of cellulose CrI caused by swelling was much lower than that by cellulose dissolution using pure IL, although cellulose CrI was reduced when microcrystalline cellulose was soaked in 20% [Emim][OAc] aqueous solution.32 In addition to cellulose swelling, considerable lignin was also removed from biomass during aqueous [Emim][OAc] pretreatment.22 The decreased cellulose CrI caused by swelling during aqueous IL pretreatment might be offset by the increase in CrI due to considerable removal of amorphous lignin, which may account for the slight changes in CrI. Therefore, the higher digestibility of the biomass pretreated with the [Emim][OAc]−seawater mixture at 150 °C might be attributed to removal of considerable lignin as well as cellulose swelling. As shown in Figure 2, the digestibility of 83% was obtained after 72 h in the enzymatic hydrolysis of wheat straw pretreated with the [Emim][OAc]− seawater mixture at 150 °C, thus furnishing a moderate reducing sugar yield (55%). [Ch][Arg] was used in the subsequent studies, due to the excellent pretreatment efficiency and a good reducing sugar yield in the subsequent enzymatic hydrolysis. Effect of IL Contents on Pretreatment and Enzymatic Hydrolysis. Then, the impacts of [Ch][Arg] contents on the IL pretreatment efficiency and the subsequent enzymatic hydrolysis were explored (Table 2), when the IL contents varied from 20% to 100%. As shown in Table 2, considerable

increased carbohydrate contents, which is in good agreement with the above quantitative data on the composition (Table 1). To understand the changes in the biomass recalcitrance before and after pretreatment with IL−seawater mixtures, the pretreated biomass was subjected to enzymatic digestion. Figure 2 shows the time courses of enzymatic hydrolysis of

Figure 2. Time courses of enzymatic hydrolysis of wheat straw pretreated with various IL−seawater mixtures. Reaction conditions: 20 mg of recovered wheat straw, 7 mL of citrate buffer (50 mM, pH 4.8), 11 FPU/g cellulase, 25 CBU/g β-glucosidase, 0.02 mg/mL NaN3, 50 °C, 200 rpm.

wheat straw pretreated with various IL−seawater mixtures, and the results of reducing sugar yields are summarized in Table 1. The native biomass was highly resistant to enzymatic degradation, in which a low reducing sugar yield (approximately 15%) was afforded. Interestingly, wheat straw became readily hydrolyzable upon pretreatment with NH2-containing ILs (Figure 2). For example, the polysaccharide digestibility of 100% and reducing sugar yields of 66−71% were obtained after wheat straw pretreated with the mixtures containing cholinium amino acid ([Ch][AA]) ILs was hydrolyzed for 24 h (Table 1, entries 2 and 3). In the case of [Ch][Tau]-pretreated biomass, high polysaccharide digestibility (97%) as well as a good reducing sugar yield (71%) was also observed (Table 1, entry 4). However, wheat straw pretreated with the [Ch][Lev]− seawater mixture remained highly recalcitrant to biodegradation

Table 2. Effect of IL Contents on Pretreatment and Enzymatic Hydrolysis of Wheat Straw pretreatmenta

composition of residues (%)c

IL content



polysaccharides

AIL

ASL

enzymatic hydrolysis of residuesd reducing sugar yields (%)

100 80 50 50b 20

80.9 71.3 69.1 73.7 56.9

48.9 52.9 56.8 63.2 64.5

84.1 77.9 81.4 67.1 74.9

5.8 8.5 8.5 6.3 10.7

0.9 0.9 0.9 0.9 0.9

71.3 71.0 73.6 70.1 77.0

Samples of wheat straw (150 mg) were incubated in [Ch][Arg]−seawater mixtures (3 g) under N2 while being stirred at 90 °C and 500 rpm for 6 h. Then, the residues were washed with deionized water prior to compositional analysis and enzymatic hydrolysis. bSamples of wheat straw (150 mg) were incubated in [Ch][Arg]−deionized water mixtures (3 g) under N2 while being stirred at 90 °C and 500 rpm for 6 h. Then, the residues were washed with deionized water prior to compositional analysis and enzymatic hydrolysis. cDetermined via the NREL protocol with a minor modification. Polysaccharide content was determined by the DNS method. Results are expressed as a percentage of the residues. AIL, acid-insoluble lignin; ASL, acid-soluble lignin. dReaction conditions: 20 mg of recovered wheat straw, 7 mL of citrate buffer (50 mM, pH 4.8), 11 FPU/g cellulase, 25 CBU/g β-glucosidase, 0.02 mg/mL NaN3, 50 °C, 200 rpm. Values calculated from the time courses shown in Figure S2. a

D


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cocktail was used as the model enzymes to evaluate the effect of seawater on the enzymatic hydrolysis of wheat straw. Prior to enzymatic hydrolysis, wheat straw was pretreated by the 50% [Ch][Arg]−seawater mixture and washed by deionized water. The recovered residues were subjected to enzymatic digestion in various media (Figure 3a). As shown in Figure 3a, the enzymes showed good catalytic performances not only in freshwater-based citrate buffer, but also in seawater-based citrate buffer; the polysaccharide digestibility of 100% was obtained, suggesting that seawater has a marginally negative effect on the enzymatic hydrolysis. Inspired by the results, use of seawater with a pH that was directly adjusted by acids as the reaction media was attempted, since the preparation of acidic seawater was much simpler and more cost-efficient than that of citrate buffer. It was interestingly found that the high polysaccharide digestibility was also obtained in pH 4.8 seawater regulated by adding citric acid. However, for the case of using HCl as the pH regulator, the poor digestibility (approximately 40%) was furnished. The decreased enzymatic hydrolytic rate was observed in 2-fold concentrated seawater, and the digestibility of 92% was achieved after 48 h, which indicates the inhibition of the concentrated seawater against the enzymes. With the development of rational design technologies of proteins, the cellulolytic enzymes with high tolerance to the concentrated seawater may emerge as a promising option for the enzymatic saccharification of biomass in seawater in the future. Then, the enzymatic hydrolysis of the residues washed by seawater was conducted (Figure 3b). The residues, after being washed by seawater followed by lyophilization, were heterogeneous, because of the presence of a considerable amount of salts; therefore, all of the recovered residues were subjected to enzymatic hydrolysis. As shown in Figure 3b, the enzymatic hydrolysis proceeded smoothly, affording the polysaccharide digestibility of 95% after 24 h. The above results indicate that the presence of seawater has a slightly negative effect on the subsequent enzymatic hydrolysis, and that the use of seawater to wash the pretreated biomass prior to the subsequent enzymatic saccharification is feasible. Pretreatment and Enzymatic Hydrolysis of Various Lignocelluloses. Encouraged by the above results, pretreatment and enzymatic hydrolysis of lignocellulosic biomass, both of which involved seawater, were conducted (Table 3). It could be found that extensive delignification of grass lignocelluloses occurred with the 50% [Ch][Arg]−seawater mixture, with the maximal lignin extractability up to 88%. After pretreatment, the composition of grass lignocelluloses changed significantly. As shown in Table 3, the polysaccharide contents increased remarkably upon pretreatment, compared to those of the native biomass, while the lignin contents decreased to 4−9%. It is in good agreement with our recent results with [Ch][Arg] as the pretreatment solvent.36 However, for woods, much lower lignin extractabilities were observed, as compared with those of grasses. In addition, almost no changes in the polysaccharide contents happened upon pretreatment. As shown in Table 3, almost all the native biomass samples were highly recalcitrant to enzymatic hydrolysis; the reducing sugar yields were generally less than 20% in the enzymatic digestion, except for the case of sugar cane bagasse (38%). After pretreatment, grass lignocelluloses became easily hydrolyzable. The polysaccharide digestibility of more than 88% was achieved after the pretreated grasses were hydrolyzed for 48 h (Figure S3, available as Supporting Information), and the reducing

lignin (57−81%) was removed within the range of IL contents tested. Generally, the increased IL content resulted in the higher lignin extractability, which is consistent with our previous results.23,24 Upon pretreatment, the biomass composition altered significantly. The polysaccharide contents increased substantially, while the lignin contents reduced significantly. Consequently, all the pretreated materials appeared to be prone to enzymatic degradation, giving the polysaccharide digestibility of >96% (Figure S2, available as Supporting Information), and good reducing sugar yields (71− 77%) were obtained in all cases (Table 2). In addition, the reducing sugar yields were comparable in the enzymatic hydrolysis of wheat straw pretreated by 50% IL−seawater and 50% IL−freshwater (74% vs 70%, Table 2). The above results have demonstrated that it is feasible to use seawater as an alternative to freshwater for IL pretreatment of lignocellulosic biomass. Effect of Reaction Media on Enzymatic Hydrolysis. Although we have demonstrated the feasibility of the use of seawater for IL pretreatment, a limited amount of water is consumed during IL pretreatment. After IL pretreatment, extensive washing of the pretreated biomass was necessary generally prior to enzymatic hydrolysis, in which a large amount of water would be consumed, because it was demonstrated that even a low amount of residual ILs present in the pretreated biomass would cause inhibitory effects on the subsequent enzymatic hydrolysis as well as microbial fermentation.33−35 If the pretreated biomass is washed by seawater, the biomass will contain a considerable amount of salts, which may affect the subsequent enzymatic hydrolysis. Therefore, the catalytic performances of the enzyme cocktail were examined in various seawater-based media (Figure 3a). Previously, Lehmann et al. reported that the cellulase variant 4D1 showed good activity in seawater.16 In this work, the commercially available enzyme

Figure 3. Time courses of enzymatic hydrolysis of the residues (a) washed by deionized water in various reaction media and the residues (b) washed by seawater in citrate buffer. Symbols: ■, freshwater-based citrate buffer (50 mM, pH 4.8); ●, seawater-based citrate buffer (50 mM, pH 4.8); ▲, pH 4.8 seawater regulated by adding citric acid; ▼, pH 4.8 seawater regulated by 4 M HCl; ⧫, pH 4.8 2-fold concentrated seawater regulated by adding citric acid. Reaction conditions for part a: 20 mg of recovered wheat straw, 7 mL of aqueous solution (pH 4.8), 11 FPU/g cellulase, 25 CBU/g β-glucosidase, 0.02 mg/mL NaN3, 50 °C, 200 rpm. Reaction conditions for part b: all recovered wheat straw, 28 mL of freshwater-based citrate buffer (50 mM, pH 4.8), 11 FPU/g cellulase, 25 CBU/g β-glucosidase, 0.02 mg/mL NaN3, 50 °C, 200 rpm. E


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Table 3. Pretreatment of Lignocellulosic Biomass with 50% [Ch][Arg]−Seawater Mixture and Subsequent Enzymatic Hydrolysis pretreatmenta biomass

IL

wheat straw [Ch][Arg] rice straw [Ch][Arg] corncob [Ch][Arg] sugar cane bagasse [Ch][Arg] pine [Ch][Arg] eucalyptus [Ch][Arg]

composition of residues (%)b



polysaccharides

AIL

ASL

enzymatic hydrolysis reducing sugar yield (%)

0 73.8 0 88.2 0 86.5 0 83.8 0 34.9 0 38.7

100 53.3 100 47.6 100 60.9 100 43.4 100 74.8 100 80.2

60.4 76.5 59.8 73.1 71.3 82.1 61.6 72.9 53.0 55.5 58.2 58.4

15.5 7.7 17.7 4.3 14.8 2.8 15.2 5.7 34.2 29.8 32.5 24.3

1.9 0.8 2.3 0.6 1.7 0.8 2.0 0.8 0.8 0.5 1.2 1.4

14.6c 59.6d 16.8c 54.3d 14.0c 71.6d 38.3c 54.5d 13.8c 14.6d 12.9c 12.2d

Samples of lignocellulosic biomass (150 mg) were incubated in 50% IL−seawater mixture (3 g) under N2 while being stirred at 90 °C and 500 rpm for 6 h. Four parallel experiments were conducted for each cases. Then, two samples were washed by deionized water for determining the composition and residue recoveries, and the other two were washed by seawater for the subsequent enzymatic digestion. bDetermined via the NREL protocol with a minor modification. Polysaccharide content was determined by the DNS method. Results are expressed as a percentage of the residues. AIL, acid-insoluble lignin; ASL, acid-soluble lignin. cReaction conditions: 20 mg of biomass, 7 mL of seawater (pH 4.8, which was regulated by adding citric acid), 11 FPU/g cellulase, 25 CBU/g β-glucosidase, 0.02 mg/mL NaN3, 50 °C, 200 rpm. Values calculated from the time courses shown in Figure S2. dReaction conditions: all the recovered biomass (approximately 2.9 mg/mL, which was washed by seawater), 22−42 mL of pH 4.8 seawater regulated by citric acid, 11 FPU/g cellulase, 25 CBU/g β-glucosidase, 0.02 mg/mL NaN3, 50 °C, 200 rpm. a

sugar yields of 54−72% were obtained (Table 3). In the enzymatic hydrolysis of the pretreated wheat straw, the reducing sugar yield obtained in pH 4.8 seawater was lower than that in freshwater-based citrate buffer (60% vs 74%). Herein, the recovered biomass was washed extensively by seawater and lyophilized prior to the enzymatic saccharification. Therefore, the lyophilized residues generally contained a considerable amount of salts, with the residue recovery of approximately 180%. So the salt concentration in the reaction mixture containing seawater and the residues washed by seawater would be much higher than that in the reaction mixture containing seawater and the residues washed by freshwater. It has been demonstrated that 2-fold concentrated seawater has a negative effect on the enzyme performance (Figure 3a). As a result, the lower reducing sugar yields might be attributed to the negative effect of the higher salt concentrations on the enzymatic hydrolysis. However, the marginal improvements in the enzymatic hydrolysis were observed after pretreatment of woods, which might be partially attributed to the presence of high contents of lignin, since lignin was known as a major obstacle for efficient enzymatic hydrolysis of lignocelluloses.37,38 Microbial Lipid Fermentation. T. fermentans is a kind of oleaginous yeast belonging to the family of Cryptococcaceae. Our previous work demonstrated that T. fermentans could efficiently produce lipid from rice straw hydrolysate as well as a model medium containing glucose and xylose.21,39 Therefore, T. fermentans was used as the model microorganism to evaluate microbial lipid production from freshwater-, seawater-, and straw-hydrolysate-based culture media (Figure 4). As shown in Figure 4a, the highest sugar consumption (approximately 22.0 g/L) was observed in wheat-straw-hydrolysate-based culture medium after 3 days of fermentation, followed by that in freshwater-based medium. The sugar consumption (approximately 14.4 g/L) in seawater-based culture medium was the poorest, suggesting the significant inhibition of seawater toward the growth of T. fermentans. Indeed, the effect of the culture

Figure 4. Lipid production from various culture media by T. fermentans. Cultivation conditions: 5% seed culture was inoculated to the culture media containing the reducing sugar of approximately 30 g/L, and incubated at 25 °C and 160 rpm for 3 days. Detailed conditions were described in the Experimental Section.

media on the biomass concentration was similar to its effect on the sugar consumption. Also, the highest biomass concentration was found in straw-hydrolysate-based culture medium, while the lowest biomass concentration is in the seawater-based medium. Recently, Takahashi and co-workers have reported the effect of post-pretreatment washing on the enzymatic saccharification and microbial fermentation, and found that the washing number had a remarkable effect on the subsequent processes, likely due to the presence of the residual ILs.40 In addition, it has been demonstrated recently that the low concentrations of [Ch][AA] ILs will result in the significant increases in the biomass concentrations during the lipid production by T. fermentans,41 due to microbial assimilation of amino acid anions of ILs as nitrogen sources. As shown in F


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ACS Sustainable Chemistry & Engineering Figure 4a, the lipid contents of T. fermentans which grew in freshwater- and seawater-based culture media were interestingly found to be comparable (47−49%), while the lipid content in the case of wheat-straw-hydrolysate-based medium was lower (approximately 33%). It is well-known that a high C/N ratio in the medium is critical for high lipid accumulation in microbial cells.42 The presence of the residual [Ch][Arg] would reduce the C/N ratio of the hydrolysate-based medium, which may account for the low lipid content. Figure 4b shows the lipid yields as well as the lipid coefficients. The lipid yields of 2.3−4.5 g/L were achieved after 3 days of fermentation, which are much lower than our previous results.21 It might be attributed to the much lower initial sugar concentrations and shorter fermentation period in this work. The highest lipid yield (4.5 g/L) was observed in wheat-straw-hydrolysate-based culture medium, which is a moderate value compared to those with the lignocellulosic hydrolysates as the media.42 As shown in Figure 4b, the lipid coefficient, a key parameter for efficient microbial lipid production,42 was very high in all cases, reaching 0.15−0.21 g/g of sugar. Because of the significant inhibitory effect of seawater against T. fermentans, microbial lipid production from wheat-straw-hydrolysate/seawater-based culture media was not conducted. It is expected that efficient lipid production by microbial fermentation from straw-hydrolysate/seawater-based culture media may be realized by using appropriate marine or highly salt-tolerant microorganisms.18,43−45

Natural Science Foundation of Guangdong Province (2014A030313263).

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(1) Tuck, C. O.; Pérez, E.; Horváth, I. T.; Sheldon, R. A.; Poliakoff, M. Valorization of Biomass: Deriving More Value from Waste. Science 2012, 337 (6095), 695−699. (2) Sheldon, R. A. Green and sustainable manufacture of chemicals from biomass: state of the art. Green Chem. 2014, 16 (3), 950−963. (3) Xu, C.; Arancon, R. A. D.; Labidi, J.; Luque, R. Lignin depolymerisation strategies: towards valuable chemicals and fuels. Chem. Soc. Rev. 2014, 43 (22), 7485−7500. (4) Ragauskas, A. J.; Beckham, G. T.; Biddy, M. J.; Chandra, R.; Chen, F.; Davis, M. F.; Davison, B. H.; Dixon, R. A.; Gilna, P.; Keller, M.; Langan, P.; Naskar, A. K.; Saddler, J. N.; Tschaplinski, T. J.; Tuskan, G. A.; Wyman, C. E. Lignin valorization: Improving lignin processing in the biorefinery. Science 2014, 344 (6185), 1246843. (5) Alvira, P.; Tomas-Pejo, E.; Ballesteros, M.; Negro, M. J. Pretreatment technologies for an efficient bioethanol production process based on enzymatic hydrolysis: A review. Bioresour. Technol. 2010, 101 (13), 4851−4861. (6) Mora-Pale, M.; Meli, L.; Doherty, T. V.; Linhardt, R. J.; Dordick, J. S. Room temperature ionic liquids as emerging solvents for the pretreatment of lignocellulosic biomass. Biotechnol. Bioeng. 2011, 108 (6), 1229−1245. (7) Silveira, M. H. L.; Morais, A. R. C.; daCostaLopes, A. M.; Olekszyszen, D. N.; Bogel-Łukasik, R.; Andreaus, J.; PereiraRamos, L. Current pretreatment technologies for the development of cellulosic ethanol and biorefineries. ChemSusChem 2015, 8 (20), 3366−3390. (8) Liu, Q. P.; Hou, X. D.; Li, N.; Zong, M. H. Ionic liquids from renewable biomaterials: synthesis, characterization and application in the pretreatment of biomass. Green Chem. 2012, 14 (2), 304−307. (9) Hou, X. D.; Xu, J.; Li, N.; Zong, M. H. Effect of anion structures on cholinium ionic liquids pretreatment of rice straw and the subsequent enzymatic hydrolysis. Biotechnol. Bioeng. 2015, 112 (1), 65−73. (10) Ren, H.; Zong, M. H.; Wu, H.; Li, N. Efficient pretreatment of wheat straw using novel renewable cholinium ionic liquids to improve enzymatic saccharification. Ind. Eng. Chem. Res. 2016, 55 (6), 1788− 1795. (11) Hou, X. D.; Liu, Q. P.; Smith, T. J.; Li, N.; Zong, M. H. Evaluation of toxicity and biodegradability of cholinium amino acids ionic liquids. PLoS One 2013, 8 (3), e59145. (12) Ninomiya, K.; Yamauchi, T.; Kobayashi, M.; Ogino, C.; Shimizu, N.; Takahashi, K. Cholinium carboxylate ionic liquids for pretreatment of lignocellulosic materials to enhance subsequent enzymatic saccharification. Biochem. Eng. J. 2013, 71, 25−29. (13) Ventura, S. P. M.; Silva, F. A. e.; Gonçalves, A. M. M.; Pereira, J. L.; Gonçalves, F.; Coutinho, J. A. P. Ecotoxicity analysis of choliniumbased ionic liquids to Vibrio fischeri marine bacteria. Ecotoxicol. Environ. Saf. 2014, 102, 48−54. (14) Domínguez de María, P. On the use of seawater as reaction media for large-scale applications in biorefineries. ChemCatChem 2013, 5 (7), 1643−1648. (15) Fang, C.; Thomsen, M. H.; Brudecki, G. P.; Cybulska, I.; Frankær, C. G.; Bastidas-Oyanedel, J.-R.; Schmidt, J. E. Seawater as alternative to freshwater in pretreatment of date palm residues for bioethanol production in coastal and/or arid areas. ChemSusChem 2015, 8 (22), 3823−3831. (16) Lehmann, C.; Sibilla, F.; Maugeri, Z.; Streit, W. R.; Dominguez de Maria, P.; Martinez, R.; Schwaneberg, U. Reengineering CelA2 cellulase for hydrolysis in aqueous solutions of deep eutectic solvents and concentrated seawater. Green Chem. 2012, 14 (10), 2719−2726. (17) Grande, P. M.; Bergs, C.; Domínguez de María, P. Chemoenzymatic conversion of glucose into 5-hydroxymethylfurfural in seawater. ChemSusChem 2012, 5 (7), 1203−1206. (18) Lin, C. S.; Luque, R.; Clark, J. H.; Webb, C.; Du, C. A seawaterbased biorefining strategy for fermentative production and chemical

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CONCLUSIONS In summary, we have demonstrated that it is feasible to utilize seawater for the biorefinery of lignocellulosic biomass in this work. Adding seawater had no significantly negative effect on the IL pretreatment efficiency. In addition, the commercially available cellulolytic enzymes remained highly active in pH 4.8 seawater. Microbial lipid production by T. fermentans from wheat straw hydrolysate furnished a good lipid coefficient (approximately 0.21 g/g of sugar). Given the problems such as overpopulation and freshwater shortage, seawater is a promising alternative to freshwater for the valorization of lignocellulosic biomass to biofuels, especially in coastal and arid areas. The discovery and engineering of highly seawatertolerant enzymes and microorganisms will significantly facilitate the success of large-scale biorefineries of lignocelluloses in seawater.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.6b01562. XRD spectra and enzymatic hydrolysis details (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*Phone/fax: +86 20 2223 6669. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the State Key Laboratory of Pulp and Paper Engineering (2015C03), and the G


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ACS Sustainable Chemistry & Engineering transformations of succinic acid. Energy Environ. Sci. 2011, 4 (4), 1471−1479. (19) Ghose, T. K. Measurement of cellulase activities. Pure Appl. Chem. 1987, 59 (2), 257−268. (20) Sluiter, A.; Hames, B.; Ruiz, R.; Scarlata, C.; Sluiter, J.; Templeton, D.; Crocker, D. Determination of Structural Carbohydrates and Lignin in Biomass; NREL/TP-510-42618; National Renewable Energy Laboratory: Golden, CO, 2008. (21) Huang, C.; Zong, M. H.; Wu, H.; Liu, Q. P. Microbial oil production from rice straw hydrolysate by Trichosporon fermentans. Bioresour. Technol. 2009, 100 (19), 4535−4538. (22) Fu, D.; Mazza, G. Aqueous ionic liquid pretreatment of straw. Bioresour. Technol. 2011, 102 (13), 7008−7011. (23) Hou, X. D.; Li, N.; Zong, M. H. Significantly enhancing enzymatic hydrolysis of rice straw after pretreatment using renewable ionic liquid-water mixtures. Bioresour. Technol. 2013, 136, 469−474. (24) Hou, X. D.; Li, N.; Zong, M. H. Facile and simple pretreatment of sugar cane bagasse without size reduction using renewable ionic liquids-water mixtures. ACS Sustainable Chem. Eng. 2013, 1 (5), 519− 526. (25) An, Y. X.; Li, N.; Zong, M. H.; Lou, W. Y. Easily measurable pH as an indicator of the effectiveness of the aqueous cholinium ionic liquid-based pretreatment of lignocellulose. RSC Adv. 2014, 4 (98), 55635−55639. (26) Hou, X. D.; Li, N.; Zong, M. H. Renewable bio ionic liquidswater mixtures-mediated selective removal of lignin from rice straw: Visualization of changes in composition and cell wall structure. Biotechnol. Bioeng. 2013, 110 (7), 1895−1902. (27) Fu, D.; Mazza, G. Optimization of processing conditions for the pretreatment of wheat straw using aqueous ionic liquid. Bioresour. Technol. 2011, 102 (17), 8003−8010. (28) Kačuráková, M.; Capek, P.; Sasinková, V.; Wellner, N.; Ebringerová, A. FT-IR study of plant cell wall model compounds: pectic polysaccharides and hemicelluloses. Carbohydr. Polym. 2000, 43 (2), 195−203. (29) Lee, S. H.; Doherty, T. V.; Linhardt, R. J.; Dordick, J. S. Ionic liquid-mediated selective extraction of lignin from wood leading to enhanced enzymatic cellulose hydrolysis. Biotechnol. Bioeng. 2009, 102 (5), 1368−1376. (30) Cheng, G.; Varanasi, P.; Arora, R.; Stavila, V.; Simmons, B. A.; Kent, M. S.; Singh, S. Impact of ionic liquid pretreatment conditions on cellulose crystalline structure using 1-ethyl-3-methylimidazolium acetate. J. Phys. Chem. B 2012, 116 (33), 10049−10054. (31) Swatloski, R. P.; Spear, S. K.; Holbrey, J. D.; Rogers, R. D. Dissolution of cellulose with ionic liquids. J. Am. Chem. Soc. 2002, 124 (18), 4974−4975. (32) Glas, D.; Paesen, R.; Depuydt, D.; Binnemans, K.; Ameloot, M.; DeVos, D. E.; Ameloot, R. Cellulose amorphization by swelling in ionic liquid/water mixtures: A combined macroscopic and secondharmonic microscopy study. ChemSusChem 2015, 8, 82−86. (33) Datta, S.; Holmes, B.; Park, J. I.; Chen, Z.; Dibble, D. C.; Hadi, M.; Blanch, H. W.; Simmons, B. A.; Sapra, R. Ionic liquid tolerant hyperthermophilic cellulases for biomass pretreatment and hydrolysis. Green Chem. 2010, 12 (2), 338−345. (34) Ouellet, M.; Datta, S.; Dibble, D. C.; Tamrakar, P. R.; Benke, P. I.; Li, C.; Singh, S.; Sale, K. L.; Adams, P. D.; Keasling, J. D.; Simmons, B. A.; Holmes, B. M.; Mukhopadhyay, A. Impact of ionic liquid pretreated plant biomass on Saccharomyces cerevisiae growth and biofuel production. Green Chem. 2011, 13 (10), 2743−2749. (35) Wahlstrom, R. M.; Suurnakki, A. Enzymatic hydrolysis of lignocellulosic polysaccharides in the presence of ionic liquids. Green Chem. 2015, 17 (2), 694−714. (36) An, Y. X.; Zong, M. H.; Wu, H.; Li, N. Pretreatment of lignocellulosic biomass with renewable cholinium ionic liquids: Biomass fractionation, enzymatic digestion and ionic liquid reuse. Bioresour. Technol. 2015, 192, 165−171. (37) Ximenes, E.; Kim, Y.; Mosier, N.; Dien, B.; Ladisch, M. Deactivation of cellulases by phenols. Enzyme Microb. Technol. 2011, 48 (1), 54−60.

(38) Zeng, Y.; Zhao, S.; Yang, S.; Ding, S.-Y. Lignin plays a negative role in the biochemical process for producing lignocellulosic biofuels. Curr. Opin. Biotechnol. 2014, 27, 38−45. (39) Zhu, L. Y.; Zong, M. H.; Wu, H. Efficient lipid production with Trichosporon fermentans and its use for biodiesel preparation. Bioresour. Technol. 2008, 99 (16), 7881−7885. (40) Ninomiya, K.; Ogino, C.; Ishizaki, M.; Yasuda, M.; Shimizu, N.; Takahashi, K. Effect of post-pretreatment washing on saccharification and co-fermentation from bagasse pretreated with biocompatible cholinium ionic liquid. Biochem. Eng. J. 2015, 103, 198−204. (41) Liu, L.; Hu, Y.; Wen, P.; Li, N.; Zong, M.; Ou-Yang, B.; Wu, H. Evaluating the effects of biocompatible cholinium ionic liquids on microbial lipid production by Trichosporon fermentans. Biotechnol. Biofuels 2015, 8 (1), 119. (42) Jin, M.; Slininger, P. J.; Dien, B. S.; Waghmode, S.; Moser, B. R.; Orjuela, A.; Sousa, L. d. C.; Balan, V. Microbial lipid-based lignocellulosic biorefinery: feasibility and challenges. Trends Biotechnol. 2015, 33 (1), 43−54. (43) Goncalves, F. A.; Santos, E. S. d.; de Macedo, G. R. Alcoholic fermentation of Saccharomyces cerevisiae, Pichia stipitis and Zymomonas mobilis in the presence of inhibitory compounds and seawater. J. Basic Microbiol. 2015, 55 (6), 695−708. (44) Ahammed, S.; Prema, P. Influence of media nutrients on synthesis of lignin peroxidase from Aspergillus sp. Appl. Biochem. Biotechnol. 2002, 102 (1), 327−336. (45) Gonzalez, J.; Mayer, F.; Moran, M.; Hodson, R.; Whitman, W. Sagittula stellata gen. nov., sp. nov., a lignin-transforming bacterium from a coastal environment. Int. J. Syst. Bacteriol. 1997, 47 (3), 773− 780.

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Utilization of Seawater for the Biorefinery of Lignocellulosic Biomass

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