Enhanced Ethanol Production from Pomelo Peel Waste by Integrated

May 6, 2014 - ABSTRACT: Pomelo peel is an abundant pectin-rich biomass waste in China and has the potential to serve as a source of fuels and chemical...
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Enhanced Ethanol Production from Pomelo Peel Waste by Integrated Hydrothermal Treatment, Multienzyme Formulation, and Fed-Batch Operation Renliang Huang,‡,∥ Ming Cao,†,∥ Hong Guo,† Wei Qi,†,# Rongxin Su,*,†,# and Zhimin He† †

State Key Laboratory of Chemical Engineering, Tianjin Key Laboratory of Membrane Science and Desalination Technology, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, People’s Republic of China ‡ School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, People’s Republic of China # Collaborative Innovation Center of Chemical Science and Engineering (Tianjin),Tianjin 300072, People’s Republic of China ABSTRACT: Pomelo peel is an abundant pectin-rich biomass waste in China and has the potential to serve as a source of fuels and chemicals. This study reports a promising way to deal with pomelo peel waste and to utilize it as raw material for ethanol production via simultaneous saccharification and fermentation (SSF). An integrated strategy, incorporating hydrothermal treatment, multienzyme formulation, and fed-batch operation, was further developed to enhance the ethanol production. The results show that hydrothermal treatment (120 °C, 15 min) could significantly reduce the use of cellulase (from 7 to 3.8 FPU g−1) and pectinase (from 20 to 10 U g−1). A multienzyme complex, which consists of cellulase, pectinase, β-glucosidase, and xylanase, was also proven to be effective to improve the hydrolysis of pretreated pomelo peel, leading to higher concentrations of fermentative sugars (36 vs 14 g L−1) and galacturonic acid (23 vs 9 g L−1) than those with the use of a single enzyme. Furthermore, to increase the final ethanol concentration, fed-batch operation by adding fresh substrate was employed in the SSF process. A final solid loading of 25% (w/v), which is achieved by adding 15% fresh substrate to the SSF system at an initial solid loading of 10%, produced 36 g L−1 ethanol product in good yield (73.5%). The ethanol concentration is about 1.73-fold that at the maximum solid loading of 14% for batch operation, whereas both of them have a closed ethanol yield. The results indicate that the use of the fed-batch mode could alleviate the decrease in ethanol yield at high solid loading, which is caused by significant mass transfer limitation and increased inhibition of toxic compounds in the SSF process. The integrated strategy demonstrated in this work could open a new avenue for dealing with pectin-rich biomass wastes and utilization of the wastes to produce ethanol. KEYWORDS: pomelo peel, ethanol, cellulase, pectinase, enzymatic hydrolysis, SSF

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sugar-based chemicals.2,8 Ethanol, being one of the most promising biofuels, has received great attention with the increasing fuel demand and environmental concerns globally. In recent years, much effort has been invested to develop secondgeneration ethanol fuel from nonfood crops. Most of this research focuses on the use of recalcitrant lignocellulosic biomass, such as corn cobs,9,10 corn stover,11−13 wood chips,14,15 switchgrass,16 and rice straw,17 due to their largescale availability and low raw material cost. In these cases, the high content of lignin and the recalcitrant structure of these feedstocks greatly hinder enzymatic hydrolysis and fermentation. Generally, harsh pretreatment and high enzyme loading are required in ethanol production. In comparison with recalcitrant lignocellulosic biomass, citrus wastes, such as citrus peel, contain less lignin and more pectin, which enable a facile hydrolysis of the wastes into fermentable sugars by the use of several hydrolytic enzymes in combination.18,19 With the large sugar content generated from highly degradable polysaccharides and the elimination of harsh pretreatment, citrus wastes could prove to be an attractive source of bioethanol.20 This

INTRODUCTION Citrus production and consumption worldwide have grown strongly over the past few decades. According to the statistical data from the Food and Agriculture Organization of the United Nations (FAOSTAT), the world production of citrus fruits in 2010 was over 100 million tons.1 With the heavy consumption of these fruits (e.g., juice/sugar production), a large amount of citrus waste is generated, which accounts for about 50% of the fruit weight. The wastes include fruit peel, membrane residues, and other byproducts.2 Traditionally, the wastes are disposed of through a burning process or used as raw material for active substance extraction and animal feed manufacturing.3−6 However, these means are not satisfactory as the market demand may be limited or secondary contamination and lowvalue products result. Up to now, the majority of citrus wastes are still deposited or disposed of simply as mentioned before, leading to serious environmental pollution and great wastage of resources. Therefore, it is highly desirable to develop green and economical ways to deal with citrus wastes, as well as to utilize the wastes to produce staple or value-added products. Citrus wastes mainly consist of soluble mono- and disaccharides (e.g., glucose, fructose, sucrose), insoluble polysaccharides (e.g., cellulose, hemicellulose, pectin), and lignin.7 This chemical composition renders the wastes to be a potential candidate for the production of biofuels and other © 2014 American Chemical Society

Received: Revised: Accepted: Published: 4643

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Table 1. Chemical Compositions (Percent of Dry Matter) of Different Citrus Wastes citrus waste

cellulose

hemicellulose

pectin

lignin

soluble sugars

ash

orange peel47 orange waste23 Kinnow mandarin peel27 mandarin peel28 grapefruit waste47 pomelo peel (this work)

37.08 22.0 10.72 20.8 26.57 16.50

11.04 11.1 3.88 17.2 5.59 6.86

23.02 25.0 22.88 14.2 8.53 35.42

7.52 2.19 1.91 8.9 11.56 3.16

9.57 22.9 29.66 21.6 8.02 12.62

2.56 3.73 3.52 3.0 8.09 4.14

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study aims to provide a practical approach to utilize citrus wastes as biomass source for ethanol production. In previous studies, citrus wastes from orange (Citrus sinensis),21−25 Kinnow mandarins (Citrus reticulata),26−28 and grapefruit (Citrus paradisi)29 have been successfully used to produce ethanol via enzymatic hydrolysis and fermentation. As we know, the technological package (e.g., pretreatment, enzymatic hydrolysis, fermentation) utilized in ethanol production is generally specific to the feedstocks, due to the significant structural differences among various biomass materials. The technology, together with the properties of the feedstock (e.g., degradability, availability, locality, price), determine the economic feasibility of ethanol production. Pomelo (Citrus maxima), a characteristic fruit of China, has a large annual production of >2 million tons (pomelo and grapefruit production are estimated at 3.6 million tons in 2011 from FAOSTAT, with China being the largest producer). Pomelo has more peel and segment membrane than most other citrus fruits, generating a significant quantity of pomelo waste in China. However, to date, no report has discussed the utilization of these wastes for ethanol production. Pomelo peel has a higher pectin content than other citrus wastes (see Table 1). Although pectin can be easily hydrolyzed, the high pectin content could still limit the hydrolysis of cellulose and hemicellulose because it acts as a physical barrier to restrict the access of enzymes to the substrate. 30 Furthermore, pectin can increase the viscosity of substrate slurry and limit mass transfer during hydrolysis and fermentation. The lower sugar content (∼36%, including cellulose, hemicellulose, and soluble sugars) in pomelo peel as compared to other citrus wastes could also result in a lower final ethanol concentration. Therefore, to focus on using pomelo peel as the feedstock for ethanol production, it is essential to develop efficient and economic approaches to improve the hydrolysis efficiency and increase the final ethanol concentration. Herein, we report a green and economical way to deal with pomelo peel waste and utilize it for ethanol production via a simultaneous saccharification and fermentation (SSF) process. An integrated strategy, incorporating hydrothermal treatment, multienzyme formulation, and fed-batch operation, was proposed to enhance ethanol production. Specifically, to reduce the enzyme dosage in the process, we introduced a simple hydrothermal treatment at a high temperature (120 °C) before performing SSF. Multienzyme formulation and fed-batch operation were further employed to improve the hydrolysis efficiency and increase the final ethanol concentration. In addition, the effects of the initial D-limonene concentration, pH value, and dry yeast concentration on ethanol production were also investigated.

MATERIALS AND METHODS

Chemicals, Enzymes, and Yeast. Glucose (>99.5%), cellobiose (>99%), sucrose (>99.5%), fructose (>99%), xylose (>99%), galactose (>99%), arabinose (>99%), rhamnose (>99%), and galacturonic acid (>97%) were purchased from Sigma. D-Limonene (>97%) was obtained from Aladdin Industrial Corp. (Shanghai, China). Cellulase (GC220, 123 FPU mL−1) was purchased from Genencor International (Palo Alto, CA, USA) in 2009. Pectinase (Multifect pectinase, 597 U mL−1) was graciously provided by Genencor International in 2009. Commercial ethanol instant active dry yeast powder (Saccharomyces cerevisiae, yeast cells concentration ∼ 20 billion cells/g, water content ∼ 6%) was purchased from Angel Yeast Co., Ltd. (Wuhan, China). All of the other chemicals, such as ethanol, sulfuric acid, hydrochloric acid, and oxalate ammonium, were of analytical grade and were obtained from commercial sources. Sample Preparation. Mature yellow pomelos (C. maxima) were collected from a local fruit market in Tianjin, China. Pomelo peels were manually separated and washed with deionized water to remove any extraneous matter. Then the peels were cut into small pieces (∼1 cm) and dried in a hot-air oven at 70 °C to a constant weight. The dried pomelo peels were ground to a fine powder (500−600 μm) using an electric mill and stored in an enclosed container for subsequent use. Hydrothermal Treatment. To facilitate the operation of pretreatment and subsequent enzymatic hydrolysis or SSF, as well as to enhance the break of biomass structure, sodium acetate buffer (50 mM, pH 4.8) was chosen as acidic medium to pretreat pomelo peels. In a typical experiment, 10 g of dried pomelo peels was placed in a screw-capped laboratory bottle (Pyrex bottles) and mixed with sodium acetate buffer (50 mM, pH 4.8) solution at a final concentration of 10% (w/v). The solid/liquid slurry was then put into a stainless steel autoclave at 120 °C for 15 min. After cooling, the treated pomelo peels were directly used for subsequent enzymatic hydrolysis or SSF processing. To prepare the sample for compositional analysis, the solid residue after hydrothermal treatment was separated by filtering, washed with deionized water, and dried in a hot-air oven at 70 °C to a constant weight. Enzymatic Hydrolysis by Multienzyme Complex. The pomelo peels were first treated under different solid loadings, 5, 7, 10, and 14% (w/v), according to the procedure mentioned above. After hydrothermal treatment, two commercial enzymes, GC220 and Multifect pectinase, were added into the resulting pomelo peel samples over loading ranges of 0−4.6 FPU g−1 dry matter (DM) and 0−25 U g−1 DM, respectively. Digestive reactions were carried out in an air-bath shaker at 150 rpm and 50 °C for the appropriate time. Five hundred microliter aliquots were taken out at different time points for highperformance liquid chromatography (HPLC) analysis. To investigate the effect of hydrothermal treatment on sugar release, raw pomelo peels without pretreatment were used as a control sample for enzymatic hydrolysis. Ethanol Production via SSF Process. All of the SSF experiments with a loading volume of 30 mL were carried out in 100 mL glass bottles. The pomelo peels was mixed with sodium acetate buffer (50 mM, pH 4.8) containing 1 g L−1 (NH4)2HPO4, 1 g L−1 MgSO4·7H2O, and 2.0 g L−1 yeast extract at the desired initial DM concentrations (5−14%, w/v, based on the dry weight of raw pomelo peels). After hydrothermal treatment, GC220 (3.8 FPU g−1 DM) and Multifect pectinase (15 U g−1 DM) were added into the treated pomelo peel samples and then incubated at 50 °C for 10 h with continuous shaking 4644

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at 150 rpm. After prehydrolysis, the temperature was reduced to 30 °C, and the pH of the medium was controlled at 5.0. The initial pH of the prehydrolyzed medium was between 4.0 and 4.3, and sodium hydroxide was added to the mixture to adjust the pH. The SSF experiments were started by adding 2−6 g L−1 dry yeast at 30 °C and 150 rpm. The time of yeast addition was referred to as time zero. The experiments were run for another 48 h. Five hundred microliter aliquots were taken out at different time points for HPLC analysis (e.g., ethanol, sugars, galacturonic acid). After SSF processing, the residue solid was washed, filtered, dried, and weighed. Ethanol volumetric productivity (grams per liter per hour, g L−1 h−1) was calculated as the ratio of the final ethanol concentration (g L−1) to the fermentation time (h). The fed-batch SSF experiments were started with an initial 10% solid loading. Another 10 or 15% dry matter was added during the SSF process, giving a final solid loading of 20 or 25%. The final solid loading of 20% is achieved by adding 0.6 g (3%, w/v), 0.6 g (3%), 0.6 g (3%), and 0.2 g (1%) of fresh pretreated pomelo peel at 8, 18, 24, and 30 h, respectively. The final solid loading of 25% is achieved by adding 0.6 g (3%, w/v), 0.6 g (3%), 0.6 g (3%), 0.5 g (2.5%) 0.5 g (2.5%), and 0.2 g (1%) fresh pretreated pomelo peel at 8, 18, 24, 30 , 36, and 42 h, respectively. The fermentation conditions, enzyme loadings, and nutrient concentrations were the same as in the batch experiments. Analytical Methods. The ethanol-soluble fraction was extracted by using repeated extractions of pomelo peels at 10% solid-to-liquid ratio using 80% ethanol at 80 °C. All of the filtrate was collected and centrifuged at 13000 rpm for 10 min and then analyzed for sugars by using HPLC. The contents of cellulose, hemicellulose, lignin, and ash in pomelo peels were determined in accordance with the standardized methods of the National Renewable Energy Laboratory (NREL, Golden, CO, USA).31 Pectin content was determined by sequential extraction of water (60 °C, 2 h, two times), 0.5% oxalate ammonium (60 °C, 2 h, two times), and 0.05 M HCl (80 °C, 1 h).32 The extracts were recovered by filtration and precipitated by ethanol with 4 times volume, respectively. The resulting pectin were then dried and weighed. D-Limonene content in pomelo peels was estimated using the Scott oil method.33 Sugars, such as glucose, xylose, galactose, and arabinose, were quantified by a HPLC system (Agilent, USA) using a refractive index detector and Aminex HPX-87P column (Bio-Rad, Hercules, CA, USA) at 85 °C with ultrapure water as the mobile phase at a flow rate of 0.8 mL min−1. Peaks were detected and quantified on the basis of the area and retention time of the sugar standards (e.g., sucrose, glucose, fructose, arabinose, galactose, xylose, and rhamnose). Ethanol and galacturonic acid were determined by HPLC equipped with a refractive index detector on an Aminex HPX-87H column (Bio-Rad) at 65 °C. The mobile phase was 0.005 mol L−1 sulfuric acid, and the flow rate was 0.6 mL min−1. All of the samples were centrifuged and filtered through 0.45 μm membranes. The activities of GC220 and Multifect pectinase were determined on the basis of the different substrates because both of them contain many different enzymes. Cellulase and β-glucosidase activities of enzymes were determined using filter paper and cellobiose as substrate, respectively, according to the methods suggested by Ghose.34 Xylanase and pectinase activities (U mL−1, 1 U = 1 μmol min−1 of substrate converted) of enzymes were assayed using birchwood xylan and polygalacturonic acid as substrate, respectively, as previously described.35,36 The protein content in enzyme solution was measured using Bradford’s method with bovine serum albumin as the standard.37 Each datum was represented as the mean of three replicate analyses. Especially, cellulase activity (FPU mL−1) and βglucosidase activity (CBU mL−1) were given by34

cellulase activity =

β‐glucosidase activity 0.0926 = CBU mL−1 [enzyme] releasing 1.0 mg of glucose

where [enzyme] represents the proportion of original enzyme solution present in the directly tested enzyme dilution.

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RESULTS AND DISCUSSION Chemical Composition of Pomelo Peel. We have determined the chemical composition of raw pomelo peel and its compositional changes after hydrothermal treatment. As shown in Figure 1, raw pomelo peel consists of cellulose

Figure 1. Compositional change of pomelo peel after hydrothermal treatment. Treatment conditions: solid loading 10% (w/v), 50 mM sodium acetate buffer (pH 4.8), 120 °C, 15 min.

(16.5%, based on dry matter), hemicellulose (6.86%), pectin (35.42%), lignin (3.16%), soluble sugars (12.62%), and ash (4.14%). Especially, the soluble sugars in ethanol extract are mainly composed of glucose (5.6% of dry matter) and fructose (6.9%). After treatment, the overall weight loss of solid components is 29.88%. Compositional analysis shows that the pectin content decreased to 31.65%, indicating the partial hydrolysis of pectin during the treatment. On the basis of the change in soluble sugars contents (12.62 vs 13.71%, Figure 1) and overall weight loss of solid components, we found that 23.8% of soluble sugars were released from solid fractions to liquid phase. In addition, cellulose and hemicellulose, generally, are difficult to be degraded during the treatment process (120 °C, 15 min). Their content in residual solid increased to 23.14 and 8.27% after treatment, respectively, remaining 98.2 and 84.4% of their initial weight in raw materials. In general, no significant change in chemical composition of pomelo peel was found during the hydrothermal treatment. Enzymatic Hydrolysis of Pomelo Peel via Multienzyme Complex. In this study, two commercial enzymes, namely, GC220 and Multifect pectinase, were used to hydrolyze the pomelo peel waste. Protein concentrations and specific activities of the enzymes are presented in Table 2. GC220 has a high cellulase activity of 123 FPU mL−1, and Multifect pectinase has a high pectinase activity of 597.4 U mL−1. Both of them have β-glucosidase and xylanase activities, which are sufficient to degrade cellobiose and xylan, making the addition of β-glucosidase and xylanase for this hydrolysis process unnecessary. When the cellulase and pectinase loadings

0.37 [enzyme] releasing 2.0 mg of glucose FPU mL−1

(2)

(1) 4645

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Table 2. Protein Concentrations and Specific Activities of Enzymes enzyme

protein concn (mg mL−1)

cellulase (FPU mL−1)

β-glucosidase (CBU mL−1)

xylanase (U mL−1)

pectinase (U mL−1)

GC220 Multifect pectinase

64.9 23.8

123 0.1

74.5 232.1

908.6 207.1

0.1 597.4

Figure 2. (a, b) Effect of cellulase loading on the release of fermentable sugars and galacturonic acid from raw pomelo peel (a) and treated pomelo peel (b) at 10% (w/v) solid concentration. Other default conditions unless otherwise noted: pectinase 20 U g−1, pH 4.8, 50 °C, 150 rpm, 24 h. (c, d) Effect of pectinase loading on the release of fermentable sugars and galacturonic acid from raw pomelo peel (c) and treated pomelo peel (d) at 10% (w/v) solid concentration. Other default conditions unless otherwise noted: cellulase 3.8 FPU g−1, pH 4.8, 50 °C, 150 rpm, 24 h.

were 3.8 FPU g−1 and 15 U g−1, respectively, the hydrolysate after 24 h of reaction at 5% solid loading contained glucose (13.7 g L−1), fructose (5.9 g L−1), galactose (2.6 g L−1), rhamnose (1.0 g L−1), xylose (1.0 g L−1), arabinose (1.3 g L−1), and galacturonic acid (10.8 g L−1). To calculate the sugars content for ethanol production, we defined the sum of glucose, fructose, galactose, and rhamnose as fermentable sugars, which can be transformed into ethanol by S. cerevisiae. Figure 2 shows the effect of cellulase and pectinase loading on the release of fermentable sugars and galacturonic acid from pomelo peel at a 10% solid concentration. When the pectinase loading was 20 U g−1, the concentrations of fermentable sugars increased with increasing cellulase loading for both untreated and treated pomelo peel (Figure 2a,b). The sugars concentration was measured at about 15 g L−1 only by using pectinase alone (zero points in Figure 2a,b). The increased fermentable sugars after cellulase addition are mainly attributed to the release of glucose from cellulose chains on the basis of the HPLC results (data not shown). In the case of treated pomelo peel, 3.8 FPU g−1 cellulase loading can produce a high sugar concentration (∼36 g L−1), and this loading is lower than that for raw pomelo peel (∼7 FPU g−1) to produce the same sugar

concentration. In addition, the treated pomelo peel has a higher galacturonic acid concentration than raw pomelo peel (23 vs 15 g L−1) at a low cellulase loading (e.g., 1 FPU g−1). Generally, galacturonic acid is localized in pectic fractions of citrus peels.38 Higher galacturonic acid concentration suggests that hydrothermal treatment together with cellulose degradation may allow pectinase to become more accessible to pectin and thus improve its hydrolysis. The changes in the concentration of fermentative sugars and galacturonic acid with increasing pectinase loading are shown in Figure 2c,d, and the cellulase loading was kept at 3.8 FPU g−1. When cellulase was used alone, the sugars concentration was about 14 g L−1 (zero points in Figure 2c,d), which mainly resulted from cellulose and hemicellulose. As the pectinase loading was increased to 20 U g−1, the sugar concentration for raw pomelo peel increased to about 35 g L−1 (Figure 2c). It is noted that the same concentration can be reached using a lower enzyme loading of 10 U g−1 for pretreated pomelo peel sample (Figure 2d). The higher sugar concentration after pectinase addition is attributed to the release of glucose and galactose in pectin-branched chains, as well as the enhanced hydrolysis of cellulose and hemicellulose. On the other hand, the 4646

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cellulase and 15 U g−1 pectinase loadings; the temperature was then reduced to 30 °C, and the yeast was added to initiate SSF process. Previous studies had demonstrated that D-limonene in citrus wastes was toxic to yeast cells and thus reduced the alcoholic fermentation ability.22 Therefore, citrus wastes are often required to remove D-limonene below inhibitory levels for yeasts prior to SSF. For example, steam explosion can be used to remove >90% of the D-limonene in citrus waste because it is highly volatile.22 In this work, the hydrothermally treated pomelo peel contains 0.045% (v/w, of dry matter) D-limonene, which could lead to a maximum concentration of 0.0045% during the SSF process at a 10% solid loading. To determine the inhibition threshold amount of D-limonene to S. cerevisiae, we investigated the effect of initial concentration of D-limonene on ethanol production. The commercial D-limonene (97%) was added to pomelo peel at the beginning of SSF to achieve different D-limonene concentrations, ranging from 0.01 to 0.15% (v/v). As shown in Figure 4a, ethanol concentrations in fermentations decreased for initial D-limonene concentrations >0.06% (v/v). Because the D-limonene concentration in pomelo peel slurry (0.0045%) is much lower the critical value (0.06%), additional removal of D-limonene is not required in further study. To obtain high alcoholic fermentation ability, two key operating parameters, the initial pH value and the yeast concentration, were optimized in this study. As shown in Figure 4b, the initial pH had a significant effect on both ethanol and galacturonic acid concentrations. Alcoholic fermentation at an initial pH of 4.5 produced the least ethanol. In this case, the final pH at the end of fermentation was 4.2. The result indicated that pH 4.2−4.5 greatly repressed yeast vitality and thus reduced the ethanol production. When the initial pH increased to 5.0 or 5.5, the final pH values in fermentation broth were 4.71 and 5.05, respectively. In both cases, we could obtain high ethanol concentrations within the pH range of 4.7− 5.5. However, further increase of initial pH to ≥6.0 could reduce the ethanol yield. Meanwhile, we found that the galacturonic acid concentrations decreased as the pH increased, because pectinase is more effective at a lower pH (e.g., pH 4.5). Therefore, the initial pH of 5.0 is ideal for both S. cerevisiae and pectinase and was used in the following study. A series of SSF experiments were further carried out using different dry yeast inoculations to determine the most appropriate yeast concentration. As shown in Figure 4c, when the yeast concentration was 2 g L−1, 9.83 g L−1 ethanol was produced after 36 h of SSF process using 3.8 FPU g−1 cellulase and 15 U g−1 pectinase loadings. Final ethanol levels increased with increasing yeast concentration up to 5 g L −1. When using 5 g L−1 yeast, we obtained 17.3 g L−1 ethanol product after 36 h of SSF. Nevertheless, as the yeast amount reached 6 g L−1, the production rate and concentration of ethanol decreased. This finding can be explained by increased sugar consumption for cell growth under an oxygen circumstance and a large amount of seed. During the SSF process, glucose, fructose, and galactose can be fermented to ethanol by S. cerevisiae cells (no sucrose was detected in the samples). The glucose consumption rate is higher than that of fructose and galactose.26 The maximum concentration of fermentable sugars was 38.4 g L−1 at a 10% solid loading. A theoretical ethanol concentration of 19.6 g L−1 could be obtained if all fermentable sugars were fermented to ethanol. Using the optimized parameters as described before,

concentration of galacturonic acid was directly correlated with pectinase loading. For raw and pretreated pomelo peel, the saturated pectinase dosages were 20 and 15 U g−1 respectively. These results indicate that the application of hydrothermal treatment could significantly reduce the use of cellulase (from 7 to 3.8 FPU g−1) and pectinase (from 20 to 10 U g−1). This treatment process, combined with a multienzyme complex, could further improve the hydrolysis of pectin and cellulose, leading to high concentrations of fermentative sugars (from 14 to 36 g L−1) and galacturonic acid (from 9 to 23 g L−1). Enzymatic Hydrolysis of Pomelo Peel at High Solid Loadings. High concentrations of fermentable sugars are often required to obtain high ethanol concentrations. A promising approach to increase sugar concentrations involves the operation of enzymatic hydrolysis at high solid loadings. For pomelo peel, when the solid loading exceeded 14%, almost all of the buffer solution was absorbed. The high viscosity will prevent efficient mixing and mass transfer, resulting in more power consumption, poor solid and enzyme distribution, and localized product buildup. In this work, we investigated the enzymatic hydrolysis of pretreated pomelo peel over the solid loading range of 5−14%. As shown in Figure 3, the final

Figure 3. Time course of the release of fermentable sugars from pretreated pomelo peel at different solid loadings. Conditions: cellulase 3.8 FPU g−1, pectinase 15 U g−1, pH 4.8, 50 °C, 150 rpm.

concentrations of fermentable sugar obtained after 24 h of hydrolysis could reach 19, 28, 36, and 44 g L−1 at solid loadings of 5, 7, 10, and 14%, respectively. The hydrolysis rates were notably higher within the first 12 h of hydrolysis than at the later stage. The enzymatic hydrolysis could be nearly finished within 12 h when the solid loading was 30 h was necessary for the entire hydrolysis reaction. The results suggest that high solid loading (e.g., 14%) could cause significant mass transfer limitation, leading to decreased hydrolysis efficiency. Therefore, in this case, a solid loading of 10% is appropriate for enzymatic hydrolysis of pomelo peel. Ethanol Production from Pomelo Peel via a SSF Process. SSF is a promising process for ethanol production from lignocellulosic biomass due to its reduced catabolite repression, simplicity of operation, low capital investment, and improved process economics. In this study, we therefore employed SSF to produce ethanol using pectin-rich pomelo peel as substrate. Prior to the SSF process, a prehydrolysis operation was performed at 50 °C for 10 h with 3.8 FPU g−1 4647

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Figure 4. (a−c) Effects of initial D-limonene concentrations (a), pH values (b), and yeast concentrations (c) on ethanol production in the SSF process. (d) Time course of ethanol production and sugars consumption in the SSF process. Default conditions unless otherwise noted: (1) prehydrolysis conditions: cellulase 3.8 FPU g−1, pectinase 15 U g−1, pH 4.8, 50 °C, 150 rpm, 10 h; (2) SSF conditions: solid loading 10%, yeast 5 g L−1, pH 5.0, 30 °C, 150 rpm, 48 h.

Figure 5. Mass flow for key components of pomelo peels during the hydrothermal pretreatment and SSF process. Pretreatment conditions: solid loading 10% (w/v), 50 mM sodium acetate buffer (pH 4.8), 120 °C, 15 min. SSF conditions: (1) prehydrolysis: 10% solid loading, cellulase 3.8 FPU g−1, pectinase 15 U g−1, pH 4.8, 50 °C, 150 rpm, 10 h; (2) SSF: yeast 5 g L−1, pH 5.0, 30 °C, 150 rpm, 48 h.

17.35 g L−1 ethanol was obtained after 48 h of SSF (Figure 4d), accounting for 88.4% of the theoretical yield. Within the first 16 h, >90% of the total ethanol could be produced (the volumetric ethanol productivity is 0.98 g L−1 h−1), which suggests that yeast cells may have utilized most of the sugars at this stage and then switched into stationary mode due to nutrient exhaustion and changes in environmental conditions in the medium. Figure 5 shows the mass flow for key components of pomelo peels during the hydrothermal pretreatment and SSF process. As shown in Figure 5, the main products are ethanol (17.35 g,

equivalent to 34.02 fermentable sugars) and galacturonic acid (22.1 g), indicating that approximately 80.5% of the original components (cellulose, pectin, and soluble sugars) was transferred to product. Ethanol Production via SSF with Batch and Fed-Batch Operation. For ethanol production, an important objective is to increase the final ethanol concentration, thus reducing the costs of downstream processing. Generally, operating SSF at high solid loadings is a promising way to increase ethanol concentrations. In this work, we investigated the ethanol 4648

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35−38 g L−1, 0.42−0.8 g L−1 h−142). In this study, SSF with batch operation could obtain higher volumetric ethanol productivity (0.98 g L−1 h−1) compared with the SHF process. However, batch SSF produces only 17.35 g L−1 ethanol due to the low sugar content in pomelo peel. Other citrus wastes and the mixed fruit wastes have higher sugar content and lower pectin content (Table 1); therefore, simple batch SSF could be used for ethanol production, achieving high ethanol concentration and high ethanol productivity (e.g., Kinnow waste and banana peel, 26.84 g L−1, 0.56 g L−1 h−1;43 citrus peel, 38.68 g L−1, 1.61 g L−1 h−1;22 Kinnow mandarin peel, 33.87 g L−1, 2.82 g L−1 h−1;27 Kinnow waste, 42 g L−1, 3.5 g L−1 h−126). For pomelo peel, however, it is difficult to obtain high ethanol concentration in the batch SSF process, as mentioned before, due to the high viscosity at high solid loading. Here, we proposed fed-batch SSF strategy, combined with hydrothermal treatment and multienzyme formulation, to enhance the ethanol production, obtaining 36 g L−1 ethanol product as close to that from other citrus peels. Additionally, the fed-batch SSF had an ethanol productivity of 0.75 g L−1 h−1 within 48 h, which is higher than that in batch-SSF with 14 wt % solid loading (0.43 g L−1 h−1, within 48 h). In this study, S. cerevisiae, a robust yeast strain, was used to produce ethanol from pomelo peel. It presents many advantages, including high ethanol tolerance and favorable performance at relatively low pH and oxygen levels. However, S. cerevisiae has no ability to naturally ferment galacturonic acid and pentose sugars.44 Citrus wastes, such as pomelo peel in this work, contain significantly large amounts of galacturonic acid. If galacturonic acid can be converted to ethanol, it would further increase the final ethanol concentration and thus increase the feasibility of these feedstocks for large-scale ethanol production. Whereas engineering S. cerevisiae to utilize galacturonic acid is a solution, it is still currently a challenge to develop efficiently engineered S. cerevisiae for ethanol production from galacturonic acid.45 However, successful attempts have been made to ferment galacturonic acid to ethanol using recombinant bacteria, such as Escherichia coli KO11.19,46 Therefore, these strains can be employed to utilize the galacturonic acid in the residual fermentation broth after SSF process. In summary, we have developed an integrated strategy, incorporating hydrothermal treatment, multienzyme formulation, and fed-batch operation, to enhance ethanol production from pomelo peel. Hydrothermal treatment helped in partial hydrolysis of pectin and helped in reducing the use of cellulase and pectinase. Multienzyme complex was also proven to be effective in improving the hydrolysis of pretreated pomelo peel to fermentative sugars and galacturonic acid. Moreover, to overcome the drawback of low ethanol concentration, we employed fed-batch SFF to produce ethanol from pomelo peel, achieving 36 g L−1 ethanol product in good yield (73.5%) and ethanol productivity (0.75 g L −1 h −1 ). The strategy demonstrated in our work, combined with subsequent galacturonic acid conversion, could open a new avenue for utilization of the abundant pectin-rich biomass available to produce ethanol.

production with batch operation at different solid loadings. As described before, the maximum solid loading for batch operation is 14% due to the high viscosity of pomelo peel slurry. The ethanol concentrations and yields at the solid loadings of 5−14% are shown in Figure 6. When the initial solid

Figure 6. Final ethanol concentrations and yields in the batch (5−14% solid loading) and fed-batch SSF process. Final solid loadings of 20 and 25% were achieved by adding another 10 and 15% to the SSF system with an initial solid loading of 10%, as described under Materials and Methods. Other default conditions unless otherwise noted: (1) prehydrolysis: cellulase 3.8 FPU g−1, pectinase 15 U g−1, pH 4.8, 50 °C, 150 rpm, 10 h; (2) SSF: yeast 5 g L−1, pH 5.0, 30 °C, 150 rpm, 48 h.

concentration is 5% (w/v), the ethanol concentration reached 9.24 g L−1, being 94.2% of the theoretical concentration. At the highest solid loading (14%), we can obtain 20.8 g L−1 ethanol product. However, the ethanol yield decreased significantly from 94.2 to 75.7% with increasing solid loadings. The yields would further decrease if the solid loading was increased under batch operation. The worsened performance of yeast and enzymes should be mainly attributed to the lowered mass transfer efficiency (high viscosity). Additionally, the accumulation of yeast inhibitors, such as ethanol,39,40 and D-limonene (Figure 4a),22 at high solid loading could also limit the ethanol production. To further increase the final ethanol concentration while avoiding an increase in viscosity, fed-batch operation by adding fresh substrate was used in the SSF process. As shown in Figure 6, on the basis of the initial solid concentration of 10%, an additional 10% of fresh substrate was added during the first 30 h. The final ethanol concentrations after 48 h increased to 30.45 g L−1, which is nearly 2 times higher than that at 10% solid loading (17.35 g L−1, Figure 6). The concentration could further increase to 36.06 g L−1 (the volumetric ethanol productivity is 0.75 g L−1 h−1) at a 25% final solid loading via fed-batch operation. In these cases, the yields can reached 77.6 and 73.5%, respectively, which is close to that at 14% solid loading and much higher than the predicted value (at 25% solid loading) under batch operation. Our results demonstrated that use of the fed-batch mode could alleviate the technical problem, as mentioned before, which is caused by high loading of pomelo peel in the SSF process. Previous studies had employed separate hydrolysis and fermentation (SHF) to produce ethanol from citrus wastes (e.g., orange peel41,42) hydrolysate. Although SHF could achieve high ethanol concentration, it has low volumetric ethanol productivity (e.g., 37−40 g L−1, 0.50−0.55 g L−1 h−1;41

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*(R.S.) E-mail: [email protected]. Phone: +86 22 27407799. Fax: +86 22 27407599. Author Contributions ∥

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R.H. and M.C. contributed equally to this work. dx.doi.org/10.1021/jf405172a | J. Agric. Food Chem. 2014, 62, 4643−4651

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Funding

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We acknowledge the financial supports received from the National Natural Science Foundation of China (No. 21276192), the Ministry of Science and Technology of China (Nos. 2012BAD29B05 and 2013AA102204), Open Funding Project of the State Key Laboratory of Chemical Engineering (No. SKL-ChE-11B01), and the Ministry of Education (Nos. NCET-11-0372, 20110032130004, and B06006). Notes

The authors declare no competing financial interest.

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