A low-energy, cost-effective approach to fruit and citrus peel wasteprocessing for bioethanol production

In Seong Choi a, Yoon Gyo Lee a, Sarmir Kumar Khanal b, Bok Jae Park c, Hyeun-Jong Bae a,d,aDepartment of Wood Science and Landscape Architecture, Chonnam National University, Gwangju 500-757, Republic of KoreabDepartment of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI 96822, United StatescDivision of Business and Commerce, Chonnam National University, Yeosu 550-749, Republic of KoreadDepartment of Bioenergy Science and Technology, Chonnam National University, Gwangju 500-757, Republic of Korea

h i g h l i g h t s

! Simple bioprocess of bioethanol production from fruit wastes containing D-limonene.! Two in-house enzymatic bioconversion rates were approximately 90%.! Limonene recovery column (LRC) was designed for absorption of D-limonene.! Ethanol production by immobilized yeast fermentation and LRC was 12-fold greater.

a r t i c l e i n f o

Article history:Received 22 March 2014Received in revised form 17 November 2014Accepted 29 November 2014Available online 13 December 2014

Keywords:Citrus peel wasteBio ethanolEnzymatic hydrolysisD-Limonene extractContinuous immobilized yeast fermentation

a b s t r a c t

Large quantities of fruit waste are generated from agricultural processes worldwide. This waste is oftensimply dumped into landfills or the ocean. Fruit waste has high levels of sugars, including sucrose, glu-cose, and fructose, that can be fermented for bioethanol production. However, some fruit wastes, such ascitrus peel waste (CPW), contain compounds that can inhibit fermentation and should be removed forefficient bioethanol production. We developed a novel approach for converting single-source CPW (i.e.,orange, mandarin, grapefruit, lemon, or lime) or CPW in combination with other fruit waste (i.e., bananapeel, apple pomace, and pear waste) to produce bioethanol. Two in-house enzymes were produced fromAvicel and CPW and were tested with fruit waste at 1215% (w/v) solid loading. The rates of enzymaticconversion of fruit waste to fermentable sugars were approximately 90% for all feedstocks after 48 h. Wealso designed a D-limonene removal column (LRC) that successfully removed this inhibitor from the fruitwaste. When the LRC was coupled with an immobilized cell reactor (ICR), yeast fermentation resulted inethanol concentrations (14.429.5 g/L) and yields (90.293.1%) that were 12-fold greater than productsfrom ICR fermentation alone.

! 2014 Elsevier Ltd. All rights reserved.

1. Introduction

The world consumed approximately 89 million barrels of crudeoil per day in 2013. Consumption of liquid fuels (mainly petro-leum) is expected to increase to 115 million barrels per day by2040, which is a 63% overall increase in total liquid fuel consumed.The consumption of liquid fuel by the transportation sector willincrease by 57% by 2040 [1]. The transportation sector is a sourceof emissions of carbon dioxide (CO2) and other greenhouse gases

(GHG) such as nitrogen oxide (NOx) and sulfur oxide (SOx). Biofuelsare and alternative energy source that reduce the production ofpollution gases [2]. The production of nonpetroleum liquid fuels,such as biofuels, from food crops is not sustainable due to compe-tition for materials and high production costs. Therefore, cheap andabundant nonfood materials are required as alternative biomassfeedstocks (e.g., agricultural byproducts, woody biomass, or energycrops) and processes must be developed that can efficiently andeconomically convert these types of lignocellulosic and cellulosicbiomass into biofuels, such as bioethanol [3].

Fruit waste is generated in large quantities from the processingof agricultural products. Examples of such waste include citrus,banana, apple, and pear residues remaining after industrial pro-cessing. Citrus, which includes oranges, grapefruits, lemons, limes,

http://dx.doi.org/10.1016/j.apenergy.2014.11.0700306-2619/! 2014 Elsevier Ltd. All rights reserved.

Corresponding author at: Department of Bioenergy Science and Technology,Chonnam National University, Gwangju 500-757, Republic of Korea. Tel.: +82 62530 2097; fax: +82 62 530 0029.

E-mail address: baehj@chonnam.ac.kr (H.-J. Bae).

Applied Energy 140 (2015) 6574

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mandarins, are the most abundant crops in the world. Over 115million tons of citrus fruits are produced annually, and about 30million tons are processed industrially for juice production. Afterindustrial processing, citrus peel waste (CPW) accounts for almost50% of the wet fruit mass. The annual production of bananas,apples, and pears are approximately 107.1, 75.5, and 24.0 milliontons, respectively, and 2540% of this mass remains as waste afterprocessing (Fig. 1A) [4]. Fruit waste serves as cattle feed, butbecause of its low protein content, it is not a high-value feedstock,and much of it is dumped into landfills or disposed of in the ocean.Because fruit waste is rich in sugars and other nutrients, theseforms of disposal may cause environmental problems. Disposal ofwaste is also becoming increasingly expensive. For example, Euro-pean Union (EU) landfill directives have caused landfill gate fees toincrease in some cases because of land limitations and transportand labor costs [5]. In America, the annual cost of apple pomacedisposal alone is $10 million USD [6]. Fruit waste is rich in ferment-able soluble sugars such as glucose, fructose, and sucrose alongwith structural cellulose and hemicellulose. These chemical con-stituents, along with the fact that fruit waste is in abundant supply,suggest that fruit waste may be an excellent source of waste bio-mass for ethanol production.

However, among the variety of fruit wastes available, CPWrequires additional processing before bioethanol production. This

is because although CPW is rich in various soluble and insolublesugars, making it an ideal feedstock, it also contains a strongmicrobial inhibitor referred to as D-limonene. The production ofD-limonene from citrus peel is economically viable, as this byprod-uct has high added value as a flavoring agent and for variousapplications in the chemical industry. Thus, removing and recover-ing D-limonene prior to the yeast fermentation process serves twopurposes: high-value utilization and enhanced fermentation ofCPW-derived sugars. The efficient removal of D-limonene fromCPW requires a pretreatment step. Most pretreatment methodsare based on thermochemical or thermophysical processes suchas milling or steam explosion as shown in Fig. 1B and Table 1[714]. A major disadvantage of these methods is the elevatedtemperature and prolonged extraction time, which can causechemical modification of the volatile molecules, including D-limonene,as well as loss of sugars for ethanol production. We developed anew technique that uses raw cotton and activated carbon toremove and recover D-limonene, and requireds with less energy.Sorbents should have high oleophilic and hydrophobic properties,and can be classified into three groups based on the materialsource (natural materials, treated cellulose, or petrochemical poly-mers). The most commonly used polymers are petrochemical poly-mers such as polypropylene, polyethylene, and polyurethane.However, these polymers are non-biodegradable materials and

Fig. 1. Citrus and fruit production and schematic representation of bioethanol production processes. (A) Annual production of citrus and major fruit worldwide, (B)traditional processes for citrus peel bioethanol production, and (C) schema of the study process. In most cases, steam explosion pretreatment is convention process to removethe fermentation inhibitor D-limonene. Citrus peel was hydrolyzed by commercial enzymes, including pectinase, cellulase, and b-glucosidase.

66 I.S. Choi et al. / Applied Energy 140 (2015) 6574

can become environmental pollutants. Raw cotton is a naturalmaterial that hydrophobic, with a high sorption capacity, and iseasily biodegradable [15]. Activated carbon possesses a highdegree of micro-porosity for absorption and is commonly usedfor water treatment, detoxification, and separation of componentsin flow systems.

In the enzymatic hydrolysis phase, cellulolytic, xylanolytic, andpectinolytic enzymes are often used to degrade plant cell walls andcatalyze the breakdown of complex carbohydrates into theirmonosaccharide components (i.e., saccharification) [12,13]. Etha-nol production from CPW has largely been conducted using com-mercial enzymes; thus, the cost of cellulosic ethanol is very high.The cost can be drastically reduced if in-house-produced enzymesare used for saccharification [16]. Trichoderma and Aspergillus areamong the most common microorganisms that produce abundantcellulolytic, xylanolytic, and pectinolytic enzymes. Trichodermaspecies have been studied for their cellulolytic enzymes content[17]. In addition, Aspergillus species often have xylanolytic and pec-tinolytic enzymes [18]. Both fungal species are considered extra-cellular producers of cell wall-degrading enzymes that havepotential for important industrial applications.

Following completion of enzymatic hydrolysis, fermentation isnecessary for bioethanol production. Generally, ethanol productionmay occur through separate hydrolysis and fermentation (SHF)processes, or simultaneous saccharification and fermentation(SSF). Continuous fermentation is also considered an efficient fer-mentation process, because it has many advantages, includingthe ability to separate immobilized yeast from the ethanol product,thus allowing the immobilized yeast to be reused for further fer-mentation. In addition, immobilizing the yeast cell wall confershigher ethanol tolerance and cell concentrations, shorter fermenta-tion time, enhanced fermentation productivity, and lower costs ofrecovery and recycling [19].

Considering the above-mentioned limitations of conventionalprocesses, we explored the possibility of directly converting fruitwaste to ethanol without pretreatment (Fig. 1B and C). Thisinvolved developing an efficient enzymatic hydrolytic process, aswell as an effective, low-cost strategy to remove the fermentationinhibitor D-limonene. The utility of this approach was examined byevaluating ethanol production efficiency during continuous fer-mentation with immobilized yeast cells. Furthermore, becausefeedstock flexibility is important for successful commercial ethanolproduction, the feasibility of using CPW alone or in combinationwith other fruit waste was also examined.

2. Materials and methods

2.1. Raw materials

Citrus (orange, mandarin, grapefruit, lemon, and lime), apple,banana, and pear were obtained from a local market (Homeplus,

Gwangju, Korea). Citrus, apple, and pear waste was collected afterjuice extraction (Hurom, Seoul, Korea). Banana waste wasremoved, lyophilized ("50 "C), and stored at "20 "C. CPW and fruitwastes, individually or mixed in equal ratios, waste were used forhydrolysis and fermentation.

2.2. Chemical composition

The content of soluble sugar was analyzed by high performanceliquid chromatography (HPLC) with a refractive index detector(2414, Waters, USA), REZEX RPM (Phenomenex, USA) column(300 # 7.8 mm) at 85 "C and eluted with deionized water at a flowrate of 0.6 mL per min. Insoluble solids were analyzed for neutralsugar content using gas chromatography (GC) [20,21]. Sampleswere hydrolyzed with 72% sulfuric acid for 45 min at room temper-ature and diluted with distilled water to 4% sulfuric acid, followedby autoclaving for 1 h at 121 "C. The neutral sugar composition wasmeasured with alditol acetates containingmyo-inositol as an inter-nal standard. Gas chromatography (GC-2010, Shimadzu, Japan)was used, and the analysis conditions, using a DB-225 capillary col-umn (30 m # 0.25 mm i.d., 0.25 lm film thickness, J&W) operatedwith He, injector temperature of 220 "C, flame ionization detector(FID) at 250 "C, and oven temperature programming, were 100 "Cfor 1.5 min and 5 "C/min to 220 "C.

The D-limonene content was determined according to a previ-ous study [13]. Briefly, CPW was homogenized in 10 mL hexane,which had a known amount of camphor as an internal standard.After treatment for 3 h, 5 mL of supernatant was transferred to atest tube. A quantity of 0.2 mL potassium hydroxide (2 N) wasadded to methanol and mixed for 1 min. After the addition of1 mL distilled water, the samples were shaken and centrifugedfor 5 min at 3000 rpm. The hexane phase was measured by GC(CP-9100, Chrompack), using a CP-Sil 5 CB fused silica capillary col-umn (25 m # 0.32 mm i.d., 1.2 lm film thickness, Chrompack)operated with He, injector temperature 280 "C, FID at 280 "C, andoven temperature programming at 110 "C for 5 min and 20 "C/min to 220 "C, which was then held constant for 10 min.

2.3. In-house enzyme production

Aspergillus citrisporus (KCCM 11449) was obtained from the Kor-ean Culture Center of Microorganisms (KCCM), and Trichodermalongibrachiatum (KCTC 6507) was purchased from the Korean Col-lection for Type Cultures (KCTC). The lyophilized fungi were revi-talized on potato dextrose broth with 1.2% (w/v) agar (PDA) andincubated for spore production for 7 days at 25oC. One hundredof potato dextrose broth (PDB) was sterilized in 500 mL Erlen-meyer flasks.

Two types of carbon sources were used to produce extracellularenzymes for fruit waste hydrolysis. The medium contained either20 g/L MP or Avicel as the carbon source. The other components

Table 1Citrus waste as substrate for bioethanol production.

Substrate Pretreatment Enzymesa Fermentation process Microorganism Ethanol production References

Orange peel Milling Pectinase, cellulase, glucosidase HF S. cerevisiae 4.7c [7]Orange peel Milling Pectinase, cellulase, glucosidase HF Escherichia coli KO11 2.76c [8]Orange peel Steam explosion Pectinase, cellulase, glucosidase SSF S. cerevisiae 3.96c [9]Orange peel Steam explosion Pectinase, cellulase, glucosidase SSF Kluyveromyces marxianus 3.45c [10]Orange peel Acidic steam explosion Pectinase, cellulase, glucosidase SSF S. cerevisiae 2.7c [11]Mandarin peel Steam explosion Pectinase, cellulase, glucosidase SSF S. cerevisiae 59.3d [12]Mandarin peel Popping Pectinase, xylanase, glucosidase SHEFb S. cerevisiae 46.2d [13]Lemon peel Steam explosion Pectinase, cellulase, glucosidase SSF S. cerevisiae 67.8d [14]

a Commercial enzymes used for hydrolysis.b SHEF, separate hydrolysis and fermentation with vacuum evaporation.c Ethanol yields presented in%, w/v.d Ethanol concentration expressed in g ethanol per g of 1000 kg of fresh substrate.

I.S. Choi et al. / Applied Energy 140 (2015) 6574 67

were similar for both media (in g/L): 40, peptone; 24, KH2PO4; 5,(NH4)2SO4; 4.7, K2C4H4O6$4H2O; 2, urea; 1.2, MgSO4$7H2O and (inmg/L) 10; ZnSO4$7H2O, 9.3; MnSO4$7H2O, 8.7; CuSO4$7H2O with1 mL Tween 80. The pH was adjusted to 5.0, using hydrochloricacid. The medium was sterilized at 121 "C for 15 min. Cultureswere conducted in a 10 L fermenter (Fermentec, Korea) equippedwith a 7 L working volume for 7 d. The culture broth was centri-fuged and the supernatant was stored at 4 "C.

2.4. In-house enzyme activity and hydrolysis

Two enzymes, produced in-house from A. citrisporus (In-houseenzyme A [HEA]) and T. longibrachiatum (In-house enzyme B[HEB]), were evaluated. The protein concentration was measuredusing the Lowry method, with bovine serum albumin (BSA) as aprotein standard [22]. Enzyme activities were assayed with a spe-cific substrate solution consisting of 50 mM citrate phosphate buf-fer, pH 4.8 (at 45 "C for 30 min), and appropriately diluted enzymeconcentrations. Endoglucanase (CMCase) and exoglucanase (Avice-lase) activities were measured with 1% carboxymethylcellulose(CMC, Sigma) and microcrystalline cellulose (Avicel, Sigma) asthe substrates, respectively. Xylanase activity was measured bythe same procedure described for endo- and exo-glucanase, butwith beechwood xylan (Sigma) as the substrate. Pectinase activitywas measured with a 0.5% polygalacturonic acid (Sigma) in 50 mMcitrate phosphate buffer (pH 4.8) at 45 "C for 5 min. Reducing sug-ars were quantified with dinitrosalicylic acid (DNS) at an absor-bance of 540 nm [23]. One unit of activity was defined as theamount of enzyme required to release one lmol of glucose, xylose,or galacturonic acid per min. Specific activities were expressed asenzyme units per milligram of protein.

HEA or HEB enzymes were added to fruit waste at concentra-tions of 1216 and 1025 mg protein/g fruit waste, respectively.Enzymatic hydrolysis was performed on 1% matter (w/v) with cit-rate phosphate buffer (pH 4.8) at 180 rpm for 48 h at 45 "C. Optimi-zation of enzyme loading volume and the influence of biomasshydrolysis time during enzymatic hydrolysis were measured usingHPLC as described in 2.2.

2.5. D-Limonene recovery column design

The D-limonene removal column (LRC) is a tubular apparatus,consisting of an internal diameter (ID) of 1.5 cm and 7.0 cm length.LRC was packed with raw cotton (100300 mg) and activated-car-bon (02 g) to optimize limonene removal. LRC was connected tothe fermentation reactor for D-limonene removal and recoveryfrom the hydrolysate prior to fermentation. The fermentation pro-cess was conducted with- or without LRC on a fermentation reac-tor. After fermentation, D-limonene was recovered from LRC usinghexane, and the recovery rate was determined using GC asdescribed in 2.2.

2.6. Continuous immobilized yeast fermentation

Saccharomyces cerevisiae KCTC 7906 was obtained from theKCTC and activated in 4 mL yeast peptone dextrose media (YPD).The yeast inoculum was placed in a 500 mL Erlenmeyer flask con-taining 100 mL autoclaved YPD media for 24 h at 30 "C.

To prepare for immobilization, 100 mL S. cerevisiae cells wereharvested at the exponential growth phase and mixed with 2%sodium alginate solution prepared by dissolving 8 g sodium algi-nate in 300 mL deionized water [24]. Using a syringe, the alginatedrops were deposited in a 0.1 M CaCl2 solution to produce beads.The beads were stored after washing with deionized water toremove any remnant CaCl2. The 3.8 mm beads were uniformlypacked and stored in deionized water at 4 "C.

The immobilized cell reactor (ICR) was used in continuous fer-mentation of the CPW hydrolysate. ICR consists of a tubular col-umn, constructed with a 2.1 cm ID and 25 cm length. About 70%of the column was packed with immobilized yeast cells. The med-ium was fed into the reactor from the feed stock, and a peristalticpump (EP-1 Econopump, Biorad) was used to transfer the feedmedium. The volumes of the reactor before and after immobilizedyeast cell packing were 80 and 42 mL, respectively. The fresh feedwas pumped in an up-flow manner and the total sugar and ethanolconcentrations were monitored during fermentation. Prior to beingfed into reactor, the pH of the CPW hydrolysates was adjusted to a

Table 2Chemical compositions of fruit waste.

(% Dry matter) Rhamnose Arabinose Xylose Mannose Galactose Sucrose Glucose Fructose FSa Total

OP 2.1 0.0 5.6 0.2 2.2 0.0 2.4 0.1 2.7 0.1 5.6 0.2 35.5 0.5 12.1 0.4 53.2 0.4 68.2 0.5MP 2.9 0.1 3.3 0.1 2.4 0.1 2.3 0.1 3.9 0.1 7.4 0.2 39.4 1.1 10.3 0.8 57.1 0.6 71.9 0.9GP 3.4 0.0 4.8 0.2 2.3 0.1 2.2 0.0 3.5 0.2 1.4 0.1 30.6 0.8 8.2 0.3 43.2 0.7 59.4 0.8LeP 2.1 0.1 5.2 0.3 2.6 0.2 2.1 0.1 4.6 0.1 ND 27.9 0.4 3.3 0.1 31.2 0.4 47.8 0.3LiP 2.5 0.2 8.5 0.4 2.5 0.1 2.0 0.1 4.3 0.1 ND 22.5 1.2 0.7 0.0 23.2 1.0 43.0 0.6AP 1.7 0.1 5.5 0.1 6.2 0.3 2.8 0.1 4.2 0.3 9.2 0.1 25.2 2.8 24.7 0.3 59.1 1.8 79.5 1.5BP 0.6 0.1 4.4 0.3 5.6 0.4 3.6 0.1 2.8 0.1 ND 30.1 0.8 15.2 0.7 45.3 0.3 71.5 0.6PP 1.3 0.1 6.0 0.3 20.2 0.9 2.4 0.0 4.5 0.3 1.9 0.1 21.1 0.6 14.1 0.5 37.1 0.8 62.3 0.7MixP 2.7 0.1 5.6 0.2 2.4 0.2 2.2 0.0 3.8 0.0 2.9 0.1 32.0 0.8 6.8 0.3 41.7 1.1 58.4 0.8TFW 2.0 0.1 5.4 0.4 5.5 0.6 2.5 0.1 3.8 0.2 3.2 0.3 29.0 1.7 11.1 0.9 43.4 1.9 62.5 1.7

Abbreviations used: OP, orange peel; MP, mandarin peel; GP, grapefruit peel; LeP, lemon peel; LiP, lime peel; AP, apple pomace; BP, banana peel; PP, pear peel; MixP, mixedcitrus peel; TFW, mixed total fruit wastes; ND, not detected.Values represent the average of three replicates.

a FS: Fermentable sugars are the sum of sucrose, glucose, and fructose, which are fermented by S. cerevisiae.

Table 3Comparison of specific activities for the in-house enzymes used in the study.

Endoglucanase (U/mg protein) Exoglucanase (U/mg protein) Xylanase (U/mg protein) Pectinase (U/mg protein)

In-house enzyme A (HEA) 8.41 0.11 0.18 0.01 170.95 1.81 17.90 0.43In-house enzyme B (HEB) 13.22 1.21 1.26 0.17 4.34 0.52 1.11 0.21

68 I.S. Choi et al. / Applied Energy 140 (2015) 6574

pH of 4.8 by the addition of CaCO3. The flow rate of feed in thepacked-bed reactor column was 0.08 mL/min. The ICR was main-tained in an incubator at 30 "C, and samples were withdrawnaseptically from the bioreactor periodically during a 10-day per-iod to analyze sugar and ethanol concentrations.

3. Results and discussion

3.1. Fruit waste composition

The carbohydrate composition of the various fruit wastes dif-fered as shown in Table 2. The total carbohydrate contents of thefruit wastes were separated into soluble sugars, which dissolveeasily in water, and insoluble sugars (cellulose and hemicellu-lose) in the cell walls. Although arabinose and xylose were pres-ent, they appeared in low concentrations in the fruit waste. Wemainly focused on fermentable sugars (FS), namely, glucose, fruc-tose, and sucrose. All fruit wastes presented were high in FS con-tent (Table 2). Sucrose and fructose were present as soluble freesugars, whereas, glucose was part of the fruit waste structuralcomponents and present as a free sugar. FS contents in the vari-ous fruit wastes ranged from 23.2% to 59.1%. Orange peel (OP),mandarin peel (MP), grapefruit peel (GP), apple pomace (AP),and banana peel (BP) waste contained 53.2%, 57.1%, 43.2%,59.1%, and 45.3% FS, respectively. Lemon peel (LeP), lime peel(LiP), and pear pomace (PP) showed moderate FS levels of31.2%, 23.2%, and 37.1%, respectively. FS contents in the CPWmixture (MixP) and CPW, in combination with other fruit waste(TFW), were 41.7% and 43.4%, respectively.

3.2. In-house enzyme production and fruit waste hydrolysis

The current cost of pretreatment and enzymes for biomasshydrolysis are major obstacles to large-scale ethanol production[25]. The cost of cellulase is estimated at a minimum of $10/kgprotein [16]. Accordingly, it is necessary to reduce the cost andamount of enzymes required for biomass hydrolysis to industrial-ize the process. Here, we produced a suitable enzyme complex forfruit waste hydrolysis using CPW or Avicel as the carbon source.In addition, we also report on the efficacy of the in-house enzymeactivities and fruit waste hydrolysis.

3.2.1. In-house enzyme activity and effective loading volumes forhydrolysis

Between the two in-house enzymes evaluated (Table 3), HEAexhibited the highest level of xylanase activity. Its pectinaseactivity was moderate, and both exoglucanase and endoglucan-ase activities were observed. The activity of xylanase and pectin-ase were lower for HEB, compared to that of HEA, butendoglucanase and exoglucanase activities were higher or HEB.Interestingly, HEA was produced using CPW as the carbon source,and it showed high xylanase activity. This may have occurredbecause hemicellulose forms a large component of the polysac-charides in CPW [13,26], and xylanase produced monosaccha-rides by CPW hydrolysis for fungal survival. Microorganismsproduce the appropriate complex enzymes for hydrolysis duringgrowth on a given substrate. The presence of hemicellulose-derived saccharides in CPW is thought to be important for HEAinduction. Based on the above mentioned HEA and HEB activities,we designed a synergistic cooperation between cellulolytic,xylanolytic and pectinolytic enzyme mixtures to hydrolysis. Todetermine the amount of enzyme necessary for fruit wastehydrolysis, different enzyme volumes were loaded onto OP, MP,GP, LeP, LiP, MixP, and TFW substrates. Data for the hydrolysisof various fruit wastes by HEA and HEB are shown in Table 4. Table4

Conv

ersion

ratesforvariou

stype

sof

citrus

peel

waste

(CPW

),alon

eor

incombina

tion

withothe

rfruitwastesaftertreatm

entwithin-hou

seen

zymes

(HEA

andHEB

)at

differen

tload

ingvolumes.

Conv

ersion

rate

(%)

12mgHEA

/gfruitwaste

16mgHEA

/gfruitwaste

MgHEB

/gfruitwaste

Group

AGroup

BGroup

AGroup

B

OP

MP

GP

MixP

TFW

LeP

LiP

OP

MP

GP

MixP

TFW

LeP

LiP

063

.21.8

65.81.2

64.00.9

66.81.5

69.11.7

70.11.9

70.61.2

70.42.2

73.11.2

72.51.6

73.11.7

73.01.0

71.62.2

70.91.0

1072

.11.2

73.10.9

71.61.5

75.41.2

72.81.6

82.12.0

81.10.8

83.43.5

82.42.9

80.11.5

84.11.5

79.61.1

85.72.1

84.12.0

1575

.43.1

76.11.8

73.63.1

77.13.3

79.82.1

89.02.1

88.71.2

85.72.1

86.11.6

82.71.7

87.82.2

84.71.9

89.01.2

88.71.6

2080

.42.2

81.22.2

78.71.6

76.22.2

84.11.8

89.11.8

88.91.7

90.21.4

90.81.2

90.11.0

90.02.0

91.12.0

89.10.9

88.41.6

2581

.22.6

82.43.3

80.11.2

83.41.1

85.83.5

89.12.1

88.92.0

90.52.0

90.71.5

90.31.9

90.41.7

91.22.1

89.41.7

88.42.1

Abb

reviations

used

:OP,

oran

gepe

el;MP,

man

darinpe

el;GP,

grap

efruitpe

el;LeP,

lemon

peel;LiP,

limepe

el;MixP,

mixed

citrus

peel;TFW,m

ixed

totalfruitwastes;

HEA

,in-ho

useen

zymeA;HEB

,in-ho

useen

zymeB.

I.S. Choi et al. / Applied Energy 140 (2015) 6574 69

Although effects on fruit waste hydrolysis rates were species-dependent, the relatively low loading of HEA supplemented withHEB achieved a high overall hydrolysis rate. An increase in FS con-centrations from OP, MP, GP, MixP, and TFW (group A) wasobserved when HEA levels were increased from 12 to 16 mg HEAwith 20 mg HEB per g fruit waste. The FS from LeP and LiP (groupB) increased at lower enzyme loadings (HEA 12 and HEB 15 mg/gfruit waste) compared to loadings required in group A. The differ-ent chemical components of fruit waste may lead to differences inenzymatic hydrolysis processes. However, FS concentration wasnot increased significantly, even added more enzymes to group Aand B. This may have occurred because the hydrolysis of hemicel-lulose and pectin increases the surface area of the fruit waste and,

therefore, increasing accessibility and probability that the cellulosewill become hydrolyzed [27,28]. A combination of 16 mg HEA and20 mg HEB, or 12 mg HEA and 15 mg HEB per g fruit waste, wasused in all further experiments for group A or B, respectively. Inthis study, treatment with HEA invertase resulted in a decreasein sucrose levels (through hydrolysis) and corresponding increasesin monomers fructose and glucose (Supplementary Fig. S1). Thehydrolysis of sucrose can be an issue in continuous bioethanol pro-duction. This is because S. cerevisiae shows preferential consump-tion of glucose and fructose over sucrose during fermentation,and, as a result, sucrose is only consumed when the former twosubstrates are exhausted. These differences in the kinetics of sugarconsumption may limit bioethanol production from fruit waste.

Fig. 2. Waste-to-FS conversion rates as influenced by substrate loadings (%, w/w).

Table 5Influence of enzymatic hydrolysis time on various kinds of citrus and mixed fruit waste.

Time (h) 3 6 9 12 15 18 21 24 48

Group A (12%) OP 53.0 2.3 64.0 2.0 76.2 2.7 84.8 3.1 86.2 1.3 87.3 1.8 88.8 2.0 89.5 2.2 90.2 1.7MP 51.8 1.9 66.3 3.0 78.8 2.1 85.7 2.7 87.4 3.1 88.5 2.0 89.3 3.3 89.7 2.6 90.8 2.1GP 51.8 2.5 65.3 3.1 75.6 2.9 85.7 2.1 87.4 3.5 88.5 2.5 89.3 2.5 89.7 3.0 90.1 2.3MixP 51.8 1.5 65.0 2.4 76.3 2.3 85.7 2.4 87.4 2.7 88.5 3.0 89.3 3.2 89.7 2.8 90.0 1.8TFW 52.1 2.1 67.2 2.0 79.1 2.6 87.8 2.1 89.2 2.3 90.1 1.8 90.3 2.7 90.8 2.1 91.4 2.1

Group B (15%) LeP 45.8 2.1 61.2 2.6 72.4 1.9 80.1 2.6 85.3 1.9 87.0 2.2 87.8 1.7 89.3 2.6 89.0 1.7LiP 47.2 2.9 64.3 1.9 73.8 1.9 79.4 3.0 86.7 2.7 87.8 1.9 88.0 1.9 88.0 2.4 88.7 2.0

Abbreviations used: OP, orange peel; MP, mandarin peel; GP, grapefruit peel; LeP, lemon peel; LiP, lime peel; MixP, mixed citrus peel; TFW, mixed total fruit wastes.Group A and B concentrations were 12% and 15% (w/w) solid loading, respectively.

Table 6Summary of ethanol production from LRCICR system.

Fermentable sugar content (g/L) Enzymatic hydrolysate (g/L, %a) Ethanol concentration (g/L) Ethanol yield (%) Productivity (g/L/h)

OP 63.8 57.5/90.2% 27.1 92.4 3.01MP 68.5 62.2/90.8% 29.5 93.1 3.28GP 51.8 46.7/90.1% 21.6 90.7 2.40MixP 50.0 44.4/90.0% 20.4 90.2 2.27TFW 47.4 43.3/91.4% 20.3 91.8 2.26LeP 46.8 42.1/89.0% 19.6 91.1 2.18LiP 34.8 31.0/88.7% 14.4 90.8 1.60

Ethanol yield was calculated based on the fermentable sugars obtained from the hydrolysis of fruit waste.Theoretical ethanol yield was assumed to be 0.51 g/g sugar.

a Enzymatic hydrolysis efficiency.

70 I.S. Choi et al. / Applied Energy 140 (2015) 6574

According to Ghorbani et al. [29], to increase fermentation effi-ciency and avoid limitations, sucrose must be hydrolyzed to glu-cose and fructose via sucrose hydrolysis enzymes.

3.2.2. Influences of fruit waste concentration and time on enzymatichydrolysis

Based on the enzyme loading results in Table 4, we evaluatedthe effects of varying fruit waste concentrations on the enzymaticconversion of waste to FS. The conversion rates were calculatedbased on the total FS of fruit waste. Fruit waste solid loadings var-ied from 3% to 18% (Fig. 2). For group A (OP, MP, GP, MixP, andTFW) and group B (LeP and LiP), conversion rates decreased onlyslightly as substrate loadings increased from 3% to 12% and 3% to15%, respectively. After this point, further increases in substrateloading significantly decreased conversion rates in group A(>12%) and group B (>15%). Based on these results, all furtherexperiments used solid waste loadings of 12% for group A wastes,and 15% for group B wastes. In addition to the influence of sub-strate loading, we examined the influence of hydrolysis time onwaste-to-FS conversion (Table 5). FS conversion rates were highwithin the first 3 h, and considerable conversion of CPW to FSwas achieved within 9 h of hydrolysis. This kinetic behavior is inagreement with our previous work [13], which showed rapid

CPW hydrolysis within the first hours of the reaction, followedby a significant decrease. An FS conversion rate of approximately85% was achieved within the first 12 h for group A. However, groupB required 15 h to reach a similar level of conversion. Conversionrates did not increase significantly after 12 and 15 h in groups Aand B, respectively. From an economic perspective, these resultsare favorable given that they permit a high degree of conversion,which is necessary to maximize yield of ethanol during fermenta-tion. Moreover, the overall hydrolysis time required was relativelyshort compared to previous studies examining ethanol productionfrom lignocellulosic biomass [19,27,30].

3.3. D-limonene recovery and continuous immobilized yeastfermentation

3.3.1. Development of a D-limonene adsorbent columnCitrus contains D-limonene, a terpenoid essential oil that inhib-

its yeast fermentation. In conventional processing, D-limonene isremoved and recovered using energy-intensive methods, such assteam explosion. In contrast, conventional pretreatment methodhas a major disadvantage, in that carbohydrate content of the feed-stock may decrease to as low as 10% after pretreatment due to

Fig. 3. Limonene removal and recovery. (A) The sorbent column was filled with raw cotton and activated carbon. (B) Citrus peel and mixed fruit waste contained differentD-limonene concentrations. (C) D-limonene from orange peel (black arrow) was detected by gas chromatography, before and after recovery, and (D) D-limonene was recoveredafter fermentation.

I.S. Choi et al. / Applied Energy 140 (2015) 6574 71

losses resulting from the Maillard reaction, caramelization, andoxidation [13,3133]. With the aim of developing a more cost-effective, low-energy solution to this issue, we devised an LRCmade of raw cotton and activated carbon, and we evaluated itsremoval efficiency through gas chromatography (GC).

To evaluate the effects of column packing on D-limoneneremoval rate, various weights of raw cotton and activated carbonwere used to construct the LRCs. Decreasing D-limonene concen-trations in the filtrate were observed when the raw cotton weightwas increased from 100 to 300 mg per column, as well as when thequantity of activated carbon was increased from 0 to 1.5 g. How-ever, further improvements in D-limonene adsorption were notobserved when using >2.0 g activated carbon. Thus LRCs containing300 mg raw cotton and 1.5 g activated carbon (as shown in Fig. 3A)were used in all further experiments. Analysis of the fresh CPWshowed contents of 0.3211.858% (w/w) D-limonene (Fig. 3B);however, after passing through the LRC, D-limonene was undetect-able. This result represented an improvement in D-limoneneremoval compared to the conventional method used in previousstudies. In previous studies, inhibition of fermentation processeswas observed at concentrations greater than or equal to 0.1% (v/w) [9]. Grohmann et al. [7] have shown inhibitory minimum con-centrations, between 0.05% and 0.15% (v/w), that can affect the fer-mentation process. Moreover, about 90% of the D-limonene wasrecovered after a 10-day fermentation period with a 0.08 mL/minflow rate (Fig. 3C and D). These removal rates could be due tothe fact that D-limonene concentration and viscosity are low inthe hydrolysate. Because oil penetration rate into the internal sur-face of sorbents is inversely proportional to oil viscosity and con-centration [34], adsorption should be high in the pores of theraw cotton and activated carbon in the LRC. Importantly, FS con-centrations remained unchanged in the LRC filtrate.

3.3.2. Immobilized yeast fermentationWhen immobilizing cells onto a solid matrix such as calcium

alginate beads, a number of factors can affect the penetration ofcells into the bead and ultimately the conversion of FS to ethanol.Factors affecting bead penetration include alginate content, theratio of yeast cells to alginate, and pore size. In a previous study,these factors were optimized and we identified a suitable alginatemicrolattice matrix for our bioethanol reactor, known as an egg-box structure [24].

Calcium alginate beads and cultured S. cerevisiae were used toconstruct an ICR, with which we evaluated fermentation efficiencyusing a number of different fruit waste hydrolysates. Total FS, eth-anol concentrations, and FS-to-ethanol conversion rates wereobtained using two types of fermentation processes: ICR aloneand LRC followed by ICR (LRCICR). The volume metric ethanolproductivity (g/L/h) was calculated by dividing final ethanol con-centration with respect to fermentation time (Table 5). The initialFS concentrations in OP, MP, GP, LeP, LiP, MixP, and TFW were57.5, 62.2, 46.7, 42.1, 31.0, 44.4, and 43.3 g/L, respectively. The rel-ative FS concentrations decreased with increasing time in the LRCICR, especially over the first 9 h, whereas ethanol concentrationsincreased. After 9 h, FS concentrations in OP, MixP, and TFW hadfallen to 2.4, 2.3, and 2.6 g/L, respectively, and ethanol concentra-tions had increased to 27.1, 29.5, and 20.3 g/L, respectively(Fig. 4AC). When using ICR alone, without prior removal of D-lim-onene, FS concentrations were subsequently lower. After 9 h, FSconcentrations for OP, MixP, and TFW were 51.9, 17.9, and20.8 g/L, respectively, whereas ethanol concentrations were 2.7,15.9, and 12.9 g/L, respectively. In the LRCICR system, the FS-to-ethanol conversion rates for OP, MixP, and TFW feedstocks were92.4%, 90.2% and 91.8%, respectively, after 10 d, whereas no furtherethanol production was observed in the ICR system after the first

Fig. 4. Comparisons of fermentable sugar conversion and ethanol concentrations in ICR vs. LRCICR fermentation. (A) Initial FS concentrations in OP, (B) MixP, and (C) TFWwere 57.5, 44.4, and 47.4 g/L, respectively. (D) The glucose-to-ethanol conversion rates obtained after 10 days of fermentation. Black solid lines indicate the amount of FS fromICR ( ) or LRCICR ( ) fermentation, and gray solid lines indicate the amount of ethanol produced from ICR ( ) or LRCICR ( ) fermentation.

72 I.S. Choi et al. / Applied Energy 140 (2015) 6574

9 h (Fig. 4D). These results are likely due to high D-limonene con-centrations and its inhibitory effect on fermentation in the ICR sys-tem. Regarding the remaining feedstocks, MP and GP showed highFS contents and low ethanol concentrations after ICR fermentation,similar to the results obtained for OP, MixP, and TFW following ICRfermentation. After ICR fermentation, LeP and LiP showed FS con-tents of 5.5 and 3.8 g/L, respectively, and ethanol concentrationsof 20.2 and 15.1 g/L, respectively (Supplementary Fig. S2AD).The FS-to-ethanol conversion rates for LeP and LiP in the ICR sys-tem were only slightly lower compared to the LRCICR system(Supplementary Fig. S2ad). This may have occurred because theinitial D-limonene concentrations in the LeP and LiP hydrolysateswere insufficient to inhibit fermentation. However, these resultsindicate that the LRCICR fermentation system improved the FS-to-ethanol conversion rates and ethanol concentrations even atlow D-limonene concentrations. Interestedly, the ethanol produc-tivity of OP and MP, which are major citrus biomass sources, were3.01 and 3.28 g/L/h, respectively, through the LRCICR system. Inother words, 1000 kg fresh OP and MP (19.8% and 20.1% of mois-ture contents) would be converted into 44.8 and 49.5 L of bioetha-nol, respectively (Table 6).

Several previous studies have examined the effects of pretreat-ment on CPW composition and subsequent bioethanol production;however, ethanol concentrations and productivities obtained inthis study were similar to or greater than those observed in previ-ous studies. For example, ethanol production from OP, using steamexplosion combined with acid pretreatment, produced 2527 g/Lethanol concentration with around 0.5 g/L/h productivity [11].Another study reported that the fermentation of MP and LeP aftersteam explosion produced approximately 60 L/1000 kg (fresh mat-ter) of ethanol concentration with 0.50.94 g/L/h productivity,respectively [12,14].

Fruit waste and other solid residues, such as coffee waste andrice, from agricultural by-products were considered bioethanolproduction materials [13,21,33]. One main obstacle to achievingefficient bioethanol production is the cost of production. The com-mercial success of ethanol production depends on productivity, interms of volume and concentration. Notably, our new processachieved high ethanol production without costly pretreatment,suggesting utility in industrial ethanol production applications.The high ethanol production during the validation experimentcould be due to several factors, including suitable inhibitorremoval conditions, enzyme production, loading volume, and con-tinuous yeast fermentation.

4. Conclusion

Fruit waste is an attractive biomass alternative for bioethanolproduction because it has high levels of FS such as sucrose, glucose,and fructose. In this study, these sugars were hydrolyzed and fer-mented without an energy-intensive conventional pretreatment.After enzymatic hydrolysis with two in-house enzymes, D-limo-nene was removed using an adsorbent column containing raw cot-ton and activated carbon and directly conducted to an immobilizedreactor (LRCICR) for fermentation. Ethanol production in thisLRCICR system was 12-fold greater than that observed withoutprior use of the sorbent column (LRC) to remove the fermentationinhibiting D-limonene. This new approach to removing D-limoneneand enhancing immobilized yeast fermentation could potentiallybe useful in more cost-effective bioethanol production.

Acknowledgements

This work was supported by Priority Research Centers Program(2010-0020141) through the National Research Foundation of

Korea (NRF) funded by the Ministry of Education, Science andTechnology, and by a grant (S211314L010120) from Forest Science& Technology Projects, Forest Service, Republic of Korea.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.apenergy.2014.11.070.

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74 I.S. Choi et al. / Applied Energy 140 (2015) 6574

A low-energy, cost-effective approach to fruit and citrus peel waste processing for bioethanol production1 Introduction2 Materials and methods2.1 Raw materials2.2 Chemical composition2.3 In-house enzyme production2.4 In-house enzyme activity and hydrolysis2.5 d-Limonene recovery column design2.6 Continuous immobilized yeast fermentation

3 Results and discussion3.1 Fruit waste composition3.2 In-house enzyme production and fruit waste hydrolysis3.2.1 In-house enzyme activity and effective loading volumes for hydrolysis3.2.2 Influences of fruit waste concentration and time on enzymatic hydrolysis

3.3 d-limonene recovery and continuous immobilized yeast fermentation3.3.1 Development of a d-limonene adsorbent column3.3.2 Immobilized yeast fermentation

4 ConclusionAcknowledgementsAppendix A Supplementary materialReferences