Release of sugar by acid hydrolysis from rice bran for single cell oil production and subsequent in-situ transesterification for biodiesel preparation

Fuel Processing Technology 167 (2017) 281–291

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Fuel Processing Technology journal homepage: www.elsevier.com/locate/fuproc

Research article

Release of sugar by acid hydrolysis from rice bran for single cell oil production and subsequent in-situ transesterification for biodiesel preparation Sylviana Sutanto a, Alchris Woo Go b, Kuan-Hung Chen a, Phuong Lan Tran Nguyen c, Suryadi Ismadji a,d, Yi-Hsu Ju a,⁎ a

Department of Chemical Engineering, National Taiwan University of Science and Technology, 43, Keelung Rd., Sec. 4, Taipei 106-07, Taiwan Department of Chemical Engineering, University of San Carlos, Talamban Campus, Nasipit Talamban, 6000 Cebu City, Philippines c Department of Mechanical Engineering, Can Tho University, 3-2 Street, Can Tho, Viet Nam d Department of Chemical Engineering, Widya Mandala Surabaya Catholic University, Kalijudan 37, Surabaya 60114, Indonesia b

a r t i c l e

i n f o

Article history: Received 23 January 2017 Received in revised form 10 July 2017 Accepted 10 July 2017 Available online xxxx Keywords: Dilute acid hydrolysis L. starkeyi Microbial oil Rice bran (Trans)esterification

a b s t r a c t Biodiesel is one of the promising alternative biofuel to petro-diesel. Efficient and effective utilization of low value feedstock is required to meet the current demand on biodiesel. Rice bran is a byproduct of rice milling that contains significant amounts of sugar (~48%) and lipids (~14%). Immediate recovery of the lipids is uneconomical owing to its moderate lipid content. In this work, hydrolysis of raw rice bran was firstly carried out to concentrate lipids in the hydrolyzed bran to ~42%. Hydrolysis of rice bran using 2% sulfuric acid at 90 °C for 3.5 h with stirring at 300 rpm resulted in hydrolysates containing ~41 g/L sugars and 3.75 g/L proteins. Rice bran hydrolysate (RBH) obtained was further utilized as growth media for Lipomyces starkeyi, an oleaginous yeast. Lipid content of 40 to 65% can be achieved, suggesting the potential of using RBH in single cell oil production. Composition of microbial oils obtained was similar to that of vegetable oils, which may potentially be adopted as a feedstock for biodiesel production. Furthermore, dried hydrolyzed rice bran was subjected to in-situ (trans)esterification with methanol under subcritical condition which resulted in high FAME yield (87.81%) and conversion (94.82%). This work demonstrated the maximized utilization of rice bran in the production of hydrolysate for fermentation and as biodiesel feedstock. This is to further support the use of direct acid hydrolysis of biomass in the recovery and use of its content of sugars and lipids. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Biodiesel is one of the renewable alternatives to reduce our dependent on petroleum based fuel. Recent life cycle analysis conducted by Argonne National Laboratory found that biodiesel emitted 74% less greenhouse gas [1] compared to petroleum diesel. Unlike bioethanol, biodiesel can be used in diesel engines without the need of engine modification. Currently, the use of biodiesel may not be economically attractive. However, it may be the best alternative in view of depleting crude oil supply. The current use of refined vegetable oil as feedstock oil accounts for ~70% of total biodiesel cost [2]. The use of low cost agro-industrial residues as feedstock is one possible way to make biodiesel production economically more attractive. Rice is a staple food in Asia with China, India and Indonesia as the top producing countries in the world [3]. Rice bran (RB) is the main byproduct of rice milling and it accounts for ~11 wt% of rice paddy [4]. ⁎ Corresponding author. E-mail address: [email protected] (Y.-H. Ju).

http://dx.doi.org/10.1016/j.fuproc.2017.07.014 0378-3820/© 2017 Elsevier B.V. All rights reserved.

Most (90%) RB is used as cattle feed [4], which is considered as an underutilization of RB. Since RB contains ~ 37% carbohydrates and 10– 20% lipids depending on the degree of milling [5], the carbohydrates can be converted into sugars for fermentation processes, while lipids can be recovered and utilized as feedstock for biodiesel production. Dilute acid hydrolysis is often employed as a pretreatment method to recover sugars from RB. In some studies, the use of defatted RB was preferred, as lipids in RB were perceived as a nuisance resulted in lower sugar yield [6,7]. Recent studies on direct dilute acid hydrolysis of lipid-containing biomass like RB and spent coffee grounds resulted in similar sugar yields to that of lipid-free biomass, while lipids remained intact and were fully recovered after hydrolysis [5,8]. Long hydrolysis time (6 h) was required [5,9], which could be reduced with the aid of stirring which was not implemented in previous studies. The effect of stirring on sugar content of hydrolysate was investigated in this work. The produced hydrolysate was then used as the medium in the growth of oleaginous yeasts, L. starkeyi. Oleaginous yeasts are capable of accumulating 20 to 70% intracellular lipids [10–14]. Lipomyces sp. is one of the oleaginous yeast that can

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grow in various substrates. L. starkeyi is capable of accumulating 30 to 65% lipid content, depending on substrate composition, growth conditions and fermentation modes [15–20]. Lipid content of L. starkeyi grown in defined medium ranged from 40 to 68% [11,15,18,21,22], while those grown in wastewater or hydrolysate only reached 20 to 40% [15,23–26] with just a few reported high lipid content (50 to 60%) [16,22]. Although the use of defined medium may be convenient in studying growth kinetics and conversion pathways, it is not practical when used in the production of single cell oils as feedstock for biodiesel [27]. Various low value sources for fermentation medium such as potato starch waste water [28], and fishmeal waste water [29] have been used in growing L. starkeyi. Unfortunately, owing to their low carbohydrates and nitrogen contents, additional carbon source or other nutrients were required, while resulting only in moderate lipid content (b20%). Thus, studies on lipid accumulation of L. starkeyi grown in undefined media such as biomass hydrolysates need to be explored to maximize the use of renewable resources. Apart from hydrolysates produced from RB, lipids concentrated in RB after hydrolysis may further be recovered and utilized. However, previous work [5] has not explored its actual utilization. Recovery of lipids often required additional processing steps, which may result in additional cost for biodiesel production. Developments in biodiesel production has led to the use of simultaneous extraction and reaction through in-situ (trans)esterification [2]. In this study, the hydrolyzed RB was subjected to in-situ (trans)esterification with methanol under subcritical conditions. This study was aimed to maximize the use of RB through direct dilute acid hydrolysis of RB to produce hydrolysates as fermentation medium for L. starkeyi. Fermentation experiments were divided into two parts: (1) improving hydrolysis productivity and yield with the aid of stirring, using one-factor-at-a-time analysis, and (2) the best hydrolysis conditions were used to produce hydolysates as fermentation medium employing various fermentation modes for cultivating L. starkeyi. Moreover, in situ (trans)esterification of hydrolyzed RB was also investigated.

2. Materials and methods RB was obtained from a local rice mill in Kaohsiung City, Taiwan. Two batches of RB were used in the experiment. Batch A was used for improving hydrolysis process, and batch B was used as fermentation nutrient. All RBs as received were stored in polypropylene bottles and kept in a freezer (−80 °C) until later use. Lipomyces starkeyi BCRC 23408 was acquired from Bioresource Collection and Research Center (Hsinchu, Taiwan). The stock culture was kept in glycerol solution and store at −80 °C. All chemicals used in this work were either analytical or HPLC grade, obtained from commercial sources.

2.1. Characterization of RB RB was weighed in a pre-weighed container, sealed with perforated paraffin film then freeze dried for at least ~24 h or until constant weight. The weight loss was used for calculating the moisture content. For lipid determination, RB was weighed and put in a thimble, plugged with cotton and extracted using n-hexane for 8 h. The weight of oil obtained was used for calculating the crude lipid content. The theoretical maximum carbohydrates present in RB was determined following the method described previously [5]. Total sugar (TS) content was analyzed using the phenol-sulphuric acid method of Dubois [30]. Briefly, a certain amount of filtrate sample was diluted to 1 mL with deionized water inside a vial where 1 mL of 5% phenol and 5 mL concentrated sulphuric acid (96%) was then added. After incubation for 30 min, absorbance of the sample was measured at 490 nm. A calibration curve using D-glucose as the standard was established prior to sample measurement.

2.2. Acid hydrolysis of RB An amount of RB was put in a screw-capped bottle and mixed with dilute sulfuric acid (2%, 3% or 4% v/v) at a solvent to solid ratio of 8 mL/g of RB on dry and lipid free basis. The mixture was stirred magnetically (300–900 rpm) and reacted for 1–6 h at 90 °C in a water bath. At predetermined time intervals, the reaction bottle was removed from the water bath and rapidly cooled to room temperature. The cooled reaction mixture was subjected to vacuum filtration to recover the hydrolysate. TS was determined according to the procedure described above. The hydrolyzed RB was dried in an oven (50 °C) until constant weight (for ~ 2 weeks) and kept for further use in in-situ (trans)esterification. 2.3. Determination of total reducing sugars, inhibitors and protein in (D)RBH Total Reducing Sugar (TRS) of (D)RBH was determined by following the method described by Miller [31], using 3,5-dinitrosalicylic acid (DNS) with slight modification. In brief, 100 μL RBH was diluted to 1 mL and 100 μL sample was taken from this dilution and was pipetted into an amber bottle. Deionized water was added until the volume was 1 mL. DNS reagent (1 mL) and 40% Rochelle salt (0.3 mL) were added to the amber bottle. The sample was heated in a boiling water bath for 5 min then rapidly cooled in an ice bath. Absorption of the sample was read at 510 nm. Sugars composition in hydrolysate was analyzed using a HPLC with a Sugar-D column (Phenomenex) with acetonitrile: water (80:20 v/v) as the eluent. 5-HMF (5-hydroxymethylfurfural) and furfural were also analyzed using a HPLC with a C-18 reverse phase column (Phenomenex, Gemini-NX). Elution was done with water-acetonitrile-acetic acid (88:11:1) as the mobile phase. UV detection was at 280 nm. Protein content (for mode HG and HX) was analyzed by taking a diluted sample and mixed it with 0.2 mL of Bradford reagent, then incubated for 5 min at room temperature. Absorbance of the samples was read at 595 nm. Calibration curve using bovine serum albumin was prepared prior to sample measurement. Nitrogen content was estimated by dividing protein content by a factor of 6.25. 2.4. Media preparation and cultivation of L. starkeyi 2.4.1. Inoculum preparation L. starkeyi was propagated in YPD agar with the following composition: 10 g/L yeast extract, 20 g/L peptone, and 20 g/L D-glucose and 20 g/L agar powder. A single colony of yeasts was streaked on agar and propagated every two weeks. Pre-culture was done aseptically by inoculating one loopfull of yeast from agar plate into 3 mL of YPD or sterilized detoxified RBH medium (contained 10 g/L yeast extract and 20 g/L of peptone) inside a test tube. The test tube was then placed in an orbital shaker 28 °C, 200 rpm until exponential growth phase (36 h). Yeast suspension was collected and its optical density (OD) was measured at 600 nm. A calibration curve of the dry cell weight vs. OD was prepared beforehand. An initial biomass concentration of 0.1 g/L was employed in subsequent fermentation experiments. 2.4.2. Preparation of DRBH as fermentation medium Hydrolysis of RB was carried out using the conditions that resulted in the highest TRS concentration and yield. RBH collected was then detoxified by adding activated carbon (30 g/L) and stirred for 30 min. The detoxified-RBH (DRBH) was then collected by filtration. The pH of the DRBH was adjusted to 6 by adding Ca(OH)2 and the precipitate (CaSO4) formed were removed by filtration. The final TRS content of DRBH was determined and the TRS concentration was adjusted using de-ionized water to 30 g/L. DRBH was then filter-sterilized using sterile PVDF membrane (Millipore 0.22 μm). The sterilized DRBH was used as

S. Sutanto et al. / Fuel Processing Technology 167 (2017) 281–291

fermentation medium. Accordingly, the term “DRBH” used in this study refers to sterilized-detoxified hydrolysate. 2.4.3. Fermentation of L. starkeyi using DRBH as medium Fermentation was carried out in one-stage (batch) and two-stage modes. Sampling was taken every 12 h to monitor biomass growth and sugar consumption. Sample (0.5 mL) was withdrawn, placed in a pre-weighed micro centrifuge tube then centrifuged (8960 × g) for 6 min. The supernatant was collected for TRS and protein analysis using the method described above, while the collected biomass was rinsed twice with deionized water and dried at 105 °C until constant weight (~24 h). Each fermentation (except for high-density and fed-batch modes) was carried out by employing 100 mL of DRBH (TRS ~ 30 g/L) in 500 mL unbaffled flask. Samples were taken every 12 h and cells were harvested once it reached early stationary phase. Initially, fermentations were carried out in batch mode to investigate whether RB hydrolysate can be utilized by L. starkeyi without prior detoxification. Batch fermentations using different inoculums (YPD vs. DRBH) were also studied. There were several studies carried out for 2-stage fermentation, such as high density cultivation, fed-batch mode, re-suspension of wet cells into second medium, and addition of powder-sugars. High density (HD) cultivation was done by transferring a small amount of inoculum (~0.1 g/L initial biomass) into 25 mL fresh DRBH in a 250 mL unbaffled flask. After 33 h, it was used as the inoculum for HD mode and transferred into 75 mL fresh RBH in a 500 mL unbaffled flask. Fed-batch (FB) mode was initially carried out with 100 mL DRBH for certain time. Feeding pulse was added twice, each with 100 mL fresh DRBH when TRS dropped below 5 g/L. In the case of re-suspension, in stage 1, fermentation was done using 100 mL DRBH (until TRS started to drop below ~5 g/L) then centrifuged to separate wet cells and supernatant. The wet cells were directly re-suspended into 100 mL fresh DRBH (coded RS), or 100 mL glucose solution (RG), or 100 mL xylose solution (RX) with initial TRS of ~30 g/L for all second stage medium. While for the other two runs, after 1-stage fermentation when TRS dropped below 5 g/L, sugar (glucose or xylose) powder were added into the flask (aseptically), while monitoring N consumption. TRS in sample was analyzed before-and-after every time sugar was added. 2.5. Lipid extraction and analysis Preliminary studies indicated that it is important to break cell wall to facilitate lipid extraction. Disruption of cell wall was done by adding 10 mL 2.5 N HCl to 1 g dry yeast and reacted at 80 °C for 2 h. The mixture was then filtered using 0.22 μm cellulose acetate membrane (Advantec) and the yeast retained was dried in an oven (50 °C) until constant weight. The hydrolyzed-yeast was sequentially extracted using hexane, methanol and then hexane, for 4 h each. Lipid extraction and analysis were done following the method described previously [5]. Fatty acid methyl ester (FAME) was analyzed using a high temperature gas chromatograph (HT-GC) Shimadzu 2010 Plus, equipped with a split injector and a flame ionized detector (FID). Injector and detector were both set at 370 °C. The temperature program was set at 180 °C, increased at 15 °C/min to 365 °C and held for 8 min. The carrier gas used was nitrogen with a linear velocity of 30 cm/s at 80 °C. Further details can be seen in the established GC analysis protocol [32].

283

matter and FFA were determined following the procedure described in AOCS Method Ca 6b-53 and AOCS Method Ca 5a-40, respectively [33]. Total fatty acids available in extracted lipids were determined according to Go et al. [34]. In-situ (trans)esterification of hydrolyzed-RB was carried out in a stainless steel reactor (Fig. 1). Hydrolyzed-RB was placed in a glass chamber (~ 101 mL) and mixed with methanol (solvent to solid ratio = 5) then placed in a reactor (290 mL). Spacer (~124 mL) was placed on top of the chamber to reduce the void inside reactor, in order to minimize the chance of methanol evaporates during reaction. Methanol served as both the extraction solvent and methylating agent for in situ (trans)esterification. In-situ (trans)esterification was carried out without pre-pressurizing. The reactor was then heated at a rate of 5 °C/min to reach the desired temperature (~30 min). The heater was turned off and reactor was immediately cooled to room temperature. Product from reactor was filtered, the solid was rinsed thrice, each with 30 mL methanol, whereas filtrate was transferred to a flask and methanol was removed by evaporation. Concentrated reaction product was re-suspended thrice with 30 mL hexane each and transferred to a separation funnel. Sodium chloride solution (5%) was added to wash the product until the washed solution was neutral. The hexane rich phase was withdrawn and concentrated in a rotary evaporator (Buchi R-114, Switzerland), until constant weight. A small amount of product was then injected and analyzed in GC to calculate the yield and conversion. Calibration curve was prepared using methyl oleate as the external standard. 2.7. Determination of acid sites in hydrolyzed RB Strong acid sites were determined according to the published method [35]. In brief, an amount of hydrolyzed-RB (~ 0.1 g) was soaked in NaCl (10 mL, 2 M) at 30 °C overnight with 250 rpm stirring. The mixture was then vacuum-filtered to separate the solid. The filtrate was titrated with standardized 0.01 M NaOH until pH 7 (pH was measured using a pH probe that was immersed in the solution). Fig. 2 shows the flowchart of this study. 2.8. Determination of extractives and lignin content Lignin determination was carried out for raw RB (batch B), hydrolyzed-RB and in-situ-RB-residue. Analysis of lignin follows the laboratory analytical procedure of NREL/TP-510-42618 [36]. Sample used for lignin determination should be an extractives-free-biomass since the presence of extractives interfere with the analysis. Briefly, sample was first extracted with hexane, followed by ethanol extraction and then

2.6. In-situ (trans)esterification of hydrolyzed RB It has been proven that lipid remained in RB after hydrolysis [5]. Since hydrolyzed- RB has high lipid content (~ 41%) similar to that of oil bearing seeds like Jatropha curcas and sunflower seeds, it could be used as feedstock for biodiesel production. Hydrolyzed-RB was characterized for its moisture and lipid content, while extracted lipids were further determined for its unsaponifiable matters, free fatty acids (FFA) and hydrolysable (total available fatty acids). Unsaponifiable

Fig. 1. Illustration of high pressure stainless steel reactor (1. Pressure gauge, 2. Inlet gas valve, 3. Thermocouple, 4. Outlet gas valve, 5. Reactor, 6. Spacer, 7. Glass chamber).

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Fig. 2. Flowchart of this study.

TS

40

TRS

30

20

10

0 1

2

3

the sample in order to dilute it to 4% H2SO4. The mixture was then autoclaved, then vacuum filtered to separate soluble and insoluble lignin. The insoluble lignin was then burned in furnace (with ramping) at 575 ± 25 °C for 3 h.

TS

Sugar yield (g glucose equivalent/100g dry RB)

Sugar yield (g glucose equivalent/00g dry RB)

water extraction. The dried-extractives-free biomass was then weighed and placed in a screw capped bottle, mixed with 72% H2SO4 and incubated in a water bath at 30 °C for 1 h with stirring. After taking the sample out of the water bath, an amount of deionized water was added to

3.5

4

4.5

5

30

20

10

0

6

300

Time (h)

TRS

30

20

10

0 2%

3%

H2SO4 concentration (%)

(C)

700

900

(B) Sugar yield (g glucose equivalent/100g dry RB)

Sugar yield (g glucose equivalent/100g dry RB)

TS

500

Stirring speed (rpm)

(A) 40

TRS

40

4%

50 TS

TRS

40 30 20 10 0 2.5

3.5

4.5

5.5

Time (h)

(D)

Fig. 3. Sugar yields after hydrolysis at a solvent to solid ratio of 8, 90 °C. (A) Effect of time with 3% acid, (B) effect of stirring speed, (C) effect of different acid concentrations, (D) effect of time at 2% acid.

S. Sutanto et al. / Fuel Processing Technology 167 (2017) 281–291

3.3. Effect of sulfuric acid concentration on RB hydrolysis

Table 1 Characteristic of non-detoxified and detoxified RBH.

Sugars (TRS) Xylose Glucose Fructose Inhibitors 5-HMF Furfural a

285

RBH (g/L)

ACD-RBHa (g/L)

46.20 ± 1.67 8.68 ± 1.15 31.51 ± 2.62 6.01 ± 1.11

43.97 ± 1.57 7.06 ± 1.37 31.12 ± 1.39 5.79 ± 0.31

1.34 ± 0.02 0.23 ± 0.04

0.05 ± 0.01 0.02 ± 0.01

Detoxified by activated carbon.

The effect of sulfuric acid concentration on sugars yield is shown in Fig. 3C. Sugar yields were found to be similar (p-value N 0.3) even at a low acid concentration of 2%. Furthermore, hydrolysis at 90 °C with 2% H2SO4 under 300 rpm stirring for 3.5 h (Fig. 3D) was found to result in a TS yields of 37.12 ± 1.49 g glucose equivalent/100 g dry RB (~57.43 ± 2.19 g/L) and TRS yields of 34.92 ± 3.21 g glucose equivalent/100 g dry RB (~54.19 ± 5.89 g/L). This hydrolysis condition was then adopted for the production of sugar-rich hydrolysates from RB (batch B) for subsequent fermentation experiment. 3.4. Characteristic of RB hydrolysate (RBH)

3. Results and discussion RB batch A had a crude lipid content of 20.34%. In the crude lipid, the FFA content was 13.25 ± 0.37%, and the content of unsaponifiable matter was 3.67 ± 0.08%; while 87.56 ± 1.32% of the crude lipid was hydrolysable. The sugar yield was 37.72 ± 6.14% (= total carbohydrates present in RB).

3.1. Effect of reaction time on RB hydrolysis Initially, dry RB was hydrolyzed using 3% H2SO4 at 90 °C and 900 rpm magnetic stirring. Yields of TS and TRS in RBH are presented in Fig. 3A. It can be observed that TS and TRS increased with time until the highest TS and TRS yields which were obtained at 4.5 h. Prolong hydrolysis time resulted in insignificant changes in sugar yields (p-value N 0.5) for both TS and TRS. On average, slightly decreases in sugar yields were observed, which could be the result of sugars degradation into furans, organic acids and humic substances.

3.2. Effect of stirring on reaction time and acid concentration during RB hydrolysis As can be observed (Fig. 3B), increasing stirring speed from 500 to 900 rpm resulted in insignificant difference in sugar yields (p-value N 0.1). However, significant decrease of TS (p-value = 0.01) was observed when the stirring speed was increased beyond 300 rpm. This could be the result of the increased hydrolysis rate leading to similar effect to that of prolonging hydrolysis time. For TRS, although it was not statistically significant (p-value = 0.08), its average value decreased. This was most-likely due to prolonged hydrolysis time and high stirring speed that resulted in harsh condition in which monomeric sugars were turned into furans resulting in lower TRS yields. Furans may decompose into acetic acid, formic acid and humic substances and resulting in overall decline in TS yields. Lowering stirring speed to 100 rpm resulted in lower TS and TRS yields (36.84 ± 1.26 and 31.85 ± 0.2 g/100 g dry RB, respectively) because of heterogeneity during hydrolysis.

RB (batch B) used in this study had moisture and lipid content of 10.3 ± 0.12% and 14.70 ± 0.16%, respectively. Sugars and inhibitors compositions in RBH are summarized in Table 1. Sugars detected in RBH are glucose (70%), xylose (17%) and fructose (13%). All these monomeric sugars were previously found to be utilizable by L. starkeyi [15,37]. The theoretical sugars present in RB batch B was 48.10%. Thus, when the best condition of hydrolysis was applied, ~97% of sugars was recovered. Fermentation employing non-detoxified RBH was carried out and compared to activated carbon-detoxified RBH, in order to know whether detoxification was necessary. It was found that biomass growth in non-detoxified RBH was inhibited, resulting in low lipid content (data not shown). Previous investigations on L. starkeyi showed that 1 g/L HMF can inhibit both biomass growth and lipid accumulation, while furfural even at 0.5 g/L may decrease lipid content up to 7% [38]. Since 5HMF concentration in RBH was higher than this limit value, suggesting that detoxification was necessary. 3.4.1. L. starkeyi grown in RBH with different inoculums Initially, L. starkeyi preculture in YPD was prepared and inoculated into DRBH without additional nutrient. It showed a lag phase in the first 24 h (Fig. 4A). Fermentation was also carried out using inoculums (abbreviated as “Ino” in Table 2) from DRBH for comparison. Preculture in DRBH medium was prepared by adding 10 g/L yeast extract and 20 g/L peptone into DRBH that has been adjusted for initial TRS, then the solution was filter-sterilized. As can be seen in Fig. 4, biomass grew faster when the yeast was inoculated from the same inoculums medium (DRBH). Slower growth from YPD inoculums was probably caused by the need of yeast to adapt in a new medium (YPD to DRBH), thus longer cultivation time was needed to reach the stationary phase. In the case of DRBH inoculums which has more nutrients (due to addition of yeast extract and peptone), more biomass was produced since more nutrients were available for biomass growth, but not lipid accumulation (Table 2). Hexane soluble lipid of yeast grown in DRBH using YPD as inoculums was higher than that of using DRBH inoculums (Table 2). However, taking into account lipid titer and productivity, it is better to use inoculums from DRBH in order to avoid lag phase

Fig. 4. Fermentation time courses of L. starkeyi in RBH at 28 °C, 200 rpm shaking speed, YPD vs DRBH inoculums.

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Table 2 L. starkeyi grown in DRBH. Run

Time (h)

Biomass (g/L)

Ino YPD

96

11.9

Ino DRBH

84

13.5

Lipid content

Lipid titer (g/L)d

Lipid productivity (g/L/day)d

Lipid conversion Y(L/S)e

%Crude lipida

% Hexane solubleb

%FFAc

Step 1: 56.77 ± 1.04 Step 2: 58.93 ± 0.83 Step 3: 59.46 ± 0.69 Step 1: 46.1 ± 0.66 Step 2: 52.8 ± 1.00 Step 3: 53.88 ± 1.05

56.89 ± 0.92

2.26 ± 0.27

7.08 (6.77)

1.77 (1.70)

0.23

52.59 ± 0.61

3.22 ± 0.01

7.27 (7.10)

2.08 (2.03)

0.24

All values were results of duplicate, independent experiments. a Cumulative percentage by weight of each extraction step based on dry cell weight (Step 1: 4 h with hexane, Step 2: 4 h with methanol, Step 3: 4 h with hexane). b Hexane soluble crude lipid obtained after washing with 5% NaCl, percent based on dry cell weight. c Percent free fatty acid (FFA) present in the hexane soluble crude lipids. d Value inside the brackets were calculated based on hexane soluble, value without the brackets were calculated based on cumulative percentage in Step 3. e Lipid conversion expressed as g lipid/g sugar consumed.

14 12

20

10 8

15

6

10

4 5

2

40

35

35

30

30

25

25 20 20 15 15 10

10

5

5

0

0 0

12

24

36

48

60

0

0

72

0

24

48

72

Time (h)

Time (h)

(HD)

(FB)

25 Biomass Concentration (g/L)

TRS Concentration (g/L)

25

Biomass Concentration (g/L)

30

96

120

144

35 30

20

25 15

20

10

15 10

5

5

TRS Concentration (g/L)

Biomass Concentration (g/L)

16

withdrawn directly after mixing (taken as 0 h) in order to analyze initial biomass and TRS concentration. TRS found in DRBH-supernatant was only 26.3 g/L which implies that ~ 14.8 g/L of sugars from the second batch preculture (25 mL DRBH inoculum) was consumed and resulted in a biomass of 2.6 g/L. HD cultivation was terminated at 72 h after inoculation of second batch preculture (a total of 105 h). Biomass obtained was similar to that of one-stage fermentation with 0.1 g/L initial inoculum (84 h), but lipid content (55.59%) and lipid titer (7.62 g/L) were higher in HD cultivation (Table 3). Comparing these values to that of 1-stage fermentation using 0.1 g/L inoculum (Run ‘Ino RBH’ in Table 2) which also utilized 15 g/L of sugars

TRS Concentration (g/L)

(from 12 to 24 h). Based on this result, inoculums from DRBH will be used in subsequent experiments. A two-stage fermentation mode was carried out to see if higher lipid content can be obtained. A few modes were tried, such as high density cultivation, fed-batch, re-suspension in a new medium (sterilized RBH or sugars solution) and addition of sugars. The fermentation timecourses for all cultivation methods are shown in Figs. 5 and 6, and summary of the results can be seen in Table 3. In HD mode (Fig. 5-HD), fermentation started after transferring 25 mL inoculums (at 33 h) into 75 mL of fresh DRBH. The total volume for HD mode was fixed at 100 mL for comparison with the one-stage fermentation. Aliquot was

0

0 0

24

48

72

96

120

Time (h)

(RS) Fig. 5. Fermentation time course of L. starkeyi in DRBH for different modes at 28 °C, 200 rpm shaking speed, on high density (HD), fed-batch (FB) cultivation, and re-suspension in RBH (RS). Filled diamond is biomass concentration, empty diamond is TRS concentration.

0.6

25

0.5 0.4

20 15

0.3

10

0.2

5

0.1 0

0 24

48

72

96

40

0.7

35

0.6

30

0.5

25

0.4

20 0.3

15 10

0.2

5

0.1

0

0 0

120

24

48

0.7

30

0.6

25

0.5

20

0.4

15

0.3

10

0.2

5

0.1

0

Biomass and TRS concentration (g/L)

35

0 72

108

120

(RX)

N content (g/L)

Biomass and TRS concentration (g/L)

(RG)

36

96

Time (h)

Time (h)

0

72

144

180

40

0.7

35

0.6

30

0.5

25

0.4

20 0.3

15

0.2

10

0.1

5 0

216

0 0

Time (h)

N content (g/L)

0

287

N content (g/L)

30

Biomass and TRS concentration (g/L)

0.7

35

N content (g/L)

Biomass and TRS concentration (g/L)

S. Sutanto et al. / Fuel Processing Technology 167 (2017) 281–291

36

72

108 144 180 216

Time (h)

(HG)

(HX)

Fig. 6. Fermentation time courses of L. starkeyi in RBH for different modes at 28 °C, 200 rpm shaking speed, re-suspension in glucose solution (RG), re-suspension in xylose solution (RS), adding glucose (HG), adding xylose (HX). Filled diamond is biomass concentration, empty diamond is TRS concentration, filled triangle is (estimated) N content in RBH.

Table 3 L. starkeyi grown in RBH, with different cultivation methods. Run

Time (h)

Biomass (g/L)

HD

72 (105)

13.7

FB

144

34.2

RS

120

23.1

RG

120

19.8

RX

120

18.6

HG

216

26.2

HX

216

25.2

a b c d e

Lipid titer (g/L)d

Lipid productivity (g/L/day)d

Lipid conversion Y(L/S)e

4.73 ± 1.06

8.00 (7.62)

1.83 (1.74)

0.29

51.57 ± 2.82

7.3 ± 2.08

18.09 (17.64)

3.01 (2.94)

0.19

56.11 ± 0.23

4.96 ± 0.18

13.17 (12.96)

2.63 (2.60)

0.21

65.55 ± 2.38

1.76 ± 0.12

13.70 (12.98)

2.74 (2.60)

0.25

60.85 ± 2.61

1.65 ± 0.15

11.84 (11.32)

2.37 (2.26)

0.22

60.35 ± 2.54

0.76 ± 0.05

16.44 (15.81)

1.83 (1.76)

0.21

59.04 ± 2.18

1.15 ± 0.21

15.58 (15.11)

1.73 (1.68)

0.18

Lipid content %Crude lipida

%Hexane solubleb

%FFAc

Step 1: 52.64 ± 1.97 Step 2: 57.63 ± 0.45 Step 3: 58.44 ± 0.04 Step 1: 43.99 ± 1.96 Step 2: 51.06 ± 1.36 Step 3: 52.89 ± 2.36 Step 1: 47.74 ± 0.91 Step 2: 55.75 ± 0.07 Step 3: 57.03 ± 0.18 Step 1: 64.87 ± 2.47 Step 2: 68.65 ± 1.89 Step 3: 69.21 ± 1.36 Step 1: 57.56 ± 4.62 Step 2: 62.50 ± 3.00 Step 3: 63.63 ± 2.33 Step 1: 60.00 ± 2.42 Step 2: 62.67 ± 2.63 Step 3: 62.74 ± 2.67 Step 1: 58.21 ± 2.69 Step 2: 60.89 ± 2.52 Step 3: 61.81 ± 1.36

55.59 ± 0.7

Cumulative percentage by weight of each extraction step based on dry cell weight (Step 1: 4 h with hexane, Step 2: 4 h with methanol, Step 3: 4 h with hexane). Hexane soluble crude lipid obtained after washing with 5% NaCl, percent based on dry cell weight. Percent free fatty acid (FFA) present in the hexane soluble crude lipids. Value inside the brackets were calculated based on hexane soluble, value without the brackets were calculated based on cumulative percentage in Step 3. Lipid conversion expressed as g lipid/g sugar consumed.

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Table 4 Comparison of lipid produced by L. starkeyi in literature. Substrates

Pretreatment and hydrolysis method of biomass

DCW (g/L)

Lipid content (%)

Lipid productivity (g/L day)

Lipid conversion (g lipid/g sugar)

Ref.

Glucose Glucose and xylose Glucose Glucose, fructose, sucrose Wheat straw hydrolysate Rice straw hydrolysate Spent cell hydrolysate Sweet sorghum stalk juice Corncob hydrolysate Willow wood sawdust hydrolysate Sugarcane bagasse hydrolysate Arundo donax hydrolysate

– – – – 2% H2SO4, 10% (w/v), 121 °C, 1 h 3.5% H2SO4, 1:10 (w/v), 121 °C, 2 h 0.5 M H2SO4, 1:10 (w/v), 120 °C, 1.5 h Saccharified (cellulase and cellobiose), 50 °C, 8.6 h Dilute H2SO4 25 mg/mL, 10% (w/v), 135 °C, 1 h 1st step: 2% H2SO4 w/w, 1:10 (w/v), 130 °C, 1 h 2nd step: dried residue +4% H2SO4 w/w, 1:10 (w/v), 150 °C, 1 h 1.5% H2SO4 w/v, 1:10 (w/v), 120 °C, 0.3 h

9.5 20.5 11.7 12.28 14.7 12.76 19.7 21.69 17.2 8.2

68 61.5 30 47.3 31.2 35.65 30.8 29.5 47 42.7

0.65 2.52 1.68 1.16 0.76 1.52 1.20 0.80 1.01 0.32

– – 0.10 0.13 – 0.18 0.12 0.08 0.21 0.14

[22] [37] [11] [15] [12] [44] [45] [15] [23] [17]

14

31

2.64

0.22

[19]

Steam explosion, enzymatic hydrolysis (cellulase and β-glucosidase) 50 °C, 72 h, 50 rpm Submerged in DI water 15% w/v, 121 °C, 1 h then 3% (cellulase and hemicellulase) w/w 55 °C, 48 h, 150 rpm Ammonium fiber expansion (AFEX) with NH3: biomass = 1:1, 90 °C, 5 min Solid state fermentation with A. oryzae 30 °C, 48 h

8.7

20.3

0.02

–

[46]

17.1 23.5 24.63

37.3 33.3 38.07

1.28 1.57 1.18

0.12 0.13 0.14

[25] [25] [20]

17.4

29.31

0.57

–

[24]

2% H2SO4, 1:8 (w/v), 90 °C, 3.5 h, 300 rpm

13.5

52.59

1.92

0.24

Present study

23.1

56.11

2.6

0.21

19.8

65.55

2.6

0.25

26.2

60.35

1.68

0.21

Wheat bran hydrolysate Corn bran hydrolysate Corn stover hydrolysate Sunflower meal hydrolysate (Rice bran hydrolysate) DRBH DRBH → resuspension in DRBH DRBH → resuspension in glucose solution RBH + glucose powder

at 36 h, it could reach a dry cell weight of 5.4 g/L. The difference in biomass possibly was due to less dissolved oxygen available in small flask, leading to less biomass obtained. The growth rate was lower than 1-stage fermentation thus HD mode needed longer time to reach maximum biomass. However, the reason behind higher lipid content obtained in HD cultivation was perhaps due to that cells had reached maximum biomass production thus it was able to accumulate lipids in earlier phase. Lipid titer in HD was higher than 1-stage mode but lipid productivity was lower. The total sugar assimilated by L. starkeyi in this mode was ~26.2 g/L, resulted in a lipid conversion of 0.29 g/g sugar in DRBH consumed. Although it seems high, but the lipid titer and productivity are not the highest compared to other modes carried out in this study. Fermentation time-course of fed-batch mode can be seen in Fig. 5FB. Based on the data in Table 3, hexane soluble lipid obtained from this mode was only 51.57% and lipid titer was 17.64 g/L. Compared to HD cultivation, it has lower lipid content, most likely was due to the addition of fresh RBH which contains balance nutrients of sugars and protein caused cells proliferate actively instead of accumulating lipids. Anschau et al. [39] did a fed batch fermentation of L. starkeyi DSM 70296 using basal medium with xylose as the C source. They reported that lipid content obtained was 37.4% after 3 feeding pulses and sugar ran out at 148 h. In the present study, higher lipid content achieved was probably due to the type of sugar consumed by the yeast. Since L. starkeyi has glucose repression mechanism, it may consume more glucose than xylose within the similar length of time (144 h cultivation of fed-batch in present study). L. starkeyi fed with glucose could reach early stationary phase earlier than that fed by xylose, resulting in earlier stage of lipid accumulation, leading to discrepancy in lipid contents. Another method studied, was by re-suspending biomass in a new batch of sterilized DRBH (Fig. 5-RS). The first stage of fermentation was harvested at 58 h, exactly when TRS left was only 5 g/L (when carbon limitation started). After the first stage of harvesting, the wet cells obtain after centrifugation were then directly re-suspended in a fresh DRBH without water rinsing, since the next medium used would be the same as the previous one. It was done to prevent yeast entering the depleted sugar phase, since yeast would need time to adapt to

new environment after it was transferred, thus maintaining the cycle short in order to obtain higher lipid productivity. This mode turned out to give very high biomass compared to other modes described previously, most likely due to more nutrients were available and assimilated by yeast, thus promoted proliferation. Slightly higher lipid content (by ~ 4%) was attained compared to the 1-stage fermentation (Run ‘Ino RBH’ in Table 2), resulting in higher lipid titer and lipid productivity. This is more advantageous over 1-stage fermentation since it could give higher lipid productivity, while utilizing merely hydrolysate without additional sugars during fermentation. To date, there is no work reported on re-suspension (in the same medium) strategy in fermentation. To find out the maximum lipid that could be produced by L. starkeyi using DRBH as medium, next trials were done by re-suspending wet cells in a sterile sugars solution (pure glucose or xylose solution without other nutrients). Cells in the first stage were harvested at 58 h by centrifugation then re-suspended directly into ~30 g/L glucose solution (RG) and xylose solution (RS). Rinsing with water was skipped to maintain and allow biomass to adapt to the new environment. In addition, without rinsing, nitrogen from the first stage (after centrifugation) mostly would remain in the supernatant and only trace amount was available together with cells. While in the second stage which consisted of only Table 5 FA profile of lipid from L. starkeyi BCRC 23408 grown in DRBH then re-suspended into glucose or xylose solution. Fatty acids

Myristic Palmitic Palmitoleic Stearic Oleic Arachidic/eicosanoic Gondoic Behenic/docosanoic Erucic

Structure

14:0 16:0 16:1 18:0 18:1 20:0 20:1 22:0 22:1

Formula

C14H28O2 C16H32O2 C16H30O2 C18H36O2 C18H34O2 C20H40O2 C20H38O2 C22H44O2 C22H42O2

wt% Glucose

Xylose

0.31 36.02 3.11 7.40 49.59 1.53 0.11 0.32 0.10

0.30 36.14 4.46 5.10 51.95 0.42 0.11 0.24 0.08

S. Sutanto et al. / Fuel Processing Technology 167 (2017) 281–291 Table 6 Characteristic of hydrolyzed-RB and its oil (in wt%). Moisture content of hydrolyzed RB Lipid content Free fatty acids (FFA) content Unsaponifiable matter Hydrolysable (total fatty acid) Theoretical FAME yield

4.03 ± 0.59 41.42 ± 0.16 32.91 ± 0.69 4.58 ± 0.08 88.19 ± 1.49 ~92.60

pure sugars, there was no nitrogen available. This was proved by the fact that there was no more nitrogen detected in both runs (Fig. 6, RG and RX) 12 h after transferring (72 h cultivation). Comparing Fig. 6 RG and RX, glucose was consumed much faster than xylose, although these two sugars ran out almost at the same time. Biomass obtained from re-suspension in glucose solution was a little higher than that of xylose solution. Further increase in biomass was not observed after 108 h despite there was excess sugar left, thus fermentations were terminated at 120 h. Total sugar consumed for re-suspension in glucose (RG) and xylose (RX) were 51.5 and 49.74 g/L respectively. Based on data in Table 3, this 2-stage fermentation method was able to achieve 65.55% and 60.85% lipid content for RG and RX, respectively. This supports the idea of carbon catabolite repression, that L. starkeyi prefers glucose to xylose [18,21]. Lipid contents obtained in these modes were higher than the rest of the data above. Lin et al. were able to obtain 64.9% lipid content and 104.6 g/L biomass when 2-stage fermentation was employed in a bioreactor (basal medium was used in stage 1, transferred to 120 g/L glucose solution, then 480 g of solid glucose was added after 16 h) [10]. Despite of using a bioreactor and large amount of glucose [10], lipid content obtained was similar to the present study. Three years later, Lin et al. [40] did a 2-stage shake flask re-suspension from basal medium with xylose in the first stage, the wet cells was then re-suspended in xylose solution after twice rinsing with water. The second stage was harvested at 48 h, where biomass and lipid content reached 11.4 g/L and 60.4%, respectively [40]. While in the present study, lipid content could reach 65.55 and 60.85% after re-suspension in glucose solution (RG) and xylose solution (RX), respectively, without water rinsing in between the 2-stages of fermentation. Another strategy investigated, was to add sugar (glucose or xylose powder) into fermentation medium (DRBH) after initial C source started to deplete. In DRBH with glucose addition (Fig. 6-Run HG), glucose powder was added 3 times at 76, 144 and 160 h during fermentation (~ 65 g/L glucose powder addition). At the end of cultivation at 216 h, all sugars were assimilated (~95 g/L, including 30 g/L TRS in initial RBH) and 60.35% lipid content was achieved. Comparing this value to that obtained by Wild et al. (27%) using a basal medium with 5 120

Yield

times glucose addition in an 1 L (fed batch) bioreactor [11], lipid content obtained in this study is significantly higher. While in the case of xylose addition (Fig. 6-Run HX), 58 g/L xylose was added during the second stage of fermentation, resulting in a total of 88 g/L sugars assimilated (adding up from 30 g/L TRS from DRBH) in this mode. Considering that cells were harvested at the same time for both HG and HX experiments, this value shows that L. starkeyi follows the glucose repression mechanism [18,21]. Nitrogen was also found in trace amount starting at 168 h for both runs, indicating that cells did consume all nitrogen available. Comparing biomass with addition of different sugars, slightly higher biomass was obtained when glucose was added as observed in Table 3. Similar hexane soluble lipids were attained in both experiments after washing with 5% NaCl. In contrast to the results in literature which reported that microbial lipid content obtained from xylose was higher than that of glucose [40,41], hexane soluble lipid in this study was not much different from that of glucose. Ratledge et al. [42] found that glucose assimilation by oleaginous yeast results in 1.1 mol acetyl-coA/100 g glucose, while xylose can undergo two different metabolism pathways in yeast: pentose phosphate pathway that produces 1.1 mol acetyl-CoA/100 g xylose or, phosphoketolase pathway (that produces 1.3 mol acetyl-CoA/100 g xylose). Therefore, the result above was probably related to L. starkeyi assimilation of xylose (in DRBH solution) that underwent pentose phosphate pathway instead of phosphoketolase pathway, thus resulted in similar lipid production as that of glucose addition. In summary, high biomass and lipid content can be achieved by employing 2-stage fermentation of L. starkeyi. The two modes that were able to give satisfactory results are re-suspension in glucose solution (19.8 g/L biomass, 65.55% hexane soluble lipid), and glucose addition to RBH (26.2 g/L dry cell weight, 60.35% hexane soluble lipid). Comparing these values with other studies investigated using lignocellulosic hydrolysate as fermentation medium, DRBH shows a superior ability to produce high lipid content and lipid conversion (summarized in Table 4), similar to those grown in complex medium with high C/N ratio. Lipid content obtained in the present study was among the highest reported for L. starkeyi which shows the potential of using RB hydrolysate. Considering that re-suspension in glucose solution without prior water washing could give satisfactory lipid content, that step is actually not necessary. The highest lipid conversion in these experiments was (0.25 g/g sugar), obtained from Run RG. Although this result is still lower than the theoretical lipid yield (0.32 g/g glucose or 0.34 g/g xylose) [43], this study proves that RBH is a good fermentation medium for culturing oleaginous yeast to produce lipids. In addition, it shows the excellent ability of L. starkeyi in converting agricultural residue hydrolysate into lipids. This lipid conversion is higher than 0.22 g lipid/g sugar consumed 120

Conversion

100

Yield

Conversion

100 FAME Yield (%)

FAME Yield (%)

289

80 60 40

80 60 40 20

20

0

0 0

30 Time (min)

(A)

60

4 5 6 Solvent to Solid Ratio (SSR)

(B)

Fig. 7. FAME yield and conversion of in-situ transesterification of hydrolyzed-RB, ~14 g dry weight hydrolyzed-RB at 185 °C (A) fixed SSR of 5 with various reaction times, (B) fixed time at 0 h, varied SSR.

290

S. Sutanto et al. / Fuel Processing Technology 167 (2017) 281–291

Fig. 8. FAME yield and conversion of hydrolyzed RB (~14 g dry weight of hydrolyzed-RB at SSR 5, 0 h, final pressure was the value obtained after the desired temperature was reached).

which was thought to be the maximum [43]. The mechanism of lipid synthesis in DRBH follows de novo lipid synthesis, where the presence of nitrogen has to be limited while sugars have to be in excess in order for the yeast to channel it for lipid accumulation [43]. Mass balance of the processes was calculated and can be found in the Supplemental files. 3.5. Fatty acids profile Crude lipid of L. starkeyi with the highest lipid content (Run RG and RX in Table 3) was subjected to analysis of unsaponifiable and hydrolysable matters. The lower part of unsaponifiable fraction was collected and hydrolyzed using sulfuric acid, the resulting fatty acids were then methylated using BF3-methanol to find the theoretical FAMEs. Unsaponifiable matters of lipid from Run RG and Run RX were 0.71 and 1.13%, respectively while hydrolysable part was account for 93.54 and 91.56% of lipids from RG and RX, respectively. Table 5 summarizes the fatty acids profile of L. starkeyi lipid, obtained from re-suspension in glucose and xylose solution. Similar lipid composition was observed and the major fatty acids present were oleic and palmitic acid, which implies that L. starkeyi grown in DRBH is a potential single cell oil source for biodiesel production. The fatty acid compositions are within the range of those reported in literature. 3.5.1. In-situ biodiesel production using hydrolyzed-RB Characteristic of hydrolyzed-RB and its oil can be seen in Table 6. Slightly higher lipid yield was obtained from hydrolyzed-RB probably due to hydrolysis of structural lipid, thus lipid recovery can reach ~ 108%. Lipid yield and recovery were calculated using formula of Go et al. [8]. In preliminary investigations, a solvent to solid ratio (SSR) of 5 and a short reaction time (0 h) was shown to give the highest FAME yield and conversion (Fig. 7a). It took ~30 min to heat the reactor content to 185 °C. The moment the temperature reached the pre-specified value was taken as 0 h. Reducing or increasing SSR both resulted in decreased FAME yield and conversion (Fig. 7b). The dry weight of hydrolyzed-RB used was ~ 14 g, which corresponds to ~ 66% reactor loading and was taken as the maximum loading. As can be seen in Fig. 8, experiments

carried out at different temperatures with a fixed SSR of 5 and 0 h, showed that maximum yield (87.81%) and conversion (94.82%) can be achieved at 185 °C. Temperature higher than 185 °C resulted in lower yield probably due to thermo-chemical degradation and the presence of strong acid sites in high temperature. All yield and conversion were calculated based on hexane extractable lipid. Hydrolyzed-RB was suspected to act as both feedstock and (solid) acid catalyst since it was the residue of dilute acid hydrolysis. Acid strength of hydrolyzed RB was determined by ion exchange in NaCl solution and subsequent titration to determine the presence of strong acid sites (SO3H groups). It was found that hydrolyzed-RB (after hydrolysis with 2% H2SO4 v/v) has 1.08 ± 0.01 mmol acid sites/g dry weight of hydrolyzed RB. These values are comparable to that reported in biomass waste (sawdust) [47]. A comparison of results of RB in-situ (trans)esterification reported in literature and in this study is given in Table 7. High amount of H2SO4 was needed as the catalyst for in-situ reaction in atmospheric condition and long reaction time (4–5 h) was required to achieve high yield even with the assist of ultrasound [49,53]. High yield can be achieved in short time but high temperature and pressure were needed in supercritical methanol method [50]. Milder condition used in subcritical solvent method could give high FAME yield, but it required longer reaction time (3 h) [51]. Another attempt of subcritical solvent using acetic acid as the catalyst gave impressive result and achieved almost complete conversion (99.7%) [52]. In the present study, methanol was used as the only reactant, no compressing gas was added for in-situ reaction, since the feedstock itself acted as solid acid catalyst. Another advantage of hydrolyzed-RB utilization was the need of lesser SSR and lower temperature to achieve high FAME yield and conversion in short time. Aside from that, moisture content as much as ~9% can also be tolerated by employing this method with comparable results to that reported in literature. 4. Conclusion Dilute acid hydrolysis can be done in short time without compromising the sugars produced. One-stage and two-stage cultivations of L. starkeyi in DRBH were carried out in order to find the highest lipid produced. The highest lipid content (65.55%) was achieved in the 2-stage fermentation after re-suspending wet cells from the 1st (RBH medium) into the 2nd stage (glucose solution), corresponding to 12.98 g/L lipid titer and 2.6 g/L/day lipid productivity. Dilute acid hydrolysis offers an advantage that it not only produced fermentable sugars in hydrolysate but also simultaneously concentrated and increased extractable lipid (lipid recovery ~108%) and converted RB into solid acid catalyst. Assessment of strong acid sites gave 1.08 mmol acid sites/g dry weight of hydrolyzed RB. In-situ (trans)esterification of dry hydrolyzed-RB using methanol as the solvent and reactant resulted in high FAME yield (87.81%) and conversion (94.82%). Biorefinery concept adapted in this study provided impressive results in better utilizing a low value-high volume by-product to produce more valuable product. This work also provides an idea of co-producing green solid acid catalyst from agricultural residue like RB.

Table 7 Comparison with other in-situ (trans)esterification of RB. Method

SSR (mL/g)

T (°C)

P (MPa)

Time (h)

FAME yieldc (%)

Ref.

18.6% H2SO4 catalyzed Ultrasound assisted, 27.6% H2SO4 catalyzed Supercritical methanol + CO2 Subcritical solvent (water + methanol) + CO2 Subcritical solvent (methanol + acetic acid) + CO2 Subcritical methanol of hydrolyzed RB (with 2% v/v H2SO4)

4 10 3 8 6 5

65 60 300 200 250 185

~0.1 ~0.1 30a 4a 12 a 2.5

5 4 0.08 (1.5)b 3.0 (3.75) 1.0 (1.75) 0 (0.55)

72.40 74.90 51.28 65.21 85.64 87.81

[48] [49] [50] [51] [52] Present study

a b c

Final pressure. Total time including heating up the reactor. FAME yield was defined as g FAME/g crude lipid available.

S. Sutanto et al. / Fuel Processing Technology 167 (2017) 281–291

Acknowledgements Financial supports from Ministry of Science and Technology of Taiwan (MOST 104-2221-E-011-146) was greatly acknowledged. The authors declare no financial and conflict of interest.

[25] [26]

[27]

Appendix A. Supplementary data

[28]

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.fuproc.2017.07.014.

[29]

References

[30]

[1] Biodiesel Vehicle Emissions, Argonne-National-Laboratory Energy Efficiency & Renewable Energy. , US Department of Energy, 2016. [2] A.W. Go, S. Sutanto, L.K. Ong, P.L. Tran-Nguyen, S. Ismadji, Y.-H. Ju, Developments in in-situ (trans)esterification for biodiesel production: a critical review, Renew. Sust. Energ. Rev. 60 (2016) 284–305. [3] Ricepedia, Rice Productivity, 2016. [4] T.S. Kahlon, Rice bran: production, composition, functionality and food applications, physiological benefits, in: Samuel C. SS (Ed.), Fiber Ingredients: Food Applications and Health Benefits, CRC Press Taylor & Francis 2009, pp. 305–318. [5] S. Sutanto, A.W. Go, K.-H. Chen, S. Ismadji, Y.-H. Ju, Maximized utilization of raw rice bran in microbial oils production and recovery of active compounds: a proof of concept, Waste and Biomass Valorization (2016) 1–14. [6] N.K.N. Al-Shorgani, M.S. Kalil, W.M.W. Yusoff, Biobutanol production from rice bran and de-oiled rice bran by clostridium saccharoperbutylacetonicum N1-4, Bioprocess Biosyst. Eng. 35 (2012) 817–826. [7] J. Lee, E. Seo, D.-H. Kweon, K. Park, Y.-S. Jin, Fermentation of rice bran and defatted rice bran for butanol production using Clostridium beijerinckii NCIMB 8052, J. Microbiol. Biotechnol. 19 (2009) 482–490. [8] A.W. Go, A.T. Conag, D.E.S. Cuizon, Recovery of sugars and lipids from spent coffee grounds: a new approach, Waste Biomass Valorization (2016) 1–7. [9] Y.A. Tsigie, C.-Y. Wang, N.S. Kasim, Q.-D. Diem, L.-H. Huynh, Q.-P. Ho, C.-T. Truong, Y.-H. Ju, Oil production from Yarrowia lipolytica PO1g using rice bran hydrolysate, J Biomed Biotechnol 10 (2012) 1–10. [10] J. Lin, H. Shen, H. Tan, X. Zhao, S. Wu, C. Hu, Z.K. Zhao, Lipid production by Lipomyces starkeyi cells in glucose solution without auxiliary nutrients, J. Biotechnol. 152 (2011) 184–188. [11] R. Wild, S. Patil, M. Popovi, M. Zappi, S. Dufreche, R. Bajpai, Lipids from Lipomyces starkeyi, Food Technol. Biotechnol. 48 (2010) 329–335. [12] X. Yu, Y. Zheng, K.M. Dorgan, S. Chen, Oil production by oleaginous yeasts using the hydrolysate from pretreatment of wheat straw with dilute sulfuric acid, Bioresour. Technol. 102 (2011) 6134–6140. [13] C. Ratledge, J. Wynn, The biochemistry and molecular biology of lipid accumulation in oleaginous microorganisms, Adv. Appl. Microbiol. 51 (2002) 1–52. [14] A. Beopoulos, J.M. Nicaud, Yeast: A New Oil Producer? Dossier Lipochimie, 2012 22–28. [15] L. Matsakas, A.-A. Sterioti, U. Rova, P. Christakopoulos, Use of dried sweet sorghum for the efficient production of lipids by the yeast Lipomyces starkeyi CBS 1807, Ind. Crop. Prod. 62 (2014) 367–372. [16] S. Tsakona, N. Kopsahelis, A. Chatzifragkou, S. Papanikolaou, I.K. Kookos, A.A. Koutinas, Formulation of fermentation media from flour-rich waste streams formicrobial lipid production by Lipomyces starkeyi, J. Biotechnol. 189 (2014) 36–45. [17] R. Wang, J. Wang, R. Xu, Z. Fang, A. Liu, Oil production by the oleaginous yeast Lipomyces starkeyi using diverse carbon sources, Bioresources 9 (2014) 7027–7040. [18] X. Yang, G. Jin, Z. Gong, H. Shen, Y. Song, F. Bai, Z.K. Zhao, Simultaneous utilization of glucose and mannose from spent yeast cell mass for lipid production by Lipomyces starkeyi, Bioresour. Technol. 158 (2014) 383–387. [19] A. Anschau, M.C.A. Xavier, S. Hernalsteens, T.T. Franco, Effect of feeding strategies on lipid production by Lipomyces starkeyi, Bioresour. Technol. 157 (2014) 214–222. [20] C.H. Calvey, Y.-K. Su, L.B. Willis, M. McGee, T.W. Jeffries, Nitrogen limitation, oxygen limitation, and lipid accumulation in Lipomyces starkeyi, Bioresour. Technol. 200 (2016) 780–788. [21] Z. Gong, Q. Wang, H. Shen, C. Hu, G. Jin, Z.K. Zhao, Co-fermentation of cellobiose and xylose by Lipomyces starkeyi for lipid production, Bioresour. Technol. 117 (2012) 20–24. [22] C. Angerbauer, M. Siebenhofer, M. Mittelbach, G.M. Guebitz, Conversion of sewage sludge into lipids by Lipomyces starkeyi for biodiesel production, Bioresour. Technol. 99 (2008) 3051–3056. [23] C. Huang, X.-F. Chen, X.-Y. Yang, L. Xiong, X.-Q. Lin, J. Yang, B. Wang, X.-D. Chen, Bioconversion of corncob acid hydrolysate into microbial oil by the oleaginous yeast Lipomyces starkeyi, Appl. Biochem. Biotechnol. 172 (2014) 2197–2204. [24] D.E. Leiva-Candia, S. Tsakona, N. Kopsahelis, I.L. Garcia, S. Papanikolau, M.P. Dorado, A.A. Koutinas, Biorefining of by-product streams from sunflower-based biodiesel

[31] [32]

[33] [34]

[35]

[36]

[37]

[38]

[39] [40]

[41]

[42] [43] [44]

[45] [46]

[47] [48] [49] [50] [51]

[52]

[53]

291

production plants for integrated synthesis of microbial oil and value added co-products, Bioresour. Technol. 190 (2015) 57–65. K.V. Probst, P.V. Vadlani, Production of single cell oil from Lipomyces starkeyi ATCC 56304 using biorefinery by-products, Bioresour. Technol. 198 (2015) 268–275. A. Yousuf, F. Sannino, V. Addorisio, D. Pirozzi, Microbial conversion of olive oil mill wastewaters into lipids suitable for biodiesel production, J. Agric. Food Chem. 58 (2010) 8630–8635. F.D. Harzevili, H. Chen, Microbial Biotechnology: Progress and Trends, CRC Press Taylor & Francis Group, 2015. J.-X. Liu, Q.-Y. Yue, B.-Y. Gao, Y. Wang, Q. Li, P.-D. Zhang, Research on microbial lipid production from potato starch wastewater as culture medium by Lipomyces starkeyi, Water Sci. Technol. 67 (2013) 1802–1808. L. Huang, B. Zhang, G. B, S. G, Application of fishmeal wastewater as a potential lowcost medium for lipid production by Lipomyces starkeyi HL, Environ. Technol. 33 (2011) (1975-1981). D. M, G. KA, H. JK, R. P, S. F, Colorimetric method for determination of sugars and related substances, Anal. Chem. 28 (1956) 350–356. G.L. Miller, Use of dinitrosalicylic acid reagent for determination of reducing sugar, Anal. Chem. 31 (1959) 426–428. S. Sutanto, A.W. Go, S. Ismadji, Y.-H. Ju, Taguchi method and grey relational analysis to improve in situ production of FAME from sunflower and Jatropha curcas kernels with subcritical solvent mixture, J. Am. Oil Chem. Soc. 92 (2015) 1513–1523. D. Firestone (Ed.), Official Methods and Recommended Practices of the American Oil Chemists' Society, The Society, 1989. A.W. Go, S. Sutanto, P.L. Tran-Nguyen, S. Ismadji, S. Gunawan, Y.-H. Ju, Biodiesel production under subcritical solvent condition using subcritical water treated whole Jatropha curcas seed kernels and possible use of hydrolysates to grow Yarrowia lipolytica, Fuel 120 (2014) 46–52. H.F. Webster, B.B. Bean, C. Estes, T.J. Fuhrer, Synthesis and Characterization of a Novel Solid Acid Catalyst for Improved Use of Waste Oil Feedstock for Biodiesel Production, United States Environmental Protection Agency, 2010. A. Sluiter, B. Hames, R. Ruiz, C. Scarlata, J. Sluiter, D. Templeton, D. Crocker, Determination of Structural Carbohydrates and Lignin in Biomass, Laboratory Analytical Procedure (LAP), National Renewable Energy Laboratory, 2012. X. Zhao, X. Kong, Y. Hua, B. Feng, Z.K. Zhao, Medium optimization for lipid production through co-fermentation of glucose and xylose by the oleaginous yeast Lipomyces starkeyi, Eur. J. Lipid Sci. Technol. 110 (2008) 405–412. X. Chen, Z. Li, X. Zhang, F. Hu, D.D.Y. Ryu, J. Bao, Screening of oleaginous yeast strains tolerant to lignocellulose degradation compounds, Appl. Biochem. Biotechnol. 159 (2009) 591–604. A. Anschau, T.T. Franco, Cell mass energetic yields of fed-batch culture by Lipomyces starkeyi, Bioprocess Biosyst. Bioeng. (2015). J. Lin, S. Li, M. Sun, C. Zhang, W. Yang, Z. Zhang, X. Lia, S. Li, Microbial lipid production by oleaginous yeast in D-xylose solution using a two-stage culture mode, RSC Adv. 4 (2014) 34944–34949. Q. Sha, A Comparative Study on Four Oleaginous Yeasts on Their Lipid Accumulating Capacity, Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala, Sweden, 2013. C. Ratledge, Biochemistry, stoichiometry, substrates and economics, in: R.S. Moreton (Ed.), Single Cell Oil, Longman Scientific & Technical, Harlow (UK) 1988, pp. 33–70. S. Papanikolaou, G. Aggelis, Lipids of oleaginous yeasts. Part I: biochemistry of single cell oil production, Eur. J. Lipid Sci. Technol. 113 (2011) 1031–1051. A.K. Azad, A. Yousuf, A. Ferdoush, M.M. Hasan, M.R. Karim, A. Jahan, Production of microbial lipids from rice straw hydrolysates by Lipomyces starkeyi for biodiesel synthesis, Microb. Biochem. Tech. (2014) 1–6. X. Yang, G. Jin, Z. Gong, H. Shen, F. Bai, Z.K. Zhao, Recycling microbial lipid production wastes to cultivate oleaginous yeasts, Bioresour. Technol. 175 (2015) 91–96. D. Pirozzi, G. Travglini, D. Sagneli, F. Sannino, G. Toscano, Study of a discontinuous fed-batch fermentor for the exploitation of agricultural biomasses to produce II-generation biodiesel, Chem. Eng. Trans. 38 (2014) 169–174. W.-J. Liu, K. Tian, H. Jiang, H.-Q. Yu, Facile synthesis of highly efficient and recyclable magnetic solid acid from biomass waste, Nat. Sci. 3 (2013) 1–7. S. Özgül-Yücel, S. Türkay, Variables affecting the yields of methyl esters derived from in situ esterification of rice bran oil, J. Am. Oil Chem. Soc. 79 (2002) 611–614. L. Yustianingsih, S. Zullaikah, Y.H. Ju, Ultrasound-assisted in-situ production of biodiesel from rice bran, J. Energy Inst. 82 (2009) 133–137. N.S. Kasim, T.-H. Tsai, S. Gunawan, Y.-H. Ju, Biodiesel production from rice bran oil and supercritical methanol, Bioresour. Technol. 100 (2009) 2399–2403. S. Zullaikah, Y.T. Rahkadima, In-situ biodiesel and sugar production from rice bran under subcritical condition, International Conference of Chemical and Material Engineering, American Institute of Physics, 2015. A.W. Go, Biodiesel production under subcritical conditions using solvent mixtures of methanol, water and acetic acid, Chemical Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan, 2015. S. Özgül-Yücel, S. Türkay, Variables affecting the yields of methyl esters derived from in situ esterification of rice bran oil, J. Am. Oil Soc. 79 (2002) 611–614.