An integrated and optimized process for cleaner production of ethanol and biodiesel from corn stover by Mucor indicus

Journal Pre-proof An integrated and optimized process for cleaner production of ethanol and biodiesel from corn stover by Mucor indicus Razieh Shafiei Alavijeh, Keikhosro Karimi, Corjan van den Berg PII:

S0959-6526(19)34191-5

DOI:

https://doi.org/10.1016/j.jclepro.2019.119321

Reference:

JCLP 119321

To appear in:

Journal of Cleaner Production

Received Date: 1 February 2019 Revised Date:

22 October 2019

Accepted Date: 13 November 2019

Please cite this article as: Shafiei Alavijeh R, Karimi K, van den Berg C, An integrated and optimized process for cleaner production of ethanol and biodiesel from corn stover by Mucor indicus, Journal of Cleaner Production (2019), doi: https://doi.org/10.1016/j.jclepro.2019.119321. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

An integrated and optimized process for cleaner production of ethanol and biodiesel from corn stover by Mucor indicus

Razieh Shafiei Alavijeha, Keikhosro Karimia,b,∗, Corjan van den Bergc

a

Department of Chemical Engineering, Isfahan University of Technology, Isfahan 84156-83111,

Iran b

Industrial Biotechnology Group, Research Institute for Biotechnology and Bioengineering,

Isfahan University of Technology, Isfahan 84156-83111, Iran c

Bioprocess Engineering, Wageningen University, PO Box 16, 6700 AA Wageningen, The

Netherlands

*

Corresponding author:

Tel: +983133915623 Fax: +983133912677 E-mail address: [email protected]

Declarations of interest: none

Graphical Abstract

Lipid extraction

Oil

Biodiesel Reactor

Biomass of M. indicus

Naturalization by NaOH

Aerobic cultivation

Liquid 100 g

Grinding

Dilute-acid pretreatmen t

Biomass of M. indicus

Solid Washing

Enzymatic hydrolysis

Solid (6.2 g)

Liquid

Anaerobic cultivation

Abstract A two-stage process was successfully developed for biodiesel and ethanol production from corn stover using zygomycetes fungus Mucor indicus. Dilute-acid pretreatment followed by enzymatic saccharification was applied to release the maximum amount of sugars (glucose and xylose) from the lignocellulosic structure of corn stover. Dilute-acid hydrolysis was optimized by a response surface design. Under the optimal reaction conditions (i.e., 1.8 % v/v H2SO4, 121 ºC for 22 min), the hydrolysis resulted in the production of 270 g glucose per kg of dry corn stover (57.8 % theoretical yield) and 100 g xylose per kg of dry corn stover (84.0 % theoretical yield). Validation of the model exhibited proper fit between predicted and observed values of glucose and xylose concentrations: 91.4 % regression adjustment for xylose and 98.2 % for glucose. In the first stage, cells fermented the enzymatic hydrolysate to the maximum amount of 74.5 % (0.38 g g-1) ethanol as the main product. In the second stage, the dilute-acid hydrolysate was used for lipid accumulation in the fungal cells of the first stage fermentation. The hydrolysates were used without detoxification since the fungus is among the most resistant microorganisms to the inhibitors available in the acid hydrolysates. Effects of addition of different nutrient sources, including fungal extract and yeast extract along with mineral salts, were also investigated to maximize lipid yield. Overall, 21.4 g ethanol and 2.2 g biodiesel (obtained from 4.00 g accumulated lipid) were produced from 100 g dry corn stover.

Keywords: Mucor indicus, Lignocellulosic substrate, Microbial lipid, Ethanol production, Biodiesel production

1

1. Introduction Production of biofuels is an effective solution for confronting the environmental and economic impacts of fossil fuels consumption (Raheem et al., 2018; Rossi et al., 2011). Biodiesel and bioethanol are two prominent renewable fuels which can be produced from a wide range of biomass substrates (Tourang et al., 2019; Pourbafrani et al., 2014). Biodiesel (fatty acid methyl esters) is produced through the transesterification of long chain fatty acids of vegetable oils and animal fats. More than 95 % of the industrial production of biodiesel is based on the transesterification of edible vegetable oils (Gui et al., 2008), which is restricted due to the conflict between food and fuel. In order to overcome this obstacle, many researchers have used non-food resources, including non-edible vegetable oils (Bateni et al., 2014; Mardhiah et al., 2017), waste oils (Hajjari et al., 2017; Leng et al., 2019; Sirisomboonchai et al., 2015), and single cell oils (Khot and Ghosh, 2017; Parsons et al., 2018; Qadeer et al., 2017). Bioethanol is another biofuel that can be produced from low-cost substrates such as lignocellulosic resources (Bhutto et al., 2015). Lignocellulosic substrates have recalcitrant polymeric structure and consist of cellulose, hemicellulose, minerals, and lignin (Taherzadeh and Karimi, 2008). Physical, chemical, and microbial pretreatments followed by enzymatic hydrolysis are used to convert polymeric carbohydrates to monomeric sugars, which are digestible by ethanolic microorganisms (Kahani et al., 2017). The most robust industrial ethanol fermenting microorganisms, e.g., Saccharomyces cerevisiae, produce ethanol from the glucose released from cellulosic components, but not from the xylose that is the most abundant fraction in the hemicellulosic part of lignocelluloses (Kim et al., 2015). The cultivation of microorganisms that can consume xylose and produce valuable biomass is one of the promising techniques to improve the economy of the bioethanol production process from lignocelluloses 2

(Satari et al., 2016). Mucor indicus is an oleaginous microorganism that produces high yield bioethanol, comparable to that of S. cerevisiae (Abedinifar et al., 2009). Also, it is among the microorganisms most resistant to the inhibitors found in acid hydrolysates (Sues et al., 2005). M. indicus consumes pentoses as well as hexoses and produces valuable biomass containing appreciable amounts of chitosan and lipid (Satari et al., 2016). Chitosan, a linear polysaccharide with antimicrobial and biodegradable properties (Jeihanipour et al., 2007), has numerous applications in wastewater treatment (Behnam et al., 2015) and as biomaterial in drug delivery and tissue engineering (Ahsan et al., 2018), agriculture, food, and pharmaceuticals (Karimi and Zamani, 2013). The production of microbial lipids, known as single cell oil, by the cultivation of oleaginous microorganisms along with ethanol production by fermentative microorganisms holds great promise for the production of biodiesel and bioethanol (Carvalho et al., 2019; Kim et al., 2015). In other words, high biofuel yields are obtained from lignocellulosic substrates by the production of ethanol from the cellulosic part and lipid/biodiesel from the hemicellulosic part. Besides, lipid accumulated in oleaginous microorganisms does not compete with food sources and does not require arable land (Diwan et al., 2018). Corn stover is one of the sustainable candidates for second-generation biofuel production (Alavijeh and Yaghmaei, 2016), in which the stover byproduct is significant. The stover part of corn stover is usually left in the field after corn grain harvesting and so is not considered a food source as is corn itself (Kemp and Stashwick, 2015; Alavijeh and Karimi, 2019). Also, the lower biomass loss during transportation and storage converts corn stover to a more favorable low-cost feedstock for biofuel production (Petrou and Pappis, 2014).

3

For efficient conversion of polymeric sugars contained in corn stover to bioethanol and biodiesel, the pretreatment should be effective in the separation of both the cellulosic and the hemicellulosic fraction. Dilute-acid pretreatment is able to solubilize the major fraction of hemicellulose into the liquid fraction, while the remaining glucan is in the solid fraction. For this reason, this pretreatment process has been used in several studies for the production of single cell oil using lignocellulosic substrates (Carvalho et al., 2019; Cai, D. et al., 2016; Kim et al., 2015). Moreover, in the cultivation of oleaginous strains for lipid production, several technical strategies are employed to increase the lipid yield. Nitrogen source limitation is among the widely used strategies for induction of higher intracellular lipid synthesis (Huang et al., 2012;; Liu et al., 2017; Rakicka et al., 2015). Kim et al. (2015) investigated the co-production of biodiesel and bioethanol from corn stover pretreated with nitric acid. They used a quasi-simultaneous saccharification and fermentation process of pretreated solid to produce ethanol by Saccharomyces cerevisiae and used dilute acid– pretreated liquid for the production of lipid by Cryptococcus curvatus. In 2013, Ruan et al. cultivated Mortierella isabellina ATCC 42613 in the combination of liquid and solid hydrolysates of corn stover and obtained a lipid yield of 150 mg g-1 consumed carbon source. Also, Chang et al. (2013) used glycerol as a carbon source and fortified the feed with additional nutrients to improve the biomass yield by 26 %, which subsequently increased the lipid yield by 53 %. Few researchers have studied the co-production of bioethanol and biodiesel and all researchers used at least two microorganisms, one for bioethanol production and another for single cell oil production. The present work deals with biodiesel and bioethanol production from corn stover using only one

4

microorganism, i.e., Mucor indicus. To improve lipid production, the aeration conditions were changed from anaerobic fermentation to aerobic cultivation. Moreover, the effect of auxiliary nutrients (fungal extract or yeast extract along with inorganic salts) was evaluated. Briefly, in the integrated batch process, first cellulosic and hemicellulosic fractions of corn stover were converted to their corresponding monomeric sugars by dilute-acid hydrolysis. Then, the xyloserich liquid phase and glucan-rich solid phase were separated. The solid phase was subjected to enzymatic hydrolysis and followed by anaerobic fermentation to reach a significant amount of ethanol as the main product. The residue of M. indicus biomass was separated and cultivated with a xylose-rich solution and essential nutrients to enable the cells to produce a high amount of lipid. The summary of this process is depicted in Fig. 1. Its low acid consumption and short residence time create the potential for industrial applications, provided growth conditions are well-controlled to avoid forming the filamentous form of fungi. To achieve a more economically feasible process on a large scale, a biorefinery producing other valuable components of M. indicus, including chitosan and protein, is also necessary.

5

Fig. 1. The overall batch process for the co-production of ethanol and biodiesel from corn stover by M. indicus as an oleaginous and ethanol-producing microorganism.

6

2. Material and methods The experiment in this study was divided into four different parts, i.e., corn stover pretreatment, enzymatic hydrolysis, ethanol production, and biodiesel production. In this section, the details of the materials and methods used in each part are presented. 2.1. Corn stover Corn stover was collected from a farm in Alavijeh, Isfahan, Iran, in fall 2017. After collection, it was air dried to achieve a moisture content of 6.5 %, then milled and screened to achieve particles with a size between 833 µm (20 mesh) and 177 µm (80 mesh). The sample was stored in resealable plastic bags at room temperature. 2.2. Dilute-acid pretreatment The experiments for dilute-acid pretreatment were designed with a response surface methodology (RSM) for cellulose separation and hemicellulosic sugars hydrolysis. Among the common experimental designs, RSM was implemented via a central composition design. Central composite response designs (CCRD) are often recommended when the design plan calls for sequential experimentation because these designs can incorporate information from a properly planned factorial experiment. A CCRD with a 2-level 2-factor was adopted to evaluate the effects of acid concentration (X1) and time (X2) on glucose and xylose recoveries. Two different levels of independent variables are shown in Table 1, namely low (-1) and high (+1). The experimental design contained 8 axial points, 8 cubic points and 2 central points (total 18 runs), shown in Table 2. All experiments were done in duplicate. The governing equation was adopted to find offset, linear, quadratic, and interaction terms of Eq (1): (1)

7

Dilute-acid pretreatment was performed in 118 mL screw cap serum bottle in an autoclave (PC 22-A, BATEC, Iran). Based on CCRD design, the sulfuric acid concentrations were 0 % (-1), 0.3 % (-0.7), 1 % (0), 1.7 % (+0.7), and 2 % (+1) (v/v) and retention times were 20 min (-1), 35 min (-0.7), 70 min (0), 105 min (+0.7), and 120 min (+1). All experiments were performed at 121 °C, and the biomass concentration for all pretreatments was 10 % dry matter. The optimal conditions for the maximum concentration of glucose and xylose were predicted by the response optimizer of MINITAB software (version 16). The input data for this prediction were provided from empirical responses obtained from the RSM experimental design. After dilute-acid pretreatment, the slurry was washed with distilled water to obtain a pH of 4.55.5 and air dried overnight. The liquid portion of the pretreatment was filtered and stored at -20 °C for further use. Table 1. Coding and levels of dilute-acid pretreatment experiment Factor

Code levels Symbol

Acid concentration

-1

-0.7

0

0.7

1

X1

0

0.3

1

1.7

2

X2

20

35

70

105

120

(%v/v) Time (min)

2.3. Enzymatic hydrolysis The pretreated corn stover was suspended in a 50 mM sodium citrate buffer (pH 4.8) at 8 % (w/w) solid loading and autoclaved at 121 °C for 20 min. To make one liter of sodium citrate buffer, 10.5 g citric acid and 3.7 g sodium hydroxide were mixed in distilled water. Then, the pH of the buffer was adjusted to 4.8 by adding 2M H2SO4 or 2M NaOH (Adney and Baker, 2008). 8

Enzymatic hydrolysis was performed by the cellulase enzyme, Cellic® CTec2, at 125 FPU mL-1, measured by the Adney and Baker (Adney and Baker, 2008) method. This enzyme was kindly provided by Novozymes (Bagsværd, Denmark). For enzymatic hydrolysis, 15 FPU cellulase per gram of biomass was added to the sterilized solution and incubated at 45 °C and 150 rpm for 72 h (Abedinifar et al. 2009). After hydrolysis, a centrifuge (TDZ5-WZ, Selecta Lab, France) was applied at 4,000 rpm for 10 min to separate the hydrolysate, to obtain a clear sugar solution. Then, the clear solution was stored at -20 °C for further use. The yield of hydrolysis was calculated based on the following equation (Salehian and Karimi, 2013): (2)

where 1.111 was the conversion factor of glucan dehydration to glucose during hydrolysis. 2.4. Microorganism and fungal biomass production Mucor indicus CCUG 22424 was obtained from the Culture Collection of the University of Gothenburg, Sweden. This strain was maintained on agar plates containing 20 g L-1 agar, 40 g L1

D-glucose, and 10 g L-1 peptone, at pH 5.5 and 32 °C. After 5 days’ cultivation, to obtain a cell

density of 3 g dry weight L-1, the spores with concentration of 6 ± 3 × 10 6 spores mL-1 were washed, suspended in sterilized distilled water, and cultivated in 50 g L-1 glucose and inorganic salt medium containing 3.5 g L-1 KH2PO4, 7.5 g L-1 (NH4)2SO4, 0.75 g L-1 MgSO4.7H2O, 1.0 g L-1 CaCl2, and 5 g L-1 yeast extract at 32 °C and 150 rpm for 24 h. Afterward, the grown cells were harvested by centrifugation at 4,000 rpm for 10 min and washed twice with distilled sterilized water in order to be used in the fermentation process.

9

2.5. Anaerobic cultivation The liquid fraction of enzymatic hydrolysis (15 mL) was transferred to 118 mL serum glass bottles and supplemented with an inorganic salt medium that was used for the fungal biomass production. The glucose solution was also used as a reference for the carbon source. In addition, to investigate the effect of a different nutrient source (yeast extract or fungal extract) on lipid and ethanol yields, the fungal extract was prepared according to the method developed by Asachi et al. (2011). The combination of the yeast extract with inorganic salts is called the auxiliary nutrient. Subsequently, the pH of the cultivation medium was adjusted to 5.5 ± 0.1 using 1M NaOH, and the bottles were autoclaved at 121 °C for 10 min. After cooling to room temperature, the biomass of M. indicus with a concentration of 1.5 g dry weight L-1 was added to the culture medium. The bottles were then closed tightly with butyl rubber seals and aluminum caps, purged with pure nitrogen and incubated at 32 °C and 150 rpm for 72 h. Liquid samples were drawn at 0, 12, 24, 48, and 72 h for sugar and bioethanol analysis. The bioethanol yield was obtained using Eq. (3) (Salehian and Karimi, 2013): (3)

2.6. Aerobic cultivation After 72 h of anaerobic fermentation, the cultivation medium was centrifuged for 10 min at 4,000 rpm, the fungal biomass separated at sterile conditions and washed twice with distilled sterile water. Afterward, the separated cells (cultivated by acidic hydrolysis of corn stover as carbon source) and the auxiliary nutrient (fungal extract or yeast extract along with inorganic salts) were used as the nutrient source. The solution of glucose and xylose was used as a reference. Aerobic cultivation was performed in a shaker incubator at 32 ºC and 120 rpm for 5 10

days in 500 mL cotton-plugged Erlenmeyer flasks. Liquid samples were drawn after 24, 72, 96, and 120 h of cultivation for analyzing sugar and bioethanol concentrations. 2.7. Estimation of cell dry weight After cultivation, the fungal biomass was harvested by centrifugation at 4,000 rpm for 10 min (3,000 × g). Then, the supernatant was discarded, and the cells were washed twice with distilled water. The biomass was dried in a freeze dryer (Alpha1-2 LD plus, CHRIST, Germany) and the weight of the dried biomass was measured. The biomass yield was calculated based on grams of dried biomass produced per gram of glucose consumed (Satari et al., 2016). 2.8. Determination and extraction of lipid Intercellular lipid was extracted by the Bligh and Dyer method (Bligh and Dyer, 1959). According to this method, chloroform:methanol:water with an optimum ratio of 2:2:1 was used for lipid extraction. The amount of lipid accumulated during fermentation was expressed as gram of lipid per gram dry biomass. Fatty acid methyl esters (FAME) were prepared by the method provided by Sabzalian et al. (2008). Fatty acids were identified by comparison of their retention times to authentic fatty acids standards. 2.9. Biodiesel production The microbial lipid was transesterified using the method presented by Laurens et al. (2012). Based on this method, 800 mg of the lipid extracted from biomass was transesterified with 48 mL of 5 % v/v HCl in methanol for 1 h at 85 °C. Subsequently, the mixture was separated into two phases by centrifugation at 4,000 rpm for 15 min. The upper phase was drawn for the determination of biodiesel yield. The conversion yield of raw oil to biodiesel (%) was calculated using following equation presented by Kılıç et al. (2013): 11

(4)

The fuels properties of biodiesel sample comprising cetane number, cold filter plugging point, cloud point, pour point, kinematic viscosity and density were predicted by Biodiesel Analyzer (Talebi et al., 2014). The fatty acid composition of the extracted lipid was experimentally measured and then inputted to this software to predict the properties of biodiesel. 2.10. Combined severity factor analysis The combined severity (CS) factor of dilute-acid pretreatment was used to convert the reaction conditions of temperature, time, and acid concentration to a single variable for comparing sugar recovery (Lloyd and Wyman, 2005): (5)

where TR was a reference temperature of 100 °C, TH was the hydrolysis temperature in °C, t was reaction time in minutes and pH was liquid pH after pretreatment. 2.11. Analytical methods Glucose, xylose, galactose, arabinose, and mannose in acidic and enzymatic hydrolysate and fermentation broths were determined by high-performance liquid chromatography (HPLC, Agilent 1100, Agilent Technologies, CA, USA) equipped with a Bio-Rad Aminex HPX-87P analytical column (Bio-Rad, CA, USA) and a refractive index (RI) detector. The eluent was deionized water at 80 °C with a flow rate of 0.6 mL min-1. To analyze ethanol, furfural, and hydroxymethylfurfural (HMF), an ion-exchange Aminex column (HPX-87H, Bio-Rad, CA, USA) was used. Sulfuric acid with a concentration of 5 mM was used as the eluent at 60 °C with a flow rate of 0.6 mL min-1. 12

FAMEs were determined by gas chromatography (Sp3420A, Beijing Beifen Ruili Analytical Instrument Co., China) equipped with a capillary splitless injection system and flame ionization detector. For separation, a SolGel-WAX column (30 m × 0.25 mm internal diameter × 1.0 µm film, SGE Analytical Science Pty Ltd., Ringwood, Australia) was used. The carrier gas was nitrogen at a constant flow rate of 1.0 mL min-1 and a split ratio of 20:1. The injector temperature was 220 °C. The flame ionization detector temperature was 250 °C. 2.12. Statistical analysis For computing significant differences among different conditions of dilute-acid hydrolysis of corn stover regarding sugar yield and lipid yield during fermentation, One-way analysis of variance (ANOVA) was employed with Tukey’s test. P-values less than 0.05 were considered significant. All statistical calculations were carried out using MINITAB software (version 16). 2.13. Mass balance The analysis of mass balance was performed based on the dilute-acid and enzymatic hydrolyses of corn stover and fermentation data. The lipid yield was calculated based on the amount of lipid extracted from M. indicus cells during the fermentation of corn stover hydrolysate divided by the dry weight of initial corn stover (Ruan et al., 2013). 3. Results The affecting parameters on dilute acid hydrolysis of corn stover were studied to optimize the glucose and xylose yield. The nondetoxified hydrolysates were then fermented by M. indicus to produce ethanol and lipid. The overall energy derived from corn stover in the form of ethanol and biodiesel was also evaluated.

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3.1. Dilute-acid pretreatment of corn stover The compositional analysis indicated that the biomass of corn stover consisted of 50.6 % glucan, 11.9 % xylan, 12.3 % lignin, and 5.7 % ash. After dilute-acid pretreatment, the liquid and solid phases were separated, and the carbohydrate composition of each phase was measured (Table 2). Depending on the pretreatment conditions, the solid recovery varied between 26.8 % and 52.0 %. The minimum of solid recovery belonged to the pretreatment with the maximum combined severity factor. Solid recovery included of the insoluble components of biomass that could not be dissolved in the aqueous phase, such as ash, lignin, and some hexose and pentose sugars, which still connect to lignin. Moreover, the lignin content was increased from 12.3 % for untreated corn stover to 21 % by increasing temperature and acid concentration. This could be explained by the fact that the more severe pretreatment resulted in more hemicellulose and cellulose solubility (Karimi and Taherzadeh, 2016).

ANOVA and Tukey’s analyses indicated that acid concentration significantly affected the glucose and xylose concentrations (P<0.05), while time was an ineffective parameter on the release of monomeric sugars. To predict the maximum glucose and xylose concentrations simultaneously, a quadratic polynomial equation was obtained using uncoded factors, where C is acid concentration (v/v, %), t is time (min), Y is glucose concentration (g L-1), and X is xylose concentration (g L-1). After ignoring insignificant parameters with low T-value and high p-value, the analysis was repeated and the final empirical models for the concentrations of glucose (Y) and xylose (X) were developed accordingly: [Y (g L-1) = 18.35 + 18.94 C - 0.1913 t- 3.547 C×C + 0.002001 t × t - 0.0680 C × t];

(6)

[X (g L-1) = 4.36 + 6.574 C - 0.1736 t +0.001564 t × t - 0.0627 C × t]

(7)

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For glucose concentration, the linear coefficient of concentration and quadratic coefficient of time offered a positive effect, while the linear coefficient of time, the quadratic coefficient of concentration and the interaction coefficient between time and acid concentration showed negative effects. For xylose concentrations, a similar trend was observed except that there was no quadratic coefficient of concentration. Based on these results, the simultaneous optimization by the response optimizer of Minitab software to obtain the maximum concentration of glucose and the maximum concentration of xylose yielded an acid concentration of 1.8 % v/v and 22 min at 121 °C. The glucose concentration of 32.5 g L-1 and a xylose concentration of 11.8 g L-1 were achieved using the aforementioned optimum operating conditions, which were higher than the results obtained from other operating conditions (7 % error for glucose and 10 % error for xylose). The statistical analysis of the empirical model for glucose and xylose concentrations and the surface and contour plots of this optimization are presented in the Supplementary Material of this paper.

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Table 2. Composition of liquid and solid phases after dilute-acid pretreatment of corn stover 1 Liquid composition (g L-1)

Code level Run

X1

X2

Glucose

Xylose

HMF2

Solid composition (%) Furfural

Untreated

Glucan

Xylan

Lignin

50.58±1.3

11.9±0.7

12.26±0.5

Solid

CSF3

yield (%)

1

-0.7

-0.7

18.4±0.4

1.14±0.1

0.84±0.2

0.61±0.2

53.0±0.9

6.17±0.3

20.63±0.4

36.0±0.1

0.64

2

0.7

-0.7

31.9±0.40

8.6±0.2

2.03±0.1

0.08±0.02

90.2±0.5

0.0±0.0

20.35±0.3

28.9±0.3

1.39

3

-0.7

0.7

24.4±0.12

3.35±0.2

1.36±0.4

0.67±0.4

74.33±0.2

2.3±0.1

21.04±0.5

28.2±0.5

1.12

4

0.7

0.7

30.8±0.10

4.59±0.5

1.96±0.3

0.96±0.1

88.8±0.1

0.0±0.0

19.96±0.3

26.4±0.2

1.87

5

-1

0

14.4±0.04

0.00±0.00

0.0±0.00

0.0±0.0

48.56±0.4

19.29±1.4

18.34±1.2

52.0±0.1

N5

6

1

0

29±0.04

3.22±0.04

0.82±0.2

0.45±0.1

90.8±0.3

0±0.0

20.6±0.4

24.0±0.1

1.77

7

0

-1

29.9±0.16

6.41±0.11

0.61±0.2

0.0±0.0

73.1±0.1

1.11±0.4

20.76±0.3

27±0.9

0.92

8

0

1

30.4±0.40

4.9±0.13

2.26±0.5

0.44±0.1

91.8±0.5

0±0.00

20.16±0.9

26.8±0.7

1.69

9

0

0

25.4±0.30

2.14±0.06

1.19±0.3

0.00±0.0

90.7±1.2

0±0.00

20.31±0.4

26.0±0.3

1.46

32.5±0.40

11.8±0.20

2.02±0.2

0.08±0.01

89.99±0.3

1.12±0.1

20.77±0.4

27±0.1

1.22

Optimum conditions 4 1

Dilute-acid pretreatment performed at solid loading 10 % (w/w) dry matter; all concentrations are the average of two replicates; 2 HMF: Hydroxymethylfurfural 3 CSF: Combined Severity Factor CSF, determined based on (CS= log (t.exp ((TH-TR)/14.25))-pH, in which TH is 121 °C, TR is 100 °C, and pH measured by pH-meter for any pretreatment. 4 Optimum conditions: 1.8 % v/v, 22 min, and 121 °C 5 Negative value

3.2. Enzymatic hydrolysis and ethanol production The concentration of glucose after 72 h enzymatic hydrolysis of corn stover pretreated at the optimum conditions was 49.5 g L-1. The concentration of glucose after 72 h enzymatic hydrolysis of untreated corn stover was 10.8 g L-1, which showed the effect of dilute-acid pretreatment on the release of glucose during enzymatic hydrolysis. The glucan content of dilute-acid pretreated solid was 89.9 % and the yield of enzymatic hydrolysis by cellulase was 100 % based on Eq. (2).

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The results of anaerobic cultivation of this glucose-rich solution by M. indicus are depicted in Fig. 2. Pure glucose at a similar concentration of sugar in the enzymatic hydrolysate was used as a reference in anaerobic cultivation. As can be seen from Fig. 2-A, glucose was assimilated in less than 24 h. The maximum yield of ethanol was 74.5 % (0.38 g g-1) for the enzymatic hydrolysate of corn stover. Ethanol maximum yield increased to 90.1 % (0.46 g g-1) when glucose was the carbon source. The maximum yield of ethanol in the presence of fungal extract alone (68.1 % for hydrolysate and 83.2 % for glucose) was slightly less than the maximum yield of ethanol using the full nutrient supplement containing yeast extract and inorganic salts (76.2 % for hydrolysate and 91.4 % for glucose).

3.3. Microbial lipid accumulation from corn stover hydrolysate The dilute-acid hydrolysate of corn stover, achieved after pretreatment at the optimum conditions, was used for cultivation of M. indicus for lipid accumulation. Moreover, the effect of different nutrient sources, including fungal extract and yeast extract along with inorganic salts on the lipid accumulation, was investigated. Figure 3-A shows glucose concentrations during aerobic cultivation, and Fig. 3-B depicts xylose concentrations. According to Figs. 3-A and 3-B, glucose was first consumed within less than 24 h, whereas xylose was assimilated during the next 72 h. Fig. 3-C shows ethanol concentration during aerobic cultivation. Ethanol produced during aerobic cultivation started to be utilized during the next 120 h (Fig. 3-C), and subsequently further fermentations were conducted for 72 h. It can be seen from Fig. 3-C that the presence of nutrient sources such as a fungal extract or yeast extract along with inorganic salts increased the yield of ethanol, while the fermentation of hydrolysate without auxiliary nutrients showed the minimum ethanol concentration.

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The yield of biomass and lipids in M. indicus through the aerobic and anaerobic cultivations are shown in Table 3. The concentration of biomass during anaerobic fermentation remained almost constant (0.002 g g-1), while the lipid yield was 16.2 mg g -1 for the synthetic medium and 17.7 mg g -1 for enzymatic hydrolysate of corn stover. The lipids accumulated in M. indicus cells during the 72 h cultivation on dilute-acid hydrolysate of corn stover as a carbon source and yeast extract along with inorganic salts as a nutrient source were 147.4 mg g-1 lipid. The lipid yield of 156 mg g-1 was achieved from the synthetic hydrolysate with similar sugar concentrations and yeast extract along with inorganic salts as a nutrient source. This yield increased considerably when the fungal extract without auxiliary nutrients was used. As can be seen from Table 3, the elimination of nutrient source not only affected the yield of lipid accumulated in M. indicus biomass but also significantly decreased the biomass concentration. The best results in Table 3 were integrated into Fig. 4. It can be concluded from these results that the cells consumed the sugars in the process of producing ethanol under anaerobic conditions, while glucose and xylose were consumed in the propagation of cells as well as in ethanol production under aerobic conditions. M. indicus produced 0.38 g g-1 ethanol and 17.2 mg g-1 lipid during 48 h of anaerobic fermentation of the hydrolysate supplemented with sufficient nutrients. The cells produced during 72 h aerobic cultivation on dilute-acid hydrolysate supplemented with yeast extract and inorganic salts accumulated significant amounts of lipid (147.4 mg g-1) and produced ethanol (0.44 g g-1).

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The yield of ethanol (%)

120

A

B

100 80 60 40 20 0 12

24

48

72

Time (h)

Fig. 2. (A) Glucose concentration (g L-1) during anaerobic cultivation and (B) the yield of ethanol (%) in anaerobic fermentation ( ) synthetic medium with fungal extract, ( ) synthetic medium with yeast extract along with inorganic salts, ( ) corn stover hydrolysate with fungal extract, and ( ) corn stover hydrolysate with yeast extract along with inorganic salts.

19

A

B

C

Fig 3. (A) Glucose concentration during aerobic cultivation, (B) xylose concentration during aerobic cultivation, and (C) the yield of ethanol yield (%) in aerobic cultivation. ( ) Synthetic medium without auxiliary nutrient, ( ) synthetic medium with yeast extract along with inorganic salts, ( ) synthetic medium with fungal extract, ( ) corn stover hydrolysate without auxiliary nutrient, ( ) corn stover hydrolysate with fungal extract, and ( ) corn stover hydrolysate with yeast extract.

20

Table 3. Biomass yield (g /g carbon source), lipid content (% wt), and lipid yield (mg/g carbon source) obtained in cultivation under different aeration conditions supplemented with different nutrients. Aeration

Carbon source

Nutrient source

conditions

Yield of

Lipid content

Yield of lipid

biomass (g g-1)

(% wt)

(mg g-1)

Anaerobic

Glucose

Yeast extract and inorganic salts

0.002±0.010

21.1±0.7

19.0±0.3

Aerobic

Glucose and xylose

Without auxiliary nutrients

0.24±0.09

17.6±0.8

78.9±0.6

Aerobic

Glucose and xylose

Yeast extract and inorganic salts

0.55±0.02

19.3±0.2

156.0±2.1

Aerobic

Glucose and xylose

Fungal extract

0.55±0.09

18.5±0.7

146.3±1.4

Anaerobic

Enzymatic hydrolysate

Yeast extract and inorganic salts

0.002±0.010

19.8±0.9

17.7±0.2

Aerobic

Dilute-acid hydrolysate

Without auxiliary nutrients

0.23±0.08

20.3±1.5

87.3±0.8

Aerobic

Dilute-acid hydrolysate

Yeast extract and inorganic salts

0.51±0.03

19.3±0.2

147.4±2.0

Aerobic

Dilute-acid hydrolysate

Fungal extract

0.42±0.09

19.6±0.7

129.5±0.9

21

A

M. indicus

B

Glucose and xylose (carbon source)

Glucose (carbon source)

Anaerobic cultivation

M. indicus (19.0 mg lipid per g consumed sugar)

Aerobic cultivation

Yeast extract and inorganic salts (nutrient source)

Yeast extract and inorganic salts (nutrient source)

Enzymatic hydrolysate of corn stover (carbon source)

Dilute acid hydrolysate of corn stover (carbon source)

M. indicus

Anaerobic cultivation

M. indicus M.mg indicus (17.7 lipid per gcells consumed sugar)

Yeast extract and inorganic salts (auxiliary nutrient)

M. indicus (156 mg lipid per g consumed sugar)

M. indicus Aerobic cultivation

(147.4 mg lipid per g consumed sugar)

Yeast extract and inorganic salts (auxiliary nutrient)

Fig. 4. The lipid yield of M. indicus cells during anaerobic and aerobic cultivation. (A) Glucose and (B) enzymatic hydrolysis of corn stover were used as the carbon source and yeast extract along with inorganic salt as the auxiliary nutrients.

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3.4. The fatty acid composition of lipid and biodiesel production The lipid sample obtained from M. indicus biomass contained 13.7 % palmitic acid (C16:0), 22.7 % palmitoleic acid (C16:1), 53.5 % stearic acid (C18:0), 6.1 % linoleic acid (C18:2), and 4.0 % nonadecanoic acid (C18:3). The lipid extracted from M. indicus biomass was subjected to transesterification reaction along with HCL/MeOH. According to Eq. (4), the biodiesel yield was 54.0 %. The properties of biodiesel are listed in Table 4 and compared to the acceptable quantity of each parameter reported by UNE-EN 14214.

Table 4. The predicted physical properties of biodiesel sample and properties of biodiesel from UNE-EN 14214. Properties

Unit

Biodiesel sample

UNE-EN 14214

Kinematic viscosity, 40 °C

(mm2/s)

4.11

3.5-5

Cetane number

-

57.7

≥51

Oxidative stability, 110 °C

h

14.27

≥6.0

Iodine value

g I2/100g

70.0

≤ 120

Cold filter plugging point

°C

23.51

-

According to the results reported in Table 4, all parameters for the derived biodiesel sample met the European biodiesel standard (UNE-EN 14214).

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3.5. Mass balance Figure 5 demonstrates the mass balance analysis of corn stover for microbial lipid and ethanol production. One hundred grams of dry corn stover contain 50.6 g cellulose, 11.9 g xylan, 12.3 g lignin, and 5.7 g ash. After dilute-acid hydrolysis, 27.3 g glucose and 9.9 g xylose were released in the liquid fraction and 24.3 g cellulose and 0.3 g xylan remained in the solid fraction. After enzymatic hydrolysis of the pretreated solid by commercial cellulase, 24.8 g glucose was obtained. Anaerobic fermentation of the glucose-rich solution, obtained from the enzymatic hydrolysis of the pretreated solid, resulted in the production of 9.4 g ethanol. Aerobic cultivation of the fungal biomass obtained from anaerobic cultivation on dilute-acid hydrolysate produced 12.0 g ethanol and 4.0 g fungal lipid, which subsequently produced 2.2 g biodiesel (total 21.4 g ethanol, 2.2 g biodiesel, and 6.2 g residual biomass per 100 g corn stover).

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Biodiesel (2.2 g)

H2SO4 (34.4 g) H2O (981.3 g)

Ethanol (12.0 g) Transesterification by HCL/MeOH

Corn stover (100 g dry): Glucan (50.6 g) Xylan (11.9 g) Lignin (12.3 g)

Dilute acid hydrolysis

Citrate buffer (546 g) Cellulase (3.9 g)

Enzymatic hydrolysis

Liquid (861 g): Glucose (27.3 g) Xylose (9.9 g)

Aerobic cultivation of M. indicus

Lipid (4.0 g)

-1

M. indicus (9.0 g dw cells of per liter) Solid (27.3 g): Distillated water Washing and Glucan (24.29 g) precipitating Xylan (0.3 g) -1 M. indicus (3.0 g dw the cells Lignin (5.6 g) cells per liter) Liquid (500 g): Glucose (24.75 g)

Anaerobic cultivation of M. indicus

Ethanol (9.4 g)

Solid residue (6.2g): glucan (1.1 g), xylan (0.3g), lignin (4.1 g)

Fig. 5. Mass balance over the process for the production of biodiesel and ethanol from corn stover.

4. Discussion Simultaneous production of biodiesel and bioethanol from corn stover was conducted by using M. indicus. This fungus is as efficient as S. cerevisiae in ethanol production, while it can also consume pentose sugars. Moreover, the fungus is highly resistant to the inhibitory compounds formed during acid hydrolysis (Goshadrou et al., 2011; Sues et al., 2005). The detoxification of acid hydrolysate is typically accompanied by the loss of some sugars and production wastes, while using M. indicus requires no detoxification of hydrolysate. Moreover, its biomass contains valuable chemicals such as chitin and chitosan. Chitosan has numerous applications in agriculture, food, and pharmaceuticals (Karimi and Zamani, 2013). In addition, this

25

microorganism can accumulate appreciable amounts of lipid in its biomass (Satari et al., 2016). Under anaerobic conditions, M. indicus ferments the hexoses to ethanol rapidly and efficiently, but without significant biomass production. On the other hand, it can consume hexoses and pentoses under aerobic conditions and produce biomass together with ethanol (Karimi and Zamani, 2013). The combination of these two features of M. indicus has been employed to investigate lipid accumulation in addition to ethanol production from corn stover as a low-cost lignocellulosic substrate. To increase lipid accumulation in the cells, a rich monomeric sugar source with a low concentration of inhibiting compounds is necessary (Lennartsson et al., 2009). Enzymatic hydrolysate of untreated corn stover is inefficient and slow. Pretreatment would overcome this barrier, enhancing the rate and yield of enzymatic hydrolysis (Abedinifar et al., 2009). Besides being an efficient pretreatment for the improvement of lignocellulosic structure, dilute-acid pretreatment is able to separate hemicellulosic sugars from cellulose. Using this concept, dilute-acid pretreatment at different conditions was used to obtain the maximum amount of xylose from corn stover in the liquid phase. In dilute-acid hydrolyzes, solid-liquid ratio, acid concentration, temperature, and time are the important factors needed to release the monomeric sugars of hemicellulose and cellulose to the liquid phase (Andrew et al., 2014). According to the reported results of dilute acid hydrolysis of corn stover (Esteghlalian et al., 1997; Schell et al., 2003), the solid-liquid ratio was considered constant and equal 100 g L-1. Acid concentration varied from 0 % to 2 % v/v, and pretreatment time from 20 to 120 min. High temperature induces the production of inhibitors such as hydroxymethylfurfural and furfural through the degradation of sugars (Taherzadeh and Karimi, 2007). In this study, diluted sulfuric acid pretreatment was carried out at a relatively low temperature (121 °C) to decrease the rate of inhibitors formation. The ANOVA analysis of xylose and glucose released during dilute-acid

26

pretreatment showed that changing the pretreatment time did not affect the yield of released sugars, while acid concentration had a significant effect on releasing the monomeric sugars. At the constant temperature of 121 °C, the lignocellulosic structure of corn stover was disrupted and hydrolyzed to the monomeric sugars by increasing the acid concentration. By increasing the hydrolysis severity, the higher amounts of sugars were released, while a part of the monomeric sugars was converted to corresponding inhibitors. The highest concentration of both glucose and xylose was obtained after the pretreatment with 1.8 % acid at 121 °C for 22 min. Under these conditions, 84 % of xylose and 54 % glucose were released. Bhandari et al. (1984) reported 78.7 % xylose yield and 18 % glucose yield in dilute-acid hydrolysis of corn stover treated at 155 °C with 1.5 % acid for 31 min. Zhi et al. (2013) achieved 78 % xylose yield and 11.8 % glucose yield through the dilute-acid pretreatment of corn stover at 100 °C with 2 % acid for 100 min. Differences in the compositional structure of corn stover, reactor type, and temperature affect the differences in the glucose yields of this study compared with previously reported results. The enzymatic hydrolysis of the solid phase was performed solely by cellulase (without hemicellulase) because the analysis showed no considerable amounts of xylan in the pretreated solids. Depending upon the pretreatment used for lignocellulosic substrates and the main object consuming the released sugars, different kinds of enzymes for enzymatic hydrolysis have been reported. For example, Josh et al. (2017) developed the mixture of cellulase and hemicellulase to hydrolyze switchgrass pretreated with dilute sulfuric acid, while focusing on lipid accumulation in Yarrowia lipolytica. In another study, Yu and Christopher (2017) focused on ethanol production from poplar wood chips using xylanase. Anaerobic fermentation of the enzymatic hydrolysate of corn stover by M. indicus was accompanied by the minimum amount of biomass production, while it produced appreciable

27

amounts of ethanol. Other researchers have reported this trend for M. indicus under anaerobic conditions (Abedinifar et al., 2009; Satari et al., 2016). Aerobic cultivation on xylose and glucose released from dilute-acid pretreatment, supplemented with different nutrients, showed that the elimination of the nutrient source not only did not affect the lipid accumulation in cells but also had a negative effect on ethanol production. On the other hand, the presence of nutrients increased the biomass production yield, and subsequently increased the amount of lipid yield. However, some reports in the literature describe the nutrient serving as a key factor for the accumulation of lipids in different microorganisms (Jin et al., 2015). Results (Table 3) showed that lipid yield from the dilute-acid hydrolysate of corn stover was lower than that from synthetic media. This may be related to the presence of compounds in the dilute-acid hydrolysate that inhibits cellular growth and propagation. Xai et al. (2012) reported 51.8 % lipid accumulation for Rhodosporidium toruloides Y4 in a synthetic medium, but only 36.4 % lipid accumulation in the ionic liquid hydrolysate of corn stover with similar cell biomass production. In addition, the yield of ethanol production from enzymatic hydrolysate under anaerobic conditions was less than that under aerobic conditions from dilute-acid hydrolysate. Lennartsson et al. (2009) observed the same trend for ethanol production by the same strain of M. indicus, concluding that the effect of inhibitors like furfural and/or acetic acid can induce cells to produce more ethanol. The two-stage process developed in this study increased lipid accumulation in M. indicus biomass from 17.7 mg g-1 carbon source under anaerobic conditions to 147.4 mg g-1 carbon source under aerobic conditions. Produced biofuels (g per 100 g substrate) and total energy derived from the co-generation of biofuels in the current study and other similar research results are summarized in Table 5. As can be seen in Table 5, the procedure proposed in this study not

28

only harvested the maximum amount of total energy but also implemented grown cells in ethanolic fermentation for the production of lipid and subsequently biodiesel. M. indicus could produce almost 150 mg g-1 lipid in addition to 84.3 % (0.43 g g-1) ethanol as a byproduct of corn stover fermentation. Implementing cells grown in anaerobic cultivation into aerobic cultivation could decrease the fermentation time of monomeric sugars by eliminating the lag phase of cells.

29

Table 5. Biofuels and total energy produced from different procedures and microorganisms using lignocellulosic substrates. Substrate

Wheat straw

Fermentation medium

Microorganism

Produced

Total energy

biofuels (g per

(MJ) per kg

100 g substrate)

sugar* 2.83

Reference

Enzymatic and detoxified

Rhodosporidium toruloides as

10.1 g ethanol

(Morikawa

dilute-acid hydrolysate of

oleaginous microorganism

and 0.8 g

et al.,

biomass

and S. cerevisiae as ethanol-

biodiesel

2014)

producer microorganism Corn stover

Enzymatic and non-

Cryptococcus curvatus as

11.9 g ethanol

detoxified dilute-acid

oleaginous microorganism

and 0.06 g

hydrolysate of biomass

and S. cerevisiae as ethanol-

microbial lipid

3.00**

(Kim et al., 2015)

producer microorganism Corn cob

Enzymatic hydrolysate of

Rhodotorula glutinis TP 13 as

17.5 g acetone-

bagasse

alkali pretreatment of

oleaginous microorganism

butanol-ethanol

biomass

and Clostridium

and 3.66 g

acetobutylicum ABE 1201 as

microbial lipid

5.57**

(Cai, D. et al., 2016)

butanol-producer microorganism Corn stover

Enzymatic and non-

M. indicus as oleaginous

21.4 g ethanol

detoxified dilute-acid

microorganism and ethanol-

and 2.2 g

hydrolysate of biomass

producer microorganism

biodiesel

*

6.203

Energy content of ethanol was calculated based on 25.1 MJ per kg of produced ethanol; energy content of biodiesel was calculated based on 37.8 MJ per kg of produced biodiesel; energy content of butanol was calculated as 36 MJ per kg of produced butanol. ** For determination of produced biofuels, the conversion factor for lipid to biodiesel was 90 %.

30

The current study

5. Conclusions Through the integrated process developed in this study, high yields of ethanol and biodiesel were obtained from corn stover by the combination of dilute acid, enzymatic hydrolysis, and fermentation. Dilute-acid pretreatment of corn stover at optimum conditions (1.8 % v/v H2SO4, 22 min, and 121 °C) followed by enzymatic hydrolysis was applied to convert efficiently polymeric sugars to monomeric sugars. The hexose available in the enzymatic hydrolysate was consumed by M. indicus cells, producing ethanol at a high yield. Then, the grown cells were separated and introduced to undetoxified dilute-acid hydrolysate containing glucose and xylose to produce fungal lipid. The distinct advantage of the process is using only one kind of microorganism for both ethanol production and lipid accumulation without losing any of the sugars anchored in lignocellulosic substrate. Very few researches have proposed more than one kind of microorganisms producing at maximum yield both ethanol and lipid (Jin et al., 2015). This process also prescribes using non-detoxified hydrolysate, because M. indicus is among the microorganisms most resistant to the inhibitory compounds available in the acid hydrolysate (Karimi and Zamani, 2013). Overall, this integrated process was able to generate 6.2 MJ energy from each kg of dry corn stover. Further researches could improve the process economically by focusing on the extraction of other valuable components of M. indicus such as chitosan and protein.

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Highlights

A process was developed for biodiesel and ethanol production from corn stover Dilute-acid pretreatment followed by enzymatic saccharification was employed One microorganism, i.e., Mucor indicus, was used for oil and ethanol production Optimization of dilute-acid hydrolysis was performed by a response surface design From 100 g of corn stover, 21.4 g ethanol and 2.2 g biodiesel were produced

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: