Bioconversion of barley straw lignin into biodiesel using Rhodococcus sp. YHY01

Bioconversion of barley straw lignin into biodiesel using Rhodococcus sp. YHY01

Bioresource Technology 289 (2019) 121704 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/...

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Bioresource Technology 289 (2019) 121704

Contents lists available at ScienceDirect

Bioresource Technology journal homepage: www.elsevier.com/locate/biortech

Bioconversion of barley straw lignin into biodiesel using Rhodococcus sp. YHY01

T

Shashi Kant Bhatiaa,b, Ranjit Gurava, Tae-Rim Choia, Yeong Hoon Hana, Ye-Lim Parka, Jun Young Parka, Hye-Rim Junga, Soo-Yeon Yanga, Hun-Suk Songa, Sang-Hyoun Kimc, ⁎ Kwon-Young Choid, Yung-Hun Yanga,b, a

Department of Biological Engineering, College of Engineering, Konkuk University, Seoul, South Korea Institute for Ubiquitous Information Technology and App1ications (CBRU), Konkuk University, Seoul, South Korea c School of Civil and Environmental Engineering, Yonsei University, Seoul, South Korea d Department of Environmental Engineering, Ajou University, Suwon, Gyeonggi-do, South Korea b

GRAPHICAL ABSTRACT

ARTICLE INFO

ABSTRACT

Keywords: Aromatic compounds Biodiesel Biomass Barley straw Lignin Rhodococcus

Rhodococcus sp. YHY01 was studied to utilize various lignin derived aromatic compounds. It was able to utilize pcoumaric acid, cresol, and 2,6 dimethoxyphenol and resulted in biomass production i.e. 0.38 g dcw/L, 0.25 g dcw/L and 0.1 g dcw/L, and lipid accumulation i.e. 49%, 40%, 30%, respectively. The half maximal inhibitory concentration (IC50) value for p-coumaric acid (13.4 mM), cresol (7.9 mM), and 2,6 dimethoxyphenol (3.4 mM) was analyzed. Dimethyl sulfoxide (DMSO) solubilized barley straw lignin fraction was used as a carbon source for Rhodococcus sp. YHY01 and resulted in 0.130 g dcw/L with 39% w/w lipid accumulation. Major fatty acids were palmitic acid (C16:0) 51.87%, palmitoleic acid (C16:l) 14.90%, and oleic acid (C18:1) 13.76%, respectively. Properties of biodiesel produced from barley straw lignin were as iodine value (IV) 27.25, cetane number (CN) 65.57, cold filter plugging point (CFPP) 14.36, viscosity (υ) 3.81, and density (ρ) 0.86.

1. Introduction

bioalcohol) (Awasthi et al., 2019; Bhatia et al., 2017c; Hazeena et al., 2019). It is composed of cellulose, hemicellulose and aromatic polymer lignin (Sahoo et al., 2018). Utilization of lignocellulosic material involved production of free sugars using chemical based pretreatment

Lignocellulosic biomass is considered as a renewable and sustainable raw material for the production of energy resource (biodiesel and



Corresponding author at: Department of Biological Engineering, College of Engineering, Konkuk University, Seoul, South Korea. E-mail address: [email protected] (Y.-H. Yang).

https://doi.org/10.1016/j.biortech.2019.121704 Received 29 May 2019; Received in revised form 24 June 2019; Accepted 25 June 2019 Available online 27 June 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.

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methods and enzyme saccharification followed by fermentation of free sugars (Abraham et al., 2016; Bhatia et al., 2018b; Saini et al., 2015). Most of the biorefineries operating worldwide are based on sugars (cellulose and hemicellulose) for biochemical and bioenergy production (Bhatia et al., 2017c; Flores-Gómez et al., 2018; Singhania et al., 2015). Lignin is a least investigated and unutilized part of biomass and there is need to develop process and technologies for its utilization to improve the economics of commercially valuable products production. According to a report only 2% of the lignin produced throughout the world was industrially utilized in 2010 and most of the lignin disposed of by burning and landfilling (Brzonova et al., 2017). Lignin is a recalcitrant polymer and composed of high energy dense aromatic compounds and various techniques have been reported for its extraction from lignocellulosic biomass (Das et al., 2018; Meyer et al., 2018). Lignin and its derived aromatic compounds can be used to produce various valuable chemicals e.g. vanillin, phenolics derivatives, benzene, toluene, biochar and biobased polymer (polyethylene terephthalate) (Cao et al., 2018; Mialon et al., 2010; Wang et al., 2019; Wang et al., 2018). Biodiesel has higher energy density (38 MJ kg−) as compared to bioethanol (26.8 MJ kg−) and can be directly used to run existing diesel engine (Le et al., 2017). Use of oleaginous microbes to produce biodiesel is a rapidly growing research area and many microbes have been reported for biodiesel production from different carbon sources e.g., Cryptococcus curvatus, Yarrowia lipolytica, Chlorella sp., Rhodococcus sp., etc. (Bhatia et al., 2019a,b, 2015; Kurosawa et al., 2015; Patel et al., 2019). Most of the biofuel production processes are based on the utilization of food crops which hinders their scale up due to the food security issue. Lignin is the second most abundantly available raw material but very few reports are available on its utilization for microbial fermentation and biodiesel production. Lignin is derived from mainly three types of monomer units p-coumaryl, coniferyl, and sinapyl alcohol which give rise to p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) subunits when incorporated into the lignin polymer (Weng et al., 2008). Monomer units are linked through (β-O-4) aryl ether bonds. Depending on the lignin source ratio between different monomer units may vary significantly. Angiosperm plants lignin main components are guaiacyl/ syringyl with the minor content of p-hydroxyphenyl, while gymnosperm and fern lignin is mainly composed of guaiacyl monomer with a small amount of p-hydroxyphenyl unit (Weng et al., 2008). Various catalytic, thermal and biological methods have been reported to depolymerize lignin and resulted in heterogenous product state which made lignin valorization process a challenging task (Linger et al., 2014). Naturally few microbes have evolved metabolic pathways to utilize various aromatic compounds as a carbon source e.g. Oceanimonas doudoroffii, Pseudomonas putida, Pandoraea sp., Ralstonia eutropha, Rhodococcus opacus and utilized for various biochemicals production (Kumar et al., 2017; Le et al., 2017; Linger et al., 2014; Numata & Morisaki, 2015; Wei et al., 2015). Rhodococcus is a well reported oleaginous microbe have pathway for ring opening of aromatic compounds via β-ketoadipate pathway and channel the aromatic derived intermediate carbons into central carbon metabolic pathways (TCA cycle) (Kosa & Ragauskas, 2012). Kosa et al. proposed a theoretical pathway for the degradation of lignin model compound (4-hydroxybenzoic acid) into acetyl-CoA in Rhodococcus sp. which involves various enzymes i.e. protocatechuate 3,4-dioxygenase; β-carboxy-ciscis-muconate lactonizing enzyme; γ-carboxymuconolactone decarboxylase; enol-lactone hydrolase; β-ketoadipate/succinyl-CoA transferase; β-ketoadipyl-CoA thiolase; and acetyl-CoA carboxylase (Kosa & Ragauskas, 2012; Patrauchan et al., 2005). Biodiesel production using various aromatic compounds (p-hydroxybenzoic acid, vanillic acid, vanillin, furfural, hydroxymethylfurfural (HMF) etc.,) derived from lignocellulosic waste during the pretreatment methods have been reported in Rhodococcus (Bhatia et al., 2017b; Kosa & Ragauskas, 2012; Kurosawa et al., 2015). The present study investigated lipid accumulation in Rhodococcus

sp. YHY01 from lignin-derived aromatic compounds p-coumaric acid, cresol and 2,6 dimethoxyphenol which represents lignin model compounds p-coumaryl, coniferyl and sinapyl, respectively. Utilization of different aromatic compounds as a sole carbon source by Rhodococcus sp. YHY01 and their role in growth and lipid accumulation was studied. In the final step, barley straw lignin was used as a carbon source to evaluate the Rhodococcus sp. YHY01 potential for lipid accumulation. Biodiesel produced from the accumulated lipids was further analyzed for its properties. 2. Materials and methods 2.1. Media components, microorganisms, and culture conditions All the media components used in this study were purchased from Difco Laboratories (Becton–Dickinson Franklin Lakes, NJ, USA) and other chemicals; i.e., p-coumaric acid, cresol, 2,6 dimethoxyphenol and all the derivatizing agents were procured from Sigma–Aldrich (St. Louis, MO, USA). Barley straw lignin was a gift from SugarEn company (South Korea). Rhodococcus sp. YHY01 previously isolated, maintained in our laboratory and explored for biodiesel production was used in this study (Bhatia et al., 2019c). Seed culture of Rhodococcus sp. YHY01 was prepared at 5 mL scale in Luria-bertani media by inoculating a loop full culture from LB agar plate and incubating at 30 °C for 24 h (200 rpm). Minimal media (M9-5X) of following composition g/L (Na2HPO4 (33.9), KH2PO4 (15), NH4Cl (5), NaCl (2.5)) was used as a production media with various aromatic compound (p-coumaric acid, cresol, 2,6 dimethoxyphenol) or barley straw lignin as a carbon source. For lipid production Rhodococcus sp. YHY01 was cultured in production media at 10 mL scale in a 25 mL capacity glass tube at 30 °C for 96 h (200 rpm). 2.2. Analytical methods On completion of growth Rhodococcus sp. YHY01 cell culture was centrifuged at 11200g for 10 min to recover the cell biomass. The cell pellet was washed thrice with deionized water to remove any media components. Collected biomass was subjected for lyophilization and dry cell powder was further used for biomass, and lipid accumulation analysis. Dry cell weight (DCW) was quantified by using the gravimetric method and lipid accumulation was analyzed by methylation method by converting lipids into fatty acid methyl ester (FAME). To identify and quantify FAME, GC–MS chromatography (Perkin Elmer Clarus 500) method was used under conditions as already reported (Bhatia et al., 2019b). Fatty acid methyl ester peaks were identified by the mass spectrometric fragmentation data and confirmed by comparison to spectral data that was available from the online libraries of Wiley (http://www.palisade.com) and NIST (http://www.nist.gov). For analysis of aromatic compounds produced by depolymerization of lignin during fermentation, N-methyl-N-trimethylsilyltrifluoroacetamide (MSTFA) method was used. Extraction was performed with ethyl acetate and after nitrogen evaporation residual were subjected to derivatization. Derivatization was performed by dissolving samples in 100 ul pyridine followed by the addition of 500 μL MSTFA with 1% trimethylchlorosilane (TMCS). Samples were heated for one hour at 70 °C to generate trimethylsilyl (TMS) derivatives (Yee et al., 2012). GC–MS analysis of the samples was performed and derivatized compounds were identified using the NIST library. Total phenolics was determined by using UV spectrophotometric method at 280 nm (Aleixandre-Tudo et al., 2017). Sugars were analyzed by using HPLC method. A HPLC system equipped with a Bio-Rad Aminex HPX-87H column (Bio-Rad Co., Hercules, CA, USA) was used to analyze the sugar present in barley hydrolysate. A mobile phase of 5 mM H2SO4 at flow rate of 0.6 mL/min was used, and the column temperature was maintained at 50 °C (Bhatia et al., 2018a). 2

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2.3. Effect of lignin-derived aromatic compounds on Rhodococcus sp. YHY01 growth and lipid accumulation Lignin is basically composed of three types of aromatic precursors pcoumryl, coniferyl, and sinapyl alcohol. Keeping in view this composition three aromatic compounds p-coumaric acid, cresol and 2,6 dimethoxyphenol representing p-coumryl, coniferyl, and sinapyl alcohol respectively were used to study their utilization as a carbon source and role in lipid accumulation in Rhodococcus sp. YHY01. Rhodococcus sp. YHY01 was cultured in M9 production media in the presence of various aromatic compounds (0–0.1% w/v) as a sole carbon source for 96 h and samples were collected at the interval of 24 h to analyze growth and lipid accumulation as mentioned in Section 2.2. 2.4. Role of additional carbon source on aromatic compound utilization Plant biomass is composed of cellulose, hemicellulose, and lignin. During the processing of plant biomass glucose and xylose are the main sugars which are present with the lignin (Bhatia et al., 2016a). Rhodococcus strain is able to utilize only glucose as a carbon source while it have no ability to utilize xylose (Bhatia et al., 2018a; Kurosawa et al., 2013). Glucose was used as an additional carbon source to check its effect on aromatic compound utilization and lipid accumulation. Rhodococcus sp. YHY01 was cultured in the presence of various aromatic compound (0.05%) and 0.05% additional glucose in M9 media under above mentioned conditions and utilization profile of all the aromatic compound and glucose was studied. 2.5. DMSO solubilized lignin fraction preparation Lignin enriched biomass (glucan 37.7%, xylan 2.3%, and lignin 52.8%) prepared from barley straw by H2SO4 catalyzed hydrothermal process followed by enzymatic hydrolysis was provided by SugarEn company (South Korea). To separate additional carbohydrate present in the lignin biomass, it was dissolved in 0.1 M NaOH (pH 12.5). Solubilized lignin was filtered using Whatman filters and the filtrate was precipitated by lowering the pH to 3 with 2 M H2SO4 (He et al., 2017). Precipitates were washed with deionized water and dried to powder using lyophilizer. Lignin powder 0.5% was dissolved in the minimum volume of DMSO and incubated at 37 °C for 2 h under shaking conditions (200 rpm) and then double distilled water was added to adjust final DMSO concentration to 2%. Insoluble lignin fraction was separated by centrifugation at 4000g for 20 min and the supernatant was sterile filtered. The DMSO solubilized lignin fraction was used as a carbon source in further experiments.

Fig. 1. Effect of different concentrations of p-coumaric acid on Rhodococcus sp. YHY01 (a) growth (b) biomass production, fatty acid production, accumulation, and (c) composition of accumulated fatty acids.

2.6. Utilization of DMSO solubilized barley straw lignin as raw material for biodiesel production

calculate the biodiesel properties (Talebi et al., 2014). Various biodiesel properties related to fuel stability and engine performance (iodine value (IV), cetane number (CN), oxidation stability (OS), higher heating value (HHV), kinematic viscosity (υ), and density (ρ), and cold flow related properties (cold filter plugging point (CFPP) were studied and compared with biodiesel produced using other carbon sources and international standards.

Rhodococcus sp. YHY01 was able to utilize various aromatic compounds as a carbon source for growth and lipid accumulation. Rhodococcus sp. YHY01 was cultured in M9 production media at 50 mL scale with DMSO solubilized barley straw lignin fraction (0.05%) as a carbon source under above mentioned conditions for 96 h. Samples were collected at the interval of 24 h and analyzed for depolymerization of lignin using derivatization and GC–MS method. After completion of growth cell biomass was recovered and analyzed for biomass quantification, lipid productivity, and composition.

3. Results and discussions

2.7. Biodiesel properties analysis

3.1. Utilization of different aromatic compounds as a carbon source by Rhodococcus sp. YHY01

Lipids accumulated by the Rhodococcus sp. YHY01 converted into FAMEs using methods mentioned in section 2.2 and analyzed for composition using GC–MS method. Properties of biodiesel depend on the composition of fatty acids. Biodiselanalyzer v1. 1 software (publicly available at http://www.brteam.ir/biodieselanalyzer) was used to

Different aromatic compounds i.e. p-coumaric acid (0–0.1% w/v), cresol (0–0.1% w/v), and 2,6 dimethoxyphenol (0–0.1% w/v) were studied for their utilization as a carbon source for growth by Rhodococcus sp. YHY01 (Figs. 1–3). Rhodococcus sp. YHY01 was able to utilize all the phenolic compounds as a carbon source up to certain 3

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Fig. 3. Effect of different concentrations of 2,6 dimethoxyphenol on Rhodococcus sp. YHY01 (a) growth (b) biomass production, fatty acid production, accumulation, and (c) composition of accumulated fatty acids.

Fig. 2. Effect of different concentrations of cresol on Rhodococcus sp. YHY01 (a) growth (b) biomass production, fatty acid production, accumulation, and (c) composition of accumulated fatty acids.

dimethoxyphenol was the most toxic phenolic compound which effect Rhodococcus sp. YHY01 growth adversely. Kosa et al. studied the effect of different aromatic compounds i.e. 4-hydroxybenzoic acid (4-HB) and vanillic acid on Rhodococcus opacus DSM 1069 growth and reported an increase in lag phase in case of vanillic acid (32 h) (Kosa & Ragauskas, 2012). Inhibitory effect of various aromatic compounds have also been reported for Gluconacetobacter xylinus where coniferyl aldehyde was reported as most potent inhibitor, followed by vanillin, ferulic acid, and 4-HB (Zhang et al., 2014). Ravi et al. studied utilization of various aromatic compounds by Pseudomonas putida KT2440 and reported that specific growth rate on benzoate, p-coumarate, and 4-HB was higher than those on ferulate and vanillic acid (Ravi et al., 2017). Vanillin was reported as inhibitory compound for Streptomyces coelicolor as it inhibit mycelial formation and completely abolished antibiotic production (Bhatia et al., 2016b).

concentration. An increase in Rhodococcus sp. YHY01 growth was observed with the increasing concentration of p -coumaric acid (0.08% w/ v) without any toxic effect (Fig. 1a). Further increase in p-coumaric acid concentration (0.1%) exhibited a toxic effect on Rhodococcus sp. YHY01 growth as an increase in lag phase was observed and it took 48 h to attain maximum growth. In the case of cresol Rhodococcus sp. YHY01 showed an increase in growth up to 0.04% w/v concentration after that an increase in lag phase and reduction in growth was observed (Fig. 2a). Rhodococcus sp. YHY01 was able to achieve the stationary phase only after 72 h. Phenolic compounds 2,6 dimethoxyphenol appear as most toxic as a continues decrease in Rhodococcus sp. YHY01 growth was observed and very little growth was observed above 0.06% w/v (Fig. 3a). Maximum growth i.e. 0.38 g dcw/L, 0.25 g dcw/L and 0.1 g dcw/L was observed at p-coumaric acid (0.08% w/v), cresol (0.06% w/v) and 2,6 dimethoyphenol (0.02%), respectively. The half maximal inhibitory concentration (IC50) for various phenolics was recorded as i.e. p-coumaric acid (13.4 mM), cresol (7.9 mM), and 2,6 dimethoxyphenol (3.4 mM). This study concluded that 2,6 4

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3.2. Role of different aromatic compounds in lipid accumulation in Rhodococcus sp. YHY01 All the phenolics compound studied here also play a role in lipid accumulation. An increase in fatty acid production (0.17 g/L) was recorded up to certain concentration of p-coumaric acid (0.08% w/v) (Fig. 1b). Maximum fatty acid accumulation (49% w/w) was recorded at 0.1% w/v p-coumaric acid. Further increase in p-coumaric acid concentration led to a decrease in fatty acid accumulation. In case of cresol maximum fatty acid production (0.1 g/L) and accumulation (40% w/w) was recorded at 0.06% w/v (Fig. 2b). Further increase in cresol concentration resulted in a rapid decrease in fatty acid production and accumulation. For 2,6 dimethoxyphenol maximum fatty acid production (0.018 g/L) was recorded at 0.04% w/v, while maximum fatty acid accumulation (30% w/w) was at 0.06% w/v (Fig. 3b). Composition profile of fatty acids accumulated in Rhodococcus sp. YHY01 using various phenolics compound was also analyzed. Main fatty acids accumulated in Rhodococcus sp. YHY01 were myristic acid (C14:0), pentadecanoic acid (C15:0), palmitic acid (C16:0), palmitoleic acid (C16:1), heptadecanoic acid (C17:0), cis-10-heptadecanoic acid (cis-10-C17:1), stearic acid (C18:0) and oleic acid (C18:1) (Figs. 1c, 2c, 3c). Composition profile of fatty acids accumulated in Rhodococcus sp. YHY01 with different phenolic compounds as carbon source was same with change in the content of different fatty acids. p-Coumaric acid led to the production of higher content of saturated fatty acid (SFA) (> 80%) and their content increase with the p-coumaric acid concentration in growth media (Fig. 1c). Cresol also favored higher content of SFA over unsaturated fatty acid (UFA), but a decrease in SFA content was observed with the increase of cresol concentration (Fig. 2c). In case of 2,6 dimethoxyphenol, low content of SFA (< 75%) was recorded as compared to other carbon sources (Fig. 3c). At higher concentration of 2,6 dimethoxyphenol an increase in SFA was observed. Almost same fatty acid composition profile was reported in strains Rhodococcus opacus MITXM-61 with increased content of SFA using other aromatic compounds (furfural, hydroxymethylfurfural (HMF), and vanillin) as a carbon sources (Kurosawa et al., 2015).

Fig. 5. Effect of glucose (glu) on (a) utilization of different aromatic compounds (p-coumaric acid (cou), cresol (cre), 2,6 dimethoxyphenol (DMP)), biomass production and lipid accumulation, and (b) composition of fatty acids in Rhodococcus sp. YHY01.

3.3. Biomass and fatty acid productivity using different aromatic compounds as a sole carbon source

was a continues decrease in biomass and fatty acid yield was observed with the increase of phenolics compound concentration in the media due to their toxic effect on Rhodococcus sp. YHY01 at higher concentration. Kosa et al. reported Yx/s 0.59 g/g 4-HB and Yp/s 0.1 g/g 4-HB for Rhodococcus opacus DSM 1069 with 4-HB as a sole carbon source in a nitrogen limited media (Kosa & Ragauskas, 2012).

Rhodococcus sp. YHY01 was able to utilize all the phenolics compounds as a carbon source for growth and fatty acid production. Maximum biomass yield (Yx/s) was observed at lower concentration (0.02%) of phenolics compound i.e. 0.85 g/g p-coumaric acid, 0.65 g/g cresol, and 0.5 g/g 2,6 dimethoxyphenol (Fig. 4). Phenolic compounds also support the production of fatty acids and maximum fatty acid yield (Yp/s) was obtained at 0.04% w/v of p-coumaric acid (0.31 g/g), cresol (0.18 g/g) and 0.02% w/w of 2,6 dimethoxyphenol (0.08 g/g). There

3.4. Effect of additional carbon source on aromatic compounds utilization Effect of glucose (0.05% w/v) was studied on utilization of various phenolics compounds (0.05% w/v) by Rhodococcus sp. YHY01 and their potential to accumulate lipids. Additional glucose source resulted in increased biomass production (glucose: p-coumaric acid (0.53 g dcw/L), glucose: cresol (0.44 g dcw/L), and glucose: 2,6 dimethoxyphenol (0.37 g dcw/L)) and fatty acid production (glucose: p-coumaric acid (0.25 g/L), glucose: cresol (0.19 g/L), and glucose: 2,6 dimethoxyphenol (0.14 g/L) which was higher as compared to pure phenolic compound as a carbon source (Fig. 5a). Rhodococcus sp. YHY01 was able to utilize p-coumaric acid, cresol, and 2,6 dimethoxyphenol up to 72%, 64%, and 30%, respectively in 96 h. Addition of glucose reduced utilization percentage of various phenolics compound and only 32%, 28% and 22% utilization of p-coumaric, cresol and 2,6 dimethoxyphenol was observed. Fatty acid accumulation was also affected by additional glucose and 46% w/w, 44% w/w, and 37% w/w fatty acid accumulation was recorded in case of glucose: p-coumaric, glucose: cresol and glucose: 2,6 dimethoxyphenol, respectively which was higher as compared to pure phenolic compounds as a carbon source

Fig. 4. Growth yield coefficient (Yx/s) and fatty acid yield coefficient (Yp/s) of Rhodococcus sp. YHY01 with different aromatic carbon sources. 5

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broth was analyzed for the presence of aromatic compounds produced during the depolymerization of lignin. Using MSTFA derivatization and GC–MS analysis 3,5 dimethoxy-4-hydroxybenzaldehyde (syringaldehyde, related to lignin model compound syringyl (S)) was observed in the fermented broth. Which indicates the capacity of Rhodococcus sp. YHY01 to depolymerize lignin polymer into its monomer units. Rhodococcus sp. YHY01 able to utilize this as a carbon source as in screening experiments we have observed that it has capability to utilize structurally related syringyl alcohol (2, 6 dimethoxyphenol). Barley lignin resulted in 0.130 g dcw/L biomass production, 0.051 g/L lipid production and 39% w/w lipid accumulation (Fig. 6). Wei et al. reported 0.067 g/L lipid yield in Rhodococcus opacus 1069 from oxygen pretreated kraft lignin as a carbon source (Wei et al., 2015). Composition profile of lipids accumulated by Rhodococcus sp. YHY01 using barley straw lignin as a carbon source was analyzed. Major FAMEs were C16:0 (51.87%), C16:1 (14.90%), C18:1 (13.76%) followed by minor content of C18:0 (9.26%), C14:0 (4.46%), C17:0 (2.54%), C17:1 (1.38%), and C15:0 (1.83%). Same composition profile of fatty acids was observed with barley straw lignin as observed with other aromatic carbon sources (p-coumaric acid, cresol, and 2,6 dimethoxyphenol) with the slight change in content of various fatty acids (Table 1). A decrease in total SFA acid (69.96%) content was observed in the case of barley straw lignin in comparison to other aromatic compounds as a carbon sources. Composition profile of lipids accumulated by Rhodococcus sp. YHY01 is almost same as reported for different Rhodococcus strains with other plants biomass hydrolysate (Table 1) (Kurosawa et al., 2014; Li et al., 2019; Wells et al., 2015). Composition of accumulated biodiesel directly control the properties of biodiesel (Atabani et al., 2012). Properties of biodiesel were analyzed and recorded as iodine value (IV) 27.25, cetane number (CN) 65.57, cold filter plugging point (CFPP) 14.36, viscosity (υ) 3.81, density (ρ) 0.86 (Table 2). The CN value of biodiesel produced from barely straw lignin in between 15 and 100 which ensure proper combustion of

Fig. 6. Time course of biomass production and lipid accumulation in Rhodococcus sp. YHY01 using DMSO solubilized barley straw lignin fraction as a carbon source.

(42% w/w, 41% w/w, 30% w/w, respectively). Addition of glucose also affected the composition profile of fatty acids accumulated in Rhodococcus sp. YHY01. An increase in SFA content was observed in glucose supplemented samples in comparison to pure phenolics as a carbon source (Fig. 5b). This study showed that Rhodococcus sp. YHY01 preferred glucose over other aromatic compounds for growth and fatty acid accumulation as a decrease in phenolic consumption was recorded in the presence of glucose. 3.5. Biodiesel production using DMSO solubilized barley straw lignin and its properties analysis For biodiesel production Rhodococcus sp. YHY01 was cultured in M9 media with DMSO solubilized barley lignin fraction. The fermented

Table 1 Comparison of fatty acid composition profile accumulated in different strain of Rhodocosscus using different carbon sources. Fatty acid

p-coumaric

Cresol

2,6 DMP

Barley lignin

Rhodococcus sp. YHY01 Myristic acid (C14:0) Pentadecanoic acid (C15:0) Palmitic acid (C16:0) Palmitoleic acid (C16:l) Heptadecanoic acid (C17:0) cis-10-Heptadecenoic acid (C17:l) Stearic acid (C18:0) Oleic acid (C18:1) SFA UFA

1.96 0.48 71.63 18.47 2.09 1.37 2.49 1.51 78.65 21.35 This study

2.41 1.82 36.69 13.77 20.54 4.53 13.95 6.29 75.41 24.59

5.85 2.16 52.84 11.42 2.58 1.90 12.42 10.83 75.85 24.15

4.46 1.83 51.87 14.9 2.54 1.38 9.26 13.76 69.96 30.04

Polar wood

Pine hydrolysate

Rhodococcus opacus PD630

Rhodococcus opacus 1069

5 – 41.87 11.25 – – 17.50 24.38 64.37 35.63 (Li et al., 2019)

4.5 5.0 20.0 7.5 9.0 7.0 17.0 30.0 55.5 44.5 (Wells et al., 2015)

Table 2 Properties of Rhodococcus sp. YHY01 biodiesel produced from barley straw lignin and various aromatic compounds and comparison with international biodiesel standards. Properties

DU IV (g I2/100 g) CN CFPP (°C) OS (h) HHV (MJ/kg) υ (mm2/s) ρ (kg/m3)

Rhodococcus sp. YHY01 Coumaric acid

Cresol

2,6 DMP

Barley lignin

21.32 19.81 66.90 9.94 Infinity 39.11 3.70 0.86

24.56 19.37 75.13 16.97 Infinity 30.17 2.8 0.66

24.13 21.12 66.94 19.63 Infinity 39.21 3.83 0.86

30.04 27.25 65.57 14.36 Infinity 39.24 3.81 0.86

6

Algal oil

Petro diesel

EN14214

IS 15,607

– – 52.6 – 1.2 38.25 4.25 0.87

– – 40–51 – – 40–45 1.9–4.1 0.83

– 120 max > 51 – 6.0 – 3.5–5.0 0.86-0.90

– – 48 – – – 2.5–6 0.86–0.90

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engine (Bhatia et al., 2017a). Properties of biodiesel produced from barley straw lignin were similar to the properties of algal fuel, petrodiesel and satisfy the international standard ENI4214, IS15607 (Bhatia et al., 2017a; Pascal Schlagermann et al., 2012).

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4. Conclusions Lignin is the second most abundant unutilized raw material. This is the first study related to the utilization of lignin derived aromatic compounds (p-coumaric acids, cresol and 2,6 dimethoxyphenol) and barley straw lignin as a carbon source for biodiesel production. Rhodococcus sp. YHY01 was able to utilize all the investigated aromatic compounds at lower concentration with different rate and an inhibitory effect was observed as the concentration increased. Different plants lignin have different compositions and led to different aromatic compounds on depolymerization. Such studies will be helpful to design the biodiesel production process from different plants lignin. Acknowledgements The authors would like to acknowledge the KU Research Professor Program of Konkuk University, Seoul, South Korea. This work was supported by Research Program to solve social issues of the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2017M3A9E4077234) and National Research Foundation of Korea (NRF) (NRF-2015M1A5A1037196, 2017R1D1A1B03030766 and NRF-2019M3E6A1065160). In addition, this work was also supported by polar academic program (PAP, PE18900). This work was also supported by Next-Generation BioGreen21 Program (SSAC, PJ01312801), Rural Development Administration. The consulting service of the Microbial Carbohydrate Resource Bank (MCRB, Seoul, South Korea) is greatly appreciated. References Abraham, A., Mathew, A.K., Sindhu, R., Pandey, A., Binod, P., 2016. Potential of rice straw for bio-refining: an overview. Bioresour. Technol. 215, 29–36. Aleixandre-Tudo, J.L., Buica, A., Nieuwoudt, H., Aleixandre, J.L., du Toit, W., 2017. Spectrophotometric analysis of phenolic compounds in grapes and wines. J. Agric. Food. Chem. 65, 4009–4026. Atabani, A.E., Silitonga, A.S., Badruddin, I.A., Mahlia, T.M.I., Masjuki, H.H., Mekhilef, S., 2012. A comprehensive review on biodiesel as an alternative energy resource and its characteristics. Renew. Sustain. Energy Rev. 16, 2070–2093. Awasthi, A., Dhyani, V., Biswas, B., Kumar, J., Bhaskar, T., 2019. Production of phenolic compounds using waste coir pith: estimation of kinetic and thermodynamic parameters. Bioresour. Technol. 274, 173–179. Bhatia, S.K., Yi, D.-H., Kim, Y.-H., Kim, H.-J., Seo, H.-M., Lee, J.-H., Kim, J.-H., Jeon, J.M., Jang, K.-S., Kim, Y.-G., Yang, Y.-H., 2015. Development of semi-synthetic microbial consortia of Streptomyces coelicolor for increased production of biodiesel (fatty acid methyl esters). Fuel 159, 189–196. Bhatia, S.K., Lee, B.-R., Sathiyanarayanan, G., Song, H.S., Kim, J., Jeon, J.-M., Yoon, J.-J., Ahn, J., Park, K., Yang, Y.-H., 2016b. Biomass-derived molecules modulate the behavior of Streptomyces coelicolor for antibiotic production. 3 Biotech 6, 223. Bhatia, S.K., Lee, B.-R., Sathiyanarayanan, G., Song, H.-S., Kim, J., Jeon, J.-M., Kim, J.-H., Park, S.-H., Yu, J.-H., Park, K., Yang, Y.-H., 2016a. Medium engineering for enhanced production of undecylprodigiosin antibiotic in Streptomyces coelicolor using oil palm biomass hydrolysate as a carbon source. Bioresour. Technol. 217, 141–149. Bhatia, S.K., Kim, J., Song, H.-S., Kim, H.J., Jeon, J.-M., Sathiyanarayanan, G., Yoon, J.-J., Park, K., Kim, Y.-G., Yang, Y.-H., 2017b. Microbial biodiesel production from oil palm biomass hydrolysate using marine Rhodococcus sp. YHY01. Bioresour. Technol. 233, 99–109. Bhatia, S.K., Kim, S.-H., Yoon, J.-J., Yang, Y.-H., 2017c. Current status and strategies for second generation biofuel production using microbial systems. Energy Convers. Manage. 148, 1142–1156. Bhatia, S.K., Bhatia, R.K., Yang, Y.-H., 2017a. An overview of microdiesel—a sustainable future source of renewable energy. Renew. Sustain. Energy Rev. 79, 1078–1090. Bhatia, S.K., Joo, H.-S., Yang, Y.-H., 2018b. Biowaste-to-bioenergy using biological methods–a mini-review. Energy Convers. Manage. 177, 640–660. Bhatia, S.K., Gurav, R., Choi, T.-R., Jung, H.-R., Yang, S.-Y., Moon, Y.-M., Song, H.-S., Jeon, J.-M., Choi, K.-Y., Yang, Y.-H., 2018a. Bioconversion of plant biomass hydrolysate into bioplastic (polyhydroxyalkanoates) using Ralstonia eutropha 5119. Bioresour. Technol. 271, 306–315. Bhatia, S.K., Gurav, R., Choi, T.-R., Jung, H.-R., Yang, S.-Y., Song, H.-S., Kim, Y.-G., Yoon, J.-J., Yang, Y.-H., 2019b. Effect of synthetic and food waste-derived volatile fatty acids on lipid accumulation in Rhodococcus sp. YHY01 and the properties of produced

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