Production of biodiesel utilizing laccase pretreated lignocellulosic waste liquor: An attempt towards cleaner production process

Energy Conversion and Management 196 (2019) 979–987

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Production of biodiesel utilizing laccase pretreated lignocellulosic waste liquor: An attempt towards cleaner production process

T

Lohit K.S. Gujjalaa, Tapas K. Bandyopadhyayb, Rintu Banerjeea,c,

⁎

a

P.K. Sinha Centre for Bioenergy and Renewables, Advanced Technology Development Centre, Indian Institute of Technology Kharagpur, India Metallurgical and Materials Engineering Department, Indian Institute of Technology Kharagpur, India c Agricultural and Food Engineering Department, Indian Institute of Technology Kharagpur, India b

ARTICLE INFO

ABSTRACT

Keywords: Biodiesel Aspergillus awamori Kinetic modelling Ricinus communis

Substituting conventional fossil-based energy with eco-friendly and renewable option is inevitable owing to the harmful environmental emissions and steep increase in energy demand. In this context, biodiesel is a clean fuel considering fewer particulate and carbon emissions in comparison to fossil fuels. However, it still faces concerns towards commercialization due to the high cost of raw materials. Thus, in this study, laccase treated waste liquor of Ricinus communis was utilized for lipid production employing Aspergillus awamori. Detailed characterization of laccase treated waste liquor of Ricinus communis revealed that it is favourable for fungal lipid production owing to C/N molar ratio of 132.45 and phenolic content of 0.609 g Gallic Acid Equivalent per L. Major components of phenolics include cinnamic acid, 3-Bromo-5-ethoxy-4-hydroxybenzaldehyde and benzoic acid. Central composite design based response surface methodology was further adopted to maximize lipid content where optimal solution was predicted as 31 °C, carbon to nitrogen molar ratio 156 and inoculum volume 11.5% v/v resulting in 134 mg lipid/g sugar consumed. Further, validation of the process was carried out at pilot scale to justify the feasibility of the process and its applicability for industrial application. Biodiesel, thus produced was characterized for iodine value, acid value and gross calorific value which has been measured to be 73.68 g /100 g, 0.25 mg KOH/g and 37 MJ/kg respectively and conforms to EN 14214 and ASTM D6751. Thus, the present study led to the development of an efficient and clean process for valorization of laccase treated Ricinus communis waste liquor towards biodiesel production.

1. Introduction Post industrialization, concentration of CO2 has increased approximately by 42.8% [1] with major contribution from fossil fuel consumption. Critical issue with elevated CO2 levels is that a great bulk of it remains in the atmosphere for millions of years [2] thus leading to rise in global temperatures due to greenhouse effect. This alarming situation necessitates the adoption of environment friendly and renewable carbon neutral processes [3]. Biodiesel is one such fuel source which is eco-friendly, renewable in nature [4,5] and has the potential to address energy security issues without harming environment [6]. Recent advances in this field of research led to the development of microbial oil based biodiesel production processes [7], salient features of which includes renewability of the microbial lipid, short growth cycles of the microbes involved, etc. Lipid produced from oleaginous microbes are similar in composition to that of vegetable oils with majority of fatty acids constituting neutral lipid [8,9]. Microbes convert the external

⁎

carbon sources into intracellular carbohydrates and then to lipid which generally exists in the form of triacylglycerols (TAGs). Filamentous fungi viz., Mucor sp. [10,11], Cunninghamella sp. [11,12], Aspergillus sp. [11,13,14], Rhizopus sp. [15], etc., have been extensively studied for microbial oil production. Major advantage with the utilization of filamentous fungi is the easy recovery of biomass from the fermentation broth considering its pellet forming ability. Although microbial lipid is used for biodiesel production, still the issue with commercialization of the process is high cost of production medium. In this context, several industrial residues/effluents viz., glycerol, sewage sludge, monosodium glutamate wastewater, food waste, acid hydrolysates of lignocellulosics, etc., have been investigated as media for microbial lipid production. Glycerol, a by-product of biodiesel production process has been reported to be utilized for lipid accumulation in Aspergillus niger (41–57% w/w lipid [16]) and Rhodosporidium toruloides ATCC 10788 [17]. Sewage sludge has also been investigated for cultivating lipid corresponding to Lipomyces starkeyi

Corresponding author at: Agricultural and Food Engineering Department, Indian Institute of Technology Kharagpur, India. E-mail address: [email protected] (R. Banerjee).

https://doi.org/10.1016/j.enconman.2019.06.036 Received 27 March 2019; Received in revised form 27 May 2019; Accepted 16 June 2019 0196-8904/ © 2019 Published by Elsevier Ltd.

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[18]. Industrial effluents from food processing sector have been used for producing microbial lipid (Rhodotorula glutinis 25% w/w lipid [19,20]) thus opening a revenue stream while simultaneously treating wastewater. Integrated approach of wastewater treatment and biomass production has also been reported for microalgae. Freely available nitrogen and phosphorous are taken up as nutrients for microalgal biomass accumulation along with CO2 sequestration [21]. Wastewater from refining units of soybean oil have also been utilized for microbial lipid production along with simultaneous reduction of chemical oxygen demand and oil content [22]. In addition to conventional effluents from industries, hydrolysates from second generation bioethanol refinery have been targeted frequently for microbial lipid accumulation owing to their rich organic content. Conventional acid mediated lignin degradation processes i.e., the first step of bioethanol production, leads to the generation of significant amount of waste liquor rich in sugar moieties. These residues have been utilized for lipid production in a host of filamentous fungi viz., A. niger, A. terreus, C. elegans, C. globosum, M. circinelloides, M. isabelline, M. plumbeus, M. vinacea, N. fischeri, R. oryzae and T. lanuginosus (Zheng et al. [23]). Apart from fungi, oleaginous yeast viz., Trichosporon cutaneum has also been cultivated in acid hydrolysate of lignocellulosics for lipid production (Hu et al. [24]; T. cutaneum cultured in acid hydrolysate of corn stover yielding a lipid coefficient of 150 mg lipid/ g sugar consumed). Cellulosic ethanol wastewater has been utilized as a medium for lipid accumulation in Rhodotorula glutinis along with simultaneous chemical oxygen demand, total organic carbon and total nitrogen removal efficiency of 83.15%, 81.81% and 85.49% respectively [25]. Although this venture of waste to wealth has unlocked a revenue stream, still it faces a major drawback when acid hydrolysates of lignocellulosics are utilized owing to the presence of dehydrated sugars viz., furfurals and hydroxymethyl furfurals which are toxic for microbial growth [26]. In this context, enzymatically delignified waste liquor is of importance since it doesn’t lead to any such dehydrated sugars formation [27,28]. Waste liquor generated subsequent to laccase treatment of lignocellulosic biomass is rich in organic matter and thus it requires proper treatment prior to disposal. Therefore, in the present study, an attempt is made for development of a waste to wealth valorization process using the waste liquor towards biodiesel production as a clean energy source. Considering this scenario, the present manuscript deals with utilization of the waste liquor of Ricinus communis (LTB) for lipid production in Aspergillus awamori. Aspergillus sp., have frequently been chosen for lipid production study owing to their capability to utilize wide range of substrates ranging from industrial wastewaters viz., potato processing wastewater [29], sugarcane distillery wastewater [30] etc. Ricinus communis, castor oil plant, is non-edible and non-grazable in nature owing to the presence of ricin toxin and hence its biomass devoid of its seeds have been extensively utilized for biofuel production [31]. Moreover, the fact that Ricinus communis is fast growing and abundantly available throughout the world majorly in India, Brazil and China makes it a useful resource [32]. In this context, waste streams emanating from Ricinus communis based ethanol production process can be available in industrial quantities to serve a secondary biorefinery. Till date, utilization of laccase treated hydrolysate of Ricinus communis for lipid accumulation and subsequently biodiesel production has not been reported. In the present study, process optimization was conducted to maximize lipid yield using non-linear central composite design (CCD) based response surface methodology (RSM) technique. Further to understand the process in details, time profile of biomass production, lipid production, sugar utilization and nitrogen utilization was studied. Based on the physiology of the fungus, kinetic modelling study was conducted which led to estimation of kinetic constants and yield coefficients. Further, variation in the fatty acid composition of the fungal biomass was studied as a function of time along with characterization of the biodiesel produced. Pilot scale studies on fungal lipid

production and consequent conversion to biodiesel was carried out and has been documented in the present manuscript justifying its relevance for industrial application. 2. Materials and methods 2.1. Microorganisms Aspergillus awamori [33] was used for lipid production studies utilizing laccase treated waste liquor of Ricinus communis. This organism was maintained on potato dextrose agar medium at 4 °C. Laccase used in the study has been extracted from the solid-state fermentation culture of Pleurotus djamor (Bhattacharya and Banerjee [34]). 2.2. Preparation and characterization of LTB of Ricinus communis Ricinus communis has been collected from the campus of IIT Kharagpur and oven dried at 60 °C until constant weight followed by its milling to obtain a particle size of < 0.2 mm. The fine powdered biomass thus obtained was used during the experiments. To prepare LTB of Ricinus communis, powdered biomass was incubated with laccase where the process conditions have been as maintained as suggested by Mukhopadhyay et al. [31]. After delignification process, solid and liquid separation was carried out and the leachate obtained has been referred to as LTB. Proximate analysis viz., total solids, volatile solids, fixed carbon, ash [35] and biochemical characteristics viz., pH, reducing sugar content [36], glucose, xylose, arabinose, total phenolic content and nitrogen content of LTB was measured according to the standard protocols [37,38]. Gas chromatography-mass spectrometry (GC–MS) analysis of LTB was conducted to probe into the lignin derived compounds (Mukhopadhyay and Banerjee [39]). 2.3. Lipid production studies on LTB of Ricinus communis Aspergillus awamori was cultivated on LTB of Ricinus communis by maintaining process conditions viz., temperature, carbon to nitrogen (C/N) ratio and inoculum volume at their optimum values which were determined during the optimization study (Section 3.2) conducted through CCD based RSM approach performed in MINITAB 17 software. The spore count of inoculum and incubation time was maintained as 1 × 106 spores per mL and 5 days respectively. 2.4. Analytical methods Microbial biomass accumulated over time was determined by harvesting it through filtration over Whatman No. 1 filter paper followed by drying in a hot air oven maintained at 60 °C till constant weight. The lipid content (% w/w) was determined according to the protocol of Bligh and Dyer [40] representing % of lipid accumulated in the biomass. Parameters viz., lipid yield (g/L) representing its concentration in the reaction mixture was also measured during the process. Fatty acid composition of the biomass was measured using GC–MS analysis following the protocol of Kumar and Banerjee [33]. Sample for GC–MS analysis was prepared by mixing methanol, fungal biomass and sulphuric acid in the molar ratio 245:1:3.8 while incubating the mixture at 70 °C for 4 h. The resulting solution was allowed to stand till the formation of fatty acid methyl ester (FAME) and glycerol layer. Further, a representative sample from the FAME layer was used for analysis. During analysis, HP-5 column (dimension: 30 m length × 0.25 mm internal diameter × 0.25 μ film thickness; Agilent make) and MS detector were used. Whereas, oven program was maintained as follows: initial oven temperature 150 °C held for 2 min followed by a ramp to 230 °C at 4 °C /min and held for 5 min. GC was run in split mode at a ratio of 1:50 with flow rate of helium at 0.8 mL/min. Temperature for injector and detector was maintained at 240 and 260 °C respectively. MS was 980

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operated in electron impact mode set at 70 eV with scan mode in the range of 50–550 m/z.

Table 1 Characterization of LTB of Ricinus communis.

2.5. Kinetic modelling study In the lifecycle of an oleaginous microorganism, there are two major phases:

• Exponential growth phase, where nitrogen and carbon sources are •

both available in abundance and thus the fungus grows and accumulates the membrane lipid till the nitrogen source is depleted; Stationary phase, where nitrogen source has already been exhausted and hence there is no more observable growth, rather the microbe accumulates lipid and carbohydrates in this phase for cell maintenance. Once the carbon source is depleted, the storage lipid and carbohydrates are utilized for cell maintenance [41,42].

(2)

µ ds Q = SN + L X dt YXs YLS

µ dN = sN X dt YXN µsN = µsNmax

QL = QLmax·

s N ks + s kN + N

s k2 kLs + S k2 + N

pH Total solids Volatile solids Fixed carbon Ash Reducing sugar Glucose Xylose Arabinose Nitrogen Total Phenolic content

– % w/w (wet weight basis) % w/w (dry weight basis of TS) % w/w (dry weight basis of TS) % w/w (dry weight basis of TS) g/L g/L g/L g/L mg/L μg Gallic acid equivalent/ mL

6.0–6.4 1.70 ± 0.076 27.54 ± 1.25 67.15 ± 2.97 5.31 ± 0.24 3.77 ± 0.250 0.82 ± 0.034 2.92 ± 0.142 ND 80 ± 3.6 609.27 ± 29.77

Pilot scale experiments were conducted at P.K. Sinha Centre for Bioenergy and Renewables, IIT Kharagpur premises. Pilot reactor made up of stainless steel with a working volume of 40 L was used for production of Aspergillus awamori utilizing LTB. During the study, parameters viz., lipid yield, biomass titer, etc., have been estimated according to the protocols as mentioned in Section 2.4. Lipid obtained herewith have been transesterified using lipase catalysed process and the subsequent biodiesel produced has been characterized as mentioned in Section 3.6. 3. Results and discussion

The model adopted in the study has been shown in Eqs. (1)–(6).

dL = QL x dt

Value

2.6. Pilot plant study of biodiesel production

• Growth of Aspergillus awamori takes place in a homogenous reactor containing LTB with well mixed environment; • The growth of fungal biomass depends on sugar and nitrogen source available from LTB; • Sugars available in LTB was used for both biomass and lipid synthesis; • Nitrogen was consumed only for fat free biomass synthesis (1)

Unit

ND: Not Detected.

In this paper, kinetic model as proposed by Economou et al. [43] was adopted for studying the kinetics of fungal growth, lipid production, sugar utilization and nitrogen utilization. Utilization of lipid for cell maintenance after sugar reserve gets exhausted has not been taken into consideration. The assumptions of the model are as follows:

dx = µ sN x dt

Components

3.1. Characterisation of LTB To initiate the experimental work on microbial lipid production, detailed characterization of LTB was carried out and is tabulated in Table 1. Based on the results obtained, it can be concluded that LTB is a potential medium for culturing oleaginous microbes which may be due to its mild acidic pH which is favourable for the growth of Aspergillus awamori. This fact is well supported by the reported literature on utilization of lignocellulosic hydrolysates for microbial lipid production [24]. Passamani et al. [45] had studied the effect of temperature, water activity and pH on the growth profile of Aspergillus niger and Aspergillus carbonarius and reported optimal pH range as 5–6.5 and 4–6.5 respectively. LTB was further analysed for reducing sugar and nitrogen content which has been estimated to be 3.77 g/L and 80 mg/L respectively. Thus, C/N molar ratio of LTB can be estimated to be 132.8 approximately. High values of carbon to nitrogen tends to drive majority of carbon towards lipid accumulation and hence will be suitable for lipid production. Composition of reducing sugar has been analysed through high performance liquid chromatography (HPLC) technique which revealed that LTB comprises xylose and glucose in the ratio of 3.56:1. Xylose rich LTB can be utilized by Aspergillus sp. since it possesses xylose transporter, xylose reductase and xylitol dehydrogenase in its metabolic machinery [46]. Total phenolic content of LTB was estimated to be 0.609 g gallic acid equivalent (GAE)/L which majorly comprises cinnamic acid, 3-bromo-5-ethoxy-4-hydroxybenzaldehyde and benzoic acid. Other organic acids detected during GC–MS study include propanoic acid, butanoic acid, acetic acid, 2-Ethyl-3-hydroxypropionic acid, butanedioic acid and 3-Bromo-5-ethoxy-4-hydroxybenzaldehyde. The organic acids detected in this study have been reported to stimulate biomass growth and lipid accumulation in T. fermentans according to a study conducted by Huang et al. [47]. Based on the detailed characterization, it can be inferred that LTB can be an efficient medium for oleaginous lipid production which can be subsequently utilized for biodiesel production.

(3) (4) (5) (6)

where, x is the concentration of biomass g/L, L is the concentration of lipid in the biomass g/L, S is the concentration of reducing sugar in the broth g/L, N is the concentration of nitrogen in the broth g/L, μSN is the specific growth rate of lipid free biomass h−1 while μSNmax represent its maximum value, QL is the specific rate of lipid production h−1 while QLmax is its maximum value, YXS is the yield of lipid free biomass on reducing sugar g/g, YLS is the yield of lipid on reducing sugars g/g, YXN is the yield of lipid free biomass on nitrogen source g/g, ks, kN and kLS are the saturation coefficients in g/L. k2 is a constant (g/L) which makes sure that at high nitrogen content within the broth, lipid content is low. To fit the experimental observation onto the model, “least square error non-linear algorithm” was adopted and the values of kinetic constants and yield coefficients viz., μSNmax, QLmax, YXS, YLS, YXN, ks, kN and k2 were derived [44]. All the simulations were performed in MATLAB 10.0 software. 981

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has subsequently been used for experimentation. Lipid titer, g/L was chosen as the response and fed to MINITAB 17 software for developing a model based on non-linear RSM technique. The predicted and corresponding experimental values of lipid titer have been tabulated in Table 2. From Table 2, it can be inferred that predicted values of lipid titer, g/L are in coherence with the experimental observation which is evident from the correlation coefficient values (R2 = 0.99; R2 adjusted = 0.98; R2 predicted = 0.94). The regression equation obtained from the simulation study has been shown in Eq. (7).

Table 2 Predicted values of lipid, g/L obtained from RSM study along with the experimental results. Sl. No.

Temperature °C

C/N molar ratio

Inoculum volume % v/v

Lipid titer, g/L Experimental

Lipid titer, g/L Predicted

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

25 35 25 35 25 35 25 35 25 35 30 30 30 30 30 30 30 30 30 30

100 100 200 200 100 100 200 200 150 150 100 200 150 150 150 150 150 150 150 150

5 5 5 5 15 15 15 15 10 10 10 10 5 15 10 10 10 10 10 10

0.11 0.10 0.15 0.20 0.19 0.27 0.15 0.36 0.32 0.42 0.35 0.41 0.34 0.45 0.49 0.51 0.52 0.53 0.49 0.52

0.11 0.087 0.13 0.20 0.17 0.27 0.15 0.35 0.34 0.42 0.36 0.41 0.35 0.46 0.50 0.50 0.50 0.50 0.50 0.50

Lipid titer, g/ L=

4.691 + 0.2600 × (Temp) + 0.01081 × (C/N) + 0.0524 × (IV)

× (Temp) × (Temp)

0.000042 × (C/N) × (C/N)

+ 0.000095 × (Temp) × (C/N) + 0.001250 × (Temp) × (IV)

0.004636

0.003636 × (IV) × (IV)

0.000045 × (C/N) × (IV)

(7) Where, Temp represents temperature °C, C/N represents carbon to nitrogen ratio (molar ratio) and IV represents inoculum volume % (v/ v). The statistical significance of the predictions from the model have been checked through ANOVA analysis which has been shown in Table 3. From the ANOVA analysis, it can be suggested that temperature, C/ N ratio and inoculum volume exhibited statistical significance at α = 0.05 level of significance for the linear, square and interaction terms except the interaction between C/N ratio and inoculum volume. The p-value for lack of fit has been observed to be 0.24 which suggests that this specific term is insignificant and hence the overall model can be judged to be significant. The model F-value calculated during ANOVA analysis i.e., 114.02 is much larger than Fcritical value (F 9,10 = 3.02) for α = 0.05 which infers that null hypothesis can be rejected thus signifying the adequacy of the model. Considering the adequacy of the model in deciphering the experimental observation of lipid titer, response optimizer option of RSM has been utilized for predicting input conditions for maximum lipid yield. Optima has been predicted as 31 °C, C/N ratio of 156 (molar ratio) and inoculum volume of 11.5% v/v leading to a lipid titer of 0.51 g/L. The accuracy of prediction has been validated by performing experiments at the derived conditions where lipid titer of 0.508 ± 0.025 g/L has been obtained which translates to a lipid coefficient of 134 mg/g of reducing sugar utilized. Lipid coefficient obtained from this study in corroboration with the findings of Zheng et al. [23] where different fungal strains viz., A. terreus, C. elegans, M. isabellina, M. vinacea, R. oryzae, T. lanuginosus have been utilized for lipid accumulation on acid pretreated hydrolysates of wheat straw with experimental lipid coefficient values observed in the range of 38–123 mg/g of sugar consumed. When compared with synthetic medium for culturing oleaginous microbes for

3.2. Selection of influential parameters for maximizing lipid production of Aspergillus awamori cultured on LTB Process parameters viz., temperature, C/N ratio and inoculum size play a crucial role in lipid accumulation ability of any oleaginous microbe. Temperature is an important stress factor which determines the type of lipid accumulated by the microbe. The type of fatty acid constituting microbial lipid determine the fuel quality of biodiesel since abundance of saturated fatty acids are predominantly desired. C/N ratio is one of the most influential factors in lipid accumulation of oleaginous microbes since its high values (i.e., nitrogen stress) drives the carbon flux towards lipid accumulation once the nitrogen source has been depleted. Inoculum size is another crucial factor which affects the growth of oleaginous microbe and ultimately the lipid reserve since an optimum value will lead to maximum biomass growth. Thus, to study the effect of temperature, C/N ratio and inoculum volume on biomass growth and lipid accumulation ability of Aspergillus awamori, non-linear CCD based RSM model was utilized. In this model, range of input variables were set as temperature (25–35 °C), C/N ratio (100–200, molar ratio) and inoculum size (5–15, %v/v). Design of experiments were generated using central composite design algorithm (Table 2) and

Table 3 ANOVA analysis to assess the adequacy of the second order non-linear RSM model in predicting experimental lipid, g/L. Source

Degrees of freedom

Adj. SS

Adj. MS

F-value

P-value

Model Linear Temperature C/N ratio Inoculum volume Square Temperature × Temperature C/N ratiox C/N ratio Inoculum volume × Inoculum volume 2-Way interaction Temperature × C/N ratio Temperature × Inoculum volume C/N ratio × Inoculum volume Error Lack of fit Pure error Total

9 3 1 1 1 3 1 1 1 3 1 1 1 10 5 5 19

0.424 0.051 0.018 0.006 0.027 0.359 0.036 0.03 0.022 0.013 0.004 0.007 0.001 0.0041 0.002 0.0014 0.428

0.047 0.017 0.018 0.006 0.027 0.119 0.036 0.03 0.022 0.004 0.004 0.007 0.001 0.000414 0.0005 0.000280

114.02 41.74 44.71 15.11 65.39 289.57 89.34 74.59 54.96 10.75 10.91 18.89 2.45

< 0.001 < 0.001 < 0.001 < 0.05 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001 < 0.05 < 0.05 < 0.05 0.149

1.95

0.240

982

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Fig. 1. Contour plots showing the interactions between input variables and their influence on lipid content. Experimental points are represented by dots on the contour plots. Hold values: 150C/N molar ratio, 10% v/v inoculum volume.

Fig. 2. Profile of pH, sugar utilization, nitrogen utilization, lipid accumulation and biomass accumulation with time.

lipid production, LTB shows similar lipid coefficient per unit gram of sugar consumed. This is evident from the report of Ruan et al. [48] who had used synthetic medium for culturing Mortierella isabelline – a filamentous fungus and had obtained lipid coefficient of 140 mg/g sugar. The predicted C/N ratio for maximum lipid content is in coherence with the report of Matsakas et al. [49] who had reported C/N ratio of 100 for maximizing lipid accumulation in Fusarium oxysporum. High C/N ratio tend to direct the carbon flux in the metabolic cycle of an oleaginous microbe mostly towards accumulation of triacylglycerols. However, beyond a certain limit, growth of the organism tends to get affected considering low nitrogen concentration thus affecting the overall lipid titer [49]. A graphical representation of the interaction between input variables and their influence on lipid titer has been shown in Fig. 1. It can be observed that simultaneous increase of C/N ratio and temperature led to an increase in lipid titer up to 0.51 g/L (31 °C and 156C/N molar ratio) beyond which it decreased since optimal temperature for growth of Aspergillus sp. ranges from 25 to 33 °C [45]. Thus, to summarize, an efficient strategy was developed in this study where LTB has been valorized for biodiesel production through a green route which otherwise is a waste stream emanating in the bioethanol production process. Major finding from this study is the production of 134 mg lipid/ g reducing sugar consumed which is in line with the reported literature on microbial lipid accumulation [50] (190 mg lipid/g sugar consumed as accumulated by R. toruloides DSMZ 4444 utilizing corn stover hydrolysate). Higher yields of lipid have been reported by Soccol et al. [51] where 213 mg/g of lipid have been reported for a pilot scale biodiesel production study utilizing sugarcane juice as the carbon source. Although the reported yield is 1.58 folds higher than the present study, still there lies an issue of food vs. fuel controversy if first generation sources viz., sugarcane juice are utilized for microbial lipid production. Thus, the findings from the present study relates closely to the processes which intends to valorize lignocellulosic hydrolysates through a clean and green route.

incubation (Fig. 2). The nitrogen starved conditions might have channelized the excess carbon towards storage lipid accumulation. This trend has been observed till 4th day, beyond which biomass titer and lipid content started decreasing. This may be due to exhaustion of sugar reserves and subsequent utilization of stored carbohydrates and lipid for cell maintenance (Fig. 2). Variation of pH as a function of time (Fig. 2) revealed a rapid decrease in pH value from 6.4 to 5.4 on 2nd day of incubation followed by a stagnation till 5th day (pH 5.1). Similar profile of pH decrease has also been reported by Wild et al. [52] within 48 h of incubation. Detailed analysis of growth and lipid accumulation of Aspergillus awamori gives a comprehensive picture of the variation in biomass growth, lipid accumulation, reducing sugar and nitrogen utilization with time. Major findings from the study reveals that on 2nd day of incubation, nitrogen source gets exhausted beyond which the carbon source is channelized towards lipid accumulation. 3.4. Kinetics of Aspergillus awamori on LTB Kinetic model on biomass and lipid accumulation along with reducing sugar and nitrogen utilization has been studied by fitting experimental data onto the simulated profile as predicted by the coupled model (Eqs. (1)–(6)). Least squared error nonlinear algorithm was used for minimizing error between experimental and predicted trend along with estimation of kinetic and yield constants. Goodness of fit between experimental and simulated profile of process variables have been shown in Fig. 3. From Fig. 3, it can be inferred that simulated profile of process variables (biomass, lipid, reducing sugar and nitrogen) are in good agreement with the experimental results (r2 > 0.96). However, anomaly (i.e., over prediction) was observed while predicting reducing sugar beyond 48 h due to which YLS has been overestimated (predicted value 0.304 g/g; experimental observation 0.134 g/g). The kinetic and yield constants thus estimated from the modelling study have been tabulated in Table 4. The value of maximum specific growth rate estimated from the study corroborates with the reported literature viz., Carlsen et al. [53] (μmax of Aspergillus oryzae: 0.27–0.29 h−1) and Hellendoorn et al. [54] (μmax of Aspergillus awamori: 0.17 h−1). Yield of lipid free biomass per unit of sugar consumed has been estimated to be 0.40 g/g (i.e., 0.623 g of biomass/g sugar consumed considering 35.8% lipid content) which is in coherence with the report of Hellendoorn et al. [54] i.e., 0.6 g/g when grown on a synthetic medium as proposed by Tanaka et al. [55]. Thus, from the kinetic modelling study, it can be inferred that the growth profile of the fungus is being mimicked as evidenced through validation with experimental observation (r2 > 0.96 for all the

3.3. Growth and lipid accumulation ability of Aspergillus awamori on LTB The profile of biomass accumulation, lipid content, reducing sugar and nitrogen utilization of Aspergillus awamori cultured on LTB was studied for a period of 5 days. During experimentation, 31 °C, C/N molar ratio of 156 and inoculum volume of 11.5% v/v was maintained. The results obtained are shown graphically in Fig. 2. From Fig. 2, it can be inferred that maximum biomass titer of 1.51 g/L was obtained after 4 days of incubation with a lipid content of 35.80% w/w. A rapid increase in the biomass titer was observed till 2nd day (0.85 g/L) followed by decelerated growth till 4th day (1.51 g/L) which may be due to exhaustion of nitrogen source on 2nd day of 983

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Fig. 3. Simulated (continuous lines) and experimental profile of biomass (o), lipid (*), reducing sugar (+) and nitrogen (◄) as a function of time.

been observed to be C18:1 which has also been reported by Fakas et al. [56] to be the major fatty acid in C. echinulata and M. isabelline at all stages of growth. The profile of fatty acids as a function of time (Table 5; Supplementary Fig. 1) depicts that till 96 h of incubation, there was a gradual increase in the concentration of C18:1 beyond which no significant increase was observed. Presence of C18:1 as a major constituent proves the candidature of fungal lipid for biodiesel production. Based on fatty acid composition, theoretical estimate of the prospected biodiesel viz., viscosity, cold filter plugging point and oxidative stability have been estimated using stoichiometric equations [57,58]. Viscosity is one of the crucial flow properties of biodiesel since it is an indicator of atomization property and the energy requirement to pump the fuel through the engine. High viscosity biodiesel has to face problems viz., inferior combustion and harmful exhaust emissions. Cold flow properties of biodiesel estimated in terms of cold filter plugging point (CFPP) gives an indication of lowest temperature at which the produced biodiesel tends to get crystallized within the engine pipelines. Oxidative stability is another crucial factor which determines the shelf life of biodiesel. Using stoichiometric equations, theoretical estimate of viscosity, CFPP and oxidative stability have been derived to be 3.99 mm2/s, 12.6 °C and 9.17 h. The derived estimate of viscosity and oxidative stability conforms to the prescribed international standards i.e., ASTM 6751–3 and EN 14214.

Table 4 Kinetic constants derived from the simulation study. Sl. No.

Kinetic parameter

Observed value from this study

1 2 3 4 5 6 7 8 9

μmax, h−1 ks, g/L kn, g/L YXS, g/g qLmax, g/(L*h) YLS, g/g YXN, g/g kLS, g/L k2, g/L

0.30 1.00 0.052 0.40 0.583 0.304 9.54 50.00 1.00

variables investigated in the study). 3.5. Fatty acid composition of the fungal lipid Fatty acid composition of the lipid fraction of Aspergillus awamori cultured on LTB has been analysed through GC–MS study and the obtained results have been tabulated in Table 5. From Table 5, it can be inferred that fungal biomass is rich in C16:0, C18:0, C18:1 and C18:2 all of which are major constituents of neutral lipid desirable for biodiesel production. Predominant fatty acid has Table 5 Fatty acid composition of the lipid fraction of Aspergillus awamori cultured on LTB Time, h

C16:0 (% w/w of total fatty acids)

C16:1 (% w/w of total fatty acids)

C18:0 (% w/w of total fatty acids)

C18:1 (% w/w of total fatty acids)

C18:2 (% w/w of total fatty acids)

C15:0 (% w/w of total fatty acids)

C17:0 (% w/w of total fatty acids)

C20:0 (% w/w of total fatty acids)

24 48 72 96 120

35.32 19.86 21.13 19.94 17.38

ND 0.22 0.34 0.48 0.45

15.99 32.40 18.42 15.32 13.55

30.41 33.30 39.98 47.78 50.58

18.09 14.02 16.83 15.84 17.9

ND ND 3.10 0.44 0.01

ND ND ND 0.067 0.067

ND 0.10 0.08 0.05 0.01

ND: Not detected. 984

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accumulated was further transesterified using lipase following the protocol of Dash and Banerjee [13]. Biodiesel thus obtained was characterized and the results obtained are tabulated in Table 6. From Table 6, it can be inferred that lipid yield in the range of 136.19–137.63 mg lipid/g sugar consumed was obtained. All the process indicators have shown consistency across different scales thus justifying the feasibility of the process. Similar trend was observed in case of biodiesel characteristics viz., acid value, iodine value and gross calorific value. Till date, there have been very few reports on pilot scale studies with lipid from oleaginous microbes utilizing lignocellulosic hydrolysates so the current study will be an addition to the literature. When compared with first generation biodiesel process (Soccol et al. [51]; Pilot scale study (1500 L) for lipid accumulation in Rhodosporidium toruloides utilizing sugarcane juice with a reported lipid coefficient of 213 mg lipid/g sugar consumed), lipid coefficient value obtained in the current study (134 mg lipid/g sugar consumed) is much lower. However, it is not logical to compare lipid coefficient value obtained in the present study with a first-generation process which involves food vs. fuel controversy. Moreover, sugarcane juice is majorly composed of a disaccharide (molecular weight 342.3 g/mol, sucrose equivalent), and hence it is not reasonable to compare the lipid coefficient obtained in the process with an equimolar concentration of monosaccharide. The major emphasis which authors would like to put forward is the utilization of reducing sugar in laccase treated waste liquor of lignocellulosics which contains a mixture of monosaccharides (hexoses and pentoses). Thus, an attempt to utilize pentose sugars along with hexoses for lipid accumulation by Aspergillus awamori is another important issue which has been addressed through the present study. A preliminary economic analysis of biodiesel production revealed the production cost to be US$ 2.12 per litre based on laboratory and pilot scale experiments. When compared with microbial lipid-based biodiesel processes, the current finding shows promise (Satyanarayana et al. [63] reported biodiesel cost as US$ 2.8 per litre). However, firstgeneration processes are still cost effective (Rico and Sauer [64] reported biodiesel production cost to be US$ 0.81/L) but have limitations of being seasonal, having limited productivity/availability, requires large acreage. In this context, the present study is promising since it is independent of any seasonal variation and with a production cycle of five days only. However, studies are still continuing to make the process further cost-effective.

Table 6 Pilot plant study (40 L working volume) of biodiesel production utilizing lipids from Aspergillus awamori grown on LTB

Biomass, g Lipid content, % w/w Lipid coefficient, mg/g sugar consumed Density, g/mL Acid value, mg KOH/g Iodine value, g /100 g Gross calorific value, MJ/kg

1L

40 L

1.51 35.0 137.63 0.865 0.250 73.60 37

59.6 35.1 136.19 0.868 0.27 72.33 36.8

3.6. Lipase mediated transesterification of lipid obtained from Aspergillus awamori Lipid extracted from Aspergillus awamori has been subjected to lipase mediated transesterification [59] for biodiesel production. Biodiesel thus produced has been characterized for density, acid value, iodine value and gross calorific value following the protocol of Kumar and Banerjee [59] and A.O.A.C [60]. Detailed characterization of biodiesel revealed the following properties: density 0.865 g/mL, acid value 0.25 mg KOH/g, iodine value 73.6 g /100 g and gross calorific value 37 MJ/kg. The acid and iodine values observed in the study indicate the non-corrosive nature and low risk of polymerization of the fuel. Moreover, the characteristics of the biodiesel comply with both the European and the American standards for biodiesel quality i.e., EN 14214 [61] and ASTM D6751 [62] respectively. 3.7. Pilot plant study for biodiesel production Pilot plant study has been carried out to validate the process of biodiesel production utilizing lipid accumulated in Aspergillus awamori grown on LTB. A pilot run of 40 L working capacity has been conducted for large scale fungal lipid production where process indicators viz., biomass titer, lipid content (% w/w) and lipid coefficient (mg/g) have been estimated and tabulated in Table 6. A pictorial representation of the pilot plant set up has been shown in Fig. 4. Characteristics of LTB for pilot scale studies were reducing sugar 3.84 g/L, inoculum size 1 × 106 spores per mL with an incubation period of 5 days. Lipid

Fig. 4. Pictorial representation of the pilot study; (A) Pilot reactor, (B) Top view of fungal biomass accumulated over time, (C) Side view of fungal biomass accumulated over time.

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3.8. Conclusion

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[16]

Laccase treated waste liquor of Ricinus communis was found to be a potential medium for lipid production owing to its favourable biochemical characteristics for Aspergillus awamori cultivation. CCD based non-linear RSM model led to maximum lipid yield of 134 mg/g sugar consumed. The predicted optimal solution has been validated at pilot scale justifying the feasibility of the process for industrial application. Detailed profiling of biomass production, lipid accumulation, sugar utilization and nitrogen utilization revealed that beyond 2nd day of incubation, majority of the carbon source has been channelized for lipid accumulation which is evident from the increase in C18:1 content beyond 48 h of incubation. Biodiesel produced from lipase mediated transesterification has been characterized and the results revealed that they are in corroboration with ASTM and EN standards which further substantiate the potentiality of Aspergillus awamori biodiesel in commercial application.

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Declaration of Competing Interest

[23]

The authors declare that they have no conflict of interest.

[24]

Acknowledgement

[25]

Authors acknowledge Indian Institute of Technology Kharagpur for providing infrastructural facility to conduct the presented work.

[26]

Appendix A. Supplementary data

[27]

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.enconman.2019.06.036.

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