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Novel integration of biohydrogen production with fungal biodiesel production process
Bioresource Technology 288 (2019) 121603
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Novel integration of biohydrogen production with fungal biodiesel production process Supratim Ghosha, Shantonu Royb, a b
T
⁎
Porter School of the Environment and Earth Sciences, Tel Aviv University, Israel Department of Biotechnology, National Institute of Technology, Arunachal Pradesh 791112, India
ARTICLE INFO
ABSTRACT
Keywords: Mixture design Continuous hydrogen production Oleaginous yeast cultivation Biodiesel production
An integration of bio-H2 with fungal biodiesel production process was investigated. Highest cumulative H2 production of 3.3 ± 0.20 L L−1 was observed during media optimization using mixture design. Using optimized media composition, continuous H2 production at 0.2 h−1 dilution rate, showed highest H2 production rate, H2 yield and biomass yield of 1020 ± 23 mL L−1 h−1, 2.8 ± 0.1 mols mol−1 reducing sugar and 1.2 ± 0.06 g L−1, respectively. Using the spent media generated from the dark fermentation, oleaginous yeast cultivation was done. Highest biomass and total lipid yield of 6.4 ± 0.20 g L−1 and 0.46 ± 0.04 g g−1 was observed at initial 15% v/v inoculums strength, pH of 5, 1.5 L min−1 aeration rate and 25 °C temperature of cultivation, respectively. Energy recovery improved by 90.3% in integrated process when compared with single stage hydrogen production.
1. Introduction Non-judicious use of fossil fuels has disrupted the global climatic pattern. Use of renewable sources of energy for mitigation of such problem has gained importance in recent times. Renewable fuels such as biofuels which have lower carbon footprint could provide an alternative to conventional fossil-based sources. Hydrogen (H2) has the highest energy density (143 GJ ton−1) and is truly a carbon neutral fuel (Das and Veziroglu, 2008). It can be produced through various convention methods such as water gas process, methane reforming, electrolysis, etc. The biological H2 production processes require less energy and can be produced using renewable feedstock; thereby making the overall process sustainable (Das, 2017; Khanal et al., 2004). The H2 production through dark fermentation has shown higher rates, substrate conversion efficiency and yields when compared with other biological routes. This biohydrogen can be directly converted into electricity through fuel cells. This would certainly help in development of a standalone electricity generation system from organic wastes. Biohydrogen produced can be directly converted into electricity through fuel cells. This would certainly help in development of a standalone electricity generation system from organic wastes. (Ghosh et al., 2018). The various physicochemical parameters such as the type of substrate, pre-processing and fermentation conditions influence H2
⁎
production, which accentuates the necessity for optimization (Rai et al., 2014). Statistical methodologies are often used for process optimization where suitable combinations of physicochemical parameters are studied for predicting maximum product yield (Roy et al., 2014, 2012). There are different statistical optimization methodologies which are widely used for process optimization studies viz. Box-Behnken, Taguchi design, factorial design etc. (Roy et al., 2014). The mixture design is a statistical optimization method which belongs to the class of response surface designs. In this design, the factors are considered in the form of proportions but not magnitude. Such designs are thus quite useful for optimization of formulations or mixtures (Didier et al., 2007). A mixture design also helps in understanding the interaction among the factors in response to the output (Sathish et al., 2008). The volatile fatty acids (VFAs) are the byproducts of dark fermentative hydrogen production. These VFAs includes majorly acetate, butyrate, ethanol and a small fraction of propionate. The energy entrapped in these VFAs remains untapped in the single stage systems. The potential use of VFAs rich spent media for further energy recovery has been explored by various routes such as photo-fermentation to produce biohydrogen (Basak et al., 2014), in the microbial fuel cells to produce electricity (Varanasi et al., 2016), for enhancement of lipids and algal biomass production (Ghosh et al., 2017b) and for hydrogen and methane production (Bolzonella et al., 2018). Cultivation of oleaginous microorganisms has shown a lot of
Corresponding author. E-mail address: [email protected] (S. Roy).
https://doi.org/10.1016/j.biortech.2019.121603 Received 17 April 2019; Received in revised form 31 May 2019; Accepted 2 June 2019 Available online 04 June 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
Bioresource Technology 288 (2019) 121603
S. Ghosh and S. Roy
promise when compared with vegetable oil production. They require a lesser area of land, higher growth rate and high lipid yields (Beopoulos and Nicaud, 2012; Ratledge, 2004). Various organic carbon sources as feedstock (e.g., sugars and organic acids) can be suitably used by oleaginous microorganisms such as microalgae, yeasts, fungi etc. thereby accumulating lipids within the cells (> 20%). The general pathway of lipid synthesis using glucose as a substrate involves a cascade of enzymes which convert it to acetate via mitochondria and then it’s channelized to lipogenesis in the cytoplasm. Whereas, acetate can be directly subjected to lipogenesis if it is provided as a sole substrate to the oleaginous microorganisms. Acetic acid is one of the major components of VFAs which could be used as a suitable substrate for cultivation of oleaginous microorganisms. The composition of lipids produced by oleaginous microorganisms is similar to vegetable oils such as soybean and rapeseed oil. The fatty acids with the chain length of 16 or 18 carbons are prominent in oleaginous microbes (Xu et al., 2015). The feedstock used for cultivation of these oleaginous microbes accounts for 40–80% of the total cost of production thus making microbial oils production economically unfeasible (Cho et al., 2015). Cultivation of oleaginous microbes on low-cost substrates such as volatile fatty acids, lignocellulosic biomass, De-oiled cakes (DOCs) can make the overall production process economically viable (Venkata Mohan and Prathima Devi, 2012). Cultivating the oleaginous microorganisms on the spent medium produced during of dark fermentative H2 production can potentially help in integrating both the processes. Such integration was widely studied using algal biomass cultivation using spent media (Boboescu et al., 2016). Such integration showed potential in achieving both wastewater remediation and simultaneous energy production (Béligon et al., 2018). Cultivation of oleaginous red yeast on spent media generated during of dark fermentative hydrogen production has not been studied extensively. The present study investigates the potential integration of dark fermentative hydrogen production with oleaginous fungal cultivation. For maximization of H2 production, Mixture design was used to optimize the composition of the media components viz. concentration of cane molasses, di-ammonium phosphate concentration and DOC. Based on the optimized parameters, batch and continuous bio-H2 production were studied. The spent medium generated during dark fermentation was further used for cultivation of oleaginous yeast. Various parameters such as adjustment of initial pH, inoculums strength, aeration rate and temperature of cultivation were optimized for maximization of biomass and lipid yield. Confocal microscopic studies were performed to observe the lipid accumulation profile. Overall energy recovery was also studied for the combined process. To best of our knowledge, no previous reports are available regarding the integration of dark fermentative hydrogen production with oleaginous fungal cultivation. Such integration could certainly improve the overall energy recovery from a single substrate.
2.1.2. Setup for batch hydrogen production The batch fermentation was performed for biohydrogen production using a cylindrical double jacked reactor with working volume of 400 mL (Mishra et al., 2017) at 37 °C and 200 rpm. The temperature was maintained by using circulating water bath and rpm was maintained using magnetic stirrer. The anaerobic condition was created by flushing nitrogen gas (N2) through the reactor for 30 mins. 2.1.3. Setup for continuous hydrogen production Continuous cultivation was performed in a cylindrical double jacked reactor (500 mL) with working volume of 400 mL. The feed was infused into the reactor and simultaneously spent media was expelled out using peristaltic pumps. The dilution rates were varied from 0.1 to 0.3 h−1. The fermentation was performed at 37 °C and 200 rpm. Stable hydrogen production rate with respect to each dilution rate was considered as steady state (Ghosh et al., 2018). 2.1.4. Oleaginous yeast cultivation The Rhodotorula minuta MTCC 2518 was used to study lipid production process studies. The cultivation media contains glucose (SRL, India) 1% (w/v), yeast extract (SRL, India) 1% (w/v) and Peptone (SRL Laboratories, India) 0.4% (w/v) at 32 °C in an incubator shaker (New Brunswick Scientific, New Jersey, USA) at 160 rpm. 2.2. Maximization of biohydrogen production using mixture design Cane molasses, Di-ammonium phosphate (DAP) and deoiled cake (DOC) were three components of fermentation media from which a suitable concoction was formulated for maximum gaseous energy recovery. For formulation, a mixture design was used where the effects of the components in a blend was observed on an output variable (H2 production in this case). The mixture design could be as expressed mathematically by below mentioned equation (Eq. (1)): p
Y=
Xi = X1+X2 + X3 + .....Xp = 1;
Xi > 0; i = 1,2,3,4. ...,p
i=1
(1)
where ‘p’ represents the number of ingredients, and Xi denotes the proportions of the components. A simplex-lattice design was employed to study the effect of cane molasses, Di-ammonium phosphate (DAP) and DOC proportion on bio-H2 production. The respective proportions were fermented in 500 mL double jacketed glass reactor with 400 mL working volume. In this design, the total proportions of the different components were made to 100% i.e., 6 g. Total 10 experiments were proposed by the design. This includes three experiments having respective pure components, whereas other three experiments were with binary blends for each possible two-component. Moreover, additional three experiments were performed with complete concoction, where the centroid point represents equal proportions of all three components. The H2 output values with respect to all the array of 10 experiments proposed by the design was shown in Table 1. Analysis of the mixture design was done by MINITAB 15 using following equations:
2. Materials and methods 2.1. Microorganism and culture conditions
Table 1 Array of experiments proposed by Mixture design.
2.1.1. Growth and culture conditions The H2 production studies were performed using Clostridium acetobutylicum MTCC 11276. The growth media composition includes glucose 1% (w/v), Tryptone) 1% (w/v), DOC 0.4% (w/v) and Cysteine HCl 0.1% (w/v) at 37 °C in an incubator shaker (New Brunswick Scientific, New Jersey, USA) at 160 rpm. The organism was grown in 100 mL serum bottles (Sigma Aldrich, USA) with a working volume of 70 mL. A strict anaerobic condition was maintained by flushing nitrogen gas (N2) through the reactor. For bio H2 production, cane molasses, di-ammonium phosphate (DAP) (SRL Laboratories, India), DOC and Cysteine HCl 0.1% (w/v) was used. 2
Exp No.
Molasses
DAP
DOC
Bio-H2 production (L L−1)
1 2 3 4 5 6 7 8 9 10
1.00000 1.00000 1.33333 0.00000 0.66667 0.33333 2.00000 0.33333 0.00000 0.00000
0.00000 1.00000 0.33333 2.00000 0.66667 0.33333 0.00000 1.33333 0.00000 1.00000
1.00000 0.00000 0.33333 0.00000 0.66667 1.33333 0.00000 0.33333 2.00000 1.00000
2.30 2.40 3.40 0.00 3.20 2.50 0.60 2.10 0.20 0.22
Bioresource Technology 288 (2019) 121603
S. Ghosh and S. Roy
acid methyl esters (FAMEs) along with unreacted triglycerides. The hexane layer was washed until neutrality with deionized water (5 mL) and was analyzed using a gas chromatograph equipped with a FID detector (Clarus 500, PerkinElmer) (Ghosh et al., 2017a). On comparing the respective retention times and fragmentation patterns with standards of fatty acid methyl ester (FAME) mixture were identified (37mix, Supelco Inc., Bellefonte, PA).
p
Y=
i Xi
Linear Model
(2)
i=1 p
Y=
i Xi
p
+
i
i=1 p
Y=
i Xi
+
i=1
p i
i Xi Xj
i Xi Xj
+
Quadratic Model p i
i Xi Xj Xk
(3) Special Cubic
(4)
2.4.4. Fluorescence microscopy study of lipid accumulation Nile red (0.1 mg dissolved in 1 mL acetone) was used to stain the neutral lipids accumulated in the microorganism. A100 µL of Rhodotorula sp., cell suspension was mixed with 10 µL of Nile Red (0.1 mg/mL) and 2 mL PBS. The stained cells were kept in dark for 5 min (Kimura et al., 2004). Leica Dmi8 live cell imaging microscope was used for microscopic images, with Leica DFC365 FX camera and Las X software version, using the 40× lens. Nile red stains neutral lipid droplets in red color but for selectivity, it can be viewed yellow gold fluorescence (450–500 nm excitation; > 528 nm emission). The fluorescence images were acquired using Leica Led module 530for Nile Red and the transmitted light images were acquired using differential interference contrast.
2.3. Lipid extraction from the fungal biomass Lipid extraction was performed using the modified Bligh and Dyer method (1959) (Bligh and Dyer, 1959). The fungal cells were sonicated (20 kHz, 5 min) and chloroform: methanol (2:1; v/v) solution was added. The resulting solution was stirred at room temperature for 2 h at 150 rpm. The mixture was centrifuged at 3000×g for 5 min to obtain the supernatant which was further mixed with 0.034% (w/v) MgCl2 and 0.88% v/v 2 N KCl. The chloroform: methanol: water (2:2:1.8 v/v/v) was mixed with the upper phase and followed by centrifugation at 6000×g for 4 min led to phase separation. The organic phase was aspirated out and vacuum dried to obtain the total lipids. Gravimetric measurements were utilised to determine the biomass content (g L−1) and lipid yield (g g−1). The lipid content (% w/w) of the oleaginous yeast was measured by the equations followed as:
CL (\%) =
CT (g L 1) × 100 X (g L 1)
2.4.5. Reducing sugar estimation Reducing sugar content (glucose) of the sample was estimated spectrophotometrically using DNS (dinitro salicylic acid) reagent method (Lorenz, 1959). DNS reagent was prepared by mixing solution A and solution B in a fixed concentration. Solution A contained 300 mg of Rochelle salt (Sodium potassium tartarate) dissolved in 500 mL of distilled water. Solution B contained 10 g of dinitrosalycylic acid dissolved in 200 mL of 2 N NaOH solution. DNS reagent was prepared by mixing 10 mL of solution A, 4 mL of solution B and 6 mL of distilled water. Calibration curve of glucose was prepared by using various known glucose concentrations as standards. Reagents were added as follows and OD (optical density) was read at a wave length of 540 nm.
(5)
where CL = Lipid content; CT = Total Lipids; and X = Biomass content. 2.4. Analysis 2.4.1. Biogas and fatty acid analysis Hydrogen concentration was measured by gas chromatography (GC Agilent Technology 7890A U.S.A) using thermal conductivity detector (TCD) equipped with a stainless steel packed with Porapak Q (80/100). The oven and the detector operational temperatures of the injection port were 80 °C, 150 °C and 200 °C, respectively. The carrier gas (N2) was used at a flow rate of 20 mL min−1. Flame ionization detector (FID) was used for estimation of volatile fatty acids and FAME. The column used was a capillary column (0.5 mm diameter × 30 m length) at preset injector, oven and detector temperatures of 250 °C, 200 °C and 280 °C respectively. The FID operational temperatures of the injection port, the detector and programmed column were 220 °C, 240 °C, and 130–175 °C, respectively. For flame ignition, a mixture of H2 and air at a flow rate of 30 mL min−1 was used (Ghosh et al., 2018).
2.4.6. Volatile fatty acids (VFA) analysis The fermentation broth was centrifuged at 10,000 rpm for 5 min in order to separate the cell debris from the supernatant. The supernatant was then distilled to concentrate the VFAs. VFA and alcohols present in the spent media were estimated by GC using FID Detector, capillary column (0.5 mm diameter × 30 m length) at preset injector, oven and detector temperatures of 250 °C, 200 °C and 280 °C respectively. The FID operational temperatures of the injection port, the detector and programmed column were 220 °C, 240 °C, and 130–175 °C, respectively. For flame generation a mixture of hydrogen and air at a flow rate of 30 mL min−1 was used.
2.4.2. Biomass estimation The biomass production profile of Rhodotorula minuta was measured using the gravimetric method. A known volume of fungal culture was collected periodically and was centrifuged at 6500g for 10 min. The supernatant was discarded and the pellet was then washed twice with deionized water followed by drying at 60 °C for till the constant weight was observed.
2.5. Calculation for net energy recovery Palmitate has been used as common fatty acid based on which the estimation of total energy content of biodiesel was calculated. The stoichiometric equation representing complete combustion of oleate methyl ester and hydrogen was shown in Eqs. (6) and (7).
2.4.3. Biodiesel production and FAME analysis About 1 g of freshly extracted fungal oil was used for transesterification study. The transesterification was carried on in 50 cc glass vials with airtight caps (Schott Duran, Hattenbergstrasse, Germany) that retained any vaporized methanol to the reacting mixture. The vials were kept in a temperature-controlled incubator. The mixture was stirred by using a cyclomixture at every 30 min interval. Transesterification was performed with acidified methanol (10 mL) at 90 °C for analysis of fatty acids in the fungal biomass. Phase separation was performed using hexane (5 mL) as a solvent in order to obtain fatty
C18 H36 O2 + 26
2H2 (g)+ O2 (g)
1 O2 2
18H2 O + 18CO2 + (11670 kJ mol 1)
2H2 O(l) + (241 kJ mol 1)
(6) (7)
Along with byproducts, energy is also released in terms of heat of enthalpy. So theoretically, 11790.5 kJ per mole was the total energy that can be recovered from complete combustion of 1 mol of palmitate methyl ester and hydrogen. 3
Bioresource Technology 288 (2019) 121603
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3. Results and discussions
Table 2 ANOVA analysis of the mixture design models.
3.1. Maximization of H2 production using mixture design All the 10 experiments showed variations in H2 production. The experiment no. 3, gave highest cumulative production of 3.4 L L−1. In all the experiments (3, 5, 6 and 8) where cane molasses, DAP and DOC were present in the concoction showed substantial improvement in H2 production. Experiment no. 5 had all the components in equal proportion and showed cumulative production of 3.2 L L−1. Experiment no. 9 and 10 had no cane molasses content and showed very little H2 production probably from the nutrients available in DOC. Whereas, experiment no. 4 had di-ammonium phosphate (DAP) as sole component in the concoction showed no H2 production. Many volatile fatty acids were produced during fermentation that leads to decrease in pH of the media thereby hindering further growth (Grzelak et al., 2018). The use of DAP in the concoction was to provide a buffering environment so as to prevent sudden decrease in pH of the fermentation media (Lin and Lay, 2004). The DAP also acts as inorganic nitrogen source. Combination of only cane molasses and DAP (Experiment no. 2) showed less H2 production due to absence of valuable micronutrients provided by DOC. The DOC provides valuable vitamins and nutrients that are indispensable for H2 production (Ghosh et al., 2018). A surface plot and contour plot showed the optimum zones of combinations that would maximize the H2 production (Fig. 1a and b). From the model optimum combination of the components (in terms of proportion) for maximization of H2 production was predicted to be 0.863717 of cane molasses, 0.545455 of Di-ammonium phosphate (DAP) and 0.59082 of DOC, respectively with desirability of 1. This corresponds to 5.182 g of total reducing sugar in cane molasses, 3.27 g of Di-ammonium phosphate (DAP) and 3.55 g of DOC, respectively in 400 mL of water. The predicted model showed R2 = 0.98 with F = 17.61 value showing significance at P < 0.05. The ANOVA analysis showed significantly higher F values than P values for quadratic and special cubic models (Table 2). Linear model showed poor significance when compared with other models. The residual error was also significantly less. Similar application of mixture design for H2 production using different agro residues had shown significant improvement (Prakasham et al., 2009).
Source
DF
Seq SS
Adj SS
Adj MS
F
P
Regression Linear Quadratic Special cubic Residual Error Total
6 2 3 1 3 9
14.9066 2.0556 11.4982 1.3528 0.4232 15.3298
14.90655 0.27440 5.01015 1.35279 0.42321
2.48443 0.13720 1.67005 1.35279 0.14107
17.61 0.97 11.84 9.59
0.019 0.473 0.036 0.053
which was near to the mixture design values. A 79% consumption of reducing sugar was also observed during 20 h fermentation. The fermentation broth has pH of was 3.6 indicating production of soluble acidic metabolites such as acetate (A), butyrate (B), propionate and ethanol. Highest butyrate, acetate and ethanol concentration of 1105 mg L−1, 574 mg L−1 and 152 mg L−1 was observed respectively. Butyrate production superseded acetate from 8th h onwards. The B/A ratio were below 1 up to 4th h and increased from 1.13 to 1.92 as fermentation proceeded. 3.2. Continuous biohydrogen production using CSTR The optimum proportion of media components which was estimated in above section was used for continuous H2 production was studied in a continuous stirred tank reactor (CSTR). The influence of varying dilution rates on steady state biomass concentration, H2 production rate, H2 yield and VFAs concentration was shown in Fig. 3. At 0.2 h−1 dilution rate, a highest rate of H2 production, H2 yield and biomass yield of 1020 ± 23 mL L−1 h−1, 2.8 ± 0.1 mol mol−1 reducing sugar and 1.18 ± 0.042 g L−1, respectively was observed. The observed yield was 70% of the theoretical yield indicating the significant energy recovery. A decrement in H2 production rate, yields and biomass concentration was observed on increasing dilution rate beyond 0.2 h−1. The H2 production rate, yields and suspended cells concentration showed a decreasing trend beyond 0.2 h−1 dilution rate. The H2 production ceased at a dilution rate of 0.3 h−1. This could be probably due to inadequate time for microbes inside the reactor to convert substrate to product, low hydraulic retention time, and eventually occurrence of washout of metabolically cells from the reactor (Pugazhendhi et al., 2017). In dark fermentative H2 production, acetate, butyrate, propionate, ethanol, etc. were produced as byproducts (Venkata Mohan and Prathima Devi, 2012). The steady state metabolite concentration of the respective dilution rates was estimated from the effluents exiting from the reactor. The volatile fatty acids (VFAs) were in the order: butyrate > acetate > ethanol for all the dilution rates. The concentration of 1098 ± 84 mg L−1 butyrate, 470 ± 25 mg L−1 acetate, and 150 ± 30 mg L−1 ethanol, respectively was observed in the spent media. A concurrent variation of
3.1.1. Batch fermentation using optimized formulation derived from mixture design Based on the optimized values predicted by the mixture model, batch fermentation was performed to validate the output experimentally. The cumulative H2 production, pH profile, reducing sugar profile, volatile fatty acids (VFAs) profile and B/A ratio was shown in Fig. 2a and b. Maximum cumulative H2 production of 3.62 L L−1 was observed
Fig. 1. a) Response surface plot and b) Contour plot of mixture design representing effect of proportional mixing of media components on hydrogen production. 4
Bioresource Technology 288 (2019) 121603
S. Ghosh and S. Roy
Fig. 2. Batch fermentation studies using optimized media composition: a) H2 production, pH and reducing sugar concentration profile; b) Volatile fatty acids profile and B/A ratio. Error bar represents standard deviation of triplicate experiments. Temperature 37 °C and 200 rpm was maintained in all the experiments.
VFAs concentrations and hydrogen yield was observed on varying dilution rates. The VFAs profile indicated that a mixed acid fermentation. The VFAs profile is indicative of the dynamic changes in the ratio of NAD+/ (NADH + H+). If ethanol: acetate ratio is 1:1 then it indicates a balance between NAD+/(NADH + H+). Moreover, if this dynamic equilibrium gets disturbed due to shifts from 1:1 ratio then it implies an unbalanced fermentation process. In one such study, H2 production using Enterobacter aerogenes MTCC 2822 showed an unstable ethanol: acetate ratio (Rai et al., 2014).
cultivation of yeast which includes adjustment of initial pH, inoculums strength, aeration rate and temperature of cultivation. 3.3.1. Maximization of biomass and lipid yields by optimizing process parameters The growth of Rhodotorula minuta was studied using spent media (generated during 0.2 h−1 dilution rate of continuous process) which was adjusted to various pH values using 1 N HCl (pH 4.5, 5, 5.5, 6 and 6.5) (Fig. 4a). The cultivation conditions were as follows: temperature of 25 °C, aeration rate of 1 L per minute (LPM), 5% (v/v) inoculum strength. Initial pH of the medium had an influence on the yield of biomass and lipids. Biomass and lipid yield of 4.5 ± 0.21 g L−1 and 0.13 ± 0.04 g g−1 was observed on using spent media with adjusted pH of 5. The biomass yields increased (∼60% improvement) when the pH of media was adjusted. Initial pH of 6 and 6.5 did not show any significant improvement in biomass yield. Literature reports also suggest that Rhodotorula sp. grow favorably in the pH range of 5–6
3.3. Second stage integration for cultivation of Rhodotorula minuta using spent media The VFAs rich spent media was used for cultivation of oleaginous yeast Rhodotorula minuta. The acetate and butyrate were the primary substrate metabolized into lipids. The spent media had pH of 3.5–4 which needs further fortification before it can be used further
Fig. 3. Continuous hydrogen production study using optimized media composition. 5
Bioresource Technology 288 (2019) 121603
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Fig. 4. Valorization studies of spent media produced during biohydrogen production process: a) Biomass and lipid yield using pH adjusted spent media; b) Biomass and lipid yield on using different temperatures; c) Biomass and lipid yield on using different inoculum strengths and d) Biomass and lipid yield on using different aeration rate. Error bar represents standard deviation of triplicate experiments.
(Tchakouteu et al., 2017). The pH adjusted spent medium (pH 5) was utilized for further cultivation of oleaginous yeast. The aeration rate was set to 1 LPM with inoculum strength of 5% (v/v) (Fig. 4b). Temperature of cultivation was varied from 20 °C to 40 °C. The variation in biomass and lipid yield was observed with changing. Biomass and lipid yield of 4.5 ± 0.21 g L−1 and 0.13 ± 0.04 g g−1 was observed at 25 °C. Growth and lipid metabolism are essential for proper activity of enzymes. At optimum temperature, there is maximum activity of the enzyme system which leads to maximum biomass and lipid content (Yang and Hu, 2019). For any bioprocess, inoculum strength also plays an important role in determination of product yield. Optimum inoculum strength is vital for kick starting the growth of any microorganism as it provides the active cells in certain concentration (Juanssilfero et al., 2018). This was studied by varying the inoculum strength from 10% to 30% (v/v). Aeration rate of 1 LPM, optimized temperature of 25 °C and initial pH of 5 was maintained for aerobic cultivation of yeast (Fig. 4c). Highest biomass and lipid yield of 5.3 ± 0.21 g L−1 and 0.22 ± 0.04 g g−1 was observed at 15% (v/v) inoculum strength. Higher inoculum strengths (25% and 30% (v/v)) displayed a decrease in biomass productivity possibly due to quick substrate exhaustion and media dilution (Juanssilfero et al., 2018). The effect of aeration rates was also studied by varying it from 0.5 LPM to 3 LPM for biomass and lipid production in oleaginous yeast (Fig. 4d). Highest biomass and lipid yield of 6.4 ± 0.20 g L−1 and 0.46 ± 0.04 g g−1 was observed at 1.5 LPM. As the aeration rates were increased, it was observed that there was no significant increment in biomass concentration. This could be due to the effects of sheer stress on the cells due to increased aeration rates. Moreover, higher agitation rates lead to foaming of the medium thereby leading to difficulty in operation conditions (Osadolor et al., 2017). Dissolved oxygen is
provided by sparging of air in the medium which influences aerobic fermentation and helps in homogeneous mixing of media components. Moreover, oxygen acts as the terminal electron acceptor which in turns helps in generation of ATP. This energy is required for lipid production in endoplasmic reticulum (Athenaki et al., 2018). 3.3.2. Batch fermentation study with optimized parameters lipid yield and VFA profile change The VFA profiles were estimated for various phases of growth and lipid production. The profiles showed good similarity with the growth of oleaginous yeast which signified that the VFAs were efficiently consumed during the process (Fig. 5). The major VFAs in the spent medium of biohydrogen production were acetate and butyrate. Ethanol was also present in negligible quantities. The final acetate and butyrate concentrations in the spent media were 43 ± 2 mg L−1 and 140 ± 4 mg L−1 respectively. The acetate and butyrate concentration reduced by a percentage of 95% ± 0.32 and 68.35% ± 0.64, respectively. The acetate consumption was relatively greater than butyrate. The growth and lipid production profiles showed similarity with the substrate degradation pattern. The complex structure of butyrate (CH3CH2CH2COOH) might be the reason for lesser utilization as compared to acetate. A maximum biomass concentration of 6.4 ± 0.04 g L−1 was observed with a lipid yield of 0.48 g g−1. Under elongated cultivation conditions, an increase in biomass as well as lipid productivity was observed. Similar results for biomass and lipid accumulation were observed by Jiru et al. (2018) where they utilized cane molasses as a substrate. Prolonged cultivation periods increased the lipid content by ∼30% (Jiru et al., 2018). Other studies using VFAs as a substrate have reported a maximum biomass concentration of 6.75 g L−1 with a lipid yield of 0.51 g g−1 (Huang et al., 2016). These values were similar to the values obtained in our studies utilizing VFAs as a substrate for biomass production and lipid accumulation. 6
Bioresource Technology 288 (2019) 121603
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Fig. 5. Batch cultivation of oleaginous yeast using optimized parameters: profile of lipid yields, biomass concentration, acetate and butyrate consumption.
3.3.2.1. Study of lipid accumulation profile using Confocal microscopy. Nile red staining of cells at different time interval time interval showed gradual accumulation of neutral lipids inside the cells. The Nile red binds with the neutral lipids only and gives fluorescence at 530 nm. The phase contrast image on overlapping with fluorescence image gives DIC images. Initial phase of the culture i.e. 2nd–8th h showed very few populations having lipid accumulation. As the fermentation time proceeds, accumulation of lipids intensifies. The 18th and 22nd h samples showed almost all the cells enriched in neutral lipids. Similar observation regarding accumulation of neutral lipids in Rhodosporidium toruloides was made when cultivated using glucose as substrate (Kimura et al., 2004).
3.5. Energy analysis The energy obtained from both the processes were calculated and analyzed as the total energy evolved. The hydrogen yield was 2.8 mol molsubstrate−1 and the maximum lipid content was found to be 0.48 g g−1. Hydrogen has a higher heating value (HHV) of 241 KJ mol−1. The major free fatty acid was found to be oleic acid which has a heat of combustion equivalent to 11,670 kJ mol−1. The gross energy obtained from the process was calculated to be 6976.6 kJ mol−1. There was a significant increase in total energy content as compared to single stage biohydrogen production using dark fermentation. This was comparable to other reports of two stage energy production (Bagy et al., 2014). Previous studies with biodiesel followed by biohydrogen production reported a maximum total energy yield of 3250.12 kJ mol−1 utilizing the microorganism Epicoccum nigrum. Significant increase in total energy content was observed. This might be due to the higher content of oleic acid in Rhodotorula minuta MTCC 2518 as compared to E. nigrum. The most abundant fatty acid in E. nigrum was palmitic acid which had a lower heat of combustion as compared to oleic acid. Also, the higher content of oleic acid in our samples could be a reason for a higher gross energy content. This suggests that energy efficiency of the hydrogen production process could be increased by combining with biodiesel production using oleaginous microorganisms. This could in turn be beneficial in a biorefinery concept for energy generation from biomass as well as remediation of wastewater.
3.4. Study of biodiesel yields Oleaginous microorganisms have promise as a potential feedstock for 3rd generation biofuels. This owes to the similarity in fatty acid composition as compared to vegetable oils. Biodiesel was produced from the wet biomass of fungus by alkali catalysed trans-esterification showed a yield of 92%. Major fatty acids obtained after trans-esterification were long chain fatty acids (C16-C18): palmitic, stearic, oleic and linoleic acids. The present study showed that the oleic acid as the major lipid constituent with 54.52 ± 0.66%. Whereas, the palmitic acid, linoleic acid, stearic acid, linolenic acid and palmitoleic acid was also observed with values of 19.71 ± 1.41%, 13.31 ± 1.2%, 3.4 ± 0.3%, 4.8 ± 0.99% and 0.59 ± 0.1%, respectively. A relatively higher content of monounsaturated fatty acids (MUFAs) (up to 79.18 ± 2.56%) demonstrated the suitability of lipids from Rhodotorula minuta MTCC 2518 as a feedstock for biodiesel production (Byreddy et al., 2015). Higher levels of unsaturation in oil content can lead to instability of biodiesel blended fuel. Moreover, such instability might hamper the internal combustion engine performance (Metzger and Bornscheuer, 2006). Similar fatty acid profiles on using oleaginous yeasts was observed where molasses was used as carbon source (Vieira et al., 2014). Moreover, fatty acid compositions of oleaginous yeasts grown on complex substrates such as spent media of dark fermentation (Béligon et al., 2018), amaranth seeds (Deeba et al., 2018) and food waste leachate (Johnravindar et al., 2018) showed higher percentage of mono-unsaturated fatty acids (oleic and palmitic acids). The linoleic and stearic acids concentrations were also detected but at low concentrations. The fatty acid profiles of Rhodotorula minuta MTCC corroborated with other reported oleaginous yeasts and also with various other vegetable oils (Christophe et al., 2012).
4. Conclusions For maximization of H2 production by Clostridium acetobutylicum MTCC 11276, mixture design was used to formulate media composing using cane molasses, di-hydrogen ammonium phosphate and de-oiled cakes. A high rate of H2 production was observed in continuous process. The VFAs rich spent media generated during dark fermentation was then used for cultivation of oleaginous yeast. The relative percentage of saturated and monounsaturated fatty acids produced by Rhodotorula minuta MTCC 2518 was significantly high, making it suitable for biodiesel production. A significant improvement in energy recovery was also observed using such integrated process when compared with single stage H2 production. Acknowledgements The authors thankfully acknowledge the financial support of DST INSPIRE Faculty Scheme, DST-SERB (ECR/2017/000501), Government 7
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of India and NIT Arunachal Pradesh for providing all the essential facilities.
Huang, X.F., Liu, J.N., Lu, L.J., Peng, K.M., Yang, G.X., Liu, J., 2016. Culture strategies for lipid production using acetic acid as sole carbon source by Rhodosporidium toruloides. Bioresour. Technol. 206, 141–149. Jiru, T.M., Steyn, L., Pohl, C., Abate, D., 2018. Production of single cell oil from cane molasses by Rhodotorula kratochvilovae (syn, Rhodosporidium kratochvilovae) SY89 as a biodiesel feedstock. Chem. Cent. J. 12, 91. Johnravindar, D., Karthikeyan, O.P., Selvam, A., Murugesan, K., Wong, J.W.C., 2018. Lipid accumulation potential of oleaginous yeasts: a comparative evaluation using food waste leachate as a substrate. Bioresour. Technol. 248, 221–228. Juanssilfero, A.B., Kahar, P., Amza, R.L., Miyamoto, N., Otsuka, H., Matsumoto, H., Kihira, C., Thontowi, A., Ogino Yopi, C., Prasetya, B., Kondo, A., 2018. Effect of inoculum size on single-cell oil production from glucose and xylose using oleaginous yeast Lipomyces starkeyi. J. Biosci. Bioeng. 125, 695–702. Khanal, S.K., Chen, W.H., Li, L., Sung, S., 2004. Biological hydrogen production: effects of pH and intermediate products. Int. J. Hydrogen Energy 29, 1123–1131. Kimura, K., Yamaoka, M., Kamisaka, Y., 2004. Rapid estimation of lipids in oleaginous fungi and yeasts using Nile red fluorescence. J. Microbio. Methods 56 (3), 331–338. Lin, C.Y., Lay, C.H., 2004. Carbon/nitrogen-ratio effect on fermentative hydrogen production by mixed microflora. Int. J. Hydrogen Energy 29, 41–45. Lorenz, M.G., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31, 426–428. Metzger, J.O., Bornscheuer, U., 2006. Lipids as renewable resources: current state of chemical and biotechnological conversion and diversification. Appl. Microbiol. Biotechnol. 71, 13–22. Mishra, P., Balachandar, G., Das, D., 2017. Improvement in biohythane production using organic solid waste and distillery effluent. Waste Manage. 66, 70–78. Osadolor, O.A., Nair, R.B., Lennartsson, P.R., Taherzadeh, M.J., 2017. Empirical and experimental determination of the kinetics of pellet growth in filamentous fungi: a case study using Neurospora intermedia. Biochem. Eng. J. 124, 115–121. Prakasham, R.S., Sathish, T., Brahmaiah, P., Subba Rao, C., Sreenivas Rao, R., Hobbs, P.J., 2009. Biohydrogen production from renewable agri-waste blend: optimization using mixer design. Int. J. Hydrogen Energy 34, 6143–6148. Pugazhendhi, A., Anburajan, P., Park, J.H., Kumar, G., Sivagurunathan, P., Kim, S.H., 2017. Process performance of biohydrogen production using glucose at various HRTs and assessment of microbial dynamics variation via q-PCR. Int. J. Hydrogen Energy 42, 27550–27557. Rai, P.K., Singh, S.P., Asthana, R.K., Singh, S., 2014. Biohydrogen production from sugarcane bagasse by integrating dark- and photo-fermentation. Bioresour. Technol. 152, 140–146. Ratledge, C., 2004. Fatty acid biosynthesis in microorganisms being used for Single Cell Oil production. Biochimie 86, 807–815. Roy, S., Ghosh, S., Das, D., 2012. Improvement of hydrogen production with thermophilic mixed culture from rice spent wash of distillery industry. Int. J. Hydrogen Energy 37, 15867–15874. Roy, S., Vishnuvardhan, M., Das, D., 2014. Continuous thermophilic biohydrogen production in packed bed reactor. Appl. Energy 136, 51–58. Sathish, T., Lakshmi, G.S., Rao, C.S., Brahmaiah, P., Prakasham, R.S., 2008. Mixture design as first step for improved glutaminase production in solid-state fermentation by isolated Bacillus sp. RSP-GLU. Lett. Appl. Microbiol. 47, 256–262. Tchakouteu, S.S., Kopsahelis, N., Chatzifragkou, A., Kalantzi, O., Stoforos, N.G., Koutinas, A.A., Aggelis, G., Papanikolaou, S., 2017. Rhodosporidium toruloides cultivated in NaCl-enriched glucose-based media: adaptation dynamics and lipid production. Eng. Life Sci. 17, 237–248. Varanasi, J.L., Nayak, A.K., Sohn, Y., Pradhan, D., Das, D., 2016. Improvement of power generation of microbial fuel cell by integrating tungsten oxide electrocatalyst with pure or mixed culture biocatalysts. Electrochim. Acta 199, 154–163. Venkata Mohan, S., Prathima Devi, M., 2012. Fatty acid rich effluent from acidogenic biohydrogen reactor as substrate for lipid accumulation in heterotrophic microalgae with simultaneous treatment. Bioresour. Technol. 123, 627–635. Vieira, J.P.F., Ienczak, J.L., Rossell, C.E.V., Pradella, J.G.C., Franco, T.T., 2014. Microbial lipid production: screening with yeasts grown on Brazilian molasses. Biotechnol. Lett. 36, 2433–2442. Xu, X., Kim, J.Y., Cho, H.U., Park, H.R., Park, J.M., 2015. Bioconversion of volatile fatty acids from macroalgae fermentation into microbial lipids by oleaginous yeast. Chem. Eng. J. 264, 735–743. Yang, Y., Hu, B., 2019. Investigation on the cultivation conditions of a newly isolated fusarium fungal strain for enhanced lipid production. Appl. Biochem. Biotechnol. 187 (4), 1220–1237.
Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.121603. References Athenaki, M., Gardeli, C., Diamantopoulou, P., Tchakouteu, S.S., Sarris, D., Philippoussis, A., Papanikolaou, S., 2018. Lipids from yeasts and fungi: physiology, production and analytical considerations. J. Appl. Microbiol. 124, 336–367. Bagy, M.M.K., Abd-Alla, M.H., Morsy, F.M., Hassan, E.A., 2014. Two stage biodiesel and hydrogen production from molasses by oleaginous fungi and Clostridium acetobutylicum ATCC 824. Int. J. Hydrogen Energy 39, 3185–3197. Basak, N., Jana, A.K., Das, D., 2014. Optimization of molecular hydrogen production by Rhodobacter sphaeroides O.U.001 in the annular photobioreactor using response surface methodology. Int. J. Hydrogen Energy 39, 11889–11901. Béligon, V., Noblecourt, A., Christophe, G., Lebert, A., Larroche, C., Fontanille, P., 2018. Proof of concept for biorefinery approach aiming at two bioenergy production compartments, hydrogen and biodiesel, coupled by an external membrane. Biofuels 9, 163–174. Beopoulos, A., Nicaud, J.M., 2012. Yeast: a new oil producer? OCL – Ol. Corps Gras Lipides 19, 22–28. Bligh, E., Dyer, W., 1959. Canadian journal of biochemistry and physiology. J. Biochem. Physiol. 37, 911–917. Boboescu, I.Z., Gherman, V.D., Lakatos, G., Pap, B., Bíró, T., Maróti, G., 2016. Surpassing the current limitations of biohydrogen production systems: the case for a novel hybrid approach. Bioresour. Technol. 204, 192–201. Bolzonella, D., Battista, F., Cavinato, C., Gottardo, M., Micolucci, F., Lyberatos, G., Pavan, P., 2018. Recent developments in biohythane production from household food wastes: a review. Bioresour. Technol. 257, 311–319. Byreddy, A.R., Gupta, A., Barrow, C.J., Puri, M., 2015. Comparison of cell disruption methods for improving lipid extraction from thraustochytrid strains. Mar. Drugs 13, 5111–5127. Cho, H.U., Kim, Y.M., Choi, Y.N., Xu, X., Shin, D.Y., Park, J.M., 2015. Effects of pH control and concentration on microbial oil production from Chlorella vulgaris cultivated in the effluent of a low-cost organic waste fermentation system producing volatile fatty acids. Bioresour. Technol. 184, 245–250. Christophe, G., Kumar, V., Nouaille, R., Gaudet, G., Fontanille, P., Pandey, A., Socco, C.R., Larroche, C., 2012. Recent developments in microbial oils production: a possible alternative to vegetable oils for biodiesel without competition with human food? Brazilian Arch. Biol. Technol. 55, 29–46. Das, D., 2017. A road map on biohydrogen production from organic wastes. Ina. Lett. 2, 153–160. Das, D., Veziroglu, T.N., 2008. Advances in biological hydrogen production processes. Int. J. Hydrogen Energy 33, 6046–6057. Deeba, F., Patel, A., Arora, N., Pruthi, V., Pruthi, P.A., Negi, Y.S., 2018. Amaranth seeds (Amaranthus palmeri L.) as novel feedstock for biodiesel production by oleaginous yeast. Environ. Sci. Pollut. Res. 25, 353–362. Didier, C., Etcheverrigaray, M., Kratje, R., Goicoechea, H.C., 2007. Crossed mixture design and multiple response analysis for developing complex culture media used in recombinant protein production. Chemom. Intell. Lab. Syst. 86, 1–9. Ghosh, S., Banerjee, S., Das, D., 2017a. Process intensification of biodiesel production from Chlorella sp. MJ 11/11 by single step transesterification. Algal Res. 27, 12–20. Ghosh, S., Dutta, B., Banerjee, A., Roy, S., 2018. Bio-H2 production using de-oiled cake as cheap nitrogen source and subsequent electricity generation by hybrid system. Bioresour. Technol. Rep. 4, 1–8. Ghosh, S., Roy, S., Das, D., 2017b. Enhancement in lipid content of Chlorella sp. MJ 11/11 from the spent medium of thermophilic biohydrogen production process. Bioresour. Technol. 223, 219–226. Grzelak, J., Ślęzak, R., Krzystek, L., Ledakowicz, S., 2018. Effect of pH on the production of volatile fatty acids in dark fermentation process of organic waste. Ecol. Chem. Eng. S 25, 295–306.
8
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Novel integration of biohydrogen production with fungal biodiesel production process Supratim Ghosha, Shantonu Royb, a b
T
⁎
Porter School of the Environment and Earth Sciences, Tel Aviv University, Israel Department of Biotechnology, National Institute of Technology, Arunachal Pradesh 791112, India
ARTICLE INFO
ABSTRACT
Keywords: Mixture design Continuous hydrogen production Oleaginous yeast cultivation Biodiesel production
An integration of bio-H2 with fungal biodiesel production process was investigated. Highest cumulative H2 production of 3.3 ± 0.20 L L−1 was observed during media optimization using mixture design. Using optimized media composition, continuous H2 production at 0.2 h−1 dilution rate, showed highest H2 production rate, H2 yield and biomass yield of 1020 ± 23 mL L−1 h−1, 2.8 ± 0.1 mols mol−1 reducing sugar and 1.2 ± 0.06 g L−1, respectively. Using the spent media generated from the dark fermentation, oleaginous yeast cultivation was done. Highest biomass and total lipid yield of 6.4 ± 0.20 g L−1 and 0.46 ± 0.04 g g−1 was observed at initial 15% v/v inoculums strength, pH of 5, 1.5 L min−1 aeration rate and 25 °C temperature of cultivation, respectively. Energy recovery improved by 90.3% in integrated process when compared with single stage hydrogen production.
1. Introduction Non-judicious use of fossil fuels has disrupted the global climatic pattern. Use of renewable sources of energy for mitigation of such problem has gained importance in recent times. Renewable fuels such as biofuels which have lower carbon footprint could provide an alternative to conventional fossil-based sources. Hydrogen (H2) has the highest energy density (143 GJ ton−1) and is truly a carbon neutral fuel (Das and Veziroglu, 2008). It can be produced through various convention methods such as water gas process, methane reforming, electrolysis, etc. The biological H2 production processes require less energy and can be produced using renewable feedstock; thereby making the overall process sustainable (Das, 2017; Khanal et al., 2004). The H2 production through dark fermentation has shown higher rates, substrate conversion efficiency and yields when compared with other biological routes. This biohydrogen can be directly converted into electricity through fuel cells. This would certainly help in development of a standalone electricity generation system from organic wastes. Biohydrogen produced can be directly converted into electricity through fuel cells. This would certainly help in development of a standalone electricity generation system from organic wastes. (Ghosh et al., 2018). The various physicochemical parameters such as the type of substrate, pre-processing and fermentation conditions influence H2
⁎
production, which accentuates the necessity for optimization (Rai et al., 2014). Statistical methodologies are often used for process optimization where suitable combinations of physicochemical parameters are studied for predicting maximum product yield (Roy et al., 2014, 2012). There are different statistical optimization methodologies which are widely used for process optimization studies viz. Box-Behnken, Taguchi design, factorial design etc. (Roy et al., 2014). The mixture design is a statistical optimization method which belongs to the class of response surface designs. In this design, the factors are considered in the form of proportions but not magnitude. Such designs are thus quite useful for optimization of formulations or mixtures (Didier et al., 2007). A mixture design also helps in understanding the interaction among the factors in response to the output (Sathish et al., 2008). The volatile fatty acids (VFAs) are the byproducts of dark fermentative hydrogen production. These VFAs includes majorly acetate, butyrate, ethanol and a small fraction of propionate. The energy entrapped in these VFAs remains untapped in the single stage systems. The potential use of VFAs rich spent media for further energy recovery has been explored by various routes such as photo-fermentation to produce biohydrogen (Basak et al., 2014), in the microbial fuel cells to produce electricity (Varanasi et al., 2016), for enhancement of lipids and algal biomass production (Ghosh et al., 2017b) and for hydrogen and methane production (Bolzonella et al., 2018). Cultivation of oleaginous microorganisms has shown a lot of
Corresponding author. E-mail address: [email protected] (S. Roy).
https://doi.org/10.1016/j.biortech.2019.121603 Received 17 April 2019; Received in revised form 31 May 2019; Accepted 2 June 2019 Available online 04 June 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
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promise when compared with vegetable oil production. They require a lesser area of land, higher growth rate and high lipid yields (Beopoulos and Nicaud, 2012; Ratledge, 2004). Various organic carbon sources as feedstock (e.g., sugars and organic acids) can be suitably used by oleaginous microorganisms such as microalgae, yeasts, fungi etc. thereby accumulating lipids within the cells (> 20%). The general pathway of lipid synthesis using glucose as a substrate involves a cascade of enzymes which convert it to acetate via mitochondria and then it’s channelized to lipogenesis in the cytoplasm. Whereas, acetate can be directly subjected to lipogenesis if it is provided as a sole substrate to the oleaginous microorganisms. Acetic acid is one of the major components of VFAs which could be used as a suitable substrate for cultivation of oleaginous microorganisms. The composition of lipids produced by oleaginous microorganisms is similar to vegetable oils such as soybean and rapeseed oil. The fatty acids with the chain length of 16 or 18 carbons are prominent in oleaginous microbes (Xu et al., 2015). The feedstock used for cultivation of these oleaginous microbes accounts for 40–80% of the total cost of production thus making microbial oils production economically unfeasible (Cho et al., 2015). Cultivation of oleaginous microbes on low-cost substrates such as volatile fatty acids, lignocellulosic biomass, De-oiled cakes (DOCs) can make the overall production process economically viable (Venkata Mohan and Prathima Devi, 2012). Cultivating the oleaginous microorganisms on the spent medium produced during of dark fermentative H2 production can potentially help in integrating both the processes. Such integration was widely studied using algal biomass cultivation using spent media (Boboescu et al., 2016). Such integration showed potential in achieving both wastewater remediation and simultaneous energy production (Béligon et al., 2018). Cultivation of oleaginous red yeast on spent media generated during of dark fermentative hydrogen production has not been studied extensively. The present study investigates the potential integration of dark fermentative hydrogen production with oleaginous fungal cultivation. For maximization of H2 production, Mixture design was used to optimize the composition of the media components viz. concentration of cane molasses, di-ammonium phosphate concentration and DOC. Based on the optimized parameters, batch and continuous bio-H2 production were studied. The spent medium generated during dark fermentation was further used for cultivation of oleaginous yeast. Various parameters such as adjustment of initial pH, inoculums strength, aeration rate and temperature of cultivation were optimized for maximization of biomass and lipid yield. Confocal microscopic studies were performed to observe the lipid accumulation profile. Overall energy recovery was also studied for the combined process. To best of our knowledge, no previous reports are available regarding the integration of dark fermentative hydrogen production with oleaginous fungal cultivation. Such integration could certainly improve the overall energy recovery from a single substrate.
2.1.2. Setup for batch hydrogen production The batch fermentation was performed for biohydrogen production using a cylindrical double jacked reactor with working volume of 400 mL (Mishra et al., 2017) at 37 °C and 200 rpm. The temperature was maintained by using circulating water bath and rpm was maintained using magnetic stirrer. The anaerobic condition was created by flushing nitrogen gas (N2) through the reactor for 30 mins. 2.1.3. Setup for continuous hydrogen production Continuous cultivation was performed in a cylindrical double jacked reactor (500 mL) with working volume of 400 mL. The feed was infused into the reactor and simultaneously spent media was expelled out using peristaltic pumps. The dilution rates were varied from 0.1 to 0.3 h−1. The fermentation was performed at 37 °C and 200 rpm. Stable hydrogen production rate with respect to each dilution rate was considered as steady state (Ghosh et al., 2018). 2.1.4. Oleaginous yeast cultivation The Rhodotorula minuta MTCC 2518 was used to study lipid production process studies. The cultivation media contains glucose (SRL, India) 1% (w/v), yeast extract (SRL, India) 1% (w/v) and Peptone (SRL Laboratories, India) 0.4% (w/v) at 32 °C in an incubator shaker (New Brunswick Scientific, New Jersey, USA) at 160 rpm. 2.2. Maximization of biohydrogen production using mixture design Cane molasses, Di-ammonium phosphate (DAP) and deoiled cake (DOC) were three components of fermentation media from which a suitable concoction was formulated for maximum gaseous energy recovery. For formulation, a mixture design was used where the effects of the components in a blend was observed on an output variable (H2 production in this case). The mixture design could be as expressed mathematically by below mentioned equation (Eq. (1)): p
Y=
Xi = X1+X2 + X3 + .....Xp = 1;
Xi > 0; i = 1,2,3,4. ...,p
i=1
(1)
where ‘p’ represents the number of ingredients, and Xi denotes the proportions of the components. A simplex-lattice design was employed to study the effect of cane molasses, Di-ammonium phosphate (DAP) and DOC proportion on bio-H2 production. The respective proportions were fermented in 500 mL double jacketed glass reactor with 400 mL working volume. In this design, the total proportions of the different components were made to 100% i.e., 6 g. Total 10 experiments were proposed by the design. This includes three experiments having respective pure components, whereas other three experiments were with binary blends for each possible two-component. Moreover, additional three experiments were performed with complete concoction, where the centroid point represents equal proportions of all three components. The H2 output values with respect to all the array of 10 experiments proposed by the design was shown in Table 1. Analysis of the mixture design was done by MINITAB 15 using following equations:
2. Materials and methods 2.1. Microorganism and culture conditions
Table 1 Array of experiments proposed by Mixture design.
2.1.1. Growth and culture conditions The H2 production studies were performed using Clostridium acetobutylicum MTCC 11276. The growth media composition includes glucose 1% (w/v), Tryptone) 1% (w/v), DOC 0.4% (w/v) and Cysteine HCl 0.1% (w/v) at 37 °C in an incubator shaker (New Brunswick Scientific, New Jersey, USA) at 160 rpm. The organism was grown in 100 mL serum bottles (Sigma Aldrich, USA) with a working volume of 70 mL. A strict anaerobic condition was maintained by flushing nitrogen gas (N2) through the reactor. For bio H2 production, cane molasses, di-ammonium phosphate (DAP) (SRL Laboratories, India), DOC and Cysteine HCl 0.1% (w/v) was used. 2
Exp No.
Molasses
DAP
DOC
Bio-H2 production (L L−1)
1 2 3 4 5 6 7 8 9 10
1.00000 1.00000 1.33333 0.00000 0.66667 0.33333 2.00000 0.33333 0.00000 0.00000
0.00000 1.00000 0.33333 2.00000 0.66667 0.33333 0.00000 1.33333 0.00000 1.00000
1.00000 0.00000 0.33333 0.00000 0.66667 1.33333 0.00000 0.33333 2.00000 1.00000
2.30 2.40 3.40 0.00 3.20 2.50 0.60 2.10 0.20 0.22
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acid methyl esters (FAMEs) along with unreacted triglycerides. The hexane layer was washed until neutrality with deionized water (5 mL) and was analyzed using a gas chromatograph equipped with a FID detector (Clarus 500, PerkinElmer) (Ghosh et al., 2017a). On comparing the respective retention times and fragmentation patterns with standards of fatty acid methyl ester (FAME) mixture were identified (37mix, Supelco Inc., Bellefonte, PA).
p
Y=
i Xi
Linear Model
(2)
i=1 p
Y=
i Xi
p
+
i
i=1 p
Y=
i Xi
+
i=1
p i
i Xi Xj
i Xi Xj
+
Quadratic Model p i
i Xi Xj Xk
(3) Special Cubic
(4)
2.4.4. Fluorescence microscopy study of lipid accumulation Nile red (0.1 mg dissolved in 1 mL acetone) was used to stain the neutral lipids accumulated in the microorganism. A100 µL of Rhodotorula sp., cell suspension was mixed with 10 µL of Nile Red (0.1 mg/mL) and 2 mL PBS. The stained cells were kept in dark for 5 min (Kimura et al., 2004). Leica Dmi8 live cell imaging microscope was used for microscopic images, with Leica DFC365 FX camera and Las X software version, using the 40× lens. Nile red stains neutral lipid droplets in red color but for selectivity, it can be viewed yellow gold fluorescence (450–500 nm excitation; > 528 nm emission). The fluorescence images were acquired using Leica Led module 530for Nile Red and the transmitted light images were acquired using differential interference contrast.
2.3. Lipid extraction from the fungal biomass Lipid extraction was performed using the modified Bligh and Dyer method (1959) (Bligh and Dyer, 1959). The fungal cells were sonicated (20 kHz, 5 min) and chloroform: methanol (2:1; v/v) solution was added. The resulting solution was stirred at room temperature for 2 h at 150 rpm. The mixture was centrifuged at 3000×g for 5 min to obtain the supernatant which was further mixed with 0.034% (w/v) MgCl2 and 0.88% v/v 2 N KCl. The chloroform: methanol: water (2:2:1.8 v/v/v) was mixed with the upper phase and followed by centrifugation at 6000×g for 4 min led to phase separation. The organic phase was aspirated out and vacuum dried to obtain the total lipids. Gravimetric measurements were utilised to determine the biomass content (g L−1) and lipid yield (g g−1). The lipid content (% w/w) of the oleaginous yeast was measured by the equations followed as:
CL (\%) =
CT (g L 1) × 100 X (g L 1)
2.4.5. Reducing sugar estimation Reducing sugar content (glucose) of the sample was estimated spectrophotometrically using DNS (dinitro salicylic acid) reagent method (Lorenz, 1959). DNS reagent was prepared by mixing solution A and solution B in a fixed concentration. Solution A contained 300 mg of Rochelle salt (Sodium potassium tartarate) dissolved in 500 mL of distilled water. Solution B contained 10 g of dinitrosalycylic acid dissolved in 200 mL of 2 N NaOH solution. DNS reagent was prepared by mixing 10 mL of solution A, 4 mL of solution B and 6 mL of distilled water. Calibration curve of glucose was prepared by using various known glucose concentrations as standards. Reagents were added as follows and OD (optical density) was read at a wave length of 540 nm.
(5)
where CL = Lipid content; CT = Total Lipids; and X = Biomass content. 2.4. Analysis 2.4.1. Biogas and fatty acid analysis Hydrogen concentration was measured by gas chromatography (GC Agilent Technology 7890A U.S.A) using thermal conductivity detector (TCD) equipped with a stainless steel packed with Porapak Q (80/100). The oven and the detector operational temperatures of the injection port were 80 °C, 150 °C and 200 °C, respectively. The carrier gas (N2) was used at a flow rate of 20 mL min−1. Flame ionization detector (FID) was used for estimation of volatile fatty acids and FAME. The column used was a capillary column (0.5 mm diameter × 30 m length) at preset injector, oven and detector temperatures of 250 °C, 200 °C and 280 °C respectively. The FID operational temperatures of the injection port, the detector and programmed column were 220 °C, 240 °C, and 130–175 °C, respectively. For flame ignition, a mixture of H2 and air at a flow rate of 30 mL min−1 was used (Ghosh et al., 2018).
2.4.6. Volatile fatty acids (VFA) analysis The fermentation broth was centrifuged at 10,000 rpm for 5 min in order to separate the cell debris from the supernatant. The supernatant was then distilled to concentrate the VFAs. VFA and alcohols present in the spent media were estimated by GC using FID Detector, capillary column (0.5 mm diameter × 30 m length) at preset injector, oven and detector temperatures of 250 °C, 200 °C and 280 °C respectively. The FID operational temperatures of the injection port, the detector and programmed column were 220 °C, 240 °C, and 130–175 °C, respectively. For flame generation a mixture of hydrogen and air at a flow rate of 30 mL min−1 was used.
2.4.2. Biomass estimation The biomass production profile of Rhodotorula minuta was measured using the gravimetric method. A known volume of fungal culture was collected periodically and was centrifuged at 6500g for 10 min. The supernatant was discarded and the pellet was then washed twice with deionized water followed by drying at 60 °C for till the constant weight was observed.
2.5. Calculation for net energy recovery Palmitate has been used as common fatty acid based on which the estimation of total energy content of biodiesel was calculated. The stoichiometric equation representing complete combustion of oleate methyl ester and hydrogen was shown in Eqs. (6) and (7).
2.4.3. Biodiesel production and FAME analysis About 1 g of freshly extracted fungal oil was used for transesterification study. The transesterification was carried on in 50 cc glass vials with airtight caps (Schott Duran, Hattenbergstrasse, Germany) that retained any vaporized methanol to the reacting mixture. The vials were kept in a temperature-controlled incubator. The mixture was stirred by using a cyclomixture at every 30 min interval. Transesterification was performed with acidified methanol (10 mL) at 90 °C for analysis of fatty acids in the fungal biomass. Phase separation was performed using hexane (5 mL) as a solvent in order to obtain fatty
C18 H36 O2 + 26
2H2 (g)+ O2 (g)
1 O2 2
18H2 O + 18CO2 + (11670 kJ mol 1)
2H2 O(l) + (241 kJ mol 1)
(6) (7)
Along with byproducts, energy is also released in terms of heat of enthalpy. So theoretically, 11790.5 kJ per mole was the total energy that can be recovered from complete combustion of 1 mol of palmitate methyl ester and hydrogen. 3
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3. Results and discussions
Table 2 ANOVA analysis of the mixture design models.
3.1. Maximization of H2 production using mixture design All the 10 experiments showed variations in H2 production. The experiment no. 3, gave highest cumulative production of 3.4 L L−1. In all the experiments (3, 5, 6 and 8) where cane molasses, DAP and DOC were present in the concoction showed substantial improvement in H2 production. Experiment no. 5 had all the components in equal proportion and showed cumulative production of 3.2 L L−1. Experiment no. 9 and 10 had no cane molasses content and showed very little H2 production probably from the nutrients available in DOC. Whereas, experiment no. 4 had di-ammonium phosphate (DAP) as sole component in the concoction showed no H2 production. Many volatile fatty acids were produced during fermentation that leads to decrease in pH of the media thereby hindering further growth (Grzelak et al., 2018). The use of DAP in the concoction was to provide a buffering environment so as to prevent sudden decrease in pH of the fermentation media (Lin and Lay, 2004). The DAP also acts as inorganic nitrogen source. Combination of only cane molasses and DAP (Experiment no. 2) showed less H2 production due to absence of valuable micronutrients provided by DOC. The DOC provides valuable vitamins and nutrients that are indispensable for H2 production (Ghosh et al., 2018). A surface plot and contour plot showed the optimum zones of combinations that would maximize the H2 production (Fig. 1a and b). From the model optimum combination of the components (in terms of proportion) for maximization of H2 production was predicted to be 0.863717 of cane molasses, 0.545455 of Di-ammonium phosphate (DAP) and 0.59082 of DOC, respectively with desirability of 1. This corresponds to 5.182 g of total reducing sugar in cane molasses, 3.27 g of Di-ammonium phosphate (DAP) and 3.55 g of DOC, respectively in 400 mL of water. The predicted model showed R2 = 0.98 with F = 17.61 value showing significance at P < 0.05. The ANOVA analysis showed significantly higher F values than P values for quadratic and special cubic models (Table 2). Linear model showed poor significance when compared with other models. The residual error was also significantly less. Similar application of mixture design for H2 production using different agro residues had shown significant improvement (Prakasham et al., 2009).
Source
DF
Seq SS
Adj SS
Adj MS
F
P
Regression Linear Quadratic Special cubic Residual Error Total
6 2 3 1 3 9
14.9066 2.0556 11.4982 1.3528 0.4232 15.3298
14.90655 0.27440 5.01015 1.35279 0.42321
2.48443 0.13720 1.67005 1.35279 0.14107
17.61 0.97 11.84 9.59
0.019 0.473 0.036 0.053
which was near to the mixture design values. A 79% consumption of reducing sugar was also observed during 20 h fermentation. The fermentation broth has pH of was 3.6 indicating production of soluble acidic metabolites such as acetate (A), butyrate (B), propionate and ethanol. Highest butyrate, acetate and ethanol concentration of 1105 mg L−1, 574 mg L−1 and 152 mg L−1 was observed respectively. Butyrate production superseded acetate from 8th h onwards. The B/A ratio were below 1 up to 4th h and increased from 1.13 to 1.92 as fermentation proceeded. 3.2. Continuous biohydrogen production using CSTR The optimum proportion of media components which was estimated in above section was used for continuous H2 production was studied in a continuous stirred tank reactor (CSTR). The influence of varying dilution rates on steady state biomass concentration, H2 production rate, H2 yield and VFAs concentration was shown in Fig. 3. At 0.2 h−1 dilution rate, a highest rate of H2 production, H2 yield and biomass yield of 1020 ± 23 mL L−1 h−1, 2.8 ± 0.1 mol mol−1 reducing sugar and 1.18 ± 0.042 g L−1, respectively was observed. The observed yield was 70% of the theoretical yield indicating the significant energy recovery. A decrement in H2 production rate, yields and biomass concentration was observed on increasing dilution rate beyond 0.2 h−1. The H2 production rate, yields and suspended cells concentration showed a decreasing trend beyond 0.2 h−1 dilution rate. The H2 production ceased at a dilution rate of 0.3 h−1. This could be probably due to inadequate time for microbes inside the reactor to convert substrate to product, low hydraulic retention time, and eventually occurrence of washout of metabolically cells from the reactor (Pugazhendhi et al., 2017). In dark fermentative H2 production, acetate, butyrate, propionate, ethanol, etc. were produced as byproducts (Venkata Mohan and Prathima Devi, 2012). The steady state metabolite concentration of the respective dilution rates was estimated from the effluents exiting from the reactor. The volatile fatty acids (VFAs) were in the order: butyrate > acetate > ethanol for all the dilution rates. The concentration of 1098 ± 84 mg L−1 butyrate, 470 ± 25 mg L−1 acetate, and 150 ± 30 mg L−1 ethanol, respectively was observed in the spent media. A concurrent variation of
3.1.1. Batch fermentation using optimized formulation derived from mixture design Based on the optimized values predicted by the mixture model, batch fermentation was performed to validate the output experimentally. The cumulative H2 production, pH profile, reducing sugar profile, volatile fatty acids (VFAs) profile and B/A ratio was shown in Fig. 2a and b. Maximum cumulative H2 production of 3.62 L L−1 was observed
Fig. 1. a) Response surface plot and b) Contour plot of mixture design representing effect of proportional mixing of media components on hydrogen production. 4
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Fig. 2. Batch fermentation studies using optimized media composition: a) H2 production, pH and reducing sugar concentration profile; b) Volatile fatty acids profile and B/A ratio. Error bar represents standard deviation of triplicate experiments. Temperature 37 °C and 200 rpm was maintained in all the experiments.
VFAs concentrations and hydrogen yield was observed on varying dilution rates. The VFAs profile indicated that a mixed acid fermentation. The VFAs profile is indicative of the dynamic changes in the ratio of NAD+/ (NADH + H+). If ethanol: acetate ratio is 1:1 then it indicates a balance between NAD+/(NADH + H+). Moreover, if this dynamic equilibrium gets disturbed due to shifts from 1:1 ratio then it implies an unbalanced fermentation process. In one such study, H2 production using Enterobacter aerogenes MTCC 2822 showed an unstable ethanol: acetate ratio (Rai et al., 2014).
cultivation of yeast which includes adjustment of initial pH, inoculums strength, aeration rate and temperature of cultivation. 3.3.1. Maximization of biomass and lipid yields by optimizing process parameters The growth of Rhodotorula minuta was studied using spent media (generated during 0.2 h−1 dilution rate of continuous process) which was adjusted to various pH values using 1 N HCl (pH 4.5, 5, 5.5, 6 and 6.5) (Fig. 4a). The cultivation conditions were as follows: temperature of 25 °C, aeration rate of 1 L per minute (LPM), 5% (v/v) inoculum strength. Initial pH of the medium had an influence on the yield of biomass and lipids. Biomass and lipid yield of 4.5 ± 0.21 g L−1 and 0.13 ± 0.04 g g−1 was observed on using spent media with adjusted pH of 5. The biomass yields increased (∼60% improvement) when the pH of media was adjusted. Initial pH of 6 and 6.5 did not show any significant improvement in biomass yield. Literature reports also suggest that Rhodotorula sp. grow favorably in the pH range of 5–6
3.3. Second stage integration for cultivation of Rhodotorula minuta using spent media The VFAs rich spent media was used for cultivation of oleaginous yeast Rhodotorula minuta. The acetate and butyrate were the primary substrate metabolized into lipids. The spent media had pH of 3.5–4 which needs further fortification before it can be used further
Fig. 3. Continuous hydrogen production study using optimized media composition. 5
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Fig. 4. Valorization studies of spent media produced during biohydrogen production process: a) Biomass and lipid yield using pH adjusted spent media; b) Biomass and lipid yield on using different temperatures; c) Biomass and lipid yield on using different inoculum strengths and d) Biomass and lipid yield on using different aeration rate. Error bar represents standard deviation of triplicate experiments.
(Tchakouteu et al., 2017). The pH adjusted spent medium (pH 5) was utilized for further cultivation of oleaginous yeast. The aeration rate was set to 1 LPM with inoculum strength of 5% (v/v) (Fig. 4b). Temperature of cultivation was varied from 20 °C to 40 °C. The variation in biomass and lipid yield was observed with changing. Biomass and lipid yield of 4.5 ± 0.21 g L−1 and 0.13 ± 0.04 g g−1 was observed at 25 °C. Growth and lipid metabolism are essential for proper activity of enzymes. At optimum temperature, there is maximum activity of the enzyme system which leads to maximum biomass and lipid content (Yang and Hu, 2019). For any bioprocess, inoculum strength also plays an important role in determination of product yield. Optimum inoculum strength is vital for kick starting the growth of any microorganism as it provides the active cells in certain concentration (Juanssilfero et al., 2018). This was studied by varying the inoculum strength from 10% to 30% (v/v). Aeration rate of 1 LPM, optimized temperature of 25 °C and initial pH of 5 was maintained for aerobic cultivation of yeast (Fig. 4c). Highest biomass and lipid yield of 5.3 ± 0.21 g L−1 and 0.22 ± 0.04 g g−1 was observed at 15% (v/v) inoculum strength. Higher inoculum strengths (25% and 30% (v/v)) displayed a decrease in biomass productivity possibly due to quick substrate exhaustion and media dilution (Juanssilfero et al., 2018). The effect of aeration rates was also studied by varying it from 0.5 LPM to 3 LPM for biomass and lipid production in oleaginous yeast (Fig. 4d). Highest biomass and lipid yield of 6.4 ± 0.20 g L−1 and 0.46 ± 0.04 g g−1 was observed at 1.5 LPM. As the aeration rates were increased, it was observed that there was no significant increment in biomass concentration. This could be due to the effects of sheer stress on the cells due to increased aeration rates. Moreover, higher agitation rates lead to foaming of the medium thereby leading to difficulty in operation conditions (Osadolor et al., 2017). Dissolved oxygen is
provided by sparging of air in the medium which influences aerobic fermentation and helps in homogeneous mixing of media components. Moreover, oxygen acts as the terminal electron acceptor which in turns helps in generation of ATP. This energy is required for lipid production in endoplasmic reticulum (Athenaki et al., 2018). 3.3.2. Batch fermentation study with optimized parameters lipid yield and VFA profile change The VFA profiles were estimated for various phases of growth and lipid production. The profiles showed good similarity with the growth of oleaginous yeast which signified that the VFAs were efficiently consumed during the process (Fig. 5). The major VFAs in the spent medium of biohydrogen production were acetate and butyrate. Ethanol was also present in negligible quantities. The final acetate and butyrate concentrations in the spent media were 43 ± 2 mg L−1 and 140 ± 4 mg L−1 respectively. The acetate and butyrate concentration reduced by a percentage of 95% ± 0.32 and 68.35% ± 0.64, respectively. The acetate consumption was relatively greater than butyrate. The growth and lipid production profiles showed similarity with the substrate degradation pattern. The complex structure of butyrate (CH3CH2CH2COOH) might be the reason for lesser utilization as compared to acetate. A maximum biomass concentration of 6.4 ± 0.04 g L−1 was observed with a lipid yield of 0.48 g g−1. Under elongated cultivation conditions, an increase in biomass as well as lipid productivity was observed. Similar results for biomass and lipid accumulation were observed by Jiru et al. (2018) where they utilized cane molasses as a substrate. Prolonged cultivation periods increased the lipid content by ∼30% (Jiru et al., 2018). Other studies using VFAs as a substrate have reported a maximum biomass concentration of 6.75 g L−1 with a lipid yield of 0.51 g g−1 (Huang et al., 2016). These values were similar to the values obtained in our studies utilizing VFAs as a substrate for biomass production and lipid accumulation. 6
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Fig. 5. Batch cultivation of oleaginous yeast using optimized parameters: profile of lipid yields, biomass concentration, acetate and butyrate consumption.
3.3.2.1. Study of lipid accumulation profile using Confocal microscopy. Nile red staining of cells at different time interval time interval showed gradual accumulation of neutral lipids inside the cells. The Nile red binds with the neutral lipids only and gives fluorescence at 530 nm. The phase contrast image on overlapping with fluorescence image gives DIC images. Initial phase of the culture i.e. 2nd–8th h showed very few populations having lipid accumulation. As the fermentation time proceeds, accumulation of lipids intensifies. The 18th and 22nd h samples showed almost all the cells enriched in neutral lipids. Similar observation regarding accumulation of neutral lipids in Rhodosporidium toruloides was made when cultivated using glucose as substrate (Kimura et al., 2004).
3.5. Energy analysis The energy obtained from both the processes were calculated and analyzed as the total energy evolved. The hydrogen yield was 2.8 mol molsubstrate−1 and the maximum lipid content was found to be 0.48 g g−1. Hydrogen has a higher heating value (HHV) of 241 KJ mol−1. The major free fatty acid was found to be oleic acid which has a heat of combustion equivalent to 11,670 kJ mol−1. The gross energy obtained from the process was calculated to be 6976.6 kJ mol−1. There was a significant increase in total energy content as compared to single stage biohydrogen production using dark fermentation. This was comparable to other reports of two stage energy production (Bagy et al., 2014). Previous studies with biodiesel followed by biohydrogen production reported a maximum total energy yield of 3250.12 kJ mol−1 utilizing the microorganism Epicoccum nigrum. Significant increase in total energy content was observed. This might be due to the higher content of oleic acid in Rhodotorula minuta MTCC 2518 as compared to E. nigrum. The most abundant fatty acid in E. nigrum was palmitic acid which had a lower heat of combustion as compared to oleic acid. Also, the higher content of oleic acid in our samples could be a reason for a higher gross energy content. This suggests that energy efficiency of the hydrogen production process could be increased by combining with biodiesel production using oleaginous microorganisms. This could in turn be beneficial in a biorefinery concept for energy generation from biomass as well as remediation of wastewater.
3.4. Study of biodiesel yields Oleaginous microorganisms have promise as a potential feedstock for 3rd generation biofuels. This owes to the similarity in fatty acid composition as compared to vegetable oils. Biodiesel was produced from the wet biomass of fungus by alkali catalysed trans-esterification showed a yield of 92%. Major fatty acids obtained after trans-esterification were long chain fatty acids (C16-C18): palmitic, stearic, oleic and linoleic acids. The present study showed that the oleic acid as the major lipid constituent with 54.52 ± 0.66%. Whereas, the palmitic acid, linoleic acid, stearic acid, linolenic acid and palmitoleic acid was also observed with values of 19.71 ± 1.41%, 13.31 ± 1.2%, 3.4 ± 0.3%, 4.8 ± 0.99% and 0.59 ± 0.1%, respectively. A relatively higher content of monounsaturated fatty acids (MUFAs) (up to 79.18 ± 2.56%) demonstrated the suitability of lipids from Rhodotorula minuta MTCC 2518 as a feedstock for biodiesel production (Byreddy et al., 2015). Higher levels of unsaturation in oil content can lead to instability of biodiesel blended fuel. Moreover, such instability might hamper the internal combustion engine performance (Metzger and Bornscheuer, 2006). Similar fatty acid profiles on using oleaginous yeasts was observed where molasses was used as carbon source (Vieira et al., 2014). Moreover, fatty acid compositions of oleaginous yeasts grown on complex substrates such as spent media of dark fermentation (Béligon et al., 2018), amaranth seeds (Deeba et al., 2018) and food waste leachate (Johnravindar et al., 2018) showed higher percentage of mono-unsaturated fatty acids (oleic and palmitic acids). The linoleic and stearic acids concentrations were also detected but at low concentrations. The fatty acid profiles of Rhodotorula minuta MTCC corroborated with other reported oleaginous yeasts and also with various other vegetable oils (Christophe et al., 2012).
4. Conclusions For maximization of H2 production by Clostridium acetobutylicum MTCC 11276, mixture design was used to formulate media composing using cane molasses, di-hydrogen ammonium phosphate and de-oiled cakes. A high rate of H2 production was observed in continuous process. The VFAs rich spent media generated during dark fermentation was then used for cultivation of oleaginous yeast. The relative percentage of saturated and monounsaturated fatty acids produced by Rhodotorula minuta MTCC 2518 was significantly high, making it suitable for biodiesel production. A significant improvement in energy recovery was also observed using such integrated process when compared with single stage H2 production. Acknowledgements The authors thankfully acknowledge the financial support of DST INSPIRE Faculty Scheme, DST-SERB (ECR/2017/000501), Government 7
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of India and NIT Arunachal Pradesh for providing all the essential facilities.
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