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Exploring Pongamia seed cake hydrolysate as a medium for microbial lipid production by Aspergillus ochraceus
Biocatalysis and Agricultural Biotechnology 24 (2020) 101543
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Biocatalysis and Agricultural Biotechnology
Exploring Pongamia seed cake hydrolysate as a medium for microbial lipid production by Aspergillus ochraceus Harshitha Madhusoodan Jathanna *, Chandrayan Vaman Rao, Louella Concepta Goveas Visvesvaraya Technological University, Faculty, Department of Biotechnology Engineering, NMAM Institute of Technology, Nitte, Karkala, 574110, India
ARTICLE INFO
ABSTRACT
Keywords: de-oiled Pongamia seed cake Aspergillus ochraceus Microbial lipid Biodiesel Acid hydrolysate
Biofuels and their production is the need of the hour considering the current fuel crisis. In this regard, ways of mitigating biodiesel production costs is being actively researched upon. With the same intention, in the present work, an investigation was carried out to test the capability of utilizing the hydrolysates of de-oiled Pongamia seed cake, a byproduct of biodiesel, for microbial lipid production using Aspergillus ochraceus. This is the first time A. ochraceus is being reported to be used to evaluate its oleaginous property. Also, Pongamia seed cake is the novel source being used for microbial lipid production. The different variants of the seed cake based medium proved acid hydrolysate of de-oiled seed cake to be the effective medium for lipid production. The values recorded for maximum biomass titer, lipid titer and lipid content were 3.065 ± 0.248 g/L, 0.849 ± 0.056 g/L and 28.93%, respectively, upon using acid hydrolysate of the seed cake. The fatty acid composition obtained by GC-MS analysis revealed the presence of linoleic, oleic, stearic and palmitic acids as the major fatty acid components in the lipids produced proving their similarity to the vegetable oils. The fuel properties of the lipids produced were found be in accordance with the standard specifications making them a very good source for biodiesel production. The findings from the current study reveal the effectiveness of Pongamia seed cake for microbial lipid production and simultaneous value addition to the biodiesel industry.
1. Introduction Energy crisis and environmental risks associated with the usage of conventional fuels has led to the shift of research focus towards energy generation from renewable sources (Yen et al., 2015). In this regard, biofuel serves to be an alternative, with a few forms of it being bioethanol, biobutanol and biodiesel. Of these, biodiesel is being accepted globally as a transport fuel in recent years since it enables production from inexpensive renewable feedstocks, is more environmental friendly and has fuel properties almost comparable to that of conventional diesel (Cea et al., 2015; Taskin et al., 2016; Bhatia et al., 2017; Annamalai et al., 2018). Current production of biodiesel is mainly from plant oils such as rapeseed, soybean, sunflower, Jatropa, Pongamia, etc, by means of transesterification (Harde et al., 2016). However, plant derived lipids cannot be relied upon as sole sources of biodiesel production in the years to come due to raising concerns of demand for land areas for plantation, food inflation due to competition with existing food plantations, seasonal dependency, etc, (Amaretti et al., 2012; Poli et al., 2014). Although appreciable
*
Corresponding author. E-mail address: [email protected] (H.M. Jathanna).
https://doi.org/10.1016/j.bcab.2020.101543 Received 4 February 2020; Accepted 12 February 2020 Available online 15 February 2020 1878-8181/© 2020 Elsevier Ltd. All rights reserved.
amount of work has been carried out towards utilizing waste cooking oil and animal fats for biodiesel generation, these sources are scarce and would not be sufficient to meet the continuously growing demands for green fuels (Chang et al., 2015; Dias et al., 2015). Also, these presently used sources in the production of biodiesel account to a significant proportion of the total production cost, necessitating the exploration of newer alternatives for production (Liu et al., 2015; Cea et al., 2015; Bhatia et al., 2017). In this context, utilization of oleaginous microorganisms as a promising substitute for conventional sources was considered (Patel et al., 2015; Deeba et al., 2017; Annamalai et al., 2018). Among the different classes of oleaginous microorganisms, microalgae are the ones which have been majorly researched upon (Devi et al., 2012; Yen et al., 2015). However, microalgae cannot serve the purpose due to certain shortcomings associated with the cultivation such as requirement of sunlight for growth, lesser titer, inefficiency to be mass cultivated in conventional bioreactors, etc. (Ma, 2006; Liu et al., 2015). Therefore, the current attention is on utilizing oleaginous fungi, mainly oleaginous yeasts, including the genera Candida, Cryptococcus, Lipomyces, Rhodotorula, Rhodosporidium,
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Trichosporan, Yarrowia, etc. (Patel et al., 2015; Taskin et al., 2016; Chang et al., 2015), and a very few classes of oleaginous bacteria for biodiesel production since they have the capacity to store more than 20% of their biomass as triglycerides, (Subramaniam et al., 2010; Peng et al., 2013; Poli et al., 2014); the studies have also found out that the oil produced is similar in nature to vegetable oils thus proving microorganisms as ideal sources for biodiesel production (Chang et al., 2015; Taskin et al., 2016; Poontawee et al., 2017; Annamalai et al., 2018). It has been estimated that raw materials used account for almost 75% of the total biodiesel production cost (Meng et al., 2009; Yan et al., 2014; Chang et al., 2015). This has necessitated the search for cheaper raw materials for the production so as to bring down the costs. In this regard, quite a good amount of work has been done in utilizing agro-industrial residues as substrates for microbial lipid production (Subhash and Mohan, 2015; Qadeer et al., 2017; Cho and Park, 2018). Conventional bio-diesel production using non-edible oil seeds results in the oil-seed cake as one of the major byproducts, a major proportion of which remains unutilized other than being used for manure purpose. Jatropha oil that is extensively used in India is alone reported to generate around 70–75% of the seed content as oil seed cake (Singh et al., 2008). Although seed cake can be used as animal fodder, it needs to be treated before being used to serve the purpose. The treatment process being economically not feasible, results in the seed cake waste remaining underutilized. However, this byproduct being a rich source of nutrients, could be exploited for its effective usage as source of microbial lipid production. Thus an effort towards utilizing de-oiled Pongamia seed cake for microbial oil production using the fungal species, Aspergillus ochraceus was undertaken in the present work with the view of utilizing an inexpensive raw material and providing value addition to the underutilized biodiesel industrial residue. To the best of our knowledge, this is the first study reporting the utilization of Pongamia seed cake as a medium for microbial lipid production. Also, the microbial species used has not been reported to have been used earlier.
seed cultures were obtained from Potato Dextrose Broth (PDB) medium maintained at 30 °C and 150 rpm for 3 days.
2. Materials and methods
2.4. Lipid productivity studies
2.1. Materials The primary raw material used for the study was de-oiled Pongamia seed cake. The seed cake was procured from the Biodiesel Centre of the institution, NMAMIT, Nitte; it was stored at −20 °C; prior to use, the cake was dried, powdered and weighed as required. All the chemicals and reagents used for the study were of analytical grade.
For the three media used, shake flask studies were carried out in batch mode. The sterilized media were taken in 250 mL conical flasks considering a working volume of 100 mL. Initial pH of all the media was maintained at 5.5. Each of the media was inoculated with 8% (v/ v) of the seed culture (corresponding to a spore count of approximately 9 × 1010 spores/mL). The flasks were incubated for a period of 5 days at 150 rpm and 30 °C. The experiments were carried out in triplicates.
2.2. Isolation and culturing of strain for microbial lipid production
2.5. Biomass and lipid quantification
The biodiesel oil sludge procured from the Biofuel Centre was used as a source for the isolation of microbial colonies. From the sludge that was rich in fungal growth, pure colonies were isolated on PDA (Potato Dextrose Agar) plates incubated at 30 °C for 72 h. From the colonies observed, a single colony was selected owing to better growth compared to other colonies. Genotypic characterization of the selected strain was carried out by sequencing the ITS region of the 18s rDNA using the fungal universal primers ITS4 and ITS5. The sequences were compared with those in the NCBI database (http://www.ncbi.nlm.nih.gov/) using BLAST programme and analyzed to reach identity. The evolutionary relationship of the species used for the study was obtained by constructing a phylogenetic tree using BioEdit software by considering other sequences which shared the similarity index. The working stock was maintained in PDA slants at 4 °C and was revived every week. For inoculation into the media under study, the
For the measurement of the biomass, the fungal pellets formed in the culture medium were filtered using muslin cloth following the incubation period. These were later dried at nearly 60 °C to 80 °C for about 12–24 h until constant weight was obtained, following which, the biomass was measured gravimetrically. The entire dried biomass was then used for lipid extraction. Lipid extraction was performed according to modified Folch method. To determine the lipid concentration, the release of intracellular lipids is essential. To attain this, the dried biomass was homogenized in an aqueous medium using a glass homogenizer. The homogenized cells were subjected to solvent extraction using chloroformmethanol mixture (2:1) with proper mixing. After phase separation, the chloroform layer was collected and subjected to lipid estimation using phospho-vanillin method (Frings and Dunn, 1970). The microbial lipid was reported as lipid titer (gram of lipid/liter of culture broth).
2.3. Media preparation Three different media based on de-oiled Pongamia seed cake were used for the study. Acid hydrolysate of the de-oiled seed cake (AH) was used as the first medium and was prepared by treating 2.5% (w/ v) of the powdered seed cake with 2% H2SO4 (Yu et al., 2011). This solid loading rate was chosen based on the earlier studies performed in the lab showing 2.5% as the ideal substrate concentration for acid hydrolysis (using 2% H2SO4) of Pongamia seed cake (data not shown). The cake suspension was heated on a low flame for a temperature of around 75 °C for 30 min. The clear supernatant obtained upon sedimentation was neutralized using NaOH flakes; autoclaved and used as the medium for lipid production. The media was chosen considering the fact that acids act upon the recalcitrant lignocellulosic biomass resulting in better release of reducing sugars that can easily be taken up by microorganisms (Mosier et al., 2005). The second medium used was the aqueous hydrolysate of the seed cake (H); this was obtained by boiling 2.5% of the powdered de-oiled seed cake in distilled water at similar conditions as that used for acid treatment, i.e., at a temperature of 75 °C for 30 min. Upon cooling, the clear sediment obtained was autoclaved and used as the medium. An attempt to use this medium was done in the present work in order to evaluate if water treatment alone would be sufficient to release the nutrients from the seed cake which is a rich source of carbon and nitrogen. This was carried out with the intention of attempting to bring down the overall pretreatment cost by avoiding the usage of acid and in turn the base for the neutralization of acidified hydrolysate. The third variant of the Pongamia seed cake based medium used was based on the second medium. Here, the supernatant obtained upon aqueous hydrolysis of the powdered seed cake was added with 1.5 g/L of yeast extract with the view of evaluating if addition of a nitrogen source could positively influence biomass and lipid production.
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Based on the biomass and lipid concentrations obtained, biomass productivity (mg/L/h), lipid productivity (mg/L/h) and biomass content (% dry weight) were calculated.
the following strains (with 99% sequence identity and e-value score of zero): Aspergillus ochraceus strain IHEM 18887 isolate ISHAM-ITS_ID MITS290 (Accession number KP131611.1), A. melleus strain UOA/ HCPF 14021 isolate ISHAM-ITS_ID MITS251 (Acc. No. KC253959.1), Aspergillus sp. 245A (Acc. No. GQ120974.1), A.ochraceus strain AS III (Acc. No. EU805804.2) and A. ochraceus strain UOA/HCPF 9349A isolate ISHAM-ITS_ID MITS291 (Acc. No. FJ878632.1). The sequence homology study revealed that the partial sequence of rDNA gene of 457 bp of the fungal strain used corresponds to Aspergillus ochraceus and a GenBank Accession number of MK483340 was assigned. The taxonomic relationship of the strain used for the study with other related strains was obtained using BioEdit software. The evolutionary history of the taxa was inferred using the Neighbor Joining method. The tree obtained was as shown in Fig. 1. The dendrogram shows that although the organism under study is related to strains belonging to Aspergillus genus, it stands out as a separate clade possibly due to its adaptation with time to the source from which it is being isolated.
2.6. Analysis of extracted lipids Fractionation of lipid samples was performed using the procedure reported by Gong et al. (2016). Sample containing about 100 mg of the lipid was loaded on a silica gel column and eluted using 100 mL of 1,2-dichloroethane, 70 mL of 1,2-dichloroethane/acetone (1:10, v/ v) and 35 mL of methanol in sequence. The respective solvents were evaporated and the weight of each fraction was determined. The fractions in the order of elution were neutral lipids, glycolipids and sphingolipids, and phospholipids. For the analysis of fatty acid composition profiles of the lipid samples, the lipids extracted from the homogenized fungal mass using chloroform-methanol mixture were subjected to transesterification followed by n-hexane extraction of methyl esters. The fatty acid composition of the methyl esters was analyzed by GC-MS (Shimadzu) using a BP-X5 capillary column (30 m × 0.25 mm ID X 0.25 μm). 1 μL of the sample was injected using helium (1 mL/min) as the carrier gas. The oven temperature was initially set to 70 °C, was then raised to 280 °C at the rate of 10 °C/min. The peaks of fatty acids were identified by comparing their retention times to those of known standards. In order to check the utility of lipids produced in biodiesel applications, physical properties of the FAMEs were determined using experimental equations as reported previously by Tanimura et al. (2014). Average unsaturation (AU) was calculated from the compositional profiles as AU = ∑ N X Ci
3.2. Biomass and lipid production in different media The composition of the de-oiled Pongamia seed cake used for the study, as represented in Table 1, was adopted from Kamath et al. (2018) since the present study was carried out in the same laboratory using the same source. The de-oiled seed cake based media used in the study were at first used to evaluate their efficiencies to support the fungal growth. Thus the growth patterns of the fungus A. ochraceus were studied in the three media. The variations of biomass with incubation time observed were as represented in Fig. 2. The consistent growth observed in the acid hydrolysate (AH) medium during the incubation period, reaching a biomass concentration of 3.065 ± 0.248 g/L at 120th hour of incubation, is an indication of the fact that acid hydrolyzed de-oiled seed cake medium, even with-
(1)
where N is the number of carbon-carbon double bonds of unsaturated fatty acids and Ci is the concentration (mass fraction of the component). Viscosity = −0.6316 AU + 5.2065
(2)
Specific gravity = 0.0055 AU + 0.8726
(3)
Cloud point = −13.356 AU + 19.994
(4)
Cetane number = −6.6684 AU + 62.876
(5)
Iodine number = 74.373 AU + 12.71
(6)
Higher Heating Value (HHV) = 17.601 AU + 38.536
(7)
2.7. Analytical methods Estimation of the nutrient content of the medium was carried out for each day of incubation following the separation of fungal cells by filtration. The content of total sugars was estimated using phenol sulfuric acid method (Dubois et al., 1956), reducing sugars by DNSA method (Miller, 1959), proteins by Lowry's method (Lowry et al., 1951) and lipids using the method mentioned earlier. Nutrient estimation was also carried out for each of the media samples prior to inoculation.
Fig. 1. Phylogenetic analysis of ITS sequence of Aspergillus ochraceus strain used for the study. Table 1
Composition of the de- oiled Pongamia seed cake used for the study.
3. Results and discussion 3.1. Strain isolation and identification The identification of the strain used in the present study at the genus level was performed using 18s rDNA based sequencing. The nucleotide sequence of length 457 bp obtained upon amplifying the ITS regions of rDNA when searched for similarity with other sequences available in the public database, revealed sequence homology with 3
Components
%(w/w)
Cellulose Hemicellulose Starch Lignin Protein Lipid Moisture Ash
27.60 ± 1.17 31.20 ± 0.85 1.10 ± 0.10 4.20 ± 0.51 13.10 ± 1.15 16.45 ± 1.13 3.16 ± 0.96 5.32 ± 0.92
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Fig. 2. Comparison of biomass titer, lipid titer, nutrient estimation profiles for different media: AH (Acid Hydrolysate), H (Aqueous hydrolysate), HY (aqueous hydrolysate supplemented with yeast extract).
out the addition of any media supplements, contains enough nutrients to support the fungal growth. Fig. 2(a) represents the initial nutrient content of the medium which indicates higher concentrations of sugars (8.962 ± 0.052 g/L of total sugars and 7.38 ± 0.046 g/L of reducing sugars) when compared to proteins (1.896 ± 0.032 g/L) or lipids (2.106 ± 0.155 g/L). There are reports available wherein acid hydrolysates of the lignocelluloses have been used as media for microbial lipid production but with added salts (Economou et al., 2011; Yu et al., 2011; Huang et al., 2009) which proves the cost effectiveness of the current study. In the study, an attempt was made to utilize the aqueous hydrolysate of the de-oiled seed cake as a source for microbial lipid production as a means of investigating the potential of the hydrolysate to support microbial growth without any acid pretreatment. Interestingly, a constant increase in the biomass production was observed even in case of aqueous hydrolysate (H) medium with the biomass concentration increasing from 0.31 ± 0.028 g/L to 2.62 ± 0.077 g/L during 24th to 96th hour of incubation with a slight drop to 2.31 ± 0.12 g/L towards the end of incubation period which may be due to depletion of nutrients in the medium as represented in Fig. 2 (b), towards the end of the incubation period. Although the biomass production during the total incubation period was lesser when compared to the acid hydrolysate medium, which could be due to comparatively lower nutritional value of the medium, the aqueous hydrolysate medium did not inhibit microbial growth as such, instead supported the growth to a good extent. These results demonstrate that even mild water treatment can solubilize the cellulosic content of the de-oiled seed cake and release sufficient nutrients to support the microbial growth. This paves the way to explore the possibility of optimizing the culture conditions for this medium so as to enhance biomass production, thus eliminating the need of utilizing acid for hy-
drolysis in turn bringing down the overall costs of producing microbial lipids from de-oiled seed cake source. However, considering that the addition of media supplements to the aqueous hydrolysate would enhance the nutrient value of the medium in turn promoting better fungal growth and lipid production, the aqueous hydrolysate was supplemented with yeast extract (1.5 g/L) and was used as the third medium As expected, there was significant enhancement in the nutritional value of the medium both with respect to the initial concentration of total sugars and proteins, as is evident from Fig. 2(c). In accordance with this, the maximum biomass concentration attained in this medium was higher compared to the other two media studied, with the concentration reaching a maximum of 3.762 ± 0.157 g/L during 72nd hour of incubation that slightly dropped to 3.282 ± 0.162 g/L towards 120th hour of incubation. The results for biomass production obtained in all the three variants of the seed cake based media thus indicate the potential of the unconventional media studied in supporting the growth of the fungus, A. ochraceus. At the same time, it also proves the potential of this indigenous fungus in adapting itself to the undefined media studied and showing considerable growth. Following biomass estimation, the media used in the study were investigated for their potential to support fungal lipid production in addition to supporting fungal growth. Lipid production profiles in the different media studied indicate that higher lipid titer values were obtained in acid hydrolysate (AH) medium when compared to the other two media. The maximum lipid titer of 0.849 ± 0.056 g/L was obtained for the AH medium during 72nd hour of incubation while that obtained for aqueous hydrolysate medium was 0.622 ± 0.017 g/L during 96th hour and for HY medium was 0.2133 ± 0.011 g/L during 72nd hour of incubation. A drop in the lipid production towards the end of the incubation period observed in each of the media studied could be attributed to the utilization of storage lipids by the fungus to 4
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maintain sustained growth till the end. On the whole, the results obtained suggest that the acid hydrolysate of the de-oiled Pongamia seed cake serves as a favorable medium for the fungal lipid production when compared to either the aqueous hydrolysate or the aqueous hydrolysate supplemented with yeast extract. Lower lipid production in aqueous hydrolysate medium compared to the acid hydrolysate medium is in agreement with the poor nutritional value of the medium as compared to the acid hydrolysate medium. However, till date, there are no reports of utilization of aqueous hydrolysate alone of any seed cake for microbial lipid production. In this regard, the efficiency of the medium to facilitate lipid production could not be ruled out completely owing to the fact that in spite of its restricted nutritional value, it still could result in some lipid production in addition to supporting microbial growth. On the other hand, in the medium containing aqueous hydrolysate supplemented with yeast extract, the lipid production was much lesser compared to all other media throughout the incubation period. Although enhancing the nutrient value of aqueous hydrolysate by adding yeast extract supplement resulted in increased biomass production compared to the other media, it did not have any positive influence on lipid production. Yeast extract, being a source of nitrogen, results in the decrease of C/N content of the medium. The fact that the extent of lipid production is pro-
portional to the C/N ratio in the medium (Papanikolaou and Aggelis, 2002) could be the reason for the decreased lipid production observed upon supplementation with yeast extract. Fig. 3 shows comparison of biomass productivity (mg/L/h), lipid productivity (mg/L/h) and lipid content (%) obtained for different test media; the values of maximum biomass productivity, lipid productivity, lipid content obtained for acid hydrolysate medium were 40.76 mg/L/h and 11.79 mg/L/h and 28.93%, respectively. From the results it may be concluded that excess carbon in the media results in the distribution of the available carbon source between two metabolic pathways – biomass production and lipid accumulation (Subhash and Mohan, 2015; Gong et al., 2016). As evident from the figure, although biomass productivity was higher in the aqueous hydrolysate medium supplemented with yeast extract, both lipid productivity and lipid content were the minimum. Since the focus of the study was to produce microbial lipids, the results further confirm that the medium containing acid hydrolysate of the seed cake is most favorable for the purpose. Estimation of nutrients in the media performed during the incubation period for all the media under study demonstrate that it was sugars that was utilized by the fungi to the maximum extend to support the cell growth and corresponding lipid production. In HY media, although the initial concentration of total sugars, proteins and lipids was higher compared to the other two media owing to media supplementation, this did not have any significant effect on fungal lipid production which could be due to low C/N ratio as discussed earlier. The continuous drop in the concentration of total sugars with time is well in agreement with the constant increase in biomass production during the incubation period in all the media as observed in the figure. Maximum biomass and lipid production in AH medium compared to the other two medium could be attributed to the higher concentration of reducing sugars present in the AH medium compared to the others (at 0th hour, the concentration of the reducing sugars in AH, H and HY media were 7.38 ± 0.047 g/L, 1.464 ± 0.033 g/L and 1.46 g/L respectively), leading to their effective utilization towards biomass as well as lipid production along with the utilization of total sugars as is evident from the nutrient utilization profiles. The results obtained are consistent with the studies carried out with acid hydrolysates of feed stocks such as wheat straw (Yu et al., 2011), corncob (Xavier and Franco, 2014), carop pulp (Freitas et al. (2014), etc, as mentioned in Table 2. The results are also in agreement with other works where Aspergillus strains have been reported to be used for biodiesel production exhibiting FAME profiles containing 30.2%, 31.5%, 38.3% of satu-
Fig. 3. Comparison of biomass productivity, lipid productivity and lipid content profiles for different media: AH (Acid Hydrolysate), H (Aqueous hydrolysate), HY (Aqueous hydrolysate supplemented with yeast extract). Table 2
Data on biomass and lipid productivities using different sources. Source
Pretreatment Method
Organism
Lipid Content (%)
Lipid productivity (g/L/h)
Fatty Acid Profiles
Ref
Detoxified rice straw hydrolysate
Dilute acid
Trichosporan fermentans CICC 1368
40.1
0.06
palmitate> stearate> oleiate> linoleiate
Huang et al. (2009)
Detoxified sugarcane bagasse hydrolysate
Dilute acid
Yarrowia lipolytica Po1g
58.5
0.073
oleiate> palmitate> palmitoleiate>
Y.A.Tsigie et al. (2011)
stearate Wheat straw hydrolysate
Dilute acid
Cryptococcus curvatus
33.5
0.04
palmitate> stearate> oleiate> linoleiate
Yu et al. (2011)
(ATCC, 20509) Detoxified corncob hydrolysate
Dilute acid
Trichosporan cutaneum
36
0.041
palmitate> stearate> oleiate> linoleiate
Chen et al. (2012)
Sugarcane bagasse
Dilute acid
Lipomyces starkeyi
26.6
0.026
oleiate>palmitate> stearate> linoleiate
Xavier and Franco
Carob pulp syrup
Aqueous
Rhodosporidium toruloides
17.27
0.019
Not performed
Freitas et al. (2014)
53.18
0.033
palmitate> stearate> oleiate
A.Patel et al. (2015)
(2014) NCYC 921 Aqueous extract of Cassia fistula L. fruit pulp
Aqueous
Rhodosporidium kratochvilovae HIMPA1
Corncob hydrolysate+6% glucose
Dilute acid
Cryptococcus sp. SM5S05
60.2
0.079
oleiate> palmitate> stearate> linoleiate
Chang et al. (2015)
Sonicated papermill sludge extract
Aqueous
Cryptococcus vishniaccii (MTCC 232)
53.4
0.054
oleiate> palmitate> linoleiate>stearate
Deeba et al. (2016)
Cellulosic ethanol fermentation wastewater
NIL
Trichosporon cutaneum ACCC 20271
13.3
0.018
oleiate> palmitate> stearate
Wang et al. (2017)
Office
Hydrogen peroxide
Cryptococcus curvatus
37.8
0.08
oleiate> palmitate> stearate> linoleiate
Annamalai et al. (2018)
Aspegillus ochraceus MK483340
28.93
0.012
Linoleate> oleate> palmitate> stearate
Present study
waste paper hydrolysate (enzyme hydrolysis) Acid hydrolysate of Pongamia seed cake
treatment Dilute acid
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rated, monounsaturated and polyunsaturated methyl esters, respectively (Kakkad et al., 2015). The media used in the current study was not an enriched one, instead it comprised of a small concentration of dilute acid pretreated de-oiled seed cake (2.5%) without any nutrient enhancement. This could be one of the reasons for lipid content and productivity obtained in the present study to be comparatively lesser the other studies mentioned above. Optimization of the pretreatment conditions with respect to the seed cake and the acid concentration for effective solubilization of the source could be one of the strategies employed to enhance lipid yield and content. In addition, acid pretreatment of the lignocellulosic biomass is known to release inhibitors in to the medium, majorly, furfural and 5-hydroxymethylfurfural (HMF) from the degradation of pentoses and hexoses (Mosier et al., 2005) which restrict the growth of microorganisms in turn restricting microbial lipid production. In this regard, efforts have been made in some of the works to detoxify the medium to bring down the effect of toxic inhibitors (Tsigie et al., 2011; Yu et al., 2011; Ahmad et al., 2016). Accordingly, detoxification of the medium following acid pretreatment could be another strategies followed to improve the overall biomass and lipid productivity.
Table 3
Table representing the biodiesel properties of the FAMEs obtained from the lipids produced by Aspergillus ochraceus in the medium containing acid hydrolysate of Pongamia seed cake and comparison with the fuel properties of Jatropha oil and rapeseed oil and the standard specifications (ASTM 6751 and EN 14214). Biodiesel properties
ASTM D6751
EN 14214
Jatropha oil
Rapeseed oil
Present study (acid hydrolysate of Pongamia seed cake)
Kinematic Viscosity (mm2/s) Density (g/cm3) Cloud point (°C) Cetane number
1.9–6.0
3.5–5.0
4.48
4.40
4.53
–
0.86–0.90 0.8789
0.8796
0.8784
–
–
4.67
2.93
5.68
47 (min) –
51 (min)
55.23
54.35
55.73
120 (max) –
98.02
107.76
92.42
40.55
40.78
40.42
Iodine number High heating value (MJ/kg)
3.3. Analysis of lipids produced by Aspergillus ochraceus in acid hydrolysate medium
–
All the properties calculated such as kinematic viscosity, density and iodine number (iodine value) that depends on the degree of unsaturation, as well as cetane number that depends on the degree of saturation, are well in agreement with the specified standard limits and the corresponding values for Jatropha oil and rapeseed oil thus indicating a optimum ratio of both saturated and unsaturated FAMEs required for improved biodiesel quality (Deeba et al., 2017). The cloud point value which refers to the temperature, below which the biodiesel gels or solidifies thus clogging the filter, also is in close agreement with the value for biodiesel obtained from Jatropha oil thus indicating the optimum temperature properties of the oil obtained in the present study. In addition, the gross energy content of the FAMEs obtained from the present study is equivalent to those of rapeseed and Jatropha oil. Although the fungal lipids produced in the present study are rich in unsaturated fatty acids when compared to saturated fatty acids, these oils can still be used as good sources for biodiesel production with the use of certain chemical or enzyme catalysts (Chang et al., 2013). In addition, the oil produced is rich in monounsaturated fatty acid (30.27%) which makes it an excellent source for production of biodiesel (Sankh et al., 2013). This is because although the linoleic content is higher, high amount of oleic acid balances the amount of saturated fatty acids and polyunsaturated fatty acids. All these properties prove the possibility of exploiting the oil produced by Aspergillus ochraceus from de-oiled Pongamia seed cake based medium for biodiesel applications.
Owing to the maximum production of fungal lipid in the medium containing acid hydrolysate as compared to the other two media, further analysis of the lipids produced in the acid hydrolysate medium was carried out to characterize and to determine the quantity of triacylglycerols (TAG) in the lipids produced. Upon characterization of lipids it was observed that the percentages of neutral lipids, glycolipids and sphingolipids, and phospholipids were 92.86%, 5% and 2.14%, respectively. Thus, it is clear that, the neutral lipids contribute to significant proportion of the lipids produced by A. ochraceus. The FAME profiles of lipids produced by A.ochraceus grown on acid hydrolysate medium were analyzed using GC-MS which revealed that the lipids mainly comprised of methyl esters of linoleic acid (C18:2; 38.25%), oleic acid (C18:1; 30.27%), palmitic acid (C16:0; 18.27%), stearic acid (C18:0; 10.43%); traces of behenic acid (0.91%), lignoceric acid (0.71%), arachidic acid (0.48%) and myristic acid (0.28%). The results are well in agreement with the fatty acid contents of sunflower oil, soybean oil and Jatropha oil that are commonly used as a source for biodiesel production with the methyl ester profiles being linoleic acid (66.2%, 53.2%, 32.48%), oleic acid (21.1%, 23.4%, 39.08%), stearic acid (4.5%, 4%, 6.86%), palmitic acid (0%, 11%, 14.66%), for sunflower oil, soybean oil and Jatropha oil, respectively (Patel et al., 2015). Also, the results obtained are consistent with the previous studies (as mentioned in Table 2) wherein a similar lipid compositional profile has been obtained for the cultivation of various yeast strains in different unconventional media. The results thus indicate the presence of fatty acids mainly with 18 and 16 carbon atoms thus proving the fatty acid profile to be similar to those of vegetable oils (Gong et al., 2016). Thus the fungal lipid produced can be considered as a potential feedstock for biodiesel production.
4. Conclusions In the present study, an unconventional medium, Pongamia seed cake was investigated as a cost-effective source for the production of lipids using a novel fungal strain of A.ochraceus. The growth of fungus A. ochraceus in all the variants of the source used indicates the potential of the source to support microbial growth. A maximum lipid content of 28.93% obtained in the medium containing the acid hydrolysate of the seed cake confirms the effectiveness of both the source as well as the microbe in offering a cost effective resource for biodiesel production as well as providing value addition to residues generated in biodiesel industries. Optimization of the culture conditions may possibly result in further enhancement in lipid production.
3.4. Biodiesel properties of the microbial lipid In order to evaluate if the lipids produced by A. ochraceus could be utilized as a source for biodiesel production, important biodiesel characteristics of the lipid produced were obtained by theoretical means and compared with the US and European specifications for biodiesel, ASTM 6751 and EN 14214 and those of routinely used plant oils for the production of biodiesel as represented in Table 3. The lipids produced in the medium containing acid hydrolysate medium were used for calculation purpose.
Declaration of competing interest The authors declare that they have no conflict of interest. 6
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Acknowledgements
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Biocatalysis and Agricultural Biotechnology
Exploring Pongamia seed cake hydrolysate as a medium for microbial lipid production by Aspergillus ochraceus Harshitha Madhusoodan Jathanna *, Chandrayan Vaman Rao, Louella Concepta Goveas Visvesvaraya Technological University, Faculty, Department of Biotechnology Engineering, NMAM Institute of Technology, Nitte, Karkala, 574110, India
ARTICLE INFO
ABSTRACT
Keywords: de-oiled Pongamia seed cake Aspergillus ochraceus Microbial lipid Biodiesel Acid hydrolysate
Biofuels and their production is the need of the hour considering the current fuel crisis. In this regard, ways of mitigating biodiesel production costs is being actively researched upon. With the same intention, in the present work, an investigation was carried out to test the capability of utilizing the hydrolysates of de-oiled Pongamia seed cake, a byproduct of biodiesel, for microbial lipid production using Aspergillus ochraceus. This is the first time A. ochraceus is being reported to be used to evaluate its oleaginous property. Also, Pongamia seed cake is the novel source being used for microbial lipid production. The different variants of the seed cake based medium proved acid hydrolysate of de-oiled seed cake to be the effective medium for lipid production. The values recorded for maximum biomass titer, lipid titer and lipid content were 3.065 ± 0.248 g/L, 0.849 ± 0.056 g/L and 28.93%, respectively, upon using acid hydrolysate of the seed cake. The fatty acid composition obtained by GC-MS analysis revealed the presence of linoleic, oleic, stearic and palmitic acids as the major fatty acid components in the lipids produced proving their similarity to the vegetable oils. The fuel properties of the lipids produced were found be in accordance with the standard specifications making them a very good source for biodiesel production. The findings from the current study reveal the effectiveness of Pongamia seed cake for microbial lipid production and simultaneous value addition to the biodiesel industry.
1. Introduction Energy crisis and environmental risks associated with the usage of conventional fuels has led to the shift of research focus towards energy generation from renewable sources (Yen et al., 2015). In this regard, biofuel serves to be an alternative, with a few forms of it being bioethanol, biobutanol and biodiesel. Of these, biodiesel is being accepted globally as a transport fuel in recent years since it enables production from inexpensive renewable feedstocks, is more environmental friendly and has fuel properties almost comparable to that of conventional diesel (Cea et al., 2015; Taskin et al., 2016; Bhatia et al., 2017; Annamalai et al., 2018). Current production of biodiesel is mainly from plant oils such as rapeseed, soybean, sunflower, Jatropa, Pongamia, etc, by means of transesterification (Harde et al., 2016). However, plant derived lipids cannot be relied upon as sole sources of biodiesel production in the years to come due to raising concerns of demand for land areas for plantation, food inflation due to competition with existing food plantations, seasonal dependency, etc, (Amaretti et al., 2012; Poli et al., 2014). Although appreciable
*
Corresponding author. E-mail address: [email protected] (H.M. Jathanna).
https://doi.org/10.1016/j.bcab.2020.101543 Received 4 February 2020; Accepted 12 February 2020 Available online 15 February 2020 1878-8181/© 2020 Elsevier Ltd. All rights reserved.
amount of work has been carried out towards utilizing waste cooking oil and animal fats for biodiesel generation, these sources are scarce and would not be sufficient to meet the continuously growing demands for green fuels (Chang et al., 2015; Dias et al., 2015). Also, these presently used sources in the production of biodiesel account to a significant proportion of the total production cost, necessitating the exploration of newer alternatives for production (Liu et al., 2015; Cea et al., 2015; Bhatia et al., 2017). In this context, utilization of oleaginous microorganisms as a promising substitute for conventional sources was considered (Patel et al., 2015; Deeba et al., 2017; Annamalai et al., 2018). Among the different classes of oleaginous microorganisms, microalgae are the ones which have been majorly researched upon (Devi et al., 2012; Yen et al., 2015). However, microalgae cannot serve the purpose due to certain shortcomings associated with the cultivation such as requirement of sunlight for growth, lesser titer, inefficiency to be mass cultivated in conventional bioreactors, etc. (Ma, 2006; Liu et al., 2015). Therefore, the current attention is on utilizing oleaginous fungi, mainly oleaginous yeasts, including the genera Candida, Cryptococcus, Lipomyces, Rhodotorula, Rhodosporidium,
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Trichosporan, Yarrowia, etc. (Patel et al., 2015; Taskin et al., 2016; Chang et al., 2015), and a very few classes of oleaginous bacteria for biodiesel production since they have the capacity to store more than 20% of their biomass as triglycerides, (Subramaniam et al., 2010; Peng et al., 2013; Poli et al., 2014); the studies have also found out that the oil produced is similar in nature to vegetable oils thus proving microorganisms as ideal sources for biodiesel production (Chang et al., 2015; Taskin et al., 2016; Poontawee et al., 2017; Annamalai et al., 2018). It has been estimated that raw materials used account for almost 75% of the total biodiesel production cost (Meng et al., 2009; Yan et al., 2014; Chang et al., 2015). This has necessitated the search for cheaper raw materials for the production so as to bring down the costs. In this regard, quite a good amount of work has been done in utilizing agro-industrial residues as substrates for microbial lipid production (Subhash and Mohan, 2015; Qadeer et al., 2017; Cho and Park, 2018). Conventional bio-diesel production using non-edible oil seeds results in the oil-seed cake as one of the major byproducts, a major proportion of which remains unutilized other than being used for manure purpose. Jatropha oil that is extensively used in India is alone reported to generate around 70–75% of the seed content as oil seed cake (Singh et al., 2008). Although seed cake can be used as animal fodder, it needs to be treated before being used to serve the purpose. The treatment process being economically not feasible, results in the seed cake waste remaining underutilized. However, this byproduct being a rich source of nutrients, could be exploited for its effective usage as source of microbial lipid production. Thus an effort towards utilizing de-oiled Pongamia seed cake for microbial oil production using the fungal species, Aspergillus ochraceus was undertaken in the present work with the view of utilizing an inexpensive raw material and providing value addition to the underutilized biodiesel industrial residue. To the best of our knowledge, this is the first study reporting the utilization of Pongamia seed cake as a medium for microbial lipid production. Also, the microbial species used has not been reported to have been used earlier.
seed cultures were obtained from Potato Dextrose Broth (PDB) medium maintained at 30 °C and 150 rpm for 3 days.
2. Materials and methods
2.4. Lipid productivity studies
2.1. Materials The primary raw material used for the study was de-oiled Pongamia seed cake. The seed cake was procured from the Biodiesel Centre of the institution, NMAMIT, Nitte; it was stored at −20 °C; prior to use, the cake was dried, powdered and weighed as required. All the chemicals and reagents used for the study were of analytical grade.
For the three media used, shake flask studies were carried out in batch mode. The sterilized media were taken in 250 mL conical flasks considering a working volume of 100 mL. Initial pH of all the media was maintained at 5.5. Each of the media was inoculated with 8% (v/ v) of the seed culture (corresponding to a spore count of approximately 9 × 1010 spores/mL). The flasks were incubated for a period of 5 days at 150 rpm and 30 °C. The experiments were carried out in triplicates.
2.2. Isolation and culturing of strain for microbial lipid production
2.5. Biomass and lipid quantification
The biodiesel oil sludge procured from the Biofuel Centre was used as a source for the isolation of microbial colonies. From the sludge that was rich in fungal growth, pure colonies were isolated on PDA (Potato Dextrose Agar) plates incubated at 30 °C for 72 h. From the colonies observed, a single colony was selected owing to better growth compared to other colonies. Genotypic characterization of the selected strain was carried out by sequencing the ITS region of the 18s rDNA using the fungal universal primers ITS4 and ITS5. The sequences were compared with those in the NCBI database (http://www.ncbi.nlm.nih.gov/) using BLAST programme and analyzed to reach identity. The evolutionary relationship of the species used for the study was obtained by constructing a phylogenetic tree using BioEdit software by considering other sequences which shared the similarity index. The working stock was maintained in PDA slants at 4 °C and was revived every week. For inoculation into the media under study, the
For the measurement of the biomass, the fungal pellets formed in the culture medium were filtered using muslin cloth following the incubation period. These were later dried at nearly 60 °C to 80 °C for about 12–24 h until constant weight was obtained, following which, the biomass was measured gravimetrically. The entire dried biomass was then used for lipid extraction. Lipid extraction was performed according to modified Folch method. To determine the lipid concentration, the release of intracellular lipids is essential. To attain this, the dried biomass was homogenized in an aqueous medium using a glass homogenizer. The homogenized cells were subjected to solvent extraction using chloroformmethanol mixture (2:1) with proper mixing. After phase separation, the chloroform layer was collected and subjected to lipid estimation using phospho-vanillin method (Frings and Dunn, 1970). The microbial lipid was reported as lipid titer (gram of lipid/liter of culture broth).
2.3. Media preparation Three different media based on de-oiled Pongamia seed cake were used for the study. Acid hydrolysate of the de-oiled seed cake (AH) was used as the first medium and was prepared by treating 2.5% (w/ v) of the powdered seed cake with 2% H2SO4 (Yu et al., 2011). This solid loading rate was chosen based on the earlier studies performed in the lab showing 2.5% as the ideal substrate concentration for acid hydrolysis (using 2% H2SO4) of Pongamia seed cake (data not shown). The cake suspension was heated on a low flame for a temperature of around 75 °C for 30 min. The clear supernatant obtained upon sedimentation was neutralized using NaOH flakes; autoclaved and used as the medium for lipid production. The media was chosen considering the fact that acids act upon the recalcitrant lignocellulosic biomass resulting in better release of reducing sugars that can easily be taken up by microorganisms (Mosier et al., 2005). The second medium used was the aqueous hydrolysate of the seed cake (H); this was obtained by boiling 2.5% of the powdered de-oiled seed cake in distilled water at similar conditions as that used for acid treatment, i.e., at a temperature of 75 °C for 30 min. Upon cooling, the clear sediment obtained was autoclaved and used as the medium. An attempt to use this medium was done in the present work in order to evaluate if water treatment alone would be sufficient to release the nutrients from the seed cake which is a rich source of carbon and nitrogen. This was carried out with the intention of attempting to bring down the overall pretreatment cost by avoiding the usage of acid and in turn the base for the neutralization of acidified hydrolysate. The third variant of the Pongamia seed cake based medium used was based on the second medium. Here, the supernatant obtained upon aqueous hydrolysis of the powdered seed cake was added with 1.5 g/L of yeast extract with the view of evaluating if addition of a nitrogen source could positively influence biomass and lipid production.
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Based on the biomass and lipid concentrations obtained, biomass productivity (mg/L/h), lipid productivity (mg/L/h) and biomass content (% dry weight) were calculated.
the following strains (with 99% sequence identity and e-value score of zero): Aspergillus ochraceus strain IHEM 18887 isolate ISHAM-ITS_ID MITS290 (Accession number KP131611.1), A. melleus strain UOA/ HCPF 14021 isolate ISHAM-ITS_ID MITS251 (Acc. No. KC253959.1), Aspergillus sp. 245A (Acc. No. GQ120974.1), A.ochraceus strain AS III (Acc. No. EU805804.2) and A. ochraceus strain UOA/HCPF 9349A isolate ISHAM-ITS_ID MITS291 (Acc. No. FJ878632.1). The sequence homology study revealed that the partial sequence of rDNA gene of 457 bp of the fungal strain used corresponds to Aspergillus ochraceus and a GenBank Accession number of MK483340 was assigned. The taxonomic relationship of the strain used for the study with other related strains was obtained using BioEdit software. The evolutionary history of the taxa was inferred using the Neighbor Joining method. The tree obtained was as shown in Fig. 1. The dendrogram shows that although the organism under study is related to strains belonging to Aspergillus genus, it stands out as a separate clade possibly due to its adaptation with time to the source from which it is being isolated.
2.6. Analysis of extracted lipids Fractionation of lipid samples was performed using the procedure reported by Gong et al. (2016). Sample containing about 100 mg of the lipid was loaded on a silica gel column and eluted using 100 mL of 1,2-dichloroethane, 70 mL of 1,2-dichloroethane/acetone (1:10, v/ v) and 35 mL of methanol in sequence. The respective solvents were evaporated and the weight of each fraction was determined. The fractions in the order of elution were neutral lipids, glycolipids and sphingolipids, and phospholipids. For the analysis of fatty acid composition profiles of the lipid samples, the lipids extracted from the homogenized fungal mass using chloroform-methanol mixture were subjected to transesterification followed by n-hexane extraction of methyl esters. The fatty acid composition of the methyl esters was analyzed by GC-MS (Shimadzu) using a BP-X5 capillary column (30 m × 0.25 mm ID X 0.25 μm). 1 μL of the sample was injected using helium (1 mL/min) as the carrier gas. The oven temperature was initially set to 70 °C, was then raised to 280 °C at the rate of 10 °C/min. The peaks of fatty acids were identified by comparing their retention times to those of known standards. In order to check the utility of lipids produced in biodiesel applications, physical properties of the FAMEs were determined using experimental equations as reported previously by Tanimura et al. (2014). Average unsaturation (AU) was calculated from the compositional profiles as AU = ∑ N X Ci
3.2. Biomass and lipid production in different media The composition of the de-oiled Pongamia seed cake used for the study, as represented in Table 1, was adopted from Kamath et al. (2018) since the present study was carried out in the same laboratory using the same source. The de-oiled seed cake based media used in the study were at first used to evaluate their efficiencies to support the fungal growth. Thus the growth patterns of the fungus A. ochraceus were studied in the three media. The variations of biomass with incubation time observed were as represented in Fig. 2. The consistent growth observed in the acid hydrolysate (AH) medium during the incubation period, reaching a biomass concentration of 3.065 ± 0.248 g/L at 120th hour of incubation, is an indication of the fact that acid hydrolyzed de-oiled seed cake medium, even with-
(1)
where N is the number of carbon-carbon double bonds of unsaturated fatty acids and Ci is the concentration (mass fraction of the component). Viscosity = −0.6316 AU + 5.2065
(2)
Specific gravity = 0.0055 AU + 0.8726
(3)
Cloud point = −13.356 AU + 19.994
(4)
Cetane number = −6.6684 AU + 62.876
(5)
Iodine number = 74.373 AU + 12.71
(6)
Higher Heating Value (HHV) = 17.601 AU + 38.536
(7)
2.7. Analytical methods Estimation of the nutrient content of the medium was carried out for each day of incubation following the separation of fungal cells by filtration. The content of total sugars was estimated using phenol sulfuric acid method (Dubois et al., 1956), reducing sugars by DNSA method (Miller, 1959), proteins by Lowry's method (Lowry et al., 1951) and lipids using the method mentioned earlier. Nutrient estimation was also carried out for each of the media samples prior to inoculation.
Fig. 1. Phylogenetic analysis of ITS sequence of Aspergillus ochraceus strain used for the study. Table 1
Composition of the de- oiled Pongamia seed cake used for the study.
3. Results and discussion 3.1. Strain isolation and identification The identification of the strain used in the present study at the genus level was performed using 18s rDNA based sequencing. The nucleotide sequence of length 457 bp obtained upon amplifying the ITS regions of rDNA when searched for similarity with other sequences available in the public database, revealed sequence homology with 3
Components
%(w/w)
Cellulose Hemicellulose Starch Lignin Protein Lipid Moisture Ash
27.60 ± 1.17 31.20 ± 0.85 1.10 ± 0.10 4.20 ± 0.51 13.10 ± 1.15 16.45 ± 1.13 3.16 ± 0.96 5.32 ± 0.92
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Biocatalysis and Agricultural Biotechnology 24 (2020) 101543
Fig. 2. Comparison of biomass titer, lipid titer, nutrient estimation profiles for different media: AH (Acid Hydrolysate), H (Aqueous hydrolysate), HY (aqueous hydrolysate supplemented with yeast extract).
out the addition of any media supplements, contains enough nutrients to support the fungal growth. Fig. 2(a) represents the initial nutrient content of the medium which indicates higher concentrations of sugars (8.962 ± 0.052 g/L of total sugars and 7.38 ± 0.046 g/L of reducing sugars) when compared to proteins (1.896 ± 0.032 g/L) or lipids (2.106 ± 0.155 g/L). There are reports available wherein acid hydrolysates of the lignocelluloses have been used as media for microbial lipid production but with added salts (Economou et al., 2011; Yu et al., 2011; Huang et al., 2009) which proves the cost effectiveness of the current study. In the study, an attempt was made to utilize the aqueous hydrolysate of the de-oiled seed cake as a source for microbial lipid production as a means of investigating the potential of the hydrolysate to support microbial growth without any acid pretreatment. Interestingly, a constant increase in the biomass production was observed even in case of aqueous hydrolysate (H) medium with the biomass concentration increasing from 0.31 ± 0.028 g/L to 2.62 ± 0.077 g/L during 24th to 96th hour of incubation with a slight drop to 2.31 ± 0.12 g/L towards the end of incubation period which may be due to depletion of nutrients in the medium as represented in Fig. 2 (b), towards the end of the incubation period. Although the biomass production during the total incubation period was lesser when compared to the acid hydrolysate medium, which could be due to comparatively lower nutritional value of the medium, the aqueous hydrolysate medium did not inhibit microbial growth as such, instead supported the growth to a good extent. These results demonstrate that even mild water treatment can solubilize the cellulosic content of the de-oiled seed cake and release sufficient nutrients to support the microbial growth. This paves the way to explore the possibility of optimizing the culture conditions for this medium so as to enhance biomass production, thus eliminating the need of utilizing acid for hy-
drolysis in turn bringing down the overall costs of producing microbial lipids from de-oiled seed cake source. However, considering that the addition of media supplements to the aqueous hydrolysate would enhance the nutrient value of the medium in turn promoting better fungal growth and lipid production, the aqueous hydrolysate was supplemented with yeast extract (1.5 g/L) and was used as the third medium As expected, there was significant enhancement in the nutritional value of the medium both with respect to the initial concentration of total sugars and proteins, as is evident from Fig. 2(c). In accordance with this, the maximum biomass concentration attained in this medium was higher compared to the other two media studied, with the concentration reaching a maximum of 3.762 ± 0.157 g/L during 72nd hour of incubation that slightly dropped to 3.282 ± 0.162 g/L towards 120th hour of incubation. The results for biomass production obtained in all the three variants of the seed cake based media thus indicate the potential of the unconventional media studied in supporting the growth of the fungus, A. ochraceus. At the same time, it also proves the potential of this indigenous fungus in adapting itself to the undefined media studied and showing considerable growth. Following biomass estimation, the media used in the study were investigated for their potential to support fungal lipid production in addition to supporting fungal growth. Lipid production profiles in the different media studied indicate that higher lipid titer values were obtained in acid hydrolysate (AH) medium when compared to the other two media. The maximum lipid titer of 0.849 ± 0.056 g/L was obtained for the AH medium during 72nd hour of incubation while that obtained for aqueous hydrolysate medium was 0.622 ± 0.017 g/L during 96th hour and for HY medium was 0.2133 ± 0.011 g/L during 72nd hour of incubation. A drop in the lipid production towards the end of the incubation period observed in each of the media studied could be attributed to the utilization of storage lipids by the fungus to 4
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Biocatalysis and Agricultural Biotechnology 24 (2020) 101543
maintain sustained growth till the end. On the whole, the results obtained suggest that the acid hydrolysate of the de-oiled Pongamia seed cake serves as a favorable medium for the fungal lipid production when compared to either the aqueous hydrolysate or the aqueous hydrolysate supplemented with yeast extract. Lower lipid production in aqueous hydrolysate medium compared to the acid hydrolysate medium is in agreement with the poor nutritional value of the medium as compared to the acid hydrolysate medium. However, till date, there are no reports of utilization of aqueous hydrolysate alone of any seed cake for microbial lipid production. In this regard, the efficiency of the medium to facilitate lipid production could not be ruled out completely owing to the fact that in spite of its restricted nutritional value, it still could result in some lipid production in addition to supporting microbial growth. On the other hand, in the medium containing aqueous hydrolysate supplemented with yeast extract, the lipid production was much lesser compared to all other media throughout the incubation period. Although enhancing the nutrient value of aqueous hydrolysate by adding yeast extract supplement resulted in increased biomass production compared to the other media, it did not have any positive influence on lipid production. Yeast extract, being a source of nitrogen, results in the decrease of C/N content of the medium. The fact that the extent of lipid production is pro-
portional to the C/N ratio in the medium (Papanikolaou and Aggelis, 2002) could be the reason for the decreased lipid production observed upon supplementation with yeast extract. Fig. 3 shows comparison of biomass productivity (mg/L/h), lipid productivity (mg/L/h) and lipid content (%) obtained for different test media; the values of maximum biomass productivity, lipid productivity, lipid content obtained for acid hydrolysate medium were 40.76 mg/L/h and 11.79 mg/L/h and 28.93%, respectively. From the results it may be concluded that excess carbon in the media results in the distribution of the available carbon source between two metabolic pathways – biomass production and lipid accumulation (Subhash and Mohan, 2015; Gong et al., 2016). As evident from the figure, although biomass productivity was higher in the aqueous hydrolysate medium supplemented with yeast extract, both lipid productivity and lipid content were the minimum. Since the focus of the study was to produce microbial lipids, the results further confirm that the medium containing acid hydrolysate of the seed cake is most favorable for the purpose. Estimation of nutrients in the media performed during the incubation period for all the media under study demonstrate that it was sugars that was utilized by the fungi to the maximum extend to support the cell growth and corresponding lipid production. In HY media, although the initial concentration of total sugars, proteins and lipids was higher compared to the other two media owing to media supplementation, this did not have any significant effect on fungal lipid production which could be due to low C/N ratio as discussed earlier. The continuous drop in the concentration of total sugars with time is well in agreement with the constant increase in biomass production during the incubation period in all the media as observed in the figure. Maximum biomass and lipid production in AH medium compared to the other two medium could be attributed to the higher concentration of reducing sugars present in the AH medium compared to the others (at 0th hour, the concentration of the reducing sugars in AH, H and HY media were 7.38 ± 0.047 g/L, 1.464 ± 0.033 g/L and 1.46 g/L respectively), leading to their effective utilization towards biomass as well as lipid production along with the utilization of total sugars as is evident from the nutrient utilization profiles. The results obtained are consistent with the studies carried out with acid hydrolysates of feed stocks such as wheat straw (Yu et al., 2011), corncob (Xavier and Franco, 2014), carop pulp (Freitas et al. (2014), etc, as mentioned in Table 2. The results are also in agreement with other works where Aspergillus strains have been reported to be used for biodiesel production exhibiting FAME profiles containing 30.2%, 31.5%, 38.3% of satu-
Fig. 3. Comparison of biomass productivity, lipid productivity and lipid content profiles for different media: AH (Acid Hydrolysate), H (Aqueous hydrolysate), HY (Aqueous hydrolysate supplemented with yeast extract). Table 2
Data on biomass and lipid productivities using different sources. Source
Pretreatment Method
Organism
Lipid Content (%)
Lipid productivity (g/L/h)
Fatty Acid Profiles
Ref
Detoxified rice straw hydrolysate
Dilute acid
Trichosporan fermentans CICC 1368
40.1
0.06
palmitate> stearate> oleiate> linoleiate
Huang et al. (2009)
Detoxified sugarcane bagasse hydrolysate
Dilute acid
Yarrowia lipolytica Po1g
58.5
0.073
oleiate> palmitate> palmitoleiate>
Y.A.Tsigie et al. (2011)
stearate Wheat straw hydrolysate
Dilute acid
Cryptococcus curvatus
33.5
0.04
palmitate> stearate> oleiate> linoleiate
Yu et al. (2011)
(ATCC, 20509) Detoxified corncob hydrolysate
Dilute acid
Trichosporan cutaneum
36
0.041
palmitate> stearate> oleiate> linoleiate
Chen et al. (2012)
Sugarcane bagasse
Dilute acid
Lipomyces starkeyi
26.6
0.026
oleiate>palmitate> stearate> linoleiate
Xavier and Franco
Carob pulp syrup
Aqueous
Rhodosporidium toruloides
17.27
0.019
Not performed
Freitas et al. (2014)
53.18
0.033
palmitate> stearate> oleiate
A.Patel et al. (2015)
(2014) NCYC 921 Aqueous extract of Cassia fistula L. fruit pulp
Aqueous
Rhodosporidium kratochvilovae HIMPA1
Corncob hydrolysate+6% glucose
Dilute acid
Cryptococcus sp. SM5S05
60.2
0.079
oleiate> palmitate> stearate> linoleiate
Chang et al. (2015)
Sonicated papermill sludge extract
Aqueous
Cryptococcus vishniaccii (MTCC 232)
53.4
0.054
oleiate> palmitate> linoleiate>stearate
Deeba et al. (2016)
Cellulosic ethanol fermentation wastewater
NIL
Trichosporon cutaneum ACCC 20271
13.3
0.018
oleiate> palmitate> stearate
Wang et al. (2017)
Office
Hydrogen peroxide
Cryptococcus curvatus
37.8
0.08
oleiate> palmitate> stearate> linoleiate
Annamalai et al. (2018)
Aspegillus ochraceus MK483340
28.93
0.012
Linoleate> oleate> palmitate> stearate
Present study
waste paper hydrolysate (enzyme hydrolysis) Acid hydrolysate of Pongamia seed cake
treatment Dilute acid
5
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Biocatalysis and Agricultural Biotechnology 24 (2020) 101543
rated, monounsaturated and polyunsaturated methyl esters, respectively (Kakkad et al., 2015). The media used in the current study was not an enriched one, instead it comprised of a small concentration of dilute acid pretreated de-oiled seed cake (2.5%) without any nutrient enhancement. This could be one of the reasons for lipid content and productivity obtained in the present study to be comparatively lesser the other studies mentioned above. Optimization of the pretreatment conditions with respect to the seed cake and the acid concentration for effective solubilization of the source could be one of the strategies employed to enhance lipid yield and content. In addition, acid pretreatment of the lignocellulosic biomass is known to release inhibitors in to the medium, majorly, furfural and 5-hydroxymethylfurfural (HMF) from the degradation of pentoses and hexoses (Mosier et al., 2005) which restrict the growth of microorganisms in turn restricting microbial lipid production. In this regard, efforts have been made in some of the works to detoxify the medium to bring down the effect of toxic inhibitors (Tsigie et al., 2011; Yu et al., 2011; Ahmad et al., 2016). Accordingly, detoxification of the medium following acid pretreatment could be another strategies followed to improve the overall biomass and lipid productivity.
Table 3
Table representing the biodiesel properties of the FAMEs obtained from the lipids produced by Aspergillus ochraceus in the medium containing acid hydrolysate of Pongamia seed cake and comparison with the fuel properties of Jatropha oil and rapeseed oil and the standard specifications (ASTM 6751 and EN 14214). Biodiesel properties
ASTM D6751
EN 14214
Jatropha oil
Rapeseed oil
Present study (acid hydrolysate of Pongamia seed cake)
Kinematic Viscosity (mm2/s) Density (g/cm3) Cloud point (°C) Cetane number
1.9–6.0
3.5–5.0
4.48
4.40
4.53
–
0.86–0.90 0.8789
0.8796
0.8784
–
–
4.67
2.93
5.68
47 (min) –
51 (min)
55.23
54.35
55.73
120 (max) –
98.02
107.76
92.42
40.55
40.78
40.42
Iodine number High heating value (MJ/kg)
3.3. Analysis of lipids produced by Aspergillus ochraceus in acid hydrolysate medium
–
All the properties calculated such as kinematic viscosity, density and iodine number (iodine value) that depends on the degree of unsaturation, as well as cetane number that depends on the degree of saturation, are well in agreement with the specified standard limits and the corresponding values for Jatropha oil and rapeseed oil thus indicating a optimum ratio of both saturated and unsaturated FAMEs required for improved biodiesel quality (Deeba et al., 2017). The cloud point value which refers to the temperature, below which the biodiesel gels or solidifies thus clogging the filter, also is in close agreement with the value for biodiesel obtained from Jatropha oil thus indicating the optimum temperature properties of the oil obtained in the present study. In addition, the gross energy content of the FAMEs obtained from the present study is equivalent to those of rapeseed and Jatropha oil. Although the fungal lipids produced in the present study are rich in unsaturated fatty acids when compared to saturated fatty acids, these oils can still be used as good sources for biodiesel production with the use of certain chemical or enzyme catalysts (Chang et al., 2013). In addition, the oil produced is rich in monounsaturated fatty acid (30.27%) which makes it an excellent source for production of biodiesel (Sankh et al., 2013). This is because although the linoleic content is higher, high amount of oleic acid balances the amount of saturated fatty acids and polyunsaturated fatty acids. All these properties prove the possibility of exploiting the oil produced by Aspergillus ochraceus from de-oiled Pongamia seed cake based medium for biodiesel applications.
Owing to the maximum production of fungal lipid in the medium containing acid hydrolysate as compared to the other two media, further analysis of the lipids produced in the acid hydrolysate medium was carried out to characterize and to determine the quantity of triacylglycerols (TAG) in the lipids produced. Upon characterization of lipids it was observed that the percentages of neutral lipids, glycolipids and sphingolipids, and phospholipids were 92.86%, 5% and 2.14%, respectively. Thus, it is clear that, the neutral lipids contribute to significant proportion of the lipids produced by A. ochraceus. The FAME profiles of lipids produced by A.ochraceus grown on acid hydrolysate medium were analyzed using GC-MS which revealed that the lipids mainly comprised of methyl esters of linoleic acid (C18:2; 38.25%), oleic acid (C18:1; 30.27%), palmitic acid (C16:0; 18.27%), stearic acid (C18:0; 10.43%); traces of behenic acid (0.91%), lignoceric acid (0.71%), arachidic acid (0.48%) and myristic acid (0.28%). The results are well in agreement with the fatty acid contents of sunflower oil, soybean oil and Jatropha oil that are commonly used as a source for biodiesel production with the methyl ester profiles being linoleic acid (66.2%, 53.2%, 32.48%), oleic acid (21.1%, 23.4%, 39.08%), stearic acid (4.5%, 4%, 6.86%), palmitic acid (0%, 11%, 14.66%), for sunflower oil, soybean oil and Jatropha oil, respectively (Patel et al., 2015). Also, the results obtained are consistent with the previous studies (as mentioned in Table 2) wherein a similar lipid compositional profile has been obtained for the cultivation of various yeast strains in different unconventional media. The results thus indicate the presence of fatty acids mainly with 18 and 16 carbon atoms thus proving the fatty acid profile to be similar to those of vegetable oils (Gong et al., 2016). Thus the fungal lipid produced can be considered as a potential feedstock for biodiesel production.
4. Conclusions In the present study, an unconventional medium, Pongamia seed cake was investigated as a cost-effective source for the production of lipids using a novel fungal strain of A.ochraceus. The growth of fungus A. ochraceus in all the variants of the source used indicates the potential of the source to support microbial growth. A maximum lipid content of 28.93% obtained in the medium containing the acid hydrolysate of the seed cake confirms the effectiveness of both the source as well as the microbe in offering a cost effective resource for biodiesel production as well as providing value addition to residues generated in biodiesel industries. Optimization of the culture conditions may possibly result in further enhancement in lipid production.
3.4. Biodiesel properties of the microbial lipid In order to evaluate if the lipids produced by A. ochraceus could be utilized as a source for biodiesel production, important biodiesel characteristics of the lipid produced were obtained by theoretical means and compared with the US and European specifications for biodiesel, ASTM 6751 and EN 14214 and those of routinely used plant oils for the production of biodiesel as represented in Table 3. The lipids produced in the medium containing acid hydrolysate medium were used for calculation purpose.
Declaration of competing interest The authors declare that they have no conflict of interest. 6
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Acknowledgements
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