Improvement of Lipid Production from an Oil-Producing Filamentous Fungus, Penicillium Brevicompactum NRC 829, Through Central Composite Statistical Design

Ann Microbiol (2017) 67:601–613 DOI 10.1007/s13213-017-1287-x

ORIGINAL ARTICLE

Improvement of lipid production from an oil-producing filamentous fungus, Penicillium brevicompactum NRC 829, through central composite statistical design

Thanaa H. Ali1 & Mamdouh S. El-Gamal2 & Dina H. El-Ghonemy1 & Ghada E. Awad3 & Amir E. Tantawy1

Received: 12 March 2017 /Accepted: 13 July 2017 /Published online: 7 August 2017 # Springer-Verlag GmbH Germany and the University of Milan 2017

Abstract In the present study, 13 filamentous fungi were commercial development for the production of LA by fer- screened for their lipid production and an oleaginous fun- mentation using cheap raw material. gus, Penicillium brevicompactum NRC 829, was found to be the highest lipid producer. Screening of various agro- Keywords Linoleic acid . Penicillium brevicompactum NRC industrial residues was performed and sunflower oil cake 829 . Response surface methodology . Unsaturated fatty acids proved to be the best substrate for lipid production. A central composite design was employed to investigate the optimum concentrations of the most significant medi- Introduction um components required to improve the lipid production by P. brevicompactum. The results clearly revealed that Polyunsaturated fatty acids (PUFAs) are long-chain fatty − the maximal lipid production of 8.014 ± 0.06 gL 1 acids containing two or more double bonds in their acyl (representing 57.6% lipid/dry biomass) was achieved by chains. Biosynthesis of PUFAs involves both methyl- the fungus when grown for 6 days at 30 °C under static directed and carboxyl-directed desaturases. The primary condition in a medium containing sunflower oil cake, product of fatty acid biosynthesis in oilseed crops is the NaNO3 and KCl at final concentrations of 8, 0.75 and 18-carbon monounsaturated oleic acid (C18:1–9). − 0.25 gL 1, respectively. Gas chromatography-mass spec- Sequential desaturation of oleic acid and its elongated trometry analysis of P. brevicompactum lipid indicated products at both ends by methyl- and carboxyl-directed that linoleic acid (LA) (C18:2–6, 9) was the most abun- desaturases results in various polyunsaturated fatty acids dant fatty acid, accounting for up to 62% of the total fatty (Hong et al. 2002). Gamma-linolenic acid (GLA; 18:3) acid profile, followed by palmitoleic acid (C16:1, 16%) is considered as an essential fatty acid in humans and and linolenic acid (C18:3, 8%). These results suggest that acts as an important intermediate in the biosynthesis of P. brevicompactum NRC 829 may have potential for prostaglandin derivatives. Linolenic acid has been re- portedtobeeffectiveforthepreventionorcuringofa variety of diseases including rheumatoid arthritis, car- * Thanaa H. Ali diovascular diseases, hyper-cholestromia, atopic eczema [email protected] and asthma (Murad et al. 2010). Although fish are the main source of long-chain polyunsaturated fatty acids,

1 there are several limitations in using fish oil as a source Department of Microbial Chemistry, Genetic Engineering and of supply of these essential fatty acids. These limitations Biotechnology Division, National Research Centre, 33 El Buhouth St., Giza 12622, Egypt involve the presence of: (1) teratogen, carcinogen and mutagen contaminants including dichlorodiphenyltrichlo- 2 Botany and Microbiology Department, Faculty of Science, Al-Azhar University, Cairo 11884, Egypt roethane and dioxin-like polychlorinated biphenyls; and 3 (2) non-carcinogen contaminants such as methyl mercu- Chemistry Natural and Microbial Products Department, +2 +2 +2 +2 +2 Pharmaceutical and Drug Industries Chemistry Division, National ry, heavy metals (Pb ,Cr ,Hg ,Cd ,andAs )and Research Centre, 33 El Buhouth St., Giza 12622, Egypt antibiotics (Abedi and Sahari 2014). 602 Ann Microbiol (2017) 67:601–613

In recent years, extensive research has been carried out for Materials and methods the production of PUFAs, such as arachidonic acid and α- linolenic acid, from oleaginous microorganisms (bacteria, al- Materials and chemicals gae, yeasts and fungi) (Ramanaiah et al. 2007). Recently, several researchers have supported the idea of using filamentous Potato dextrose medium was purchased from Liofilchem fungi for biodiesel preparation due to their rapid growth, their Bacteriology products, Italy. Chemicals and organic solvents lack of a need for light energy, easy scalability and the ability used were of analytical grade and high purity. Olive oil was to utilize different renewable substrates, such as whey, molas- obtained from a local hypermarket. ses and corncob waste liquor (Economou et al. 2011a). The major example of this biotechnological application has been Microorganisms and maintenance the exploitation of a filamentous fungus, Mortierella alpina, to produce oils containing n−1,n−3,n−4,n−6,n−7, and n−9 The 13 fungal strains used throughout this study were obtain- PUFAs as reported by Sakuradani et al. (2009). Ali and El- ed from the culture collection of the Microbial Chemistry Ghonemy (2014) reported a large amount of palmitoleic acid Department, National Research Centre, Dokki, Egypt. Each (C16:1), linoleic acid (C18:2) and linolenic acid (C18:3), up to fungus was routinely grown on potato dextrose agar medium 30, 23 and 13%, respectively, in the biomass of an oleaginous [containing (in g/L): peptone, 2.0; yeast extract, 2.0; dextrose, fungus, Trichoderma viride NRC 314. High-level production 18; potato starch, 5.0 and agar, 15 at pH 5.6 ± 0.2] at 28 °C and of α-linolenic acid by Brassica juncea hasbeenreportedby preserved at −80 °C in 50% (v/v) glycerol. using a Δ6 desaturase from the oleaginous fungus, Pythium irregulare (Hong et al. 2002).Lipidproductionbyyeast Inoculums preparation and fermentation conditions strains has also been studied for oleochemical production for over 80 years, and the results have shown that their fatty acid Seven days old slants were used for the preparation of the profiles vary with the growth conditions regardless of their inoculum; conidia of the fungus were scraped using an inoc- taxonomic affiliation (Sitepu et al. 2013). ulation needle under aseptic conditions and 5.0 mL of sterile Improvement of lipid production by oleaginous micro- distilled water was added to each slant. Aliquots of 2 mL (v/v) organism requires the optimization of culture conditions of inoculum size (1.8 × 107 spore/mL) were used to inoculate and nutritional factors during the fermentation process. 250-mL Erlenmeyer flasks each containing 50 mL of potato Lipid accumulation and fatty acid composition of oleagi- dextrose liquid medium adjusted to be slightly acidic (pH 6.0) nous microorganisms varies depending on environmental before autoclaving at 121 °C (15 min). The inoculated flasks factors (such as pH, temperature and incubation time) and were incubated for 6 days at 28 °C in a static condition. the nature of the microorganism (Ageitos et al. 2011). ‘One variable at a time’ is the classical method of approach that Determination of mycelial dry weight (biomass) permits the determination of specific requirements for growth and product formation by systematically adding After the incubation period, cultures were harvested by filtra- or deleting components from the medium. Recently, re- tion (Whatman no.1), and the mycelial mats (in triplicate) sponse surface methodology (RSM) has been extensively were rinsed thoroughly with sterile distilled water. The bio- applied in the optimization of medium composition and mass was dried in a hot-air oven at 60 °C until a constant culture conditions (Awad et al. 2011). RSM is a collection weight was achieved. Fungal growth was expressed as dry of mathematical and statistical techniques for the experi- weight of the biomass per liter of culture medium, according mental design, evaluation factors, model development and to Devi et al. (2009). optimum conditions of different biotechnological process- es. Statistical optimization not only allows quick screening Lipid estimation of large experimental domain but also reflects the role of each component. Therefore, the objectives of this work Lipid was estimated according to Mishra et al. (2014). Sulfo- were to screen different filamentous fungi for lipid pro- phospho-vanillin reagent (SPV) was freshly prepared by dis- duction and to examine how the lipid yields of the select- solving 0.6 g vanillin in 10 mL absolute ethanol and 90 mL ed fungal strain varied under different fermentation con- deionized water with continuous stirring, then 400 mL of conditions in relation to the fungal growth. In addition, the centrated phosphoric acid (85–88%) was added to the mixture influences of different agricultural wastes on the growth and the resulting reagent was stored in the dark until use. For and lipid production by P. brevicompactum NRC 829 fungal lipid quantification, a known amount of biomass in were studied. RSM was used to optimize the most effec- 100 μL water was added to 2.0 mL of concentrated sulfuric tive parameters in the fermentation conditions required for acid (98%) and heated for 10 min at 100 °C, then cooled for maximum lipid production. 5 min in an ice bath. Five mL of SPV reagent was then added Ann Microbiol (2017) 67:601–613 603 and the sample was incubated for 15 min at 37 °C under metal salts (K+,Mg+2,Co+2,Mn+2,Cd+2,Cu+2,Ca+2,Zn+2, shaking (200 rpm). A relatively stable (up to several hours) Ag+,Hg+ at a final concentration of 0.05 gL−1)ongrowthand carbonium ion (or carbocation) chromogen was formed lipid production by P. brevicompactum (Table 3). followed by the generation of a pink chromophore (Cheng et al. 2011), which was measured at 530 nm by using a Central composite design (CCD) Cary-100 UV-Vis–Spectrophotometer (Agilent Technologies, Germany). After the identification of components affecting the production of lipids by ‘one variable at a time approach’ and in order Sequential optimization of lipid production to describe the nature of response surface in the experimental by P. brevicompactum NRC 829 region, a central composite design CCD (Adinarayana et al. 2003) was conducted. A 23 factorial design with six star points Effect of medium composition on lipid production and six replicates at the central points was employed to fit the second-order polynomial model. The experimental design Seven fermentation media (Table 2)wereusedtoevaluatethe consisted of 20 runs and the independent variables were stud- ability of the selected fungus, P. brevicompactum NRC 829, ied at five different levels. The experimental design used for for accumulation of lipid under submerged fermentation con- the study is presented in Table 4 in the section BOptimization ditions. Fermentation was carried out at 28 °C for 6 days under of the culture conditions by central composite design". All the static conditions in 250-mL Erlenmeyer flasks containing experiments were carried out in triplicate and the averages of 50 mL of each medium (Table 2) adjusted to pH 5.8 before lipid content and dry mass obtained were taken as the depen- autoclaving (121 °C, 15 min). The best medium for lipid ac- dent variable or response (Y). The second-order polynomial cumulation was selected for further studies. This experiment coefficients were calculated and analyzed using the ‘SPSS’ was carried out in triplicate and the reported results are the software (Version 16.0). Second degree polynomial (Eq. 1), average value with standard deviations. which includes all interaction terms, was used to calculate the predicted response: Experimental design for optimization of lipid production ¼ þ þ þ þ 2 Y lipid content ß0 ß1X 1 ß2X 2 ß3X 3 ß11X 1 In the present study, the growth pattern, biomass and oil pro- þ ß X 2 þ ß C 2 þ ß X X duction by P.brevicompactum NRC 829 were evaluated every 22 2 33 3 12 1 2 þ þ ð Þ 24 h for 10 successive days in order to detect the optimum ß13X 1X 3 ß23X 2X 3 1 incubation time for lipid formation. The effect of incubation −1 temperatures ranging between 25 and 45 °C on dry biomass as where Y was the predicted production of lipid content (gL ), well as on lipid yields was carried out. The influence of initial X1,X2 and X3 were the independent variables corresponding to pH was investigated from pH 3.0 to 8.0 by adjusting the pH of the concentrations of sunflower oil cake, NaNO3,andKCl, the medium before autoclaving (using 0.5 M HCl/NaOH). respectively. ß0 is the intercept, ß1, ß2,andß3 are linear coef- Different agro-industrial wastes (sunflower oil cake, staff oil ficients, ß11,ß22,andß33 are quadratic coefficients and ß12, cake, jojoba oil cake, cotton seed waste, flux seed oil cake, ß13,andß23 are cross-product coefficients. Statistical analysis olive oil cake, corn cobs and rice straw) were screened for of the model was performed to evaluate the analysis of vari- their influence on lipid production by the selected fungal ance (ANOVA). Statistical significance of the model equation strain. Each waste was incorporated individually in the select- was determined by Fisher’s test value and the proportion of ed production medium (M I) at a final concentration of 6.0% variance explained by the model was given by the multiple (w/v). coefficients to determine each variable. The quadratic models To determine the influence of various carbon sources on were represented as contour plots (3D) and response surface lipid production, glucose in the production medium was re- curves were generated by using STATISTICA (0.6). placed with 20 gL−1 of each of the different carbon sources (maltose, sucrose, fructose, lactose, glucose, arabinose, galac- Extraction of lipid tose, raffinose, xylose and mannose). The influence of different nitrogen sources (peptone, yeast extracts, beef extracts, Lipid was extracted from the lyophilized mycelia dissolved in malt, soluble casein, animal peptone and tryptone; at a final chloroform (50 mg/10 mL) by using different solvent systems concentration of 0.5 gL−1) on lipid production was also eval- (Somashekar et al. 2001) as follows: chloroform/methanol uated by incorporating each nitrogen source individually into (2:1), n-hexane/isopropanol (3:2) and soxhlet-n-hexane ex- the production medium containing an optimum amount of the traction. The extract obtained was then concentrated to superior carbon source as described above. Finally, a set of 1.0 mL in a rotary evaporator (Laborota 4000; Heidolph, experiments were conducted to evaluate the effect of various Germany). 604 Ann Microbiol (2017) 67:601–613

Fatty acid analysis methylsiloxane) had dimension of length, 30 m, diameter, 320 μm, and film thickness, 0.25 μm. The flame ionization In the present study, the fatty acid profile of the lipid sample detector temperature was 280 °C and flow rate was 1.5 mL/ was estimated by converting the fatty acids in lipid to fatty min. The carrier gas was nitrogen with a flow rate of acid methyl esters (FAMEs) using acid-catalyzed esterifica- 30 mL/min, a hydrogen flow rate of 30 mL/min and the tion and the transesterification method recommended by air flow rate was 300 mL/min. Christie and Han (2010). The lipid sample (up to 500 μL) was taken in a test tube fitted with a condenser, and 2% sulfuric acid in methanol (10 mL) was added, then the mixture was refluxed for 5 h at 60 °C. After refluxing, the methanol Results and discussion was evaporated in rotary evaporator and the required esters were extracted with hexane (2 × 5 mL) by using a Pasteur Screening of different fungal strains for their lipid pipette to separate the layers. The hexane layer was washed production with water (double the sample volume) containing 2.0% potassium bicarbonate and dried over anhydrous sodium sulfate. In the present research, thirte13en filamentous fungi were The solution was filtered and the solvent removed under re- screened for their abilities to produce lipid in potato dextrose duced pressure in a rotary evaporator. Then, the samples were liquid medium after 7 days of incubation. The results shown in analyzed by gas chromatography (GC). Table 1 clearly indicate that the majority of the tested strains have the ability to accumulate lipids in different amounts rang- Gas chromatographic analysis ing from 6.3 ± 0.16 to 27.3 ± 0.52% of their dry weight. However, Penicillium brevicompactum NRC 829 cells gave The amount of fatty acid was estimated from peak areas of the highest amount of lipid accumulation (27.3 ± 0.52%) gas chromatogram compared with calibration standards. followed by P. viridicatum (20.3 ± 0.72%), Aspergillus oryzae Supelco 37 component FAME mix (Cat No. NRRL 447 (19.5 ± 0.37%), A. oryzae NRRL 480 18919 − 1AMP) was used as the FAME standard. Gas (18.8 ± 0.29%) and P. funiculosum NRC 258 chromatography–mass spectrometry (GC-MS) analysis (18.2 ± 0.47%). The least amount of lipid accumulation was performed using and Agilent Technologies 6890 N (6.3 ± 0.16%) was detected in the cells of the third strain (Net Work GC system; USA). The oven was held at an A. oryzae NRRL 3435. There have been many attempts to initial temperature of 50 °C and maintained for 2 min at obtain lipids from Aspergillus oryzae, which has been exten- successive rates of 10, 8, 5, 6 °C/min, then raised to 70, sively studied as a lipase producer, for biodiesel production by 170, 200 and 240 °C at rates of 2, 9, 5, 10 min and run time transesterification of triacylglycerols (TAG) (Adachi et al. 55 min. Injector temperature was held at 250 °C splitless. 2011). Therefore, this promising result justified the selection The capillary column HP-5MS (5.0% phenyl of P. brevicompactum NRC 829 for further studies.

Table 1 Screening of different fungal strains for their lipid accumulation

Micro-organisms Biomass Lipid Lipid/dry biomass (g/L) (g/L) (%)

Aspergillus niger NRC 877 8.8 0.81 9.4 ± 0.08 A. oryzae NRRL 447 10.2 1.99 19.5 ± 0.37 A. oryzae NRRL 3435 9.2 0.59 6.3 ± 0.16 A. oryzae NRRL 480 5.9 1.11 18.8 ± 0.29 A. phoenicis NRRL 365 9.3 0.82 8.8 ± 0.19 Fusarium oxysporum ASU 2 7.1 0.96 13.5 ± 0.42 F. moniliforme BRC 183296 8.9 0.92 10.4 ± 0.13 Penicillium brevicompactum NRC 829 10.8 2.94 27.3 ± 0.52 Penicillium chrysogenum NRC 834 7.6 1.15 15.5 ± 0.64 Penicillium funiculosum NRC 258 8.1 1.47 18.2 ± 0.47 P. purpurescens 9.8 1.79 17.5 ± 0.38 Penicillium viridicatum NRC 3712 10.2 2.08 20.3 ± 0.72 Scopulariopsis brevicaulis ASU 3 7.3 1.28 16.3 ± 0.36

Data are expressed as mean value ± SD of triplicate measurements. The micro-organism with the highest lipid production is shown in bold Ann Microbiol (2017) 67:601–613 605

Optimization of lipid production by P. brevicompactum NRC 829

In the present study, the first optimization step was carried out using the ‘one variable at a time’ experimental approach to 14.8 ± 0.13 25.8 ± 0.54 26.9 ± 0.28 23.8 ± 0.62 28.7 ± 0.83 identify the significant factors required for maximum lipid

O Lipid/dry biomass (%) production by P. brevicompactum NRC 829. Seven different 2 media were tested, and the highest lipid production of 3.27 ·6H

2 − ) 1 3 gL (representing 28.7 ± 0.83% lipid/dry biomass) was achieved in medium I (M I) (Table 2), followed by M VI

Co(NO (26.9 ± 0.28% lipid/dry biomass) and M IV (25.8 ± 0.54%

3 lipid/dry biomass), while the least amount of accumulated

FeCl lipid (10.8 ± 0.83% lipid/dry biomass) was observed with M 2

g 8 mg 0.1 mg 20.6 ± 0.39 III. Hence, medium I was selected for the subsequent μ experiments. −−− MnCl The synthesis of TAG by the fungus was influenced by the 4 supply of carbon in the growth medium. Among the various

CuSO carbon sources tested, the largest amount of lipid production 2 was obtained in glucose (28.7 ± 0.74% lipid/dry biomass)- 0.5 5 mg CaCl amended medium followed by raffinose (28.4 ± 0.53% lipid/ 4

g0.10.1mg24 dry biomass) and galactose (25.5 ± 0.87% lipid/dry biomass) μ (Table 3), while biomass production was highest with glucose −−−−−− − −−−−−− ZnSO (11.3g/L)followedbyfructose(10.6g/L)andgalactose 4 (10.2 g/L). This result is congruent with that reported for lipid 1.5 0.01 0.1 0.1 mg 0.1 mg 8 mg 0.1 mg 10.8 ± 0.18 MgSO production from Aspergillus sp. and T. viride NRC 314

4 (Kumar and Banerjee 2013; Ali and El-Ghonemy 2014). PO 2 Similarly, glucose was found to be the most suitable carbon NRC 829 7.0 1.5 KH source for lipid production (8.8% lipid/dry biomass) by 4 Cunninghamella echinulata and Mortierella isabellina as in- PO 2 vestigated by Papanikolaou et al. (2007). Generally, glucose is 2.0 7.0 1.5 55 −− 0.44 2.4 1.0 considered as the best carbon source for growth and lipid Cl Na

4 accumulation of oleaginous fungi (Saxena et al. 2008). − −− 0.5 − NH Carbon sources other than glucose, such as xylose, lactose,

4 arabinose, mannose, glycerol and waste cooking oil, have also SO 2

) been investigated as sole carbon and energy sources for the 4 Penicillium brevicompactum production of microbial lipids (Makri et al. 2010; − 2.0 (NH Papanikolaou et al. 2011). However, sugars are considered 4 rements. The most promising medium is shown in bold

PO as more effective energy sources compared to other raw ma- 2 terials (oil or raw glycerol). Fungi utilize the available carbon 0.45 1.0 − − −− − − −−−−−− 2.0 2.0 − − −− − − −−−−−− 5.0 source in the medium for growth, cell maintenance and production of lipid-free biomass. However, under conditions of excess carbon in the medium, a part of the carbon flow is − 10 − − −− −2.0 −− 0.4 −− directed toward the production of lipids (Venkata and Venkata 2014). In contrast, if the carbon is limited in the medium, or when the carbon supply becomes exhausted from 0.75 1.0 1.5 0.5 0.5 extracellular sources, the stored intracellular lipid is mobilized and utilized to sustain generations of cells and production of − −− − − − − lipid-free biomass (Park et al. 1990). As glucose emerged as the most preferred carbon source

Influence of type of medium on lipid accumulation by for lipid production by P. brevicompactum, different concen- 70 trations of glucose (1–10%) were examined to determine its optimal concentration. The results shown in Table 3 reveal Data are expressed as mean value ± SD of triplicate measu MVI 18 5.0 2.0 2.0 MV 30 MVII 40 MIV 40 MIII 30 MII 50 Component (g/L) Medium Glucose Potato Yeast ex. Peptone NaH MI Table 2 that the maximum lipid yield was obtained in culture medium 606

Table 3 Different fermentation parameters influencing lipid accumulation by P.brevicompactum NRC 829 in submerged fermentation condition

Variables

Carbon Glucose Fructose Sucrose Raffinose Maltose Lactose Galactose Mannose Xylose Lipid/dry biomass (%) 28.7 ± 0.74 22.0 ± 0.38 23.9 ± 0.46 28.4 ± 0.53 22.3 ± 0.65 20.6 ± 0.73 25.5 ± 0.87 24.8 ± 0.91 20.3 ± 0.52 Glucose (%) 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10 Lipid/dry biomass (%) 19.8 ± 0.43 21.7 ± 0.81 24.1 ± 0.62 25.9 ± 0.54 29.3 ± 0.82 31.5 ± 1.24 29.8 ± 1.32 23.7 ± 0.91 21.3 ± 0.59 16.9 ± 0.48

Variables

Organic nitrogen Yeast ex. Bacto-peptone Beef ex. Malt ex. Soluble casein Lipid/dry biomass (%) 32.7 ± 1.21 35.1 ± 0.98 26.9 ± 0.34 27.5 ± 0.82 31.5 ± 1.36

Variables

Inorganic nitrogen NaNO3 NH4(SO4)2 KNO3 (NH4)2PO4 NH4Cl Lipid/dry biomass (%) 35.3 ± 1.43 25.8 ± 1.12 27.9 ± 0.78 20.2 ± 0.61 16.5 ± 0.58 Initial pH 3.0 4.0 5.0 6.0 7.0 8.0 Lipid/dry biomass (%) 26.9 ± 0.48 31.5 ± 0.87 37.1 ± 1.42 36.2 ± 1.32 32.2 ± 1.21 22.5 ± 0.72

Variables

Temp. (°C) 25 °C 30 °C 35 °C 40 °C 45 °C Lipid/dry biomass (%) 32.5 ± 1.08 39.0 ± 1.43 34.3 ± 1.15 13.7 ± 0.61 11. 2 ± 0.29

Variables

Time (days) Two Three Four Five Six Seven Eight Nine Ten 67:601 (2017) Microbiol Ann Lipid/dry biomass (%) 11.1 ± 0.23 16.6 ± 0.71 25.3 ± 0.54 32.5 ± 1.27 39.5 ± 1.28 35.4 ± 1.42 27.2 ± 1.03 20.7 ± 0.69 13.8 ± 0.47

Variables

Agitation condition Static Shaking Lipid/dry biomass (%) 39.9 ± 1.38 36.5 ± 1.05 – 613 Ann Microbiol (2017) 67:601–613 607

supplemented with 6.0% glucose (31.5 ± 1.24% lipid/dry biomass). However, at higher concentrations, a decrease in lipid +2

Zn production was reported. Similar results were reported for lipid accumulation by T. viride (Ali and El-Ghonemy 2014). Generally, high glucose concentrations are recommended to enhance the carbon flow direction toward triacylglycerols pro- +2

Cu duction, hence improving lipid accumulation in several microorganisms (Li et al. 2007). Sugars are easily assimilated by many oleaginous microorganisms but at the same time the use of higher sugar concentrations could act as an inhibitory factor +3

Fe for microbial growth (Economou et al. 2011b). Different nitrogen sources have also been found to have a varying influence on oil production. Bacto-peptone was found to be the most suitable added organic nitrogen source for +2

Ba growth and lipid production, yielding about 35.1 ± 0.98% lipid/dry biomass followed by yeast extract (32.7 ± 1.21% lipid/dry biomass) and soluble casein (31.5 ± 1.36% lipid/ 9 ± 0.12 39.8 ± 1.57 20.9 ± 0.72 12.4 ± 0.37 39.6 ± 1.62 +2 dry biomass) (Table 3). NaNO3 has been reported as the best

Hg inorganic nitrogen supplement for maximum lipid production (35.3 ± 1.43% lipid/dry biomass) followed by potassium ni-

trate (27.9 ± 0.78% lipid/dry biomass) and NH4(SO4)2

+ (25.8 ± 1.12% lipid/dry biomass). This result is similar to that Ag reported by Nisha et al. (2010) for lipid production (42.0%) by Mortierella alpine. Venkata and Venkata (2014)reportedan increase in the lipid production of Aspergillus awamori when +2 the nitrogen concentration was decreased, reaching its maxi- Cd mum level when the nitrogen was depleted. Among the environmental factors tested, the pH of the medium showed a profound influence on biomass formation +2 and lipid productivity. From the experimental data, pH 5.0 Mn nt variable in each set is shown in bold was found to be the most suitable for lipid accumulation (37.1 ± 1.42% lipid/dry biomass) (Table 3). Similarly, maximum lipid accumulation by T. viride was observed in a medi- + um maintained at pH 5.0 (Ali and El-Ghonemy 2014). This Na result is relatively close to the optimum pH (5.5) reported by Staff oil cake Jojoba oil cake Cotton seed waste Flux seed oil cake Olive oil cake Corn cab Rice straw 42.2 ± 1.53 42.6 ± 1.51 37.9 ± 1.81 29.5 ± 0.74 42.5 ± 1.23Liet 29.6 ± 0.54 al. 27.6 ± 0.41 (2007) and Venkata and Venkata (2014) for lipid

± 1.67 38.5 ± 1.17 34.2 ± 1.05 3.8 ± 0.04 8.4 ± 0.08 15. production from Rhodosporidium toruloides and Aspergillus + 41.6 K awamori, respectively. In this regard, Amanullah et al. (2001) investigated the influence of broth pH values on the growth kinetics of different micro-organisms and concluded that the

plicate measurements. The most importa pH of the medium is an important environmental factor affect- +2 Sunflower oil cake 46.4 ± 1.88 Ca ing cell growth and product formation. In addition, they reported that pH values between 5 and 6 at the time of inoculation were the most favorable for fungal growth. The incubation temperature had an impact on the biomass +2 formation and lipid synthesis. The results indicate that there was an increase in growth biomass as well as lipid accumulation with the increase of incubation temperature from 25 °C to 35 °C (Table 3). However, the highest lipid productivity was reported at 30 °C (39.0 ± 1.43% lipid/dry biomass) during the preliminary screening, while a decrease in biomass and lipid accumulation was noticed with lower/higher incubation temperature compared Variables Metal ions (50 mg/L)Lipid/dry biomass (%) Mg 40.3 ± 1.84 38.8 ± 1.23 Variables Agricultural residue Control Lipid/dry biomass (%) 42.8 ± 1.46 Data are expressed as mean value ± SD of tri with the optimum temperature. These results are in accordance 608 Ann Microbiol (2017) 67:601–613 with those reported by Li et al. (2007), Ali and El-Ghonemy respectively). The effects of metal ions on lipid production by (2014) and Venkata and Venkata (2014). In this regard, Carlile Cunninghamella sp. 2A1 have been studied by Muhid et al. et al. (2001) reported the optimum temperature for maximum (2008), who reported that the addition of Mg2+,Fe2+ and Zn2+ fungal biomass to be 30 °C, which might be attributed to the resulted in 64, 43 and 33% increases in lipid content, respec- natural environments of fungi. At high temperatures, an increase tively, when compared to the basal medium. On the other in nutritional requirements is sometimes observed in hand, the supplementation of the fermentation medium with

Saccharomyces (Carlile et al. 2001). 0.0005 g/L CuSO4 and 0.0075 g/L ZnSO4 increased lipid The time of incubation is also an important factor and showed accumulation in Morterella ramanniana by 13.9% when com- an influence on the fungal biomass formation and lipid accumu- pared to the culture medium which lacked the respective metal lation. Lipid production of each strain differs depending upon the ions, as reported by Hansson and Dostalekm (1988). specific growth rate of the strain, whereas the maximum lipid Carbon sources obtained from lignocellulosic biomasses production could be obtained only after a certain incubation time (forest residues, agricultural residues, food wastes, municipal which allows the culture to grow at a steady state. In the current wastes, and animal wastes) are one of the most important study, the highest biomass and lipid accumulation were noticed potential feed stocks that can be utilized for the production on the 6th day of incubation (39.5 ± 1.28% lipid/dry biomass) of lignocellulosic-based fungal lipids (Economou et al. (Table 3). In contrast, the highest biomass and lipid accumulation 2011b). The suitability of agro-industrial substrates for lipid of Rhodosporidium toruloides and T. viride were reported on the production by P.brevicompactum was studied. Data in Table 3 5th day of incubation (Li et al. 2007; Ali and El-Ghonemy 2014). shown that the order of substrate suitability for lipid produc- In contrast, El-Fadaly et al. 2009 reported 2.2 gL−1 of microbial tion was sunflower oil cake > olive oil cake > jojoba oil cake > oil with 59.5% oil percentage after 72 h of incubation from staff oil cake > cotton seed waste > flax seed oil cake > corn Cryptococcus curvatus. They suggested that, after inoculation, cob > rice straw. Therefore, sunflower oil cake (at a final up to 72 h the fungus consumed all the available nitrogen, and concentration of 6.0% w/v) was found to be the most suitable then the reserve lipid was synthesized in distinct oil droplets. substrate for lipid accumulation (46.4 ± 1.88% lipid/dry bio- A comparable study between shaking and static culture mass). These results suggest that lipid accumulation is directly conditions showed that the latter fermentation condition was related to the raw starch content in the agro-industrial wastes, characterized by higher levels of biomass (12.3 gL−1) and which may be due to the preferred utilization of starch for lipid lipid accumulation (39.9 ± 1.38% lipid/dry biomass) when production over hemicellulose. In this regard, Ali and El- compared with shaking conditions (10.9 gL−1, 36.5 ± 1.05% Ghonemy (2014) reported that the addition of molasses or lipid/dry biomass), as shown in Table 3.Thisresultmightbe wheatbrantogrowthmediumwerebothpoorinsupporting related to the aeration level which is considered as a very growth and lipid production of T. viride NRC 314 when com- important factor for fungal cell growth as well as total lipid pared to the level produced by organisms in potato dextrose level. The dissolved oxygen amount in the culture can greatly liquid medium. influence fatty acid composition in lipids. In static conditions, From the reported results of the first step of sequential the glyceride fraction varies considerably and the amounts of optimization, we can conclude that the maximum lipid accu- phospholipids and sterol decrease. These variations result di- mulation by Penicillium brevicompactum was achieved after rectly in an increase in the amount of saturated fatty acids, 6 days of incubation in static fermentation conditions at 30 °C, which become the main component of the lipids. Similar re- with an initial medium pH value of 5. Among various agro- sults have been reported for lipid accumulation in Chlorella industrial wastes tested, sunflower oil cake was found to be pyrenoidosa and Chlorococcum sp. when grown under static the most suitable substrate for lipid accumulation. In addition, culture conditions (Nigam et al. 2011;Kirroliaetal.2013). NaNO3 and KCl were found tobe the best nitrogen and metal The effect of various metal ions, in the form of chloride ion sources, respectively. Therefore, sunflower oil cake, salts, on lipid accumulation by P. brevicompactum NRC 829 NaNO3 and KCl were chosen for further investigation in order was investigated by individually incorporating each metal ion to optimize their concentrations in the fermentation medium (0.05 g/L) in the optimized medium. The results cited in by using CCD. Table 3 clearly indicate that K+,Mg+2 and Ba+2 enhanced the lipid accumulation and growth formation, yielding Optimization of the culture conditions by central 41.6 ± 1.67, 40.3 ± 1.84 and 39.8 ± 1.57% lipid content/dry composite design biomass, respectively. This might be related to their role as cofactors which are required by key enzymes implicated in the In order to select the optimum concentrations of the most signif- lipid biosynthesis pathway, while the lowest lipid levels ac- icant medium components (sunflower oil cake, NaNO3 and KCl) companied by poor growth formation were obtained in Fe+3, that supported the highest production of lipid, CCD experiments Hg+2,Cu+2,Ag+ and Cd+2 media (20.9 ± 0.72, 15.9 ± 0.12, were performed. Table 4 shows the CCD experimental plan, the 12.4 ± 0.37, 8.4 ± 0.08 and 3.8 ± 0.04% lipid/dry biomass, coded and un-coded level of the independent investigated Ann Microbiol (2017) 67:601–613 609

Table 4 Central composite design (CCD) consisting of 20 experiments for three experimental factors in coded and actual values for the production of lipid by P. brevicompactum NRC 829

Factor levels

Trial Sunflower NaNO3 KCl Lipid content Dry mass No. (X1) (X2) (X3) (g/L) (g/L)

Coded Actual coded Actual Coded Actual Observed Predicted (g/L) (g/L) (g/L)

1a −14 −10.25−1 0.5 4.35 ± 0.18 3.83 11.52 ± 0.35 2a +1 8 −10.25−1 0.5 4.29 ± 0.19 3.15 12.37 ± 0.18 3a −1 4 +1 0.75 −1 0.5 5.98 ± 0.07 5.13 11.87 ± 0.15 4a +1 8 +1 0.75 −1 0.5 8.01 ± 0.06 7.81 13.91 ± 0.27 5a −14 −1 0.25 +1 1.5 5.16 ± 0.07 4.69 11.07 ± 0.12 6a +1 8 −1 0.25 +1 1.5 5.81 ± 0.12 4.37 13.04 ± 0.24 7a −1 4 +1 0.75 +1 1.5 3.63 ± 0.14 0.58 11.92 ± 0.31 8a +1 8 +1 0.75 +1 1.5 3.77 ± 1.82 2.62 10.67 ± 0.42 9b −2 2 0 0.5 0 1 2.44 ± 0.07 3.22 9.12 ± 0.16 10b +2 10 0 0.5 0 1 3.45 ± 0.19 4.59 10.29 ± 0.14 11b 06−2 0.1 0 1 3.1 ± 0.02 4.39 10.40 ± 0.23 12b 0 6 +2 1 0 1 1.98 ± 0.21 3.26 10.41 ± 0.58 13b 0600.5−2 0.25 3.38 ± 0.31 5.17 10.02 ± 0.11 14b 0 6 0 0.5 +2 2 2.56 ± 0.22 1.035 8.94 ± 0.047 15c 0 6 0 0.5 0 1 5.42 ± 0.16 5.51 11.67 ± 0.19 16c 0 6 0 0.5 0 1 5.34 ± 0.11 5.51 11.08 ± 0.21 17c 0 6 0 0.5 0 1 5.44 ± 0.11 5.51 11.72 ± 0.12 18c 0 6 0 0.5 0 1 5.22 ± 0.13 5.51 12.20 ± 0.49 19C 0 6 0 0.5 0 1 5.28 ± 0.09 5.51 12.10 ± 0.26 20c 0 6 0 0.5 0 1 5.34 ± 0.12 5.51 11.68 ± 0.35

Data are expressed as mean value ± SD of triplicate measurements a Fractional 23 factorial design b Star points c Central points variables and the observed and predicted lipid production. effects of the factors on the response value. Fig. 1a–cshows

Multiple regression analysis of the experimental data gave the the response surface of sunflower oil cake and NaNO3,sun- following second-order polynomial Eq. (2). flower oil cake and KCl, and NaNO3 and KCl concentrations on lipid production, respectively, keeping the other component at the fixed zero level. Table 5 shows the regression Y lipid content ¼ −3:393 þ 0:689X 1 þ 11:392X 2 results from the data of the CCD experiments, which revealed that the NaNO concentration (X ) had a significant effect on þ 8:833X −0:103X 2−8:081X 2−2:807X 2 3 2 3 1 2 3 lipid production, as it had the largest coefficient, followed by

þ 1:183X 1X 2 þ 0:094X 1X 3−10:830X 2X 3 ð2Þ KCl concentration (X3) and sunflower oil cake concentration (X1). The results obtained by ANOVA analysis show a significant Fisher value (F value) (4.123) which implies that the where Y is the response of lipid production and X1,X2 and X3 are model is significant. Model terms had probability values of the coded values of the test variables (sunflower oil cake, NaNO3 Prob > F = 0.001 which are less than 0.05 and considered and KCl, respectively). highly significant. The determination of the coefficient (R2) was calculated as 0.888 for lipid production (a value of The three-dimensional response surface plots are the graph- R2 > 0.75 indicating the aptness of the model, demonstrating ical representations of the regression equation. They were that the statistical model could explain 84.9% of variability in helpful in understanding both the main and the interaction the response. The R2 value is always between 0 and 1. The 610 Ann Microbiol (2017) 67:601–613

Fig. 1 Response surface plot of lipid content by P. brevicompactum NRC 829 showing the interaction between (a) different concentrations of sunflower oil cake and NaNO3 at X3 = 0; (b) different concentrations of sunflower oil cake and KCl at X2 = 0; and (c) different concentrations of NaNO3 and KCl at X1 =0

goodness of fit of the model can be checked by the determi- reasonable agreement with the adjusted R2 of 0.788, which nation of the coefficient R2 and adjusted R2. The closer R2 is to ensures a satisfactory adjustment of the quadratic model to 1, this indicates that the model is strong and a better predicted the experimental data. response (Munk et al. 1993). The observed R2 are in

Table 5 Model coefficients estimated by multiple linear regression Validation of the model (significance of regression coefficients) The validation of the model was carried out in triplicate under Term Regression coefficient Standard error t test P value optimum medium conditions and predicted by the polynomial Intercept −3.393 4.724 −.718 0.489 model. The results clearly revealed that the maximal lipid production of 8.014 ± 0.06 gL−1 (representing 57.6% lipid/ X1 0.689 0.883 0.780 0.453 dry biomass) was achieved by the fungus when grown for X2 11.392 7.372 1.545 0.153 6 days at 30 °C in a medium containing sunflower oil cake, X3 8.833 3.757 2.351 0.041 X 2 −0.103 0.055 −1.849 0.094 NaNO3 and KCl at final concentrations of 8, 0.75 and 0.25 1 −1 2 gL , respectively. This result was close to its predicted value X2 −8.081 4.222 −1.914 0.085 2 (7.81 ± 0.05 g/L) after 6 days of fermentation, validating the X3 −2.807 1.096 −2.561 0.028 proposed model. An increase of about 273% in lipid content X1X2 1.183 0.796 1.486 0.168 was achieved after CCD application. This reflected the neces- X1X3 0.094 0.398 0.236 0.818 sity and value of the optimization process. Many reports have X2X3 −10.830 3.182 −3.403 0.007 been mentioned in the optimization of lipid production by F value = 4.123; P > F =0.001;R2 = 0 .888; Adjusted R2 =0.788 response surface methodology. The fermentation condition Ann Microbiol (2017) 67:601–613 611

Table 6 Lipid profile of P. brevicompactum NRC 829 and its fatty acid percentage

Peak No Carbon No RT Scientific name Percentage (%)

2 C 8: (0) 11.371 Caprylic acid 0.043 3 C10: (0) 15.106 Decanoic acid (capric acid) 0.0483 4 C11: (0) 17.018 Undecanoic acid 0.077 5 C13: (0) 19.508 Tridecylic acid 0.064 6 C14: (1) 22.056 Mysristoleic 0.147 7 C15: (1) 25.090 Cis-10-pentadecenoic 0.100 8 C16: (0) 28.409 Palmitatic acid 0.78 9 C16: (1) 29.658 Palmitoleic 15.72 11 C17: (0) 31.809 Margaric acid 0.213 12 C17: (1) 32.593 Hepta-decanoic acid 0.201 13 C18: (2) 35.505 Linoleic 61.83 14 C18: (3) 36.032 Linolenic 7.97 17 C20: (0) 40.351 Arachidic acid 0.462 18 C20: (1) 40.998 Cis-5-Eicosenic 0.449 20 C20: (4) 42.22 Cis-5,8,11,14,17-Eicosatetraenoic Arachidonic 0.23 24 C20: (5) 44.794 Cis-5,8,11,14,17-Eicosapentaenoic 0.539 25 C22: (0) 45.671 Behenic acid 2.67 26 C22: (1) 46.456 Erucic 1.050 28 C23: (0) 48.753 Tricosylic acid 0.119 29 C24: (0) 50.631 Lignoceric acid 0.199 30 C24: (1) 51.750 Nervonic acid 1.583

of oleaginous yeast Psuedozyma sp. was optimized by CCD, chloroform:methanol (2:1) was the most suitable solvent resulting in 5.17 g/L of lipid production after optimization, as for maximum lipid extraction from Mucor rouxii MTCC investigated by Areesirisuk et al. (2015). Towijit et al. (2014) 386. reported the highest lipid production of 16 ± 1.41 g/L by – Rhodotorula graminis TISTR 5124 using a Box Behnken Fatty acids analysis of the extracted fungal lipid (SCOs) design. Maximum lipid production of 1.53 ± 0.12 g/L was obtained by Cryptococcus laurentii biomass when cultivated In the present work, the fatty acids profile of P. brevicompactum in a cheese whey medium supplemented with sugarcane mo- NRC 829 lipid revealed that it is composed of high fractions of lasses and using a full factorial design (Castanha et al. 2014). mono- and polyunsaturated fatty acids, mainly 62% C18:2 (linoleic acid), 8.0% C18:3 (α- linolenic acid) 16% Lipid extraction and identification C16:1(palmitoleic acid), and a limited percentage of saturated fatty acids such as C: 8, C: 18, C: 24 and C: 25 (Table 6). This In the present study, extraction of lipids and fatty acids from an result is closed to that reported for fatty acids composition from oleaginous fungus P. brevicompactum NRC 829 was carried microalgae oil which composed of a mixture of unsaturated out by using different solvents such as chloroform:methanol fatty acids, such as palmitoleic (C16:1), oleic (C18:1), linoleic (2:1), n-Hexan:isopropanol (3:2) and Soxhlet n-Hexane. The (C18:2) and linolenic acid (C18:3) with a small extent of satu- results showed that chloroform:methanol (2:1) was the rated fatty acids, such as palmitic (C16:0) and stearic (C18:0) optimum extracted solvent yielding about 5.1 g/L (42%) (Halim et al. 2012). The fatty acid composition of followed by n-Hexan:isopropanol (3:2) (3.7 g/L). While P. brevicompactum NRC 829 lipid is similar, with about the least amount of lipid accumulation (2.3 g/L) was de- 60% of the composition of fatty acids of A. awamori lipid tected with Soxhlet n-Hexane. This result is in agreement but differs in the ratio of saturated to unsaturated fatty with Ali and El-Ghonemy (2014), who found that the acids (Venkata and Venkata 2014). In contrast to our re- chloroform:methanol (2:1) was the most suitable solvent sults, the fatty acid composition of lipid obtained from for lipid and fatty acid extraction from T. viride NRC 314 oleaginous algae and Yanobacteria showed a dominance biomass. Similarly, Somashekar et al. (2001) reported that of C:14 and C:16 fatty acids, respectively (Hu et al. 612 Ann Microbiol (2017) 67:601–613

2008). Our result was also found to be different from the marinensis under solid-state fermentation. Process Biochem 38: – fatty acid composition of vegetable oils in being rich in 1565 1572. doi:10.1016/S0032-9592(03)00057-8 Ageitos JM, Vallejo JA, Veiga-Crespo P, Villa TG (2011) Oily yeasts as polyunsaturated fatty acids (PUFAs); hence, it may be oleaginous cell factories,Appl Microbiol Biotechnol 90:1219–1227 exploited for PUFA production (Chisti 2007). The high Ali TH, El-Ghonemy DH (2014) Optimization of culture conditions for degree of unsaturation would indicate lower oxidative sta- the highest lipid production from some oleaginous fungi for biodie- bility, but excellent fuel properties at low temperatures, sel preparation. Asian J Appl Sci 2:601–609 which is an advantage in winter operation (Vicente et al. Amanullah A, McFarlane CM, Emery AN, Nienow AW (2001) Scale- down model to simulate spatial pH variations in large-scale biore- 2004). actors. 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