Improvement of Lipid Production from an Oil-Producing Filamentous Fungus, Penicillium Brevicompactum NRC 829, Through Central Composite Statistical Design
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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, sev- eral 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 con- ditions 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 produc- tion 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.