Microbial Production of γ-linolenic Acid: Submerged versus Solid-state Fermentations

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1 Food Sci. Biotechnol. 21(4): (2012) DOI /s RESEARCH MINIREVIEW Microbial Production of γ-linolenic Acid: Submerged versus Solid-state Fermentations Milan Čertík, Zuzana Adamechová, and Kobkul Laoteng Received: 5 January 2012 / Revised: 19 March 2012 / Accepted: 20 March 2012 / Published Online: 31 August 2012 KoSFoST and Springer 2012 Abstract One of the major interests of lipid biotechnology is targeted on natural manufacturing of healthy oils containing polyunsaturated fatty acids. Of them, γ-linolenic acid (C18:3 n-6; GLA) as the key intermediate in the n-6 fatty acid family is involved to maintain the proper mammalian cell functions. Insufficient supply of GLA from agricultural and animal sources resulted in hunting for appropriate microorganisms suitable to produce this essential fatty acid in high yield. Extensive studies on oleaginous lower filamentous fungi have led to development of two basic fermentation techniques for GLA production: submerged and solid- state fermentations. Each of the processes provide specific advantages in various applications depending on the GLA product form (GLA-rich oil, whole cells, and fermented mass) and might bring new prospects to fill marketing claims in food, feed, pharmaceutical, and veterinary fields. Keywords: γ-linolenic acid, microbial oil, oleaginous fungi, solid-state fermentation, submerged fermentation Introduction Polyunsaturated fatty acids (PUFAs) have a number of uses Milan Čertík( ), Zuzana Adamechová Department of Biochemical Technology, Faculty of Chemical and Food Technology, Slovak University of Technology, Radlinského 9, Bratislava, Slovak Republic Tel: ; Fax: milan.certik@stuba.sk Kobkul Laoteng Bioresources Technology Unit, National Center for Genetic Engineering and Biotechnology, National Sciences and Technology Development Agency, Khong Luang, Pathumthani 12120, Thailand in various biomedical and nutraceutical fields due to their structural and functional properties. They regulate architecture, dynamics, phase transition, and permeability of membranes as well as behavior of some membrane-bound proteins. In addition, PUFAs as essential compounds are precursors of diverse metabolites, such as prostaglandins, leukotrienes, and hydroxy-fatty acids. γ-linolenic acid (C18:3 n-6; GLA) is synthesized from the first n-6 fatty acid, linoleic acid (C18:2 n-6) by the rate-limiting 6 -desaturase. Rate of endogenous conversion of linoleic acid to GLA is low or failures in a variety of diseases and pathological conditions, such as atopic dermatitis, diabetes neuropathy, premenstrual syndrome, and rheumatoid arthritis (1). Indeed, GLA provides numerous applications in various medical, pharmaceutical, and cosmetic arenas (2). Nevertheless, there are only a few sources of GLA particularly in plant kingdom. The major commercially available GLA-rich oils are derived from seeds of evening primrose (8-10% GLA), borage seeds (24-25% GLA), and black currant seeds (16-17% GLA). Because these plants can not meet increasing market demand for GLA-oils, attention has been oriented on seeking other suitable natural source(s) of this fatty acid. Biotechnological production of GLA by oleaginous microorganisms, as promising alternatives to agricultural commodities, has been studied intensively for several years (3). However, production cost of microbial oils is currently high compared to plantderived oils. Therefore, this fact justifies the search for more efficient strains enable to accumulate large amounts of lipids with an appropriate GLA composition, for increasing the product value, effective fermentation systems and lowering the crucial downstream process. The present paper compares submerged and solid-state fermentation techniques that have been developed for biotechnological GLA production.

2 922 Čertík et al. Microbial Producers of GLA Production of single cell oils enriched with polyunsaturated fatty acids, such as GLA, is one of the main tasks for biotechnological production of nutritionally important lipids. Certain fungi, microalgae, and mosses synthesize GLA, and may represent potential microbial sources of this essential fatty acid (4-6). However, special cultivation conditions as well as techniques for oil recovery and purification are required for microalgae, so their practical large-scale production as single cell oils is constantly reconsidered. Zygomycetes are other group of microorganisms that have been often reported for their proficiency to effectively produce GLA. Significant activities in the biosynthesis of GLA have been described for species of the lower oleaginous fungi belonging to the genera Cunninghamella, Mortierella, Mucor, Rhizopus, and Thamnidium (4,7-10). These oleaginous fungi could be economically and practically valuable because their GLA assemblies predominantly in the triacylglycerol form. There are also several natural advantages that can be derived from these GLA-synthesizing fungi: (a) enormous growth rate on huge numbers of agroindustrial substrates and byproducts is useful due to rapid utilization and transformation of very low-cost materials; (b) active lipid-synthesizing apparatus makes them as perspective oil sources; (c) simple physiological regulation of active lipidmodifying apparatus under controlled conditions; (d) ability to simultaneously perform various transformation reactions allows production of GLA with other purposeful compounds; (e) they could be as appropriate hosts for cloning foreign genes for upgrading of existing fatty acids; (f) GLA-forming fungi are rich in proteins, dietary fibres, trace elements, vitamins, antioxidants, and cholesterol-free oil, so they could be apply as additives to functional food and feed; (g) fermentation process is not dependent on seasonal or climatic variations, thus microbial GLA production can be carried out during whole year. These oleaginous fungi also fulfil characteristics of industrial producing microorganisms, such as genetic stability (genetically tailor-made the desirable oil-producing attributes of fungal cells remains constant throughout all fermentation batches), non-pathogenic and nontoxin-forming properties (the product must be safe for its supplementation into feeds, foods, and pharmaceuticals), and easy downstream of the process (oil extraction and purification). Fermentation Processes for GLA Production Fermentation techniques have been considered for microbial oil production for several decades. During such period, an interest has been primarily focused on submerged processes for GLA production, however solid-state fermentations have been successfully developed for production of GLAenriched products as well (3,4). Depending on purpose of usages, the GLA products with different forms (whole cell, fermented mass, or extracted oil) obtained from each fermentation process would be targets specific to individual applications, such as feed and food supplement, medical and pharmaceutical uses, and cosmetic additives. At the same time, attention must be directed not only to fermentation process economy, but also safety of the final microbial products in terms of strains used and their undesired toxic metabolites. Submerged Fermentation for GLA Production Submerged cultivation process for industrial development of oleaginous microorganisms commonly requires 3 key operation units: fermentation, cell separation, and oil extraction and refining. Compositional design of growth media is an important factor for industrial fermentations from the economic standpoint and it is strictly affected by the cost of incoming materials. The most cost-proficient substrates are the waste materials and byproducts from food industry and agroindustry. Substrates after their appropriate pre-treatments are utilized by suitable fungi under favorable conditions in batch, fed-batch, or continuous fermentation systems. Fermentation physiology of oleaginous microorganisms is based on application of media rich with carbon source and restricted amounts of other nutrients, nitrogen especially. It typically results in biphasis process, where the first phase is characterized by rapid fungal growth until a growth nutrient, other than carbon source, is exhausted. Lipid is then accumulated during the second phase by efficient conversion of carbon substrate into fatty acids and their subsequent incorporation into triacylglycerols. Adequate oxygen supply and its optimal maintenance in fermentation tanks by controlling agitation and fungal morphology are important factors to attain high GLA content in microbial oil. Another useful strategy is based on 2-stage fermentation process, where mycelial cultivation after the first stage is ended and the culture is subsequently switched to conditions promoting lipid and GLA synthesis. In addition, ability of lower filamentous fungi to utilize exogenous oils

3 Microbial Production of γ-linolenic Acid 923 Table 1. GLA production by fungal submerged fermentations 1) Strain C-source N-source GLA yield (mg/l) Lipid/d.c.w. (%) GLA/TFA (%) Ref Mucor rouxii Glucose (NH 4 ) 2 SO 4 nr Mucor circinelloides Glucose nr 216 nr Mucor mucedo Glucose+SO nr Cunninghamella echinulata Glucose+SO nr Cunninghamella echinulata Starch NH 4 NO nr Mortierella ramanniana Glucose nr Mortierella isabellina Glucose (NH 4 ) 2 SO Cunninghamella echinulata Xylose (NH 4 ) 2 SO 4 1, Rhizopus nigricans Soluble starch urea 1, Thamnidium elegans Glycerol (NH 4 ) 2 SO 4 +YE ) d.c.w., dry cell weight; TFA, total fatty acids; SO, sunflower oil; YE, yeast extract; nr, not reported is advantageous because there are a number of natural oils or oil-related waste materials containing linoleic acid, which is a GLA precursor. Consumption of such oil-rich substrates resulted in high lipid accumulation in microbial cells. However, it should be emphasized that although relative level of GLA in total cellular fatty acids is often reduced after microbial transformation of external oils, the total yield of GLA is significantly higher compared to the cultivation without oil supplementation (11). Thus, physiological regulation of lipogenesis in fungi by rational cultivation strategy is essential for achievement of the maximal GLA productivity. Biotechnological production of GLA was the first process developed for microbial polyunsaturated fatty acids. Some Zygomycetes fungi (Mucor, Cunninghamella, and Mortierella) are able under appropriate fermentation conditions to grow rapidly with acceptable biomass densities (50 g/l in submerged culture), high oil content (more than 20% of d.w.) and suitable GLA levels in total fatty acids (20-25%), that results in satisfied GLA amount in fungal cells (about 4% of d.w.) (4,5). Such promising GLA yields in oleaginous microorganisms triggered attempts in many researchers to commercially produce the fungal GLA-oils. It led not only to isolation of new strains with increased capacities to synthesize GLA, but also to optimizing cultivation parameters and strategies for particular fungal strain (Table 1). For example, it was considered, that Mucor mucedo and Cunninghamella echinulata were perspective GLA producers because under suitable conditions they formed up to 30 mg GLA/g mycelia (12). The fungus C. echinulata utilizing glucose with a C/ N ratio of 160 produced up to 720 mg GLA/L after lipid accumulation was completed (8). The cost-effective strategy of microbial GLA-oil production has been proposed by application of simultaneous formation of GLA and other useful metabolite(s). Roux et al. (13) developed the technique employing Mucor circinelloides for fermentation production of both GLA and cocoa butter equivalents. Rhizopus arrhizus effectively converted saccharides to biomass rich in GLA and extracellular L(+)-lactic acid (14,15) based on calculations constructed fungal model and it was successfully applied it in large-scale fermentations. Furthermore, experimental design using statistical approach has been successfully applied to determine optimal parameters including medium composition and culture condition for GLA production in oleaginous fungi. Using the central composite design in conjunction with response surface methodology, the optimal medium components (glucose, yeast extract, and ammonium nitrate) for maximizing GLA production in Mucor rouxii CFR-G15 were derived yielding about 18.6% GLA of total fatty acid and 35% lipid in biomass (16). The similar statistical method was employed for investigation of both agitation speed and dissolved oxygen concentration for GLA production by Mucor sp. RRL001 culture grown in a 5-L stirred tank bioreactor. Optimization of these 2 parameters yielded about 350 mg GLA/L indicating that agitation rate has more pronounced effect on GLA productivity than dissolved oxygen concentration (17). Not only the C:N ratio, the type of either carbon or nitrogen source also affect the GLA production yield of these fungi as shown in Table 1. Effort to scale-up fermentation production of GLA resulted in the first commercial microbial process by JE Sturge, UK (1985) with M. circinelloides (Mucor javanicus) in a 200-kL fermentor. Under appropriate conditions accumulated oil in fungal cells (20%) contained 15-18% GLA in total fatty acids (18). This microbial GLA-oil has been approved for human consumption because the fungus is widely used for preparation of oriental fermented food. However, although GLA content in the fungal oil was 2 times higher than that of evening primrose oil, uncompetitive price, and other marketing difficulties with such specialized microbial oil were the major reasons for deferment of this industrial production in the Europe. The other biotechnological process based on 2-stage continuous fermentor system for production of microbial GLA-rich oil has been expanded

4 924 Čertík et al. in Japan. The fermentation of Mortierella isabellina yielded up to 3.4 g GLA/L (83 g lipid/l containing 4.5% GLA) (19). The productivity of GLA was improved by using fermentor equipped with a special mixing system for highly viscous media, and cultivation of Mortierella ramanniana ended up to 5.5 g GLA/L (30 g lipid/l with 18% GLA in oil) (20). This oil has commercial applications as a food and cosmetic additives and also as healthier foods in the form of drinks, candy, and jelly. Solid-state Fermentation for GLA Production As it was emphasized above, marketing of microbial GLAoil derived by submerged cultivation has been more critical step than developing large-scale fermentation and the downstream process. An alternative technology for preparation of microbial GLA is based on application of solid-state fermentation (SSF). SSF is a prospective bioprocess combining both fungal utilization of moist solid materials and production of valuable metabolites at low cost approach (21). The main task for successful development of GLA production by SSF is particularly focused on both selections of fungal strains with high efficiency to utilize and enrich low-priced substrates with high GLA content and reducing the processing. The advantage of the process is that GLA-oil is not necessary to extract from the fermented materials but these newly formed GLA-materials could be directly used for applications as e.g., food and feed additives. SSF is therefore advised as a powerful platform for the effective transformation of agro-materials and byproducts from agro-food industries and thus increase the value of diverse varieties of fermented bioproducts with requisite properties. Various types of Zygomycetes fungi (Thamnidium spp., Cunninghamella spp., Mucor spp., Mortierella spp., and Rhizopus spp.) have been screened for their ability to form GLA during their growth on plenty of substrates (22,23). There are several advantages why these filamentous molds are considered as convenient microbial candidates for SSF processes: (a) the fungal mycelia rapidly cover the surface of materials; (b) due to fungal enzymes the hyphae rapidly penetrate into the substrates (e.g., cereal grains) resulting in efficient consumption of these materials; (c) they produce the enzymes necessary for hydrolysis of sources bound in biopolymers; (d) they decrease amounts of anti-nutrient compounds in substrates; (e) their growth at reduced water activity avoids bacterial pollution. SSF can be performed with a variety of agroindustrial materials covering cereals and cereal-derived wastes (e.g., rice bran, wheat bran, oat flakes, and peeled barley), soybean meal, spent malt grain, orange peel, tomato, and apple pomace (8,23-25). Because of nutritional diversity of these substrates, it is necessary to optimize their utilization and subsequent transformation to GLA-oil by suitable microorganisms. Cereals are suitable materials providing necessary nutrients for fungal growth and lipid biosynthesis, with high carbon content in starch, and sufficient amounts of organic nitrogen having C/N ratio generally from 20 to 60. However, GLA accumulation in fungal cells could be further improved by following strategies (26): (a) accessibility of carbon source is increased by hydrolysis of cereal substrates; (b) addition of appropriate carbon source will elevate carbon/nitrogen ratio; (c) well balanced substrate/internal support ratio is required for improving respiration and aeration efficiency, for elimination the heat formed during fermentation and for reduction of substrate particles agglomeration; (d) appropriate substrate moistening is significant as a solvent for nutrients and is also obligatory for satisfied fungal growth, for evaporative cooling of fermentation mass; (e) proper water activity prevents growth of undesired microorganisms; (f) optimized oxygen availability is necessary for high activity of 6 -desaturase transforming linoleic acid to GLA; (g) substrate supplementation by GLA precursors (e.g., oils containing linoleic acid); (h) addition of minerals and other compounds that might stimulate GLA biosynthesis. SSF processes with lower filamentous fungi utilizing various types of agroindustrial sources have been tested for GLA production as shown in Table 2. Depending on the strain, the substrates were wrapped by mycelium after 1-3 days of cultivation. Cultivation of M. isabellina utilized barley led to fermented product containing 18.6% oil and 1.8% GLA (27). Mortierella isabellina growing on pear pomace yielded to significant amounts of 12% lipid and 2.9 g GLA/kg of in dry fermented mass, respectively (28). Cunninghamella echinulata efficiently transformed orange peel enriched with inorganic nitrogen and glucose to produce 1.5 g GLA/kg of dry fermented orange peel (8). Mucor rouxii was studied for growth on various types of rice materials (polished rice, broken rice, and rice bran), where high GLA accumulation was detected in stationary phase. Of them, rice bran considerably maximized content of GLA up to 6 g/kg of fermented mass (24). It should be emphasized that although the M. rouxii utilizing soybean meal resulted in reduced biomass and GLA content in fermented product than the rice bran culture, the high

5 Microbial Production of γ-linolenic Acid 925 Table 2. GLA production by solid state fermentations of fungi utilizing various substrates Strain Substrate 1) GLA yield (g/kg BP) 2) Ref Thamnidium elegans Spelt flakes/smg Crushed corn Wheat bran/smg/so Wheat bran/smg/so/plant extracts Mortierella isabellina barley Pear pomace Cunninghamella elegans Barley/SMG/peanut oil Cunninghamella echinulata Orange peel Mucor rouxii Rice bran ) SMG, spent malt grains; SO, sunflower oil BP, bioproduct/fermented mass proportion of GLA in total fatty acids (23.9%) and short cultivation period of 3 days might be remarkable standpoint from the view of GLA-oil applications and operating cost. Oppositely, C. elegans reached 14.2 g GLA/kg dry substrate (mix of barley, spent malt grain, and peanut oil) after 11 days of cultivation (22). Interesting results have also been obtained with Thamnidium elegans that effectively utilized a variety of cereals and the cultivation basically led up to 5 g GLA/kg fermented mass (23). However, supplementation of cereals with spent malt grains as an internal support rapidly enhanced bioconversion of linoleic acid presented in substrates to GLA. It should be noted that spent malt grain as a sole substrate also improved GLA biosynthesis in Mucor rouxii (24). Because T. elegans possesses active oil-biotransforming system, this strain converted successfully either extracellularly added plant oils rich in linoleic acid as a GLA precursor (e.g., sunflower oil) or oil-rich substrates to the maximal yield of 10 g GLA/kg fermented mass (26). Further, supplementation of oil-rich substrates with selected plant extracts yielded maximally up to 20 g GLA/kg product (28). Thus, GLA production by SSF could be a valuable biotechnological platform for a newly developing market with low investment cost and practical applications of GLA-enriched agricultural commodities. Conclusions The extensive research and progress in GLA manufacturing carried out over the past years has been focused on biotechnological workhorse. Successes in developing of submerged and SSF with oleaginous fungi have opened new possibilities for natural production of GLA-oils or GLA-rich products. However, such fermentation production of GLA will definitely depend on its acceptability in the market, regulatory approval, and the investment cost required for these technologies. Thus, future prospects of microbial GLA-oils should be directed not only how to economically improve fermentation GLA production but also how to effectively commercialize such euphonically sounded bio-based GLA products variously ranging through feeds, foods, cosmetics, and pharmaceuticals. Acknowledgments The work was supported by grant VEGA 1/0975/12 from the Grant Agency of the Ministry of Education and by grants VVCE and APVV from the Slovak Research and Development Agency, Slovak Republic, and from the National Science and Technology Development Agency, Thailand. References 1. Horrobin DF. Medical roles of metabolites of precursor EFA. Inform 6: (1995) 2. Gill I, Valivety R. Polyunsaturated fatty acids. I. Occurrence, biological activities, and applications. Trends Biotechnol. 15: (1997) 3. Laoteng K, Čertík M. Biotechnological production and application of high-value microbial oils. pp In: Industrial Fermentation: Food Processes, Nutrient Sources, and Production Strategies. Krause J, Fleischer O (eds). Nova Science Publisher, Inc., Hauppauge, NY, USA (2010) 4. Čertík M, Shimizu S. Biosynthesis and regulation of microbial polyunsaturated fatty acid production. J. Biosci. Bioeng. 87: 1-14 (1999) 5. Ward OP, Singh A. Omega-3/6 fatty acids: Alternative sources of production. Process Biochem. 40: (2005) 6. Guschina IA, Harwood JL. Lipids and lipid metabolism in eukaryotic algae. Prog. Lipid Res. 45: (2006) 7. Kennedy MJ, Reader SL, Davies RJ. Fatty acid production characteristics of fungi with particular emphasis on γ-linolenic acid production. Biotechnol. Bioeng. 42: (1993) 8. Gema H, Kavadia A, Dimou D, Tsagou V, Komaitis M, Aggelis G. Production of γ-linolenic acid by Cunninghamella echinulata cultivated on glucose and orange peel. Appl. Microbiol. Biot. 58: (2002) 9. Papanikolaou S, Michael Komaitis M, Aggelis G. Single cell oil (SCO) production by Mortierella isabellina grown on high-sugar content media. Bioresource Technol. 95: (2004) 10. Fakas S, Čertík M, Papanikolaou S, Aggelis G, Komaitis M, Galiotou-Panayotou M. γ-linolenic acid production by Cunninghamella echinulata growing on complex organic nitrogen sources. Bioresource Technol. 99: (2008) 11. Čertík M, Balteszova L, Sajbidor J. Lipid formation and γ-linolenic

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