Production of arachidonic acid by Mortierella alpina using wastes from potato chips industry.

AIMS: This research aimed to determine the potential use of wastes from the potato chips industry as a carbon source to develop an economical culture medium for the production of biomass, lipids and arachidonic acid (ARA) by Mortierella alpina. METHODS AND RESULTS: A synthetic culture medium was optimized using a Plackett-Burman and central composite rotatable design, and used as a base to evaluate and characterize the potential use of wastes from the potato chips industry as carbon sources for the production of biomass, lipids and ARA by M. alpina. The waste was selected among other solid and liquid hydrolysed residues/by-products, and local low-cost alternatives for nitrogen sources were also evaluated. After 6 days of fermentation, the biomass concentration reached 20 g l-1 with 40% of total lipids, and a 35% ARA content in the lipids fraction. Savings in production were calculated using a sensitivity analysis for the alternative culture medium in different scenarios. CONCLUSIONS: This study showed a 7% savings in culture media expenses in the production of ARA-enriched biomass of M. alpina, compared to the conventional synthetic culture medium, when waste from the potato chips industry was used as an alternative source of carbon and macro/microelements, supplemented with a low-cost yeast extract alternative. SIGNIFICANCE AND IMPACT OF THE STUDY: The demonstration of the use of potato chips wastes as a low-cost carbon source for the biomass, lipids and ARA production, suggesting an eco-friendly alternative for the use of agri-food wastes for valuable metabolites production.

Arachidonic acid production by Mortierella alpina using raw crop materials.

BACKGROUND: Arachidonic acid (ARA) is one of the three essential fatty acids, and it is important for human body to keep healthy and is widely used. At present, expensive materials such as glucose and yeast extract are generally reported to be optimal for ARA production. A new cost-effective fermentation process including cheaper material for ARA production is of great significance. METHODS: Feasibility of using corn meal and powdered soybean for fungal growth and lipid accumulation was evaluated by means of single factor test. N-hexadecane concentration was optimized, and the effect of temperature on biomass and ARA content was examined. RESULTS: Mortierella alpina made better use of the aforementioned material as carbon and nitrogen sources for both hyphae growth and ARA production compared with glucose and yeast extract. Maximal levels of 10.9 g/L ARA and 26.1 g/L total lipids were obtained when 66 g/L corn meal, 54 g/L soybean meal and 6% (v/v) n-hexadecane were supplemented. A temperature-shift strategy involved three steps, namely, 30°C (3 days) - 25°C (4 days) - 20°C (4 days), which further improved ARA production by 24.7%. CONCLUSIONS: Several factors such as carbon and nitrogen sources, temperature and dissolved oxygen had great influence on biomass and microbial oil production. Mortierella alpina preferred corn and soybean meal compared with glucose and yeast extract, which would surely alleviate the high cost of ARA production. Based on this study, the new process is both low cost and practicable.

Production and enhancement of omega-3 fatty acid from Mortierella alpina CFR-GV15: its food and therapeutic application.

Mortierella sp. has been known to produce polyunsaturated fatty acids (PUFAs) such as GLA and AA under normal growth medium conditions. Similarly, under the stress condition, this fungus produces EPA and DHA in their mycelial biomass. Among the 67 soil samples screened from the Western Ghats of India, 11 Mortierella isolates showed the presence of omega-6 and omega-3 fatty acid, mainly GLA, AA, EPA, and DHA in starch, yeast-extract medium. Nile red and TTC strains were used for screening their qualitative oleaginesity. Among the representative isolates, when Mortierella sp. is grown in a fat-producing basal medium, a maximum lipid content of 42.0 ± 1.32% in its mycelia, 6.72 ± 0.5% EPA, and 4.09 ± 0.1% DHA was obtained. To understand the Mortierella sp. CFR-GV15, to the species level, its morphology was seen under the light microscope and scanning electron microscope, respectively. These microscopic observations showed that isolate Mortierella sp. CFR-GV15 produced coenocytic hyphae. Later on, its 18S rRNA and the internal transcribed spacer (ITS) sequences were cloned, sequenced, and analyzed phylogenetically to 18S rRNA and ITS1 and ITS4 sequences of related fungi. This newly isolated Mortierella alpina CFR-GV15 was found to be promising culture for the development of an economical method for commercial production of omega-3 fatty acid for food and therapeutical application.

Metabolism and synthesis of lipids in the polyunsaturated fatty acid-producing fungus Mortierella alliacea.

The fungal strain Mortierella alliacea YN-15 is a promising industrial producer of polyunsaturated fatty acids (PUFAs), in particular arachidonic acid. In order to more efficiently produce PUFAs, the metabolism of an externally supplied plant oil, α-linolenic acid (ALA)-rich linseed triacylglycerol (TAG), was examined, and time-dependent changes in the composition of its lipid and fatty acid metabolites were traced. Addition of linseed TAG to growing cultures resulted in a transient increase in extracellular 1,2-diacylglycerol (DAG), and even more so of 1,3-DAG, in the mycelia. This was followed by a decrease in both DAGs and an increase in TAG. Eicosapentaenoic acid (EPA), a desaturated and elongated product of ALA, accumulated to a greater extent in cellular phospholipids than in neutral lipids. Moreover, the addition of ALA in free fatty acid form to the culture led to the generation of EPA. However, EPA production was not observed upon addition of ALA-rich 1,2- or 1,3-DAG, indicating that fatty acids released from exogenous lipids were used for resynthesis of mycelial TAG. These results suggested that TAG might be hydrolyzed by extracellular lipases, whereas its synthesis might be catalyzed by intracellular enzymes. Appropriate regulation of such enzymes might be an effective strategy to enhance PUFA production under plant oil supplementation.

Safety evaluation of arachidonic acid rich Mortierella alpina biomass in albino rats--a subchronic study.

Safety evaluation of arachidonic acid rich Mortierella alpina biomass was carried out in Wistar rats by acute and subchronic oral toxicity studies. A preliminary acute toxicity study revealed that the biomass was safe at acute doses and that the LD50 exceeded 5000mg/kg BW, the highest dose used in the study. In subchronic study, rats were fed diet containing 0, 2500, 5000, 10,000, 20,000 and 30,000mg/kg, M. alpina biomass for a period of 13 weeks. Results indicated that biomass fortification had a positive influence on growth with no overt toxic effects on the survival, food consumption and body weight gain throughout the treatment interlude. The statistically significant changes in relative organ weights, serum biochemical and hematological indices in M. alpina fed groups' viz., higher relative weights of spleen, liver, brain and ovary in females, reduced hemoglobin concentration in males, elevated WBC counts at highest dose, reduction in serum triglycerides and increased alkaline phosphatase activity were not concomitant with pertinent histopathological changes and hence toxicologically inconsequential. No microscopic or macroscopic lesions attributable to the treatment were manifested in the experimental groups. The results of the present study strongly advocate the safety of M. alpina biomass in rats at levels used in the study.

Polyunsaturated fatty acids (PUFAs) content of the fungus Mortierella alpina isolated from soil.

Twenty-five isolates of Mortierella spcies were prepared, which can be used for the production of polyunsaturated fatty acids (PUFAs)-rich oil for nutritional supplements. The fatty acid contents were determined after heterotrophic fermentation. The content of total fatty acids (TFAs) in the cell dry weight of all isolates including two commercially purchased Mortierella alpina strains ranged from 207.51 to 370.11 mg/g, whereas PUFAs were the dominant fatty acid type. The highest PUFA-containing strain, M. alpina SC9, was identified and confirmed as a new strain of M. alpina through comparison analysis of the sequences of internal transcribed spacers 1 and 2 (ITS1 and ITS2) and the 5.8S rDNA region. During a 7-day fermentation, the PUFAs content of M. alpina SC9 varied between 189.83 and 240.00 mg/g, with a remarkable correlation between the oleic acid (C18:1, OA) and arachidonic acid (C20:4n-6, AA) contents and between the linoleic acid (C18:2n-6, LA) and AA contents, suggesting the PUFA content in the fungus is tightly regulated. This study provides a framework of isolation, identification, and characterization of an important PUFA-producing species, M. alpina.

Repression of reserve lipid turnover in Cunninghamella echinulata and Mortierella isabellina cultivated in multiple-limited media.

AIMS: To study patterns of reserve lipid biosynthesis and turnover (degradation) in two oleaginous Zygomycetes, namely Cunninghamella echinulata and Mortierella isabellina under various growth conditions. Fatty acid composition of the reserve lipid of both strains was also studied in all growth steps. METHODS AND RESULTS: Cunninghamella echinulata and Mortierella isabellina were grown in carbon-excess batch cultures. In the investigated strains, accumulation of reserve lipid occurred only when the activity of both NAD(+)-isocitrate dehydrogenase (ICDH) and NADP(+)-ICDH were not detectable in the cell-free extract. Specifically, in C. echinulata, NAD(+)-ICDH activity was detected even after depletion of ammonium nitrogen in the medium, resulting in a delay of the initiation of lipid accumulation period. On the contrary, in M. isabellina, lipid accumulation occurred simultaneously with ammonium nitrogen exhaustion in the growth medium, as the activity of both NAD(+)- and NADP(+)-ICDH were not detectable after nitrogen depletion. In C. echinulata reserve lipid was not degraded after glucose had been exhausted. Supplementations of the medium with Fe(3+), yeast extract or Mg(2+) induced, however, reserve lipid breakdown and formation of lipid-free material. In M. isabellina after glucose exhaustion, notable lipid degradation occurred, accompanied by a significant lipid-free material biosynthesis. Nevertheless, in multiple-limited media, in which Mg(2+) or yeast extract, besides carbon and nitrogen, were limiting nutrients, reserve lipid breakdown was repressed. In both strains, the quantity of gamma-linolenic acid (GLA) in the reserve lipids [varying between 9 and 16% (w/w) in C. echinulata and 1.5-4.5% (w/w) in M. isabellina] was proportional to lipid-free biomass. CONCLUSIONS: Lipid accumulation period in Zygomycetes is initiated by the attenuation of ICDH activity in the mycelium while the regulation of ICDH from ammonium nitrogen is strain specific. While a single nitrogen limitation was enough to induce lipid accumulation, however, multiple limitations were needed in order to repress lipid turnover in oleaginous Zygomycetes. As for GLA, its biosynthesis in the mycelium seemed proportional to lipid-free biomass synthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: Several nutrients are indispensable for functioning the mechanisms involved in the mobilization of reserve lipid in oleaginous moulds. Therefore, reserve lipid turnover in oleaginous moulds could be repressed in multiple-limited media.

Improved arachidonic acids production from the fungus Mortierella alpina by glutamate supplementation.

The effect of various concentrations of glutamate on arachidonic acid (AA) production from Mortierella alpina in shaker flask culture was studied. Glutamate supplementation promoted Mortierella growth, accelerated substrate metabolism, and increased AA production, and a concentration of 0.8 g/l glutamate resulted in the greatest AA yield (1.41 g/l). In 10 l airlift stirred fermenter culture, AA yield in the cultures exposed to 0.8 g/l glutamate was also greater than that in the control (0.56 g/l).

Production of gamma-linolenic acid by disrupted mycelia of Mortierella isabellina.

AIMS: To optimize the production of linolenic acid by disrupted mycelia of Mortierella isabellina. METHODS AND RESULTS: Effects of incubation conditions such as incubation time, pH of reaction mixture, concentration of Mg2+ or malate and incubation temperature on production of linolenic acid were studied. The production of gamma-linolenic acid reached 224 mg g-1 dry cells when the reaction mixture was composed of 1.0 g (dry mycelial mass) of disrupted mycelia of M. isabellina, 50 ml (50 mmol l(-1)) potassium phosphate buffer supplemented with 0.312 mmol l(-1) of Mg2+ and 10 mmol l(-1) of malate, pH 7.0 and incubated at 5 degrees C for 1 day. CONCLUSIONS: Incubation temperature, concentration of Mg2+ and malate showed major effects on the increased linolenic acid production. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlights conditions for increasing gamma-linolenic acid production by cell-free mycelia of M. isabellina and an insight into rapidly gaining high production of polyunsaturated fatty acids.

An inexpensive medium for production of arachidonic acid by Mortierella alpina.

The production of arachidonic acid was studied in the fungus Mortierella alpina using an inexpensive medium. Glucose derived from maize starch hydrolysate was the sole carbon source and defatted soybean meal and sodium nitrate were the nitrogen sources. Optimal arachidonic acid yield (1.47 g l(-1)) was observed at a glucose concentration of 100 g l(-1). Various treatments of defatted soybean meal to extract soluble nitrogen nutrients were evaluated. Alkali extract was the most effective for arachidonic acid production. A mixture of soybean alkali-extract protein and sodium nitrate was an excellent nitrogen source for fungal growth, lipid accumulation, and arachidonic acid production. A maximum yield of 1.87 g arachidonic acid l(-1) was obtained with a soybean protein concentration of 4.6 g l(-1) and a sodium nitrate concentration of 2.3 g l(-1).