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Mortierella alpina is known as a saprobic soil inhabiting filamentous fungus. It is considered an oleaginous fungus that has an incredible capability of accumulating unsaturated fatty acids such as arachidonic acid (ARA) and conducting biotransformation process producing some beneficial substances for the human health (Zhu and others 2002). Streekstra 1997 described M. alpina as the most efficient ARA productive organism, which made it a captivating field for biotechnological researches (Wagner 2013; Streekstra1997).
Mortierella alpina has been classified as a member of the family Mortierellaceae, order Mortierellales, under the phylum of Zygomycetes . M alpina belongs to the subphylum of Mucoromycotina (Wang 2011). The first Mortierella species was described by Coeman in 1863, and the name Mortierella was given in respect to M. Du Mortier, the president of the Society of Botanique of Belgium (Lee 2014). However, the second section of the name (alpina) is originated from the first isolated strains from alpine grassland mountains. M. alpina species’ mnemonic is MORAP, synonym is Mortierella renispora, and according to the references it has no common name. However, it has other names, Mortierella alpina and Mortierella sp. JZ-157. These species include several strains: 1S-4, ATCC 16266 / C 112, ATCC 16266, ATCC 32221 / CBS 527.72 / M135, ATCC 32221, ATCC 32222 / CBS 528.72 / M136, CBS 528.72, CBS 210.32 / CCRC 32738, CBS 210.32, and NCBI. 64518 taxon have been identified (UniPort 2002).
Phylogenetic analyses including sequences analysis of an extensive types and reference strains shows that the genus Mortierella is divided into nine sections of specifically morphologically related species. Alpina is among the clade number six (Wagner 2013). According to this phylogenetic tree the closest relatives to Mortierella alpina species were Mortierella amoeboidea and Mortierella Antarctica (Wang 2011; Wagner 2013).
Morphology and biology
M. alpine is known to be Heterothallic, filamentous fungus, which can grow fast and sporulates well in a wide diversity environment (Sakuradani and others 2009; Ho and others 2008; Yadav and others 2014). It can reach 35-45 mm diameter after incubation for 7days at 28 ºC. M. alpina has milky-white color, and Mortierella-like odor. M. alpina has erected, coenocytic, septate, and unbranched mycelia. However, sometimes the mycelium could be dichotomously branched with terminal sporangia. Sporangia is hyaline, obovoid when its in the young phase, but spherical at maturity phase, with 12-15 µm in diameter. It is multispored with a delitescent wall. Sporangiophores which arising from the mycelia have 60-110 µm in size. M. alpina has asexual sporangiospores, usually cylindrical 5-7 µm in size. However, sometimes they have been curved and irregularly shaped (Yadav and others 2014). M. alpina asexual germination path.https://www.flickr.com/photos/136241512@N06/21963216228/in/album-72157657493607393/ https://www.flickr.com/photos/136241512@N06/21529972293/in/album-72157657493607393/
Zygospores could be formed at 10-20 ºC on hemp seed agar (HAS) in the aerial mycelium between two compatible strains. When Zygospores are all together they looked white, but they looked hyaline when they are individual spores. They are Globose to sub- globose, (42-) 54 (-80) x (40-) 52 (-70) µm, have distinguished walls; 2-4, 4-8, and 2-4 µm thick, respectively. Also the large suspensor is globose (30-) 40-43 (-65) µm in diam. The outer wall is smooth, and the hypha is not covered. M. alpine has abundant anastomoses, which is considered gametangia stages, and it produces zygospore as numerous hyphal masses. This structure is recorded as uncovered zygospores (Kuhlman 1975).
M. alpina is an ubiquitous saprophytic fast-growing filamentous, and originally widespread in soil environments, however its spores could be found in air and rain. These species could adapt to variant environmental conditions. M. alpine has isolated from different geographical locations, such as the tropical areas, alpine areas, and salt mashes (HO 2008). M. alpina can grow on glucose as the single carbon source (Wang 2011). Accordingly, observers found that most species in the Mortierella alpine group have maximum rates of isolation in the summer (Bissett and others 1979). Despite that fact, Mortierella alpine also found all over Alaska on trees or logs, which present it as a freezing tolerant species ( Nowoisky and others 2015).
Pathogenicity of Mortierella alpine
All Mortierella alpine strains have been originally found in soil without association with animal material. There is one exception: strain CBS 396.91 has been isolated from the air bladder of juvenile fish, so that association with pathogenesis of cold-blooded animals cannot be excluded entirely. However, the environmental risk of the release of biomass may be regarded as exceptionally low: M. alpina is a commonly isolated fungus from the soil of temperate climates, so that exposure to the spores of this organism occurs frequently. Thus, to confirm that M. alpina are incapable of causing any harm to warm-blooded animals, including humans, all M. alpine species were tested to determine their maximum and optimum growth temperature. M. alpina maximum growth was at 30 ºC, and the optimal growth was at 21-24 ºC (Streekstra 1997). The results shows that none of M. alpine even the tropical strains can grow at 36 ºC, which prove its disability to be pathogenic to warm blood animals.
Mortierella alpine biotechnology and economically importance
Mortierella alpine is an oleaginous fungus. Through extended screening tests M. alpine showed that it could produce lipids reaching up to 50% of its dry weight. These lipids usually are triacylglycerol connected to polyunsaturated fatty acids (PUFAs), which used in a wide range in pharmaceutical and food applications because of their incredible effect on human health. For M. alpina the main production is one of the most useful unsaturated fatty acids, arachidonic acid C20 (Lee 2014; Thevenieau 2013). Arachidonic acid (ARA; 5,8,11,14-cis-eicosatetraenoic acid) is considered very important fatty acids because ARA plays a role of keeping the membrane fluidity in biological cells. It has many significant physiological actions such as uterine muscle contraction, vasodilatation, antihypertensive, and relaxation action (Higashiyama 2002). In addition, ARA can: protect the gastric mucosa, reduce the fatty liver, kill the tumor cells, and improve the lipid metabolism of cirrhotic patients. ARA can play an important role as a natural ligand for the cannabinoid receptor, which is explicated in the central nervous system in the areas that control memories, cognition, movement and pain perception. ARA’s function in the human body grabbed scientists’ attention as unfamiliar function of PUFAs (Higashiyama 2002). Some M. alpine strains such as (ITA1-CCMA 952) have a massive potential as a biotechnological producer, and M. alpina ATCC 32222 particularly had the highest production rate of ARA, γ-linolenic acid and linoleic acid among 11 test species (Jang 2005). Furthermore, several strains of M. alpine produce antioxidant substances, and antibiotics besides the production of PUFAs (Melo and others 2014). Thus, M. alpine could be an economically beneficial source if the optimum condition were provided for it.https://www.flickr.com/photos/136241512@N06/22152253425/in/dateposted-public/
Factors affect M. alpine growth and ARA production
For the industrial production of ARA, various studies, such as isolation of a high-potential strain and optimization of culture conditions, have been conducted. Studies, including the investigation of morphology are important because ARA is accumulated in the mycelia, and to increase the biomass concentration, which is essential for yielding a high ARA amount (Higashiyama 2002). Thus, these culture affective factors should be controlled.
The optimum dissolved oxygen (DO) concentration for M. alpina growth was found to be between15-20 ppm. When DO retained at 20–50 ppm, the morphology changed from filaments to pellets, and the ARA production decreased dramatically because of the stress that accrued as a result of the limitation in mass transfer through the pellet walls (Higashiyama 1999).
2-carbon, nitrogen amount and sources
Some studies indicated that a medium containing 3.1% soy flour and 1.8% glucose is the optimum for M.alpina growth and ARA production. The ARA yield reached 9.8g/L/7 d, and the major morphology was small pellets (1–2 mm) (Higashiyama 1998). Jang in 2005 presented that the mixture of KNO3 and yeast extracts at 2:1 (w/w) with soluble starch at 10% was the best carbon and nitrogen sources for total PUFAs and arachidonic acid production. The optimum C/N ratio was ranged from 5.1 to 9.0. Each gram of carbonyeilded194.2 mg of total PUFAs, 103.0 mg of arachidonic acid at 20 °C, While it yielded 218.4 mg of total PUFAs, 110.3 mg of arachidonic acid at 12 °C (Jang 2005). In addition Chen’S in his article concluded that the superior carbon and nitrogen sources for M.alpine growth and fatty acid production were soluble starch, urea at 24°C (Chen 1997).
Minerals such as potassium, phosphorus, and magnesium particularly are found to be affective to fungi's growth. Many studies have shown that the M. alpine’s cell mass yielded from incubated in optimum conditions was corresponding in size and amount to the quantity of minerals. These minerals in some forms could stimulate the fungi growth. For instance, adding KH2PO4, MgSO4·7H2O and CaCl2·2H2O compounds can increase the cell mass yielded (Totani 2002; Jang 2005).
4- pH and temperature
After long observation researches have found that the culture pH affects the production of polyunsaturated fatty acids, especially arachidonic acid (AA). Thus, to optimize the condition for rapid biomass (maximize the fungi growth) and lipid production, the best condition was pH 6.5 at 25°C which yielded production reach to 31% ARA. However. 18°C also provides relatively good growth condition for good fungus morphology, such as pellets forming. This study found that the best lipid production achieved (ARA up to 33%) by using glucose as a carbon source (Lindberg 1993).
As concluded, M. alpine morphology and mycelia size are both affected by many factors such as the ratio of carbon to nitrogen and the dissolved oxygen concentration. Following the changing in the environment M. alpine’s filaments may be changed to pellets (Park and other 2001, Higashiyama, 1999).