Milan Čertík

Use of several waste substrates for carotenoid-rich yeast biomass production

Carotenoids are industrially significant pigments produced in many bacteria, fungi, and plants. Carotenoid biosynthesis in yeasts is involved in stress response mechanisms. Thus, controlled physiological and nutrition stress can be used for enhanced pigment production. Huge commercial demand for natural carotenoids has focused attention on developing of suitable biotechnological techniques including use of liquid waste substrates as carbon and/or nitrogen source. In this work several red yeast strains (Sporobolomyces roseus, Rhodotorula glutinis, Rhodotorula mucilaginosa) were enrolled into a comparative screening study. To increase the yield of these pigments at improved biomass production, several types of exogenous as well as nutrition stress were tested. Each strain was cultivated at optimal growth conditions and in medium with modified carbon and nitrogen sources. Synthetic media with addition of complex substrates (e.g. yeast extract) and vitamin mixtures as well as some waste materials (whey, potato extract) were used as nutrient sources. Peroxide and salt stress were applied too. The production of carotene enriched biomass was carried out in flasks as well as in laboratory fermentor. The best production of biomass was obtained in inorganic medium with yeast extract. In optimal conditions tested strains differ only slightly in biomass production. All strains were able to use most of waste substrates. Biomass and pigment production was more different according to substrate type. In laboratory fermentor better producers of enriched biomass were both Rhodotorula strains. The highest yields were obtained in R. glutinis CCY 20-2-26 cells cultivated on whey medium (cca 45 g per liter of biomass enriched by 46 mg/L of beta-carotene) and in R. mucilaginosa CCY 20-7-31 grown on potato medium and 5% salt (cca 30 g per liter of biomass enriched by 56 mg/L of beta-carotene). Such dried carotenoid-enriched red yeast biomass could be directly used in feed industry as nutrition supplement.

Arachidonic acid production byMortierella sp. S-17 influence of C/N ratio

Several fungal strains of the genus Mortierella accumulate high amounts of arachidonic acid (ARA), one of the most interesting polyunsaturated fatty acid (PUFA) applicable in pharmacy and medicine. Totani and Oba (1987) reported 68.5-78.8~ ARA in total fatty acids of M.alpina. According to Yamada et a1.(1987) the production of ARA by M. elongata reached 0.99 mg/ml (22 rag/ g dry ceils). A very important factor affecting lipid over-production is the metabolic shift caused by nitrogen limitation. Yokochi and Suzuki (1987) found that the lipid accumulation in Mortierella isabelina increased from 7.9 to 62.97,; the highest rate of lipogenesis was found at C/N = 20 and glucose concentration 270 g/l i tre (Yokochi et al., 1989). Because the isolate of Mortierella sp. used in our experiment can be regarded a potential ARA producer, the influence of various ratio of carbon and nitrogen concentration on lipid and ARA production was studied.

Effect of extraction methods on lipid yield and fatty acid composition of lipid classes containing γ-linolenic acid extracted from fungi

The effect of extraction procedures on the lipid yield and fatty acid composition of total lipid and main lipid structures (phospholipids, diacylglycerols, triacylglycerols, free fatty acids, and sterol esters) of fungal biomass (Mucor mucedo CCF-1384) containing γ-linolenic acid (GLA) was investigated. Seventeen extraction methods, divided into three groups, were tested: six with chloroform/methanol, five with hexane/alcohols, and six with common solvents or mixtures. The chloroform/methanol procedure (2∶1) was selected as standard, where lipid yield (TL/DCW, total lipid per dry cell weight) was 17.8%, considered to be 100% of lipids present. All chloroform/methanol extractions yielded more than 83% recorvey of lipids. Use of hexane/isopropanol solvent systems led to a maximum of 75% recovery. The best lipid yield was achieved by a two-step extraction with ethanol and hexane (120%). Extraction efficiency of the other solvent systems reached a maximum of 73%. Triacylglycerols were the main structures of lipid isolated; only methanol-extracted lipid contained 58.5% phospholipids. The fatty acid content of total recovered lipid was variable and depended on both the lipid class composition and the solvent system. GLA concentrations in total lipids isolated by hexane/alcohol procedures (7.3–10.7%) are comparable with classical chloroform/methanol systems (6.5–10.0%). The maximal GLA yield was obtained with chloroform/methanol/n-butanol/water/0.1 M ethylenediaminetetraacetic acid (EDTA) (2∶1∶1∶1∶0.1, by vol) and after two-step extraction with ethanol and hexane (14.3 and 13.7 g GLA/kg DCW, respectively). The highest GLA content was analyzed in the phospholipid fraction (16.1%) after using chloroform/methanol/n-butanol/water/0.1 M EDTA (2∶1∶1∶1∶0.1, by vol). Remarkably low concentrations of polyunsaturated fatty acids were determined in the free fatty acid fraction.

Selection ofRhizopus strains forl(+)-lactic acid andγ-linolenic acid production

The production ofl(+)-lactic acid and formation ofγ-linolenic acid by 50Rhizopus strains growing on saccharidic substrates were investigated. Formation of acids was observed on solid cultivation media but mainly during submerged fermentation. Strains with the highest selectivity of bothl(+)-lactic acid production andγ-linolenic acid formation were tested in a laboratory fermenter. The best producer was treated by UV irradiation to increase the fatty acid content in the biomass, especially that ofγ-linolenic acid. The conversion of 10% saccharidic substrate by this newly prepared strainRhizopus arrhizus CCM 8109 results in more than 95% of theoretical yield ofl(+)-lactic acid and permits a volume productivity of 0.4 gγ-linolenic acid per liter.

Simultaneous enrichment of cereals with polyunsaturated fatty acids and pigments by fungal solid state fermentations.

Four Mucor strains were tested for their ability to grow on four cereal substrates and enriched them with gamma-linolenic acid (GLA) and β-carotene. M. circinelloides CCF-2617 as the best producer accumulated of both GLA and β-carotene in high amounts during utilization of rye bran/spent malt grains (3:1). The first growth phase was characterized by rapid GLA biosynthesis, while distinct β-carotene formation was found in the stationary fungal growth. Therefore various cultivation conditions were tested in order to optimize the yield of either GLA or β-carotene. The fungus grown on cereal substrate supplemented with glucose produced maximal 8.5 mg β-carotene and 12.1 g GLA in 1 kg fermented substrate, respectively. On the other hand, the highest amount of GLA in the fermented substrate (24.2 g/kg) was achieved when 30% of sunflower oil was employed to the substrate. Interestingly, β-carotene biosynthesis was completely inhibited when either whey or linseed oil were added to the substrate.

Modulation of phenolic content and antioxidant activity of maize by solid state fermentation with Thamnidium elegans CCF 1456

Thamnidium elegans CCF 1456, a filamentous fungus, was used to enhance the total phenolic content and radical scavenging activity of maize via solid-state fermentation. Thamnidium fermented maize (TFM) and unfermented maize (UFM) grains were extracted with 65% ethanol and dimethylsulfoxide (DMSO). Total phenolic content (TPC), and radical scavenging capacity — determined with 1,1-diphenyl-2-picrylhydrazyl (DPPH) and radical cations of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS·+) found for TFM — were significantly (P < 0.05) higher on the 5th day of incubation than that of UFM extracts. A linear correlation was observed among TPC, DPPH and ABTS scavenging activities, and also among TPC and various carbohydrate-cleaving enzymes (α-amylase, β-glucosidase and xylanase), suggesting that this? strategy may help to enrich? TFM with improved phytochemical properties and antioxidant activities.

Supercritical CO2 extraction of fungal oil containing γ-linolenic acid

Supercritical CO2 has been used to extract an oil containing γ-linolenic acid (GLA) from Cunninghamella echinulata. The highest oil recovery from dry biomass (26.4%, w/w) and GLA yield (26.1 g/kg biomass) has been achieved at 30 MPa and 50 °C after 180 min using fungal particles smaller than 0.5 mm and mass flow of 50 kg CO2/kg dry biomass. Extractions with hexane/ethanol and chloroform/methanol methods gave less than 90% of the GLA/kg reached with the supercritical CO2 method.

Lipid analysis of baker's yeast

Abstract Commercial bakers yeast was analysed for lipids by TLC and GC. The TLC system of a silica gel plate with n -hexane-diethyl ether-acetate acid (70:30:1, v/v/v) allowed the total lipids to be divided into sterol esters (ES), free fatty acids (FFA), triacylglycerols (TAG), diacylglycerols (DAG), free sterols (FS), monoacylglycerols (MAG) and phospholipids (PL). Phospholipids were separated by two-step chromatography with the systems SiO 2 with acetone and SiO 2 with chloroform-methanol-acetic acid-water (25:15:4:2, v/v). The following phospholipids were detected: phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylcholine (PC) and lysophospholipids (LPL). The fatty acid composition of TAG, ES, FFA, DAG, MAG, PC, PE, PS and PI was investigated by using packed column (15% DEGJ + 3% H 3 PO 4 ) and capillary column (SP 2340) GC, Twenty-two individual fatty acids with carbon chain lengths in the range 12–24 were detected by capillary GC. The presence of squalene, zymosterol, ergosterol and lanosterol in non-saponifiable lipid was proved and the compounds were determined by GC (SE-30).

Isolation, structure elucidation and biological activity of angucycline antibiotics from an epiphytic yew streptomycete.

In the course of study of epiphytic microorganisms occurring on the surface of roots of Taxus baccata L. a new strain Streptomyces sp. AC113 was isolated. According to 16S ribosomal DNA‐based identification the new strain is 99% identical with Streptomyces flavidofuscus. This strain cultivated in an arginine glycerol medium produced three major metabolites identified as (–)‐8‐O ‐methyltetrangomycin (1), 8‐O ‐methyltetrangulol (2) and 8‐O ‐methyl‐7‐deoxo‐7‐hydroxytetrangomycin (3). The chemical structures of these angucyclines were elucidated by 1D and 2D NMR as well as by mass spectrometry. Isolated angucycline metabolites showed significant antimicrobial activity against Bacillus cereus and Listeria mocytogenes. Cytotoxic activities of compounds 1, 2 and 3 against four cell lines (B16, HT‐29 and non – tumor V79, L929) were evaluated. Compound 3 was the most potent anticancer agents with IC50 0.054 μg/ml against cell line B16. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Overexpression of diacylglycerol acyltransferase in Yarrowia lipolytica affects lipid body size, number and distribution

In the oleaginous yeast Yarrowia lipolytica, the diacylglycerol acyltransferases (DGATs) are major factors for triacylglycerol (TAG) synthesis. The Q4 strain, in which the four acyltransferases have been deleted, is unable to accumulate lipids and to form lipid bodies (LBs). However, the expression of a single acyltransferase in this strain restores TAG accumulation and LB formation. Using this system, it becomes possible to characterize the activity and specificity of an individual DGAT. Here, we examined the effects of DGAT overexpression on lipid accumulation and LB formation in Y. lipolytica Specifically, we evaluated the consequences of introducing one or two copies of the Y. lipolytica DGAT genes YlDGA1 and YlDGA2 Overall, multi-copy DGAT overexpression increased the lipid content of yeast cells. However, the size and distribution of LBs depended on the specific DGAT overexpressed. YlDGA2 overexpression caused the formation of large LBs, while YlDGA1 overexpression generated smaller but more numerous LBs. This phenotype was accentuated through the addition of a second copy of the overexpressed gene and might be linked to the distinct subcellular localization of each DGAT, i.e. YlDga1 being localized in LBs, while YlDga2 being localized in a structure strongly resembling the endoplasmic reticulum.