Production of high yields of docosahexaenoic acid by Schizochytrium sp. strain SR21

The culture conditions for high-yield production of docosahexaenoic acid (DHA) by Schizochytrium sp. strain SR21 were investigated in a fermenter. With increasing carbon (glucose) and nitrogen (corn steep liquor and ammonium sulfate) sources (up to 12% glucose) in the medium, DHA productivity increased without a decrease in growth rate, i.e., 2.0, 2.7, and 3.3 g DHA/L/d with 6, 10, and 12% glucose, respectively. Eventually, 48.1 g dry cells/L and 13.3 g DHA/L were produced in 4 d with 12% glucose. DHA productivity was decreased with 15% glucose, i.e., 3.1 g/L/d. With 12% glucose, the lipid content was 77.5% of dry cells, and DHA content was 35.6% of total fatty acids. The lipid was composed of about 95% neutral lipid and 5% polar lipid. In polar lipids, the contents of phosphatidylcholine (PC), phosphatidylethanolamine, and phosphatidylinositol were 74, 11, and 5%, respectively. The PC profile was simple, 70% of PC molecules were 1-palmitoyl-2-DHA-PC and 1.2-di-DHA-PC. These results indicate that Schizochytrium sp. strain 21 is an excellent source for microbial DHA production, including not only the acid form of DHA but also 2-DHA-PC.     

Inulin as a Promising Alternative Feedstock for Docosahexaenoic Acid Production by Schizochytrium sp. ATCC 20888

Schizochytrium sp. is considered as a promising alternative commercial source of docosahexaenoic acid (DHA), but the production is hindered by the high feedstock cost. In this study, inulin is used as a cheap and readily available feedstock for Schizochytrium sp. ATCC 20888 to produce DHA. The strain could not utilize inulin directly and therefore inulin first needed to be hydrolyzed. Compared with the acidic hydrolyzate by HCl and hydrolyzate by endo‐inulinase, the hydrolyzate by exo‐inulinase serves as the most effective carbon source for microalgal growth. Hydrolysis of inulin by exo‐inulinase is further optimized, and up to 97.8% of inulin conversion is obtained under the optimal conditions of 40 °C, pH 7.0, substrate concentration of 80 g L^?1 and exo‐inulinase loading of 2 g kg^?1 substrate for 12 h. The resulting hydrolyzate containing mainly fructose is used for the DHA production by the microalga. The lipid content in biomass, DHA content in total fatty acids, DHA yield, and DHA productivity at 72 h reach 45.26%, 35.59%, 5.64 g L^?1 and 1.88 g L^?1 d^?1, respectively. The results suggest that inulin is an excellent feedstock for Schizochytrium sp. suitable for commercial DHA production. Practical Applications: DHA is an essential nutrient for human health and is widely used in infant formula and functional food. As a reserve carbohydrate, inulin present in plants represents a cheap, abundant, and readily available bioresource. This study describes the suitability of inulin as a promising alternative to glucose for DHA production by Schizochytrium sp. Hence, a practical bioprocess for commercial DHA production from inulin by Schizochytrium sp. could be developed. As far as it is known, this is the first report of inulin as a feedstock for Schizochytrium sp. to produce DHA.     

Cost-cutting of nitrogen source for economical production of cellulolytic enzymes by Trichoderma inhamatum KSJ1

For saccharifying food wastes, cellulolytic enzymes were produced using Trichoderma inhamatum KSJ1 in modified Mandel’s medium. In a previous study, 0.1% bacto peptone in Mandel’s medium was established as the best organic nitrogen source for the production of cellulolytic enzymes using strain KSJ1. However, economically, peptone was too expensive. Therefore, soybean, yeast and Chunggookjang (fermented soybean paste) were substituted for peptone in this research. Also, yeast or ground soybean hydrolyzed by sulfuric acid or from a culture broth of Bacillus alcalophilus, a strain producing protease, was added to the medium as the nitrogen source to the production of cellulolytic enzyme. In the cultivation using 0.5% yeast hydrolyzed with a culture solution of B. alcalophilus as the nitrogen source, the activities of FPase and amylase were 0.20 and 2.17 U/mL in a 100 mL flask, compared to 0.35 and 1.24 U/mL with the 0.1% peptone as control, respectively. In a 10 L jar fermenter, the activities of FPase and amylase were improved to 0.40 and 4.82 U/mL in the cultivation, respectively, using 0.5% yeast hydrolyzed with the culture broth, compared with 0.38 and 3.79 U/mL, respectively, for the 0.1% peptone as control. Therefore, hydrolyzed yeast was established as an available nitrogen source for the industrial scale production of cellulolytic enzymes by strain KSJ1, resulting in a 52.3% cost reduction in the production of cellulolytic enzyme by substitution of the expensive nitrogen sources.     

The Delta 5,7-Sterols and Astaxanthin in the Marine Microheterotroph Schizochytrium sp. S31

The thraustochytrids, the heterotrophic marine microorganisms, have received increasing attention because of their capacity for the production of high-value products such as docosahexaenoic acid (DHA), carotenoids, and sterols. In this study, three potential Δ^5,7-sterols, showing similar UV absorption spectra to that of ergosterol, in the thraustochytrid Schizochytrium sp. S31, were estimated by the ultraperformance liquid chromatography (UPLC) − APCI−MS/MS method. The proposed structures of three Δ^5,7-sterols were ergosta-5,7,24(28)-trien-3β-ol, ergosta-5,7-dien-3β-ol, and 24-ethylcholesta-5,7,22-trien-3β-ol. The contents of astaxanthin, ergosterol, and the other three Δ^5,7-sterols in Schizochytrium sp. S31 during the cultivation performed in three separate batches were analyzed by a fast UPLC–PDA method. The results demonstrated that astaxanthin, ergosterol, and ergosta-5,7-dien-3β-ol were the predominant carotenoid and Δ^5,7-sterols, respectively, and their contents kept increasing during the growth and reached a maximum after 23 days of cultivation. The estimated maximum contents of astaxanthin and four Δ^5,7-sterols, including ergosterol, ergosta-5,7-dien-3β-ol, ergosta-5,7,24(28)-trien-3β-ol, and 24-ethylcholesta-5,7,22-trien-3β-ol were 2.31, 4.02, 3.43, 0.30, and 0.97 mg g⁻¹ dry biomass, respectively. These findings are different to other strains reported in the composition and accumulation of Δ^5,7-sterols and carotenoids. The present study suggests that Schizochytrium sp. S31 might be a promising marine microorganism for the productions of high-value natural pigment astaxanthin, the provitamin D₂ ergosterol, and the provitamin D₄ ergosta-5,7-dien-3β-ol, and taken as a model organism to study the sterologenesis of the microorganisms because of its complexity of sterol compositions.     

EPA and DHA in microalgae: Health benefits,biosynthesis, and metabolic engineering advances

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are ω-3 very long-chain polyunsaturated fatty acids (VLC-PUFAs) that offer a wide range of human health benefits impacting cardiovascular, anti-inflammatory, and neurological health. It is widely known that humans inefficiently synthesize these compounds and as such rely on exogenous dietary sources, such as marine fish oils. Unfortunately, the production of marine fish oils is an unsustainable process and has suffered a dramatic fall in recent years due to overfishing and climate change, as the demand for EPA and DHA continues to rise. Therefore, there is an urgent need to develop alternative, sustainable sources for consumable EPA and DHA. Metabolic engineering of marine microalgae to improve their EPA and DHA productivity is regarded as a promising option that has received increasing commercial attention in recent years. In this mini-review, we describe several notable health benefits of EPA and DHA, summarize the natural sources and biosynthesis of VLC-PUFAS, as well as the recent advances in metabolic engineering of EPA and DHA production in representative microalgal and protist species, including Schizochytrium sp., Phaeodactylum tricornutum, and Nannochloropsis oceanica.     

Production of docosahexaenoic and docosapentaenoic acids bySchizochytrium sp. isolated from Yap Islands

A marine microbe (strain SR21) from the coral reef area of the Yap Islands was isolated by a screening test for polyunsaturated fatty acids and was found to accumulate lipid that contained 22:5n-6 docosapentaenoic acid (DPA) as well as 22:6n-3 docosahexaenoic acid (DHA). Strain SR21 was identified as genusSchizochytrium in Labyrinthulomycota, owing to its ultrastructural character and life cycle, which is composed of vegetative cell, zoosporangium, and zoospore stages. After cultural optimization, both in flask and fermenter, the highest DHA and DPA productivities of 2.0 and 0.44 g/L per day, respectively, were obtained in a medium of 60 g/L glucose and corn steep liquor/ammonium sulfate in a half salt concentration of seawater in fermenter culture at 28°C and pH 4. This productivity was almost twice that obtained with flask culture, indicating its high resistance to mechanical stirring. The lipid extracted from the cell was about 50% of the dry cell weight and was composed of 93% triacylglycerol (TG). DHA content of the lipid was 34% of total fatty acids. The TG profile was simple, and the content of the most dominant TG, 1,3-dipalmitoyl-2-DHA-TG, was 27%. TG that contained DHA and n-6-DPA amounted to 57 and 17%, respectively, of total TG molecules. Strain SR21 was revealed to be an excellent source of microbial DHA and n-6 DPA.     

Utilization of cane molasses for docosahexaenoic acid production by Schizochytrium sp. CCTCC M209059

Cane molasses (CM), an agro-industrial by-product, was first examined for docosahexaenoic acid (DHA) production by Schizochytrium sp. Cell dry weight as 21.94 g/L at treated CM cultivation was similar to that at pure glucose (26.7 g/L) cultivation. Batch fermentation at different initial CM concentration showed that DHA percentage could reach 47.51% at 10 g/L CM but only 37.90% at 70 g/L CM. By analyzing the fermentation process, monosodium glutamate might be a positive agent for effective DHA production. Finally, monosodium glutamate and malic acid were introduced to the fed-batch fermentation for effective DHA production.     

Fatty acid production in Schizochytrium sp.: Involvement of a polyunsaturated fatty acid synthase and a type I fatty acid synthase

Schizochytrium sp. is a marine microalga that has been developed as a commercial source for docosahexaenoic acid (DHA, C22∶6 ω−3), enriched biomass, and oil. Previous work suggested that the DHA, as well as docosapentaenoic acid (DPA, C22∶5 ω−6), that accumulate in Schizochytrium are products of a multi-subunit polyunsaturated fatty acid (PUFA) synthase (1). Here we show data to support this view and also provide information of other aspects of fatty acid synthesis in this organism. Three genes encoding subunits of the PUFA synthase were isolated from genomic DNA and expressed in E. coli along with an essential accessory gene encoding a phosphopantetheinyl transferase (PPTase). The resulting transformants accumulated both DHA and DPA. The ratio of DHA to DPA was approximately the same as that observed in Schizochytrium. Treatment of Schizochytrium cells with certain levels of cerulenin resulted in inhibition of ¹⁴C acetate incorporation into short chain fatty acids without affecting labeling of PUFAs, indicating distinct biosynthetic pathways. A single large gene encoding the presumed short chain fatty acid synthase (FAS) was cloned and sequenced. Based on sequence homology and domain organization, the Schizochytrium FAS resembles a fusion of fungal FAS β and α subunits.     

Eicosapentaenoic acid and docosahexaenoic acid production potential of microalgae and their heterotrophic growth

Twenty microalgal strains were investigated in photoautotrophic flask cultures for their potential for eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) production. The highest EPA proportion (% of total fatty acids) was produced by Monodus subterraneus UTEX 151 (34.2%), followed by Chlorella minutissima UTEX 2341 (31.3%) and Phaeodactylum tricornutum UTEX 642 (21.4%). The highest DHA proportion (% of total fatty acids) was obtained in Crypthecodinium cohnii UTEX L1649 (19.9%), followed by Amphidinium carterae UTEX LB 1002 (17.0%) and Thraustochytrium aureum ATCC 28211 (16.1%). Among the 20 strains screened, the EPA yield was high in M. subterraneus UTEX 151 (96.3 mg/L), P. tricornutum UTEX 642 (43.4 mg/L), Chl. minutissima UTEX 2341 (36.7 mg/L), and Por. cruentum UTEX 161 (17.9 mg/L) owing to their relatively high biomass concentrations. The DHA yield was high in C. cohnii UTEX L1649 (19.5 mg/L) and A. carterae UTEX LB 1002 (8.6 mg/L). Heterotrophic growth of these 20 microalgae was also tested on two different carbon sources, acetate and glucose. All microalgae except Nannochloropsis oculata UTEX LB 2164 showed growth on glucose (5 g/L) under heterotrophic conditions. Twelve of them could grow heterotrophically when acetate (1 g/L) was used as their sole carbon and energy source.     

Fermentation strategy for the production of poly(3-hydroxyhexanoate) by <Emphasis Type="Italic">Aeromonas sp.</Emphasis> KC014

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(3HB-co-3HHx)) was produced by Aeromonas sp. KC014 strain isolated from Taiwan in soil environment in the flask and fermentor cultures. The medium optimization, such as carbon source and nitrogen source, carbon-nitrogen ratio was conducted to obtain the optimum 3-hydroxyhexanoate content. The defined medium with dodecanoic acid as the carbon source and (NH₄)₂SO₄ as the nitrogen source was obtained as the main medium. When cells grown in medium containing 30 g/L dodecanoic acid, 15 g/L sodium gluconate and 1 g/L soytone (C/N was 30/1) as final PHA concentration, the cell dry weight and HB content of 5.16 g/L, 14.0 g/L and 36.0%, respectively, were obtained. The maximum HHx/PHA content increased from 0.1% to 1.3% nearly 12-fold when the dissolved oxygen was decreased from 40% to 20%. P(3HB-co-3HHx) biosynthesis was triggered by the addition of limited nitrogen, phosphorus and magnesium to get a maximum HHx/PHA content of 14% in 95 hours. This work was presented at 13 ^th YABEC symposium held at Seoul, Korea, October 20–22, 2007.     

Thermal‐ and Photo‐Induced Isomerization of All‐E‐ and Z‐Isomer‐Rich Xanthophylls: Astaxanthin and Its Structurally‐Related Xanthophylls,Adonirubin, and Adonixanthin

The effects of heating and photo‐irradiation on the stability of all‐E‐isomer‐rich and Z‐isomer‐rich xanthophylls, astaxanthin and its structurally related xanthophylls, adonirubin, and adonixanthin, are investigated. The xanthophylls with high Z‐isomer content are prepared from their high‐purity all‐E‐isomers by thermal isomerization and filtering techniques, that is, total Z‐isomer ratios of adonirubin, astaxanthin, and adonixanthin are 80.9%, 89.5%, and 72.5%, respectively. The all‐E‐ and Z‐isomer‐rich xanthophylls dissolved in ethanol are stored at 4, 30, and 50 °C in the dark and at 30 °C under photo‐irradiation using a fluorescent light for 21 days. In the all‐E‐isomer‐rich xanthophylls, as the storage temperature increases, the total Z‐isomer ratio becomes higher, whereas in the Z‐isomer‐rich xanthophylls, the all‐E‐isomer ratio becomes higher. Photo‐irradiation slightly promotes Z‐isomerization in (all‐E)‐xanthophylls, but highly promotes all‐E‐isomerization in Z‐isomer‐rich xanthophylls. In addition, photo‐irradiation prevents thermal Z‐isomerization of (all‐E)‐xanthophylls. Moreover, it is found that some xanthophyll Z‐isomers such as (9Z)‐astaxanthin are more stable than that of the other Z‐isomers against heating and photo‐irradiation. These findings can contribute not only to establishing suitable storage conditions for Z‐isomer‐rich xanthophylls, but also to developing control techniques for the E/Z‐isomer ratio of the xanthophylls. Practical Applications: The fundamental data on the stability of xanthophyll isomers against heating and photo‐irradiation and finding stable xanthophyll Z‐isomers are very important to develop xanthophyll materials rich in the Z‐isomers. Moreover, this study clearly shows that the heat treatment enhances the Z‐isomerization of xanthophylls, whereas the photo‐irradiation enhances the all‐E‐isomerization and prevents thermal Z‐isomerization of them. This information can be utilized in technology for arbitrarily controlling E/Z‐isomerization of xanthophylls.     

Optimization of production of docosahexaenoic acid (DHA) byThraustochytrium aureum ATCC 34304

By varying culture carbon source, lipid content in mycelium ofThraustochytrium aureum ATCC 34304 varied widely in the range 1–25% of biomass weight. Docosahexaenoic acid (DHA) content of mycelium lipid was higher (65–76%) when biomass lipid content was very low (1–2%) and lower (40–50%) when biomass contained a high lipid content (14–18%). DHA yields from glucose, starch and maltose were 270, 325 and 334 mg/L, respectively. DHA yield and content of biomass was optimal at an initial culture pH of 6.0. During the culture cycle ofT. aureum, DHA content in lipids remained relatively constant with optimal DHA yield being observed after six days. Biomass, lipid content in biomass, DHA content in biomass and DHA yield were all optimal at a cultivation temperature of 28°C. However, the proportion of DHA in lipids declined with increase in temperature. Biomass, lipids in biomass and DHA yields were 13%, 42% and 47% higher, respectively, din light-exposed cultures as compared to dark cultures. A maximum yield of DHA of 511 mg/L was observed in light- exposed cultures containing 2.5% starch, where lipids accounted for over 20% of biomass dry weight. To whom correspondence should be addressed.     

Electron microscopy may reveal structure of docosahexaenoic acid-rich oil within Schizochytrium sp

Schizochytrium sp. is an algae-like microorganism utilized for commercial production of docosahexaenoic acid (DHA)-rich oil and dried microalgae for use as a source of DHA in foods, feeds, and nutritional supplements. Electron microscopic analysis of whole cells of Schizochytrium sp. employing sample preparation by high-pressure freeze substitution suggests the presence of secondary and tertiary semicrystalline structures of triacylglycerols within the oil bodies in Schizochytrium sp. A fine secondary structure consisting of alternating light-and darkstaining bands was observed inside the oil bodies. Dark bands were 29±1 Å in width, and light bands were 22±1 Å in width. The tertiary (three-dimensional) structure may be a multilayered ribbon-like structure which appears coiled and interlaced within the oil body. In freeze-fracture photomicrographs, Schizochytrium oil bodies exhibited fracture planes with terraces averaging 52±7 Å in height which could correspond to the combined width of two halves of two light bands and one dark band observed in the high-pressure freeze substitution photomicrographs. The results suggest that triacyglycerols within Schizochytrium sp. oil bodies may be organized in a triple chainlength structure. High-pressure freeze substitution electron micrographs of two other highly unsaturated oil-producing species of microalgae, Thraustochytrium sp. and Isochrysis galbana, also revealed this fine structure, whereas microalgae containing a higher proportion of saturated oil did not. The results suggest that the staining pattern is not an artifact of preparation and that the triple chain-length conformation of triacylglycerols in Schizochytrium sp. oil bodies may be caused by the unique fatty acid composition of the triacylglycerols.     

Studies on cultivation and biological activities ofPleurotus nebrodensis inzenga

Environmental factors affecting mycelial growth and exo-polysaccharide production fromPleurotus nebrodensis Inzenga (PN) and biological activities of PN extractsin vitro were studied. The culture conditions for effective mycelial growth and exo-polysaccharide production were found to be 25 ‡C, 5% of inoculum size, and an initial pH from 6.5 to 7.0. When 5% of glucose was used, the maximum mycelial growth and exo-polysaccharide concentrations were 8.3 and 3.07 g/L, respectively. Among the various nitrogen sources, the mycelial growth and exo-polysaccharide production were very strong when polypeptone was used. In the case of the minerals sources, K₂HPO₄ and MgSO₄·7H₂O were found to best support for mycelial growth and exo-polysaccharide production. Under optimal conditions and methods, the maximum mycelial growth and exo-polysaccharide production were obtained after 10 days of culture, 12.84 and 4.85 g/L, respectively, in a jar fermentor. The effects of the PN extracts on the viability of three human cancer cell lines and antioxidant activity were investigatedin vitro. When cancer cells of the lung (A549), cervical region (HeLa) and colon (KM12C) were incubated at 6 mg/mL of the PN ethanol extracts, the viabilities of the HeLa and KM12C cells were slightly decreased. However, the growth of the A549 cells was remarkably inhibited when the PN ethanol extract was over 4 mg/mL. The antioxidant activity showed 46.2% at 40 μL, which was about 3.2 fold higher than that of the PN methanol extract.     

Lipid and γ-linolenic acid accumulation in strains of zygomycetes growing on glucose

Strains of Zygomycetes, belonging to the genera Zygorhynchus, Mortierella, Rhizopus, Mucor, and Cunninghamella, when cultivated on glucose produced significant quantities of γ-linolenic acid (GLA). After exhaustion of the nitrogen source from the culture medium, all strains accumulated cellular lipids in concentrations ranging from 10 to 28% (oil/dry mycelium). However, in some strains after the depletion of the carbon source (glucose) from the culture medium, a reconsumption of the accumulated oil and synthesis of fat-free cell material was observed. Accumulation of large amounts of oil in the mycelium resulted in the production of oil with low GLA content. Rhizopus stolonifer strain LGAM (9)1, and Cunninghamella sp. strain LGAM (9)2 produced more than 30 mg GLA/g of dry cellular mass. Cunninghamella sp. accumulated 28.1% oil/dry cellular mass, which contained 11.9% GLA. The production of GLA was 260 mg/L of culture medium.     

Isolation and Characterization of a Docosahexaenoic Acid-Phospholipids Producing Microorganism Crypthecodinium sp. D31

Thirty-four strains of docosahexaenoic acid (DHA)-producing microorganisms were newly isolated from brackish areas in Japan. These strains showing various compositions of fatty acids. Especially, the fatty acids produced by one of the strains, named D31, had a high DHA content (over 60 % of the total fatty acids) and the simple fatty acid composition (16:0, 18:0, 18:1 and DHA without any other polyunsaturated acids). Although most oleaginous microorganisms accumulate DHA as triacylglycerol, the strain D31 accumulated DHA mainly as a polar lipid (79.4 % of total DHA), especially as phosphatidylcholine (71.4 % of polar DHA). This strain D31 was identified as a related species of Crypthecodinium cohnii on the basis of phylogenetic analysis. Crypthecodinium sp. D31 showed high DHA productivity when cultivated in a medium containing glycerol as the carbon source and a mixture of yeast extract and polypeptone as the nitrogen sources, with a salinity that was equivalent to 50 % of that of seawater and a pH in the acidic range (<pH 6.0). Crypthecodinium sp. D31 is considered as a promising producer of high-purity DHA-containing phospholipids.     

Production of eicosapentaenoic acid bySaprolegnia sp. 28YTF-1

Saprolegnia sp. 28YTF-1, isolated from a freshwater sample, is a potent producer of 5,8,11,14,17-cis-eicosapentaenoic acid (EPA). The fungus used various kinds of carbon sources, such as starch, dextrin, sucrose, glucose, and olive oil for growth, and olive oil was the best carbon source for EPA production. The EPA content reached 17 mg/g dry mycelium (0.25 mg/L) when the fungus was grown in a medium that contained 2.5% olive oil and 0.5% yeast extract, at pH 6.0 and 28°C for 6 d with shaking. Accompanying production of arachidonic acid (AA; 3.2 mg/g dry mycelia, EPA/AA = 5.1) and other ω6 polyunsaturated fatty acids was low. Both EPA content and EPA/AA ratio increased in parallel by lowering growth temperature. Triglyceride was the major mycelial lipid (ca. 84%), but EPA comprised only 2.2% of the total fatty acids of this lipid. About 40% of the EPA produced was found in polar lipids, such as phosphatidylethanolamine (EPA content, 28.2%), phosphatidylcholine (13.6%), and phosphatidylserine (21.2%).     

Production of docosahexaenoic acid by Crypthecodinium cohnii grown in a pH-auxostat culture with acetic acid as principal carbon source

Crypthecodinium cohnii, a marine alga used for the commercial production of docosahexaenoic acid (DHA), was cultivated in medium containing sodium acetate as principal carbon source; the pH was maintained at a constant value by addition of acetic acid, which also provided an additional carbon source in a controlled manner. The accumulation of lipid by C. cohnii in this pH-auxostat culture was significantly greater than previously reported for batch cultures using glucose as principal carbon source. Of six strains tested in pH-auxostat cultures, C. cohnii ATCC 30772 was the best, with the cells reaching 20 to 30 g dry weight per liter after 98 to 144 h and containing in excess of 40% (w/w) total lipid, with DHA representing approximately half of the total fatty acids in the triacylglycerol fraction. A productivity of 36 mg DHAL⁻¹ h⁻¹ was achieved during cultivation for 98 h using a 5% (vol/vol) inoculum, and DHA production was in excess of 3 g per liter of culture. Most of the DHA was present in neutral lipids.     

Enhanced production of cellobiose dehydrogenase and β-glucosidase by Phanerochaete chrysosporium

The production of cellobiose dehydrogenase (CDH) and β-glucosidase by Phanerochaete chrysosporium ATCC 32629 was assessed during submerged fermentation. The maximum concentrations of CDH and β-glucosidase were obtained using rice straw as the carbon source. Organic nitrogen sources were more effective in enzyme production than inorganic nitrogen sources. Corn steep liquor (CSL) for CDH production and soy bean meal (SBM) for β-glucosidase production were the most appropriate organic nitrogen sources. Using optimum medium obtained by response surface methodology (RSM), the maximum concentrations of CDH and β-glucosidase achieved in the stirred-tank reactor (STR) were 204 U/L and 140 U/L, respectively. CDH productivity (22.7 U/L·day) was the highest at 9 days.     

Optimization of arachidonic acid production by Mortierella alpina Wuji-H4 isolate

A fungal isolate Wuji-H4 with a dense-lobe rosette growth pattern on malt extract agar was identified as Mortierella alpina Peyronel. It was capable of producing 504 mg/L of arachidonic acid (AA) in the screening medium. Its AA content accounted for 42.4% of the total fatty acids. The AA yield was raised to 1,817 mg/L by a step-by-step approach, which uncovered that the preferred carbon source, nitrogen source, and temperature for fungal growth and lipid production were soluble starch, urea, and 24°C, respectively. Productivity was further optimized by exploiting the interactions between the constituents of the medium by the response surface method. A partial factorial design, followed by steepest ascent analysis, was carried out to locate the general vicinity of the optimal level of each nutrient. The response surface of AA production in this optimal region was then approximated with a full quadratic equation obtained from a three-factor/five-level central composite rotatable design. Maximum AA yield was predicted to occur in a medium that contained 99.7 g/L of soluble starch, 12.6 g/L of yeast extract, and 3.0 g/L of KH₂PO₄. Upon verification, the average experimental yield of AA (3,885 mg/L) was not significantly different from the predicted AA yield (3,940 mg/L), indicating that the response surface method had succeeded in exploiting the AA production potential of this new fungal isolate.