Degradation of Distillery Lees (Shochu kasu) by Cellulase-Producing Thraustochytrids.

Single cell oils produced by oleaginous microorganisms have attracted increasing interests as a petroleum alternative energy. Marine eukaryotes, thraustochytrids were heterotrophic, and can grow rapidly and accumulate large amount of lipids containing functional fatty acids, such as docosahexaenoic acid (DHA) in their cells body. In this investigation, thraustochytrids isolated from marine environment were cultured in the medium containing an industrial waste and an unused resource, distillery lees (Shochu kasu) to produce biofuel or functional fatty acids by microorganisms. Sixty-nine thraustochytrids and Schizochytrium aggregatum ATCC 28209 were screened for cellulase production, and the activities were detected using sodium carboxymethyl cellulose (CMC) as a substrate. Based on the screening test, strain TM02Bc identified to Schizochytrium sp. was selected for the Shochu kasu degradation test and compared with S. aggregatum ATCC 28209 previously known as a cellulase-producing thraustochytrid. Strains TM02Bc and ATCC 28209 were cultured in artificial seawater containing Shochu kasu for 15 days. The two strains could degrade Schochu kasu, especially that from sweet potato Shochu (Imo Shochu). Cellulase (CMCase) and protease activities were detected in culture supernatant of both strains, and the ratio of polyunsaturated fatty acids (PUFAs) significantly increased as a result of incubation of Shochu kasu with two strains. This preliminary study indicated that strain TM02Bc was a potent candidate for Shochu kasu treatment and fatty acid production.

Effect of trace elements on growth of marine eukaryotes, tharaustochytrids.

We determined the effect of trace elements on the growth of thraustochytrids. The growth of the strains cultured with the trace elements was much higher than that of the strains cultured without any trace element. Iron and zinc were particularly important to obtaining the optimum growth of thraustochytrids.

Versatile transformation system that is applicable to both multiple transgene expression and gene targeting for Thraustochytrids.

A versatile transformation system for thraustochytrids, a promising producer for polyunsaturated fatty acids and fatty acid-derived fuels, was established. G418, hygromycin B, blasticidin, and zeocin inhibited the growth of thraustochytrids, indicating that multiple selectable marker genes could be used in the transformation system. A neomycin resistance gene (neo(r)), driven with an ubiquitin or an EF-1α promoter-terminator from Thraustochytrium aureum ATCC 34304, was introduced into representatives of two thraustochytrid genera, Aurantiochytrium and Thraustochytrium. The neo(r) marker was integrated into the chromosomal DNA by random recombination and then functionally translated into neo(r) mRNA. Additionally, we confirmed that another two genera, Parietichytrium and Schizochytrium, could be transformed by the same method. By this method, the enhanced green fluorescent protein was functionally expressed in thraustochytrids. Meanwhile, T. aureum ATCC 34304 could be transformed by two 18S ribosomal DNA-targeting vectors, designed to cause single- or double-crossover homologous recombination. Finally, the fatty acid Δ5 desaturase gene was disrupted by double-crossover homologous recombination in T. aureum ATCC 34304, resulting in an increase of dihomo-γ-linolenic acid (C(20:3n-6)) and eicosatetraenoic acid (C(20:4n-3)), substrates for Δ5 desaturase, and a decrease of arachidonic acid (C(20:4n-6)) and eicosapentaenoic acid (C(20:5n-3)), products for the enzyme. These results clearly indicate that a versatile transformation system which could be applicable to both multiple transgene expression and gene targeting was established for thraustochytrids.

Detection of genes involved in fatty acid elongation and desaturation in thraustochytrid marine eukaryotes.

Heterotrophic marine protists known as thraustochytrids can synthesize polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA). The biosynthetic pathways of PUFAs in thraustochytrids are poorly understood, however. In this study, we attempted to reveal the enzymes involved in DHA synthesis in thraustochytrids. Nine thraustochytrid strains representing 3 genera (Aurantiochytrium, Schizochytrium, and Thraustochytrium) were used for PCR-based detection of the genes encoding Δ5-elongase and Δ4-desaturase and for fatty acid analysis. The degenerate primers were designed to amplify the Δ5-elongase and Δ4-desaturase genes, and the partial sequences of the enzymes were obtained from the genera Thraustochytrium and Schizochytrium. These fragments were identical to those of known Δ5-elongase and Δ4-desaturase. Neither Δ5-elongase nor Δ4-desaturase was detected in the strains belonging to the genus Aurantiochytrium, however, suggesting that this group likely synthesizes DHA not via the elongation/desaturation pathway but via an alternate pathway such as the polyketide synthase pathway. The fatty acid profiles of thraustochytrids were consistent with the presence of genes involved in PUFA biosynthesis in thraustochytrid genera. Thus, our findings suggest that two biosynthetic pathways for PUFAs exist in these organisms.

Effects of cold shock treatment on total lipid content and fatty acid composition of Aurantiochytrium limacinum strain mh0186.

To examine the effect of cold shock treatment on the fatty acid composition of Aurantiochytrium limacinum strain mh0186, a marine thraustochytrid, we cultivated this strain at 28°C for 72 h with shaking and stored the obtained biomass at 10°C for 72 h. A growth experiment was carried out for comparison, wherein strain mh0186 was grown at 10 and 15°C for 72 h with shaking, and it was found that the unsaturation of fatty acids was accelerated relative to that at 28°C. In the cold shock experiment, the total lipid content significantly increased during storage at 10°C for 72 h. Overall, the percentage of unsaturated fatty acids such as docosahexaenoic acid was almost stable while that of n-6 docosapentaenoic acid decreased slightly, but significantly, relative to that in the growth experiment.

Effect of Tween 80 on the growth, lipid accumulation and fatty acid composition of Thraustochytrium aureum ATCC 34304.

Thraustochytrium aureum ATCC 34304 was grown in the presence and absence of polyoxyethylene sorbitan monooleate (Tween 80). The aim of this work was to obtain basic knowledge about the effect of Tween 80 on growth, lipid accumulation and fatty acid composition in T. aureum. The addition of Tween 80 to a culture medium significantly enhanced the growth of T. aureum, and the biomass increased with an increase of Tween 80 content. Total lipid content and total fatty acid content were significantly higher in 1.0% Tween 80 in comparison with the control (absence of Tween 80). The fatty acid profile showed that the content of C18:1n-9 (oleic acid) significantly increased as a result of the addition of Tween 80. These results indicated that part of the Tween 80 added to the medium was utilized as a carbon source or that the oleate included in Tween 80 was directly incorporated into T. aureum cells as a fatty acid. Neither the DHA content nor the percentage of DHA did not change in spite of the addition of Tween 80. However, the DHA yield significantly increased because the biomass increased due to the addition of Tween 80.

The distribution of extracellular cellulase activity in marine Eukaryotes, thraustochytrids.

Cellulolytic ability was evaluated in 19 strains of thraustochytrids, representing nine genera, using carboxymethylcellulose (CMC) as a substrate. Extracellular cellulolytic enzyme activity was determined in the culture supernatants during cell growth. CMC hydrolysis was observed in 14 out of the 19 strains examined. These belonged to the genera Aplanochytrium, Botryochytrium, Oblongichytrium, Parietichytrium, Schizochytrium, Sicyoidochytrium, Thraustochytrium and Ulkenia. On the other hand, cellulolytic enzyme activity was not detected in any strains belonging to the genus Aurantiochytrium.

Use of an antifungal drug, amphotericin B for isolation of thraustochytrids.

The inhibitory effect of amphotericin B (AMPH) on the growth of fungi during the isolation of thraustochytrids was examined. The growth of fungi was significantly inhibited by addition of AMPH, and therefore colonies of thraustochytrids were not overlaid with fungal mycelia, which resulted in increased efficiency of thraustochytrids isolation.

Optimization of culture conditions for growth and docosahexaenoic acid production by a marine thraustochytrid, Aurantiochytrium limacinum mh0186.

The effects of carbon sources, seawater concentration and seawater component in a culture medium were investigated to optimize culture conditions for growth by a marine thraustochytrid Aurantiochytrium limacinum strain mh0186. Strain mh0186 could utilize D-glucose, D-fructose and D-mannose as carbon sources. Seawater concentrations between 12.5 - 200% were required for good growth, and a single omission of magnesium sulfate from the seawater reduced the growth of the cells. Jar fermentor trials were carried out for the purpose of docosahexaenoic acid (DHA) production by strain mh0186. The total fatty acid content of the cell was 466.5 mg/g dry cells, and biomass and DHA yield were estimated as 23.1 g/L and 4.3 g/L, respectively, at 26 h. The daily production of DHA by the strain was 4.0 g/L/d, suggesting that the higher DHA production rate of our strain mh0186 should be appropriate for industrial production of DHA.

Extracellular enzymes produced by marine eukaryotes, thraustochytrids.

Extracellular enzymes produced by six strains of thraustochytrids, Thraustochytrium, Schizochytrium, and Aurantiochytrium, were investigated. These strains produced 5 to 8 kinds of the extracellular enzymes, depending on the species. Only the genus Thraustochytrium produced amylase. When insoluble cellulose was used as substrate, cellulase was not detected in the six strains of thraustochytrids. This study indicates that marine eukaryotes, thraustochytrids, produced a wide variety of extracellular enzymes.

Influences of culture temperature on the growth, lipid content and fatty acid composition of Aurantiochytrium sp. Strain mh0186.

The growth, lipid content, and fatty acid composition of Aurantiochytrium sp. strain mh0186 at different temperatures were investigated. Strain mh0186 grew well at 15-30 degrees C, but weakly at 10 degrees C. The biomass at 15-30 degrees C was significantly higher than at 10 and 35 degrees C, and the total lipid at 15-35 degrees C was significantly higher than that at 10 degrees C. The amount of DHA in the total fatty acid was highest at 10 degrees C and decreased in response to temperature increase. The content of DHA (mg/g-dry cell weight) at 15-30 degrees C were significantly higher than those at 35 degrees C and those at 15-25 degrees C were significantly higher than those at 10 and 35 degrees C. The DHA yield at 15-35 degrees C was significantly higher than those at 10 and 35 degrees C. Unsaturation of fatty acid was regulated by temperature and was enhanced in response to temperature decrease. The ratio of DHA to DPA varied at different temperatures.

Effect of addition of Tween 80 and potassium dihydrogenphosphate to basal medium on the isolation of marine eukaryotes, thraustochytrids.

Tween 80, KH(2)PO(4) and tomato juice were added to basal medium for the isolation of thraustochytrids. By the addition of Tween 80 and KH(2)PO(4), the number of thraustochytrids isolated from seawater increased. KH(2)PO(4) and Tween 80 were considered to be useful for isolating thraustochytrids.