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Aurantiochytrium sp. is a heterotrophic microorganism that accumulates high amounts of fatty acids (FAs), including odd-chain FAs and an essential FA, docosahexaenoic acid (DHA). Since complex nutrient sources such as tryptone and yeast extract are conventionally utilized in the growth medium for Aurantiochytrium, it is difficult to investigate which individual vitamins affect growth and FA production. Thus, in the present study, we simplified the nitrogen source and defined the vitamins required for Aurantiochytrium sp. cultivation. Glutamate was found to be the most effective simple nitrogen source in cultivation media and thiamine (vitamin B1) was identified as a unique essential vitamin for this organism. Media supplemented with thiamine, as the only member of the vitamin B complex added, allowed for propagation of four strains of Aurantiochytrium. The ratio of odd-chain FAs significantly decreased in the medium containing glucose, glutamate, and thiamine when compared with the conventional medium containing glucose, tryptone, and yeast extract. This is likely caused by a decreased availability of branched-chain amino acids. Under thiamine starvation, FA content per dry cell weight gradually decreased throughout cultivation, and the specific activity of pyruvate dehydrogenase (PDH), a thiamine-dependent enzyme, was severely repressed. These results indicate that when thiamine is absent, the cells compensate some amount of acetyl-CoA through β-oxidation of FAs in vivo, instead of from the reaction by pyruvate, as occurs under normal growth conditions. Providing experimental evidence that the activity of vitamin-dependent enzymes affects biosynthesis of FAs is critical to a better understanding of the effect of vitamin supplementation in Aurantiochytrium sp. media. In the present study, we demonstrated the close relationship between PDH activity, FA content and the importance of thiamine supplementation in the culture medium in Aurantiochytrium sp.

Defatted heterotrophic microalgae (Aurantiochytrium limacinum SR21) was treated with high temperature water (175-350°C, 10-90min) to obtain nitrogen and phosphorous nutrients as a water soluble fraction (WS). Yields of nitrogen and phosphorous recovered in WS varied from 38 to 100% and from 57 to 99%, respectively. Maximum yields of nitrogen containing compounds in WS were proteins (43%), amino acids (12%) and ammonia (60%) at treatment temperatures of 175, 250 and 350°C, respectively. Maximum yield of phosphorous in WS was 99% at a treatment temperature of 250°C. Cultivation experiments of microalgae (A. limacinum SR21) using WS obtained at 200 and 250°C showed positive growth. Water soluble fractions from hydrothermal treatment of defatted microalgae are effective nitrogen and phosphorous nutrient sources for microalgae cultivation.

Coupling of hydrothermal treatment methods with microalgae cultivation within a municipal wastewater treatment process can allow internal recycle of chemicals to improve overall waste treatment efficiency and provide biofuel as by-product. In this work, municipal sludge was treated with high temperature water in two steps to selectively recover nutrients and cellulose-rich fractions to avoid Maillard reactions. In the first step, nitrogen and phosphorous compounds were recovered in the liquid product and cellulose was recovered in the solid residue. In the second step, the cellulose-rich solid residue (ca. 60 wt%), was treated with sulfuric acid to obtain glucose. Cultivation of a mixotrophic microalgae (Euglena gracilis) and a heterotrophic microalgae (Aurantiochytrium) with the liquid product and the isolated glucose product gave positive growth indicating that the proposed two-step hydrothermal liquefaction process allows production of oils and recycle of nutrients such as sugars, nitrogen and phosphorous derived from municipal sludge. Hydrothermal treatment is an effective solid reduction technique and is a feasible method for recycling nutrients for microalgae cultivation.

Microalgal lipids and fatty acids are important components for achieving biofuel because of their potential high productivity. Although fatty acids that have a hydroxy group adjacent to the end of the acyl chain might be an important chemical feedstock, most algae do not accumulate it. To produce (ω−1)-hydroxy fatty acids from 3-hydroxybutylyl-CoA, an intermediate for polyhydroxybutylate biosynthesis in the cyanobacterium Synechocystis sp. PCC 6803, we expressed a gene for the promiscuous 3-ketoacyl-ACP synthase III from Alicyclobacillus acidocalderius (aaKASIII) by the cpc560 promoter. To supply 3-hydroxybutyryl-CoAs for aaKASIII, the phaC gene for polyhydroxybutylate polymerase was deleted, and the phaAB genes for 3-hydroxybutyryl-CoA synthesis from Cupriavidus necator were overexpressed. The genetically modified strain synthesized 15-hydroxyhexadecanoic acid, 17-hydroxyoctadecanoic acid, and 17-hydroxyoctadec-9-enoic acid, and accumulated approximately 2.1 mol% of (ω−1)-hydroxy fatty acids in total fatty acids under illumination with 70 μmol photons m−2 s−1, although its growth was severely retarded. Under weak light (35 μmol photons m−2 s−1) conditions, the strain grew as well as the wild-type and showed lower hydroxy fatty acids (0.04 mol%) accumulation than that at higher illumination levels. The photosynthetic activity of this strain was lower than that of wild-type cells, suggesting that high light conditions enhanced hydroxy fatty acids production and inhibited photosynthesis. (ω−1)-Hydroxy fatty acids were not predominantly observed in the galactolipids from thin-layer chromatography, which are the major lipid classes in cyanobacteria. To the best of our knowledge, this is the first report on photoautotrophic production of fatty acids possessing a functional group near the end of the acyl chain in cyanobacteria.

Cyanobacterium is an attractive host for the production of various chemicals and alternative fuels using solar energy and carbon dioxide. In previous study, we succeeded to produce isopropanol using engineered Synechococcus elongatus PCC 7942 under dark and anaerobic conditions (0.43 mM, 26.5 mg/l). In the present study, we report the further optimization of this isopropanol producing condition. We then optimized growth conditions for production of isopropanol by the engineered cyanobacteria, including the use of cells in early stationary phase and buffering of the production medium to neutral pH. We observed that shifting of cultures from dark and anaerobic to light and aerobic conditions during the production phase dramatically increased isopropanol production by conversion to isopropanol from acetate, byproduct under dark and anaerobic condition. Under the optimized production conditions, the titer of isopropanol was elevated 6-fold, to 2.42 mM (146 mg/l).

The hydrocarbons in cultures of marine haptophytes Emiliania huxleyi NIES837 and Gephyrocapsa oceanica NIES1315 were analyzed, and nonacosadienes and hentriacontadienes were detected as the major compounds in both strains. C29 and C31 monoenes and di-, tri- and tetra-unsaturated C33 alkenes were also detected as minor compounds but not C37 and C38 alkenes. The positions of the double bonds in the C29 and C31 alkenes were determined by mass spectrometry of their dimethyl disulfide (DMDS) adducts. Among the four C29 alkenes identified, the most abundant isomer was 2,20-nonacosadiene, and the other three compounds were 1,20-nonacosadiene, 3,20-nonacosadiene and 9-nonacosene, respectively. Hitherto, 2,20-nonacosadiene and 3,20-nonacosadiene were unknown to be natural products. The double bond at the n-9 (ω9) position in these C29 alkenes is hypothesized to be derived from precursors of unsaturated fatty acids possessing an n-9 double bond, such as (9Z)-9-octadecenoic acid. Nonacosadienes have the potential for being used as distinct haptophyte biomarkers.

Thraustochytrids are osmoheterotrophic protists that are emerging as a promising group of oleaginous microorganisms due to their high DHA, EPA and palmitic acid production. Squalene, a triterpene hydrocarbon known for its antioxidant, antitumorigenic and emollient properties, is accumulated in some strains of thraustochytrids especially in the genus Aurantiochytrium. Thraustochytrids can produce extracellular enzymes to degrade organic matter as their means of nutrition. Many studies attempt to grow thraustochytrids in cost-effective substrates such as lignocellulosic biomass. As the first step towards cellulose utilization, we genetically modified Aurantiochytrium sp. strain 18W-13a to express and secrete a highly active cellulase, β-glucosidase from Aspergillus aculeatus (AaBgl). The transformant strain, AaBgl+, exhibited increasing growth and enzyme activity under cellobiose as the sole carbon source. Native PAGE zymogram showed an active β-glucosidase enzyme in the supernatant of the AaBgl+ strain. Both wild-type and AaBgl+ strains can produce squalene and fatty acids DHA and DPA. This work is one of the first reports in targeted secretion of a functional enzyme in thraustochytrids. Moreover, enhanced expression of cellulases has never been done in thraustochytrids. We hope that this study could pave way for establishing consolidated bioprocessing thraustochytrid strains.

The compositions of long chain alkenes, alkenones and alkenoates in a cultured strain of the haptophyte Chrysotila lamellosa CCMP1307, isolated from a salt marsh, were investigated. The biomarker patterns were distinctive and showed a high proportion of tetraunsaturated alkenones and alkenoates, with a pronounced proportion of C 40 alkenones and a lack of C 38 methyl and C 39 ethyl alkenones. Linear regression of the alkenone unsaturation degree ( U 37 K ) with growth temperature ( T ) was obtained over the range of possible CCMP1307 growth temperature values (4–20 °C): U 37 K = 0.045 × T (°C) − 1.016, ( n = 13, r 2 0.96), while the U 37 K ′ values were weakly correlated with T : U 37 K ′ = 0.0035 T (°C) + 0.0511 ( n = 18, r 2 0.70). The U 37 K calibration exhibited a low y-intercept in comparison with that of a Chinese inland lake strain reported previously. The data show significant intraspecific variation in U 37 K for C. lamellosa between strains from different geographic origins. C. lamellosa CCMP1307 reproduced typical U 37 K values observed in C 37:4 rich lakes, especially at lower temperature ( 29:2 , C 31:1 , C 31:2 and C 31:3 alkenes, and found that the unsaturation degree of C 31 alkenes ( U 31 en ), calculated with C 31:1 and C 31:2 alkenes, might also be a useful index of growth temperature for the haptophyte C. lamellosa .

Optimization of the light conditions for biofuel production by the microalga Botryococcus braunii BOT-22 (race B) was performed using monochromatic red light. The lipid and sugar contents were approximately 40% and 20-30% of the cell dry weight, respectively, and about half of the lipids were liquid hydrocarbons. The half-saturation intensities for the production rate of lipids, hydrocarbons, and sugars were 63, 49, and 44μmolm(-2)s(-1), respectively. Fluorescence microscopic images of Nile Red-stained cells showed an increased number of intracellular neutral lipid granules due to increased light intensity. After 16days of incubation in the dark, lipid and sugar, but not hydrocarbon content decreased. Growth, metabolite production, and photosynthesis were saturated at 100, 200 and 1000μmolm(-2)s(-1), respectively. These results indicate that photosynthetically captured energy is not used efficiently for metabolite production; thus, improvements in metabolic regulation may increase hydrocarbon production.

Long-chain n-3 fatty acids can lower the risk of lifestyle-related diseases, therefore, we introduced a plant fatty acid desaturation3 (FAD3) gene into mammalian cells. The FAD3 cDNA was isolated from the immature seeds of scarlet flax and optimized to human high-frequency codon usage for enhancement of its expression levels in mammalian cells (hFAD3). We introduced the gene into bovine muscle satellite cells, which can be differentiated into multilocular adipocytes in vitro. After hFAD3 transfection, the cells were differentiated into adipocytes and their fatty acid composition was analyzed by gas chromatography. The level of alpha-linolenic acid (18:3n-3) in transfected adipocytes increased about ten-fold compared with non-transfected adipocytes. In addition, the levels of docosapentaenoic acid (DPA, 22:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) in transfected adipocytes were significantly higher than those in non-transfected adipocytes. Moreover, we produced bovine cloned embryos from the hFAD3 cells by somatic cell nuclear transfer. Blastocyst rates of hFAD3 clones were the same as the control clones using the non-transfected cells (21% vs 27%, P > 0.05). hFAD3 transcripts were detected in all of the blastocysts. These results demonstrate the functional expression of a plant hFAD3 in mammalian adipocytes, and normal development of cloned embryos carrying the hFAD3 gene.

In previous studies, we characterized five histidine kinases (Hiks) and the cognate response regulators (Rres) that control the expression of approximately 70% of the hyperosmotic stress-inducible genes in the cyanobacterium Synechocystis sp. PCC 6803. In the present study, we screened a gene knock-out library of Rres by RNA slot-blot hybridization and with a genome-wide DNA microarray and identified three Hik-Rre systems, namely, Hik33-Rre31, Hik10-Rre3, and Hik16-Hik41-Rre17, as well as another system that included Rre1, that were involved in perception of salt stress and transduction of the signal. We found that these Hik-Rre systems were identical to those that were involved in perception and transduction of the hyperosmotic stress signal. We compared the induction factors of the salt stress- and hyperosmotic stress-inducible genes that are located downstream of each system and found that these genes responded to the two kinds of stress to different respective extents. In addition, the Hik33-Rre31 system regulated the expression of genes that were specifically induced by hyperosmotic stress, whereas the system that included Rre1 regulated the expression of one or two genes that were specifically induced either by salt stress or by hyperosmotic stress. Our observations suggest that the perception of salt and hyperosmotic stress by the Hik-Rre systems is complex and that salt stress and hyperosmotic stress are perceived as distinct signals by the Hik-Rre systems.

Microorganisms respond to hyperosmotic stress via changes in the levels of expression of large numbers of genes. Such responses are essential for acclimation to a new osmotic environment. To identify factors involved in the perception and transduction of signals caused by hyperosmotic stress, we examined the response of Synechocystis sp. PCC 6803, which has proven to be a particularly useful microorganism in similar analyses. We screened knockout libraries of histidine kinases (Hiks) and response regulators (Rres) in Synechocystis by DNA microarray and slot-blot hybridization analyses, and we identified several two-component systems, which we designated Hik-Rre systems, namely, Hik33-Rre31, Hik34-Rre1, and Hik10-Rre3, as well as Hik16-Hik41-Rre17, as the transducers of hyperosmotic stress. We also identified Hik2-Rre1 as a putative additional two-component system. Each individual two-component system regulated the transcription of a specific group of genes that were responsive to hyperosmotic stress.