Culturable diversity and biochemical features of thraustochytrids from coastal waters of Southern China

Thraustochytrids are ubiquitous marine osmo-heterotrophic fungi-like microorganisms with only about 40 identified species till now. In this study, a total of 60 thraustochytrid strains were isolated from marine coastal habitats. Analysis of 18S rRNA gene sequences revealed that they belonged to three genera, i.e., Schizochytrium, Aurantiochytrium, and Thraustochytrium. All of the isolates were found to show considerable cellulolytic and lipolytic activities. Strains of Aurantiochytrium sp. and Thraustochytrium sp. were found to produce the highest levels of extracellular polysaccharides (EPS), which reached 345 μg ml^?1 in the growth media. Fourier transform infrared (FTIR) spectra of the EPS samples derived from two thraustochytrids (PKU#Sed1 and #SW1) displayed peaks for carbohydrates, proteins, lipids, uronic acids, and nucleic acids. Fatty acid profiles of four thraustochytrids comprised of palmitic acid (C16:0) and docosahexaenoic acid (DHA) as their major constituents. Schizochytrium sp. demonstrated the highest DHA production at 44 % of total fatty acids (TFA) with biomass and DHA yield of 7.1 and 1.6 g l^?1, respectively, on the fourth day of growth. All the four isolates exhibited considerable production of palmitic acid (16:0) in their fatty acid profiles ranging from 35 to 50 % TFA. This is the first report on extracellular enzymes, EPS, and DHA production from thraustochytrids isolated from the coastal habitats of China.     

Comparison of Thraustochytrids Aurantiochytrium sp., Schizochytrium sp., Thraustochytrium sp., and Ulkenia sp. for Production of Biodiesel,Long-Chain Omega-3 Oils,and Exopolysaccharide

Heterotrophic growth of thraustochytrids has potential in coproducing biodiesel for transportation, as well as producing a feedstock for omega-3 long-chain (≥C20) polyunsaturated fatty acids (LC-PUFA), especially docosahexaenoic acid (DHA) for use in nutraceuticals. In this study, we compared eight new endemic Australian thraustochytrid strains from the genera Aurantiochytrium, Schizochytrium, Thraustochytrium, and Ulkenia for the synthesis of exopolysaccharide (EPS), in addition to biodiesel and LC-PUFA. Aurantiochytrium sp. strains readily utilized glucose for biomass production, and increasing glucose from 2 to 4 % w/v of the culture medium resulted in increased biomass yield by an average factor of 1.7. Ulkenia sp. strain TC 010 and Thraustochytrium sp. strain TC 033 did not utilize glucose, while Schizochytrium sp. strain TC 002 utilized less than half the glucose available by day 14, and Thraustochytrium sp. strain TC 004 utilized glucose at 4 % w/v but not 2 % w/v of the culture suggesting a threshold requirement between these values. Across all strains, increasing glucose from 2 to 4 % w/v of the culture medium resulted in increased total fatty acid methyl ester content by an average factor of 1.9. Despite an increasing literature demonstrating the capacity of thraustochytrids for DHA synthesis, the production of EPS from these organisms is not well documented. A broad range of EPS yields was observed. The maximum yield of EPS was observed for Schizochytrium sp. strain TC 002 (299 mg/L). High biomass-producing strains that also have high lipid and high EPS yield may be better candidates for commercial production of biofuels and other coproducts.     

Effects of molten-salt/ionic-liquid mixture on extraction of docosahexaenoic acid (DHA)-rich lipids from Aurantiochytrium sp. KRS101

In this study, lipid extraction from Aurantiochytrium sp. was performed using a molten-salt/ionic-liquid mixture. The total fatty acid content of Aurantiochytrium sp. was 478.8 mg/g cell, from which 145 mg/g cell (30.3 % of total fatty acids) of docosahexaenoic acid (DHA) was obtained. FeCl₃·6H₂O showed a high lipid extraction yield (207.9 mg/g cell), when compared with that of [Emim]OAc, which was only 118.1 mg/g cell; notably however, when FeCl₃·6H₂O was mixed with [Emim]OAc (5:1, w/w), the yield was increased to 478.6 mg/g cell. When lipid was extracted by the FeCl₃·6H₂O/[Emim]OAc mixture at a 5:1 (w/w) blending ratio under 90 °C, 30 min reaction conditions, the fatty acid content of the extracted lipid was a high purity 997.7 mg/g lipid, with most of the DHA having been extracted (30.2 % of total fatty acids). Overall, lipid extraction from Aurantiochytrium sp. was enhanced by the synergistic effects of the molten-salt/ionic-liquid mixture with different ions.     

High cell density cultivation of a novel Aurantiochytrium sp. strain TC 20 in a fed-batch system using glycerol to produce feedstock for biodiesel and omega-3 oils

A recently isolated Australian Aurantiochytrium sp. strain TC 20 was investigated using small-scale (2 L) bioreactors for the potential of co-producing biodiesel and high-value omega-3 long-chain polyunsaturated fatty acids. Higher initial glucose concentration (100 g/L compared to 40 g/L) did not result in markedly different biomass (48 g/L) or fatty acid (12–14 g/L) yields by 69 h. This comparison suggests factors other than carbon source were limiting biomass production. The effect of both glucose and glycerol as carbon sources for Aurantiochytrium sp. strain TC 20 was evaluated in a fed-batch process. Both glucose and glycerol resulted in similar biomass yields (57 and 56 g/L, respectively) by 69 h. The agro-industrial waste from biodiesel production—glycerol—is a suitable carbon source for Aurantiochytrium sp. strain TC 20. Approximately half the fatty acids from Aurantiochytrium sp. strain TC 20 are suitable for development of sustainable, low emission sources of transportation fuels and bioproducts. To further improve biomass and oil production, fortification of the feed with additional nutrients (nitrogen sources, trace metals and vitamins) improved the biomass yield from 56 g/L (34 % total fatty acids) to 71 g/L (52 % total fatty acids, cell dry weight) at 69 h; these yields are to our knowledge around 70 % of the biomass yields achieved, however, in less than half of the time by other researchers using glycerol and markedly greater than achieved using other industrial wastes. The fast growth and suitable fatty acid profile of this newly isolated Aurantiochytrium sp. strain TC 20 highlights the potential of co-producing the drop-in biodiesel and high value omega-3 oils.     

Pollen baiting facilitates the isolation of marine thraustochytrids with potential in omega-3 and biodiesel production

Marine heterotrophic microbes are capable of accumulating large amounts of lipids, omega-3 fatty acids, carotenoids, and have potential for biodiesel production. Pollen baiting using Pinus radiata pollen grain along with direct plating techniques were used in this study as techniques for the isolation of oil-producing marine thraustochytrid species from Queenscliff, Victoria, Australia. Thirteen isolates were obtained using either direct plating or using pine pollen, with pine pollen acting as a specific substrate for the surface attachment of thraustochytrids. The isolates obtained from the pollen baiting technique showed a wide range of docosahexaenoic acid (DHA) accumulation, from 11 to 41 % of total fatty acid content (TFA). Direct plating isolates showed a moderate range of DHA accumulation, from 19 to 25 % of TFA. Seven isolates were identified on the basis of 18S rRNA sequencing technique as Thraustochytrium species, Schizochytrium species, and Ulkenia species. Although both methods appear to result in the isolation of similar strains, pollen baiting proved to be a simpler method for the isolation of these relatively slow-growing organisms.     

Fatty acid production of thraustochytrids from Saudi Arabian mangroves

This is the first report of thraustochytrids from Saudi Arabia. A total of 108 isolates of thraustochytrid were cultured from Syhat mangroves, Arabian Gulf, Saudi Arabia. Isolated thraustochytrids belonged to five genera: Aplanochytrium, Aurantiochytrium, Schizochytrium, Thraustochytrium and Ulkenia. Cultured thraustochytrids isolated from decaying leaves of Avicennia marina (77 isolates), sediment (15), seawater (10) and decaying thalli of Sargassum (6). Of the 108 isolates, three strains (SY25, SY38 and SY52) were selected based on their high biomass productivity and high percentages of PUFAs. Phylogenetic analyses based on 18S rDNA placed the three strains within the Aurantiochytrium clade with high statistical support. Species of Aurantiochytrium formed six separate clades, the two strains (SY38 and SY52) formed a separate clade that is a sister clade to the one that contains the type species A. limacinum, while SY25 grouped with Aurantiochytrium sp. TA4, that is also isolated from mangroves in Iran, Arabian Gulf. The strains (SY38 and SY52) shared the phylogenetic placement, their morphology and fatty acid profile. The strain SY25 have different shape of sporangia that divide to give zoospores directly, sporogenous cells are surrounded by thick gelatinous sheath and produce high levels of Linoleic and Oleic essential unsaturated fatty acids. The three studied strain produced high levels of Palmitic acid (ranged between 31.1 and 65.3 % of total fatty acids) that can be further optimized for biofuel production.     

Production of lipids containing high levels of docosahexaenoic acid from empty palm fruit bunches by Aurantiochytrium sp. KRS101

The oleaginous microalga Aurantiochytrium sp. KRS101 was cultivated in enzymatic hydrolysates of alkali-pretreated empty palm fruit bunches (EFBs), without prior detoxification process. The maximal levels of lipid and docosahexaenoic acid synthesized were 12.5 and 5.4 g L^?1 after cultivation for 36 h. Similar lipid levels were also obtained via simultaneous saccharification and cultivation. The results suggested that EFB is a promising source for production of useful lipids by the microalgal strain.     

Long-chain n−3 polyunsaturated fatty acid production by members of the marine protistan group the thraustochytrids: screening of isolates and optimisation of docosahexaenoic acid production

Following an isolation programme for thraustochytrids (marine fungoid protists) from three different locations, 57 isolates were screened for biomass, oil and docosahexaenoic acid production (DHA). Although a common fatty acid profile for the thraustochytrid isolates emerged, there was considerable variation in the DHA content of the oil. In some isolates from a cold temperate environment, DHA represented almost 50% of the total fatty acids present. Although isolates from a sub-tropical environment produced higher levels of biomass, with up to 37% (w/w) oil, the DHA fraction of the fatty acids was low. Cool temperate isolates gave intermediate values. Studies to optimise biomass and DHA production by manipulation of growth medium composition were carried out on a tropical strain. Results indicated that medium with a high C:N ratio stimulated DHA production. The use of such media in bioreactor cultivations gave maximum biomass, lipid and DHA content of 14 g l⁻¹, 78 and 25% (w/w), respectively. Optimum DHA production was 2.17 g l⁻¹ after 107 h cultivation.     

高产DHA破囊壶菌Aurantiochytrium sp. PKU#SW7诱变株的筛选

【目的】对野生菌株Aurantiochytrium sp.PKU#SW7诱变育种,筛选高产DHA突变株。【方法】采用UV诱变和化学药物胁迫筛选方式,以菌株的生物量、油脂产量、DHA产量作为筛选指标,获得高产DHA突变株。【结果】经鉴定获得一株DHA高产突变株PKU#PM003,该菌株传代4次后仍保持较好的遗传稳定性。摇瓶发酵后,PKU#PM003生物量产量高达6.62 g/L,比原始菌株5.95 g/L提高了11.26%,脂肪酸含量高达4.01 g/L,比原始菌株3.18 g/L提高了26.1%,DHA在脂肪酸中所占比例由29.97%增加到33.43%,产量提高了41.01%,油脂突变效果显著。【结论】突变株PKU#PM003可作为性状优良的工业化发酵生产菌种,并在DHA产量提升上仍具有巨大的空间。     

A new strategy for strain improvement of Aurantiochytrium sp. based on heavy-ions mutagenesis and synergistic effects of cold stress and inhibitors of enoyl-ACP reductase

Developing a strain with high docosahexaenoic acid (DHA) yield and stable fermenting-performance is an imperative way to improve DHA production using Aurantiochytrium sp., a microorganism with two fatty acid synthesis pathways: polyketide synthase (PKS) pathway and Type I fatty acid synthase (FAS) pathway. This study investigated the growth and metabolism response of Aurantiochytrium sp. CGMCC 6208 to two inhibitors of enoyl-ACP reductase of Type II FAS pathway (isoniazid and triclosan), and proposed a method of screening high DHA yield Aurantiochytrium sp. strains with heavy ion mutagenesis and pre-selection by synergistic usage of cold stress (4 °C) and FAS inhibitors (triclosan and isoniazid). Results showed that (1) isoniazid and triclosan have positive effects on improving DHA level of cells; (2) mutants from irradiation dosage of 120 Gy yielded more DHA compared with cells from 40 Gy, 80 Gy treatment and wild type; (3) DHA contents of mutants pre-selected by inhibitors of enoyl-ACP reductase of Type II FAS pathway (isoniazid and triclosan)at 4 °C, were significantly higher than that of wild type; (4) compared to the wild type, the DHA productivity and yield of a mutant (T-99) obtained from Aurantiochytrium sp. CGMCC 6208 by the proposed method increased by 50% from 0.18 to 0.27 g/Lh and 30% from 21 to 27 g/L, respectively. In conclusion, this study developed a feasible method to screen Aurantiochytrium sp. with high DHA yield by a combination of heavy-ion mutagenesis and mutant-preselection by FAS inhibitors and cold stress.     

Batch, fed-batch and repeated fed-batch fermentation processes of the marine thraustochytrid Schizochytrium sp. for producing docosahexaenoic acid

Different fermentation processes, including batch, fed-batch and repeated fed-batch processes by Schizochytrium sp., were studied and compared for the effective DHA-rich microbial lipids production. The comparison between different fermentation processes showed that fed-batch process was a more efficient cultivation strategy than the batch process. Among the four different feeding strategies, the glucose concentration feed-back feeding strategy had achieved the highest fermentation results of final cell dry weight, total lipids content, DHA content and DHA productivity of 72.37, 48.86, 18.38 g l^?1 and 138.8 mg l^?1 h^?1, respectively. The repeated fed-batch process had the advantages of reducing the time and cost for seed culture and inoculation between each fermentation cycles. The results of fermentation characteristics and lipid characterization of the repeated fed-batch process indicated that this repeated fed-batch process had promising industrialization prospect for the production of DHA-rich microbial lipids.     

Exploring potential use of Australian thraustochytrids for the bioconversion of glycerol to omega-3 and carotenoids production

Marine microbes have the potential for accumulating large quantities of lipids and are therefore suitable candidate as feedstock in unsaturated fatty acid production. The efficient utilisation of glycerol as an alternative carbon source to glucose was demonstrated in the fermentation of newly isolated thraustochytrid strains from the Queenscliff, Victoria, Australia. The isolates exhibited the presence of omega-3 and omega-6 polyunsaturated fatty acids, with the major fatty acids for all isolates being (as percent total fatty acid), palmitic acid (25.1–40.78%), stearic acid (4.24–13.2%), eicosapentaenoic acid EPA (2.31–8.5%) and docosapentaenoic acid (7.24–10.9%). Glycerol as a carbon source gave promising biomass growth with significant lipid and DHA productivity. An approximate three-fold increase in carotenoid content in all isolates was achieved when glycerol was used as a carbon source in the production medium.     

深圳海域6株破囊壶菌的生长特性及油脂成分分析

【目的】从深圳海域分离得到6株破囊壶菌,对其基本形态特征、生活史和油脂含量等进行研究,开发其应用潜力。【方法】使用松花粉垂钓法对破囊壶菌进行分离,通过18S r RNA基因测序的方法对破囊壶菌进行鉴定,用显微镜观察其基本形态特征,通过使用尼罗红(Nile Red)染色法对油脂含量进行定性检测,并用GC-MS分析菌株的油脂含量和组成情况。【结果】18S r RNA基因鉴定其属于Aurantiochytrium sp.、Schizochytrium sp.和Thraustochytrium sp.三个属。破囊壶菌的脂肪酸主要成分为十六碳饱和脂肪酸和二十二碳六烯酸(DHA),其中Mn11和Mn15的饱和脂肪酸含量达到总脂肪酸含量的70%以上,Mn16和Sw7的DHA产量分别达到1.29 g/L和1.26 g/L。【结论】Mn11和Mn15菌株适合用于生物柴油的生产,Mn16和Sw7是DHA发酵生产的潜力菌株。     

Screening and characterization of Isochrysis strains and optimization of culture conditions for docosahexaenoic acid production

Isochrysis is a genus of marine unicellular microalgae that produces docosahexaenoic acid (DHA, C22:6), a very long chain polyunsaturated fatty acid (PUFA) of significant health and nutritional value. Mass cultivation of Isochrysis for DHA production for human consumption has not been established due to disappointing low DHA productivity obtained from commonly used Isochrysis strains. In this study, 19 natural Isochrysis strains were screened for DHA yields and the results showed that the cellular DHA content ranged from 6.8 to 17.0 % of total fatty acids with the highest DHA content occurring in the exponential growth phase. Isochrysis galbana #153180 exhibited the greatest DHA production potential and was selected for further investigation. The effects of different light intensities, forms, and concentrations of nitrogen, phosphorus, and salinity on growth and DHA production of I. galbana #153180 were studied in a bubble column photobioreactor (PBR). Under favorable culture conditions, I. galbana #153180 contained DHA up to 17.5 % of total fatty acids or 1.7 % of cell dry weight. I. galbana #153180 was further tested in outdoor flat-plate PBRs varying in light path length, starting cell density (SCD), and culture mode (batch versus semicontinuous). When optimized, record high biomass and DHA productivity of I. galbana #153180 of 0.72 g L^?1?day^?1 and 13.6 mg?L^?1?day^?1, or 26.4 g?m^?2?day^?1 and 547.7 mg?m^?2?day^?1, respectively, were obtained, suggesting that I. galbana #153180 may be a desirable strain for commercial production of DHA.     

CFD investigation of <Emphasis Type="Italic">Schizochytrium</Emphasis> sp. impeller configurations on cell growth and docosahexaenoic acid synthesis

Effects of impeller configurations on docosahexaenoic acid production and flow characteristics were investigated by Schizochytrium sp. in a 15 L bioreactor. 6-straight blade disc turbine (6-SBDT), 6-arrowy-blade disc turbine (6-ABDT) and down-pumping propeller (DPP) were combined to form different impeller configurations. Simulated results showed that configuration SSA consisting of upper two 6-SBDT and one bottom 6-ABDT possessed the worst oxygen supply capacity. But it obtained the highest DHA percentage of 48.17 % and DHA yield of 21.42 g/L, indicating that it was beneficial for DHA synthesis and converting glucose to biomass and lipids. Configuration SAS consisting of one middle 6-ABDT and two 6-SBDT provided better mixing capacity, which resulted in the maximum glucose consumption rate of 2.86 g/L h and the highest biomass of 108.09 g/L. This study would improve insight into understanding the relationship between flow field and the physiology of Schizochytrium sp. for the scale-up of industrial DHA production.     

Alkaline lipase activity from the marine protists,thraustochytrids

Two thraustochytrid protists of the genus Thraustochytrium isolated from coastal and mangrove habitats of Goa, India were studied for extracellular alkaline lipase production. Maximum lipase production was supported by a combination of peptone and yeast extract in the growth medium while strong inhibition of enzyme production was observed in presence of glucose. The inducible nature of the enzyme production was evidenced by the requirement of olive oil in the medium. Lipase production was salt-dependent and optimum production required 3.4% (w/v) crude sea salt. Ideal conditions for maximum production of lipases were therefore adopted as incubation at 30 ± 2°C for 168 h at an initial pH of 6.0 in a medium consisting of 0.5% peptone, 0.01% yeast extract, 0.5% olive oil and 3.4% crude salt. Extracellular lipase production by the two thraustochytrid isolates [designated TZ (ATCC #PRA-295) and AH-2 (ATCC #PRA-296)] was increased threefold under these optimized culture conditions. This appears to be the first report on optimization of cultivation conditions for the production of alkaline lipases by thraustochytrids.     

Replacement of fish oil with thraustochytrid Schizochytrium sp. L oil in Atlantic salmon parr (Salmo salar L) diets

Replacing fish oil with that from a docosahexaenoic acid (22:6omega3, DHA) rich single cell micro-organism, thraustochytrid Schizochytrium sp. L, in diets for Atlantic salmon (Salmo salar) was investigated. Four experimental diets containing 100% thraustochytrid oil (TO), 100% palm oil (PO) and a 4:1 palm and thraustochytrid oil mixture (MX) were compared to a fish oil (FO) diet over 9 weeks. A saltwater transfer challenge occurred at the end of the trial for 14 days to test the diet treatments on the ability of salmon to smolt. There were no significant differences in the feed consumption of the diets or the digestibility of the omega3 or omega6 PUFA, indicating no differences in the digestibility of fatty acids between diets. No significant differences were noted between the growth of fish on the four diet treatments. Significant differences were noted in the fatty acid profiles of the fish muscle tissues between all diets. Fish on the TO diet had a significantly greater percentage of DHA in muscle tissue compared with fish on all other diets. Blood osmolarity, which is inversely related to the ability of salmon to smolt, from the TO and FO fed fish was significantly lower than that of fish on the PO diet. This study showed that thraustochytrid oil can be used to replace fish oil in Atlantic salmon diets without detriment to the growth of parr. Including thraustochytrid oil in fish diets significantly increases the amount of DHA in Atlantic salmon muscle and therefore is a candidate for use in oil blends for salmon diets. Thraustochytrid oil provides a renewable source of essential fatty acids, in particular DHA, for aquafeeds.     

Effects of light intensity,light quality,and illumination period on cell growth,TFA accumulation,and DHA production in <Emphasis Type="Italic">Crypthecodinium</Emphasis> sp. SUN

Crypthecodinium strains are ideal candidates for DHA production. In a previous study, light was found to be efficient in inducing total fatty acid accumulation in Crypthecodinium sp. SUN. In order to further analyze the light-inducing behavior of this microalga, experiments were conducted to elucidate the influence of different light intensities, light qualities, and illumination periods on DHA production. The results showed that an irradiance of 30 μmol photons m^?2 s^?1 was most suitable for DHA production. Compared to red light and blue light, green light was more efficient in elevating the total fatty acid content in the cells. It was also found that illumination at the first 24 h promoted cell growth, whereas it favored total fatty acid accumulation only during 48–96 h. This is the first systematic investigation of the influence of light on total fatty acid accumulation and DHA production in Crypthecodinium sp. SUN, providing a solid foundation for further research on DHA production.     

The effect of temperature on growth and lipid and fatty acid composition on marine microalgae used for biodiesel production

Cultivation temperature is one of the major factors affecting the growth and lipid accumulation of microalgae. In this study, the effects of temperature on the growth, lipid content, fatty acid composition and biodiesel properties of the marine microalgae Chaetoceros sp. FIKU035, Tetraselmis suecica FIKU032 and Nannochloropsis sp. FIKU036 were investigated. These species were cultured at different temperatures (25, 30, 35 and 40 °C). The results showed that the specific growth rate, biomass and lipid content of all microalgae decreased with increasing temperature. With regards to fatty acids, the presence of saturated fatty acids (SFAs) in T. suecica FIKU032 and Nannochloropsis sp. FIKU036 decreased with increasing temperature, in contrast with polyunsaturated fatty acids (PUFAs). Moreover, Chaetoceros sp. FIKU035 was the only species that could grow at 40 °C. The highest lipid productivity was observed in Chaetoceros sp. FIKU035 when cultivated at 25 °C (66.73 ± 1.34 mg L^?1 day^?1) and 30 °C (61.35 ± 2.89 mg L^?1 day^?1). Moreover, the biodiesel properties (cetane number, cold filter plugging point, kinematic viscosity and density) of the lipids obtained from this species were in accordance with biodiesel standards. This study indicated that Chaetoceros sp. FIKU035 can be considered as a suitable species for biodiesel production in outdoor cultivation.     

Efficient docosahexaenoic acid production by Schizochytrium sp. via a two-phase pH control strategy using ammonia and citric acid as pH regulators

The cell growth and docosahexaenoic acid (DHA) synthesis of Schizochytrium sp. are closely related to the culture pH. A two-phase pH control strategy based on nitrogen consumption was developed in which pH 7.0 was used for biomass accumulation and pH 5.0 for DHA synthesis. Using this strategy, the cell dry weight and DHA content reached 98.07 and 25.85 g/L, respectively. Furthermore, ammonia and citric acid were used as pH regulators. Application of citric acid further resulted in 7.88 and 4.87% improvements of total lipids and the ratio of DHA to total fatty acids, respectively. Ammonia, as a suitable nitrogen source, promoted non-lipid biomass accumulation. Using this method, a maximum DHA yield of 32.75 g/L was obtained with non-lipid biomass (58.01 g/L) and the ratio of DHA to total fatty acids (52.36%). This study provides an easy strategy for large-scale industrial production of DHA via high-cell-density fermentation of Schizochytrium sp.