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.     

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.     

Biodiscovery of new Australian thraustochytrids for production of biodiesel and long-chain omega-3 oils

Heterotrophic growth of thraustochytrids has potential in co-producing a feedstock for biodiesel and long-chain (LC, ≥C₂₀) omega-3 oils. Biodiscovery of thraustochytrids from Tasmania (temperate) and Queensland (tropical), Australia, covered a biogeographic range of habitats including fresh, brackish, and marine waters. A total of 36 thraustochytrid strains were isolated and separated into eight chemotaxonomic groups (A–H) based on fatty acid (FA) and sterol composition which clustered closely with four different genera obtained by 18S rDNA molecular identification. Differences in the relative proportions (%FA) of long-chain C₂₀, C₂₂, omega-3, and omega-6 polyunsaturated fatty acids (PUFA), including docosahexaenoic acid (DHA), docosapentaenoic acid, arachidonic acid, eicosapentaenoic acid (EPA), and saturated FA, as well as the presence of odd-chain PUFA (OC-PUFA) were the major factors influencing the separation of these groups. OC-PUFA were detected in temperate strains of groups A, B, and C (Schizochytrium and Thraustochytrium). Group D (Ulkenia) had high omega-3 LC-PUFA (53% total fatty acids (TFA)) and EPA up to 11.2% TFA. Strains from groups E and F (Aurantiochytrium) contained DHA levels of 50–61% TFA after 7 days of growth in basal medium at 20 °C. Groups G and H (Aurantiochytrium) strains had high levels of 15:0 (20–30% TFA) and the sum of saturated FA was in the range of 32–51%. β,β-Carotene, canthaxanthin, and astaxanthin were identified in selected strains. Phylogenetic and chemotaxonomic groupings demonstrated similar patterns for the majority of strains. Our results demonstrate the potential of these new Australian thraustochytrids for the production of biodiesel in addition to omega-3 LC-PUFA-rich oils.     

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.     

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.     

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.     

Efficient production of triacylglycerols rich in docosahexaenoic acid (DHA) by osmo-heterotrophic marine protists

Thraustochytrids have recently emerged as a promising source for docosahexaenoic acid (DHA) production due to their high growth rate and oil content. In this study, two thraustochytrid isolates, Aurantiochytrium sp. PKU#SW7 and Thraustochytriidae sp. PKU#Mn16 were used for DHA production. Following growth parameters were optimized to maximize DHA production: temperature, pH, salinity, and glucose concentration. Both isolates achieved the highest DHA yield at the cultivation temperature of 28 °C, pH 6, 100 % seawater, and 2 % glucose. A DHA yield of 1.395 g/l and 1.426 g/l was achieved under the optimized culture conditions. Further investigation revealed that both isolates possess simple fatty acids profiles with palmitic acid and DHA as their dominant constituents, accounting for ～79 % of total fatty acids. To date, very few studies have focused on the DHA distribution in various lipid fractions which is an important factor for identifying strains with a potential for industrial DHA production. In the present study, the lipids profiles of each strain both revealed that the majority of DHA was distributed in neutral lipids (NLs), and the DHA distribution in NLs of PKU#SW7 was exclusively in the form of triacylglycerols (TAGs) which suggest that PKU#SW7 could be utilized as an alternative source of DHA for dietary supplements. The fermentation process established for both strains also indicating that Aurantiochytrium sp. PKU#SW7 was more suitable for cultivation in fermenter. In addition, the high percentage of saturated fatty acids produced by the two thraustochytrids indicates their potential application in biodiesel production. Overall, our findings suggest that two thraustochytrid isolates are suitable candidates for biotechnological applications.     

Zoospore production and motility of mangrove thraustochytrids from Hong Kong under various salinities

We investigated the effects of salinity on the zoospore production of four mangrove thraustochytrid isolates, Schizochytrium sp. KF1, Aurantiochytrium mangrovei KF6, Thraustochytrium striatum KF9 and Ulkenia sp. KF13. The zoospore motilities, which were based on curvilinear velocity (VCL) and straight-line velocity (VSL), were monitored using the Computer-Assisted Sperm Motility Analysis (CASA) Software system. The zoospore production of four isolates was suppressed at salinity above 15‰. Schizochytrium sp. produced the greatest number of zoospores at 15‰, while Aurantiochytrium mangrovei and Ulkenia sp. produced abundant zoospores in diluted sea water ranging from 7.5 to 15‰. Thraustochytrium striatum performed relatively poorly under all salinities. Salinity and exposure time, as well as their interactions, had significant impacts on most zoospore velocity measurements. The optimal velocities of zoospore motility also varied among isolates. Zoospores of Schizochytrium sp. and A. mangrovei had similar responses to salinity, with the highest motility at 7.3‰, followed by a decrease in velocities with increasing salinity. In contrast, the zoospore of T. striatum had optimal motility at 12‰ and remained highly motile from 15 to 20‰. The velocities of zoospores of Ulkenia sp. were the lowest among the tested thraustochytrids and had optimal motility at 12‰. Zoospores of all the isolates remained active after 4 h of exposure to aqueous medium, but the optimal salinity for each mode of swimming changed. The ecological significance of these data are discussed.     

TLC screening of thraustochytrid strains for squalene production

Screenings of thraustochytrids (Labyrinthulomycetes) have been conducted for 176 strains isolated from various sites in the Asian region to investigate what type of species and strains accumulate high levels of squalene. Thin layer chromatography (TLC) screening for squalene production revealed that 38 strains were rated as “+” (high), 29 as “±” (medium), and 109 as “?” (low). Further, high performance liquid chromatography analysis strongly supported the TLC screening results. Besides the 18W-13a strain of Aurantiochytrium sp., which was previously recognized as a squalene-rich strain, several strains produced squalene at approximately 1 g L^?1 of culture volume. Squalene production was strongly related to locality, colony color, and phylogenetic clade. Most strains with “+” squalene spots were isolated from Okinawa, a subtropical region of Japan, while the strains with “±” and “?” squalene spots were isolated from wide geographical regions from tropical to subarctic. Approximately half the strains with orange colonies on GTY medium plates produced a high amount of squalene, whereas the other strains with different colors showed less or no squalene spots on TLC. All the squalene-rich strains were assigned to the Aurantiochytrium clade. Overall, our results suggest that (1) the thraustochytrids show tendentious locality in terms of squalene production, (2) a relationship exists between the metabolic synthesis of carotenoid pigments and squalene production, and (3) the Aurantiochytrium clade may have evolved to accumulate squalene.     

Docosahexaenoic acid (DHA) production by Thraustochytrium sp. ATCC 20892

Thraustochytrium sp. ATCC 20892 produced high yields of docosahexaenoic acid (DHA), more than four other strains of Thraustochytrium and Schizochytrium tested, but insignificant amounts of other polyunsaturated fatty acids. Glucose and sodium glutamate were the preferred carbon and nitrogen sources, respectively, and the optimum conditions for growth and DHA production were pH 7.0 at 25°C with 40 g glucose 1^-1 for 4 days. Temperature profiling under these optimum conditions further enhanced the yield and volumetric productivity of DHA.     

Biomass and oil content of Chlorella sp., Haematococcus sp., Nannochloris sp. and Scenedesmus sp. under mixotrophic growth conditions in the presence of technical glycerol

The growth of algae strains Chlorella sp., Haematococcus sp., Nannochloris sp. and Scenedesmus sp. under mixotrophic conditions in the presence of different concentrations of technical glycerol was investigated with the aim of increasing biomass growth and algae oil content. The highest concentration of lipid obtained in media with 5 g L^?1 glycerol for Chlorella sp., Scenedesmus sp., Nannochloris sp. and Haematococcus sp. was 17.77, 22.34, 27.55 and 34.22 % larger than during the autotrophic growth of these species. Increases in triacylglycerols of up to ten times was observed for Scenedesmus sp. under mixotrophic conditions (using 10 g L^?1 glycerol), whereas an increase of 2.28 times was found for Haematococcus sp. The content of saturated fatty acids of Scenedesmus, Chlorella, Haematococcus and Nannochloris was 67.11, 34.63, 23.39 and 24.23 %, and the amount of unsaturated fatty acids was 32.9, 65.06, 79.61 and 75.78 % of total fatty acids, respectively. Growth on technical glycerol of these strains with light produced higher biomass concentrations and lipid content compared with autotrophic growth. The fatty acid content of oils from these species suggests their potential use as biodiesel feedstock.     

Cloning and functional analysis of putative malonyl-CoA:acyl-carrier protein transacylase gene from the docosahexaenoic acid-producer Schizochytrium sp. TIO1101

Malonyl-CoA:acyl-carrier protein transacylase (MCAT), which transfers the malonyl group from malonyl-CoA to holo-acyl carrier protein (ACP), is a key enzyme in fatty acid biosynthesis. Schizochytrium sp. TIO1101 is a marine protist with high levels of docosahexaenoic acid accumulation. In this study, the putative fabD gene coding MCAT was isolated from Schizochytrium sp. TIO1101. The Schizochytrium MCAT gene (ScTIOfabD) contained an 1176 bp open reading frame encoding a protein of 391 amino acids. The ScTIOfabD gene exhibited high novelty in nucleotide and amino acid sequence. The highest amino acid identity was only 35 % between ScTIOMCAT and the reported MCATs. Further studies demonstrated that ScTIOMCAT could bind malonyl-CoA directly and transfer malonyl group from malonyl-CoA to the ACP domain in vitro. Phylogenetic analysis suggested that ScTIOMCAT was relative close to MCATs of yeast strains. Overexpression of ScTIOMCAT in Saccharomyces cereviseae significantly increased the MCAT activity, without negative effects on the growth rate of the host strain. In addition, ScTIOMCAT generated 16.8 and 62 % increase in biomass and fatty acid accumulation, respectively, and did not alter the profile of fatty acid. Our results indicated that the novel MCAT gene from Schizochytrium sp. TIO1101 was crucial for fatty acid synthesis and had potential applications for genetic modifications of oil-producing species.     

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.     

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.     

Yamadazyma siamensis sp. nov., Yamadazyma phyllophila sp. nov. and Yamadazyma paraphyllophila sp. nov., three novel yeast species isolated from phylloplane in Thailand and Taiwan

Four strains representing three novel anamorphic yeast species were isolated from the external surface of sugarcane leaves (DMKU-RK254^T), corn leaves (DMKU-RK548^T), bean leaves (K129) in Thailand and hengchun pencilwood leaves (TrB1-1^T) in Taiwan. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, the sequence analysis of the D1/D2 region of the large subunit (LSU) rRNA gene, the internal transcribed spacer (ITS) region, the actin gene (ACT1) and the elongation factor 2 gene (EF2), the four strains were determined to represent novel Yamadazyma species although formation of ascospores was not observed. Strain DMKU-RK254^T was determined to be related to Candida diddensiae, Candida naeodendra and Candida kanchanaburiensis but with 1.8, 1.8 and 2.0 % nucleotide substitutions in the D1/D2 region of the LSU rRNA gene, respectively. It was assigned to Yamadazyma siamensis sp. nov. (type strain DMKU-RK254^T = BCC 50730^T = NBRC 108901^T = CBS 12573^T). The sequences of the D1/D2 region of the LSU rRNA gene, the ITS region, ACT1 gene and EF2 gene of two strains (DMKU-RK548^T and K129) were identical but differed from that of strain TrB1-1^T by 0.6, 1.0, 3.3 and 5.9 % nucleotide substitutions, respectively. Therefore, the two strains (DMKU-RK548^T and K129) and strain TrB1-1^T were assigned to be two separate species. The closest species in terms of pairwise sequences similarity of the D1/D2 region to the two novel species was Yamadazyma philogaea but with 1.1–1.7 % nucleotide substitutions. The two strains (DMKU-RK548^T and K129) were assigned to Yamadazyma phyllophila sp. nov. (type strain DMKU-RK548^T = BCC 50736^T = NBRC 108906^T = CBS 12572^T) and the strain TrB1-1^T was named Yamadazyma paraphyllophila sp. nov. (type strain TrB1-1^T = BCRC 23030^T = CCTCC AY 204005^T = CBS 9928^T).     

Chryseobacterium zeae sp. nov., Chryseobacterium arachidis sp. nov., and Chryseobacterium geocarposphaerae sp. nov. isolated from the rhizosphere environment

Four yellow pigmented strains (91A-561^T, 91A-576, 91A-593^T, and JM-1085^T) isolated from plant materials, showed 97.2–98.7 % 16S rRNA gene sequence similarities among each other and were studied in a polyphasic approach for their taxonomic allocation. Cells of all four isolates were rod-shaped and stained Gram-negative. Comparative 16S rRNA gene sequence analysis showed that the four bacteria had highest sequence similarities to Chryseobacterium formosense (97.2–98.7 %), Chryseobacterium gwangjuense (97.1–97.8 %), and Chryseobacterium defluvii (94.6–98.0 %). Sequence similarities to all other Chryseobacterium species were below 97.5 %. Fatty acid analysis of the four strains showed Chryseobacterium typical profiles consisting of major fatty acids C_15:0 iso, C_15:0 iso 2-OH/C_16:1 ω7c, C_17:1 iso ω9c, and C_17:0 iso 3-OH, but showed also slight differences. DNA–DNA hybridizations with type strains of C. gwangjuense, C. formosense, and C. defluvii resulted in values below 70 %. Isolates 91A-561^T and 91A-576 showed DNA–DNA hybridization values >80 % indicating that they belonged to the same species; but nucleic acid fingerprinting showed that the two isolates represent two different strains. DNA–DNA hybridization results and the differentiating biochemical and chemotaxonomic properties showed, that both strains 91A-561^T and 91A-576 represent a novel species, for which the name Chryseobacterium geocarposphaerae sp. nov. (type strain 91A-561^T=LMG 27811^T=CCM 8488^T) is proposed. Strains 91A-593^T and JM-1085^T represent two additional new species for which we propose the names Chyrseobacterium zeae sp. nov. (type strain JM-1085^T=LMG 27809^T, =CCM 8491^T) and Chryseobacterium arachidis sp. nov. (type strain 91A-593^T=LMG 27813^T, =CCM 8489^T), respectively.     

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.     

Haloplanus litoreus sp. nov. and Haloplanus ruber sp. nov., from a marine solar saltern and an aquaculture farm,respectively

Two halophilic archaea, strains GX21^T and R35^T, were isolated from a marine solar saltern and an aquaculture farm in China, respectively. Cells of the two strains were observed to be pleomorphic, flat, to contain gas vesicles, stain Gram-negative and produce red-pigmented colonies. Strain GX21^T was found to be able to grow at 25–50 °C (optimum 37 °C), at 2.6–4.8 M NaCl (optimum 3.4 M NaCl), at 0.05–1.0 M MgCl₂ (optimum 0.1 M MgCl₂) and at pH 6.0–8.5 (optimum pH 6.5) while strain R35^T was found to be able to grow at 25–45 °C (optimum 37 °C), at 2.1–4.8 M NaCl (optimum 3.1 M NaCl), at 0–0.7 M MgCl₂ (optimum 0.03 M MgCl₂) and at pH 5.5–9.5 (optimum pH 6.5–7.0). The cells of both isolates were observed to lyse in distilled water. The minimum NaCl concentrations that prevented cell lysis were determined to be 15 % (w/v) for strain GX21^T and 12 % (w/v) for strain R35^T. The major polar lipids of the two strains were identified as phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, one major glycolipid and a minor lipid chromatographically identical to sulfated mannosyl glucosyl diether and mannosyl glucosyl diether, respectively. 16S rRNA gene sequence analysis revealed that strains GX21^T and R35^T show 97.1 % sequence similarity to each other and are closely related to Haloplanus aerogenes TBN37^T (96.8 and 95.8 %), Haloplanus vescus RO5-8^T (96.7 and 96.1 %), Haloplanus salinus YGH66^T (96.4 and 95.8 %) and Haloplanus natans JCM 14081^T (96.3 and 95.4 %). The rpoB′ gene similarity between strains GX21^T and R35^T is 90.5 % and show 88.5–90.8 % similarity to the Haloplanus species with validly published names. The DNA G+C content of strain GX21^T and R35^T were determined to be 65.8 and 66.0 mol%, respectively. The DNA–DNA hybridization values between strain GX21^T and strain R35^T, and the two strains with the Haloplanus species with validly published names, showed less than 50 % DNA–DNA relatedness. It was concluded that strain GX21^T (=CGMCC 1.10456^T = JCM 17092^T) and strain R35^T (=CGMCC 1.10594 ^T = JCM 17271^T) represent two new species of Haloplanus, for which the names Haloplanus litoreus sp. nov. and Haloplanus ruber sp. nov. are proposed.     

Physiological characterization of lipid accumulation and in vivo ester formation in Gordonia sp. KTR9

Previous work has demonstrated the feasibility of in vivo biodiesel synthesis in Escherichia coli, however, ethyl ester formation was dependent on an external fatty acid feedstock. In contrast to E. coli, actinomycetes may be ideal organisms for direct biodiesel synthesis because of their capacity to synthesize high levels of triacylglcerides (TAGs). In this study, we investigated the physiology and associated TAG accumulation along with the in vivo ability to catalyze ester formation from exogenous short chain alcohol sources in Gordonia sp. KTR9, a strain that possesses a large number of genes dedicated to fatty acid and lipid biosynthesis. Total lipid fatty acids content increased by 75 % and TAG content increased by 50 % under nitrogen starvation conditions in strain KTR9. Strain KTR9 tolerated the exogenous addition of up to 4 % methanol, 4 % ethanol and 2 % propanol in the media. Increasing alcohol concentrations resulted in a decrease in the degree of saturation of recovered fatty acid alcohol esters and a slight increase in the fatty acid chain length. A linear dose dependency in fatty alcohol ester synthesis was observed in the presence of 0.5–2 % methanol and ethanol compared to control KTR9 strains grown in the absence of alcohols. An inspection of the KTR9 genome revealed the presence of several putative wax ester synthase/acyl-coenzyme A?:?diacylglycerol acyltransferase (WS/DGAT) enzymes, encoded by atf gene homologs, that may catalyze the in vivo synthesis of fatty acid esters from short chain alcohols. Collectively, these results indicate that Gordonia sp. KTR9 may be a suitable actinomycete host strain for in vivo biodiesel synthesis.