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.     

At high temperature lipid production in Ettlia oleoabundans occurs before nitrate depletion

Temperature and light intensity effects on biomass and lipid production were investigated in Ettlia oleoabundans to better understand some fundamental properties of this potentially useful but poorly studied microalgal species. E. oleoabundans entered dormant state at 5 °C, showed growth at 10 °C, and when exposed to light at 70 μmol photons per square meter per second at 10 °C, cells reached a biomass concentration of >2.0 g?L^?1 with fatty acid methyl esters of 11.5 mg?L^?1. Highest biomass productivity was at 15 °C and 25 °C regardless of light intensity, and accumulation of intracellular lipids was stimulated by nitrate depletion under these conditions. Although growth was inhibited at 35 °C, at 130 μmol photons per square meter per second lipid content reached 10.37 mg?L^?1 with fatty acid content more favorable to biodiesel dominating; this occurred without nitrate depletion. In a two-phase temperature shift experiment at two nitrate levels, cells were shifted after 21 days at 15 °C to 35 °C for 8 days. Although after the shift growth continued, lipid productivity per cell was less than that in the 35 °C cultures, again without nitrate depletion. This study showed that E. oleoabundans grows well at low temperature and light intensity, and high temperature can be a useful trigger for lipid accumulation independent of nitrate depletion. This will prove useful for improving our knowledge about lipid production in this and other oleaginous algae for modifying yield and quality of algal lipids being considered for biodiesel production.     

Optimization of cultivation conditions for fatty acid composition and EPA production in the eustigmatophycean microalga Trachydiscus minutus

We determined the effects of cultivation conditions (nitrogen source, salinity, light intensity, temperature) on the composition of polyunsaturated fatty acids (PUFAs) and the production of eicosapentaenoic acid (EPA) in the laboratory cultured eustigmatophycean microalga, Trachydiscus minutus. T. minutus was capable of utilizing all nitrogen compounds tested (potassium nitrate, urea, ammonium nitrate, ammonium carbonate) with no differences in growth and only minor differences in fatty acid (FA) compositions. Ammonium carbonate was the least appropriate for lipid content and EPA production, while urea was as suitable as nitrates. Salinity (0.2 % NaCl) slightly stimulated EPA content and inhibited growth. Increasing salinity had a marked inhibitory effect on growth and PUFA composition; salinity at or above 0.8 % NaCl was lethal. Both light intensity and temperature had a distinct effect on growth and FA composition. The microalga grew best at light intensities of 470–1,070 μmol photons m^?2 s^?1 compared to 100 μmol photons m^?2 s^?1, and at 28 °C; sub-optimal temperatures (20, 33 °C) strongly inhibited growth. Saturated fatty acids increased with light intensity and temperature, whereas the reverse trend was found for PUFAs. Although the highest level of EPA (as a proportion of total FAs) was achieved at a light intensity of 100 μmol photons m^?2 s^?1 (51.1?± 2.8 %) and a temperature of 20 °C (50.9?±?0.8 %), the highest EPA productivity of about 30 mg L^?1?day^?1 was found in microalgae grown at higher light intensities, at 28 °C. Overall, for overproduction of EPA in microalgae, we propose that outdoor cultivation be used under conditions of a temperate climatic zone in summer, using urea as a nitrogen source.     

Inhibition of pathogenic Vibrio by the microalgae Isochrysis galbana

Vibrio alginolyticus, Vibrio campbellii, and Vibrio harveyi were inhibited by Isochrysis galbana in batch cultures. I. galbana reduced the V. alginolyticus, V. campbellii, and V. harveyi counts to undetectable levels in 2, 4, and 7 days (<0.01 Vibrio spp. mL^?1), respectively, remaining so until the end of the experiment on day 15. Other heterotrophic bacteria reached counts of 10⁶ CFU mL^?1 on ZoBell medium at the end of the experiment. Vibrio parahaemolyticus was not inhibited by I. galbana. In all mixed I. galbana and Vibrio spp. cultures, the algal density increased from 3.5 to 4.0?×?10⁷ cells mL^?1, higher than that in I. galbana cultures alone, indicating a lack of an inhibitory effect on microalgae in the mixed cultures. The predominant fatty acids (>82 %) of I. galbana during the stationary growth phase were estearidonic (24.3 %), oleic (15.7 %), myristic (13.8 %), docosahexaenoic (11.0 %), palmitic (10.3 %), and α-linolenic (7.2 %) acids. These results demonstrate that I. galbana synthesizes antibacterial fatty acids that inhibit the growth of pathogenic bacteria such as V. alginolyticus, V. campbellii, and V. harveyi.     

Isolation and characterization of novel ginsenoside-hydrolyzing glycosidase from Microbacterium esteraromaticum that transforms ginsenoside Rb2 to rare ginsenoside 20(S)-Rg3

Ginsenoside Rb2 was transformed by recombinant glycosidase (Bgp2) into ginsenosides Rd and 20(S)-Rg3. The bgp2 gene consists of 2,430 bp that encode 809 amino acids, and this gene has homology to the glycosyl hydrolase family 2 protein domain. SDS-PAGE was used to determine that the molecular mass of purified Bgp2 was 87 kDa. Using 0.1 mg ml^?1 of enzyme in 20 mM sodium phosphate buffer at 40 °C and pH 7.0, 1.0 mg ml^?1 ginsenoside Rb2 was transformed into 0.47 mg ml^?1 ginsenoside 20(S)-Rg3 within 120 min, with a corresponding molar conversion yield of 65 %. Bgp2 hydrolyzed the ginsenoside Rb2 along the following pathway: Rb2 → Rd → 20(S)-Rg3. This is the first report of the biotransformation of ginsenoside Rb2 to ginsenoside 20(S)-Rg3 using the recombinant glycosidase.     

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.     

Fatty acid composition and biological activities of Isochrysis galbana T-ISO,Tetraselmis sp. and Scenedesmus sp.: possible application in the pharmaceutical and functional food industries

Organic and water extracts of Isochrysis galbana T-ISO (=Tisochrysis lutea), Tetraselmis sp. and Scenedesmus sp. were evaluated for their antioxidant activity, acetylcholinesterase (AChE) inhibition, cytotoxicity against tumour cell lines, and fatty acids and total phenolic content (TPC). I. galbana T-ISO had the highest TPC (3.18 mg GAE g^?1) and radical scavenging activity, with an IC₅₀ value of 1.9 mg mL^?1 on the acetone extract. The extracts exhibited a higher ability to chelate Fe²⁺ than Cu²⁺, and the maximum Fe²⁺ chelating capacity was observed in the hexane extract of Scenedesmus sp. (IC₅₀=0.73 mg mL^?1) and Scenedesmus sp. (IC₅₀?=?0.73 mg mL^?1). The highest ability to inhibit AChE was observed in the water and ether extracts of Scenedesmus sp., with IC₅₀ values of 0.11 and 0.15 mg mL^?1, respectively, and in the water extract of I. galbana (IC₅₀?=?0.16 mg mL^?1). The acetone extract of I. galbana T-ISO significantly reduced the viability of human hepatic carcinoma HepG2 cells (IC₅₀?=?81.3 μg mL^?1) as compared to the non-tumour murine stromal S17 cell line, and displayed a selectivity index of 3.1 at the highest concentration tested (125 μg mL^?1). All species presented a highly unsaturated fatty acids profile. Results suggest that these microalgae, particularly I. galbana T-ISO, could be a source of biomolecules for the pharmaceutical industry and the production of functional food ingredients and can be considered as an advantageous alternative to several currently produced microalgae.     

Cloning,over-expression and characterization of a thermo-tolerant xylanase from Thermotoga thermarum

The xyn10B gene, encoding the endo-1,4-β-xylanase Xyn10B from Thermotoga thermarum, was cloned and expressed in Escherichia coli. The ORF of the xyn10B was 1,095 bp and encoded to mature peptide of 344 amino acids with a calculated MW of 40,531 Da. The recombinant xylanase was optimally active at 80 °C, pH 6.0 and retained approx. 60 % of its activity after 2 h at 75 °C. Apparent K _m , k _cat and k _cat /K _m values of the xylanase for beechwood xylan were 1.8 mg ml^?1, 520 s^?1 and 289 ml mg^?1 s^?1, respectively. The end products of the hydrolysis of beechwood xylan were mainly oligosaccharides but without xylose after 2 h hydrolysis.     

Growth,pigments, and biochemical composition of marine red alga Gracilaria crassa

The marine red alga Gracilaria crassa was investigated for its proximate composition, minerals, fatty acids, amino acids, and agar content to decipher its nutritional implications. The growth performance and pigments were studied under different combinations of temperature and salinity. On a dry weight basis the total lipid content was 1.30?±?0.05 %, protein was 5.18?±?0.64 %, carbohydrate was 42.0?±?1.2 %, ash was 43.18?±?1.15 %, and agar content was 21.52?±?0.73 %. Appreciable amounts of macro-, micro-nutrients (K?>?Na, Ca, Mg, and Fe), and essential amino acids (Ileu, His, Thr, Leu, and Lys) were found. Palmitic, stearic acid, and arachidonic acid were major fatty acids detected. The alga showed maximum daily growth rate (DGR %) 5.8?±?0.09 % at 25 °C, 35 ‰ salinity. The highest content of pigment R-phycoerythrin (444.7?±?1.9 μg g^?1 fresh weight (FW) basis) was obtained at 25 ‰ salinity at 35 °C while that of R-phycocyanin (476.3?±?2.3 μg g^?1 DW) at 30 ‰ salinity at 30 °C. This study revealed that this alga can be utilized as a potential source for food and feed. The data generated on best growth conditions will be very useful for farming of G. crassa in open sea. This alga could be used for production of natural colorants at defined control condition.     

Culture medium optimization of a new bacterial extracellular polysaccharide with excellent moisture retention activity

A new kind of extracellular polysaccharide (EPS) from Pseudomonas fluorescens PGM37 was obtained and culture media was optimized using the statistical methods single factor experiments and response surface methodology (RSM) design. As a result, the optimum cultivation conditions initial pH value, medium volume, inoculum size, temperature, and rotation speed were 7.5, 100 mL/250 mL, 5 %, 28 °C, and 180 rpm, respectively. The optimized media: sucrose 36.23 g?L^?1, yeast extract 3.32 g?L^?1, sodium chloride 1.13 g?L^?1, and calcium chloride 0.20 g?L^?1. The maximum predicted yield of EPS was 10.1163 g?L^?1 under these conditions. The validation data was 10.012 g?L^?1, which could strongly confirm the correlation between the experimental and theoretical values. Gas Chromatography analysis revealed that the polymer was made up of mannose and glucose in the ratio of 1:1. Infrared spectroscopy showed that the polysaccharide had β-D-pyranoid configuration and contained no other substituent. Graded by different multiples of alcohol after specific degradation by enzyme and then detected by LC-ESI-MS, the EPS structure was β-D-Glcp-(1, 4)-β-D-Manp-(1, 4)-β-D-Glcp-(1, 4)-β-D-Manp. The moisture retention ability of the EPS was found to be superior to glycerol and only a little inferior to hyaluronic acid (HA), which presented potential application value in cosmetics and clinical medicine fields.     

The effect of sodium bicarbonate supplementation on growth and biochemical composition of marine microalgae cultures

The addition of bicarbonate (NaHCO₃; 0, 1, or 2 g L^?1) to microalgal cultures has been evaluated for two species (Tetraselmis suecica and Nannochloropsis salina) in respect of growth and biochemical composition. In batch cultures, addition of bicarbonate (1 g L^?1) resulted in significantly (P?<?0.05) higher final mean cell abundances for both species. No differences in specific growth rates (SGRs) were recorded for T. suecica between treatments; however, increasing bicarbonate addition decreased SGR values in N. salina cultures. Bicarbonate addition (1 g L^?1) significantly improved nitrate utilisation from the external media and photosynthetic efficiency (F _v /F _m ) in both species. For both T. suecica and N. salina, bicarbonate addition significantly increased the cellular concentrations of total pigments (3,432–3,587 and 19–37 fg cell^?1, respectively) compared to cultures with no additional bicarbonate (1,727 and 11 fg cell^?1, respectively). Moreover, final concentrations of total cellular fatty acids in T. suecica and N. salina cultures supplemented with 2 g L^?1 bicarbonate (7.6?±?1.2 and 1.8?±?0.1 pg cell^?1, respectively) were significantly higher than those cells supplemented with 0 or 1 g L^?1 bicarbonate (3.2–3.5 and 0.9–1.0 pg cell^?1, respectively). In nitrate-deplete cultures, bicarbonate addition caused species-specific differences in the rate of cellular lipid production, rates of change in fatty acid composition and final lipid levels. In summary, the addition of sodium bicarbonate is a viable strategy to increase cellular abundance and concentrations of pigments and lipids in some microalgae as well as the rate of lipid accumulation in nitrate-deplete cultures.     

A novel cold-active and salt-tolerant α-amylase from marine bacterium Zunongwangia profunda: molecular cloning,heterologous expression and biochemical characterization

A novel gene (amyZ) encoding a cold-active and salt-tolerant α-amylase (AmyZ) was cloned from marine bacterium Zunongwangia profunda (MCCC 1A01486) and the protein was expressed in Escherichia coli. The gene has a length of 1785 bp and encodes an α-amylase of 594 amino acids with an estimated molecular mass of 66 kDa by SDS-PAGE. The enzyme belongs to glycoside hydrolase family 13 and shows the highest identity (25 %) to the characterized α-amylase TVA II from thermoactinomyces vulgaris R-47. The recombinant α-amylase showed the maximum activity at 35 °C and pH 7.0, and retained about 39 % activity at 0 °C. AmyZ displayed extreme salt tolerance, with the highest activity at 1.5 M NaCl and 93 % activity even at 4 M NaCl. The catalytic efficiency (k _cat/K _m) of AmyZ increased from 115.51 (with 0 M NaCl) to 143.30 ml mg^?1 s^?1 (with 1.5 M NaCl) at 35 °C and pH 7.0, using soluble starch as substrate. Besides, the thermostability of the enzyme was significantly improved in the presence of 1.5 M NaCl or 1 mM CaCl₂. AmyZ is one of the very few α-amylases that tolerate both high salinity and low temperatures, making it a potential candidate for research in basic and applied biology.     

Biomass,Lipid and Fatty Acid Production in Large-Scale Cultures of the Marine Macroalga Derbesia tenuissima (Chlorophyta)

Biomass productivity was quantified for the marine macroalga Derbesia tenuissima cultivated outdoors at seven stocking densities from 0.25 to 8 g L^?1 for 5 weeks. Total lipids and fatty acid quantity and quality was measured from samples that were freeze-dried, dried by oven (75 °C), food dehydrator (60 °C), or outdoor in the sun (40 °C) or shade (38 °C). Stocking densities of 0.25 to 2 g L^?1 yielded the highest biomass productivities (>20 g dry weight m^?2 day^?1) with no effect on total lipid quantity (11 %), or fatty acid quantity (5.3 %) or quality at any density tested. However, there was an interactive effect of stocking density and drying technique, with a decrease of up to 40 % in polyunsaturated fatty acids in sun-dried compared to freeze-dried biomass. Notably, while fatty acid and biomass productivity may be inseparable in macroalgae, cultivation conditions have a significant carryover effect in the post-harvest delivery of high-quality bio-oils.     

Gene cloning and characterization of recombinant L-Asparaginase from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain R5

L-asparaginase gene from Bacillus subtilis strain R5 (Asn-R5), comprising 990 nucleotides corresponding to a polypeptide of 329 amino acids, was cloned and expressed in Escherichia coli. Recombinant Asn-R5 was produced in soluble and active form exhibiting a specific activity of 223 μmol min^?1 mg^?1. The optimal temperature and pH for L-asparaginase activity of Asn-R5 were 35 °C and 9.0, respectively. Asn-R5 displayed a 50% activity with D-asparagine and 2% with L-glutamine compared to 100% with L-asparagine. No activity could be detected when D-glutamine was used as substrate. Half-life of the enzyme was 180 min at 35 °C and 40 min at 50 °C. There was no effect of metal ions and EDTA on the activity indicating that Asn-R5 enzyme activity is not metal ion dependent. The K_m and V_max values were 2.4 mM and 265 μmol min^?1 mg^?1, respectively. Activation energy for reaction catalyzed by Asn-R5 was 28 kJ mol^?1. High L-asparaginase activity and thermostability of recombinant Asn-R5 may be beneficial for industrial production and application.     

Evaluation of growth and phycobiliprotein composition of cyanobacteria isolates cultivated in different nitrogen sources

Phycobiliproteins, light-harvesting pigments found in cyanobacteria and in some eukaryotic algae, have numerous commercial applications in food, cosmetic, and pharmaceutical industries. Colorant production from cyanobacteria offers advantages over their production from higher plants, as cyanobacteria have fast growth rate and high photosynthetic efficiency and require less space. In this study, three cyanobacteria strains were studied for phycobiliprotein production and the influence of sodium nitrate, potassium nitrate and ammonium chloride on the growth and phycobiliprotein composition of the strains were evaluated. In the batch culture period of 12 days, Phormidium sp. and Pseudoscillatoria sp. were able to utilize all tested nitrogen sources; however, ammonium chloride was the best nitrogen source for both strains to achieve maximum growth rate μ?=?0.284?±?0.03 and μ?=?0.274?±?0.13 day^?1, chlorophyll a 16.2?±? 0.5 and 12.2?±? 0.2 mg L^?1, and phycobiliprotein contents 19.38?±?0.09 and 19.99?±?0.14% of dry weight, whereas, for Arthrospira platensis, the highest growth rate of μ?=?0.304?±?0.0 day^?1, chlorophyll a 19.1?±?0.5 mg L^?1, and phycobiliprotein content of 22.27?±?0.21% of dry weight were achieved with sodium nitrate. The phycocyanin from the lyophilized cyanobacterial biomass was extracted using calcium chloride and food grade purity (A₆₂₀/A₂₈₀ ratio >?0.7) was achieved. Furthermore, phycocyanin was purified using two-step chromatographic method and the analytical grade purity (A₆₂₀/A₂₈₀ ratio >?4) was attained. SDS-PAGE demonstrated the purity and presence of two bands corresponding to α- and β-subunits of the C-phycocyanin. The results showed that Phormidium sp. and Pseudoscillatoria sp. could be good candidates for phycocyanin production.     

Characterization and preliminary mutation analysis of a thermostable alanine racemase from Thermoanaerobacter tengcongensis MB4

A thermostable alanine racemase from Thermoanaerobacter tengcongensis MB4 was successfully expressed in Escherichia coli and characterized. The full-length gene MBalr2 (1164 bp) encodes 388 amino acid residues including 6 out of 8 highly conserved amino acid residues at the entryway to the active site of alanine racemase. Recombinant MBAlr2 and three mutants (S171A, H359Y and double mutation S171A/H359Y) of MBAlr2 were purified by His₆-tag affinity column and gel filtration chromatography. The purified protein MBAlr2 was a dimeric PLP-dependent enzyme with broad substrate specificity. The optimal racemization temperature and pH were 70–75 °C and 11.0, respectively. The kinetic parameters K _m and V _max of MBAlr2 at 70 °C, determined by HPLC, were 20.16 mM and 1414 μmol min^?1 for l-alanine, and 9.95 mM and 702.6 μmol min^?1 for d-alanine, respectively. Enzymatic assays showed that the activity of both mutants (S171A and H359Y) was lost, but the activity of mutant S171A/H359Y was recovered to 69.8 % of wild type, which suggested that residues Ser171 and His359 might be the important residues for catalytic mechanisms of MBAlr2.     

Genetic Evaluation of Physiological Functions of Thiolase Isozymes in the n-Alkane-Assimilating Yeast Candida tropicalis

The n-alkane-assimilating diploid yeast Candida tropicalis possesses three thiolase isozymes encoded by two pairs of alleles: cytosolic and peroxisomal acetoacetyl-coenzyme A (CoA) thiolases, encoded by CT-T1A and CT-T1B, and peroxisomal 3-ketoacyl-CoA thiolase, encoded by CT-T3A and CT-T3B. The physiological functions of these thiolases have been examined by gene disruption. The homozygous ct-t1aΔ/t1bΔ null mutation abolished the activity of acetoacetyl-CoA thiolase and resulted in mevalonate auxotrophy. The homozygous ct-t3aΔ/t3bΔ null mutation abolished the activity of 3-ketoacyl-CoA thiolase and resulted in growth deficiency on n-alkanes (C₁₀ to C₁₃). All thiolase activities in this yeast disappeared with the ct-t1aΔ/t1bΔ and ct-t3aΔ/t3bΔ null mutations. To further clarify the function of peroxisomal acetoacetyl-CoA thiolases, the site-directed mutation leading acetoacetyl-CoA thiolase without a putative C-terminal peroxisomal targeting signal was introduced on the CT-T1A locus in the ct-t1bΔ null mutant. The truncated acetoacetyl-CoA thiolase was solely present in cytoplasm, and the absence of acetoacetyl-CoA thiolase in peroxisomes had no effect on growth on all carbon sources employed. Growth on butyrate was not affected by a lack of peroxisomal acetoacetyl-CoA thiolase, while a retardation of growth by a lack of peroxisomal 3-ketoacyl-CoA thiolase was observed. A defect of both peroxisomal isozymes completely inhibited growth on butyrate. These results demonstrated that cytosolic acetoacetyl-CoA thiolase was indispensable for the mevalonate pathway and that both peroxisomal acetoacetyl-CoA thiolase and 3-ketoacyl-CoA thiolase could participate in peroxisomal β-oxidation. In addition to its essential contribution to the β-oxidation of longer-chain fatty acids, 3-ketoacyl-CoA thiolase contributed greatly even to the β-oxidation of a C₄ substrate butyrate.