Studies on Core-Shell Nanocapsules of Felodipine: <Emphasis Type="Italic">In Vitro</Emphasis>-<Emphasis Type="Italic">In Vivo</Emphasis> Evaluations

The present study aimed for in vitro-in vivo-in silico simulation studies of experimentally designed (3²-factorial) Capmul PG-8-cored, Eudragit RSPO-Lutrol F 127 nanocapsules to ferry felodipine using GastroPlus?. The in silico parameter sensitivity analysis for pharmacokinetic parameters was initially assessed to justify the preparation of felodipine-loaded nanocapsules (FLNs) with enhanced solubility to overcome the bioavailability issues of felodipine. The overall integrated desirability ranged between 0.8187 and 0.9488 for three optimized FLNs when analyzed for mean particle size, zeta potential, encapsulation efficiency, and in vitro dissolution parameters. The morphological evaluation (SEM, TEM, and AFM) demonstrated spherical nanoparticles (200–300 nm). Validated LC-MS/MS analysis demonstrated enhanced relative bioavailability (13.37-fold) of optimized FLN as compared to suspension. The simulated regional absorption of the FLN presented significant absorption from the cecum (26.3%) and ascending colon (20.1%) with overall absorption of 67.4% from the GIT tract. Furthermore, in vitro-in vivo correlation demonstrated the Wagner-Nelson method as the preferred model as compared to mechanistic and numerical deconvolution on the basis of least mean absolute prediction error, least standard error of prediction, least mean absolute error, and maximum correlation coefficient (r ² = 0.920). The study demonstrated enhanced oral absorption of felodipine-loaded nanocapsules, and GastroPlus? was found to be an efficient simulation tool for in vitro-in vivo-in silico simulations.     

Single point mutations enhance activity of <Emphasis Type="Italic">cis</Emphasis>-epoxysuccinate hydrolase

Objectives

To enhance activity of cis-epoxysuccinate hydrolase from Klebsiella sp. BK-58 for converting cis-epoxysuccinate to tartrate.

Results

By semi-saturation mutagenesis, all the mutants of the six important conserved residues almost completely lost activity. Then random mutation by error-prone PCR and high throughput screening were further performed to screen higher activity enzyme. We obtained a positive mutant F10D after screening 6000 mutations. Saturation mutagenesis on residues Phe10 showed that most of mutants exhibited higher activity than the wild-type, and the highest mutant was F10Q with activity of 812 U mg^?1 (k _cat /K _m , 9.8 ± 0.1 mM^?1 s^?1), which was 230 % higher than that of wild-type enzyme 355 U mg^?1 (k _cat /K _m , 5.3 ± 0.1 mM^?1 s^?1). However, the thermostability of the mutant F10Q slightly decreased.

Conclusions

The catalytic activity of a cis-epoxysuccinate hydrolase was efficient improved by a single mutation F10Q and Phe10 might play an important role in the catalysis.

     

Inheritance pattern and genetic correlations among growth and wood quality traits in Para rubber tree (<Emphasis Type="Italic">Hevea brasiliensis</Emphasis>) and implications for breeding

Inheritance pattern of wood traits viz. specific gravity, fibre dimensions and fibre-derived biometrical indices and their interactions among themselves and with that of growth are reported in Hevea brasiliensis. Girth (h² =???0.02?±?0.44 to h² =?0.35?±?0.24) showed moderate genetic control. Among wood traits, specific gravity (h²?=?0.15?±?0.31 to h² =?0.33?±?0.28) was found to be under moderate genetic control. Fibre traits viz., fibre length (h² =???0.26?±?0.30 to h² =?0.50?±?0.34), fibre diameter (h² =?0.19?±?0.49 to h² =?0.70?±?0.11), fibre lumen diameter (h² =???0.18?±?0.35 to h² =?0.56?±?0.47) and fibre wall thickness (h² =???5.17?±?5.26 to h² =?0.50?±?0.50) were under moderate to strong genetic control. Among fibre-derived indices, flexibility coefficient (h² =?0.48?±?0.21 to h² =?0.89?±?0.29) showed moderate to very strong genetic control. The Runkel ratio (h² =???0.40?±?0.27 to h² =?0.42?±?0.29) and slenderness ratio (h² =???0.36?±?0.29 to h² =?0.43?±?0.28) showed moderate genetic control. Girth showed very strong positive genetic correlation with fibre wall thickness and strong positive correlation with fibre width indicating scope of indirect selection potential for these traits. Wood specific gravity was not correlated with either girth or fibre traits. Hence, it would be possible to concomitantly improve growth and fibre traits without adversely affecting wood specific gravity. Moderate to very high estimates of heritability for fibre traits, girth and specific gravity indicated that considerable genetic gain can be realised for these traits. Implications of the above findings in genetic improvement of wood in Hevea are discussed.     

<Emphasis Type="Italic">Acorus calamus</Emphasis> Linn.: phytoconstituents and bactericidal property

Acorus calamus Linn. of the family Araceae (Acoraceae), commonly known as Sweet Flag and Vacha. The rhizome of this plant has medicinal properties against bugs, moths, lice and emetic stomach in dyspepsia. Chemical composition of the hydro-distilled essential oil obtained from the rhizomes of A. calamus was analyzed by gas chromatography equipped with flame ionization detector and gas chromatography coupled with mass spectrometry. The essential oil of A. calamus and its major compound β-asarone were tested against five Gram-positive, eight Gram-negative bacteria, and three fungi by the tube-dilution method at a concentration rang of 5.0–0.009 mg/mL. Forty constituents were identified which comprised 98.3 % of the total oil. The major compound β-asarone (80.6 %) was identified and confirm by NMR (¹H– & ¹³C–) in rhizome oil of A. calamus. The organism Micrococcus luteus was found to be more susceptible to the oil with minimum bactericidal concentration (MBC) value of 0.032 ± 0.004 mg/mL, followed by Aspergillus fumigatus, Aspergillus niger and Micrococcus flavus with MBC values of 0.104 ± 0.016, 0.117 ± 0.017 and 0.143 ± 0.013 mg/mL, respectively. The compound β-asarone was susceptible to the microorganism A. niger with MBC value 0.416 ± 0.065 mg/mL. The present study revealed that tetraploid variety of A. calamus is growing in this region with substantial amount of β-asarone. The oil showed bactericidal property against tested bacteria and fungi. The β-asarone exhibited poorer bactericidal activity against test microorganisms.     

<Emphasis Type="Italic">Scopulibacillus cellulosilyticus</Emphasis> sp. nov., a cellulose-degrading bacterium isolated from tea

A Gram-stain positive, aerobic, non-motile, endospore-forming and rod-shaped strain (THG-NT9^T) was isolated from a green tea sample. Growth occurred at 20–45 °C (optimum 28–35 °C), at pH 6.0–8.0 (optimum 7.0) and at 0–2.0% NaCl (optimum 0%). Based on 16S rRNA gene sequence analysis, the near phylogenetic neighbours of strain THG-NT9^T were identified as Scopulibacillus daqui DSM 28236^T (98.6%), Scopulibacillus darangshiensis DSM 19377^T (97.4%), Pullulanibacillus pueri CGMCC 1.12777^T (96.7%) and Pullulanibacillus camelliae CGMCC 1.15371^T (96.3%). The DNA G?+?C content of strain THG-NT9^T was determined to be 47.5 mol %. DNA–DNA hybridization values between strain THG-NT9^T and S. daqui DSM 28236^T, S. darangshiensis DSM 19377^T, P. pueri CGMCC 1.12777^T, P. camelliae CGMCC 1.15371^T and Pullulanibacillus naganoensis DSM 10191^T were 41.3?±?0.1 (39.4?±?0.4% reciprocal analysis), 39.1?±?0.1 (37.3?±?0.1%), 21.4?±?0.7 (20.1?±?0.3%), 20.7?±?0.1 (20.1?±?0.4%) and 12.1?±?0.2% (8.3?±?0.2%). The polar lipids were identified as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and three unidentified lipids. The quinone was identified as MK-7. The major fatty acids were C_18:3 ω7c, iso-C_15:0, iso-C_16:0, iso-C_17:0 and anteiso-C_17:0. The cell wall type was determined to be A1γ peptidoglycan with meso-diaminopimelic acid as the diagnostic diamino acid plus alanine and glutamic acid and glucose as the cell wall sugar. On the basis of the phylogenetic analysis, chemotaxonomic data, physiological characteristics, and DNA–DNA hybridization data, strain THG-NT9^T represents a novel species of the genus Scopulibacillus, for which the name Scopulibacillus cellulosilyticus sp. nov. is proposed. The type strain is THG-NT9^T (=?KCTC 33918^T?=?CGMCC 1.16305^T).     

The nitrite reductase activity of horse heart carboxymethylated-cytochrome <Emphasis Type="Italic">c</Emphasis> is modulated by cardiolipin

Horse heart carboxymethylated cytc (CM-cytc) displays myoglobin-like properties. Here, the effect of cardiolipin (CL) liposomes on the nitrite reductase activity of ferrous CM-cytc [CM-cytc-Fe(II)], in the presence of sodium dithionite, is reported between pH 5.5 and 7.6, at 20.0 °C. Cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO₂ ^?-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO [k _on = (7.3 ± 0.7) × 10^?2 M^?1 s^?1; at pH 7.4], whereas the value of k _on for NO₂ ^? reduction by CM-cytc-Fe(II) is 1.1 ± 0.2 M^?1 s^?1 (at pH 7.4). CL facilitates the NO₂ ^?-mediated nitrosylation of CM-cytc-Fe(II) in a dose-dependent manner, the value of k _on for the NO₂ ^?-mediated conversion of CL–CM-cytc-Fe(II) to CL–CM-cytc-Fe(II)-NO (5.6 ± 0.6 M^?1 s^?1; at pH 7.4) being slightly higher than that for the NO₂ ^?-mediated conversion of CL–cytc-Fe(II) to CL–cytc-Fe(II)-NO (2.6 ± 0.3 M^?1 s^?1; at pH 7.4). The apparent affinity of CL for CM-cytc-Fe(II) is essentially pH independent, the average value of B being (1.3 ± 0.3) × 10^?6 M. In the absence and presence of CL liposomes, the nitrite reductase activity of CM-cytc-Fe(II) increases linearly on lowering pH and the values of the slope of the linear fittings of Log k _on versus pH are ?1.05 ± 0.07 and ?1.03 ± 0.03, respectively, reflecting the involvement of one proton for the formation of the transient ferric form, NO, and OH^?. These results indicate that Met80 carboxymethylation and CL binding cooperate in the stabilization of the highly reactive heme-Fe atom of CL–CM-cytc.     

<Emphasis Type="Italic">Novosphingobium profundi</Emphasis> sp. nov. isolated from a deep-sea seamount

A marine bacterial strain, F72^T, was isolated from a solitary scleractinian coral, collected in Yap seamounts in the Pacific Ocean. Strain F72^T is a Gram-negative, light-yellow-pigmented, motile, rod-shaped bacterium. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain F72^T is related to the genus Novosphingobium and has high 16S rRNA gene sequence similarities with the type strains of Novosphingobium pentaromativorans US6-1^T (97.7 %), Novosphingobium panipatense SM16^T (97.6 %), Novosphingobium mathurense SM117^T (97.2 %) and Novosphingobium barchaimii LL02^T (97.1 %). Ubiquinone Q-10 was detected as the dominant quinone. The predominant cellular fatty acids were C_18:1ω7c and C_17:1ω6c. The genomic DNA G+C content of strain F72^T was 63.4 mol %. The polar lipids profile contained phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylmethylethanolamine, phosphatidylcholine, sphingoglycolipid and one uncharacterized lipid. Strain F72^T shared DNA relatedness of 25 % with N. pentaromativorans JCM 12182^T, 31 % with N. panipatense DSM 22890^T, 21 % with N. mathurense DSM 23374^T and 26 % with N. barchaimii DSM 25411^T. Combined data from phenotypic, phylogenetic and DNA–DNA relatedness studies demonstrated that the strain F72^T is a representative of a novel species of the genus Novosphingobium, for which we propose the name Novosphingobium profundi sp. nov. (type strain F72^T = KACC 18566^T = CGMCC 1.15390^T).     

Diversity of low-molecular weight organic acids synthesized by <Emphasis Type="Italic">Salix</Emphasis> growing in soils characterized by different Cu,Pb and Zn concentrations

The aim of the study was to evaluate the biosynthesis and exudation of 10 low-molecular weight organic acids (LMWOAs) into the rhizosphere with a simultaneous analysis of the acid contents in the roots and leaves of 9 Salix taxa growing on two experimental areas, differing in their concentrations of copper (Cu), lead (Pb) and zinc (Zn) in the soil (Area 1—low, Area 2—high concentration). The obtained results reveal a significant difference in the phytoextraction of the tested Salix taxa for the analysed metals in both areas. The highest contents of Cu, Pb and Zn were observed for all Salix collected from Area 2, especially in S. × smithiana roots (116 ± 8.76, 87.84 ± 7.30 and 203.42 ± 14.62 mg kg^?1 DW, respectively). The results obtained in Area 2 also revealed acidification of the rhizosphere and a higher concentration of acids, mainly oxalic, malic, malonic, acetic and citric acids. Contents of oxalic, malic, acetic and citric acids increased in the roots of Salix taxa from Area 2, while in the leaves formic and succinic acids were also present. S. × smithiana was the taxon with the highest concentration of acids in the rhizosphere and roots (73.48 ± 6.77 and 49.79 ± 2.65 μM 100 g^?1 DW, respectively), while in leaves a higher content was observed for S. alba and S. viminalis ‘PR’ taxa (78.12 ± 3.95 and 71.12 ± 3.75 μM 100 g^?1 DW, respectively).     

<Emphasis Type="Italic">Halomonas heilongjiangensis</Emphasis> sp. nov., a novel moderately halophilic bacterium isolated from saline and alkaline soil

A moderately halophilic bacterium, designated strain 9-2^T, was isolated from saline and alkaline soil collected in Lindian county, Heilongjiang province, China. The strain was observed to be strictly aerobic, Gram-negative, rod-shaped, oxidase-positive, catalase-positive and motile. It was found to require NaCl for growth and to grow at NaCl concentrations of 0.5–14 % (w/v) (optimum, 7–10 %, w/v), at temperatures of 10–45 °C (optimum 25–30 °C) and at pH 5.0–10.0 (optimum pH 8.0). Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain 9-2^T is a member of the genus Halomonas and is closely related to Halomonas desiderata DSM 9502^T (96.68 %), Halomonas campaniensis DSM 1293^T (96.46 %), Halomonas ventosae DSM 15911^T (96.27 %) and Halomonas kenyensis DSM 17331^T (96.27 %). The DNA–DNA hybridization value was 38.9 ± 0.66 % between the novel isolate 9-2^T and H. desiderata DSM 9502^T. The predominant ubiquinones were identified as Q9 (75.1 %) and Q8 (24.9 %). The major fatty acids were identified as C_16:0 (22.0 %), Summed feature 8 (C_18:1 ω6c/C_18:1 ω7c, 19.6 %), Summed feature 3 (C_16:1 ω6c/C_16:1 ω7c, 12.6 %), C_12:0 3-OH (12.0 %) and C_10:0 (11.7 %). The DNA G+C content was determined to be 69.7 mol%. On the basis of the evidence presented in this study, strain 9-2^T is considered to represent a novel species of the genus Halomonas, for which the name Halomonas heilongjiangensis sp. nov. is proposed. The type strain is 9-2^T (=DSM 26881^T = CGMCC 1.12467^T).     

Toxin Gene Contents and Activity of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Strains Against Two Sugarcane Borer Species, <Emphasis Type="Italic">Diatraea saccharalis</Emphasis> (F.) and <Emphasis Type="Italic">D. flavipennella</Emphasis> (Box)

Bacillus thuringiensis (Berliner) bears essential characteristics in the control of insect pests, such as its unique mode of action, which confers specificity and selectivity. This study assessed cry gene contents from Bt strains and their entomotoxicity against Diatraea saccharalis (F.) and Diatraea flavipennella (Box) (Lepidoptera: Crambidae). Bioassays with Bt strains were performed against neonates to evaluate their lethal and sublethal activities and were further analyzed by PCR, using primers to identify toxin genes. For D. saccharalis and D. flavipennella, 16 and 18 strains showed over 30% larval mortality in the 7th day, respectively. The LC₅₀ values of strains for D. saccharalis varied from 0.08 × 10⁵ (LIIT-0105) to 4104 × 10⁵ (LIIT-2707) spores + crystals mL^?1. For D. flavipennella, the LC₅₀ values of strains varied from 0.40 × 10⁵ (LIIT-2707) to 542 × 10⁵ (LIIT-2109) spores + crystals mL^?1. For the LIIT-0105 strain, which was the most toxic to D. saccharalis, the genes cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C, cry1D, cry1F, cry1I, cry2Aa, cry2Ab, cry8, and cry9C were detected, whereas for the strain LIIT-2707, which was the most toxic to D. flavipennella, detected genes were cry1Aa, cry1Ab, cry1Ac, cry1B, cry1D, cry1F, cry1I, cry2Aa, cry2Ab, and cry9. The toxicity data and toxin gene content in these strains of Bt suggest a great variability of activity with potential to be used in the development of novel biopesticides or as source of resistance genes that can be expressed in plants to control pests.     

Artificial induction of genetic competence in <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> isolates

Objectives

To induce natural genetic competence in Bacillus amyloliquefaciens isolates through overexpression of the master regulator, ComK, from B. subtilis (ComK _Bsu ).

Results

Plasmid pUBXC carrying the xylose-inducible comK expression cassette was constructed using plasmid pUB110 as a backbone. Plasmid pUBXC could be transferred from B. subtilis to B. amyloliquefaciens through plasmid pLS20-mediated biparental conjugation. After being induced by xylose, four B. amyloliquefaciens strains harbouring plasmid pUBXC developed genetic competence. Under optimal conditions, the transformation efficiencies of plasmid DNA ranged from 129 ± 20.6 to 1.7 ± 0.1 × 10⁵ cfu (colony-forming units) per μg DNA, and the transformation efficiencies of PCR-assembled deletion constructs ranged from 3.2 ± 0.76 to 3.5 ± 0.42 × 10⁴ cfu per μg DNA in the four tested strains.

Conclusion

Artificial induction of genetic competence through overexpressing ComK _Bsu in B. amyloliquefaciens completed the tasks of replicative plasmid delivery and gene knockout via direct transformation of PCR-generated deletion cassettes.

     

Development of a powder formulation based on <Emphasis Type="Italic">Bacillus</Emphasis><Emphasis Type="Italic">cereus</Emphasis> sensu lato strain B25 spores for biological control of <Emphasis Type="Italic">Fusarium verticillioides</Emphasis> in maize plants

Maize is an economically important crop in northern Mexico. Different fungi cause ear and root rot in maize, including Fusarium verticillioides (Sacc.) Nirenberg. Crop management of this pathogen with chemical fungicides has been difficult. By contrast, the recent use of novel biocontrol strategies, such as seed bacterization with Bacillus cereus sensu lato strain B25, has been effective in field trials. These approaches are not without their problems, since insufficient formulation technology, between other factors, can limit success of biocontrol agents. In response to these drawbacks, we have developed a powder formulation based on Bacillus B25 spores and evaluated some of its characteristics, including shelf life and efficacy against F. verticillioides, in vitro and in maize plants. A talc-based powder formulation containing 1 × 10⁹ c.f.u. g^?1 was obtained and evaluated for seed adherence ability, seed germination effect, shelf life and antagonism against F. verticillioides in in vitro and in planta assays. Seed adherence of viable bacterial spores ranged from 1.0 to 1.41 × 10⁷ c.f.u. g^?1. Bacteria did not display negative effects on seed germination. Spore viability for the powder formulation slowly decreased over time, and was 53 % after 360 days of storage at room temperature. This formulation was capable of controlling F. verticillioides in greenhouse assays, as well as eight other maize phytopathogenic fungi in vitro. The results suggest that a talc-based powder formulation of Bacillus B25 spores may be sufficient to produce inoculum for biocontrol of maize ear and root rots caused by F. verticillioides.     

<Emphasis Type="Italic">Microbacterium rhizosphaerae</Emphasis> sp. nov., isolated from a Ginseng field,South Korea

A novel Gram-stain positive, aerobic, short rod-shaped, non-motile bacterium, designated strain CHO1^T, was isolated from rhizosphere soil from a ginseng agriculture field. Strain CHO1^T was observed to form yellow colonies on R2A agar medium. The cell wall peptidoglycan was found to contain alanine, glycine, glutamic acid, d-ornithine and serine. The cell wall sugars were identified as galactose, mannose, rhamnose and ribose. Strain CHO1^T was found to contain MK-11, MK-12, MK-13 as the predominant menaquinones and anteiso-C_15:0, iso-C_16:0, and anteiso-C_17:0 as the major fatty acids. Diphosphatidylglycerol, phosphatidylglycerol, phosphoglycolipid, an unidentified phospholipid and three unidentified glycolipids were found to be present in strain CHO1^T. Based on 16S rRNA gene sequence analysis, strain CHO1^T was found to be closely related to Microbacterium mangrovi DSM 28240^T (97.81 % similarity), Microbacterium immunditiarum JCM 14034^T (97.45 %), Microbacterium oryzae JCM 16837^T (97.33 %) and Microbacterium ulmi KCTC 19363^T (97.10 %) and to other species of the genus Microbacterium. The DNA G+C content of CHO1^T was determined to be 70.1 mol %. The DNA–DNA hybridization values of CHO1^T with M. mangrovi DSM 28240^T, M. immunditiarum JCM 14034^T, M. oryzae JCM 16837^T and M. ulmi KCTC 19363^T were 46.7 ± 2, 32.4 ± 2, 32.0 ± 2 and 29.2 ± 2 %, respectively. On the basis of genotypic, phenotypic and phylogenetic properties, it is concluded that strain CHO1^T represents a novel species within the genus Microbacterium, for which the name Microbacterium rhizosphaerae sp. nov. is proposed. The type strain of M. rhizosphaerae is CHO1^T (= KEMB 7306-513^T = JCM 31396^T).     

Hemato-Immunological Responses and Disease Resistance in Siberian Sturgeon <Emphasis Type="Italic">Acipenser baerii</Emphasis> Fed on a Supplemented Diet of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis>

A feeding trial was conducted to investigate the effects of different levels of dietary Lactobacillus plantarum on hemato-immunological parameters and resistance against Streptococcus iniae infection in juvenile Siberian sturgeon Acipenser baerii. Fish (14.6 ± 2.3 g) were fed three experimental diets prepared by supplementing a basal diet with L. plantarum at different concentrations [1 × 10⁷, 1 × 10⁸ and 1 × 10⁹ colony-forming units (cfu) g^?1] and a control (non-supplemented basal) diet for 8 weeks. Innate immune responses (immunoglobulin (Ig), alternative complement activity (ACH50) and lysozyme activity) were significantly higher in fish fed the 1 × 10⁸ and 1 × 10⁹ cfu g^?1 L. plantarum diet compared to the other groups (P < 0.05). Furthermore, fish fed on various levels of L. plantarum significantly showed higher red blood cell (RBC), hemoglobin (Hb), white blood cell (WBC) and monocyte compared to those of the control group (P < 0.05). At the end of the feeding experiment, some fish were challenged with S. iniae to quantify the level of disease resistance. The mortality after S. iniae challenge was decreased in fish fed a probiotic. These results indicated that dietary supplementation of L. plantarum improved immune response and disease resistance of Siberian sturgeon juvenile.     

Growth kinetics and fucoxanthin production of <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> and <Emphasis Type="Italic">Isochrysis galbana</Emphasis> cultures at different light and agitation conditions

Fucoxanthin is a carotenoid that exerts multiple beneficial effects on human health. However, reports comparing microalgae culture conditions and their effect on growth and fucoxanthin production are still limited. Isochrysis galbana and Phaeodactylum tricornutum cultures in different light (62.0, 25.9, 13.5, or 9.1 μmol photons m^-2 s^-1), mixing conditions (1 vvm aeration or 130 rpm agitation), and media compositions (F/2 and Conway medium) were studied for comparison of cellular growth and fucoxanthin production on F/2 medium. I. galbana showed a better adaptation to tested culture conditions in comparison with P. tricornutum, reaching 2.15?×?10⁷?±?4.07?×?10⁶ cells mL^-1 and a specific growth rate (μ) of 1.12?±?0.05 day^-1 under aerated conditions and 62.0 μmol photons m^-2 s^-1 light intensity. Fucoxanthin concentration was about 25 % higher in P. tricornutum cultures under 13.5 μmol photons m^-2 s^-1 light intensity and aerated conditions, but the highest fucoxanthin total production was higher in I. galbana, where 3.32 mg can be obtained from 1 L batch cultures at the 16th day under these conditions. Moreover, higher cell densities (~32.41 %), fucoxanthin concentration (~42.46 %), and total production (~50.68 %) were observed in I. galbana cultures grown in Conway medium, if compared with cultures grown in F/2 medium. The results show that the best growth conditions did not result in the best fucoxanthin production for either microalgae, implying that there is not a direct relationship between cellular growth and fucoxanthin production. Moreover, the results suggest that I. galbana cultures on Conway medium are strong candidates for fucoxanthin production, where 1.2 to 15 times higher fucoxanthin concentration are observed in comparison to macroalgal sources.     

Growth and phosphorus removal by <Emphasis Type="Italic">Synechococcus elongatus</Emphasis> co-immobilized in alginate beads with <Emphasis Type="Italic">Azospirillum brasilense</Emphasis>

This study examined the co-immobilization of the cyanobacterium Synechococcus elongatus with the plant growth-promoting bacterium Azospirillum brasilense in alginate beads and its potential application for the removal of phosphorus from aquaculture wastewater. Co-immobilization of both microorganisms significantly increased the cell density of S. elongatus (2852.5?×?10⁴ cells mL^?1) compared with that of immobilization of cyanobacteria alone (1325.2?×?10⁴ cells mL^?1). Chlorophyll a content was similar in co-immobilized (11.1?±?3.5 pg cell^?1) and immobilized S. elongatus (14.5?±?4.9 pg cell^?1). Azospirillum brasilense showed continuous growth until day 2, after which its cell concentration declined until the end of the assay. Co-immobilized S. elongatus removed more phosphorus (44.8 %) than immobilized cyanobacteria cells alone (32.0 %). In conclusion, phosphate removal was greater with free cells of S. elongatus but overlapped with the values that were obtained with the treatment of co-immobilization of cells. Our results demonstrate that A. brasilense enhances the growth of S. elongatus and improves its removal of phosphorus when they are co-immobilized in alginate beads compared with only immobilization of cyanobacteria cells alone.     

<Emphasis Type="Italic">Rhizobium hedysari</Emphasis> sp. nov., a novel species isolated from a root nodule of <Emphasis Type="Italic">Hedysarum multijugum</Emphasis> in China

A strain 5-1-2^T was isolated from a root nodule of Hedysarum multijugum collected from Zhangye city, Gansu province, north-west China. Phylogenetic analysis based on the 16S rRNA gene sequence and other housekeeping genes (recA and atpD) indicated that the strain represents a novel species in the genus Rhizobium close to the strain Rhizobium subbaraonis JC85^T with similarities of 98.27, 88.92 and 89.62%, respectively. Strain 5-1-2^T contained Q-10 as the predominant ubiquinone. Our results showed that the major fatty acids were feature 8 (C_18:1 ω7c and/or C_18:1 ω6c; 38.90%). In addition, the DNA–DNA hybridizations with the type strains R. subbaraonis JC85^T and Rhizobium halophytocola YC6881^T were 39.2 ± 2.1 and 44.3 ± 1.9, respectively. Therefore, a novel species Rhizobium hedysari sp. nov. is proposed, and 5-1-2^T (=CGMCC1.15677^T = NBRC112532^T) is designated as the type strain.     

Comparison of bioethanol production from cultivated versus wild <Emphasis Type="Italic">Gracilaria verrucosa</Emphasis> and <Emphasis Type="Italic">Gracilaria gigas</Emphasis>

The seaweed genus Gracilaria is a potential candidate for the production of bioethanol due to its high carbohydrate content. Gracilaria is abundant throughout the world and can be found in both wild and cultivated forms. Differences in the ecological factors such as temperature, salinity, and light intensity affecting wild and cultivated specimens may influence the biochemical content of seaweeds, including the carbohydrate content. This study aimed to investigate the proximate composition and potential bioethanol production of wild and cultivated G. gigas and G. verrucosa. Bioethanol was produced using separate hydrolysis fermentation (SHF), employing a combination of enzymatic and acid hydrolysis, followed by fermentation with Saccharomyces cerevisiae ATCC 200062. The highest carbohydrate content was found in wild G. gigas. The highest galactose and glucose contents (20.21 ± 0.32 and 9.70 ± 0.49 g L^?1, respectively), as well as the highest production of bioethanol (3.56 ± 0.02 g L^?1), were also found in wild G. gigas. Thus, we conclude that wild G. gigas is the most promising candidate for bioethanol production. Further research is needed to optimize bioethanol production from wild G. gigas. Domestication of wild G. gigas is a promising challenge for aquaculture to avoid overexploitation of this wild seaweed resource.     

Phosphoenolpyruvate-supply module in <Emphasis Type="Italic">Escherichia coli</Emphasis> improves <Emphasis Type="Italic">N</Emphasis>-acetyl-<Emphasis Type="SmallCaps">d</Emphasis>-neuraminic acid biocatalysis

Objectives

N-Acetyl-d-neuraminic acid (Neu5Ac) is often synthesized from exogenous N-acetylglucosamine (GlcNAc) and excess pyruvate. We have previously constructed a recombinant Escherichia coli strain for Neu5Ac production using GlcNAc and intracellular phosphoenolpyruvate (PEP) as substrates (Zhu et al. Biotechnol Lett 38:1–9, 2016).

Results

PEP synthesis-related genes, pck and ppsA, were overexpressed within different modes to construct PEP-supply modules, and their effects on Neu5Ac production were investigated. All the PEP-supply modules enhanced Neu5Ac production. For the best module, pCDF-pck-ppsA increased Neu5Ac production to 8.6 ± 0.15 g l^?1, compared with 3.6 ± 0.15 g l^?1 of the original strain. Neu5Ac production was further increased to 15 ± 0.33 g l^?1 in a 1 l fermenter.

Conclusions

The PEP-supply module can improve the intracellular PEP supply and enhance Neu5Ac production, which benefited industrial Neu5Ac production.