Molecular characterization of Alr1105 a novel arsenate reductase of the diazotrophic cyanobacterium Anabaena sp. PCC7120 and decoding its role in abiotic stress management in Escherichia coli

This paper constitutes the first report on the Alr1105 of Anabaena sp. PCC7120 which functions as arsenate reductase and phosphatase and offers tolerance against oxidative and other abiotic stresses in the alr1105 transformed Escherichia coli. The bonafide of 40.8 kDa recombinant GST+Alr1105 fusion protein was confirmed by immunoblotting. The purified Alr1105 protein (mw 14.8 kDa) possessed strong arsenate reductase (Km 16.0 ± 1.2 mM and Vmax 5.6 ± 0.31 μmol min^?1 mg protein^?1) and phosphatase activity (Km 27.38 ± 3.1 mM and Vmax 0.077 ± 0.005 μmol min^?1 mg protein^?1) at an optimum temperature 37 °C and 6.5 pH. Native Alr1105 was found as a monomeric protein in contrast to its homologous Synechocystis ArsC protein. Expression of Alr1105 enhanced the arsenic tolerance in the arsenate reductase mutant E. coli WC3110 (?arsC) and rendered better growth than the wild type W3110 up to 40 mM As (V). Notwithstanding above, the recombinant E. coli strain when exposed to CdCl₂, ZnSO₄, NiCl₂, CoCl₂, CuCl₂, heat, UV-B and carbofuron showed increase in growth over the wild type and mutant E. coli transformed with the empty vector. Furthermore, an enhanced growth of the recombinant E. coli in the presence of oxidative stress producing chemicals (MV, PMS and H₂O₂), suggested its protective role against these stresses. Appreciable expression of alr1105 gene as measured by qRT-PCR at different time points under selected stresses reconfirmed its role in stress tolerance. Thus the Alr1105 of Anabaena sp. PCC7120 functions as an arsenate reductase and possess novel properties different from the arsenate reductases known so far.     

Characterization and application of a newly synthesized 2-deoxyribose-5-phosphate aldolase

A codon-optimized 2-deoxyribose-5-phosphate aldolase (DERA) gene was newly synthesized and expressed in Escherichia coli to investigate its biochemical properties and applications in synthesis of statin intermediates. The expressed DERA was purified and characterized using 2-deoxyribose-5-phosphate as the substrate. The specific activity of recombinant DERA was 1.8 U/mg. The optimum pH and temperature for DERA activity were pH 7.0 and 35 °C, respectively. The recombinant DERA was stable at pH 4.0–7.0 and at temperatures below 50 °C. The enzyme activity was inhibited by 1 mM of Ni²⁺, Ba²⁺ and Fe²⁺. The apparent K _m and V _max values of purified enzyme for 2-deoxyribose-5-phosphate were 0.038 mM and 2.9 μmol min^?1 mg^?1, for 2-deoxyribose were 0.033 mM and 2.59 μmol min^?1 mg^?1, respectively, which revealed that the enzyme had similar catalytic efficiency towards phosphorylated and non-phosphorylated substrates. To synthesize statin intermediates, the bioconversion process for production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose from chloroacetaldehyde and acetaldehyde by the recombinant DERA was developed and a conversion of 94.4 % was achieved. This recombinant DERA could be a potential candidate for application in production of (3R, 5S)-6-chloro-2,4,6-trideoxyhexose.     

In vitro and in vivo analyses of the role of the carboxysomal β-type carbonic anhydrase of the cyanobacterium Synechococcus elongatus in carboxylation of ribulose-1,5-bisphosphate

The carboxylase activities of crude carboxysome preparations obtained from the wild-type Synechococcus elongatus strain PCC 7942 strain and the mutant defective in the carboxysomal carbonic anhydrase (CA) were compared. The carboxylation reaction required high concentrations of bicarbonate and was not even saturated at 50 mM bicarbonate. With the initial concentrations of 50 mM and 25 mM for bicarbonate and ribulose-1,5-bisphosphate (RuBP), respectively, the initial rate of RuBP carboxylation by the mutant carboxysome (0.22 μmol mg^?1 protein min^?1) was only 30 % of that observed for the wild-type carboxysomes (0.71 μmol mg^?1 protein min^?1), indicating the importance of the presence of CA in efficient catalysis by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). While the mutant defective in the ccmLMNO genes, which lacks the carboxysome structure, could grow under aeration with 2 % (v/v) CO₂ in air, the mutant defective in ccaA as well as ccmLMNO required 5 % (v/v) CO₂ for growth, indicating that the cytoplasmically localized CcaA helped utilization of CO₂ by the cytoplasmically localized Rubisco by counteracting the action of the CO₂ hydration mechanism. The results predict that overexpression of Rubisco would hardly enhance CO₂ fixation by the cyanobacterium at CO₂ levels lower than 5 %, unless Rubisco is properly organized into carboxysomes.     

Electrochemical selective detection of dopamine on microbial carbohydrate-doped multiwall carbon nanotube-modified electrodes

Microbial carbohydrate-doped multiwall carbon nanotube (MWNT)-modified electrodes were prepared for the purpose of determining if 4-(2-aminoethyl)benzene-1,2-diol (3,4-dihydroxyphenylalanine; dopamine) exists in the presence of 0.5 mM ascorbic acid, a representative interfering agent in neurotransmitter detection. The microbial carbohydrate dopants were α-cyclosophorohexadecaose (α-C16) from Xanthomonas oryzae and cyclic-(1 → 2)-β-d-glucan (Cys) from Rhizobium meliloti. The cyclic voltammetric responses showed that the highest sensitivity (5.8 × 10^?3 mA cm^?2 μM^?1) is attained with the Cys-doped MWNT-modified ultra-trace carbon electrode, and that the α-C16-doped MWNT-modified glassy carbon electrode displays the best selectivity to dopamine (the approximate peak potential separation is 310 mV).     

First Thermostable Endo-β-1,4-Glucanase from Newly Isolated Xanthomonas sp. EC102

A novel gene encoding thermostable endoglucanase was identified in Xanthomonas sp. EC102 from soil. The gene had 1,458 base pairs of open reading frame, which encode a 52-kDa protein of 486 amino acid residues. Sequence of the amino acid residues was similar with the endoglucanase from Xanthomonas campestris pv. campestris ATCC33913 (GenBank Accession No. NP_638867.1) (94 % identity). The endoglucanase was overexpressed in Escherichia coli BL21 and purified. Temperature for the highest enzymatic activity was 70 °C and pH optima was pH 5.5. The specific activity of the endoglucanase toward carboxymethylcellulose (CMC) was approximately 2 μmol min^?1 mg^?1, V _max for CMC was 1.44 μmol mg^?1 min^?1, and K _m values was 25.6 mg mL^?1. The EC102 endoglucanase was stable at temperatures up to 60 °C, and it was activated by 0.1 mM of Mn²⁺ and Co²⁺. This is the first report about thermostable endoglucanase from Xanthomonas sp.     

THE SYNTHESIS OF GLUTAMATE BY RAT BRAIN MITOCHONDRIA

The synthesis of glutamate from 2-oxoglutarate generated by the citric acid cycle and ammonium acetate has been studied in brain mitochondria of synaptic or non synaptic origin. Non synaptic brain mitochondria synthesise glutamate at twice the rate (1.3 nmol. min^?1. mg protein^?1) of synaptic mitochondria (0.65 nmol. min^?1. mg protein^?1) when pyruvate is the precursor for 2-oxoglutarate, but at a similar rate (0.9 and 0.7 nmol. min^?1, mg protein^?1) when 3 hydroxybutyrate is the precursor. Glutamate synthesis from ammonium acetate and extramitochondrially addcd 2-oxoglutarate (5 mM) by both synaptic and nonsynaptic mitochondria was 5-fold higher (5-6nmol. min^?1. mg protein^?1) than glutamate synthesis from endogenously produced 2-oxoglutarate. In the uncoupled state (or un-coupler + oligomycin) the rate was reduced by half. (2.5-3 nmol. min^?1. mg protein^?1) as compared to mitochondria synthesising glutamate in states 3 or 4 (± oligomycin). The changes in brain mitochondrial nicotinamide nucleotide redox state have been monitored by fluorimetric, spectrophotometric and enzymatic techniques during glutamate synthesis and compared with liver mitochondria under similar conditions. On the instigation of glutamate synthesis by NH⁺₄ addition a significant NAD(P)H oxidation occurs with liver mitochondria but no detectable change occurs with brain mitochondria. Leucine (2 mM) causes a doubling of glutamate synthesis by both synaptic and non synaptic brain mitochondria with no detectable change in the NAD(P)H redox state. The results are discussed with respect to the control of glutamate synthesis by mitochondrial redox potential and the possible intramitochondrial compartmentation of this process.     

Feeding and digestive responses to fatty acid intake in two South American passerines with different food habits

Specific fatty acids (FA) such as unsaturated (UFA) and saturated (SFA) fatty acids contained in foods are key factors in the nutritional ecology of birds. By means of a field and experimental approach, we evaluated the effect of diet on the activity of three esterases involved in FA hydrolysis; carboxylesterase (CE: 4-NPA-CE and a-NA-CE) and butyrylcholinesterase, in two South American passerines: the omnivorous rufous-collared sparrow (Zonotrichia capensis) and the granivorous common diuca-finch (Diuca diuca). The activity of the three esterases was measured in the intestines of freshly caught individuals over two distinct seasons and also after a chronic intake of a UFA-rich or SFA-rich diet in the laboratory. In turn, we assessed the feeding responses of the birds choosing amongst diets contrasting in the kind of specific FA (UFA- vs. SFA-treated diets). During summer, field CE activities (4-NPA-CE and a-NA-CE) in the small intestine were higher in the rufous-collared sparrow (25.3 ± 3.3 and 81.4 ± 10.8 µmol min^?1 g tissue^?1, respectively) than in the common diuca-finch (10.0 ± 3.0 and 33.9 ± 13.1 µmol min^?1 g tissue^?1, respectively). Two hour feeding trial test indicated that both species exhibited a clear preference for UFA-treated diets. On average, the rufous-collared sparrow consumed 0.46 g 2 h^?1 of UFA-rich diets and 0.12 g 2 h^?1 of SFA-rich diets. In turn, the consumption pattern of the common diuca-finch averaged 0.73 and 0.16 g 2 h^?1 for UFA-rich and SFA-rich diets, respectively. After a month of dietary acclimation to UFA-rich and SFA-rich diets, both species maintained body mass irrespective of the dietary regime. Additionally, the intestinal 4-NPA-CE activity exhibited by birds fed on a UFA-rich or SFA-rich diet was higher in the rufous-collared sparrow (39.0 ± 5.3 and 44.2 ± 7.3 µmol min^?1 g tissue^?1, respectively) than in the common diuca-finch (13.3 ± 1.9 and 11.2 ± 1.4 µmol min^?1 g tissue^?1, respectively). Finally, the intestinal a-NA-CE activity exhibited by the rufous-collared sparrow was about two times higher when consuming an UFA-rich diet. Our results suggest that the rufus-collared sparrow exhibits a greater capacity for intestinal FA hydrolysis, which would allow it to better deal with fats from different sources.     

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.     

Arginine Metabolising Enzymes as Therapeutic Tools for Alzheimer’s Disease: Peptidyl Arginine Deiminase Catalyses Fibrillogenesis of β-amyloid Peptides

The accumulation of arginine in the cerebrospinal fluid and brains of patients suffering from acute neurodegenerative diseases like Alzheimer’s disease, point to defects in the metabolic pathways involving this amino acids. The deposits of neurofibrillary tangles and senile plaques perhaps as a consequence of fibrillogenesis of β-amyloid peptides has also been shown to be a hallmark in the aetiology of certain neurodegenerative diseases. Peptidylarginine deiminase (PAD II) is an enzyme that uses arginine as a substrate and we now show that PAD II not only binds with the peptides Aβ_1-40, Aβ_22-35, Aβ_17-28, Aβ_25-35 and Aβ_32-35 but assists in the proteolytic degradation of these peptides with the concomitant formation of insoluble fibrils. PAD was purified in 12.5% yield and 137 fold with a specific activity of 59 μmol min^?1?mg^?1 from bovine brain by chromatography on diethylaminoethyl (DEAE)-Sephacel. Characterisation of the enzyme gave a pH and temperature optima of 7.5°C and 68°C, respectively, and the enzyme lost 50% activity within 38 min at this temperature. Michaelis-Menten kinetics established a V _max and K _m of 1.57 μmol min^?1?ml^?1 and 1.35 mM, respectively, with N-benzoyl arginine ethyl ester as substrate. Kinetic analysis was used to measure the affinity (K _i) of the amyloid peptides to PAD with values between 1.4 and 4.6 μM. The formation of Aβ fibrils was rate limiting involving an initial lag time of about 24 h that was dependent on the concentration of the amyloid peptides. Turbidity measurements at 400 nm, Congo Red assay and Thioflavin-T staining fluorescence were used to establish the aggregation kinetics of PAD-induced fibril formation.     

Identification and characterization of cytosolic fructose-1,6-bisphosphatase in Euglena gracilis

Euglena gracilis has the ability to accumulate a storage polysaccharide, a β-1,3-glucan known as paramylon, under aerobic conditions. Under anaerobic conditions, E. gracilis cells degrade paramylon and synthesize wax esters. Cytosolic fructose-1,6-bisphosphatase (FBPase) appears to be a key enzyme in gluconeogenesis and position branch point of carbon partitioning between paramylon and wax ester biosynthesis. We herein identified and characterized cytosolic FBPase from E. gracilis. The K_m and V_max values of EgFBPaseIII were 16.5 ± 1.6 μM and 30.4 ± 7.2 μmol min^?1 mg protein^?1, respectively. The activity of EgFBPaseIII was not regulated by AMP or reversible redox modulation. No significant differences were observed in the production of paramylon in transiently suppressed EgFBPaseIII gene expression cells by RNAi (KD-EgFBPaseIII); nevertheless, FBPase activity was markedly decreased in KD-EgFBPaseIII cells. On the other hand, the growth of KD-EgFBPaseIII cells was slightly higher than that of control cells.     

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.     

Ecosystem respiration derived from 222Rn measurements on Rishiri Island, Japan

We evaluated the nighttime CO₂ flux (ecosystem respiration) on Rishiri Island, located at the northern tip of Hokkaido, Japan, from 2009 to 2011, by using the relationship between atmospheric ²²²Rn and CO₂ concentrations. The annual mean CO₂ flux was 1.8 μmol m^?2 s^?1, with a maximum monthly mean in July (4.6 ± 2.6 μmol m^?2 s^?1) and a broad minimum from December to March (0.33 ± 0.29 μmol m^?2 s^?1). The annual mean was comparable to fluxes at the JapanFlux sites in northern Japan. During the season of snow cover (mid-December to early April), the CO₂ flux was low (0.45 ± 0.43 μmol m^?2 s^?1). Total annual respiration was estimated at 679 ± 174 g cm^?2, about 8 % of which occurred during the season of snow cover.     

Effects of floating vegetation on denitrification,nitrogen retention,and greenhouse gas production in wetland microcosms

Wetlands are biogeochemical hotspots that have been identified as important sites for both nitrogen (N) removal from surface waters and greenhouse gas (GHG) production. Floating vegetation (FV) commonly occurs in natural and constructed wetlands, but the effects of such vegetation on denitrification, N retention, and GHG production are unknown. To address this knowledge gap, we used microcosm experiments to examine how FV affects N and GHG dynamics. Denitrification and N retention rates were significantly higher in microcosms with FV (302 μmol N m^?2 h^?1 and 203 μmol N m^?2 h^?1, respectively) than in those without (63 μmol N m^?2 h^?1 and 170 μmol N m^?2 h^?1, respectively). GHG production rates were not significantly different between the two treatments. Denitrification rates were likely elevated due to decreased dissolved oxygen (DO) in microcosms with FV. The balance of photosynthesis and respiration was more important in affecting DO concentrations than decreased surface gas exchange. The denitrification fraction (N₂-N production: N retention) was higher in microcosms with FV (100 %) than those without (33 %) under increased (tripled) N loading. A 5 °C temperature increase resulted in significantly lower denitrification rates in the absence of FV and significantly lowered N₂O production with FV, but did not significantly change CH₄ production or N retention in either treatment. These results suggest that intentional introduction of FV in constructed wetlands could enhance N removal while leaving GHG production unchanged, an insight that should be further tested via in situ experiments.     

Immobilization and characterization of carbonic anhydrase purified from E. coli MO1 and its influence on CO2 sequestration

The present investigation entails the immobilisation and characterisation of Escherichia coli MO1-derived carbonic anhydrase (CA) and its influence on the transformation of CO₂ to CaCO₃. CA was purified from MO1 using a combination of Sephadex G-75 and DEAE cellulose column chromatography, resulting in 4.64-fold purification. The purified CA was immobilised in chitosan-alginate polyelectrolyte complex (C-A PEC) with an immobilisation potential of 94.5 %. Both the immobilised and free forms of the enzyme were most active and stable at pH 8.2 and at 37 °C. The K _m and V _max of the immobilised enzyme were found to be 19.12 mM and 416.66 μmol min^?1 mg^?1, respectively; whereas, the K _m and V _max of free enzyme were 18.26 mM and 434.78 μmol min^?1 mg^?1, respectively. The presence of metal ions such as Cu²⁺, Fe²⁺, and Mg²⁺ stimulated the enzyme activity. Immobilised CA showed higher storage stability and maintained its catalytic efficiency after repeated operational cycles. Furthermore, both forms of the enzyme were tested for targeted application of the carbonation reaction to convert CO₂ to CaCO₃. The amounts of CaCO₃ precipitated over free and immobilised CA were 267 and 253 mg/mg of enzyme, respectively. The results of this study show that immobilised CA in chitosan-alginate beads can be useful for CO₂ sequestration by the biomimetic route.     

Effect of photoperiod,light intensity and carbon sources on biomass and lipid productivities of Isochrysis galbana

Biomass and lipid productivities of Isochrysis galbana were optimized using nutrients of molasses (4, 8, 12 g l^?1), glucose (4, 8, 12 g l^?1), glycerol (4, 8, 12 g l^?1) and yeast extract (2 g l^?1). Combinations of carbon sources at different ratios were evaluated in which the alga was grown at three different light intensities (50, 100 and 150 μmol m^?2 s^?1) under the influence of three different photoperiod cycles (12/12, 18/6 and 24/0 h light/dark). A maximum cell density of 8.35 g l^?1 with 32 % (w/w) lipid was achieved for mixotrophic growth at 100 μmol m^?2 s^?1 and 18/6 h light/dark with molasses/glucose (20:80 w/w). Mixotrophic cultivation using molasses, glucose and glycerol was thus effective for the cultivation of I. galbana.     

Nutrient uptake rates by the alien alga Undaria pinnatifida (Phaeophyta) (Nuevo Gulf, Patagonia, Argentina) when exposed to diluted sewage effluent

In the early nineties, Undaria pinnatifida has been accidentally introduced to Nuevo Gulf (Patagonia, Argentina) where the environmental conditions would have favored its expansion. The effect of the secondary treated sewage discharge from Puerto Madryn city into Nueva Bay (located in the western extreme of Nuevo Gulf) is one of the probable factors to be taken into account. Laboratory cultures of this macroalgae were conducted in seawater enriched with the effluent. The nutrients (ammonium, nitrate and phosphate) uptake kinetics was studied at constant temperature and radiation (16?°C and 50 μE m^?2 s^?1 respectively). Uptake kinetics of both inorganic forms of nitrogen were described by the Michaelis–Menten model during the surge phase (ammonium: V _max ^sur: 218.1 μmol h^?1 g^?1, K _s ^sur: 476.5 μM and nitrate V _max ^sur: 10.7 μmol h^?1 g^?1, K _s ^sur: 6.1 μM) and during the assimilation phase (ammonium: V _max ^ass: 135.6 μmol h^?1 g^?1, K _s ^ass: 407.2 μM and nitrate V _max ^ass: 1.9 μmol h^?1 g^?1, K _s ^ass: 2.2 μM), with ammonium rates always higher than those of nitrate. Even though a net phosphate disappearance was observed in all treatments, uptake kinetics of this ion could not be properly estimated by the employed methodology.     

Decolorization of azo dyes by Geobacter metallireducens

Geobacter metallireducens was found to be capable of decolorizing several azo dyes with different structures to various extents. Pyruvate, ethanol, acetate, propionate, and benzoate could support 66.3?±?2.6?93.7?±?2.1 % decolorization of 0.1 mM acid red 27 (AR27) in 40 h. The dependence of the specific decolorization rate on AR27 concentration (25 to 800 μM) followed Michaelis–Menten kinetics (K _m?=?186.9?±?1.4 μΜ, V _max?=?0.65?±?0.02 μmol?mg protein^?1 h^?1). Enhanced AR27 decolorization was observed with the increase of cell concentrations ranging from 7.5 to 45 mgL^?1. AR27 decolorization by G. metallireducens was retarded by the presence of goethite, which competed electrons with AR27 and was reduced to Fe(II). The addition of low concentrations of humic acid (1?100 mgL^?1) or 2-hydroxy–1,4-naphthoquinone (0.5?50 μM) could improve the decolorization performance of G. metallireducens. High-performance liquid chromatography analysis suggested reductive pathway to be responsible for decolorization. This was the first study on azo dye decolorization by Geobacter strain and might improve our understanding of natural attenuation and bioremediation of environments polluted by azo dyes.     

Comparison of growth and biochemical parameters of two strains of <Emphasis Type="Italic">Rhodomonas salina</Emphasis> (Cryptophyceae) cultivated under different combinations of irradiance,temperature, and nutrients

Growth and biochemical parameters of two strains of Rhodomonas salina (Cryptophyceae), cultivated under different combinations of irradiance, temperature, and nutrients, were compared. The microalgae were grown in batch mode for 10 days, in f/2 medium at 33‰ salinity. The experimental design was a 2⁵ factorial design with the following variables: nitrate [0.441 mM (N1) and 3.529 mM (N2)], phosphate [0.018 mM (P1) and 0.144 mM (P2)], temperature [19 and 29 °C], continued irradiance [100 μmol photons m^?2 s^?1 (low light, LL), and 200 μmol photons m^?2 s^?1 (high light, HL)] and microalgae strains (CS-174 and CS-24). Growth parameters, protein and lipid content, and fatty acids profiles were analyzed. Principal component analysis showed that combined high nitrate, high phosphate availability, and high light, regardless of temperature, achieved the best growth in both strains; while combined high nitrate and high phosphate, regardless of irradiance or temperature, resulted in the highest protein accumulation in both strains. On the other hand, the content of total lipid, arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, as well as EPA/DHA ratio, were strongly influenced by temperature in both strains. Strain CS-174 grew better and achieved significantly higher (p?<?0.05) total lipid content (maximum 25.4?±?1.5 %), ARA, EPA and DHA content (maximum 3.5, 13.2 and 6.5 %, respectively), and EPA / DHA ratio (maximum 2.03), than strain CS-24, being thus more suitable for use in aquaculture nutrition.     

Effects of manganese on the microstructures of Chenopodium ambrosioides L., A manganese tolerant plant

Chenopodium ambrosioides L. can tolerate high concentrations of manganese and has potential for its use in the revegetation of manganese mine tailings. Following a hydroponic investigation, transmission electron microscopy (TEM)-energy disperse spectroscopy (EDS) was used to study microstructure changes and the possible accumulation of Mn in leaf cells of C. ambrosioides in different Mn treatments (200, 1000, 10000 μmol·L^?1). At 200 μmol·L^?1, the ultrastructure of C. ambrosioides was clearly visible without any obvious damage. At 1000 μmol·L^?1, the root, stem and leaf cells remained intact, and the organelles were clearly visible without any obvious damage. However, when the Mn concentration exceeded 1000 μmol·L^?1 the number of mitochondria in root cells decreased and the chloroplasts in stem cells showed a decrease in grana lamellae and osmiophilic granules. Compared to controls, treatment with 1000 μmol·L^?1 or 10000 μmol·L^?1 Mn over 30 days, gave rise to black agglomerations in the cells. At 10000 μmol·L^?1, Mn was observed to form acicular structures in leaf cells and intercellular spaces, which may be a form of tolerance and accumulation of Mn in C. ambrosioides. This study has furthered the understanding of Mn tolerance mechanisms in plants, and is potential for the revegetation of Mn-polluted soils.     

Inorganic nitrogen uptake kinetics of sugarcane (Saccharum spp.) varieties under in vitro conditions with varying N supply

An in vitro system was established for the characterisation of inorganic nitrogen uptake by sugarcane plantlets of variety NCo376. After multiplication and rooting, plantlets (0.27–0.3 g fresh mass) were placed on N-free medium for 4 days, and then supplied with 2–20 mM N as NO₃ ^?-N only, NH₄ ⁺-N only or NO₃ ^?-N + NH₄ ⁺-N (as 1:1). With few exceptions, on all the tested N media, the in vitro plants always had a higher V_max for NH₄ ⁺-N (28.69–66.51 μmol g^?1 h^?1) than for NO₃ ^?-N uptake (10.24–30.19 μmol g^?1 h^?1) and the K_m indicated a higher affinity for NO₃ ^?-N (0.02–7.38 mM) than for NH₄ ⁺-N (0.06–9.15 mM). When N was applied as 4 and 20 mM to varieties N12, N19 and N36, the interaction between variety, N form and concentration resulted in differences in the V_max and K_m. The high N-use efficient varieties (N12 and N19), as determined in previous pot and field trials, behaved similarly under all tested conditions and displayed a lower V_max and K_m than the low N-use efficient ones (NCo376 and N36). Based on this finding, it was suggested that the N-use efficient designation (from pot and field trials) may not be ascribed solely to N uptake. Assessment of the relative preference index (RPI) for NO₃ ^?-N and NH₄ ⁺-N uptake revealed that, at present, the RPI has no application in sugarcane due to its preferential uptake of NH₄ ⁺-N.