Improvement of succinate production by overexpression of a cyanobacterial carbonic anhydrase in Escherichia coli

Succinate fermentation was investigated in Escherichia coli strains overexpressing cyanobacterium Anabaena sp. 7120 ecaA gene encoding carbonic anhydrase (CA). In strain BL21 (DE3) bearing ecaA, the activity of CA was 21.8 U mg⁻¹ protein, whereas non-detectable CA activity was observed in the control strain. Meanwhile, the activity of phosphoenolpyruvate carboxylase (PEPC) increased from 0.2 U mg⁻¹ protein to 1.13 U mg⁻¹ protein. The recombinant bearing ecaA reached a succinate yield of 0.39 mol mol⁻¹ glucose at the end of the fermentation. It was 2.1-fold higher than that of control strain which was just 0.19 mol mol⁻¹ glucose. EcaA gene was also introduced into E. coli DC1515, which was deficient in glucose phosphotransferase, lactate dehydrogenase and pyruvate:formate lyase. Succinate yield can be further increased to 1.26 mol mol⁻¹ glucose. It could be concluded that the enhancement of the supply of HCO₃⁻ in vivo by ecaA overexpression is an effective strategy for the improvement of succinate production in E. coli.     

Evaluation of distant carbon sources in biosurfactant production by a gamma ray-induced Pseudomonas putida mutant

Pseudomonas putida 33 wild strain, subjected to gamma ray mutagenesis and designated as P. putida 300-B mutant was used as microbial rhamnolipid-producer by using distant carbon sources (viz. hydrocarbons, waste frying oils ‘WFOs’, vegetable oil refinery wastes and molasses) in the minimal media under shake flask conditions. The behavior of glucose as co-substrate and growth initiator was examined. The 300-B mutant strain showed its ability to grow on all the substrates tested and produced rhamnolipid surfactants to different extents however; soybean and corn WFOs were observed to be preferred carbon sources followed by kerosene and paraffin oils, respectively. The best cell biomass (3.5 g l⁻¹) and rhamnolipids yield (4.1 g l⁻¹) were obtained with soybean WFO as carbon source and glucose as growth initiator under fed-batch cultivation showing an optimum specific growth rate (μ) of 0.272 h⁻¹, specific product yield (q_p) of 0.318 g g⁻¹ h and volumetric productivity (P_V) of 0.024 g l⁻¹ h. The critical micelle concentration of its culture supernatant was observed to be 91 mg rhamnolipids l⁻¹ and surface tension as 31.2 mN m⁻¹.     

Effects of overexpressing photosynthetic carbon flux control enzymes in the cyanobacterium Synechocystis PCC 6803

Synechocystis PCC 6803 is a model unicellular cyanobacterium used in e.g. photosynthesis and CO₂ assimilation research. In the present study we examined the effects of overexpressing Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), sedoheptulose 1,7-biphosphatase (SBPase), fructose-bisphosphate aldolase (FBA) and transketolase (TK), confirmed carbon flux control enzymes of the Calvin-Bassham-Benson (CBB) cycle in higher plants, in Synechocystis PCC 6803. Overexpressing RuBisCO, SBPase and FBA resulted in increased in vivo oxygen evolution (maximal 115%), growth rate and biomass accumulation (maximal 52%) under 100 μmol photons m⁻² s⁻¹ light condition. Cells overexpressing TK showed a chlorotic phenotype but increased biomass by approximately 42% under 100 μmol photons m⁻² s⁻¹ light condition. Under 15 μmol photons m⁻² s⁻¹ light condition, cells overexpressing TK showed enhanced in vivo oxygen evolution. This study demonstrates increased growth and biomass accumulation when overexpressing selected enzymes of the CBB cycle. RuBisCO, SBPase, FBA and TK are identified as four potential targets to improve growth and subsequently also yield of valuable products from Synechocystis PCC 6803.     

Optimisation and scale-up of α-glucuronidase production by recombinant Saccharomyces cerevisiae in aerobic fed-batch culture with constant growth rate

α-Glucuronidase (EC 3.2.1.139) of family GH 115 from Scheffersomyces stipitis is a valuable enzyme for the modification of water-soluble xylan into insoluble biopolymers, due to its unique ability to act on polymeric xylans. The influence of growth rate on the production of α-glucuronidase by recombinant Saccharomyces cerevisiae MH1000pbk10D-glu in glucose-limited fed-batch culture was studied at 14 and 100 L scale. At and below the critical specific growth rate (μ_crit) of 0.12 h⁻¹ at 14 L scale, the biomass yield coefficient (Y_x/s) remained constant at 0.4 g g⁻¹ with no ethanol production, whereas ethanol yields relative to biomass (k_eth/x) of up to 0.54 g g⁻¹ and a steady decrease in Y_x/s were observed at μ > 0.12 h⁻¹. Production of α-glucuronidase was growth associated at a product yield (k_α-glu/x) of 0.45 mg g⁻¹, with the highest biomass (37.35 g/L) and α-glucuronidase (14.03 mg/L) concentrations, were recorded during fed-batch culture at or near to μ_crit. Scale-up with constant k_La from 14 to 100 L resulted in ethanol concentrations of up to 2.5 g/L at μ = 0.12 h⁻¹. At this scale, α-glucuronidase yield could be maximised at growth rates below μ_crit, to prevent localised high glucose concentration pockets at the feed entry zone that would induce oxido-reductive metabolism. This is the first report where recombinant production of α-glucuronidase (EC 3.2.1.139) by S. cerevisiae was optimised for application at pilot scale.     

Effect of oxygen supply on biomass and helvolic acid production in submerged fermentation of Cordyceps taii

The entomogenous fungus Cordyceps taii, a traditional Chinese medicinal mushroom, exhibits potent important pharmacological effects and it has great potential for health foods and medicine. In this work, the effects of oxygen supply on production of biomass and bioactive helvolic acid were studied in shake-flask fermentation of C. taii mycelia. The value of initial volumetric oxygen transfer coefficient (K_La) within 10.1–33.8 h⁻¹ affected the cell growth, helvolic acid production and expression levels of biosynthetic genes. The highest cell concentration of 17.2 g/L was obtained at 14.3 h⁻¹ of initial K_La. The highest helvolic acid production was 9.6 mg/L at 10.1 h⁻¹ of initial K_La. The expression levels of three genes encoding hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA reductase and squalene synthase were down-regulated on day 2 and day 8 but up-regulated on day 14 at an initial K_La value of 10.1 h⁻¹ vs. 33.8 h⁻¹, which well corresponded to the helvolic acid biosynthesis in those conditions. The information obtained would be helpful for improving the biomass and helvolic acid production in large-scale fermentation of C. taii.     

Osmoregulation in Dunaliella,Part II: Photosynthesis and starch contribute carbon for glycerol synthesis during a salt stress in Dunaliella tertiolecta

In response to an osmotic stress, Dunaliella tertiolecta osmoregulates by metabolizing intracellular glycerol as compatible solute. Upon the application of a salt stress to 0.17 M or 0.7 M NaCl grown D. tertiolecta cells, rates of total glycerol synthesis were substantially higher than that arising from photosynthetic ¹⁴CO₂ fixation into glycerol. The source of this extra carbon is the reserve starch pool. The contribution of carbon from the starch breakdown to glycerol synthesis was estimated from the difference between the total glycerol synthesized and that arising from ¹⁴CO₂ fixation. The maximum observed flux of carbon from ¹⁴CO₂ to glycerol from photosynthesis was of the order of 15–20 μmol ¹⁴C-glycerol mg⁻¹ Chl h⁻¹, whereas the total glycerol synthesis reached about 70 μmol glycerol mg⁻¹ Chl h⁻¹. The contribution of products of starch breakdown to glycerol synthesis increased progressively with increasing salt stress. In light, contrary to prevailing assumptions, both the photosynthesis and the starch breakdown contribute carbon to glycerol biosynthesis. The relative contributions of these two processes in the light, while cells were actively photosynthesizing, depended on the magnitude of the salt stress. On application of dilution stress, the flux of carbon from newly photosynthetically fixed ¹⁴CO₂ into glycerol was reduced progressively with increasing dilution stress that was also accompanied by a decline in total glycerol contents of the cell. The maximum observed rate of glycerol dissimilation was about 135 μmol glycerol mg⁻¹ Chl h⁻¹.     

The effects of NH4+ and NO3− on growth,resource allocation and nitrogen uptake kinetics of Phragmites australis and Glyceria maxima

The effects of NH₄⁺ or NO₃⁻ on growth, resource allocation and nitrogen (N) uptake kinetics of two common helophytes Phragmites australis (Cav.) Trin. ex Steudel and Glyceria maxima (Hartm.) Holmb. were studied in semi steady-state hydroponic cultures. At a steady-state nitrogen availability of 34 μM the growth rate of Phragmites was not affected by the N form (mean RGR = 35.4 mg g⁻¹ d⁻¹), whereas the growth rate of Glyceria was 16% higher in NH₄⁺-N cultures than in NO₃⁻-N cultures (mean = 66.7 and 57.4 mg g⁻¹ d⁻¹ of NH₄⁺ and NO₃⁻ treated plants, respectively). Phragmites and Glyceria had higher S/R ratio in NH₄⁺ cultures than in NO₃⁻ cultures, 123.5 and 129.7%, respectively.Species differed in the nitrogen utilisation. In Glyceria, the relative tissue N content was higher than in Phragmites and was increased in NH₄⁺ treated plants by 16%. The tissue NH₄⁺ concentration (mean = 1.6 μmol g fresh wt⁻¹) was not affected by N treatment, whereas NO₃⁻ contents were higher in NO₃⁻ (mean = 1.5 μmol g fresh wt⁻¹) than in NH₄⁺ (mean = 0.4 μmol g fresh wt⁻¹) treated plants. In Phragmites, NH₄⁺ (mean = 1.6 μmol g fresh wt⁻¹) and NO₃⁻ (mean = 0.2 μmol g fresh wt⁻¹) contents were not affected by the N regime. Species did not differ in NH₄⁺ (mean = 56.5 μmol g⁻¹ root dry wt h⁻¹) and NO₃⁻ (mean = 34.5 μmol g⁻¹ root dry wt h⁻¹) maximum uptake rates (V_max), and V_max for NH₄⁺ uptake was not affected by N treatment. The uptake rate of NO₃⁻ was low in NH₄⁺ treated plants, and an induction phase for NO₃⁻ was observed in NH₄⁺ treated Phragmites but not in Glyceria. Phragmites had low K_m (mean = 4.5 μM) and high affinity (10.3 l g⁻¹ root dry wt h⁻¹) for both ions compared to Glyceria (K_m = 6.3 μM, affinity = 8.0 l g⁻¹ root dry wt h⁻¹). The results showed different plasticity of Phragmites and Glyceria toward N source. The positive response to NH₄⁺-N source may participates in the observed success of Glyceria at NH₄⁺ rich sites, although other factors have to be considered. Higher plasticity of Phragmites toward low nutrient availability may favour this species at oligotrophic sites.     

Effects of feed sugar concentration on continuous ethanol fermentation of cheese whey powder solution (CWP)

Cheese whey powder (CWP) solution with different CWP or sugar concentrations was fermented to ethanol in a continuous fermenter using pure culture of Kluyveromyces marxianus (DSMZ 7239). Sugar concentration of the feed CWP solution varied between 55 and 200 g l⁻¹ while the hydraulic residence time (HRT) was kept constant at 54 h. Ethanol formation, sugar utilization and biomass formation were investigated as functions of the feed sugar concentration. Percent sugar utilization and biomass concentrations decreased and the effluent sugar concentration increased with increasing feed sugar concentrations especially for the feed sugar contents above 100 g l⁻¹. Ethanol concentration and productivity (DP) increased with increasing feed sugar up to 100 g l⁻¹ and then decreased with further increases in the feed sugar content. The highest ethanol concentration (3.7%, v v⁻¹) and productivity (0.54 gE l⁻¹ h⁻¹) were obtained with the feed sugar content of 100 g l⁻¹ or 125 g l⁻¹. The ethanol yield coefficient (Y_P/S) was also maximum (0.49 gE gS⁻¹) when the feed sugar was between 100 and 125 g l⁻¹. The growth yield coefficient (Y_X/S) decreased steadily from 0.123 to 0.063 gX gS⁻¹ when the feed sugar increased from 55 to 200 g l⁻¹ due to adverse effects of high sugar contents on yeast growth. The optimal feed sugar concentration maximizing the ethanol productivity and sugar utilization was between 100 and 125 g l⁻¹ under the specified experimental conditions.     

Purification and characterization of cyanogenic β-glucosidase from wild apricot (Prunus armeniaca L.)

β-Glucosidase catalyzes the sequential breakdown of cyanogenic glycosides in cyanogenic plants. The β-glucosidase from Prunus armeniaca L. was purified to 8-fold, and 20% yield was obtained, with a specific activity of 281 U/mg protein. The enzyme showed maximum activity in 0.15 M sodium citrate buffer, pH 6, at 35 °C with p-nitrophenylglucopyranoside as substrate. The β-glucosidase from wild apricot was used successfully for the saccharification of cellobiose into D-glucose. This enzyme has a V_max of 131.6 μmol min⁻¹ mg⁻¹ protein, K_m of 0.158 mM, K_cat of 144.8 s⁻¹, K_cat/K_m of 917.4 mM⁻¹ s⁻¹, and K_m/V_max of 0.0012 mM min mg μmole⁻¹, using cellobiose as substrate. The half-life, deactivation rate coefficient, and activation energy of this β-glucosidase were 12.76 h, 1.509 × 10⁻⁵ s⁻¹, and 37.55 kJ/mol, respectively. These results showed that P. armeniaca is a potential source of β-glucosidase, with high affinity and catalytic capability for the saccharification of cellulosic material.     

Geosmin production and polyphasic characterization of Oscillatoria limosa Agardh ex Gomont isolated from the open canal of a large drinking water system in Tianjin City,China

Taste and odor (T & O) episodes always cause strong effects on drinking water supply system. Luanhe River diversion into Tianjin City in China is an important drinking water resource. Massive growth of a benthic filamentous cyanobacterium with geosmin production in the open canal caused a strong earthy odor episode in Tianjin. On the basis of the morphological and molecular identification of this cyanobacterium as Oscillatoria limosa Agardh ex Gomont, the genetic basis for geosmin biosynthesis and factors influencing growth and geosmin production of O. limosa CHAB 7000 were studied in this work. A 2268-bp open reading frame, encoding 755 amino acids, was amplified and characterized as the geosmin synthase gene (geo), followed by a cyclic nucleotide-binding protein gene (cnb). Phylogenetic analysis implied that the evolution of the geosmin genes in O. limosa CHAB 7000 might involve a horizontal gene transfer event. Examination on the growth and geosmin production of O. limosa CHAB 7000 at different light intensities showed that the maximum geosmin production was observed at 10 μmol photons m⁻² s⁻¹, while the optimum growth was at 60 μmol photons m⁻² s⁻¹. Under three temperature conditions (15 °C, 25 °C, and 35 °C), the maximum growth and geosmin production were observed at 25 °C. Most amounts of geosmin were retained in cells during the growth phase, but high temperature and low light intensity increased the release of geosmin into the medium, implying that O. limosa CHAB 7000 had a high potential harm for the release of geosmin from its cells at these adverse conditions.     

Investigation on the properties and kinetics of glucose-fed aerobic granular sludge

Aerobic granulation is a process in which suspended biomass aggregate and form discrete well-defined granules in aerobic systems. To investigate the properties and kinetics of aerobic granular sludge, aerobic granules were cultivated with glucose synthetic wastewater in a series of sequencing batch reactors (SBR). The spherical shaped granules were observed on 8th day with the mean diameter of 0.1 mm. With the organic loading rate (OLR) being increased to 4.0 g COD L⁻¹ d⁻¹, aerobic granules grew matured with spherical shape. The size of granules ranged from 1.2 to 1.8 mm, and the corresponding settling velocity of individual granule was 24.2–36.4 m h⁻¹. The oxygen utilization rate (OUR) of mature granules was 41.90 g O₂ kg MLSS⁻¹ h⁻¹, which was two times higher than that of activated sludge (18.32 g O₂ kg MLSS⁻¹ h⁻¹). The experimental data indicated that the substrate utilization and biomass growth kinetics generally followed Monod's kinetics model. The corresponding kinetic coefficients of k (maximum specific substrate utilization rate), K_s (half velocity coefficient), Y (growth yield coefficient) and K_d (decay coefficient) were determined as follows, k_c = 23.65 d⁻¹, K_c = 3367.05 mg L⁻¹, K_N = 0.038 d⁻¹, K_N = 29.65 mg L⁻¹, Y = 0.1927–0.2022 mg MMLS (mg COD)⁻¹ and K_d = 0.00845–0.0135 d⁻¹, respectively. Those properties of aerobic granules made aerobic granules system had a short setup period, high substrate utilization rate and low sludge production.     

Sex-specific light acclimation of Chara canescens (Charophyta)

Bisexual populations of the charophyte Chara canescens (Desv. et Loisel. in Loisel., 1810) containing male and female individuals are rarely found. Two experiments were carried out to study whether male and female algae from the same site exhibit different physiological capacities, especially with respect to light acclimation.Algae from two different shore levels and from laboratory cultures acclimated to six irradiance conditions (35–500 μmol photons m⁻² s⁻¹) were compared. Field measurements showed that both female and male algae of C. canescens are able to acclimate to daily changes in solar irradiance. The quantum yield of Photosystem II (PSII) decreased with increasing irradiance in the morning and increased with decreasing irradiance in the afternoon. Growth experiments showed increasing growth rates from 35 μmol photons m⁻² s⁻¹ (∼7 mg FW) up to 500 μmol photons m⁻² s⁻¹ (∼27 mg FW) in female and male C. canescens. The irradiance saturation point for photosynthesis (E_k) was about 140 μmol m⁻² s⁻¹ for both sexes within the whole range of acclimation irradiances. The maximum photosynthesis rate at saturating irradiances (P_max) of male algae was highest at E_k, whereas P_max of female algae was highest at 500 μmol photons m⁻² s⁻¹. The photosynthetic efficiency in the light-limited range (α) increased in female C. canescens and decreased in male C. canescens. The ratio of the non-photochemical quenching parameter (NPQ) to the relative electron transport rates rETR_(MT) increased in both sexes with irradiance, but showed a steeper increase in male than in female algae. Pigment analysis showed similar acclimation pattern for male and female C. canescens. Chl a/Chl b ratios of both sexes were constant over the whole range of E_g, whereas Chl a/carotenoid ratios in male and female C. canescens decreased from 70 μmol photons m⁻² s⁻¹ upwards. Pigment analysis pointed out that the carotenes α-, β- and γ-carotene were more prominent in male than in female algae.Our results indicate that female C. canescens are more efficient in light acclimation than male algae from the same site. Nevertheless, further investigations of bisexual C. canescens populations resolving CO₂-uptake mechanisms and/or genetic differences are needed.     

Effects of flooding on photosynthesis and root respiration in saltcedar (Tamarix ramosissima), an invasive riparian shrub

The introduced shrub Tamarix ramosissima invades riparian zones, but loses competitiveness under flooding. Metabolic effects of flooding could be important for T. ramosissima, but have not been previously investigated. Photosynthesis rates, stomatal conductance, internal (intercellular) CO₂, transpiration, and root alcohol dehydrogenase (ADH) activity were compared in T. ramosissima across soil types and under drained and flooded conditions in a greenhouse. Photosynthesis at 1500 μmol quanta m⁻² s⁻¹ (A₁₅₀₀) in flooded plants ranged from 2.3 to 6.2 μmol CO₂ m⁻² s⁻¹ during the first week, but A₁₅₀₀ increased to 6.4–12.7 μmol CO₂ m⁻² s⁻¹ by the third week of flooding. Stomatal conductance (g_s) at 1500 μmol quanta m⁻² s⁻¹ also decreased initially during flooding, where g_s was 0.018 to 0.099 mol H₂O m⁻² s⁻¹ during the first week, but g_s increased to 0.113–0.248 mol H₂O m⁻² s⁻¹ by the third week of flooding. However, photosynthesis in flooded plants was reduced by non-stomatal limitations, and subsequent increases indicate metabolic acclimation to flooding. Root ADH activities were higher in flooded plants compared to drained plants, indicating oxygen stress. Lower photosynthesis and greater oxygen stress could account for the susceptibility of T. ramosissima at the onset of flooding. Soil type had no effect on photosynthesis or on root ADH activity. In the field, stomatal conductance, leaf water potential, transpiration, and leaf δ¹³C were compared between T. ramosissima and other flooded species. T. ramosissima had lower stomatal conductance and water potential compared to Populus deltoides and Phragmites australis. Differences in physiological responses for T. ramosissima could become important for ecological concerns.     

Evaluation of the association of mercury(II) with some dicysteinyl tripeptides

The present study was undertaken to gain insight into the associations of mercury(II) with dicysteinyl tripeptides in buffered media at pH 7.4. We investigated the effects of increasing the distance between cysteinyl residues on mercury(II) associations and complex formations. The peptide–mercury(II) formation constants and their associated thermodynamic parameters in 3-(N-morpholino)propanesulfonic acid (MOPS) buffered solutions were evaluated by isothermal titration calorimetry. Complexes formed in different relative ratios of mercury(II) to cysteinyl peptides in ammonium formate buffered solutions were characterized by LTQ Orbitrap mass spectrometry. The results from these studies show that n-alkyl dicysteinyl peptides (CP 1–4), and an aryl dicysteinyl peptide (CP 5) can serve as effective “double anchors” to accommodate the coordination sites of mercury(II) to form predominantly one-to-one Hg(peptide) complexes. The aryl dicysteinyl peptide (CP 5) also forms the two-to-two Hg₂(peptide)₂ complex. In the presence of excess peptide, Hg(peptide)₂ complexes are also detected. Notably, increasing the distance between the ligating groups or “anchor points” in CP 1–5 does not significantly affect their affinity for mercury(II). However, the enthalpy change (ΔH) values (ΔH₁ ∼ −91 kJ mol⁻¹ and ΔH₂ ∼ −66 kJ mol⁻¹) for complex formation between CP 4 and 5 with mercury(II) are about one and a half times larger than the related values for CP 1, 2 and 3 (ΔH₁ ∼ −66 kJ mol⁻¹ and ΔH₂ ∼ 46 kJ mol⁻¹). The corresponding entropy change (ΔS) values (ΔS₁ ∼ −129 J K⁻¹ mol⁻¹ and ΔS₂ ∼ −116 J K⁻¹ mol⁻¹) of the structurally larger dicysteinyl peptides CP 4 and 5 are less entropically favorable than for CP 1, 2 and 3 (ΔS₁ ∼ −48 J K⁻¹ mol⁻¹ and ΔS₂ ∼ −44 J K⁻¹ mol⁻¹). Generally, these associations result in a decrease in entropy, indicating that these peptide–mercury complexes potentially form highly ordered structures. The results from this study show that dicysteinyl tripeptides are effective in binding mercury(II) and they are promising motifs for the design of multi-cysteinyl peptides for binding more than one mercury(II) ion per peptide.     

Salt effects on β-glucosidase: pH-profile narrowing

Salts inhibit the activity of sweet almond β-glucosidase. For cations (Cl⁻ salts) the effectiveness follows the series: Cu⁺², Fe⁺² > Zn⁺² > Li⁺ > Ca⁺² > Mg⁺² > Cs⁺ > NH₄⁺ > Rb⁺ > K⁺ > Na⁺ and for anions (Na⁺ salts) the series is: I⁻ > ClO₄⁻ > ⁻SCN > Br⁻ ≈ NO₃⁻ > Cl⁻ ≈ ⁻OAc > F⁻ ≈ SO₄^− 2. The activity of the enzyme, like that of most glycohydrolases, depends on a deprotonated carboxylate (nucleophile) and a protonated carboxylic acid for optimal activity. The resulting pH-profile of k_cat/K_m for the β-glucosidase-catalyzed hydrolysis of p-nitrophenyl glucoside is characterized by a width at half height that is strongly sensitive to the nature and concentration of the salt. Most of the inhibition is due to a shift in the enzymic pK_as and not to an effect on the pH-independent second-order rate constant, (k_cat/K_m)^lim. For example, as the NaCl concentration is increased from 0.01 M to 1.0 M the apparent pK_a1 increases (from 3.7 to 4.9) and the apparent pK_a2 decreases (from 7.2 to 5.9). With p-nitrophenyl glucoside, the value of the pH-independent (k_cat/K_m)^lim (= 9 × 10⁴ M^− 1 s^− 1) is reduced by less than 4% as the NaCl concentration is increased. There is a similar shift in the pK_as when the LiCl concentration is increased to 1.0 M. The results of these salt-induced pK_a shifts rule out a significant contribution of reverse protonation to the catalytic efficiency of the enzyme. At low salt concentration, the fraction of the catalytically active monoprotonated enzyme in the reverse protonated form (i.e., proton on the group with a pK_a of 3.7 and dissociated from the group with a pK_a of 7.2) is very small (≈ 0.03%). At higher salt concentrations, where the two pK_as become closer, the fraction of the monoprotonated enzyme in the reverse protonated form increases over 300-fold. However, there is no increase in the intrinsic reactivity, (k_cat/K_m)^lim, of the monoprotonated species. For other enzymes which may show such salt-induced pK_a shifts, this provides a convenient test for the role of reverse protonation.     

Pilot-scale production of 1,3-propanediol using Klebsiella pneumoniae

The conversion of glycerol to 1,3-propanediol (PDO) using Klebsiella pneumoniae M5al under anaerobic condition was scaled up from scale 5 to 5000 l in series. A simple strategy for scale-up was to transfer the optimized conditions of a lab scale bioreactor to pilot-scale fermentation. Multistage inocula were developed and their fermentation abilities were assessed in a small-scale fermenter. The experimental results showed that inoculum development in the early steps of a scale-up process could influence the outcomes of a large scale fermentation. Through three-stage liquid inoculum development and a pulse addition of (NH₄)₂SO₄ and yeast extract at 30 h of fermentation, the best results in a 5000 l fermentation were achieved leading to 58.8 g l⁻¹ 1,3-propanediol with a yield of 0.53 mol mol⁻¹ glycerol and productivity of 0.92 g l⁻¹ h⁻¹. This is the first report on pilot-scale 1,3-propanediol production using K. pneumoniae.     

Seasonal variations in photosynthetic irradiance response curves of macrophytes from a Mediterranean coastal lagoon

The main photosynthesis and respiration parameters (dark respiration rate, light saturated production rate, saturation irradiance, photosynthetic efficiency) were measured on a total of 23 macrophytes of the Thau lagoon (2 Phanerogams, 5 Chlorophyceae, 10 Rhodophyceae and 6 Phaeophyceae). Those measurements were performed in vitro under controlled conditions, close to the natural ones, and at several seasons. Concomitantly, measurements of pigment concentrations, carbon, phosphorous and nitrogen contents in tissues were performed. Seasonal intra-specific variability of photosynthetic parameters was found very high, enlightening an important acclimatation capacity. The highest photosynthetic capacities were found for Chlorophyceae (e.g. Monostroma obscurum thalli at 17 °C, 982 μmol O₂ g⁻¹ dw h⁻¹ and 9.1 μmol O₂ g⁻¹ dw h⁻¹/μmol photons m⁻² s⁻¹, respectively for light saturated net production rate and photosynthetic efficiency) and Phanerogams (e.g. Nanozostera noltii leaves at 25 °C, 583 μmol O₂ g⁻¹ dw h⁻¹ and 2.6 μmol O₂ g⁻¹ dw h⁻¹/μmol photons m⁻² s⁻¹ respectively for light saturated net production rate and photosynthetic efficiency). As expected, species with a high surface/volume ratio were found to be more productive than coarsely branched thalli and thick blades shaped species. Contrary to R_d (ranging 6.7–794 μmol O₂ g⁻¹ dw h⁻¹, respectively for Rytiphlaea tinctoria at 7 °C and for Dasya sessilis at 25 °C) for which a positive relationship with water temperature was found whatever the species studied, the evolution of P/I curves with temperature exhibited different responses amongst the species. The results allowed to show summer nitrogen limitation for some species (Gracilaria bursa-pastoris and Ulva spp.) and to propose temperature preferences based on the photosynthetic parameters for some others (N. noltii, Zostera marina, Chaetomorpha linum).     

Growth of the fungus Paecilomyces lilacinus with n-hexadecane in submerged and solid-state cultures and recovery of hydrophobin proteins

The filamentous fungus Paecilomyces lilacinus was grown on n-hexadecane in submerged (SmC) and solid-state (SSC) cultures. The maximum CO₂ production rate in SmC (V_max = 11.7 mg CO₂ L_g⁻¹ day⁻¹) was three times lower than in SSC (V_max = 40.4 mg CO₂ L_g⁻¹ day⁻¹). The P. lilacinus hydrophobin (PLHYD) yield from the SSC was 1.3 mg PLHYD g protein⁻¹, but in SmC, this protein was not detected. The PLHYD showed a critical micelle concentration of 0.45 mg mL⁻¹. In addition, the PLHYD modified the hydrophobicity of Teflon from 130.1 ± 2° to 47 ± 2°, forming porous structures with some filaments <1 μm and globular aggregates <0.25 μm diameter. The interfacial studies of this PLHYD could be the basis for the use of the protein to modify surfaces and to stabilize compounds in emulsions.     

Characterization of the N-oxygenase AurF from Streptomyces thioletus

AurF catalyzes the N-oxidation of p-aminobenzoic acid to p-nitrobenzoic acid in the biosynthesis of the antibiotic aureothin. Here we report the characterization of AurF under optimized conditions to explore its potential use in biocatalysis. The pH optimum of the enzyme was established to be 5.5 using phenazine methosulfate (PMS)/NADH as the enzyme mediator system, showing ∼10-fold higher activity than previous reports in literature. Kinetic characterization at optimized conditions give a K_m of 14.7 ± 1.1 μM, a k_cat of 47.5 ± 5.4 min⁻¹ and a k_cat/K_m of 3.2 ± 0.4 μM⁻¹ min⁻¹. PMS/NADH and the native electron transfer proteins showed significant formation of the p-hydroxylaminobenzoic acid intermediate, however H₂O₂ produced mostly p-nitrobenzoic acid. Alanine scanning identified the role of important active site residues. The substrate specificity of AurF was examined and rationalized based on the protein crystal structure. Kinetic studies indicate that the K_m is the main determinant of AurF activity toward alternative substrates.     

Differences in the photoacclimation and photoprotection exhibited by two species of the ciguatera causing dinoflagellate genus,Gambierdiscus

In culture, Gambierdiscus spp. have been shown to prefer irradiances that are relatively low (≤250 μmol photons m⁻² s⁻¹) versus those to which they are frequently exposed to in their natural environment (>500 μmol photons m⁻² s⁻¹). Although several behavioral strategies for coping with such irradiances have been suggested, it is unclear as to how these dinoflagellates do so on a physiological level. More specifically, how do long term exposures (30 days) affect cell size and cellular chlorophyll content, and what is the photosynthetic response to short term, high irradiance exposures (up to 1464 μmol photons m⁻² s⁻¹)? The results of this study reveal that cell size and chlorophyll content exhibited by G. carolinianus increased with acclimation to increasing photon flux density. Additionally, both G. carolinianus and G. silvae exhibited reduced photosynthetic efficiency when acclimated to increased photon flux density. Photosynthetic yield exhibited by G. silvae was greater than that for G. carolinianus across all acclimation irradiances. Although such differences were evident, both G. carolinianus and G. silvae appear to have adequate biochemical mechanisms to withstand exposure to irradiances exceeding 250 μmol photons m⁻² s⁻¹ for at least short periods of time following acclimation to irradiances of up to 150 μmol photons m⁻² s⁻¹.