Pyrolytic characteristics of heterotrophic Chlorella protothecoides for renewable bio-fuel production

Heterotrophic Chlorella protothecoides cells were pyrolyzed in athermogravimetric analyzer to investigate the pyrolytic characteristics anddetermine the kinetic parameters. Heating rates of 15, 40, 60 and 80 ^°C^-1 up to a final temperature of 800 ^°C wereused. The pyrolysis reactions mainly took place between 160–520^°C with a volatile yield of about 80%. The devolatilization stageconsisted of two main temperature zones (I and II) with a transition at300–320 ^°C. Crude lipid in cells decomposed at Zone II whileother main components at Zone I. The increase of heating rate caused alateral shift to higher temperatures in the thermograms, a decrease ofactivation energies for the devolatilization stage and an increase of both theinstantaneous maximum and average reaction rates. The difference ofactivation energies between two zones implied that more energy input forlipid pyrolysis seems needed in comparison with other main components.These data are useful for the design, operation, and modeling of thepyrolysis systems for microalgae.     

Effect of cooling rate,cryoprotectant and holding time at different transfer temperatures on the survival of cryopreserved cell suspension culture (Puccinellia distans (L.) Parl.)

Reflexed saltmarsh-grass suspension cultures produced by seed callus were frozen to the liquid nitrogen temperature. Cooling rates, cryoprotectants and holding times were taken as a function of transfer temperatures. The highest survival of cells (45%) was found at a freezing rate of 1°C min^-1, without cryoprotectant treatments. The cryoprotectants (proline, dimethyl sulphoxide, glycerol), used at different concentrations and transfer temperatures, increased the survival rate. The maximum value was 78% at 12.5% (w/v) of proline with –30°C transfer temperature. Considerable improvement of viability (from 0% to 95%) among the 12.5 and 15.0% (v/v) dimethyl sulphoxide cryopreserved cells was achieved by holding them at – 20°C for 10–30 min before plunging into the liquid nitrogen. A 20 min holding time at 15.0% (v/v) glycerol level and – 30°C transfer temperature significantly enhanced the viability of the explants from 42% to 92%. Plants were successfully regenerated from cells cryopreserved with proline (w/v) and dimethyl sulfoxide (v/v) levels of 12.5 and 15.0%, respectively.     

Influence of salinity and temperature on the germination of Kochia scoparia

Kochia scoparia is one of the most common annual halophytes foundin the Great Basin. Seeds were collected from a population growing in asalt playa at Faust, Utah and were germinated at 5 temperature regimes(12 h night/12 h day, 5–15 ^°C, 10–20 ^°C, 15–25 ^°C,20–30 ^°C and 25–35 ^°C) and 6 salinities (0, 200, 400,600, 800 and 1000 mM NaCl) to determine optimal conditions forgermination and recovery of germination from saline conditions after beingtransferred to distilled water. Maximum germination occurred in distilledwater, and an increase in NaCl concentration progressively inhibited seedgermination. Few seeds germinated at 1000 mM NaCl. A temperatureregime of 25 ^°C night and 35 ^°C day yielded maximumgermination. Cooler temperature 5–15 ^°C significantly inhibited seedgermination. Rate of germination decreased with increase in salinity.Germination rate was highest at 25–35 ^°C and lowest at5–15 ^°C. Seeds were transferred from salt solutions to distilled waterafter 20 days and those from high salinities recovered quickly at warmertemperature regimes. Final recovery germination percentages in high salttreatments were high, indicating that exposure to high concentration ofNaCl did not inhibit germination permanently.     

Outwardly rectifying chloride channels in lymphocytes

Summary Outwardly rectifying Cl^– channels in cultured human Jurkat T-lymphocytes were activated by excising a patch of membrane using the inside-out (i/o) patch-clamp configuration and holding at depolarized voltages for prolonged periods of time (1–6 min at +80 mV, 20°C). The single-channel current at +80 mV was 4.5 ± 0.3 pA and at –80 mV, it was 1.0 ± 0.4 pA. After activation, the probability of being open (P ₀)for the lymphocyte channel was voltage independent. Activation of the Cl^– channel in lymphocytes was temperature dependent. Nineteen percent of i/o recordings from lymphocytes made at 20°C exhibited Cl^– channel activity. In contrast, 49% of recordings made at 30°C showed channel activity. The number of channels in an active patch was not significantly different at the two temperatures. Channel activation in excised, depolarized patches also occurred 20-fold faster at 30°C than at 20°C. There was no marked change in the single-channel conductance at 30°C. Open-channel conductance was blocked by 200 m indanyloxyacetic acid (IAA) or 1 mm SITS when applied to the intracellular side of the patch. The characteristics of this channel are similar to epithelial outwardly rectifying Cl^– channels thought to be involved in fluid secretion     

Growth, survival, and starvation resistance of Colorado squawfish larvae

Synopsis Growth and survival of Colorado squawfish, Ptychocheilus lucius, larvae under fluctuating 18, 22, and 26° C (5° C diel fluctuations) and constant 18, 22, 26° C, and 30° C temperature conditions and ration size corresponding to 12.5, 28,64,142, 320 brine shrimp nauplii fish^–1 day^–1 determined from laboratory experiments. Growth was optimal at 31° C and high at temperatures of 26° C to 30° C, at the highest food abundance. Lowest growth was under lowest food rations and highest temperatures. Growth of Colorado squawfish larvae declined substantially at temperatures < 22° C. Neither growth nor survival was significantly different between fluctuating or constant regimes. Survival of Colorado squawfish larvae was highest (95%) at 26.2° C and 235 nauplii fish^–1 day^–1 and high at temperatures of 20 to 30° C with food abundance > 180 nauplii fish^–1 day^–1. Survival was lowest when food abundance was low and temperature was high. Highest mortality occurred more than 20 days after experiments began and mortalities occurred sooner in higher than lower temperatures. Colorado squawfish larvae denied food for 5, 10, or 15 d after first feeding could have begun (6 d), had survival greater than 87 % which was equivalent to continuously fed controls. Survival of fish denied food for 17.5 d after feeding could have begun declined from 84% before feeding to 57% after feeding. Point of no return was estimated between 17.5 and 20 d. Colorado squawfish have relatively high starvation resistance. Low, stable flows that simulate natural hydrographs may enhance growth, survival, and recruitment of early life stages of Colorado squawfish by increasing water temperature and food abundance in regulated rivers of the Colorado River basin.     

Temperature Effect on Carbachol-Induced Depression of Spontaneous Quantal Transmitter Release in Frog Neuromuscular Junction

The effects of carbachol (CCh) on the frequency (f) of the miniature endplate potentials were tested at temperatures between 5 and 30°C. Higher CCh concentrations, 1 × 10^–5 and 5 × 10^–6 M, reduced the f to 60% and the temperature dependence was negligible. However, an inverse temperature dependence was found when low concentrations 3 × 10^–7 and 6 × 10^–7 M were applied. The depression of f was 40–50% in 5–10°C but only 10–20% of the control in the 25 and 30°C. During application of CCh, the new steady of f was reached at temperatures between 5 and 30°C within 17–20 min (Q₁₀ = 1.07). Much greater temperature dependence of recovery was observed during washing out CCh (Q₁₀ = 1.6). The temperature-independence of the steady state effects of CCh, good agreement with Langmuir adsorption-desorption theory and non-steady kinetics indicate that physical rather than receptor-mediated events are responsible for the depression of f.     

High density culture of the freshwater rotifer,Brachionus calyciflorus

The freshwater rotifer, Brachionus calyciflorus is one of the live food organisms used for the mass production of larval fish. In this study possibility of obtaining high density cultures of the freshwater rotifer B. calyciflorus were investigated. The two culture systems used differed in their air and dissolved oxygen supplies using three temperatures in each case: 24, 28 and 32 °C. Rotifers were batch-cultured using 5 l-vessels and fed with the freshwater Chlorella. The growth rate of rotifers significantly increased with an increase in temperature. The maximum density of the rotifers with air-supply at 24 °C, 6500 ind. ml^–1, was significantly lower than those cultured at 28 and 32 °C, i.e. 8600 and 8100 ind. ml^–1, respectively. Dissolved oxygen levels decreased with time and ranged from 0.8 to 1.4 mg l^–1 when the density of freshwater rotifer was the highest at each temperature. The highest density (19200 ind. ml^–1) of freshwater rotifer was obtained in cultures with a supply of oxygen at 28 °C. Densities of 13500 and 17200 ind. ml^–1 were found at 24 and 32 °C, respectively. Levels of NH₃-N increased with time and a dramatic increase of NH₃-N was observed at high temperatures. Levels of NH₃-N at 24, 28 and 32 °C were 13.2, 18.5 and 24.5 mg l^–1, respectively. These levels coincided with the highest rotifer density at each of the three temperatures. When rotifers were cultured with an oxygen-supply and pH was adjusted to 7, the maximum density of rotifer reached 33500 ind. ml^–1 at 32 °C . These results suggested that high density culture of freshwater rotifer, B. calyciflorus could be achieved under optimal conditions with DO value of exceeding 5 mg l^–1 and NH₃-N values of lower than 12.0 mg l^–1.     

Galacto-oligosaccharide production by a thermostable recombinant β-galactosidase from <Emphasis Type="Italic">Thermotoga maritima</Emphasis>

Summary A β-galactosidase from Thermotoga maritima produced galacto-oligosaccharides (GOS) from lactose by transgalactosylation when expressed in Escherichia coli. The enzyme activity for GOS production was maximal at pH 6.0 and 90 °C. In thermal stability experiments, the enzyme followed first-order kinetics of pH and thermal inactivation, and half-lives at pH 5.0, pH 8.0, 80 °C, and 95 °C were 27 h, 82 h, 41 h, and 14 min, respectively, suggesting that the enzyme was stable below 80 °C and in the pH range of 5.0–8.0. Mn²⁺ was the most effective divalent cation for GOS production. Cu²⁺ and EDTA inhibited more than 84% of enzyme activity. GOS production increased with increasing lactose concentrations and peaked at 500 g lactose/l. Among tested enzyme concentrations, the highest production of GOS was obtained at 1.5 units enzyme/ml. Under the optimal conditions of pH 6.0, 80 °C, 500 g lactose/l, and 1.5 units enzyme/ml, GOS production was 91 g/l for 300 min, with a GOS productivity of 18.2 g/l · h and a conversion yield of GOS to lactose of 18%.     

Influence of light and temperature on photoinhibition of photosynthesis inSpirulina platensis

Photoinhibition of photosynthesis and its recovery in the cyanobacteriumSpirulina platensis was studied to find how photosynthetic rates were influenced by light and temperature. By exposing cell samples from a turbidostat culture to combinations of light and temperature, a connection between light, temperature and photoinhibition was found. The experiments showed that a 10 degree increase from 20 °C to 30 °C considerably reduced the photoinhibition. At 25 °C a photon flux density of 1720 µmol m^–2 s^–1 reduced the photosynthetic rate by 50 % in 1 h, but a similarly high photon flux density had nearly no negative effect at 35 °C. Reactivation in low light from 50% photoinhibition was fast and complete in 60 min at 30 °C, while at 20 °C only about 1/6 of the full capacity was regained in the same time. Addition of the protein synthesis inhibitor streptomycin to cultures undergoing photoinhibition and regeneration indicated the presence also in this organism of a repair mechanism based on protein synthesis.Author for correspondence     

Production of laccase byBotrytis cinerea and fermentation studies with strain F226

After induction, seven strains ofBotrytis cinerea released into the culture broth considerable amounts of laccase in a brief production time. The set-up of a suitable production process was studied with a selected strain in a 10-L fermenter. The optimum fermentation conditions were a 3% inoculum with a high degree of sporulation, a simple medium containing 20 g L^–1 of glucose and 2 g L^–1 of yeast extract at pH 3.5, 2 g L^–1 gallic acid as inducer, added after 2 days of growth, an agitation speed of 300 rpm, an aeration rate of 1.2 vvm and a temperature of 24°C. By optimizing the culture conditions, the enzyme activity reached 28 U ml^–1 in 5 days with a specific activity of 560 U mg^–1 protein. The best procedure to obtain a suitable crude enzyme preparation was concentration of the supernatant medium to 10% of the initial volume by ultrafiltration, followed by a fractional precipitation with ethanol. The optimum pH and temperature for laccase activity were 5.5 and 40°C, respectively, with syringaldazine as the substrate.     

Decolorization of azo dyes by Rhodobacter sphaeroides

Rhodobacter sphaeroides AS1.1737 decolorized more than 90% of several azo dyes (200 mg dyes l^–1) in 24 h. The optimal culture conditions were: anaerobic illumination (1990 lx), peptone as carbon source, temperature 35–40 °C and pH 7–8. Intracellular crude enzyme from this strain had azoreductase activity, optimized temperature as 45–50 °C, and decolorization kinetics which were consistent with a ping-pong mechanism.     

The production of cyclopiazonic acid by Penicillium commune and cyclopiazonic acid and aflatoxins by Aspergillus flavus as affected by water activity and temperature on maize grains

The combined effects of water activity (a_w) and temperature on mycotoxin production by Penicilium commune (cyclopiazonic acid — CPA) and Aspergillus flavus (CPA and aflatoxins — AF) were studied on maize over a 14-day period using a statistical experimental design. Analysis of variance showed a highly significant interaction (P 0.001) between these factors and mycotoxin production. The minimum a_w/temperature for CPA production (2264 ng g^–1 P. commune, 709 ng g^–1 A. flavus) was 0.90 a_w/30 °C while greatest production (7678 ng g^–1 P. commune, 1876 ng g^–1 A. flavus) was produced at 0.98 a_w/20 °C. Least AF (411 ng g^–1) was produced at 0.90 a_w/20 °C and most (3096 ng g^–1) at 0.98 a_w/30 °C.     

SelectingRhizobium phaseoli strains for use with beans (Phaseolus vulgaris L.) in Kenya: Tolerance of high temperature and antibiotic resistance

Forty one strains ofRhizobium phaseoli were screened for the ability to multiply at high temperatures on yeast extract-mannitol agar. Most strains were tolerant of 30°C, eight strains were tolerant of 45°C and two of 47°C although the rate of multiplication was reduced at 45–47°C. The high temperature-tolerant strains were isolated from Kenyan soils and were fast-growing. Seven of the eight strains tolerant of 45–47°C lost their infectiveness after incubation at high temperature but four strains tolerant of 40°C remained infective after incubation at that temperature.Thirty six strains were resistant to 200 g ml^–1 streptomycin sulphate and 29 strains to 200 g ml^–1 spectinomycin dihydrochloride. Eight strains were resistant to both antibiotics each at 200 g ml^–1. Two of the double-labelled antibiotic-resistant mutants lost their infectiveness onPhaseolus vulgaris. The response to acidity was unaltered and two of the mutants showed a decrease in temperature tolerance. The doublelabelled mutants were recoverable from two Kenyan soils.     

Phytase production by the yeast, Pichia anomala

Pichia anomala, isolated from dried flower buds of Woodfordia fruticosa, produced a high activity of an intracellular phytase, at 68 U per g dry biomass, when grown at 20 °C for 24 h in a medium containing glucose (40 g l^–1) and beef extract (10 g l^–1) supplemented with Fe²⁺ (0.15 mM). Partially purified phytase was optimally active at 60 °C and pH 4 with a half life of 7 days at 60 °C. It retained 85% of its activity at 80 °C for 15 min. The enzyme is suitable for supplementing animal feeds to improve the availability of phosphate from phytate.     

Photosynthetic performance of transplanted ecotypes ofEcklonia cava(Laminariales, Phaeophyta)

Young sporophytes of short-stipe ecotype ofEcklonia cavafrom a warmer locality (Tei, Kochi Pref., southern Japan) and those of long-stipe ecotype from a cooler locality (Nabeta, Shizuoka Pref., central Japan) were transplanted in 1995 to artificial reefs immersed at the habitat of long-stipe ecotype in Nabeta Bay, Shizuoka Pref., central Japan. The characteristics of photosynthesis and respiration of bladelets of the transplanted sporophytes of the two ecotypes were compared in winter and summer 1997; the results were assessed per unit area, per unit chlorophyllacontent and per unit dry weight. In photosynthesis-light curves at 10–29 °C, light saturation occurred at 200–400 mol photon m^–2s^–1in sporophytes from both Tei and Nabeta. The maximum photosynthetic rate (P _max) at 10–29 °C and the light-saturation index (I _k) at 25–29 °C in sporophytes from both localities were generally higher in winter than in summer.P _maxat 25–29 °C (per unit area and chlorophylla) were higher in sporophytes from Tei than those from Nabeta in both seasons. The optimum temperature for photosynthesis was 25 °C in winter and 27 °C in summer at high light intensities of 100–400 mol photon m^–2s^–1. However, at lower light intensities of 12.5–50 mol photon m^–2s^–1, it was 20 °C in winter and 25–27 °C in summer for sporophytes from both locations. Dark respiration increased with temperature rise in the range of 10–29 °C in sporophytes from both locations in summer and winter. The sporophytes transplanted from Tei (warmer area) showed higher photosynthetic activities than those from Nabeta (cooler area) at warmer temperatures even under the same environmental conditions. This indicates that these physiological ecotypes have arisen from genetic differentiation.     

Preparation ofN-acetylneuraminic acid from delipidated egg yolk

Egg yolk, a large proportion of the egg, was studied for the preparation ofN-acetylneuraminic acid (Neu5Ac). The delipidated hen egg yolk (DEY; 500 kg containing 0.2% w/w, Neu5Ac) was hydrolysed with HCl (pH 1.4) at 80 °C and neutralized with NaOH (pH 6.0). The mixture was filtered and electrodialysed until the conductivity was 240 µS cm^–1. The filtrate was applied on a column of Dowex HCR-W2 (20–50 mesh), followed by a column of Dowex 1-X8 (200–400 mesh). The latter column was washed with water, and then eluted with a linear gradient of HCO₂H (0–2m). The eluates containing Neu5Ac were concentrated using a reverse osmosis membrane and, finally, rotary evaporated at 40 °C. The residue was then lyophilized to yield 500 g Neu5Ac. The purity of Neu5Ac was >98% (TBA method). HPLC, NMR spectroscopy and TLC chromatography of the product obtained from the DEY showed that Neu5Ac was the sole derivative present in egg yolk. The DEY, a byproduct from egg processing plants, was found to be an excellent source for the large-scale preparation of Neu5Ac.Abbreviations Neu5Ac N-acetylneuraminic acid - Neu5Gc N-glycolylneuraminic acid - DEY delipidated egg yolk - HPLC high performance liquid chromatography - TLC thin layer chromatography - NMR nuclear magnetic resonance - IR infrared spectroscopy Presented at the 11th International Symposium on Glycoconjugates, Toronto, Canada.     

Abscisic acid and mefluidide in the regulation of cold hardiness development in Solanum tuberosum L. potato

Plants of Solanum tuberosum L. potato do not cold acclimate when exposed to low temperature such as 5°C, day/night. When ABA (45 M) was added to the culture medium, stem-cultured plantlets of S. tuberosum, cv. Red Pontiac, either grown at 20°C/15°C, day/night, or at 5°C, increased in cold hardiness from –2°C (killing temperature) to –4.5°C. The increase in cold hardiness could be inhibited in both temperature regimes if cycloheximide (70 M) was added to the culture medium at the inception of ABA treatment. Cycloheximide did not inhibit cold hardiness development, however, when it was added to the culture medium 3 days after ABA treatment.When pot-grown plants were foliar sprayed with mefluidide (50 M), ABA content increased from 10 nmol to 30 nmol g^–1 dry weight and plants increased in cold hardiness from –2°C to about –3.5°C. The increases in free ABA and cold hardiness occurred only in plants grown at 20°C/15°C; neither ABA nor cold hardiness increased in plants grown at 5°C.The results suggest that an increase in ABA and a subsequent de novo synthesis of proteins are required for the development of cold hardiness in S. tuberosum regardless of temperature regime, and that the inability to synthesize ABA at low temperature, rather than protein synthesis, appears to be the reason why S. tuberosum does not cold acclimate.     

Purification and characterization of a high molecular mass serine carboxypeptidase from <Emphasis Type="Italic">Monascus pilosus</Emphasis>

Two serine carboxypeptidases, MpiCP-1 and MpiCP-2, were purified to homogeneity from Monascus pilosus IFO 4480. MpiCP-1 is a homodimer with a native molecular mass of 125 kDa composed of two identical subunits of 61 kDa, while MpiCP-2 is a high mass homooligomer with a native molecular mass of 2,263 kDa composed of about 38 identical subunits of 59 kDa. This is unique among carboxypeptidases and distinguishes MpiCP-2 as the largest known carboxypeptidase. The two purified enzymes were both acidic glycoproteins. MpiCP-1 has an isoelectric point of 3.7 and a carbohydrate content of 11%, while for MpiCP-2 these values were 4.0 and 33%, respectively. The optimum pH and temperature were around 4.0 and 50°C for MpiCP-1, and 3.5 and 50°C for MpiCP-2. MpiCP-1 was stable over a broad range of pH between 2.0 and 8.0 at 37°C for 1 h, and up to 55°C for 15 min at pH 6.0, but MpiCP-2 was stable in a narrow range of pH between 5.5 and 6.5, and up to 50°C for 15 min at pH 6.0. Phenylmethylsulfonylfluoride strongly inhibited MpiCP-1 and completely inhibited MpiCP-2, suggesting that they are both serine carboxypeptidases. Of the substrates tested, benzyloxycarbonyl-l-tyrosyl-l-glutamic acid (Z-Tyr-Glu) was the best for both enzymes. The K_m, V_max, K_cat and K_cat/K_m values of MpiCP-1 for Z-Tyr-Glu at pH 4.0 and 37°C were 1.33 mM, 1.49 mM min^–1, 723 s^–1 and 545 mM^–1 s^–1, and those of MpiCP-2 at pH 3.5 and 37°C were 1.55 mM, 1.54 mM min^–1, 2,039 s^–1 and 1,318 mM^–1 s^–1, respectively.     

Effect of exogenous hormones on leaf yellowing in cut flowering branches of Alstroemeria pelegrina L.

Inclusion of IAA in the vase water had little effect on leaf yellowing in cut flowering branches of Alstroemeria pelegrina L. while kinetin delayed leaf yellowing at 10^–4M (continuous treatment). Chlorophyll was effectively retained by 10^–7M gibberellic acid (GA) in the vase water or by a 20h pulse at 5°C with 10^–5/10^–4M GA. After 16h of ¹⁴C-GA₃ uptake at 20°C relatively high levels of ¹⁴C were found in leaves and low levels in stems and flowers. After this treatment about half of the ¹⁴C-GA₃ in leaves was metabolized into unknown compounds.     

Production of red pigments by the insect pathogenic fungus Cordyceps unilateralis BCC 1869

Production of red pigments (naphthoquinones) by the insect pathogenic fungus Cordyceps unilateralis BCC 1869 was investigated in this study. Cultivation conditions, including temperature, intitial pH of medium, and aeration, were optimised to improve the yield of total naphthoquinones in shake-flask culture of C. unilateralis. The highest yield of total naphthoquinones (3 g L^–1) was obtained from a 28-day culture grown in potato dextrose broth with an initial pH of 7.0, at 28°C with shaking-induced aeration at 200 rpm. An extraction process for isolation of the targeted naphthoquinone, 3,5,8-trihydroxy-6-methoxy-2-(5-oxohexa-1,3-dienyl)-1,4-naphthoquinone (3,5,8-TMON), from a culture of C. unilateralis, was also developed. The yield of 3,5,8-TMON obtained was about 1.2 g L^–1 or 40% of total naphthoquinones. The stability of 3,5,8-TMON was very high, even upon exposure to strong sunlight (70,000 lx), high temperature up to 200°C, and acid and alkali solutions at concentrations of 0.1 M