Effects of thermo-chemical pre-treatment of grass silage on methane production by anaerobic digestion

Dried grass silage (GS) was pre-treated at different NaOH loading rates (1%, 2.5%, 5% and 7.5% by volatile solids (VS) mass in grass silage) and temperatures (20 °C, 60 °C, 100 °C and 150 °C) to determine effects on its bio-degradability in terms of the hydrolysis yield and degradation of ligno-cellulosic materials for biogas production. At 100 °C and the four NaOH loadings, up to 45% of the total COD was solubilised and up to 65.6%, 36.1% and 21.2% of lignin, hemicellulose and cellulose were removed, respectively; biological methane production potentials obtained were 359.5, 401.8, 449.5 and 452.5 ml CH?/g VS added, respectively, being improved by 10-38.9% in comparison with untreated GS. VS removals following anaerobic digestion were 67.6%, 76.9%, 85.3%, 95.2% and 96.7% for untreated GS and GS treated at the four NaOH loadings, respectively. 100 °C and the NaOH loading rate of 5% is recommended as a proper GS pre-treatment condition.     

Effect of physical weathering on the carbon sequestration potential of biochars and hydrochars in soil

Physical weathering can modify the stability of biochar after field exposure. The aim of our study was to determine the potential carbon sequestration of the two chars at different timescales. We investigated the modification in composition and stability resulting from physical weathering of two different chars produced (i) at low temperature (250 °C) by hydrothermal carbonization (HTC); and (ii) at high temperature (1200 °C) by gasification (GS) using contrasting feedstocks. Physical weathering of HTC and GS placed on a water permeable canvas was performed through successive wetting/drying and freezing/thawing cycles. Carbon loss was assessed by mass balance. Chemical stability of the remaining material was evaluated as resistance to acid dichromate oxidation, and biological stability was assessed during laboratory incubation. Moreover, we assessed modification in potential priming effects due to physical weathering. Physical weathering induced a carbon loss ranging between 10 and 40% of the total C mass depending on the feedstock. This C loss is most probably related to leaching of small particulate and dissolved compounds. GS produced from maize silage showed the highest C loss. The chemical stability of HTC and GS was unaffected by physical weathering. In contrast, physical weathering strongly increased the biological stability of HTC and GS char produced from maize silage. After physical weathering, the half‐life (t_1/2) of GS was doubled but only slight increase was noted for those of HTC. During the first weeks of incubation, HTC addition to soil stimulated native soil organic matter (SOM) mineralization (positive priming effect), while the GS addition led to protection of the native SOM against biologic degradation (negative priming effect). Physical weathering led to reduction in these priming effects. Model extrapolations based on our data showed that decadal C sequestration potential of GS and HTC is globally equivalent when all losses including those due to priming and physical weathering were taken into account. However, at century scale only GS may have the potential to increase soil C storage.     

Effect of temperature on low-strength wastewater treatment by UASB reactor using poly(vinyl alcohol)-gel carrier

The feasibility of treating low-strength wastewater with an up-flow anaerobic sludge blanket (UASB) reactor, using a poly(vinyl alcohol)-gel carrier, at various temperatures and hydraulic retention times (HRTs) was examined. The temperature was decreased from 35°C to 25°C and then to 15°C. The HRT was reduced from 2.0 h to 0.22 h. The COD removal rate reached 28 kg-COD m(-3)d(-1) at 35°C, 16 kg-COD m(-3)d(-1) at 25°C, and 6 kg-COD m(-3)d(-1) at 15°C. The COD removal rate was reduced by half for each temperature reduction of 10°C.     

Role of tryptophan residues in a class V chitinase from Nicotiana tabacum

Tryptophan residues located in the substrate-binding cleft of a class V chitinase from Nicotiana tabacum (NtChiV) were mutated to alanine and phenylalanine (W190F, W326F, W190F/W326F, W190A, W326A, and W190A/W326A), and the mutant enzymes were characterized to define the role of the tryptophans. The mutations of Trp326 lowered thermal stability by 5-7 °C, while the mutations of Trp190 lowered stability only by 2-4 °C. The Trp326 mutations strongly impaired enzymatic activity, while the effects of the Trp190 mutations were moderate. The experimental data were rationalized based on the crystal structure of NtChiV in a complex with (GlcNAc)(4), in which Trp190 is exposed to the solvent and involved in face-to-face stacking interaction with the +2 sugar, while Trp326 is buried inside but interacts with the -2 sugar through hydrophobicity. HPLC analysis of anomers of the enzymatic products suggested that Trp190 specifically recognizes the β-anomer of the +2 sugar. The strong effects of the Trp326 mutations on activity and stability suggest multiple roles of the residue in stabilizing the protein structure, in sugar residue binding at subsite -2, and probably in maintaining catalytic efficiency by providing a hydrophobic environment for proton donor Glu115.     

Steam reforming of bio-oil from rice husks fast pyrolysis for hydrogen production

Steam reforming of two kinds of bio-oil from rice husks fast pyrolysis was conducted for hydrogen production at three temperatures (650, 750 and 850 °C) with Ni-based catalyst in a fixed-bed reactor. The gas composition and organic compounds in liquid condensate were detected by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS), respectively. In addition, the carbon deposition was also investigated. The results showed that the mole fraction range of hydrogen was within 55.8-61.3% at all temperatures and more hydrogen was produced at the higher temperature. The highest H? efficiency of bio-oil steam reforming was 45.33% when extra water was added. The bio-oil with lower content of chemical compounds has a higher H? efficiency, but its hydrogen volume was less. Analysis of the liquid condensate showed that most of the organic compounds were circularity compounds. The carbon deposition can decrease the bio-oil conversion, and it was easier to form at the temperature of 750 °C.     

Evaluation of Integrated Anaerobic Digestion and Hydrothermal Carbonization for Bioenergy Production

Lignocellulosic biomass is one of the most abundant yet underutilized renewable energy resources. Both anaerobic digestion (AD) and hydrothermal carbonization (HTC) are promising technologies for bioenergy production from biomass in terms of biogas and HTC biochar, respectively. In this study, the combination of AD and HTC is proposed to increase overall bioenergy production. Wheat straw was anaerobically digested in a novel upflow anaerobic solid state reactor (UASS) in both mesophilic (37 °C) and thermophilic (55 °C) conditions. Wet digested from thermophilic AD was hydrothermally carbonized at 230 °C for 6 hr for HTC biochar production. At thermophilic temperature, the UASS system yields an average of 165 L_CH4/kg_VS (VS: volatile solids) and 121 L _CH4/kg_VS at mesophilic AD over the continuous operation of 200 days. Meanwhile, 43.4 g of HTC biochar with 29.6 MJ/kg_{dry_biochar} was obtained from HTC of 1 kg digestate (dry basis) from mesophilic AD. The combination of AD and HTC, in this particular set of experiment yield 13.2 MJ of energy per 1 kg of dry wheat straw, which is at least 20% higher than HTC alone and 60.2% higher than AD only.     

Two-stage acid saccharification of fractionated Gelidium amansii minimizing the sugar decomposition

Two-stage acid hydrolysis was conducted on easy reacting cellulose and resistant reacting cellulose of fractionated Gelidium amansii (f-GA). Acid hydrolysis of f-GA was performed at between 170 and 200 °C for a period of 0-5 min, and an acid concentration of 2-5% (w/v, H2SO4) to determine the optimal conditions for acid hydrolysis. In the first stage of the acid hydrolysis, an optimum glucose yield of 33.7% was obtained at a reaction temperature of 190 °C, an acid concentration of 3.0%, and a reaction time of 3 min. In the second stage, a glucose yield of 34.2%, on the basis the amount of residual cellulose from the f-GA, was obtained at a temperature of 190 °C, a sulfuric acid concentration of 4.0%, and a reaction time 3.7 min. Finally, 68.58% of the cellulose derived from f-GA was converted into glucose through two-stage acid saccharification under aforementioned conditions.     

Inhibition of chymotrypsin- and subtilisin-like serine proteases with Tk-serpin from hyperthermophilic archaeon Thermococcus kodakaraensis

A serpin homologue (Tk-serpin) from the hyperthermophilic archaeon Thermococcus kodakaraensis was overproduced in E. coli, purified, and characterized. Tk-serpin irreversibly inhibits Tk-subtilisin (TKS) from the same organism with the second-order association rate constants (k(ass)) of 5.2×103 M?1 s?1 at 40°C and 3.1×10? M?1 s?1 at 80°C, indicating that Tk-serpin inhibits TKS more strongly at 80°C than at 40°C. It also irreversibly inhibits chymotrypsin, subtilisin Carlsberg, and proteinase K at 40°C with the k(ass) values comparable to that for TKS at 80°C. Casein zymography showed that Tk-serpin inhibits these proteases by forming a SDS-resistant complex, which is typical to inhibitory serpins. The ratio of moles of Tk-serpin needed to inhibit 1 mol of protease (stoichiometry of inhibition, SI) varies from 40 to 80 at 20°C, but decreases to the minimum values of 3-7 as the temperature increases. The inhibitory activities of Tk-serpin for these proteases increase as the stabilities of these proteases decrease, suggesting that a flexibility of the active-site of protease is one of the determinants for susceptibility of protease to inhibition by Tk-serpin. This report showed for the first time that Tk-serpin inhibits both chymotrypsin- and subtilisin-like serine proteases and its inhibitory activity increases as the temperature increases up to 100°C.     

Purification, characterization and thermal inactivation kinetics of a non-regioselective thermostable lipase from a genotypically identified extremophilic Bacillus subtilis NS 8

Thermostable lipase produced by a genotypically identified extremophilic Bacillus subtilis NS 8 was purified 500-fold to homogeneity with a recovery of 16% by ultrafiltration, DEAE-Toyopearl 650M and Sephadex G-75 column. The purified enzyme showed a prominent single band with a molecular weight of 45 kDa. The optimum pH and temperature for activity of lipase were 7.0 and 60°C, respectively. The enzyme was stable in the pH range between 7.0 and 9.0 and temperature range between 40 and 70°C. It showed high stability with half-lives of 273.38 min at 60°C, 51.04 min at 70°C and 41.58 min at 80°C. The D-values at 60, 70 and 80°C were 788.70, 169.59 and 138.15 min, respectively. The enzyme's enthalpy, entropy and Gibb's free energy were in the range of 70.07-70.40 kJ mol(-1), -83.58 to -77.32 kJ mol(-1)K(-1) and 95.60-98.96 kJ mol(-1), respectively. Lipase activity was slightly enhanced when treated with Mg(2+) but there was no significant enhancement or inhibition of the activity with Ca(2+). However, other metal ions markedly inhibited its activity. Of all the natural vegetable oils tested, it had slightly higher hydrolytic activity on soybean oil compared to other oils. On TLC plate, the enzyme showed non-regioselective activity for triolein hydrolysis.     

Quantification of damage at different stages of cryopreservation of endangered North American bison (Bison bison) semen and the effects of extender and freeze rate on post-thaw sperm quality

Semen cryopreservation is an important technique for the banking of animal germplasm from endangered species and exploitation of genetically superior sires through artificial insemination. Being a member of bovidae family, bison semen has poor freezing ability as compared to dairy and beef bulls' semen. This study was designed to quantify the damage to bison sperm at different stages of cryopreservation, and to determine the effects of extender (commercial Triladyl(?) vs. custom made tris-citric acid [TCA]) and freeze rate (-10, -25 and -40°C/min) on post-thaw quality of bison semen. Semen was collected from five bison bulls (three woods and two plains) via electroejaculation. In Experiment 1, semen was diluted in Triladyl? extender and frozen with freeze rate -10°C/min. Sperm motility characteristics were recorded in fresh, diluted, cooled (4°C) and freeze-thawed semen using computer-assisted sperm analyzer (CASA). In Experiment 2, semen was diluted in Triladyl? or TCA extender, and frozen with three different freeze rates, i.e. -10, -25 or -40°C/min. Thawing was performed at 37°C for 60s. Post-thaw sperm motility characteristics were assessed using CASA, and sperm structural characteristics (plasma membrane, mitochondrial membrane potential and acrosomes) were evaluated using flow cytometer, at 0 and 3h while incubating semen at 37°C. In Experiment 1, total and progressive motilities did not differ among pre-freeze stages of cryopreservation (P>0.05). However, sperm total and progressive motilities declined (P<0.001) in freeze-thawed semen by 35% and 42%, respectively, compared to after cooling (pre-freeze) semen. In Experiment 2, Triladyl?, as compared to TCA, yielded greater (P<0.05) post-thaw sperm total motility (41% compared to 36%) and progressive motility (34% compared to 29%) at 0h, respectively. The percent change in post-thaw sperm total and progressive motilities, VAP, VCL, VSL, IPM-high ΔΨm and IPM-IACR during 3h incubation at 37°C, was less (P<0.05) in TCA than in Triladyl?. There was an effect of freeze rate on post-thaw sperm average path velocity at 0h, and total motility, progressive motility, VCL, IPM and IPM-IACR at 3h were the greatest (P<0.05) when bison semen was frozen at -40°C/min. Likewise, the percent change in post-thaw sperm total and progressive motilities, during 3h incubation at 37°C, was less (P<0.05) in bison semen frozen at -40°C/min. All post-thaw bison sperm characteristics decreased (P<0.05) from 0h to 3h, during incubation at 37°C. In conclusion, the maximum damage to bison sperm occurred during freeze-thaw processes. Post-thaw total and progressive motilities of bison sperm were greater in Triladyl? at 0h whereas sperm survival was greater in TCA extender during 3h post-thaw incubation. Bison sperm had greater survival (P<0.05) when frozen at -40°C/min freeze rate.     

Analysis of heat-induced disassembly process of three different monomeric forms of the major light-harvesting chlorophyll a/b complex of photosystem II

The temperature-dependent disassembly process of three monomeric isoforms, namely Lhcb1, Lhcb2, and Lhcb3, of the major light-harvesting chlorophyll (Chl) a/b complexes of photosystem II (LHCIIb) were characterized by observing the changes of absorption spectra, circular dichroism (CD), and dissociation processes of the bound pigments to the in vitro reconstituted complexes subjected to high temperatures. Our results suggest that the three isoforms of LHCIIb undergo conformational rearrangements, structural changes, and dissociations of the bound pigments when the ambient temperature increases from 20 to 90°C. The conformation of the complexes changed sensitively to the changing temperatures because the absorption peaks in the Soret region (436 and 471?nm) and the Qy region (650-660 and 680?nm) decreased immediately upon elevating the ambient temperatures. Analyzing temperature-dependent denaturing and pigment dissociation process, we can divide the disassembly process into three stages: The first stage, appeared from 20°C to around 50-60°C, was characterized by the diminishment of the absorption around 650-660 and 680?nm, accompanied by the blue-shift of the peak at 471?nm and disappearance of the absorbance at 436?nm, which is related to changes in the transition energy of the Chl b cluster, and the red-most Chl a cluster in the LHCIIb. The second stage, beginning at about 50-60°C, was signified by the diminishment of the CD signal between (+)483?nm and (-)490?nm, which implied the disturbance of dipole-dipole interaction of pigments, and the onset of the pigment dissociation. The last stage, beginning at about 70-80°C, indicates the complete dissociation of the pigments from the complex. The physiological aspects of the three stages in the denaturing process are also discussed.     

Purification and biochemical characterization of ionically unbound polyphenol oxidase from Musa paradisiaca leaf

An ionically unbound and thermostable polyphenol oxidase (PPO) was extracted from the leaf of Musa paradisiaca. The enzyme was purified 2.54-fold with a total yield of 9.5% by ammonium sulfate precipitation followed by Sephadex G-100 gel filtration chromatography. The purified enzyme exhibited a clear single band on native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS) PAGE. It was found to be monomeric protein with molecular mass of about 40 kD. The zymographic study using crude extract as enzyme source showed a very clear band around 40 kD and a faint band at around 15 kD, which might be isozymes. The enzyme was optimally active at pH 7.0 and 50°C temperature. The enzyme was active in wide range of pH (4.0-9.0) and temperature (30-90°C). From the thermal inactivation studies in the range 60-75°C, the half-life (t(1/2)) values of the enzyme ranged from 17 to 77?min. The inactivation energy (Ea) value of PPO was estimated to be 91.3?kJ mol(-1). It showed higher specificity with catechol (K(m)?=?8?mM) as compared to 4-methylcatechol (K(m)?=?10?mM). Among metal ions and reagents tested, Cu(2+), Fe(2+), Hg(2+), Mn(2+), Ni(2+), protocatechuic acid, and ferrulic acid enhanced the enzyme activity, while K(+), Na(+), Co(2+), kojic acid, ascorbic acid, ethylenediamine tetraacetic acid (EDTA), sodium azide, β-mercaptoethanol, and L-cysteine inhibited the activity of the enzyme.     

Production of aromatic green gasoline additives via catalytic pyrolysis of acidulated peanut oil soap stock

Catalytic pyrolysis was used to generate gasoline-compatible fuel from peanut oil soap stock (PSS), a high free fatty acid feedstock, using a fixed-bed reactor at temperatures between 450 and 550°C with a zeolite catalyst (HZSM-5). PSS fed at 81 gh(-1) along with 100 mL min(-1) inert gas was passed across a 15 g catalyst bed (WHSV=5.4h(-1), gas phase residence time=34s). Results indicate that fuel properties of PSS including viscosity, heating value, and O:C ratio were improved significantly. For PSS processed at 500°C, viscosity was reduced from 59.6 to 0.9 mm(2)s(-1), heating value was increased from 35.8 to 39.3 MJL(-1), and the O:C ratio was reduced from 0.07 to 0.02. Aromatic gasoline components (e.g., BTEX), were formed in concentrations as high as 94% (v/v) in catalytically-cracked PSS with yields ranging from 22% to 35% (v/v of PSS feed).     

Crystal structures of a type-1 ribosome inactivating protein from Momordica balsamina in the bound and unbound states

The ribosome inactivating proteins (RIPs) of type 1 are plant toxins that eliminate adenine base selectively from the single stranded loop of rRNA. We report six crystal structures, type 1 RIP from Momordica balsamina (A), three in complexed states with ribose (B), guanine (C) and adenine (D) and two structures of MbRIP-1 when crystallized with adenosine triphosphate (ATP) (E) and 2'-deoxyadenosine triphosphate (2'-dATP) (F). These were determined at 1.67?, 1.60?, 2.20?, 1.70?, 2.07? and 1.90? resolutions respectively. The structures contained, (A) unbound protein molecule, (B) one protein molecule and one ribose sugar, (C) one protein molecule and one guanine base, (D) one protein molecule and one adenine base, (E) one protein molecule and one ATP-product adenine molecule and (F) one protein molecule and one 2'-dATP-product adenine molecule. Three distinct conformations of the side chain of Tyr70 were observed with (i) χ(1)=-66°and χ(2)=165° in structures (A) and (B); (ii) χ(1)=-95° and χ(2)=70° in structures (C), (D) and (E); and (iii) χ(1)=-163° and χ(2)=87° in structure (F). The conformation of Tyr70 in (F) corresponds to the structure of a conformational intermediate. This is the first structure which demonstrates that the slow conversion of DNA substrates by RIPs can be trapped during crystallization.     

Kinetic study for Fe(NO3)3 catalyzed hemicellulose hydrolysis of different corn stover silages

Five inorganic salts, ZnCl(2), FeSO(4), Fe(2)(SO(4))(3), FeCl(3) and Fe(NO(3))(3) were chosen as catalysts to determine their effects on hemicellulose hydrolysis in control silage (no silage additive), and the results indicated that Fe(NO(3))(3) was the most efficient catalyst for hemicellulose hydrolysis. The kinetics of Fe(NO(3))(3) catalyzed hydrolysis for control silage and acid silage (treatment with HNO(3)) were investigated at various pretreatment conditions. The results demonstrated that Saeman model was well consistent with Fe(NO(3))(3) catalyzed hydrolysis reaction for corn stover silage, and kinetic parameters for this model were developed by the Arrhenius equation. Optimum pretreatment conditions were 0.05 M Fe(NO(3))(3) at 150°C for 21.2 min for control silage and 12.7 min for acid silage, which obtained the maximum xylose yields 81.66% and 93.36% of initial xylan, respectively. The activation energies for hemicellulose hydrolysis in control and acid silage ranged from 44.35 to 86.14 kJ/mol and from 3.11 to 34.11 kJ/mol, respectively.     

Resting metabolism and critical thermal maxima of vespine wasps (Vespula sp.)

Vespine wasps are known for their high endothermic capacity. Endothermic activity is directly linked to respiration. However, knowledge on wasp respiration is sparse and almost nothing is known about their resting metabolism. We investigated the yellowjackets' CO(2) production in a flow-through respirometer chamber overnight. Endothermic and behavioral activity was observed by real-time infrared thermography. Most resting wasps were ectothermic or only slightly endothermic (thoracic temperature excess against abdomen <0.6°C). In the investigated temperature range (T(a)=2.9-42.4°C) mean CO(2) production rate of resting wasps increased steeply according to an exponential function, from 5.658 μl g(-1) min(-1) at 8.3°C to 8.504 μl g(-1) min(-1) at 20.2°C, 58.686 μl g(-1) min(-1) at 35.3°C and 102.84 μl g(-1) min(-1) at 40°C. The wasps' respiratory critical thermal maximum (CT(max)), marking the upper edge of their viable temperature range, was 45.3°C. The respiratory CT(max) did not differ significantly from the activity CT(max) of 44.9°C. CT(max) values were considerably below that of honeybees (48.9 and 49.0°C for respiration and activity, respectively). This allows honeybees to kill wasps by heat-balling. Comparison with other arthropods showed that vespine wasps are among the insects with the highest mass-specific resting metabolic rate and the steepest increase of metabolism with ambient temperature.     

Lipid peroxidation and activities of tyrosine aminotransferase and glutamine synthetase in hepatoma and glioma cells grown in bovine colostrum-supplemented medium

Summary The growth stimulating properties of bovine serum and colostrum were compared in rat hepatoma (HTC) and glioma (C6) cell cultures. A colostrum concentration of 2% was optimal for HTc cells, which then reached a terminal density 40% of that in serum-supplemented medium. The corresponding figures for C6 cells were 10 and 81%, respectively. After 4 d in culture, levels of lipid hydroperoxides were measured and compared. Highest levels of lipid hydroperoxides were found in HTC and C6 cells grown in unsupplemented medium. HTC and C6 cells grown in serum supplemented medium contained levels of 52 and 64%, respectively, of that in unsupplemented medium. The corresponding levels for cells grown in presence of colostrum were 40% for HTC and 44% for C6 cells. To obtain information on any functional alterations in the cells due to the presence of colostrum the induction of tyrosine aminotransferase (EC 2.6.1.5) and glutamine synthetase (EC 6.3.1.2), by dexamethasone was studied. Although colostrum seemed to increase, the basal activities of the enzymes, no significant effects on the degree of induction could be detected. This work was supported by grants from The Wenner-Gren Foundation Arla Ekonomisk F?rening and the Nordic Society Against Painful Experiments on Animals (grant 82-07-15).     

Effect of temperature level on thermal acclimation in Large White growing pigs

The effect of temperature level (24°C, 28°C, 32°C or 36°C) on performance and thermoregulatory response in growing pigs during acclimation to high ambient temperature was studied on a total of 96 Large White barrows. Pigs were exposed to 24°C for 10 days (days -10 to -1, P0) and thereafter to a constant temperature of 24°C, 28°C, 32°C or 36°C for 20 days. Pigs were housed in individual metal slatted pens, allowing a separate collection of faeces and urine and given ad libitum access to feed. Rectal (RT) and cutaneous (CT) temperatures and respiration rate (RR) were measured three times daily (0700, 1200 and 1800 h) every 2 to 3 days during the experiment. From day 1 to 20, the effect of temperature on average daily feed intake (ADFI) and BW gain (average daily gain, ADG) was curvilinear. The decrease of ADFI averaged 90 g/day per °C between 24°C and 32°C and 128 g/day per °C between 32°C and 36°C. The corresponding values for ADG were 50 and 72 g/day per °C, respectively. The 20 days exposure to the experimental temperature was divided in two sub-periods (P1 and P2, from day 1 to 10 and from day 11 to 20, respectively). ADFI was not affected by duration of high-temperature exposure (i.e. P2 v. P1). The ADG was not influenced by the duration of exposure at 24°C and 28°C groups. However, ADG was higher at P2 than at P1 and this effect was temperature dependent (+130 and +458 g/day at 32°C and 36°C, respectively). In P2 at 36°C, dry matter digestibility significantly increased (+2.1%, P < 0.01); however, there was no effect of either duration or temperature on the digestibility of dry matter at group 24°C and 32°C. RT, CT and RR were measured three times daily (0700, 1200 and 1800 h) every 2 to 3 days during the experiment. Between 28°C and 36°C, RT and CT were lower during P2 than during P1 (-0.20°C and -0.23°C; P < 0.05), whereas RR response was not affected by the duration of exposure whatever the temperature level. In conclusion, this study suggests that the effect of elevated temperatures on performance and thermoregulatory responses is dependent on the magnitude and the duration of heat stress.     

Optimization of production and reaction conditions of polygalacturonase from Byssochlamys fulva

In the present study, the optimization of production and reaction conditions of polygalacturonase produced by a fungus Byssochlamys fulva MTCC 505 was achieved. The production of polygalacturonase with a considerable activity of 1.28 IU/ml was found when the culture was shaken at 30°C for 5 days in 100 ml of medium containing (w/v) 10 g/l pectin, 2 g/l NaNO?, 1 g/l KH?PO?, 0.5 g/l KCl, 0.5 g/l MgSO?. 7H?O, 0.001 g/l FeSO?. 7H?O, 0.001 g/l CaCl?. The best carbon and nitrogen source for this enzyme were pectin (1%) and Ca(NO?)? (0.1%), respectively. The enzyme gave maximum activity at incubation time of 72 h, temperature of 30°C and pH 4.5. During the optimization of reaction conditions, the enzyme showed maximum activity in sodium citrate buffer (50 mM) of pH 5.5 at 50°C reaction temperature for 15 minutes of incubation. The enzyme showed greater affinity for polygalacturonic acid as substrate (0.5%). Km and Vmax values were 0.15 mg/ml and 4.58 μmol/ml/min. The effect of various phenolics, thiols, protein inhibitors and metal ions on the enzyme activity was investigated. The enzyme was quite stable at 4°C and 30°C. At 40°C the half life of the enzyme was 6 h and at 60°C it was 2 h.