Fructose production by immobilizedArthrobacter cells

Summary ImmobilizedArthrobacter cells (NRRL-B-3728) were used for continuous isomerization of glucose to fructose in a bioreactor system. The system utilized stationary phase (55h) cells (2.2×10⁹ CFU/ml saline) immobilized onto K-carrageenan (3% w/v) beads [cells were heated at 65°C for 10 min to inactivate endogenous proteolytic enzymes]. Immobilized-cell preparations were hardened using three different glutaraldehyde systems. Glutaraldehyde (0.2 M) treated-immobilized cells (pH 7.0, 5°C for 30 min) exhibited good gel strength and high glucose isomerase activities. Maximal bioreactor isomerization of 44% was achieved when a buffered feedstock containing 40% glucose was fed into the column (60°C) at a flow rate of 0.2 ml/min. The biological half-life of glucose isomerase activities in this system was 400 h. Scanning electron microscopy revealed large numbers of cells distributed within the beads. A thin layer surrounding the beads following glutaraldehyde treatment was mainly due to cross-linking reactions between cell proteins and glutaraldehyde. This layer prevented leaking of cells during continuous isomerization reaction.     

Estimation of cardiac output and stroke volume from thermal equilibration and heartbeat rates in fish

Cardiac output and stroke volume were estimated for a 200 g largemouth blackbass (Micropterus salmoides) by a modified whole-body thermodilution method using the relation between thermal equilibration rates and heartbeat frequencies. The reciprocal of the thermal time constant, k (min^–1), was related to the heartbeat frequency, F (beats min^–1), by the equation k=0.00146 F + 0.309; the slope is the weight-specific stroke volume (ml g^–1) and the intercept is the weight-specific heat transfer constant (cal °C^–1 min^–1 g^–1). Stroke volume was 0.292 ml (0.00146 ml/g body weight), yielding cardiac output values ranging from 44 ml kg^–1 min^–1 (at 30 beats min) to 158 ml kg^–1 min^–1 (at 108 beats min^–1), or 4.4 to 15.8% of body weight. Active (convective) heat transfer due to blood flow constituted an estimated 11 to 34% (mean 22.5%) of total heat transfer, depending on heartbeat frequency; this variability constitutes physiological thermoregulation.     

Transesterification of divinyladipate with glucose at various temperatures by an alkaline protease of Streptomyces sp.

The transesterification of 1 M divinyladipate with 0.25 M glucose in dimethylformamide (DMF) catalyzed by 5 mg ml^–1 alkaline protease (24 units mg^–1 min^–1) from Streptomyces sp. gave 6-O-vinyladipoyl d-glucose as the main product with yields are between 60 and 90%. The optimum temperature for the reaction was about 50 °C.     

H<Subscript>2</Subscript>-Producing bacterial communities from a heat-treated soil inoculum

Hydrogen gas (60% H₂) was produced in a continuous flow bioreactor inoculated with heat-treated soil, and fed synthetic wastewater containing glucose (9.5 g l^–1). The pH in the bioreactor was maintained at 5.5 to inhibit consumption of H₂ by methanogens. The objective of this study was to characterize bacterial communities in the reactor operated under two different hydraulic retention times (HRTs of 30-h and 10-h) and temperatures (30°C and 37°C). At 30-h HRT, the H₂ production rate was 80 ml h^–1 and yield was 0.91 mol H₂/mol glucose. At 10-h HRT, the H₂ production rate was more than 5 times higher at 436 ml h^–1, and yield was 1.61 mol H₂/mol glucose. Samples were removed from the reactor under steady-state conditions for PCR-based detection of bacterial populations by ribosomal intergenic spacer analysis (RISA). Populations detected at 30-h HRT were more diverse than at 10-h HRT and included representatives of Bacillaceae, Clostridiaceae, and Enterobacteriaceae. At 10-h HRT, only Clostridiaceae were detected. When the temperature of the 10-h HRT reactor was increased from 30°C to 37°C, the steady-state H₂ production rate increased slightly to 463 ml h^–1 and yield was 1.8 mol H₂/mol glucose. Compared to 30°C, RISA fingerprints at 37°C from the 10-h HRT bioreactor exhibited a clear shift from populations related to Clostridium acidisoli (subcluster Ic) to populations related to Clostridium acetobutylicum (subcluster Ib).     

Grape skins as a natural support for yeast immobilization

Grape skins were used to immobilize Saccharomyces cerevisiae. In repeated batch fermentations of grape by immobilized and free cells, the maximum specific rate of alcohol production on glucose decreased from 7.98 h^–1 at 25 °C to 0.7 h^–1 at 5 °C. The rate was approximately twice as high as that on fructose. The rates for free cells were very low. The maximum alcohol yield (0.45 g g^–1) was obtained at 5 °C when the immobilized biocatalyst was used.     

Metabolic and hormonal responses during exercise at 20°, 0° and −20°C

This study was designed to clarify the effects of cold air exposure on metabolic and hormonal responses during progressive incremental exercise. Eight healthy males volunteered for the study. Informed consent was obtained from every participant. The following protocol was administered to each subject on three occasions in a climatic chamber in which the temperature was 20°, 0° or –20°C with relative humidity at 60%±1%. Exercise tests were conducted on an electrically braked ergocycle, and consisted of a propressive incremental maximal exercise. Respiratory parameters were continuously monitored by an automated open-circuit sampling system Exercise blood lactate (LA), free fatty acids (FFA), glucose levels, bicarbonate concentration (HCO ₃ ^– ), acidbase balance, plasma epinephrine (E) and norepinephrine (NE) were determined from venous blood samples obtained through an indwelling brachial catheter. Maximal oxygen uptake was significantly different between conditions: 72.0±5.4 ml kg^–1 min^–1 at 20°C; 68.9±5.1 ml kg^–1 min^–1 at 0°C and 68.5±4.6 ml kg^–1 min^–1 at –20°C. Workload, time to exhaustion, glucose levels and rectal Catecholamines and lactate values were not significantly altered by thermal conditions after maximal exercise but the catecholamines were decreased during rest. Bicarbonate, respiratory quotient, lactate and ventilatory thresholds increased significantly at –20°C. The data support the contention that metabolic and hormonal responses following progressive incremental exercise are altered by cold exposure and they indicate a marked decrease in maximal oxygen uptake, time to exhaustion and workload.This study was supported by grants from CSR, Univesité du Québec; FIR, Université du Québec à Trois-Rivières and NATO no, 86.0435.     

Lactulose production by β-galactosidase in permeabilized cells of<Emphasis Type="Italic"> Kluyveromyces lactis</Emphasis>

Lactulose production from lactose and fructose was investigated with several commercial -galactosidases. The enzyme from Kluyveromyces lactis exhibited the highest lactulose productivity among the -galactosidases tested. The reaction conditions for lactulose production were optimized using cells that had been permeabilized by treatment with 50% (v/v) ethanol: cell concentration, 10.4 g l^–1; concentration of substrates, 40% (w/v) lactose and 20% (w/v) fructose; temperature, 60^°C; pH 7.0. Under these conditions, the permeabilized cells produced approximately 20 g l^–1 lactulose in 3 h with a lactulose productivity of 6.8 g l^–1 h^–1. These results represent 1.3- and 2.1-fold increases in lactulose concentration and productivity compared with untreated washed cells. This is the first reported trial of enzymatic synthesis of lactulose using permeabilized yeast cells.     

Kinetics of Bifidobacterium longum ATCC 15707 fermentations: effect of the dilution rate and carbon source

The effect of the dilution rate on biomass and product synthesis in fermentations of glucose, fructose and a commercial mixture of fructooligosaccharides (FOS) by Bifidobacterium longum ATCC 15707 was studied. Kinetic parameters (maximum specific growth rate, Monod constant, maintenance, and yield coefficients) in the mathematical model of the fermentation were estimated from experimental data. In the FOS mixture fermentations, approximately 12% of the total reducing sugars (mainly fructose) in the feed were not metabolized by the bacterium. In fermentations of fructose and the FOS mixture, biomass concentration increased as the dilution rate increased and, once maximum values were reached [3.90 (D=0.20 h^–1) and 2.54 g l^–1 (D=0.15 h^–1), respectively], decreased rapidly as the culture was washed out. Formic acid was detected at low dilution rates in glucose and fructose fermentations. The main products in fermentations of the three carbon sources were lactic and acetic acids. Average values of the molar ratio between acetic and lactic acids of 1.18, 1.21 and 0.83 mol mol^–1 were obtained in glucose, fructose and FOS mixture fermentations, respectively. In batch fermentations carried out without pH control this molar ratio was lower than 1.5 only when fructose was used as the carbon source.     

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.     

Production of mannitol by a novel strain of Candida magnoliae

A novel microorganism was isolated which is able to produce mannitol when grown in the presence of fructose and glucose as carbon sources. In flask culture in a medium containing 150 g fructose l^–1, it yielded 67 g mannitol l^–1 after 168 h. In fed-batch culture with 3–12% (w/v) fructose, production reached a maximum of 209 g mannitol l^–1 after 200 h, corresponding to an 83% yield and a 1.03 g l^–1 h^–1 productivity. The isolated strain was identified as Candida magnoliae based on identical sequences in the D1/D2 domain of its 26S rDNA and a similar carbon source utilization pattern with C. magnoliae reference strains.     

Ethanol formation from cellulose by thermophilic bacteria

Summary A wild coculture of obligately thermophilic bacteria, including only a single cellulolytic species Clostridium, ferments 2% crystalline cellulose and produces 4.6–5.1 g·l^–1 of ethanol at 55°–60° C; that is, 0.96–1.1 moles of ethanol from 1 mole of glucose equivalent of cellulose degraded. However, the ethanol yield decreases with increasing cellulose concentration. Ethanolacetic acid ratio varies around 1 and cannot be influenced by substrate concentration. However, this ratio can be influenced by changing pH and temperature. For the ethanol production from cellulose, neutral and weekly alkaline media with a pH of 7.0–8.0 and a temperature of 55° C are optimal. Experiments in which the coculture was subjected to high ethanol concentrations showed that higher concentrations of added ethanol (up to 20 g·l^–1) suppress cellulose degradation by 50% and inhibit the actual production of ethanol.     

Fed-batch fermentation for production of Kluyveromyces marxianusFII 510700 cultivated on a lactose-based medium

A strain of Kluyveromyces marxianus was grown in batch culture in lactose-based media at varying initial lactose concentrations (10–60 g L^–1) at 30°C, pH 5.0, dissolved oxygen concentrations greater than 20%. Increasing the concentration of mineral salts three-fold at 40 g L^–1 and 60 g L^–1 initial lactose concentration showed only a small increase in the yield of biomass, from 0.38 g g^–1 to 0.41 g g^–1, indicating that the initial batch cultures were not significantly nutrient- (mineral salts)-limited. A relatively high biomass concentration (105 g L^–1) was obtained in fed-batch culture following extended lactose feeding. An average specific growth rate (0.27 h^–1), biomass yield (0.38 g g^–1) and overall productivity (2.9 g L^–1 h^–1) were obtained for these fed-batch conditions. This fed-batch protocol provides a strategy for achieving relatively high concentrations and productivities of K. marxianus on other lactose-based substrate streams (e.g., whey) from the dairy industry.     

Increased yield of fructose from glucose employing thermophilic xylose isomerase in water-ethanol mixtures

Summary The isomerization of D-glucose in mixed ethanol-water was studied at various reaction temperatures (40–70 °C), employing glucose isomerase fromStreptomyces phaeochromogenes andClostridium thermohydrosulfuricum, respectively. The thermophilicClostridium enzyme was considerably, more stable towards the combination of organic cosolvent and increased temperature and with this enzyme a 55% yield of fructose from glucose was obtained at relatively low concentration of ethanol (40 %).     

Optimization of thermostable lipase production from a thermophilic Geobacillus sp. using Box-Behnken experimental design

Thermostable lipase production by Geobacillus thermoleovorans was optimized in shake-flask cultures using Box-Behnken experimental design. An empirical model was developed through response surface methodology to describe the relationship between tested variables (Tween 80, olive oil, temperature and pH) and enzyme activity. Maximum enzyme activity (495 U l^–1) was attained with Tween 80 at 5 g l^–1; olive oil at 60 g l^–1; 70 °C and pH 9. Experimental verification of the model showed a validation of 95%, which is more than 4-fold increase compared to the basal medium.     

Biological conversion of wood hydrolysate to succinic acid by Anaerobiospirillum succiniciproducens

Anaerobiospirillum succiniciproducens grew on a minimal salts medium containing wood hydrolysate (equivalent to 27 g glucose l^–1) and, when supplemented with 10 g corn steep liquor l^–1 as a complex nitrogen source, succinic acid at 24 g l^–1 was obtained (yield = 88% w/w glucose). This may therefore be an economical method to produce succinic acid.     

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.     

Freeze or dehydrate: only two options for the survival of subzero temperatures in the arctic enchytraeid<Emphasis Type="Italic"> Fridericia ratzeli</Emphasis>

Hygrophilic soil animals, like enchytraeids, overwintering in frozen soil are unlikely to base their cold tolerance on supercooling of body fluids. It seems more likely that they will either freeze due to inoculative freezing, or dehydrate and adjust their body fluid melting point to ambient temperature as has been shown for earthworm cocoons and Collembola. In the present study we tested this hypothesis by exposing field-collected adult Fridericia ratzeli from Disko, West Greenland, to freezing temperatures under various moisture regimes. When cooled at –1 °C min^–1 under dry conditions F. ratzeli had a mean temperature of crystallisation (T_c) of –5.8 °C. However, when exposed to temperatures above standard T_c for 22 h, at –4 °C, most individuals (90%, n= 30) remained unfrozen. Slow cooling from –1 °C to –6 °C in vials where the air was in equilibrium with the vapour pressure of ice resulted in freezing in about 65% of the individuals. These individuals maintained a normal body water content of 2.7–3.0 mg mg^–1 dry weight and had body fluid melting points of about –0.5 °C with little or no change due to freezing. About 35% of the individuals dehydrated drastically to below 1.1 mg mg^–1 dry weight at –6 °C, and consequently had lowered their body fluid melting point to ca. –6 °C at this time. Survival was high in both frozen and dehydrated animals at –6 °C, about 60%. Approximately 25% of the animals (both frozen and dehydrated individuals) had elevated glucose concentrations, but the mean glucose concentration was not increased to any great extent in any group due to cold exposure. The desiccating potential of ice was simulated using aqueous NaCl solutions at 0 °C. Water loss and survival in this experiment were in good agreement with results from freezing experiments. The influence of soil moisture on survival and tendency to dehydrate was also evaluated. However, soil moisture ranging between 0.74 g g^–1 and 1.15 g g^–1 dry soil did not result in any significant differences in survival or frequency of dehydrated animals even though the apparent wetness and structure of the soil was clearly different in these moisture contents.Abbreviations DW dry weight - FW fresh weight - MP melting point - RH relative humidity - Tc crystallisation temperatures - WC water contentCommunicated by I.D. Hume     

o-Phthalaldehyde–N-acetylcysteine polyamine derivatives: formation and stability in solution and in C18 supports

A comparative study of different derivatization procedures has been performed in order to improve the stability of the reaction products o-phthalaldehyde–N-acetylcysteine (OPA–NAC) polyamines. Procedures such as solution derivatization, solution derivatization followed by retention on a packing support, derivatization on different packing supports and on-column derivatization, have been optimized and compared. The degradation rate constant (k) of the derivative was dependent on the procedure used and on the analyte. For the spermine (the most unstable isoindol tested) k was 8±2×10⁻² min⁻¹ in solution versus 7.7±1.1×10⁻⁴ min⁻¹ on the (C₁₈) solid support. The results obtained showed that forming the derivative on the packing support (C₁₈) gave the best results following this procedure: conditioning the cartridges with borate buffer (1 ml, 0.5 M, pH 8), retention of the analyte, addition of 0.8 ml of OPA–NAC reagent, 0.2 ml borate buffer 0.8 M (pH 8) and elution of the isoindol with 3 ml of MeOH–borate buffer (9:1). The different derivatization procedures have been used to study the stability of the reaction products OPA–NAC polyamines formed in urine matrix using spermine as model compound. Similar results were obtained for standard solutions and urine samples.     

Starch conversion by amylases fromAureobasidium pullulans

Summary A color variant strain (NRRL Y-12974) ofAureobasidium pullulans produced a saccharifying -amylase and two forms of glucoamylase extracellularly when grown on starch at 28°C for 4 days. A sugar syrup containing DP1 (degree of polymerization) and DP2 (31) was made from maltodextrin DE (dextrose equivalent) 10 (35%, w/w) at 55°C and pH 4.5 using the amylase preparation (40 U g^–1 DS (dry substance). The syrup composition was highly dependent upon substrate concentration but nearly independent of enzyme dose. Glucose syrup containing 93% glucose was made from maltodextrin DE 10 (35%, w/w) at 65°C and pH 4.5 using the same enzyme preparation at 100 U g^–1 DS. The enzyme preparation (100 U g^–1 DS) produced 98–100% glucose from raw corn starch at pH 4.5 and 50°C.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned. Abbreviations: DE, dextrose equivalent (an indication of polymerization; reducing sugars as percentage glucose); DP, degree of polymerization; DP1, glucose; DP2, disaccharide; DP3, trisaccharide; DP4, tetrasaccharide; DS, dry substance.     

Influence of environmental factors in the production of R(−)-1, 2-propanediol by clostridium thermosaccharolyticum

The effect of the most important environmental parameters, temperature, pH, gas phase composition and substrate concentration, on the production of R(–)-1,2-propanediol, was investigated. An improved yield of 0.20 g R(–)-1,2-propanediol/g glucose was reached when the fermentation was run at 60°C and pH 6.0 under a N₂ atmosphere.