Batch and continuous fermentation methods for the production of dextransucrase

The available literature concerning dextransucrase and dextran production and purification has been reviewed along with the reaction mechanisms of the enzyme. A discussion of basic fermentation theory is included, together with a brief description of bioreactor hydrodynamics and general biotechnology. The various fermenters used in this research work are described in detail, along with the various experimental techniques employed. The micro-organism Leuconostoc mesenteroides NRRL B512 (F) secretes dextransucrase in the presence of an inducer, sucrose, this being the only known inducer of the enzyme. Dextransucrase is a growth related product and a series of fed-batch fermentations have been carried out to extend the exponential growth phase of the organism. These experiments were carried out in a number of different sized vessels, ranging in size from 2.5 to 1,000 litres. Using a 16 litre vessel, dextransucrase activities in excess of 450 DSU/cm3 (21.67 U/cm3) have been obtained under non-aerated conditions. It has also been possible to achieve 442 DSU/cm3 (21.28 U/cm3) using the 1,000 litre vessel, although this has not been done consistently. A 1 litre and a 2.5 litre vessel were used for the continuous fermentations of dextransucrase. The 2.5 litre vessel was a very sophisticated MBR MiniBioreactor and was used for the majority of continuous fermentations carried out. An enzyme activity of approximately 108 DSU/cm3 (5.20 U/cm3) was achieved at a dilution rate of 0.50 h-1, which corresponds to the maximum growth rate of the cells under the process conditions. A number of continuous fermentations were operated for prolonged periods of time, with experimental run-times of up to 389 h being recorded without any incidence of contamination. The phenomenon of enzyme enhancement on hold-up of up to 100% was also noted during these fermentations, with dextransucrase of activity 89.7 DSU/cm3 (4.32 U/cm3) being boosted to 155.7 DSU/cm3 (7.50 U/cm3) following 24 hours of hold-up. These findings support the recommendation of a second reactor being placed in series with the existing vessel.

Efficiency of rumen microbial protein synthesis in cattle grazing tropical pastures as estimated by a novel technique

The efficiency of microbial protein synthesis (EMPS) in cattle grazing a range of tropical pasture types was examined using a new method of intra-jugular infusion of CrEDTA to estimate urinary excretion of purine derivatives (PD). Seven pasture types were studied in south-east Queensland, Australia, over a 13-month period. These included native tropical grass (C4) pasture (major species Heteropogon contortus and Bothriochloa bladhii) studied in the early wet, the wet/dry transition and the dry season; introduced tropical grass (C4) pasture (Bothriochloa insculpta) in the mid wet season; two introduced tropical legume species (C3), (Lablab purpureus and Clitoria ternatea); and the temperate grass (C3) pasture, ryegrass (Lolium multiflorum). There was a large range in EMPS across pasture types: 26-209 g microbial crude protein (MCP)/kg digestible organic matter intake (DOMI). Estimated rumen degradable protein (RDP) supply (42-525 g/kg DOMI) was the major factor associated with EMPS across the range of pasture types studied. EMPS in steers grazing all tropical grass pastures was low (<130 g/kg DOMI) and limited by RDP supply. Negative linear relationships (P<0.05) between EMPS and both neutral detergent fibre (NDF) and acid detergent fibre (ADF) concentrations in extrusa were evident. However, non-fibre carbohydrate in extrusa, total non-structural carbohydrate concentration in plucked pasture leaf, rumen fluid and particle dilution rate, protozoal concentration in rumen fluid and rumen fluid pH were not correlated with EMPS. It was concluded that EMPS was well below 130 g MCP/kg DOMI when cattle grazed unfertilised, tropical grass pastures in south-east Queensland and that RDP was the primary limiting nutrient. High EMPS was associated with very high RDP, vastly in excess of RDP requirements by microbes

Automatic Tracer-Dilution Method Used for Stage-Discharge Ratings and Streamflow Hydrographs on Small Iowa Streams, HR-285, 1990

An automatic system was designed to concurrently measure stage and discharge for the purpose of developing stage-discharge ratings and high flow hydrographs on small streams. Stage, or gage height, is recorded by an analog-to-digital recorder and discharge is determined by the constant-rate tracer-dilution method. The system measures flow above a base stage set by the user. To test the effectiveness of the system and its components, eight systems, with a variety of equipment, were installed at crest-stage gaging stations across Iowa. A fluorescent dye, rhodamine-WT, was used as the tracer. Tracer-dilution discharge measurements were made during 14 flow periods at six stations from 1986 through 1988 water years. Ratings were developed at three stations with the aid of these measurements. A loop rating was identified at one station during rapidly-changing flow conditions. Incomplete mixing and dye loss to sediment apparently were problems at some stations. Stage hydrographs were recorded for 38 flows at seven stations. Limited data on background fluorescence during high flows were also obtained.

Hot wire configuration for depositing device grade nano-crystalline silicon at high deposition rate

The University of Barcelona is developing a pilot-scale hot wire chemical vapor deposition (HW-CVD) set up for the deposition of nano-crystalline silicon (nc-Si:H) on 10 cm × 10 cm glass substrate at high deposition rate. The system manages 12 thin wires of 0.15-0.2 mm diameter in a very dense configuration. This permits depositing very uniform films, with inhomogeneities lower than 2.5%, at high deposition rate (1.5-3 nm/s), and maintaining the substrate temperature relatively low (250 °C). The wire configuration design, based on radicals' diffusion simulation, is exposed and the predicted homogeneity is validated with optical transmission scanning measurements of the deposited samples. Different deposition series were carried out by varying the substrate temperature, the silane to hydrogen dilution and the deposition pressure. By means of Fourier transform infrared spectroscopy (FTIR), the evolution in time of the nc-Si:H vibrational modes was monitored. Particular importance has been given to the study of the material stability against post-deposition oxidation.

Statistical inference of OH concentrations and air mass dilution rates from successive observations of non-methane hydrocarbons in single air masses

Bayesian inference has been used to determine rigorous estimates of hydroxyl radical concentrations () and air mass dilution rates (K) averaged following air masses between linked observations of nonmethane hydrocarbons (NMHCs) spanning the North Atlantic during the Intercontinental Transport and Chemical Transformation (ITCT)-Lagrangian-2K4 experiment. The Bayesian technique obtains a refined (posterior) distribution of a parameter given data related to the parameter through a model and prior beliefs about the parameter distribution. Here, the model describes hydrocarbon loss through OH reaction and mixing with a background concentration at rate K. The Lagrangian experiment provides direct observations of hydrocarbons at two time points, removing assumptions regarding composition or sources upstream of a single observation. The estimates are sharpened by using many hydrocarbons with different reactivities and accounting for their variability and measurement uncertainty. A novel technique is used to construct prior background distributions of many species, described by variation of a single parameter . This exploits the high correlation of species, related by the first principal component of many NMHC samples. The Bayesian method obtains posterior estimates of , K and following each air mass. Median values are typically between 0.5 and 2.0 × 106 molecules cm−3, but are elevated to between 2.5 and 3.5 × 106 molecules cm−3, in low-level pollution. A comparison of estimates from absolute NMHC concentrations and NMHC ratios assuming zero background (the “photochemical clock” method) shows similar distributions but reveals systematic high bias in the estimates from ratios. Estimates of K are ∼0.1 day−1 but show more sensitivity to the prior distribution assumed.

An isotope dilution model for partitioning phenylalanine uptake by the liver of lactating dairy cows

An isotope dilution model for partitioning phenylalanine uptake by the liver of the lactating dairy cow was constructed and solved in the steady state. If assumptions are made, model solution permits calculation of the rate of phenylalanine uptake from portal vein and hepatic arterial blood supply, phenylalanine release into the hepatic vein, phenylalanine oxidation and synthesis, and degradation of hepatic constitutive and export proteins. The model requires the measurement of plasma fow rate through the liver in combination with phenylalanine concentrations and plateau isotopic enrichments in arterial, portal and hepatic plasma during a constant infusion of [1-13C]phenylalanine tracer. The model can be applied to other amino acids with similar metabolic fates and will provide a means for assessing the impact of hepatic metabolism on amino acid availability to peripheral tissues. This is of particular importance for the dairy cow when considering the requirements for milk protein synthesis and the negative environmental impact of excessive nitrogen excretion.

An isotope dilution model for partitioning phenylalanine and tyrosine uptake by the mammary gland of lactating dairy cows

An isotope dilution model for partitioning phenylalanine and tyrosine uptake by the mammary gland of the lactating dairy cow is constructed and solved in the steady state. The model contains four intracellular and four extracellular pools and conservation of mass principles are applied to generate the fundamental equations describing the behaviour of the system. The experimental measurements required for model solution are milk secretion and plasma flow rate across the gland in combination with phenylalanine and tyrosine concentrations and plateau isotopic enrichments in arterial and venous plasma and free and protein bound milk during a constant infusion of [1-(13)C]phenylalanine and [2,3,5,6-(2)H]tyrosine tracer. If assumptions are made, model solution enables determination of steady state flows for phenylalanine and tyrosine inflow to the gland, outflow from it and bypass, and flows representing the synthesis and degradation of constitutive protein and hydroxylation. The model is effective in providing information about the fates of phenylalanine and tyrosine in the mammary gland and could be used as part of a more complex system describing amino acid metabolism in the whole ruminant.

Reliability of lithium dilution cardiac output in anaesthetized sheep

BACKGROUND: Cardiac output (CO) measurement with lithium dilution (COLD) has not been fully validated in sheep using precise ultrasonic flow probe technology (COUFP). Sheep generate important cardiovascular research models and the use of COLD has become more popular in experimental settings. METHODS: Ultrasonic transit-time perivascular flow probes were surgically implanted on the pulmonary artery of 13 sheep. Paired COLD readings were taken at six time points, before and after implantation of a left ventricular assist device (LVAD) and compared with COUFP recorded just after lithium injection. RESULTS: The mean COLD was 5.7 litre min(-1) (range 3.8-9.6 litre min(-1)) and mean COUFP 5.9 litre min(-1) (range 4.0-9.2 litre min(-1)). The bias (standard deviation) was 0.3 (1.0) litre min(-1) [5.1 (16.9)%] and limits of agreement (LOA) were -1.7 to 2.3 litre min(-1) (-28.8 to 39.0%) with a percentage error (PE) of 34.4%. Data to assess trending [rate (95% confidence intervals)] included a 78 (62-93)% concordance rate in the four-quadrant plot (n=27). In the half moon polar plot (n=19), the mean polar angle was +5°, the radial LOA were -49 to +35° and 68 (47-89)% of data points fell within 22.5° of the mean polar angle. Both tests indicated moderate to poor trending ability. CONCLUSION: COLD is not precise when evaluated against COUFP in sheep based on the statistical criteria set, but the results are comparable with previously published animal studies. KEYWORDS:

Grazing rate of microzooplankton measured experimentally in the North Atlantic during the Maria S. Merian cruise MSM26, spring 2013

The microzooplankton grazing dilution experiments were conducted at stations 126, 127, 131 and 133-137, following Landry & Hassett (1982). Seawater samples (whole seawater - WSW) were taken via Niskin bottles mounted on to a CTD Rosette out of the chlorophyll maximum at each station. Four different dilution levels were prepared with WSW and GF/F filtered seawater - 100% WSW, 75% WSW, 50% WSW and 25% WSW. The diluted WSW was filled in 2.4 L polycarbonate bottles (two replicates for every dilution level). Three subsamples (250 - 500 mL depending on in situ chlorophyll) of the 100% WSW were filtered on to GF/F filters (25 mm diameter) and chlorophyll was extracted in 5 mL 96% ethanol for 12-24 hours. Afterwards it was measured fluorometrically before and after the addition of HCl with a Turner fluorometer according to Jespersen and Christoffersen (1987) on board of the ship. In addition, one 250 mL subsample of the 100% WSW was fixed in 2% Lugol (final concentration), to determine the microzooplankton community when back at the Institute for Hydrobiology and Fisheries Science in Hamburg. Also, one 50 mL subsample of the 100% WSW was fixed in 1 mL glutaraldehyde, to quantify bacteria abundance. The 2.4 L bottles were put in black mesh-bags, which reduced incoming radiation to approximately 50% (to minimize chlorophyll bleaching). The bottles were incubated for 24 hours in a tank on deck with flow-through water, to maintain in situ temperature. An additional experiment was carried out to test the effect of temperature on microzooplankton grazing in darkness. Therefore, 100% WSW was incubated in the deck tank and in two temperature control rooms of 5 and 15°C in darkness (two bottles each). The same was done with bottles where copepods were added (five copepods of Calanus finmarchicus in each bottle; males and females were randomly picked and divided onto the bottles). In addition, two 100% WSW bottles with five copepods each were incubated at in situ temperature at 100% light level (without mesh-bags). All experiments were incubated for 24 hours and afterwards two subsamples of each bottle were filtered on to GF/F filters (25 mm diameter); 500 - 1000 mL depending on in situ chlorophyll. One 250 mL subsample of one of the two replicates of each dilution level and each additional experiment (temperature and temperature/copepods) was fixed in 5 mL lugol for microzooplankton determination. One 50 mL subsample of one of the two 100% WSW bottles as well as of one of the additional experiments without copepods was fixed in 1 mL glutaraldehyde for bacteria determination later on. Copepods were fixed in 4% formaldehyde for length measurements and sex determination.

Oxidation rate of thiosulfate and elemental sulfur in the Black Sea

Oxidation rate of 35S-thiosulfate under simulated natural conditions and abundance of thiosulfate-oxidizing bacteria in a redox zone of the Black Sea are lower during winter and spring than in summer, especially in halistatic regions. Oxidation of thiosulfate under natural conditions is performed chiefly by lithotropic thionic bacteria, whose activity is limited by low temperatures. Adding thiosulfate and readily available organic matter to water samples from the redox zone and raising temperature of water stimulated activity of heterotrophic thiosulfate-oxidizing bacteria. Oxidation of elemental sulfur tagged with 35S apparently invovled two stages: abiotic oxidation of thiosulfate and subsequent bacterial oxidation of thiosulfate to sulfate.