Autotrophic denitrification via a novel membrane-attached biofilm reactor

AIMS: A laboratory-scale autotrophic membrane-attached biofilm reactor was developed to remove nitrate from drinking water. METHODS AND RESULTS: Hydrogen and carbon dioxide flowed together into the lumem side of a gas-permeable silicone tube. The gases diffused through the membrane wall to feed Alcaligenes eutrophus that formed a biofilm on the surface of the silicone tube for autotrophic denitrification. Hydrogen provided the energy source, and carbon dioxide, besides serving as the carbon source, was employed to neutralize the alkalinity from denitrification. The optimal carbon dioxide concentration in the silicone tube was between 20% and 50%. CONCLUSION: This study has demonstrated that a gas-permeable silicone tube is a convenient and efficient method to feed A. eutrophus for autotrophic denitrification. Supplying a suitable amount of carbon dioxide together with hydrogen into the silicone tube solved the problem that alkalinity formation caused during denitrification. The pH of the bioreactor was maintained at about 7 to avoid nitrite accumulation, and then the nitrogen removal rate was increased. A high specific nitrogen removal rate (1.6-5.4 g Nm(2)d(-1-1) of surface area of silicone tube) was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY: In addition to combining the advantages of the hydrogenotrophic denitrification process and a membrane feeding substrate bioreactor (MFSB), this bioreactor achieved a high nitrogen removal rate and is simple to operate. It therefore is highly promising in drinking-water treatment.     

Preparation of rich handles soft cellulosic fabric using amino silicone based softener. Part-I: Surface smoothness and softness properties

A series of amino silicone based softeners with different emulsifiers were prepared and adsorbed onto the surfaces of cotton and blends of cotton/polyester fabrics. Factors affecting the performance properties of the finished substrate such as post-treatment with amino functional silicone based softener varying different emulsifiers in their formulations and its concentration on different processed fabrics were studied. Fixation of the amino-functional silicone softener onto/or within the cellulose structure is accompanied by the formation of semi-inter-penetrated network structure thereby enhancing both the extent of crosslinking and networking as well as providing very high softness. The results of the experiments indicate that the amino silicone can form a hydrophobic film on both cotton and blends of cotton/polyester fabrics and its coating reduces the surface roughness significantly. Furthermore, the roughness becomes lesser with an increase in the applied strength of amino silicone based softener.     

A new method for wide frequency range dynamic olfactory stimulation and characterization

Sensory receptors often receive strongly dynamic, or time varying, inputs in their natural environments. Characterizing their dynamic properties requires control and measurement of the stimulus over a frequency range that equals or exceeds the receptor response. Techniques for dynamic stimulation of olfactory receptors have lagged behind other major sensory modalities because of difficulties in controlling and measuring the concentration of odorants at the receptor. We present a new method for delivering olfactory stimulation that gives linear, low-noise, wide frequency range control of odorant concentration. A servo-controlled moving bead of silicone elastomer occludes the tip of a Pasteur pipette that releases odorant plus tracer gas into a flow tube. Tracer gas serves as a surrogate indicator of odorant concentration and is measured by a photoionization detector. The system has well-defined time-dependent behavior (frequency response and impulse response functions) and gives predictable control of odorant over a significant volume surrounding the animal. The frequency range of the system is about 0-100 Hz. System characterization was based on random (white noise) stimulation, which allows more rapid and accurate estimation of dynamic behavior than deterministic signals such as sinusoids or step functions. Frequency response functions of Drosophila electroantennograms stimulated by fruit odors were used to demonstrate a typical application of the system.     

Correction of infra-red gas analyser readings for changes in reference tube CO2 concentration

Abstract The theoretical basis for correcting infra-red gas analyser readings for changes in reference tube CO₂ concentration is developed, but in practice the corrections made are empirical. Analyser gain (G_A) is related to reference tube concentration (A) in hyperbolic manner. The constants in the equation are determined by plotting 1/G_A against A. Calibration and operating procedure for a particular type of analyser are described and a numerical example given.     

A modified technique for the quantitative analysis of amino acids by gas chromatography using heptafluorobutyric n-propyl derivatives.

The quantitative analysis of amino adids by gas chromatography as their heptafluorobutyric n-propyl derivatives has been examined and a modified technique developed which has been found to give improved results. A single propylation step has been adopted using 6 m HCl n-propanol at 150°C for 3.5 min, followed by a 12-min acylation step at the same temperature. Variability of response for methionine and histidine has been overcome in most cases by the introduction of an antioxidant at the acylation stage. An improved column performance on the previously used nonpolar OV silicone phase has been obtained by using a mildly polar OV phase.     

Long-size isoelectricfocusing (IEF) in flexible silicone tubes: an application to semi-preparative fractionation of proteins

Soluble proteins extracted from porcine brains were subjected to a series of optional fractionation steps on various chromatographic media including a novel device for semi-preparative isoelectrofocusing (IEF) carried in a flexible silicone tube. The dimensions of the IEF granulated gel beds can be varied from 40 to 75 cm (length) and 0.4-1.6 cm (diameter) which are dependent on the protein's concentration. An average optimal focalisation time of proteins is dependent of the tube length, its diameter and complexity of proteins' mixtures but it is usually reached during 15,000-30,000 Vh. A series of sequential protein's fractionation techniques including semi-preparative IEF carried in the flexible silicone tube with the following dimensions: 75 cm in length and 1.6 cm in diameter permitted for observation and partial characterisation of several proteins whose expression levels are specifically high in the brain.     

A combination of methods for the on-line determination of alcohols in microbial cultures

Summary A new method for the continuous on-line determination of methanol (range 0.2 to 10 gl^–1) and ethanol (0.2 to 120 gl^–1) is described. The rate limiting step is diffusion of the alcohol through the walls of a silicone tube immersed in the culture broth. A sintered SnO₂ sensor was used instead of a Flame Ionization Detector for alcohol determination. Measurement is not affected by bioreactor aeration or agitation rates, dissolved oxygen, carbon dioxide, ammonia or the concentration of cells in the medium. The assay system was tested in extended batch cultivation of Methylomonas sp. with methanol as the sole carbon source (final biomass concentration, 35 gl^–1). Sensor readings agreed well with simultaneous off-line gas chromatographic methanol determination.     

On-line monitoring and control of methanol concentration in shake-flask cultures of Pichia pastoris

The methylotrophic yeast Pichia pastoris can be used to express recombinant genes at high levels under the control of the methanol-inducible alcohol oxidase 1 (AOX1) promoter. Accurate regulation of the methanol concentration in P. pastoris cultures is necessary to maintain induction, while preventing accumulation of methanol to cytotoxic levels. We developed an inexpensive methanol sensor that uses a gas-permeable silicone rubber tube immersed in the culture medium and an organic solvent vapor detector. The sensor was used to monitor methanol concentration continuously throughout a fed-batch shake-flask culture of a P. pastoris clone producing the N-lobe of human transferrin. The sensor calibration was stable for the duration of the culture and the output signal accurately reflected the methanol concentration determined off-line by HPLC. A closed-loop control system utilizing this sensor was developed and used to maintain a 0.3% (v/v) methanol concentration in the culture. Use of this system resulted in a fivefold increase in volumetric protein productivity over levels obtained using the conventional fed-batch protocol. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 279-286, 1997.     

Simultaneous nitrification and denitrification using an autotrophic membrane-immobilized biofilm reactor

AIMS: To develop a laboratory-scale autotrophic membrane-immobilized biofilm reactor to remove nitrogen from drinking water. METHODS AND RESULTS: A polyvinyl alcohol (PVA) immobilized biofilm, attached to the surface of a silicone tube, was used as the basis of a bioreactor for simultaneous nitrification and denitrification of water. The bioreactor was aerated with air to supply oxygen for nitrification. Pure hydrogen was supplied to the silicone tube and diffused through the membrane wall to feed the biofilm for autotrophic denitrification. The bioreactor was effective for the simultaneous nitrification and denitrification of water after a short period of acclimation, while the biofilm exhibited good resistance to the inhibition of denitrification by dissolved oxygen; the denitrification rate decreased by only 8% as the dissolved oxygen increased from 2 mg l(-1) to saturation. CONCLUSIONS: By using PVA crosslinked with sodium nitrate to entrap nitrifying and denitrifying sludge on the surface of a silicone tube, a novel bioreactor for simultaneous nitrification and denitrification was developed. In addition to performing as an immobilizing agent to strengthen the biofilm, PVA protected the denitrifying microorganisms to reduce the inhibition by dissolved oxygen under aerobic condition. Therefore, nitrification and denitrification occurred simultaneously within the biofilm. Furthermore, the immobilization technique shortened the acclimation period of the bioreactor. SIGNIFICANCE AND IMPACT OF THE STUDY: The described space saving and simple to operate bioreactor for nitrogen removal performed autotrophic denitrification to solve the problem of residual carbon in heterotrophic denitrification, and thus is suitable for removing nitrogen from drinking water.     

Use of silicone fluids in studies of cellular amino acids

In numerous cellular studies, cells labeled with radioisotopes have been separated from the labeling medium by an aqueous solution in order to determine the quantity of internalized labels; however, the aqueous wash tends to remove significant labeling from the cells. Therefore, in order to preserve all of the internalized labels, non-aqueous medium such as silicone fluids may be used. The termination of the labeling is achieved in the silicone method when, upon centrifugation, the cells separate from the medium and enter the silicone fluid to sediment to the tube bottom. This sedimentation of cells placed above a layer of silicone fluid exhibits a critical dependence on the centrifugal force, and gives rise to an uncertainty of only 2 s in determining the time of separation of cells from the medium using General Electric F-50 silicone fluid and a modified Beckman J2-21 centrifuge. It is therefore possible to determine the kinetics of incorporation of labeled amino acids into intracellular pools and proteins. In particular, since this silicone wash method determines the size of the total pool and the aqueous wash method determines the size of the acid-extractable pool, the simultaneous measurements of the size of both pools leads to the determination of the kinetics of labeling of the free amino acid pool. Among many possible applications and extensions of these methods, the studies of formation of intracellular pools and relations among different pools of transported molecules, such as water and amino acids, appear promising.     

Use of a silicone tubing sensor to control methanol concentration during fed batch fermentation of Pichia pastoris

A silicone tubing sensor controlled a constant methanol concentration in a fermenter up to 72 hours without the need for on-line gas chromatography or complex feeding schemes based on dissolved oxygen spikes. Methanol concentration was controlled up to 1.0% (v/v) with control around a given set point of ± 0.24%. The length of tubing, airflow through the tubing, pump speed and medium formulation had no effect on the control of methanol concentration.     

Oxygen transfer in three-phase airlift and stirred tank reactors using silicone oil as transfer vector

The use of organic liquids as vectors to enhance mass transfer has been applied since the 1970s. However, mass transfer in three-phase reactors is only partially understood. This paper aimed to characterize oxygen transfer in three-phase reactors containing air as gas, silicone oil as vector and water as aqueous phase. A mass transfer model that considers separately air/vector, vector/water and air/water oxygen transfers was developed. The model was used to describe oxygen transfer in airlift and stirred tank reactors containing from 0 to 50% of silicone oil. Under the experimental conditions, silicone oil had a positive effect on the overall oxygen transfer. In both reactor designs, the maximum overall oxygen transfer was observed with 10% silicone oil which was increased by 65 and 84% in the airlift and stirred reactor, respectively, compared to reactors operated without silicone oil. The overall transfer increase was mainly due to an enhanced air/water transfer. With 10% silicone oil, the air/water contribution to the overall oxygen transfer was 94.7 and 93.0% for the airlift and stirred reactor, respectively.     

Monitoring and control of methanol concentration during polysaccharide fermentation using an on-line methanol sensor

Combining principles of membrane separation and semiconductor gas sensor technology, we constructed a methanol sensor to follow methanol concentrations on-line. A length of silicone tubing allowed for mass transfer of methanol from the fermentation medium to a carrier gas which then flowed over a semiconductor gas sensor for detection. The sterilizable sensor demonstrated excellent ability in following methanol concentrations during the batch production of a polysaccharide by the organism Methylomonas mucosa, even as the fermentation broth became increasingly viscous. During fed-batch control by feeding methanol to the fermentation to maintain setpoint methanol levels, a drift in the sensor signal was noted and quantified. A drift factor was determined which, after it was incorporated into the calibration calculations, improved methanol concentration control greatly. Methanol concentration was held constant over a range of set point concentrations during fedbatch fermentations.     

Oscillatory flow and gas transport through a symmetrical bifurcation

Axial gas transport due to the interaction between radial mixing and radially nonuniform axial velocities is responsible for gas transport in thick airways during High-frequency oscillatory ventilation (HFO). Because the airways can be characterized by a bifurcating tube network, the secondary flow in the curved portion of a bifurcating tube contributes to cross-stream mixing. In this study the oscillatory flow and concentration fields through a single symmetrical airway bifurcating tube model were numerically analyzed by solving three-dimensional Navier-Stokes and mass concentration equations with the SIMPLER algorithm. The simulation conditions were for a Womersley number, alpha = 9.1 and Reynolds numbers in the parent tube between 200 and 1000, corresponding to Dn2/alpha 4 in the curved portion between 2 and 80, where Dn is Dean number. For comparison with the results from the bifurcating tube, we calculated the velocity and concentration fields for fully developed oscillatory flow through a curved tube with a curvature rate of 1/10, which is identical to the curved portion of the bifurcating tube. For Dn2/alpha 4 < or = 10 in the curved portion of the bifurcating tube, the flow divider and area changes dominate the axial gas transport, because the effective diffusivity is greater than in either a straight or curved tube, in spite of low secondary velocities. However, for Dn2/alpha 4 > or = 20, the gas transport characteristics in a bifurcation are similar to a curved tube because of the significant effect of secondary flow.     

Modulation of peripheral nerve regeneration: a tissue-engineering approach. The role of amnion tube nerve conduit across a 1-centimeter nerve gap

A new type of a biodegradable nerve graft conduit material, the amnion tube, has been developed in our laboratory. To test the tube in the peripheral nerve regeneration process, it was initially applied across a 1-cm sciatic nerve gap in rats and was compared with other nerve conduit materials. We used male Sprague-Dawley rats as our animal model. The experiment included 66 rats that were randomly assigned into five groups: autograft (n = 17), amnion tube (n = 19), silicone tube (n = 20), no repair (n = 7), and sham group (n = 3). The process of peripheral nerve regeneration was evaluated at 2, 4, 10, and 17 weeks following injury and repair by using morphologic and functional assessments of the outcome of nerve regeneration in each animal. Nerve regeneration across the amnion tube nerve conduit was comparable with that seen in autograft and superior to that of the silicone group. A uniform nerve tissue was seen filling and crossing the amnion conduit, and the regenerated nerve from the proximal stump reached the distal end and was undifferentiated from the normal nerve tissues. At 4 months, the amnion tube biodegraded and no longer could be identified and differentiated from the nerve tissues. The amnion tube animal group showed a number of axons very close to that in the nerve autograft group (37,157 versus 33,054). Functional recovery at a 2- to 4-week interval was significantly statistically higher only in the amnion tube animal group (p = 0.01). However, the improvement disappeared between 10 and 17 weeks. In conclusion, the amnion tube is a potential ideal nerve conduit material secondary to its unique characteristics: it contains important neurotropic factors, is biodegradable, provokes a very weak immune response, is semiflexible, is readily available, and is easily manufactured into different sizes and diameters.     

Glucose oxidation in a dual hollow fiber bioreactor with a silicone tube oxygenator

A dual hollow fiber bioreactor, consisting of an outer silicone membrane for oxygen supply and an inner polyamide membrane for substrate permeation, was used as an immobilized enzyme reactor to carry out enzymatic glucose oxidation. Attaching a silicone tube oxygenator to provide an additional oxygen supply improved the conversion in glucose oxidation when the oxygen supply was rate-limiting. The reactor was operated in both diffusion and ultrafiltration modes. In the latter case, the conversion was much higher, but the stability of the immobilized enzyme was better maintained in the diffusion mode. As the inlet glucose concentration increased from 10mM to 500mM, the conversion decreased from 70 to 20%.     

Continuous control of water activity during biocatalysis in organic media

Summary This paper describes a newly developed technique to adjust and control the water activity in enzymatic reactions in organic media. A saturated salt solution of known water activity is circulated inside a silicone tube, submerged into the reaction medium. The circulating solution can both absorb and release water. Water activity control during lipase catalyzed esterification was demonstrated with diisopropyl ether as solvent.     

Improved synchronized accumulating radioisotope detector for gas chromatography

A synchronized accumulating radioisotope detector for radio gas chromatography was developed. It comprised seven gas-flow proportional counters each with an inner volume of 10 ml. Every counter tube was connected by a mutual anti-coincidence circuit to reduce the background. The transit time of gas particles in one counter tube could be set to an optimal value between 1 and 4 s by regulating the flow-rate of the counting gas, according to analytical requirements. The improved detector maintained high chromatographic resolution, which suggested the applicability of the apparatus to capillary gas chromatography.     

A micromethod for the analysis of free amino acids by gas chromatography and its application to biological systems.

A gas chromatographic procedure was developed for determination of minute amounts of free amino acids in natural waters and laboratory models simulating biological systems. Sample pretreatment included removal of interfering organic substances by chloroform extraction and isolation of amino acids by cation exchange. Amino acids were converted to their N-heptafluorobutyryl isobutyl ester derivatives in glass capillary tubes, permitting considerable concentration of the sample prior to gc injection. The derivatives of 19 amino acids were successfully separated on either a glass column packed with a mixture of OV-101 and OV-17 on Chromosorb W, a glass capillary column coated with OV-101, or a support-coated capillary column supported with SE-30. One to five nanograms of individual amino acids were detected using flame ionization detector. The detection limit was reduced more than 100-fold using the electron capture detector and more than 1000-fold by mass fragmentography. The procedure allowed determination of less than 1 ppb of individual amino acids in lake and river water samples and was used to estimate the exeretion of free amino acids from microbial populations.     

Transport of glycine,serine, and proline into spinach leaf mitochondria

The transport of radioactive glycine, serine, and proline into the matrix of spinach leaf mitochondria was studied using the silicone oil centrifugation technique. The uptake of all three amino acids showed a biphasic characteristic. At concentrations higher than 0.5 mm, an apparent diffusion process dominated. The uptake was not saturable at increasing amino acid concentrations, and there was no accumulation of amino acid in the matrix (i.e., concentration was similar to that in the medium). At concentrations lower than 0.5 mm, in addition to the diffusion process, an active uptake system that accumulated amino acid in the matrix became apparent. This system was partially inhibited by rotenone, antimycin A, and carbonylcyanide-m-chlorophenyl hydrazone. Also, uptake of glycine and serine was mutually inhibitory. These two amino acids exhibited comparatively less inhibitory effect on proline uptake, and proline also did not inhibit glycine or serine uptake. The results suggest that the active uptake system consists of at least two components with different degrees of amino acid specificity. The diffusion process dominates at amino acid concentrations of 0.5 mm or higher, whereas the active uptake system becomes more prominent as the amino acid concentration decreases.