Temperature effect of batch and continuous reactions in the gas phase

Summary The temperature effect on the performance of gas phase bioreactions (batch and continuous) was investigated using immobilized alcohol oxidase on Amberlite IRA-400. Reaction rates and product (acetaldehyde) compositions were compared as functions of temperature; there is a reaction temperature corresponding to a trade-off point between reaction rate and reaction extent.     

Gas phase biotransformation reaction catalyzed by baker's yeast

The gas phase continuous production of acetaldehyde from ethanol and hexanol from hexanal using dried baker's yeast was studied as an alternative approach to conventional processes. The effects of water activity, activity of substrates, and amount of yeast on the performance of the continuous bioreactor were investigated. The extent of yeast hydration and ethanol activity are the most important factors affecting yeast activity and stability.     

Bioelectronic sniffers for ethanol and acetaldehyde in breath air after drinking

Two kinds of bioelectronic gas sensors (bio-sniffer) incorporating alcohol oxidase (AOD) and aldehyde dehydrogenase (ALDH) were developed for the convenient analysis of ethanol and acetaldehyde in expired gas, respectively. The sniffer devices for gaseous ethanol and acetaldehyde were constructed by immobilizing enzyme on electrodes covered with filter paper and hydrophilic PTFE membrane, respectively. The AOD and ALDH sniffers were used in the gas phase to measure ethanol vapor from 1.0 to 500 ppm, and acetaldehyde from 0.11 to 10 ppm covering the concentration range encountered in breath after alcohol consumption. Both bio-sniffers displayed good gas selectivity which was attributed to the substrate specificity of the relevant enzymes (AOD and ALDH) as gas recognition material. From the results of physiological application, the bio-sniffers could monitor the concentration changes in breath ethanol and acetaldehyde after drinking. The ethanol and acetaldehyde concentrations in expired air from ALDH2 [-] (aldehyde dehydrogenase type 2 negative) subjects were higher than that of the ALDH2 [+] (positive) subjects. The results indicated that the lower activity of ALDH2 induced an adverse effect on ethanol metabolism, leading to ethanol and acetaldehyde remaining in the human body, even human expired air.     

On-line monitoring of ethanol,acetaldehyde and glycerol during industrial fermentations with Saccharomyces cerevisiae

Industrial fermentations carried out in a 500-1 bioreactor were monitored on-line by a prototype of a split-flow modified thermal biosensor. Acetaldehyde and glycerol in the extracellular broth were monitored over the first 48 h of fed-batch fermentations. The aim was to determine the usefulness of these secondary metabolites for on-line monitoring and control. When fermentation of the 13–16 g/l batch sugar was monitored, using immobilised aldehyde dehydrogenase, the acetaldehyde reached a peak value of 0.3 g/l. With immobilised alcohol oxidase a much larger peak of 3.5 g/l ethanol was seen immediately after the acetaldehyde peak. When glycerokinase was used a delayed peak of 1 g/l glycerol was monitored. Of the three metabolites monitored, the ethanol proved the most valuable indicator of suitable timing for the start of the feeding phase and later for controlling and preventing overfeed using the on-line biosensor system.     

Induction and stability of alcohol oxidase in the methylotrophic yeast Pichia pastoris

Summary Alcohol oxidase biosynthesis was induced when Pichia pastoris was grown in a medium containing methanol as the sole carbon and energy source. Specific activity was highest during the logarithmic phase of growth (1.22 g acetaldehyde produced/g cell dry wt. per hour), and declined steadily thereafter. The addition of 0.1% (w/v) yeast extract to the methanol growth medium promoted higher biomass production, increased alcohol oxidase specific activity, and contributed to increased enzyme stability under use conditions. When P. pastoris was used for wholecell bioconversions, 30.2 g of ethanol were oxidized to 28 g acetaldehyde in 12 h, at a carbon recovery of 97%. Acetaldehyde concentrations in excess of 1 M were achieved when the concentration of the TRIS buffer, used to chemically trap the acetaldehyde, was increased to 1 M.Issued as NRCC no. 30256Offprint requests to: W. D. Murray     

Production and application of methylotrophic yeast Pichia pastoris

Pichia pastoris is a methylotrophic yeast that makes use bf the enzyme alcohol oxidase to catalyze the first step of the dissimilatory pathway that enables it to grow on methanol. Because of its stability and low substrate specificity, alcohol oxidase is of considerable interest for a range of biotechnological processes. Various feeding regimes were evaluated in an effort to increase the biomass concentration and productivity that could be achieved from fermentations using this organism. Through continuous or semicontinuous feeding, biomass concentrations were increased 10-fold over those achieved in batch fermentations. In subsequent trials, nongrowing whole cells were applied successfully to convert ethanol to acetaldehyde. Quantitative conversions of 20-g/L solutions of ethanol have been achieved in 2 h, and acetaldehyde concentrations of up to 35 g/L have been achieved using extended reaction times of 5 h. The conversion reaction was limited by end product inhibition and by acetaldehyde holdup within the yeast cells.     

Gas-inducible product gene expression in bioreactors

Inducible transgene expression technologies are of unmatched potential for biopharmaceutical manufacturing of unstable, growth-impairing and cytotoxic proteins as well as conditional metabolic engineering to improve desired cell phenotypes. Currently available transgene dosing modalities which rely on physical parameters or small-molecule drugs for transgene fine-tuning compromise downstream processing and/or are difficult to implement technologically. The recently designed gas-inducible acetaldehyde-inducible regulation (AIR) technology takes advantage of gaseous acetaldehyde to modulate product gene expression levels. At regulation effective concentrations gaseous acetaldehyde is physiologically inert and approved as food additive by the Federal Drug Administration (FDA). During standard bioreactor operation, gaseous acetaldehyde could simply be administered using standard/existing gas supply tubing and eventually eliminated by stripping with inducer-free air. We have determined key parameters controlling acetaldehyde transfer in three types of bioreactors and designed a mass balance-based model for optimal product gene expression fine-tuning using gaseous acetaldehyde. Operating a standard stirred-tank bioreactor set-up at 10 L scale we have validated AIR technology using CHO-K1-derived serum-free suspension cultures transgenic for gas-inducible production of human interferon-beta (IFN-beta). Gaseous acetaldehyde-inducible IFN-beta production management was fully reversible while maintaining cell viability at over 95% during the entire process. Compatible with standard bioreactor design and downstream processing procedures AIR-based technology will foster novel opportunities for pilot and large-scale manufacturing of difficult-to-produce protein pharmaceuticals.     

Scaleup of ajmalicine production by plant cell cultures of Catharanthus roseus

The effect of scaleup on he production of ajmalicine by a Catharanthus roseus cell suspension culture in a selected induction medium were studied. In preliminary experiments it was observed that the culture turned brown and the production was inhibited upon transfer from a shake flask to a stirred bioreactor with forced aeration. Two factors were recognized as the potential origin of the differences between shake flask and bioreactor cultures: gas composition and mechanical shear forces. These factors were studied separately.By recirculating a large part of the exhaust gas, a comparable gas regime was obtained in a bioreactor as occurred in a shake flask cultures. This resulted in the absence of browning and a similar pattern of ajmalicine production as observed in shake flasks. The effect of shear forces could not be demonstrated. However, the experiments showed that the culture may be very sensitive to liquid phase concentrations of gaseous compounds. The effects of k(L)a, aeration rate, CO(2) production rate, and influent gas phase CO(2) concentration on the liquid phase CO(2) concentration are discussed. (c) 1993 John Wiley & Sons, Inc.     

Production of Acetaldehyde by Zymomonas mobilis

Mutants of Zymomonas mobilis were selected for decreased alcohol dehydrogenase activity by using consecutively higher concentrations of allyl alcohol. A mutant selected by using 100 mM allyl alcohol produced acetaldehyde at a level of 4.08 g/liter when the organism was grown in aerated batch cultures on a medium containing 4.0% (wt/wt) glucose. On the basis of the amount of glucose utilized, this level of acetaldehyde production represents nearly 40% of the maximum theoretical yield. Acetaldehyde produced during growth was continuously air stripped from the reactor. Acetaldehyde present in the exhaust stream was then trapped as the acetaldehyde-bisulfite addition product in an aqueous solution of sodium bisulfite and released by treatment with base. Acetaldehyde was found to inhibit growth of Z. mobilis at concentrations as low as 0.05% (wt/wt) acetaldehyde. An acetaldehyde-tolerant mutant of Z. mobilis was isolated after both mutagenesis with nitrosoguanidine and selection in the presence of vapor-phase acetaldehyde. The production of acetaldehyde has potential advantages over that of ethanol: lower energy requirements for product separation, efficient separation of product from dilute feed streams, continuous separation of product from the reactor, and a higher marketplace value.     

The role of aldehyde oxidase in ethanol-induced hepatic lipid peroxidation in the rat.

Hepatic lipid peroxidation has been implicated in the pathogenesis of alcohol-induced liver injury, but the mechanism(s) by which ethanol metabolism or resultant free radicals initiate lipid peroxidation is not fully defined. The role of the molybdenum-containing enzymes aldehyde oxidase and xanthine oxidase in the generation of such free radicals was investigated by measuring alkane production (lipoperoxidation products) in isolated rat hepatocytes during ethanol metabolism. Inhibition of aldehyde oxidase and xanthine oxidase (by feeding tungstate at 100 mg/day per kg) decreased alkane production (80-95%), whereas allopurinol (20 mg/kg by mouth), a marked inhibitor of xanthine oxidase, inhibited alkane production by only 35-50%. Addition of acetaldehyde (0-100 microM) (in the presence of 50 microM-4-methylpyrazole) increased alkane production in a dose-dependent manner (Km of aldehyde oxidase for acetaldehyde 1 mM); menadione, an inhibitor of aldehyde oxidase, virtually inhibited alkane production. Desferrioxamine (5-10 microM) completely abolished alkane production induced by both ethanol and acetaldehyde, indicating the importance of catalytic iron. Thus free radicals generated during the metabolism of acetaldehyde by aldehyde oxidase may be a fundamental mechanism in the initiation of alcohol-induced liver injury.     

A comparative study of aldehyde dehydrogenase and alcohol dehydrogenase activities in crucian carp and three other vertebrates: apparent adaptations to ethanol production

Summary In the final step of the pathway producing ethanol in anoxic crucian carp (Carassius carassius L.), acetaldehyde is reduced to ethanol by alcohol dehydrogenase. The presence of aldehyde dehydrogenase in the tissues responsible for ethanol production could cause an undesired oxidation of acetaldehyde to acetate coupled with a reduction of NAD⁺ to NADH. Moreover, acetaldehyde could competitively inhibit the oxidation of reactive biogenic aldehydes. In the present study, the distribution of aldehyde dehydrogenase (measured with a biogenic aldehyde) and alcohol dehydrogenase (measured with acetaldehyde) were studied in organs of crucian carp, common carp (Cyprinus carpio L.), rainbow trout (Salmo gairdneri Richardson), and Norwegian rat (Rattus norvegicus Berkenhout). The results showed that alcohol dehydrogenase and aldehyde dehydrogenase activities were almost completely spatially separated in the crucian carp. These enzymes occurred together in the other three vertebrates. In the crucian carp, alcohol dehydrogenase was only found in red and white skeletal muscle, while these tissues contained exceptionally low aldehyde dehydrogenase activities. Moreover, the low aldehyde dehydrogenase activity found in crucian carp red muscle was about 1000 times less sensitive to inhibition by acetaldehyde than that found in other tissues and other species. The results are interpreted as demonstrating adaptations to avoid a depletion of ethanol production, and possibly inhibition of biogenic aldehyde metabolism.Abbreviations ADH alcohol dehydrogenase - ALDH aldehyde dehydrogenase - DOPAL 3,4-dihydroxyphenylacetaldehyde - MAO monoamine oxidase - PCA perchloric acid     

Production of lignin peroxidase by Phanerochaete chrysosporium immobilized on porous poly(styrene-divinylbenzene) carrier and its application to the degrading of 2-chlorophenol

Porous poly(styrene-divinylbenzene) carriers, for the immobilization of white rot fungus Phanerochaete chrysosporium have been prepared by the concentrated emulsion polymerization method. The concentrated emulsion consists of a mixture of styrene and divinylbenzene containing a suitable surfactant and an initiator as the continuous phase, and water as the dispersed phase. The polymerization of the monomers of the continuous phase generated the polymer carrier with a porcus structure. The white rot fungus Phanerochaete chrysosporium has been immobilized on porous poly(styrene-divinylbenzene) carriers and used for the batch production and the repeated batch production of lignin peroxidase in shake cultures based on a carbon-limited medium containing veratryl alcohol. The best results were achieved when a spore inoculum was used for immobilization instead of 1-day-old mycelial pellets, for both the batch production and the repeated batch production. The porous poly(styrene-divinylbenzene) immobilized Phanerochaete chrysosporium and freely suspended mycelial pellets were used as biocatalysts for the degradation of 2-chilorophenol in a 2-L bioreactor. The porous poly(styrene-divinylbenzene) particle (diameter congruent with 0.2 cm) immobilized spores exhibited a much higher activity in the degradation of 2-chlorophenol than the freely suspended mycelial pellets. (c) 1994 John Wiley & Sons, Inc.     

Continuous production of isovaleraldehyde through extractive bioconversion in a hollow-fiber membrane bioreactor

A membrane bioreactor was developed to perform an extractive bioconversion aimed at the production of isovaleraldehyde by isoamyl alcohol oxidation with whole cells of Gluconobacter oxydans. A liquid/liquid extractive system using isooctane as extractant and assisted by a hollow-fiber hydrophobic membrane was chosen to recover the product. The aqueous bioconversion phase and the organic phase were maintained apart with the aid of the membrane. The extraction of alcohol and aldehyde was evaluated by performing equilibrium and mass transfer kinetic studies. The bioprocess was then performed in a continuous mode with addition of the substrate to the aqueous phase. Fresh solvent was added to the organic phase and exhausted solvent was removed at the same flow rate. The extractive system enabled a fast and selective in situ removal of the aldehyde from the water to the organic phase. High conversions (72–90%) and overall productivity (2.0–3.0 g l⁻¹ h⁻¹) were obtained in continuous experiments performed with different rates of alcohol addition (1.5–3.5 g l⁻¹ h⁻¹). Cell deactivation was observed after 10–12 h of operation.     

Ethane release during metabolism of aldehydes and monoamines in perfused rat liver

Infusion of aldehyde such as acetaldehyde, propionaldehyde or benzaldehyde to perfused rat liver leads to an increase in hepatic ethane production. Half-maximal effect was obtained with about 20 microM acetaldehyde, a concentration range found in plasma during ethanol metabolism. Compounds which metabolically generate aldehydes such as monoamines (benzylamine, phenylethylamine) as substrates for monoamine oxidase or ethanol as substrate for alcohol dehydrogenase [A. Müller and H. Sies (1982) Biochem. J. 206, 153-156] are also able to elicit ethane release. Results obtained with inhibitors of hepatic aldehyde metabolism (pargyline or cyanamide) or of monamine oxidase (pargyline or tranylcypromine) suggest that metabolism of the aldehydes is required for ethane production. Radical scavenging by the addition of the flavonoid, cyanidanol, or by pretreatment with vitamin E (alpha-tocopherol) abolished ethane release, in agreement with lipid peroxidation as a source of alkane production during aldehyde metabolism.     

Ethanol-induced iron mobilization: role of acetaldehyde-aldehyde oxidase generated superoxide

Superoxide radicals, a species known to mobilize ferritin iron, and their interaction with catalytic iron have been implicated in the pathogenesis of alcohol-induced liver injury. The mechanism(s) by which ethanol metabolism generates free radicals and mobilizes catalytic iron, however, is not fully defined. In this investigation the role of hepatic aldehyde oxidase in the mobilization of catalytic iron from ferritin was studied in vitro. Iron mobilization due to the metabolism of ethanol to acetaldehyde by alcohol dehydrogenase was increased 100% by the addition of aldehyde oxidase. Iron release was favored by low pH and low oxygen concentration. Mobilization of iron due to acetaldehyde metabolism by aldehyde oxidase was completely inhibited by superoxide dismutase but not by catalase suggesting that superoxide radicals mediate mobilization. Acetaldehyde-aldehyde oxidase mediated reduction of ferritin iron was facilitated by incubation with menadione, an electron acceptor for aldehyde oxidase. Mobilization of ferritin iron due to the metabolism of acetaldehyde by aldehyde oxidase may be a fundamental mechanism of alcohol-induced liver injury.     

Novel three-phase bioreactor concept for fatty acid alkyl ester production using <Emphasis Type="Italic">R. oryzae</Emphasis> as whole cell catalyst

A novel three-phase solid–gas–liquid bioreactor (SGLB) concept using gaseous alcohol and liquid rapeseed oil with immersed microorganisms overlying a nutrient agar phase (solid) is proposed for biodiesel (fatty acid alkyl esters, FAAE) production based on the high hydrophobicity and negative surface charge showed by the fungi Rhizopus oryzae. This novel bioreactor was thought to increase oil bioavailability and decrease alcohol toxicity for effective microbial growth, reaching high yields of FAAE production without any pretreatment. High growth rates were reached for R. oryzae using a SGLB simultaneously reaching a high FAAE production yield, up to 50% using methanol and up to 70% using ethanol at 144 h of incubation at 20°C. To compare the effect of gaseous alcohol, the same experiments were carried out in a three-phase solid–liquid–liquid bioreactor (SLLB), where the alcohol was added in liquid phase, showing significant R. oryzae growth but no FAAE formation. This suggests that the inhibitory effect of alcohol is more significant in lipase activity than in R. oryzae growth, and the use of alcohol in gaseous phase may decrease both of them. The experimental procedure using SGLB showed that when R. oryzae is maintained alive, it can catalyze at the same time the hydrolysis, esterification and transesterification of triglycerides from rapeseed oil, but its activity strongly depends on the used growth media. Therefore, the application of gaseous alcohol coupled with R. oryzae as immobilized whole cell catalysts may be a potential alternative to the use of commercial lipases for biodiesel production.     

Development of a novel bioreactor system for treatment of gaseous benzene

A novel, continuous bioreactor system combining a bubble column (absorption section) and a two-phase bioreactor (degradation section) has been designed to treat a gas stream containing benzene. The bubble column contained hexadecane as an absorbent for benzene, and was systemically chosen considering physical, biological, environmental, operational, and economic factors. This solvent has infinite solubility for benzene and very low volatility. After absorbing benzene in the bubble column, the hexadecane served as the organic phase of the two-phase partitioning bioreactor, transferring benzene into the aqueous phase where it was degraded by Alcaligenes xylosoxidans Y234. The hexadecane was then continuously recirculated back to the absorber section for the removal of additional benzene. All mass transfer and biodegradation characteristics in this system were investigated prior to operation of the integrated unit, and these included: the mass transfer rate of benzene in the absorption column; the mass transfer rate of benzene from the organic phase into the aqueous phase in the two-phase bioreactor; the stripping rate of benzene out of the two-phase bioreactor, etc. All of these parameters were incorporated into model equations, which were used to investigate the effects of operating conditions on the performance of the system. Finally, two experiments were conducted to show the feasibility of this system. Based on an aqueous bioreactor volume of 1 L, when the inlet gas flow and gaseous benzene concentration were 120 L/h and 4.2 mg/L, respectively, the benzene removal efficiency was 75% at steady state. This process is believed to be very practical for the treatment of high concentrations of gaseous pollutants, and represents an alternative to the use of biofilters.     

Expression of the Aspergillus niger glucose oxidase gene in Saccharomyces cerevisiae and its potential applications in wine production

There is a growing consumer demand for wines containing lower levels of alcohol and chemical preservatives. The objectives of this study were to express the Aspergillus niger gene encoding a glucose oxidase (GOX; beta- d-glucose:oxygen oxidoreductase, EC 1.1.3.4) in Saccharomyces cerevisiae and to evaluate the transformants for lower alcohol production and inhibition of wine spoilage organisms, such as acetic acid bacteria and lactic acid bacteria, during fermentation. The A. niger structural glucose oxidase (gox) gene was cloned into an integration vector (YIp5) containing the yeast mating pheromone alpha-factor secretion signal (MFalpha1(S)) and the phosphoglycerate-kinase-1 gene promoter (PGK1(P)) and terminator (PGK1(T)). The PGK1(P)- MFalpha1(S)- gox- PGK1(T) cassette (designated GOX1) was introduced into a laboratory strain (Sigma1278) of S. cerevisiae. Yeast transformants were analysed for the production of biologically active glucose oxidase on selective agar plates and in liquid assays. The results indicated that the recombinant glucose oxidase was active and was produced beginning early in the exponential growth phase, leading to a stable level in the stationary phase. The yeast transformants also displayed antimicrobial activity in a plate assay against lactic acid bacteria and acetic acid bacteria. This might be explained by the fact that a final product of the GOX enzymatic reaction is hydrogen peroxide, a known antimicrobial agent. Microvinification with the laboratory yeast transformants resulted in wines containing 1.8-2.0% less alcohol. This was probably due to the production of d-glucono-delta-lactone and gluconic acid from glucose by GOX. These results pave the way for the development of wine yeast starter culture strains for the production of wine with reduced levels of chemical preservatives and alcohol.     

Biohydrogen production in a continuous stirred tank bioreactor from synthesis gas by anaerobic photosynthetic bacterium: Rhodopirillum rubrum

Hydrogen may be considered a potential fuel for the future since it is carbon-free and oxidized to water as a combustion product. Bioconversion of synthesis gas (syngas) to hydrogen was demonstrated in continuous stirred tank bioreactor (CSTBR) utilizing acetate as a carbon source. An anaerobic photosynthetic bacterium, Rhodospirillum rubrum catalyzed water-gas shift reaction which was applied for the bioconversion of syngas to hydrogen. The continuous fermentation of syngas in the bioreactor was continuously operated at various gas flow rates and agitation speeds, for the period of two months. The gas flow rates were varied from 5 to 14 ml/min. The agitation speeds were increasingly altered in the range of 150-500 rpm. The pH and temperature of the bioreactor was set at 6.5 and 30 degrees C. The liquid flow rate was kept constant at 0.65 ml/min for the duration of 60 days. The inlet acetate concentration was fed at 4 g/l into the bioreactor. The hydrogen production rate and yield were 16+/-1.1 mmol g(-1)cell h(-1) and 87+/-2.4% at fixed agitation speed of 500 rpm and syngas flow rate of 14 ml/min, respectively. The mass transfer coefficient (KLa) at this condition was approximately 72.8h(-1). This new approach, using a biocatalyst was considered as an alternative method of conventional Fischer-Tropsch synthetic reactions, which were able to convert syngas into hydrogen.     

Bioreactor studies on the effect of dissolved oxygen concentrations on growth and differentiation of carrot (Daucus carota L.) cell cultures

A bioreactor control system was used to investigate the effects of two dissolved oxygen concentrations (10% and 100%) on the growth and differentiation of Daucus carota L. cell cultures. The strategy used allowed the dissolved oxygen concentration to be controlled without the need for changing either the agitator speed or the total gas flow rate. During the proliferation phase, reducing oxygen resulted in a lower growth rate and in a delay in sugar uptake kinetics. Nonetheless, varying levels of oxygen were observed to have no effect on the final dry biomass. The higher alcohol dehydrogenase activity obtained under reduced oxygen conditions suggests that proliferating cultures adapted to the hypoxic environment by inducing alcoholic fermentation. Cell differentiation was highly sensitive to reduced oxygen since under this condition, the somatic embryo production was inhibited by about 75%. Sugar uptake and embryo formation were also delayed.Abbreviations ADH alcohol dehydrogenase - 2,4-D 2,4-dichlorophenoxyacetic acid - DO2 dissolved oxygen - SE somatic embryos - Tris tris(hydroxymethyl)-aminoethane