Ethanolic fermentation with saccharomyces cerevisiae cells immobilized in pectate gel

High-molecular weight pectic acid with a STAUDINGER index of 210 ml/g and a degree of esterification of 3%was used as matrix material for the immobilization of Saccharomyces cerevisiae cells. In discontinuous and continuous fermentation tests the gel beads obtained exhibited the same biomass loading capacity (152–155 g dry wt. cells/kg gel) and about the same maximum specific productivity (103.0 g ethanol/kg gel · h) as alginate immobilizates. But there were distinct differences in the swelling behaviour of the two gels. Under the same experimental conditions the increase of bead volume amounted to 27% only for pectate gel in comparison to 129% for alginate gel. In continuous fermentation experiments performed in a horizontal-column packed-bed reactor with liquid recycling a mean steady-state ethanol concetration of 69.1 g/l and a mean productivity of 24.7 g ethanol/lh could be kept constant over a period of more than 10 days.     

Ammonium lactate from deproteinized alfalfa juice byStreptococcus faecium

Summary Deproteinized alfalfa juice is a by-product of the mechanical fractionation of alfalfa to obtain protein. In this work the juice was used as the substrate for the production of ammonium lactate (l-lactic acid) by a strain ofStreptococcus faecium. Batch fermentation with a constant pH of 5.8 gave 27.2 g/l of lactic acid (90% conversion and 1.1 g/l/h productivity) and 6×10¹² cells/l after 24 h. Semicontinuous fermentation allowed the conversion of 3-times the volume of deproteinized juice after 44 h, finally giving 29.7 g/l of ammonium lactate (99% conversion and 2.5 g/l/h productivity) and 4–6×10¹² cells/l.     

A study of polynucleotide phosphorylase production by <Emphasis Type="Italic">Escherichia coli</Emphasis> in a hollow fibre reactor

A 30-l hollow fibre reactor with continuous fermentation for cell recycling of Escherichia coli AS 1.183 was used to remove the inhibitory effects on cell growth and extend the fast growth phase to increase the yield of polynucleotide phosphorylase (PNPase) in E. coli cells. When the dilution rate was 1.5 h⁻¹, the cell concentration of E. coli reached 235 g/l (wet wt, 70% moisture content), with PNPase activity above 90 u/g (wet wt). With the dilution rate is 1.0 h⁻¹, the fermentor volumetric productivity of PNPase in a hollow fiber reactor can reach 974 (u/h * l) compared to 20 (u/h * l) in a conventional batch culture.     

Enzymatic synthesis of lactic acid derivatives with emulsifying properties

Novozym 435 (50 g l^–1) catalyzed the synthesis of n-octyl--d-glucopyranoside lactate by transesterification between n-octyl--d-glucopyranoside (30 g l^–1) and ethyl lactate (100 g l^–1) in acetone. In 12 h, a molar yield of 87% n-octyl -d-glucopiranoside lactate was obtained at a overall conversion of 90%.     

Cosolvent effect on Δ1-steroid-reductase activity of free and PAAH entrapped Mycobacterium Sp. NRRL B-3805 cells

Summary The effect of water miscible solvents on ¹-steroid reduction by free and polyacrylamide-hydrazide (PAAH) entrapped Mycobacterium sp. NRRL B-3805 cells was investigated. On the basis of retention of reductase activity an order of preference of diols (e.g. ethyleneglycol) > DMSO > DMF and monoalcohols (e.g. ethanol) as cosolvents was recorded. Significant increase in substrate (1,4-androstadiene-3,17-dione) solubility was attained in presence of the cosolvent of choice (ethyleneglycol), accompanied by some inhibition of the ¹-reductase activity. Optimization of ethyleneglycol concentration (10–20% (v/v)) led to specific activity in a homogeneous medium, higher than recorded in the absence of cosolvent. Immobilization in PAAH gel resulted in high retention of immobilized enzymic activity, accompanied by minor diffusional limitations. Moreover, the gel exhibited protective effect of the entrapped cells from cosolvent inhibition. Modification of gel composition (e. g. hydrophobicity) had no significant effect on reaction kinetics.     

High concentration cultivation of Bifidobacterium longum in fermenter with cross-flow filtration

Summary High concentration cultivation of Bifidobacterium longum in a fermenter with cross-flow filtration using a ceramic filter is described. Continuous cross-flow filtration allowed complete recycling of the cells to the fermenter and also continuous separation of inhibitory metabolites. The final cell concentration attained in the cultivation was 54.4 g dry wt./l; this was seven times as high as that without cross-flow filtration. The time course of the cultivation with cross-flow filtration was predicted, based on the assumption that the specific growth rate can be expressed only as a function of concentrations of metabolites (acetate and lactate) in a culture broth.Nomenclature D dilution rate (h^-1) - m maintenance coefficient (h^-1) - OD ₅₇₀ optimal density at 570 nm - P _A acetate concentration (g/l) - P _A0 initial acetate concentration (g/l) - P _L lactate concentration (g/l) - P _L0 initial lactate concentration (g/l) - S lactose (substrate) concentration (g/l) - S ₀ initial lactose (substrate) concentration (g/l) - t cultivation time (h) - Y _x/s growth yield (g/g) - X dry cell concentration (g/l) - X ₀ initial dry cell concentration (g/l) - constant - constant     

Oxidative biotransformation of thioanisole by <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> IEGM 66 cells

Comparative study of sulfoxidation activity of free and immobilized Rhodococcus rhodochrous IEGM 66 cells was performed. Free Rhodococcus cells (in the presence of 0.1 vol % n-hexadecane) displayed maximal oxidative activity towards thioanisole (0.5 g/l), a prochiral organic sulfide, added after 48-h cultivation of bacterial cells. Higher sulfide concentrations inhibited sulfoxidation activity of Rhodococcus. Use of immobilized cells allowed the 2-day preparatory stage to be omitted and a complete thioanisole bioconversion to be achieved in 24 h in the case that biocatalyst and 0.5 g/l thioanisole were added simultaneously. The biocatalyst immobilized on gel provides for complete thioanisole transformation into (S)-thioanisole sulfoxide (optical purity of 82.1%) at high (1.0–1.5 g/l) concentrations of sulfide substrate.     

Alanine production in an H+-ATPase- and lactate dehydrogenase-defective mutant of Escherichia coli expressing alanine dehydrogenase

Previously, we reported that pyruvate production was markedly improved in TBLA-1, an H⁺-ATPase-defective Escherichia coli mutant derived from W1485lip2, a pyruvate-producing E. coli K-12 strain. TBLA-1 produced more than 30 g/l pyruvate from 50 g/l glucose by jar fermentation, while W1485lip2 produced only 25 g/l pyruvate (Yokota et al. in Biosci Biotechnol Biochem 58:2164–2167, 1994b). In this study, we tested the ability of TBLA-1 to produce alanine by fermentation. The alanine dehydrogenase (ADH) gene from Bacillus stearothermophilus was introduced into TBLA-1, and direct fermentation of alanine from glucose was carried out. However, a considerable amount of lactate was also produced. To reduce lactate accumulation, we knocked out the lactate dehydrogenase gene (ldhA) in TBLA-1. This alanine dehydrogenase-expressing and lactate dehydrogenase-defective mutant of TBLA-1 produced 20 g/l alanine from 50 g/l glucose after 24 h of fermentation. The molar conversion ratio of glucose to alanine was 41%, which is the highest level of alanine production reported to date. This is the first report to show that an H⁺-ATPase-defective mutant of E. coli can be used for amino acid production. Our results further indicate that H⁺-ATPase-defective mutants may be used for fermentative production of various compounds, including alanine.     

Fed-batch two-phase production of alanine by a metabolically engineered Escherichia coli

dl-Alanine was produced from glucose in an Escherichia coli pfl pps poxB ldhA aceEF pTrc99A-alaD strain which lacked pyruvate-formate lyase, phosphoenolpyruvate (PEP) synthase, pyruvate oxidase, lactate dehydogenase, components of the pyruvate dehydogenase complex and over-produced alanine dehydrogenase (ALD). A two-phase process was developed with cell growth under aerobic conditions followed by alanine production under anaerobic conditions. Using the batch mode, cells grew to 5.3 g/l in 9 h with the accumulation of 6–10 g acetate/l, and under subsequent anaerobic conditions achieved 34 g alanine/l in 13 h with a yield of 0.86 g/g glucose. Using the fed-batch mode at μ = 0.15 h⁻¹, only about 1 g acetate/l formed in the 25 h required for the cells to reach 5.6 g/l, and 88 g alanine/l accumulated during the subsequent 23 h. This fed-batch process attained an alanine volumetric productivity of 4 g/lh during the production phase, and a yield that was essentially 1 g/g.     

Synthesis of l-tyrosine by immobilized Escherichia intermedia cells

Summary Cells of Escherichia intermedia were immobilized by entrapment in a polyacrylamide gel and used for the enzymatic production of l-tyrosine from phenol, pyruvate, and ammonia. A preparation containing 50 mg of cells/g of gel retained 60% of its original activity. The effect of temperature, pH and substrate concentration on the activity of free cells was almost identical with the effect on immobilized cells. Phenol showed inhibition and inactivation of the catalyst at high concentration. Synthesis of l-tyrosine (up to 10 g/l) was demonstrated in batch reactors with high conversion yields (95–100%) and a maximal productivity of 2 g/l/h. In continuous reactor the catalyst showed a very high operational stability (more than 54 days without losses).     

Optimum Conditions for Transformation of Maleic Acid by Immobilized Cells of Alcaligenes xylosoxidanssubsp. xylosoxidans260

Immobilized cells of Alcaligenes xylosoxidanssubsp. xylosoxidans260 transformed 98% of the maleic acid (initial concentration of 5.0 g/l medium) under periodic conditions for 48 h. Free cells transformed only 26% of the substrate in 96 h. Immobilized cells of a selected S-variant ofA. xylosoxidanstransformed the maleate (30.0 g/l) entirely in 96 h during batch cultivation and only 15.0 g/l of the maleate in continuous cultivation at a flow rate of 0.03 h^–1.     

Batch fermentation with entrapped growing cells ofLactobacillus casei

Summary Growing cells ofLactobacillus casei were entrapped in-carrageenan/locust bean gum (LBG) (2:1 or 2.75%:0.25% w/w respectively) mixed gel beads (two ranges of diameter: 0.5–1.0 and 1.0–2.0 mm) to fermentLactobacillus Selection (LBS) medium and produce biomass. The results showed significant influence of initial cell loading of the beads and bead size on the fermentation rate. The highest cell release rates were obtained with 2.75%:0.25%-carrageenan/LBG small diameter gel beads. However, 17 h fermentation of LBS medium with immobilized cells resulted in substantial softening of the gel matrix, prohibiting reuse of immobilized biocatalysts as inoculum in subsequent batch fermentation. A dynamic shear rheological study showed that the gel weakness was related to chemical interactions with the medium. Results indicated that part of the matrix-stabilizing K⁺ ions diffused back to the medium. Stabilization of the gel was obtained by adding potassium ions to the LBS medium;L. casei growth was not altered by this supplementation. Fermentation of LBS medium supplemented with KCl byL. casei showed higher cell counts in the broth medium with immobilized cells than with free cells, reaching 10¹⁰ cells/ml after about 10 h with entrapped cells in 0.5–1.0 mm diameter beads and 17 h with free cells. Counts in the gel beads after fermentation were higher than 10¹¹ cells/ml and bead integrity was maintained throughout fermentation.     

Production of molecular hydrogen with immobilized cells ofRhodospirillum rubrum

Summary Cells ofRhodospirillum rubrum have been immobilized in various gels and tested for photobiological hydrogen production. Agar proved to be the best immobilizing agent with respect to production rates as well as stability. Agar immobilized cells were also superior compared to liquid suspension cultures. Growth conditions of the cells prior to immobilization, e.g. cell age, light intensity or nutrient composition, were of primary importance for the activity in the later immobilized state. A reactor with agar immobilized cells has been operated successfully over 3000 h with a loss of the activity of about 60%. Mean rates for hydrogen production for immobilized cells in this work during the first 60 to 70 hours after immobilization were in the range of 18 to 34 μl H₂ mg⁻¹ d.w. h⁻¹ and thus by a factor of up to 2 higher than liquid cultures under the same conditions. Maximal rates of hydrogen production (57 μl H₂ ml⁻¹ immobilized cell suspension) were reached in agar gel beads with cells immobilized after 70 h growth in liquid culture in the light and a cell density of 1.0 mg ml⁻¹, 70 h after immobilization.     

On the nature of the ''nothing dehydrogenase'' reaction

Summary The biochemical mechanism underlying the nothing dehydrogenase reaction during the histochemical demonstration of dehydrogenases using tetranitro BT as the final electron acceptor has been investigated in unfixed, frozen rat liver sections. The reaction is stronger with NAD⁺ than either with NADP⁺ or in the absence of coenzyme. As much as 50% of the reaction is due to lactate dehydrogenase converting endogenous lactate and is largely inhibited by pyruvate. No NAD⁺-dependent alcohol dehydrogenase activity was detected at pH 7.45, the pH used for the incubations. The coenzyme-independent activity may be caused by SH-groups present in proteins and compounds like glutathione and cysteine and can be inhibited byN-ethylmaleimide andp-chloromercuribenzoic acid. It was also found that the nothing dehydrogenase reaction mainly occurs during the first few minutes of incubation, levelling off quickly to a slow rate. When studying the kinetics of dehydrogenase reactions with tetrazolium salts, it should be realized that the nothing dehydrogenase reaction, which as a whole is nonlinear with time, can interfere seriously with the dehydrogenase reaction to be analysed and may yield initial reaction rates that are too high. The findings of the present study reveal the nature of the reactions used for detection of necrosis in tissues with tetrazolium salts.     

Influence of pH and initial lactate concentration on the growth of Lactobacillus plantarum

Summary Fermentation yields of Lactobacillus plantarum were measured at controlled pH between 4.0 and 8.0 and initial lactate concentrations of 0–90 g/l. Optimal growth conditions at pH 6.0 without addition of lactate gave a growth rate of 0.57 h^–1 and 20 g dry biomass/mol ATP formed (Y _ATP). The pH variations resulted in a decrease in growth rate but the effect on Y _ATPwas insignificant. The addition of lactate to the medium at 0 h resulted in linear decrease in the growth rate of the culture, and all the metabolic activities were completely inhibited at 110 g/l. The Y _ATPand biomass/ substrate yield (Y _X/S) remained fairly steady up to 33 g lactate/l, beyond which both yields decreased considerably. Offsprint requests to: M. Raimbault     

Lactate Uptake and Release in the Presence of Glucose by Sympathetic Ganglia of Chicken Embryos and by Neuronal and Nonneuronal Cultures Prepared from These Ganglia

Abstract: Uptake and output of lactate were measured in lumbar sympathetic chains excised from embryos of white leghorn chickens, 14–15 days old. The chains, typically containing 30–40 μg of protein, were incubated in Eagle's minimum essential medium containing bicarbonate buffer, 6–17 mM glucose, various concentrations of lactate, and either [U-¹⁴C]lactate, [1-¹⁴C]glucose, or [6-¹⁴C]glucose. The average rate of uptake of labeled lactate was measured with incubations of 5–6 h, starting with various external lactate concentrations. From these data the instantaneous relation between lactate uptake rate and concentration was deduced with a simple computerized model. The instantaneous uptake rate increased with the concentration according to a relation that fit the Michaelis-Menten equation, with V_max = 360 μmol/g protein/h and K_m = 4.8 mM. Substantial fractions of the lactate carbon were recovered from tissue constituents and in several nonvolatile products in the medium, as well as in CO₂. Glucose uptake averaged about 108 μmol/g protein/h and did not vary greatly with external lactate concentration, although the metabolic partitioning of glucose carbon was considerably affected. Regardless of initial concentration, the lactate concentration in the medium tended to change towards approximately 0.6 mM, showing that uptake equaled output at this level, with rates at about 40 μmol/g protein/h. With the steady-state concentration of 0.6 mM lactate, about 20% of the glucose carbon was shunted out into the medium before it was reabsorbed and metabolized into various products. Lactate uptakes by neuronal and nonneuronal cultures prepared from the ganglia did not differ consistently from one another or from uptake by undissociated ganglia. The neuronal cultures tended to oxidize a greater fraction of the consumed lactate to CO₂ and to convert a smaller fraction of the lactate to products in the medium than did the nonneuronal cultures. Computer modeling, using known parameters for blood-brain transport of lactate in the adult rat and data on uptake by the ganglia, suggests that lactate may supply substantial fuel to the brain, even in the presence of abundant glucose, when the lactate concentration in the blood is raised to levels commonly observed in exercising humans, such as 10–20 mM. This is in agreement with the findings of several investigators in hypoglycemic humans and in animals with intermediate blood lactate concentrations.     

High ethanol productivities using small Ca-alginate beads of immobilized cells of Zymomonas mobilis

Summary Zymomonas mobilis cells were immobilized into small 1 mm diameter beads of Ca-alginate in order to minimize mass transfer limitations and maximize immobilized cell activity. A combination of small bead size with a high cell concentration of 58 g dry wt. cell per lit. bead volume resulted in high ethanol productivities using a newly designed packed bed bioreactor system. Steady-state dilution rates ranging from 0.4 h^-1 to 3.9 h^-1 were run resulting in a maximum productivity of 102 g ethanol/l/h for an inlet substrate concentration of 100 g glu/l and 87% conversion. The bioreactor was run continuously at a fixed dilution rate for 384 h and short intermittent treatment of the beads with CaCl₂ temporarily increased ethanol productivity to a maximum of 116 g ethanol/l/h.     

Effect of lactic acid on growth and butanediol production byKlebsiella oxytoca

Summary Production of 2,3-butanediol byKlebsiella oxytoca was enhanced in the presence of low levels (<8 g/l) of added sodium lactate. Cell growth was inhibited, however, and essentially stopped above 15 g/l added lactate. Levels of by-products (acetic acid and ethanol) were also higher. With 3 g/l lactate and an initial glucose level of 98 g/l, butanediol concentration and productivity increased 164% with 98% utilization of glucose. With high glucose concentration (219 g/l), addition of 2.64 g/l lactate after the growth phase resulted in 81 g/l butanediol, with a productivity of 0.65 g/l/h and 71% glucose utilization.     

Indirect monitoring of acetate exhaustion and cell recycle improve lactate production by non-growing Escherichia coli

A two-phase, lactate fermentation by Escherichia coli ALS974 generates succinate and ethanol anaerobically from acetate. These by-products can be minimized by monitoring acetate concentration indirectly with dissolved O₂ (DO) during the initial aerobic cell-growth phase. Without DO monitoring, 3 g succinate/l and 1 g ethanol/l were generated while, with monitoring, less than 1 g succinate/l and no detectable ethanol were generated with 130 g lactate/l being produced. Furthermore, using a cell-recycle fermentation with ultrafiltration prolonged the anaerobic lactate production phase from 22 to 34 h, thereby achieving a lactate productivity of 4.2 g/l h, nearly 20% greater than the productivity of the fed-batch process.     

Acid proteinases production by humicola lutea cells immobilized in polyhydroxyethylmethacrylate gel

The spores of Humicola lutea entrapped in polyhydroxyethylmethacrylate gel were precultivated in production medium for mycelial formation. The immobilized mycelium was reused in batch mode for acid proteinases production. The influence of precultivation time, initial inoculum gel volume, and gel particle size on the enzyme activity and proteinases production half-life were studied. After 70 h precultivation of the entrapped spores (10 ml initial inoculum volume, 12–27 mm³ gel particle size) maximum proteinases activity of 100–140% (compared with free cells) was registered in 15 reaction cycles. Under the same condition the half-life time was 18 cycles, while for the free cells it was 5 cycles. The main advantage of the polyhydroxyethylmethacylate immobilized H. lutea was the long acid proteinases production half-life at a low concentration of outgrowing cells in the medium.