Isolation and purification of acetolactate synthase and acetolactate decarboxylase from the culture ofLactococcus lactis

Enzymes catalyzing the synthesis and subsequent transformation of α-acetolactate (AcL)—acetolactate synthase (AcLS) and acetolactate decarboxylase (AcLDC)—were isolated and partially purified from the cells of lactic acid bacteriaLactococcus lactis ssp.lactis biovar.diacetylactis, strain 4. The preparation of AcLS, purified 560-fold, had a specific activity of 358 300 U/mg protein (9% yield). The preparation of AcLDC., purified 4828-fold, had a specific activity of 140 U/mg protein (4.8% yield). The enzymes exhibited optimum activity at pH 6.5 and 6.0, respectively (medium, phosphate buffer). The values of apparentK _m, determined for AcLS and AcLDC with pyruvate and AcL, respectively, were equal to 70 mM and 20 mM. AcLS appeared as an allosteric enzyme with low affinity for the substrate and a sigmoid dependence of the activity on the substrate concentration. In the case of AcLDC, this dependence was hyperbolic and the affinity of the enzyme for its substrate was high (K _m = 20 mM). Leucine, valine, and isoleucine were shown to be activators of AcDLC.     

COMPARISON OF HALF-SATURATION CONSTANTS FOR GROWTH AND NITRATE UPTAKE OF MARINE PHYTOPLANKTON 2

Growth rates and rates of nitrate uptake by N-depleted cells were measured for an oceanic diatom, Chaetoceros gracilis, and a neritic diatom, Asterionella japonica, as functions of nitrate concentration of the medium. Both growth and N-uptake rates appeared to be hyperbolic with nitrate concentration and could be fit to an equation of Michaelis-Menten form: where v is rate, V^m. is the maximum rate, S is nitrate concentration, and K_sis the half-saturation constant. K_svalues for uptake and growth were similar if not identical for each species. Uptake experiments can provide a presumptive measure of K_sfor growth, thought to be an ecologically significant characteristic of a species.     

Toxin production and competitive abilities under phosphorus limitation of Alexandrium species

The relationship between growth rate versus phosphorus concentration and cellular toxin content was determined for Alexandrium minutum AL1V, Alexandrium tamarense MDQ1096, A. tamarense EF04 and Alexandrium andersoni EF12 under different nitrogen and phosphorus supplies. The aim was to determine whether those species with a lower phosphorus uptake affinity, and hence potentially of lower competitive ability at low phosphorus concentrations, were more toxic. The range and mean of toxic content per cell (as fmol per cell) of the species were 13.5–256.5 and 140.2±50.8 for A. tamarense MDQ1096, 0.5–16.5 and 2.9±2.6 for A. minutum, 0–2.0 and 0.2±0.3 for A. tamarense EF04 and, 0–3.3 and 0.06±0.4 for A. andersoni. K_s for culture cell growth (per day),representing the phosphate concentration at which the specific culture cell growth rate is one half the maximum rate, and K_min (per day), the phosphate concentration at which the specific culture cell growth rate is zero, were used as indicators of species’ potential competitive ability at low phosphorus concentrations. Low values for both K_s and K_min indicate a high relative ability of the species to use low levels of phosphate and, hence, expected to outcompete higher K_s and K_min species under phosphorus limitation. K_s and K_min were 1.68 and 0.48 for A. tamarense MDQ1096, 1.16 and 0.39 for A. minutum, 1.0 and 0.38 for A. tamarense EF04 and, 0.74 and 0.34 for A. andersoni, respectively. There was a significant positive relationship between toxin content per cell with both K_s and K_min, indicating that those species with lower ability to compete under phosphorus limitation were more toxic. The findings support the hypothesis that toxin production by dinoflagellates species could be an adaptation evolved to offset the ecological disadvantage of having low nutrient affinity.     

<Emphasis Type="Italic">Marinococcus halophilus</Emphasis> DSM 20408<Superscript>T </Superscript>encodes two transporters for compatible solutes belonging to the betaine-carnitine-choline transporter family: identification and characterization of ectoine transporter EctM and glycine betaine transporter BetM

In response to osmotic stress, the halophilic, Gram-positive bacterium Marinococcus halophilus accumulates compatible solutes either by de novo synthesis or by uptake from the medium. To characterize transport systems responsible for the uptake of compatible solutes, a plasmid-encoded gene bank of M. halophilus was transferred into the transport-deficient strain Escherichia coli MKH13, and two genes were cloned by functional complementation required for ectoine and glycine betaine transport. The ectoine transporter is encoded by an open reading frame of 1,578 bp named ectM. The gene ectM encodes a putative hydrophobic, 525-residue protein, which shares significant identity to betaine-carnetine-choline transporters (BCCTs). The transporter responsible for the uptake of glycine betaine in M. halophilus is encoded by an open reading frame of 1,482 bp called betM. The potential, hydrophobic BetM protein consists of 493 amino acid residues and belongs, like EctM, to the BCCT family. The affinity of whole cells of E. coli MKH13 for ectoine (K_s=1.6 M) and betaine (K_s=21.8 M) was determined, suggesting that EctM and BetM exhibit a high affinity for their substrates. An elevation of the salinity in the medium resulted in an increased uptake of ectoine via EctM and glycine betaine via BetM in E. coli MKH13 cells, demonstrating that both systems are osmoregulated.Communicated by W.D. Grant     

Inhibitory effect of dihydroxyacetone onGluconobacter oxydans: Kinetic aspects and expression by mathematical equations

Summary Microbial conversion of glycerol into dihydroxyacetone (DHA) byGluconobacter oxydans was subjected to inhibition by excess substrate. Comparison of cultures containing increasing initial DHA contents (0 to 100 g l^–1) demonstrated that DHA also inhibited this fermentation process. The first effect was on bacterial growth (cellular development stopped when DHA concentration reached 67 gl^–1), and then on oxidation of glycerol (DHA synthesis only occurred when the DHA concentration in the culture medium was lower than 85 g l^–1). Productivity, specific rates and, to a lesser extent, conversion yields decreased as initial concentrations of DHA increased. The changes in the specific parameters according to increasing initial DHA contents were described by general equations. These formulae satisfactorily express the concave aspect of the curves and the reduction in biological activity when the cells were in contact with DHA concentrations of up to 96 g l^–1.Abbreviations X, S, P biomass, substrate, product concentrations - r _x,r _s,r _p rates of growth, consumption and production - ,q _s,q _p specific rates of growth, glycerol consumption and DHA production - Y _x/s, Y_p/s conversion yields of substrate into biomass and product - K _s constant of affinity of cells to the substrate - K _ip product inhibition constant - P _m threshold concentration of DHA in substrate     

A pulsing device for packed-bed bioreactors: II. Application to alcoholic fermentation

When the immobilized cells are employed in packed-bed bioreactors several problems appear. To overcome these drawbacks, a new bioreactor based on the use of pulsed systems was developed [1]. In this work, we study the glucose fermentation by immobilized Saccharomyces cerevisiae in a packed-bed bioreactor. A comparative study was then carried out for continuous fermentation in two packed-bed bioreactors, one of them with pulsed flow. The determination of the axial dispersion coefficients indicates that by introducing the pulsation, the hydraulic behaviour is closer to the plug flow model. In both cases, the residence time tested varied from 0.8 to 2.6 h. A higher ethanol concentration and productivity (increases up to 16%) were achieved with the pulsated reactors. The volumes occupied by the CO₂ were 5.22% and 9.45% for fermentation with/without pulsation respectively. An activity test of the particles from the different sections revealed that the concentration and viability of bioparticles from the two bioreactors are similar. From the results we conclude that the improvements of the process are attributable to a mechanical effect rather than to physiological changes of microorganisms.List of Symbols D m²/s dispersion coefficient - K _is l/g inhibition substrate constant - K _ip l/g inhibition ethanol constant - K _s g/l Apparent affinity constant - P g/l ethanol concentration - q _p g/(gh) specific ethanol productivity - Q _p g/(lh) overall ethanol productivity - q _s g/(gh) specific glucose consumption rate - Q _s g/(lh) glucose consumption rate - S g/l residual glucose concentration - S(in0) g/l initial glucose concentration - V _max g/(lh) maximum rate - Y _p/s g/g yield in product     

Adaptation of methane formation and enzyme contents during growth of Methanobacterium thermoautotrophicum (strain ΔH) in a fed-batch fermentor

During growth of Methanobacterium thermoautotrophicum in a fed-batch fermentor, the cells are confronted with a steady decrease in the concentration of the hydrogen energy supply. In order to investigate how the organism responds to these changes, cells collected during different growth phases were examined for their methanogenic properties. Cellular levels of the various methanogenic isoenzymes and functionally equivalent enzymes were also determined. Cells were found to maintain the rates of methanogenesis by lowering their affinity for hydrogen: the apparent K _m ^H2 decreased in going from the exponential to the stationary phase. Simultaneously, the maximal specific methane production rate changed. Levels of H₂-dependent methenyl-tetrahydromethanopterin dehydrogenase (H₂-MDH) and methyl coenzyme M reductase isoenzyme II (MCR II) decreased upon entry of the stationary phase. Cells grown under conditions that favored MCR II expression had higher levels of MCR II and H₂-MDH, whereas in cells grown under conditions favoring MCR I, levels of MCR II were much lower and the cells had an increased affinity for hydrogen throughout the growth cycle. The use of thiosulfate as a medium reductant was found to have a negative effect on levels of MCR II and H₂-MDH. From these results it was concluded that M. thermoautotrophicum responds to variations in hydrogen availability and other environmental conditions (pH, growth temperature, medium reductant) by altering its physiology. The adaptation includes, among others, the differential expression of the MDH and MCR isoenzymes.     

Turgor regulation in a novel Halomonas species

The mechanisms by which a novel eubacterium, identified as belonging to the genus Halomonas, adapted to increases in the extracellular osmotic potential were investigated. It was shown that the ability of the bacterium to grow after hyperosmotic shock was dependent on the presence of potassium ions. Growth of the bacterium in 2 M NaCl medium could be limited by low concentrations of K⁺ and this enabled the affinity for K⁺ to be determined (K _s=21.5 M). Rubidium salts could be substituted for those of potassium, but the lowest concentration of Rb⁺ that allowed growth in 2 M NaCl medium was 50-fold greater than the minimum concentration of K⁺. ¹³C-NMR spectroscopy and HPLC analysis were used to demonstrate the accumulation of organic solutes in the cytoplasm after exposure to high salinities. The major osmolyte was ectoine, but glutamate and ectoine hydroxide were also present. Addition of exogenous glycine betaine to 3.25 M NaCl medium resulted in the accumulation of high intracellular concentrations of glycine betaine in the bacterium. This reduced the level of ectoine accumulation but did not fully inhibit the synthesis of this compound in the cytoplasm.Abbreviation Specific growth rate (generations/h)     

Dependency of growth yield,maintenance and K s-values on the dissolved oxygen concentration in continuous cultures of Azotobacter vinelandii

Azotobacter vinelandii was grown diazotrophically in sucrose-limited chemostat cultures at either 12, 48, 108, 144 or 192 M dissolved oxygen. Steady state protein levels and growth yield coefficients (Y) on sucrose increased with increasing dilution rate (D). Specific rate of sucrose consumption (q) increased in direct proportion to D. Maintenance coefficients (m) extrapolated from plots of q versus D, as well as from plots of 1/Y versus 1/D exhibited a nonlinear relationship to the dissolved oxygen concentration. Constant maximal theoretical growth yield coefficients (Y ^G) of 77.7 g cells per mol of sucrose consumed were extrapolated irrespective of differences in ambient oxygen concentration. For comparison, glucose-, as well as acetate-limited cultures were grown at 108 M oxygen. Fairly identical m- and Y ^G-values, when based on mol of substrate-carbon with glucose and sucrose grown cells, indicated that both substrates were used with the same efficiency. However, acetate-limited cultures showed significantly lower m- and, at comparable, D, higher Y-values than cultures limited by either sucrose or glucose. Substrate concentrations (K _s) required for half-maximal growth rates on sucrose were not constant, they increased when the ambient oxygen concentration was raised and, at a given oxygen concentration, when D was decreased. Since biomass levels varied in linear proportion to K _s these results are interpreted in terms of variable substrate uptake activity of the culture.Abbreviations D dilution rate - K _s substrate concentration required for half maximal growth rate - m maintenance coefficient - q specific rate of substrate consumption - Y growth yield coefficient - Y ^G maximum theoretical growth yield coefficient     

Analysis of growth of Gluconobacter oxydans in glucose containing media

Gluconobacter oxydans oxidizes glucose via alternative pathways: one involves the non-phosphorylative, direct oxidation route to gluconic acid and ketogluconic acids, and the second requires an initial phosphorylation and then oxidation via the pentose phosphate pathway enzymes. During growth of G. oxydans in glucose-containing media, the activity of this pathway is strongly influenced by (1) the pH value of the environment and (2) the actual concentration of glucose present in the culture. At pH values below 3.5 the activity of the pentose phosphate pathway was completely inhibited resulting in an increased requirement of the organism for nutrient substances, and a poor cell yield. At pH 5.5 a triphasic growth response was observed when G. oxydans was grown in a defined medium. Above a threshold value of 5–15 mM glucose, oxidation of both glucose and gluconate by the pentose phosphate pathway enzymes was repressed, causing a rapid accumulation of gluconic acid in the culture medium. When growing under these conditions, a low affinity for the oxidation of glucose was found (K _s=13 mM). Below this threshold glucose concentration, pentose phosphate pathway enzymes were synthesized and glucose was actively assimilated via this pathway. It was shown that de novo enzyme synthesis was necessary for increased pentose phosphate pathway activity and that assimilation of gluconate by washed cell suspensions was inhibited by glucose.     

Purification and Characterization of Aspartic Protease Derived from Sf9 Insect Cells

An aspartic protease that is significantly produced by baculovirus-infected Spodoptera frugiperda Sf9 insect cells was purified to homogeneity from a growth medium. To monitor aspartic protease activity, an internally quenched fluoresce (IQF) substrate specific to cathepsin D was used. The purified aspartic protease showed a single protein band on SDS–PAGE with an apparent molecular mass of 40 kDa. The N-terminal amino acid sequence of the enzyme had a high homology to a Bombyx mori aspartic protease. The enzyme showed greatest affinity for the IQF substrate at pH 3.0 with a K _m of 0.85 μM. The k _cat and k _cat?K _m values were 13 s^?1 and 15 s^?1 μM^?1 respectively. Pepstatin A proved to be a potent competitive inhibitor with inhibitor constant, K _i, of 25 pM.     

Effect of increasing methanol concentrations on physiology and cytology ofCandida boidinii

Concentration of methanol in the medium strongly affected not only the physiology but also the cytology ofCandida boidinii strain 2 cells in a methanol-limited chemostat at a constant dilution rateD 0.1/h and at low pH 3.0. The formation of large cubic peroxisomes with high alcohol oxidase (AO) activity observed at low methanol concentration (S ₀ 3 g/L) disappeared on increasing the methanol concentration in the inflow medium. The AO activity in the cells sharply decreased, followed by accumulation of riboflavin phosphate and residual methanol in the medium. The activity of catalase was relatively stable. At methanol concentrationS ₀>K _I (K ₁ equal to 12 g methanol per L), which included a substantial increase in methanol dissimilation, documented by higher formaldehyde and formate dehydrogenase activities and by lower yield coefficient on methanol, the yeast cells contained large lobe-shaped peroxisomes and a smaller number of larger mitochondria. The cells formed pseudomycelium with a thick septum between the mother and daughter cells.     

Effect of light intensity and ammonium-N on carotenogenesis ofTrentepohlia odorata andDunaliella bardawil

AxenicTrentepohlia odorata was cultured at three different NH₄Cl levels (3.5 × 10^–2, 3.5 × 10^–3, 3.5 × 10^–4 M) and three different light intensities (48, 76, 122 µmol m^–2 s^–1). Chloride had no effect on growth over this range of concentration. High light intensity and high NH₄Cl concentration enhanced the specific growth rate. The carotenoid content increased under a combination of high light intensity and low N concentration. WhenD. bardawil was exposed to the same combination of growth conditions, there was an increase in its carotenoid content. The light saturation and the light inhibition constants (K _s andK _i, respectively) for growth, and the saturation constant (K _m) for NH₄Cl were determined. TheK _s andK _i values were higher inT. odorata (66.7 and> 122 mol m^–2 s^–1, respectively) than inD. bardawil (5.1 and 14.7 µmol m^–2 s^–1, respectively). TheK _m value determined at 122 µmol m^–2 s^–1, however, was lower inT. odorata (0.048 µM) than inD. bardawil (0.062 µM).Author for correspondence     

Guinea Pig Brain Histamine N-Methyltransferase: Purification and Partial Characterization

Abstract: Histamine N-methyltransferase (EC 2.1.1.8) was purified 4400–fold in 12% yield from guinea pig brain. The basic steps in the purification included differential centrifugation, calcium phosphate adsorption, DEAE-cel-lulose chromatography, and affinity chromatography on an S-adenosylhomocysteine-agarose matrix. The resulting protein was homogeneous by gel electrophoresis and was stable for at least 3 months at 80°C. It had an apparent molecular weight of 29 ,000 ± 1000 as determined by both gel filtration through Sephadex G-100 and by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The isoelectric point of the protein was found to be 5.3. The pH optima for methylation of histamine were determined to be 7.5 and 9.0; the K_ms for histamine and S-adenosyl-l-methionine were 13.57 ± 0.74 μM and 6.1 ± 0.12 μM, respectively; the K_i for S-adenosyl-l-homocysteine was 24.5 ± 1.45 μM.     

Catabolite regulation in a diauxic strain and a nondiauxic strain of Streptococcus bovis

Streptococcus bovis JB1 utilized glucose preferentially to lactose and grew diauxically, but S. bovis 581AXY2 grew nondiauxically and used glucose preferentially only when the glucose concentration was very high (greater than 5 mM). As little as 0.1 mM glucose completely inhibited the lactose transport of JB1. The lactose transport system of 581AXY2 was at least tenfold less sensitive to glucose, and 1 mM glucose caused only a 50% inhibition of lactose transport. Both strains had phosphotransferase systems (PTSs) for glucose and lactose. The glucose PTSs were constitutive, but little lactose PTS activity was detected unless lactose was the energy source for growth. JB1 had approximately threefold more glucose PTS activity than 581AXY2 (1600 versus 600 nmol glucose (mg protein)⁻¹(min)⁻¹. The glucose PTS of JB1 showed normal Michaelis Menten kinetics, and the affinity constant (K _s ) was 0.12 mM. The glucose PTS of 581AXY2 was atypical, and the plot of velocity versus velocity/substrate was biphasic. The low capacity system had a K_s of 0.20 mM, but the K_s of the high capacity system was greater than 6 mM. On the basis of these results, diauxic growth is dependent on the affinity of glucose enzyme II and the velocity of glucose transport. Received: 22 January 1996 / Accepted: 18 March 1996     

Continuous production of itaconic acid by Aspergillus terreus immobilized in a porous disk bioreactor

Summary Aspergillus terreus NRRL 1960 was grown on porous disks rotating intermittently in and out of the liquid phase. This immobilized fungal cell bioreactor was used to produce itaconic acid from glucose in a continuous operation. The effect of temperature, pH, disk rotation speed, and feed rate on the itaconic acid concentration and volumetric productivity were studied. The highest itaconic acid concentration and volumetric productivity obtained were 18.2 g/l and 0.73 g/l·h, respectively, under the following conditions: temperature at 36°C, pH 3.0, disk rotation speed at 8 rpm, and feed rate at 60 ml/h. These results are better than those by conventional fermentation or by other immobilized method.Nomenclature F feed rate (l/h) - K _1s saturation constant for immobilized cells (g/l) - K _2s saturation constant for suspended cells (g/l) - M ₁ increased mass of immobilized cells (g) - M ₂ total mass of immobilized cells (g) - P concentration of itaconic acid (g/l) - S substrate concentration in and out of the reactor (g/l) - S ₀ substrate concentration in the feed (g/l) - V liquid volume of the reactor (1) - X concentration of the suspended cells (g/l) - Y ₁ apparent yield of the immobilized cells (g cells/g substrate) - Y ₂ apparent yield of the suspended cells (g cell/g substrate) - Y ₃ apparent yield of itaconic acid (g itaconic acid/g substrate) - m ₁ maintenance and by-products coefficient of the immobilized cells (g substrate/g cell·h) - m ₂ maintenance and by-products coefficient of the suspended cells (g substrate/g cell·h) - µ_1max maximum specific growth rate of the immobilized cells (h^-1) - µ_2max maximum specific growth rate of the suspended cells (h^-1)     

Nature of the individual Ca2+ binding sites in Ca2+-regenerated bacteriorhodopsin

The binding constants, K₁ and K₂, and the number of Ca²⁺ ions in each of the two high affinity sites of Ca²⁺-regenerated bacteriorhodopsin (bR) are determined potentiometrically at different pH values in the range of pH 3.5-4.5 by using the Scatchard plot method. From the pH dependence of K₁ and K₂, it was found that two hydrogen ions are released for each Ca²⁺ bound to each of the two high affinity sites. Furthermore, we have measured by a direct spectroscopic method the association constant, K_s, for the binding of Ca²⁺ to deionized bR, which is responsible for producing the blue to purple color change. Comparing the value of K_s and its pH dependence with those of K₁ and K₂ showed that the site corresponding to K_s is to be identified with that of K₂. This is in agreement with the conclusion reached previously, using a different approach, which showed that it is the second Ca²⁺ that causes the blue to purple color change.

Our studies also show that in addition to the two distinct high affinity sites, there are about four to six sites with lower binding constants. These are attributed to the nonspecific binding in bR.

     

Performance assessment of two patent strains ofZymomonas mobilis in batch and continuous fermentations

Summary The performance ofZymomonas mobilis strains ATCC 31821 and ATCC 31823 was assessed in batch and continuous culture. In batch culture using a medium containing 250 g/l glucose, identical maximum specific growth rates of 0.16/h were found, though final biomass concentration and growth yield were significantly lower for ATCC 31 823 than for ATCC 31 821. Final ethanol concentrations in this medium were about 110 g/l vor both organisms. In continuous culture at increasing dilution rates using a medium containing 100 g/l glucose, no significant differences were seen between the two strains with respect to the fermentation parameters studied. For ATCC 31 821, maximum rates of glucose uptake (Q_s) and ethanol produktion (Q_p) of 8.7 g glu/g/h and 4.4 g eth/g/h, respectively, were found. Both strains showed a similar performance at a fixed dilution rate of 0.1/h, where maximum ethanol concentrations of about 68 g/l were reached at a feed glucose concentration of about 139 g/l. At this dilution rate the maximum values of Q_s and Q_p were about 5.8 g glu/g/h and 2.8 g eth/g/h, respectively. Test tube experiments showed that growth, measured as optical density, decreased with increasing concentrations of exogenous ethanol with complete inhibition of growth at ethanol concentrations >8% (v/v). As evidenced by the results presented here, we have been unable to practice the invention as described in U.S. Patent 4,403,034 (Rogers and Tribe 1983).Nomenclature D Dilution rate, 1/h - _max maximum specific growth rate, 1/h - S_R Initial substrate concentration, g glucose/1 - S Residual substrate concentration, g glucose/1 - S₀ Effluent substrate concentration, g glucose/1 - X Blomass concentration; g cells/l - OD₆₂₀ Optical density at 620 nm, dimensionless - [P] Product concentration, g ethanol/1 - Y_x/s Growth yield, g cells/g glucose used - Y_p/s Product yield, g ethanol/g glucose used - %, Yield Percentage yield, Y_p/sx100/Y _p ^s/max =Y_p/sx100/0.51 - Q_s Specific rate of glucose uptake, g glucose/g cells/h - Q_p Specific rate of ethanol formation, g ethanol/g cells/h - m_e Maintenance energy coefficient, g glucose/g cells/h - VP Volumetric productivity, g ethanol/l/h - t Fermentation time, h     

Ethanol production from sulphuric acid wood hydrolysate ofPinus radiata using free and immobilized cells ofPichia stipitis

Summary Ethanol was produced by a strain ofPichia stipitis adapted to an inhibitory acid wood hydrolysate ofPinus radiata. The best ethanol productivity for batch cultures was 0.21 g/l h at 0.7% ethanol. Varying culture conditions increased ethanol concentration to 0.76%, however the productivity decreased to 0.18 g/l h. A decrease in ethanol concentration in the culture fluid was noted late in the batch which suggested ethanol catabolism. Values of kinetic parameters (K _m,K _s, _max, andV _max) were evaluated for this system. The use of calcium alginate immobilized cells in a continuous-flow stirred tank reactor lead to enhanced fermentative performance, namely a maximum productivity of 0.27 g/l h and 1.13% ethanol yield. The immobilized cells in continuous flow reactors represent an attractive option for fermenting sugars released by sulphuric acid hydrolysis ofP. radiata wood.     

The role of an NAD-independent lactate dehydrogenase and acetate in the utilization of lactate byClostridium acetobutylicum strain P262

Clostridium acetobutylicum strain P262 utilized lactate at a rapid rate [600 nmol min^–1 (mg protein)^–1], but lactate could not serve as the sole energy source. When acetate was provided as a co-substrate, the growth rate was 0.05 h^–1. Butyrate, carbon dioxide and hydrogen were the end products of lactate and acetate utilization, and the stoichiometry was 1 lactate + 0.4 acetate → 0.7 butyrate + 0.6 H₂ + 1 CO₂. Lactate-grown cells had twofold lower hydrogenase than glucose-grown cells, and the lactate-grown cells used acetate as an alternative electron acceptor. The cells had a poor affinity for lactate (K_s = 1.1 mM), and there was no evidence for active transport. Lactate utilization was catabolyzed by an inducible NAD-independent lactate dehydrogenase (iLDH) that had a pH optimum of 7.5. The iLDH was fivefold more active with d-lactate than l-lactate, and the K _m for d-lactate was 3.2 mM. Lactate-grown cells had little butyraldehyde dehydrogenase activity, and this defect did not allow the conversion of lactate to butanol. Received: 17 October 1994 / Accepted: 30 January 1995