Actual and potential rates of hydrogen photoproduction by continuous culture of the purple non-sulphur bacterium Rhodobacter capsulatus

The influence of (NH₄)₂SO₄ concentration and dilution rate (D) on actual and potential H₂ photoproduction has been studied in ammonium-limited chemostat cultures of Rhodobacter capsulatus B10. The actual H₂ production in a photobioreactor was maximal (approx. 80 ml h⁻¹l⁻¹) at D = 0.06 h⁻¹ and 4 mM (NH₄)₂SO₄. However, it was lower than the potential H₂ evolution (calculated from hydrogen evolution rates in incubation vials), which amounted to 100–120 ml h⁻¹ l⁻¹ at D = 0.03–0.08 h⁻¹. Taking into account the fact that H₂ production in the photobioreactor under these conditions was not limited by light or lactate, another limiting (inhibiting) factor should be sought. One possibility is an inhibition of H₂ production by the H₂ accumulated in the gas phase. This is apparent from the non-linear kinetics of H₂ evolution in the vials or from its inhibition by the addition of H₂; initial rates were restored in both cases after the vials had been refilled with argon. The actual H₂ production in the photobioreactor at D = 0.06 h⁻¹ was shown to increase from approximately 80 ml h⁻¹ l⁻¹ to approximately 100 ml h⁻¹ l⁻¹ under an argon flow at 100 ml min⁻¹. Under maximal H₂ production rates in the photobioreactor, up to 30% of the lactate feedstock was utilised for H₂ production and 50% for biomass synthesis. Received: 22 April 1997 / Received revision: 14 July 1997 / Accepted: 27 July 1997     

Does the agitation rate and/or oxygen saturation influence exopolysaccharide production by Aureobasidium pullulans in batch culture?

  When Aureobasidium pullulans was grown at a number of agitation rates under batch conditions, exopolysaccharide yields were dramatically reduced at high rates i.e. at least 750 rpm. Investigations with gas blending, which allowed pO₂ manipulation and control independently of the agitation rate, showed that this yield reduction was due solely to the high pO₂ levels that occurred at these agitation rates. Thus, polysaccharide production at 1000 rpm could be elevated by maintaining the pO₂ at a low level during the initial phase of the fermentation. However, both the timing of the pO₂ decrease and the level at which it was maintained were crucial for obtaining yields at 1000 rpm, similar to those observed at low agitation rates. Received: 29 February 1996 / Received revision: 11 July 1996 / Accepted: 15 July 1996     

Influence of initial cellulose concentration on the carbon flow distribution during batch fermentation by Clostridium thermocellum ATCC 27405

The objective of this research was to understand how carbon loading influences hydrogen (H₂) synthesis and metabolic flow patterns in the thermophilic, cellulolytic bacterium, Clostridium thermocellum. C. thermocellum was cultivated in batch cultures with high (5 g L⁻¹) and low (1 g L⁻¹) initial concentrations of α-cellulose at 60°C. The growth rate of C. thermocellum was 22% lower (0.15 h⁻¹) in cultures with low-cellulose concentration compared with cultures with high-cellulose concentrations. Although substrate depletion coincided with the end of log-growth in low-cellulose cultures, the prime reason for growth arrest in high-cellulose cultures was not identified. Ethanol, acetate, and formate were the major soluble end-products with concomitant release of H₂ and CO₂ under both conditions. Lactate appeared during the late log phase in high-carbon cultures when pH dropped below 6.4 and became the major end-product in stationary phase. During the exponential phase of cell growth, significantly higher yields for H₂ and acetate (1.90 ± 0.14 and 1.11 ± 0.04 mol/mol glucose equivalent, respectively) were obtained from low-cellulose cultures compared to those from high-cellulose cultures. The maximum specific rate of H₂ production, 6.41 ± 0.13 mmol H₂/g dry cell/h, obtained during the exponential phase from low-carbon cultures was about 37% higher than that obtained from high-carbon cultures.     

Induction of H2-Uptake and Nitrogenase Activities in the Cyanobacterium Anabaena variabilis ATCC 29413: Effects of Hydrogen and Organic Substrate

A comparative study of the development of uptake hydrogenase and nitrogenase activities in cells of the cyanobacterium Anabaena variabilis was performed. The induction of heterocysts is followed by the induction of both in vivo hydrogen uptake and nitrogenase activities. Interestingly, a low but significant H₂-uptake [2–7 μmoles of H₂ · mg⁻¹ (Chl a) · h⁻¹] occurs in cultures with no heterocysts and with no nitrogenase activity. A slight stimulatory effect (30–40%) of H₂ on in vivo H₂-uptake was observed during the early stages of nitrogenase induction. However, exogenous H₂ does not further stimulate the induction of in vivo hydrogen uptake observed during heterocyst differentiation. Similarly, organic carbon (fructose) did not influence the induction of either in vivo hydrogen uptake or nitrogenase activities. Exogenous fructose supports higher in vivo hydrogen uptake and nitrogenase activities when the cells enter late exponential phase of growth. Received: 22 November 1995 / Accepted: 22 December 1995     

Growth characteristics of non-heterocystous cyanobacterium Plectonema boryanum with N2 as nitrogen source

Strains of filamentous, non-heterocystous cyanobacteria from the Pasteur Culture Collection (PCC), able to synthesize nitrogenase under anaerobic test conditions, were tested for growth with N₂ as sole nitrogen source at low O₂ partial pressure (less than 0.05%). Plectonema boryanum (PCC 73110) exhibited exponential growth under these conditions. This capacity was restricted to light intensities not exceeding 500 lux. Growth rates were 0.014/h at 200 and 0.023 at 500 lux and similar to those of anaerobic and aerobic control cultures with nitrate as N-source. For N₂-fixing cultures incubated at 200 and 500 lux, acetylene reduction rates were 4–8 and 5–14 nmol C₂H₄ per mg protein per min, respectively. The ratio of phycocyanine to chlorophyll was higher (200 lux) or slightly reduced (500 lux) in N₂-fixing cultures as compared to control cultures with nitrate as N-source. On the basis of epifluorescence microscopy and microfluorimetry, no differences in pigment contents were found between individual cells or filaments of N₂-fixing cultures. Also no noteworthy differences were observed between the pycobiliprotein composition of individual cells in N₂ fixing cultures as compared to nitrate-grown controls. Thus the observed exponential growth of P. boryanum at low light intensities implies simultaneous nitrogen fixation and oxygenic photosynthesis. Additional continuous culture experiments showed that N₂-fixing exponential growth was dependent on O₂ partial pressures lower than 0.2–0.4%.The other strains tested (PCC 6412, 6602, 7403, 7104) did not grow under such conditions.Abbreviations Chl chlorophyll - PBP phycobiliproteins - PC phycocyanin - PCC Pasteur Culture Collection - OD optical density     

Hydrogen production with high yield and high evolution rate by self-flocculated cells of Enterobacter aerogenes in a packed-bed reactor

Continuous hydrogen gas evolution by self-flocculated cells of Enterobacter aerogenes, a natural isolate HU-101 and its mutant AY-2, was performed in a packed-bed reactor under glucose-limiting conditions in a minimal medium. The flocs that formed during the continuous culture were retained even when the dilution rate was increased to 0.9 h⁻¹. The H₂ production rate increased linearly with increases in the dilution rate up to 0.67 h⁻¹, giving maximum H₂ production rates of 31 and 58 mmol l⁻¹ h⁻¹ in HU-101 and AY-2 respectively, at a dilution rate of more than 0.67 h⁻¹. The molar H₂ yield from glucose in AY-2 was maintained at about 1.1 at dilution rates between 0.08 h⁻¹ and 0.67 h⁻¹, but it decreased rapidly at dilution rates more than 0.8 h⁻¹. Received: 27 August 1997 / Received revision: 11 November 1997 / Accepted: 14 December 1997     

Lactobacillus sanfranciscensis CB1: manganese, oxygen, superoxide dismutase and metabolism

The latency phase, growth rate, cell yield and end-products of Lactobacillus sanfranciscensis CB1 were affected by oxygen and the supply of 225 μM Mn²⁺. Mn²⁺ was especially related to the highest substrate consumption. Aerobiosis and Mn²⁺ supply were responsible for the highest superoxide dismutase activity. An auto-inhibitory accumulation of H₂O₂ meant that the survival of air-grown cells supplied with Mn²⁺ rapidly decreased during the stationary phase. As shown by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, Mn²⁺ supply influenced protein expression. As shown by non-denaturating zymograms, Lb. sanfranciscensis CB1 expressed an approximately 12.5-kDa superoxide dismutase, which is probably Mn-dependent. The enzyme was insensitive to H₂O₂ treatment, was not induced by the presence of paraquat under aerobic conditions and was relatively stable at pH 4.0. Sourdoughs that contained high levels of oxygen enhanced cell growth, acidification and acetic acid production by Lb. sanfranciscensis CB1. Received: 24 July 1998 / Received last revision: 11 November 1998 / Accepted: 13 November 1998     

Bacterial growth in space flight: logistic growth curve parameters for Escherichia coli and Bacillus subtilis

Previous investigations have reported that bacterial suspension cultures grow to higher stationary concentrations in space flight than on Earth; however, none of these investigations included extensive ground controls under varied inertial conditions. This study includes extensive controls and cell-growth data taken at several times during lag phase, log phase, and stationary phase of Escherichia coli and Bacillus subtilis. The Marquardt-Levenberg, least-squares fitting algorithm was used to calculate kinetic growth parameters from the logistic bacterial growth equations for space-flight and control growth curves. Space-flight cultures grew to higher stationary-phase concentrations and had shorter lag-phase durations. Also, evidence was found for increased exponential growth rate in space. Received: 27 February 1998 / Received revision: 21 August 1998 / Accepted: 3 September 1998     

Rosmarinic acid production by Lavandula vera MM cell-suspension culture

The time courses of growth and rosmarinic acid production by Lavandula vera MM cell suspension were investigated. The uptake of the main nutrients (sucrose, nitrogen, phosphorus, K, Ca, Mg) was followed during cultivation and the data on the physiology of the L. vera MM cell culture are presented. It was established that the cell culture synthesizes rosmarinic acid during the linear phase of growth for a relatively short period (between the 4th and 8th days of cultivation). The influence of sucrose concentration in the nutrient medium on cell growth and accumulation of rosmarinic acid by L. vera MM cell culture was investigated. The results showed that 7% sucrose in the nutrient medium ensured a steady growth of the cell suspension and increased the yield of rosmarinic acid (29.2 g/l dry biomass and 507.5 mg/l rosmarinic acid compared to 13.0 g/l dry biomass and 68.6 mg/l rosmarinic acid for the control cultivation with 3% sucrose). Received: 17 September 1996 / Received revision: 31 January 1997 / Accepted: 1 February 1997     

Regulation of hydrogen metabolism in Butyribacterium methylotrophicum by substrate and pH

 Exogenous H₂/CO₂ and glucose were consumed simultaneously by Butyribacterium methylotrophicum when grown under glucose-limited conditions. CO₂ reduction to acetate was coupled to H₂ consumption. The addition of either H₂ or CO₂ to glucose batch fermentation resulted in an increase in cell density, hydrogenase (H₂-consuming and -producing) activities and fatty acid production by B. methylotrophicum as compared to when N₂ was the feed gas. Hydrogenase activities appeared to be tightly regulated and were produced at higher rates during the exponential phase when CO₂ was the feed gas as compared to H₂ or N₂. The increase in H₂-consuming activity and decrease in H₂-producing activity was correlated with an increase in butyrate synthesis. H₂-consuming and ferredoxin (Fd)–NAD reductase activities increased while H₂-producing and NADH–Fd reductase activities decreased in cells grown at pH 5.5 compared to those at pH 7.0. The molar ratio of butyrate/acetate was shifted from 0.35 at pH 7.0 to 1.22 at pH 5.5. The addition of exogenous H₂ did not decrease the butyrate/acetate ratio at pH 7.0 nor at pH 5.5. The results indicated that growth pH values regulated both hydrogenase and Fd–NAD oxidoreductase activities such that, at acid pH, more intermediary electron flow was directed towards butyrate synthesis than H₂ production. Received: 22 August 1995/Received revision: 18 December 1995/Accepted: 22 January 1996     

Influence of temperature and pH on production of two bacteriocins by Leuconostoc mesenteroides subsp. mesenteroides FR52 during batch fermentation

The influence of temperature and pH on growth of Leuconostoc mesenteroides subsp. mesenteroides FR52 and production of its two bacteriocins, mesenterocin 52A and mesenterocin 52B, was studied during batch fermentation. Temperature and pH had a strong influence on the production of the two bacteriocins which was stimulated by slow growth rates. The optimal temperature was 20 °C for production of mesenterocin 52A and 25 °C for mesenterocin 52B. Optimal pH values were 5.5 and 5.0 for production of mesenterocin 52A and mesenterocin 52B respectively. Thus, by changing the culture conditions, production of one bacteriocin can be favoured in relation to the other. The relationship between growth and specific production rates of the two bacteriocins, as a function of the culture conditions, showed different kinetics of production and the presence of several peaks in the specific production rates during growth. Received: 13 February 1998 / Received revision: 27 May 1998 / Accepted: 1 June 1998     

Effects of culture conditions on β-poly(l-malate) production by Physarum polycephalum

Culture conditions for the fermentative production of β-poly(l-malate) (PMLA) by microplasmodia of Physarum polycephalum were investigated and optimized. Optimal production was achieved in the presence of CaCO₃. For 1.5% (w/v) d-glucose, 1% bactotryptone and 1% CaCO₃, a maximum of 1.7 g PMLA/l was secreted in 3 days. For 4.5% glucose and otherwise the same conditions, 2.7 g PMLA/l was produced in 6 days. The contribution of carbonate was inhibited by avidin. PMLA and biomass production were not strictly coupled: PMLA was also synthesized at the beginning of the stationary phase. At pH 5.5 PMLA production was twice that at pH 4.0, while biomass was not changed. Optimal temperatures were 24–28 °C. Received: 12 November 1998 / Received revision: 10 February 1999 / Accepted: 12 February 1999     

Biological nitrogen fixation: A key to world protein

Summary 1. Characteristics and methodology of the C₂H₂-C₂H₄ assay forin situ measurement of N₂ fixation are outlined. 2. Electron micrographic analysis of the developmental morphology of the natural soybean symbiosis and C₂H₂-C₂H₄ analysis indicate that increasing N₂-fixing activity from 12–35 days of age is accompanied by an increase in bacteroid number per cell, bacteroid number per vesicle and inclusions per bacteroid. The mole ratio of leghemoglobin to nitrogenase also increases from 50 to a relatively constant plateau of 500 to 1500 during this period. The quantitative validity of the C₂H₂-C₂H₄ assay as a measure of N₂ fixation during a complete growth cycle of soybeans on nitrogen-free medium is demonstrated by Σ (C₂H₂→C₂H₄)×28/3 values which are 75–95% of the values determined for N₂ fixed by Kjeldahl analyses. 3. A technique for the establishment of the first callus N₂-fixing symbiosis in mixed cultures ofRhizobium legume provides a defined experimental system for exploration of legume symbiosis. N₂-fixing activity is about 1% of the natural system and is influenced by exogenous auxins and cytokinins. Morphology, including infection threads and vesicle enclosed bacteroids, is similar to the nodule system. 4. N₂-fixing activity of field-grown soybeans, including varieties which differed in flowering characteristics and maturity dates, and of peanuts was determined biweekly with the C₂H₂-C₂H₄ assay. Activity extended from nodule initiation to senescence and correlated with the nitrogen demands of the plant and in most cases >90% of the N₂ fixed during the 60–70 day period of fruit formation and maturation. A logarithmic relationship between N₂-fixing activity and age, and N₂ fixed and age was demonstrated as a fundamental characteristic of these annual symbionts,i.e. log N₂ fixed =k(t−t ₀), wheret ₀ is age at activity initiation. The resultant parameters: 1) age at activity initiation, 2) calculated rate of daily increase (7–9% for soybeans and 7–10% for peanuts), 3) age at end of logarithmic phase (about 80 days for soybeans), and 4) total N₂ fixed (about 250 mg per soybean plant) are useful bases for evaluation of environmental, bacterial and host effects on N₂ fixation. Various N fertilizers applied at planting and flowering inhibited N₂ fixation of soybeans by decreasing the rate of daily increase. 5. Physical and chemical characteristics of nitrogenase, including those of crystalline Mo-Fe protein, reactions of nitrogenase, and model studies are consistent with a proposed mechanism. 6. Potential utilities of N₂ fixation research include increased food protein production via initially enhanced N₂ fixation of legumes such as soybeans and eventually extension of N₂-fixing symbioses to non-legumes and new chemistry of N₂, including the direct incorporation of aerial N₂ into important organic compounds. Contribution No. 1748.     

Continuous production of l(+)-lactic acid by Lactobacillus casei in two-stage systems

A two-stage two-stream chemostat system and a two-stage two-stream immobilized upflow packed-bed reactor system were used for the study of lactic acid production by Lactobacillus casei subsp casei. A mixing ratio of D ₁₂/D ₂= 0.5 (D = dilution rate) resulted in optimum production, making it possible to generate continuously a broth with high lactic acid concentration (48 g l⁻¹) and with a lowered overall content of initial yeast extract (5  g l⁻¹), half the concentration supplied in the one-step process. In the two-stage chemostat system, with the first stage at pH 5.5 and 37 °C and a second stage at pH 6.0, a temperature change from 40 °C to 45 °C in the second stage resulted in a 100% substrate consumption at an overall dilution rate of 0.05 h⁻¹. To increase the cell mass in the system, an adhesive strain of L. casei was used to inoculate two packed-bed reactors, which operated with two mixed feedstock streams at the optimal conditions found above. Lactic acid fermentation started after a lag period of cell growth over foam glass particles. No significant amount of free cells, compared with those adhering to the glass foam, was observed during continuous lactic acid production. The extreme values, 57.5 g l⁻¹ for lactic acid concentration and 9.72 g l⁻¹ h⁻¹ for the volumetric productivity, in upflow packed-bed reactors were higher than those obtained for free cells (48 g l⁻¹  and 2.42 g l⁻¹ h⁻¹) respectively and the highest overall l(+)-lactic acid purity (96.8%) was obtained in the two-chemostat system as compared with the immobilized-cell reactors (93%). Received: 4 December 1997 / Received revision: 23 February 1998 / Accepted: 14 March 1998     

Physiology and kinetics of trimethylamine conversion by two methylotrophic strains in continuous cultivation systems

The change of dilution rate (D) on both Methylophilus methylotrophus NCIMB11348 and Methylobacterium sp. RXM CCMI908 growing in trimethylamine (TMA) chemostat cultures was studied in order to assess their ability to remove odours in fish processing plants. M. methylotrophus NCIMB11348 was grown at dilution rates of 0.012–0.084 h⁻¹ and the biomass level slightly increased up to values of D around 0.07 h⁻¹. The maximum cell production rate was obtained at 0.07 h⁻¹ corresponding to a maximum conversion of carbon into cell mass (35%). The highest rate of TMA consumption was 3.04 mM h⁻¹ occurring at D=0.076 h⁻¹. Methylobacterium sp. RXM CCMI908 was grown under similar conditions. The biomass increased in a more steep manner up to values of D around 0.06 h⁻¹. The maximum cell production rate (0.058 g l⁻¹h⁻¹) was obtained in the region close to 0.06 h⁻¹ where a maximum conversion of the carbon into cell mass (40%) was observed. The maximum TMA consumption was 2.33 mM h⁻¹ at D=0.075 h⁻¹. The flux of carbon from TMA towards cell synthesis and carbon dioxide in both strains indicates that the cell is not excreting products but directing most of the carbon source to growth. Carbon recovery levels of approximately 100% show that the cultures are carbon-limited. Values for theoretical maximum yields and maintenance coefficients are presented along with a kinetic assessment based on the determination of the substrate saturation constant and maximum growth rate for each organism. Received: 25 February 1999 / Received revision: 14 May 1999 / Accepted: 17 May 1999     

A γ-TIP cross-reacting protein is abundant in the cortex of soybean N2-fixing nodules

The distribution and abundance of tonoplast intrinsic protein (γ-TIP), a putative aquaporin which is abundant in the tonoplast of osmoregulated pulvinus motor cells, were determined in nodules of Glycine max (L.) Merr. using chemical fixation and immunolocalization. This protein was highly expressed in the tonoplast of the inner cortical cells of the nodules but poorly expressed in the vascular transfer cells and in infected cells. It is concluded that the differentiation of the inner cortical cells of the nodules like that of pulvinus motor cells, is accompanied by an increased expression of γ-TIP. This result is consistent with our previous hypothesis that a reversible exchange of intercellular water by the inner cortical cells plays a role in the regulation of nodule conductance to O₂ diffusion, and hence in subsequent N₂-fixing activity. Received: 7 February 1998 / Accepted: 22 May 1998     

[Pt(dien)]2+ migrates intramolecularly from methionine S to imidazole Nz 2 in the peptides H-His-Gly-Met-OH and Ac-His-Ala-Ala-Ala-Met-NHPh

The pH- and time-dependent reaction of [Pt(dien)(H₂O)]²⁺ with the methionine- and histidine-containing peptides H-His-Gly-Met-OH and Ac-His-Ala-Ala-Ala-Met-NHPh at 313 K has been investigated by HPLC and NMR spectroscopy. For both peptides, initial relatively rapid formation of the kinetically favoured methionine S-bound complex is followed by slow intramolecular migration of the [Pt(dien)]²⁺ fragment to imidazole N_{ε 2} (or, in the case of H-His-Gly-Met-OH, to a much lesser extent to the competing imidazole N_{δ 1}) of the histidine side chain over a period of 500 h. Time-dependent studies for the pentapeptide at pH 8.0 demonstrate that this isomerization can take place by either direct S→N_{ε 2} migration or by a two-step mechanism involving initial N_{ε 2} coordination of a second [Pt(dien)]²⁺ fragment and subsequent cleavage of the orginal Pt-S bond in the resulting dinuclear complex. The rate of κS/κN _{ε 2} isomerization is markedly reduced on lowering the pH to 5.1. Received: 26 February 1999 / Accepted: 14 April 1999     

Autolysis of Escherichia coli and Bacillus subtilis cells in low gravity

The role of gravity in the autolysis of Bacillus subtilis and Escherichia coli was studied by growing cells on Earth and in microgravity on Space Station Mir. Autolysis analysis was completed by examining the death phase or exponential decay of cells for approximately 4 months following the stationary phase. Consistent with published findings, the stationary-phase cell population was 170% and 90% higher in flight B. subtilis and E. coli cultures, respectively, than in ground cultures. Although both flight autolysis curves began at higher cell densities than control curves, the rate of autolysis in flight cultures was identical to that of their respective ground control rates. Received: 3 December 1998 / Received revision: 23 February 1999 / Accepted: 14 March 1999     

Physiological aspects of continuous lactic acid fermentations at high dilution rates

The effects of the substrate conditions on the volumetric productivity of Lactobacillus helveticus at different cell densities up to 60 g l⁻¹ in a continuous stirred-tank reactor with microfiltration to retain the biomass were investigated. At low dilution rates, D, the steady-state volumetric productivity, r _p, gradually increased to a maximum at D = 1.2–1.5 h⁻¹, because of reduced product inhibition. At higher D values, r _p unexpectedly decreased, although the substrate conditions further improved. The maxima of r _p at different cell densities coincided with a critical specific substrate utilization rate beyond which the cell metabolism seems to be controlled through a catabolic modulator factor, and r _p decreases. Received: 8 September 1997 / Received last revision: 31 December 1997 / Accepted: 2 January 1998     

Simultaneous utilization of glucose and D-xylose by Candida shehatae in a chemostat

The kinetics of biomass formation, D-xylose utilization, and mixed substrate utilization were determined in a chemostat using the yeast Candida shehatae. The maximum growth rate of C. shehatae grown aerobically on D-xylose was 0.42 h⁻¹ and the Monod constant, K _s, was 0.06 g L⁻¹. The biomass yield, Y _{X/S}, ranged from 0.40 to 0.50 g g⁻¹ over a dilution rate range of 0.2–0.3 h⁻¹, when C. shehatae was grown on pure D-xylose. Mixtures of D-xylose and glucose (∼1 : 1) were simultaneously utilized over a dilution rate from 0.15 to 0.35 h⁻¹ at pH 3.5 and 4.5, but pH 3.5 reduced μ_max and reduced the dilution rate range over which D-xylose was utilized in the presence of glucose. At pH 4.5, μ_max was not reduced with the mixed sugar feed and the overall or lumped K _s value was not significantly increased (0.058 g L⁻¹ vs 0.06 g L⁻¹), when compared to a pure D-xylose feed. Kinetic data indicate that C. shehatae is an excellent candidate for chemostat production of value added products from renewable carbon sources, since simultaneous mixed substrate utilization was observed over a wide range of growth rates on a 1 : 1 mixture of glucose and D-xylose. Received 21 August 1997/ Accepted in revised form 28 May 1998