Microbial acetate oxidation in horizontal rotating tubular bioreactor

The aim of this work was to investigate the possibility of conducting a continuous aerobic bioprocess in a horizontal rotating tubular bioreactor (HRTB). Aerobic oxidation of acetate by the action of a mixed microbial culture was chosen as a model process. The microbial culture was not only grown in a suspension but also in the form of a biofilm on the interior surface of HRTB. Efficiency of the bioprocess was monitored by determination of the acetate concentration and chemical oxygen demand (COD). While acetate inlet concentration and feeding rate influenced efficiency of acetate oxidation, the bioreactor rotation speed did not influence the bioprocess dynamics significantly. Gradients of acetate concentration and pH along HRTB were more pronounced at lower feeding rates. Volumetric load of acetate was proved to be the most significant parameter. High volumetric loads (above 2 g acetate l⁻¹ h⁻¹) gave poor acetate oxidation efficiency (only 17 to 50%). When the volumetric load was in the range of 0.60–1.75 g acetate l⁻¹ h⁻¹, acetate oxidation efficiency was 50–75%. At lower volumetric loads (0.14–0.58 g acetate l⁻¹ h⁻¹), complete acetate consumption was achieved. On the basis of the obtained results, it can be concluded that HRTB is suitable for conducting aerobic continuous bioprocesses.     

Fermentative bioconversion in a horizontal rotating tubular bioreactor

The performance of a horizontal rotating tubular bioreactor (HRTB) was investigated with a biological system under non-sterile conditions. A spontaneously developed microbial culture was cultivated in a simple glucose/yeast extract medium. A fermentative bioconversion was examined by different combinations of process parameters (bioreactor rotation speed 5–30 min⁻¹ and medium inflow rate 1–10 l h⁻¹). Bioconversion dynamics in HRTB was monitored by withdrawing the samples from five positions along the bioreactor. Investigation in HRTB showed a rapid and an efficient glucose conversion into different products of metabolism. Glucose consumption rate along the HRTB depended on medium inflow rate, while bioreactor rotation speed did not have a significant influence. Complete glucose conversion in HRTB was observed at inflow rates of up to 6.5 l h⁻¹. The pH gradient along the HRTB was detected at higher medium inflow rates (6.5 and 10 l h⁻¹), but did not significantly influence substrate conversion efficiency. A discussion of its potential use and a comparison of HRTB with other bioreactors are also presented.     

Mathematical modeling of Fe(II), Cu(II), Ni(II) and Zn(II) removal in a horizontal rotating tubular bioreactor

Industrial wastewaters polluted with toxic heavy metals are serious ecological and environmental problem. Therefore, in this study multi-heavy metals (Fe²⁺, Cu²⁺, Ni²⁺ and Zn²⁺) removal process with mixed microbial culture was examined in the horizontal rotating tubular bioreactor (HRTB) by different combinations of process parameters. Hydrodynamic conditions and biomass sorption capacity have main impact on the removal efficiency of heavy metals: Fe²⁺ 95.5–79.0%, Ni²⁺ 92.7–54.8%, Cu²⁺ 87.7–54.9% and Zn²⁺ 81.8–38.1%, respectively. On the basis of experimental results, integral mathematical model of removal heavy metals in the HRTB was established. It combines hydrodynamics (mixing), mass transfer and kinetics to define bioprocess conduction in the HRTB. Mixing in the HRTB was described by structured cascade model and metal ion removal by two combined diffusion–adsorption models, respectively. For Langmuir model, average variances between experimental and simulated concentrations of metal ions were in the range of 1.22–10.99 × 10⁻³ and for the Freundlich model 0.12–3.98 × 10⁻³, respectively. On the basis of previous facts, it is clear that developed integral bioprocess model with Freundlich model is more efficient in the prediction of concentration of metal ions in the HRTB. Furthermore, the results obtained also pointed out that the established model is at the same time accurate and robust and therefore it has great potential for use in the scale-up procedure.     

Production of 1,3-Propanediol by Clostridium butyricum VPI 3266 in continuous cultures with high yield and productivity

The effects of dilution rate and substrate feed concentration on continuous glycerol fermentation by Clostridium butyricum VPI 3266, a natural 1,3-propanediol producer, were evaluated in this work. A high and constant 1,3-propanediol yield (around 0.65 mol/mol), close to the theoretical value, was obtained irrespective of substrate feed concentration or dilution rate. Improvement of 1,3-propanediol volumetric productivity was achieved by increasing the dilution rate, at a fixed feed substrate concentration of 30, 60 or 70 g l⁻¹. Higher 1,3-propanediol final concentrations and volumetric productivities were also obtained when glycerol feed concentration was increased from 30 to 60 g l⁻¹, at D=0.05–0.3 h⁻¹, and from 60–70 g l⁻¹, at D=0.05 and 0.1 h⁻¹·30 g l⁻¹ of 1,3-propanediol and the highest reported value of productivity, 10.3 g l⁻¹ h⁻¹, was achieved at D=0.30 h⁻¹ and 60 g l⁻¹ of feed glycerol. A switch to an acetate/butyrate ratio higher than one was observed for 60 g l⁻¹ of feed glycerol and a dilution rate higher than 0.10 h⁻¹; moreover, at D=0.30 h⁻¹ 3-hydroxypropionaldehyde accumulation was observed for the first time in the fermentation broth of C. butyricum.     

Influence of temperature on process efficiency and microbial community response during the biological removal of chlorophenols in a packed-bed bioreactor

Two reactors, initially operated at 14 and 23±1°C (RA and RB, respectively), were inoculated with a bacterial consortium enriched and acclimatized to the respective temperatures over 4 months. The biofilms, formed in the reactors, were studied using scanning electron microscopy, cultivation of the biofilm microflora, and physiological analysis of the isolates. Two bacteria able to mineralize chlorophenols under a large range of temperature (10–30°C) were isolated from the biofilm communities of reactors RA and RB and characterized as Alcaligenaceae bacterium R14C4 and Cupriavidus basilensis R25C6, respectively. When temperature was decreased by 10°C, the chlorophenols removal capacity was reduced from 51.6 to 22.8 mg l⁻¹ h⁻¹ in bioreactor RA (from 14 to 4°C) and from 59.3 to 34.7 mg l⁻¹ h⁻¹ in bioreactor RB (from 23±1 to 14°C). Fluorescence in situ hybridization (FISH) of the biofilm communities showed that, in all temperatures tested, the β-proteobacteria were the major bacterial community (35–47%) followed by the γ-proteobacteria (12.0–6.5%). When the temperature was decreased by 10°C, the proportions of γ-proteobacteria and Pseudomonas species increased significantly in both microbial communities.     

Biodegradation of nonylphenol in a continuous bioreactor at low temperatures and effects on the microbial population

A packed-bed bioreactor inoculated with a mixed culture obtained from a contaminated site was used to continuously treat a saturated solution of nonylphenol. The reactor was operated at feeding rates of 13–112 ml h⁻¹ and temperatures of 5.5, 10, and 15°C. Optimal bioreactor performance was achieved at 10°C and at a feeding rate of 84 ml h⁻¹ (with a removal rate of 43 mg l⁻¹ day⁻¹ of nonylphenol). No endocrine activity was observed in the effluent of the bioreactor at any of the temperatures tested, and the only metabolic products found were branched carboxylic acids and alkanes (lacking an aromatic ring). The study of the microbial populations in the biofilm at the three temperatures tested using fluorescence in situ hybridization showed that all the bacterial species that could be identified belonged to the phylum Proteobacteria. The most abundant class identified at all three temperatures was β-Proteobacteria. The proportions of bacteria that bound to the specific probes among the total population, identified with the bacterial probe EUB338MIX, were 60, 43, and 24% at 15, 10, and 5.5°C, respectively.     

Assessment of Nitrification Potential in Ground Water Using Short Term, Single-Well Injection Experiments

Nitrification was measured within a sand and gravel aquifer on Cape Cod, MA, using a series of single-well injection tests. The aquifer contained a wastewater-derived contaminant plume, the core of which was anoxic and contained ammonium. The study was conducted near the downgradient end of the ammonium zone, which was characterized by inversely trending vertical gradients of oxygen (270 to 0 μM) and ammonium (19 to 625 μM) and appeared to be a potentially active zone for nitrification. The tests were conducted by injecting a tracer solution (ambient ground water + added constituents) into selected locations within the gradients using multilevel samplers. After injection, the tracers moved by natural ground water flow and were sampled with time from the injection port. Rates of nitrification were determined from changes in nitrate and nitrite concentration relative to bromide. Initial tests were conducted with ¹⁵N-enriched ammonium; subsequent tests examined the effect of adding ammonium, nitrite, or oxygen above background concentrations and of adding difluoromethane, a nitrification inhibitor. In situ net nitrate production exceeded net nitrite production by 3- to 6- fold and production rates of both decreased in the presence of difluoromethane. Nitrification rates were 0.02–0.28 μmol (L aquifer)⁻¹ h⁻¹ with in situ oxygen concentrations and up to 0.81 μmol (L aquifer)⁻¹ h⁻¹ with non-limiting substrate concentrations. Geochemical considerations indicate that the rates derived from single-well injection tests yielded overestimates of in situ rates, possibly because the injections promoted small-scale mixing within a transport-limited reaction zone. Nonetheless, these tests were useful for characterizing ground water nitrification in situ and for comparing potential rates of activity when the tracer cloud included non-limiting ammonium and oxygen concentrations.     

Citric acid production from sucrose using a recombinant strain of the yeast Yarrowia lipolytica

The yeast Yarrowia lipolytica is able to secrete high amounts of several organic acids under conditions of growth limitation and carbon source excess. Here we report the production of citric acid (CA) in a fed-batch cultivation process on sucrose using the recombinant Y. lipolytica strain H222-S4(p67ICL1) T5, harbouring the invertase encoding ScSUC2 gene of Saccharomyces cerevisiae under the inducible XPR2 promoter control and multiple ICL1 copies (10–15). The pH-dependent expression of invertase was low at pH 5.0 and was identified as limiting factor of the CA-production bioprocess. The invertase expression was sufficiently enhanced at pH 6.0–6.8 and resulted in production of 127–140 g l⁻¹ CA with a yield Y _CA of 0.75–0.82 g g⁻¹, whereas at pH 5.0, 87 g l ⁻¹ with a yield Y _CA of 0.51 gg⁻¹ were produced. The CA-productivity Q _CA increased from 0.40 g l ⁻¹ h⁻¹ at pH 5.0 up to 0.73 g l ⁻¹ h⁻¹ at pH 6.8. Accumulation of glucose and fructose at high invertase expression level at pH 6.8 indicated a limitation of CA production by sugar uptake. The strain H222-S4(p67ICL1) T5 also exhibited a gene–dose-dependent high isocitrate lyase expression resulting in strong reduction (<5%) of isocitric acid, a by-product during CA production.     

Microbial community of acetate utilizing denitrifiers in aerobic granules

Nitrite accumulates during biological denitrification processes when carbon sources are insufficient. Acetate, methanol, and ethanol were investigated as supplementary carbon sources in the nitrite denitrification process using biogranules. Without supplementary external electron donors (control), the biogranules degraded 200 mg l⁻¹ nitrite at a rate of 0.27 mg NO₂–N g⁻¹ VSS h⁻¹. Notably, 1,500 mg l⁻¹ acetate and 700 mg l⁻¹ methanol or ethanol enhanced denitrification rates for 200 mg l⁻¹ nitrite at 2.07, 1.20, and 1.60 mg NO₂–N g⁻¹ VSS h⁻¹, respectively; these rates were significantly higher than that of the control. The sodium dodecyl sulfate polyacrylamide gel electrophoresis of the nitrite reductase (NiR) enzyme identified three prominent bands with molecular weights of 37–41 kDa. A linear correlation existed between incremental denitrification rates and incremental activity of the NiR enzyme. The NiR enzyme activity was enhanced by the supplementary carbon sources, thereby increasing the nitrite denitrification rate. The capacity of supplementary carbon source on enhancing NiR enzyme activity follows: methanol > acetate > ethanol on molar basis or acetate > ethanol > methanol on an added weight basis.     

Glycolytic pathway and hydrogen yield studies of the extreme thermophile <Emphasis Type="Italic">Caldicellulosiruptor saccharolyticus</Emphasis>

NMR analysis of ¹³C-labelling patterns showed that the Embden–Meyerhof (EM) pathway is the main route for glycolysis in the extreme thermophile Caldicellulosiruptor saccharolyticus. Glucose fermentation via the EM pathway to acetate results in a theoretical yield of 4 mol of hydrogen and 2 mol of acetate per mole of glucose. Previously, approximately 70% of the theoretical maximum hydrogen yield has been reached in batch fermentations. In this study, hydrogen and acetate yields have been determined at different dilution rates during continuous cultivation. The yields were dependent on the growth rate. The highest hydrogen yields of 82 to 90% of theoretical maximum (3.3 to 3.6 mol H₂ per mol glucose) were obtained at low growth rates when a relatively larger part of the consumed glucose is used for maintenance. The hydrogen productivity showed the opposite effect. Both the specific and the volumetric hydrogen production rates were highest at the higher growth rates, reaching values of respectively 30 mmol g⁻¹ h⁻¹ and 20 mmol l⁻¹ h⁻¹. An industrial process for biohydrogen production will require a bioreactor design, which enables an optimal mix of high productivity and high yield.     

Nitrite-driven anaerobic ATP synthesis in barley and rice root mitochondria

Mitochondria isolated from the roots of barley (Hordeum vulgare L.) and rice (Oryza sativa L.) seedlings were capable of oxidizing external NADH and NADPH anaerobically in the presence of nitrite. The reaction was linked to ATP synthesis and nitric oxide (NO) was a measurable product. The rates of NADH and NADPH oxidation were in the range of 12–16 nmol min⁻¹ mg⁻¹ protein for both species. The anaerobic ATP synthesis rate was 7–9 nmol min⁻¹ mg⁻¹ protein for barley and 15–17 nmol min⁻¹ mg⁻¹ protein for rice. The rates are of the same order of magnitude as glycolytic ATP production during anoxia and about 3–5% of the aerobic mitochondrial ATP synthesis rate. NADH/NADPH oxidation and ATP synthesis were sensitive to the mitochondrial inhibitors myxothiazol, oligomycin, diphenyleneiodonium and insensitive to rotenone and antimycin A. The uncoupler FCCP completely eliminated ATP production. Succinate was also capable of driving ATP synthesis. We conclude that plant mitochondria, under anaerobic conditions, have a capacity to use nitrite as an electron acceptor to oxidize cytosolic NADH/NADPH and generate ATP.     

Rheology and Shear Stress of Centaurea calcitrapa Cell Suspension Cultures Grown in Bioreactor

Centaurea calcitrapa suspension cultures were grown either in Erlenmeyer flasks or in a mechanically stirred bioreactor. Its rheological behaviour, when fitted to the Oswald–de Waele model (power law), showed pseudoplastic characteristics in both cases. The flow behaviour index (n) decreased over the course of a growth cycle and the consistency index (K) increased, reached a value of 1.81 N sⁿ m⁻² run on 2 l bioreactor. Bioreactor cultivation of C. calcitrapa cells at different agitation rates (30, 60, 100 and 250 rpm), highlighted the influence of shear forces on cell viability loss (90–34%) and phenol accumulation (74–140 μg l⁻¹), due to increased stirring speeds. Analysis of these results suggests that this cell line is shear-sensitive. An empirical exponential correlation was defined between apparent viscosity and biomass concentration, under the studied conditions, giving the possibility to estimate the prevailing broth regime and to optimize bioreactor design. Revisions requested 10 October 2005; Revisions received 19 December 2005     

Anaerobic co-reduction of chromate and nitrate by bacterial cultures of Staphylococcus epidermidis L-02

Industrial wastewater is often polluted by Cr(VI) compounds, presenting a serious environmental problem. This study addresses the removal of toxic, mutagenic Cr(VI) by means of microbial reduction to Cr(III), which can then be precipitated as oxides or hydroxides and extracted from the aquatic system. A strain of Staphylococcus epidermidis L-02 was isolated from a bacterial consortium used for the remediation of a chromate-contaminated constructed wetland system. This strain reduced Cr(VI) by using pyruvate as an electron donor under anaerobic conditions. The aims of the present study were to investigate the specific rate of Cr(VI) reduction by the strain L-02, the effects of chromate and nitrate (available as electron acceptors) on the strain, and the interference of chromate and nitrate reduction processes. The presence of Cr(VI) decreased the growth rate of the bacterium. Chromate and nitrate reduction did not occur under sterile conditions but was observed during tests with the strain L-02. The presence of nitrate increased both the specific Cr(VI) reduction rate and the cell number. Under denitrifying conditions, Cr(VI) reduction was not inhibited by nitrite, which was produced during nitrate reduction. The average specific rate of chromate reduction reached 4.4 μmol Cr 10¹⁰ cells⁻¹ h⁻¹, but was only 2.0 μmol Cr 10¹⁰ cells⁻¹ h⁻¹ at 20 °C. The maximum specific rate was as high as 8.8–9.8 μmol Cr 10¹⁰ cells⁻¹ h⁻¹. The role of nitrate in chromate reduction is discussed.     

Adaptation of a <Emphasis Type="Italic">Mycobacterium</Emphasis> strain to phenanthrene degradation in a biphasic culture system: influence on interfacial area and droplet size

A phenanthrene-degrading Mycobacterium sp. strain 6PY1 was grown in an aqueous/organic biphasic culture system with phenanthrene as sole carbon source. Its capacity of degradation was studied during sequential inoculum enrichments, reaching complete phenanthrene degradation at a maximim rate of 7 mg l⁻¹ h⁻¹. Water–oil emulsions and biofilm formation were observed in biphasic cultures after four successive enrichments. The factors influencing interfacial area in the emulsions were: the initial phenanthrene concentration, the initial inoculum size, and the silicone oil volume fraction. The results showed that the interfacial area was mainly dependent on the silicone oil/mineral salts medium ratio and the inoculum size.     

Production of raw-starch-hydrolysing α-amylase from the newly isolated Geobacillus thermodenitrificans HRO10

An extracellular raw-starch-digesting α-amylase was isolated from Geobacillus thermodenitrificans HRO10. The culture conditions for the production of α-amylase by G. thermodenitrificans HRO10 was optimized in 1.2–l bioreactor using full 2⁴ and 3² factorial designs. From the optimal reaction conditions, a model (Y = − 594.206 − 0.178T² − 8.448pH² + 6.020TpH − 0.005T²pH²) was predicted, which was then used for α-amylase production. In the bioreactor studies, the enzyme yield under optimized conditions (pH 7.1, 49°C) was 30.20 U/ml, a 51% improvement over the results (19.97 U/ml) obtained when the traditional one-factor-at-a-time method was employed. This α-amylase does not require extraneous calcium ions for activity, which may be a commercially important observation.     

Particle size effects in bioleaching of pyrite by acidophilic thermophile Sulfolobus metallicus (BC)

The effect of mineral particle size on the bioleaching of pyrite by the acidophilic thermophile Sulfolobus metallicus was investigated in a batch bioreactor. Decreasing the particle size from a mean diameter of 202 micron (size fraction: 150–180 micron) to a mean diameter of 42.5 micron (size fraction: 25–45 micron) enhanced the bioleaching rate from 0.05 kg m⁻³ h⁻¹ to 0.098 kg m⁻³ h⁻¹. The particle size distribution of the mineral in this range did not influence the morphology and growth kinetics of the cells. The values of specific growth rate (μ) and yield factor (Y) were 0.018–0.025 h⁻¹ and 0.67 × 10¹¹–1.45 × 10¹¹ cells (g iron)⁻¹, respectively. Decreasing the particle size of the mineral to a mean diameter of 6.40 micron (size fraction <25 micron) adversely influenced the activity of the cells. The presence of fine particles apparently damaged the structure of the cells, resulting in their inability to oxidise pyrite. Received: 11 December 1998 / Accepted: 9 April 1999     

Large-scale cultivation of adventitious roots of Echinacea purpurea in airlift bioreactors for the production of chichoric acid, chlorogenic acid and caftaric acid

Adventitious roots of Echinacea purpurea were cultured in airlift bioreactors (20 l, 500 l balloon-type, bubble bioreactors and 1,000 l drum-type bubble bioreactor) using Murashige and Skoog (MS) medium with 2 mg indole butyric acid l⁻¹ and 50 g sucrose l⁻¹ for the production of chichoric acid, chlorogenic acid and caftaric acid. In the 20 l bioreactor (containing 14 l MS medium) a maximum yield of 11 g dry biomass l⁻¹ was achieved after 60 days. However, the amount of total phenolics (57 mg g⁻¹ DW), flavonoids (34 mg g⁻¹ DW) and caffeic acid derivatives (38 mg g⁻¹ DW) were highest after 50 days. Based on these studies, pilot-scale cultures were established and 3.6 kg and 5.1 kg dry biomass were achieved in the 500 l and 1,000 l bioreactors, respectively. The accumulation of 5 mg chlorogenic acid g⁻¹ DW, 22 mg chichoric acid g⁻¹ DW and 4 mg caftaric acids g⁻¹ DW were achieved with adventitious roots grown in 1,000 l bioreactors.     

Monoclonal antibody production: viability improvement of RC1 hybridoma cell in different types of bioreactor

The study was done to improve the viability of the RC1 hybridoma cell in order to produce more amount of monoclonal antibody (mAb). By using the optimized media, the cell had been cultured in two bioreactor systems which were the MiniPerm and Stirred Tank bioreactor (ST bioreactor), and the results were compared to the one obtained by using the T-Flask bioreactor which was used as a standard. The results showed that the ST bioreactor was able to improve the viability of the cell to the value of 91.8% which was a little bit better than the one obtained by the MiniPerm bioreactor (88.6%) and far better than that of achieved by the T-Flask bioreactor (76.4%). This was well correlated with the good growth performance of the cell in the ST bioreactor with the specific growth rate (μ) value of 0.0289 h⁻¹ followed by MiniPerm bioreactor with the value of 0.0243 h⁻¹ and then the T-Flask with the value of 0.0151 h⁻¹. The low value of doubling time (t _d ) obtained in the ST bioreactor (24 h) compared to the one obtained in the MiniPerm (29 h) and T-Flask bioreactor (46 h) had also contributed to the higher value of cell viability. As a result a higher concentration of mAb was able to be produced by the ST bioreactor (0.42 g l⁻¹) compared to that of the MiniPerm (0.37 g l⁻¹) and T-Flask bioreactor (0.23 g l⁻¹).     

Continuous pullulan fermentation in a biofilm reactor

Biofilm is a natural form of cell immobilization in which microorganisms attach onto solid support. In this study, a pigment-reduced pullulan-producing strain, Aureobasidium pullulans (ATCC 201253), was used for continuous pullulan fermentation in a plastic composite support (PCS) biofilm reactor. Optimal conditions for the continuous pullulan production were determined by evaluating the effects of the feeding medium with various concentrations of ammonium sulfate and sucrose and dilution rate. Pullulan concentration and production rate reached maximum (8.3 g/l and 1.33 g/l/h) when 15 g/l of sucrose, 0.9 g/l of ammonium sulfate, and 0.4 g/l of yeast extract were applied in the medium, and the dilution rate was at 0.16 h⁻¹. The purity of produced pullulan was 93.0%. The ratio of hyphal cells of A. pullulans increased when it was grown on the PCS shaft. Overall, the increased pullulan productivity can be achieved through biomass retention by using PCS biofilm reactor.     

Quasi steady state growth of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> in glucose-limited acceleration stat (A-stat) cultures

Quasi steady state growth of Lactococcus lactis IL 1403 was studied in glucose-limited A-stat cultivation experiments with acceleration rates (a) from 0.003 to 0.06 h⁻² after initial stabilization of the cultures in chemostat at D = 0.2–0.3 h⁻¹. It was shown that the high limit of quasi steady state growth rate depended on the acceleration rate used—at an acceleration rate 0.003 h⁻² the quasi steady state growth was observed until μ _crit = 0.59 h⁻¹, which is also the μ _max value for the culture. Lower values of μ _crit were observed at higher acceleration rates. The steady state growth of bacteria stabilized at dilution rate 0.2 h⁻¹ was immediately disrupted after initiating acceleration at the highest acceleration rate studied—0.06 h⁻². Observation was made that differences [Δ(μ − D)] of the specific growth rates from pre-programmed dilution rates were the lowest using an acceleration rate of 0.003 h⁻² (< 4% of preset changing growth rate). The adaptability of cells to follow preprogrammed growth rate was found to decrease with increasing dilution rate—it was shown that lower acceleration rates should be applied at higher growth rates to maintain the culture in the quasi steady state. The critical specific growth rate and the biomass yields based on glucose consumption were higher if the medium contained S ₀ = 5 g L⁻¹ glucose instead of S ₀ = 10 g L⁻¹. It was assumed that this was due to the inhibitory effect of lactate accumulating at higher concentrations in the latter cultures. Parallel A-stat experiments at the same acceleration and dilution rates showed good reproducibility—Δ(μ − D) was less than 5%, standard deviations of biomass yields per ATP produced (Y _ATP), and biomass yields per glucose consumed (Y _XS) were less than 15%.