Unstructured models for growth and lactic acid production during two-stage continuous cultures of Lactobacillus helveticus

During lactic acid fermentation, seed culture is usually carried out without pH control, while culture is carried out at pH controlled at the optimal value to overcome inhibitory effects. The Luedeking–Piret expression was therefore previously modified by introducing additional terms involving the undissociated form of the lactic acid, the main inhibitory species, in case of batch cultures without pH control or involving the residual lactose concentration to account for the carbon substrate limitation, responsible for cessation of production during batch cultures of Lactobacillus helveticus at controlled pH. Both expressions were also merged to deduce a generalized model. Both models, as well as the Luedeking–Piret model, were developed to describe continuous two-stage culture of L. helveticus. By considering the parameter values obtained from the fitting of batch culture data, both modified Luedeking–Piret models showed interesting predictive potential. Indeed, some rather reliable predictive calculated values were recorded in both stages; the residual standard deviations were 0.5 and less than 8.8 for the biomass and the product concentrations at steady-state in the culture stage (second stage). The optimization of the parameters for growth- and non-growth-associated parameters improved the fitting in the culture stage, leading to residual standard deviations below 2.6 for lactic acid concentrations at steady-state.     

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in continuous cultures

The oxidation and growth kinetics of ferrous iron with Thiobacillus ferrooxidans in continuous cultures was examined at several total iron concentrations. On-line off-gas analyses of O₂ and CO₂ were used to measure the oxygen and carbon dioxide consumption rates in the culture. Off-line respiration measurements in a biological oxygen monitor (BOM) were used to measure directly the maximum specific oxygen consumption rate, qO₂,max, of cells grown in continuous culture. It was shown that these reproducibly measured values of qO₂,max vary with the dilution rate. The biomass-specific oxygen consumption rate, qO₂, is dependent on the ratio of the ferric and ferrous iron concentrations in the culture. The oxidation kinetics was accurately described with a rate equation for competitive ferric iron inhibition, using the value of qO₂,max measured in the BOM. Accordingly, only the kinetic constant Ks/K _i needed to be fitted from the measurements. A new method was introduced to determine the steady-state kinetics of a cell suspension in a batch culture that only takes a few hours. The batch culture was set up by terminating the feeding of a continuous culture at its steady state. The kinetic constant K _s/K _i determined in this batch culture agreed with the value determined in continuous cultures at various steady states. Received: 8 February 1999 / Accepted: 17 February 1999     

Continuous culture of Pseudomonas fluorescens with sodium maleate as a carbon source

Pseudomonas fluorescens (ATCC 11150) was grown in batch and continuous culture in minimal media with sodium maleate as growth-limiting sole organic carbon source. Growth was followed by turbidity and dry weight measurements. Gross composition of washed cells (relative amounts of protein, lipid, RNA, and DNA) and the distribution of amino acids in protein hydrolyses of the cells were determined for cells grown in continuous culture at various dilution rates. Extracellular concentrations of the original carbon source and a number of metabolites were monitored by a total carbon analysis, ion exchange chromatography, and ultraviolet-visible scans of cell-free supernatants and chromatographic fractions, thereof. Substrate inhibition by maleate was a major factor in the growth kinetics of both batch and continuous cultures. Excessive maleate concentration caused instability in continuous cultures. By appropriate operation, much higher specific growth rates (0.305/hr) could ultimately be achieved in continuous culture compared to batch culture (0.174/hr). Adaptation was responsible for only part of the differences between batch and continuous cultures; the differing distribution of metabolites were also major factors.     

Toxicity evaluation of 2-hydroxybiphenyl and other compounds involved in studies of fossil fuels biodesulphurisation

The acute toxicity of some compounds used in fossil fuels biodesulphurisation studies, on the respiration activity, was evaluated by Gordonia alkanivorans and Rhodococcus erythropolis. Moreover, the effect of 2-hydroxybiphenyl on cell growth of both strains was also determined, using batch (chronic bioassays) and continuous cultures. The IC₅₀ values obtained showed the toxicity of all the compounds tested to both strains, specially the high toxicity of 2-HBP. These results were confirmed by the chronic toxicity data. The toxicity data sets highlight for a higher sensitivity to the toxicant by the strain presenting a lower growth rate, due to a lower cells number in contact with the toxicant. Thus, microorganisms exhibiting faster generation times could be more resistant to 2-HBP accumulation during a BDS process. The physiological response of both strains to 2-HBP pulse in a steady-state continuous culture shows their potential to be used in a future fossil fuel BDS process.     

A comparative study using the dual staining flow cytometric protocol applied to Lactobacillus rhamnosus and Bacillus licheniformis batch cultures

During microbial bioprocesses development, it is crucial monitoring cell stress response as such information determines the process efficiency.Multi-parameter flow cytometry coupled with fluorescent stains [PI and DiOC₆(3) (3,3′-dihexylocarbocyanine iodide)] was used to monitor bacterial batch cultures. It was observed that the most significant differences in the cell physiological responses of these microorganisms were observed at the early stationary phase. While Bacillus licheniformis batch culture displayed 91% of healthy cells, 1.3% of cells with depolarised cytoplasmic membrane and 8% of cells with both depolarised and permeabilised cytoplasmic membrane as the culture reached this phase, Lactobacillus rhamnosus batch culture displayed 66% of healthy cells, 29% of cells with depolarised cytoplasmic membrane and 5% of depolarised and permeabilised cytoplasmic membrane, at that stage. However, at the late stationary phase, both bacterial cultures showed a high proportion of polarised healthy cells, suggesting that the microorganisms could be using storage materials as a survival strategy.It was found that the double staining protocol using PI and DiOC₆(3) could be used to monitor cell physiological response of both bacterial batch cultures and provided important physiological information at the individual cell level that is difficult to obtain in any other way, which can be used to enhance bioprocesses efficiency.     

Growth and enzymological characteristics of a pink-pigmented facultative methylotrophMethylobacterium sp. MB1

Growth characteristics of batch and continuous cultures of the pink facultative methylotrophMethylobacterium sp. MB1 were determined. The response of a chemostat culture to a pulse increase of methanol concentration was studied. Malate, succinate and oxaloacetate additions to the methanol-supplemented medium decreased batch culture growth inhibition by methanol. The carotenoid content in cells grown in a chemostat decreased with increasing growth rate. The key enzyme activities of C₁-metabolism were measured in a chemostat culture at different dilution rates.     

Improved tolerance to bile salts of aggregated Bifidobacterium longum produced during continuous culture with immobilized cells

The effect of cell immobilization and continuous culture was studied on selected physiological and technological characteristics of Bifidobacterium longum NCC2705 cultivated for 20 days in a two stage continuous fermentation system. Continuous immobilized cell (IC) cultures with and without glucose limitation exhibited formation of macroscopic cell aggregates after 12 and 9 days, respectively. Auto-aggregation resulted in underestimation of viable cell counts by plate counts by more than 2 log units CFU/ml compared with qPCR method. Modifications of cell membrane composition might partially explain aggregate formation in IC cultures. Decreases in the ratio of unsaturated to saturated fatty acid content from 1.74 to 0.58 might also contribute to the enhanced tolerance of IC cells to porcine bile salts and aminoglycosidic antibiotics compared with free cells from batch cultures.The enhanced resistance against bile salts in combination with auto-aggregation may confer an advantage to probiotic bacteria produced by IC technology.     

Optimization of growth and production of toxins by three dinoflagellates in photobioreactor cultures

A bioreactor system for biotoxin production was appraised against traditional methods of growing dinoflagellate cultures. In an optimised bioreactor culture (5.4?L) operated in batch mode, growth of Karenia selliformis was more efficient than in 15-L bulk carboy culture in terms of growth rate (μ?=?0.07?day^?1 versus 0.05?day^?1) and growth maximum (G _max, 169.10⁶ versus 41.10⁶ cells L^?1). Maximal gymnodimine concentration (1200?μg L^?1) in bioreactor culture was 8-fold higher than in bulk carboy culture, and the yield per cell (pg cell^?1) was 2-fold higher. Similarly the bioreactor batch culture of Alexandrium ostenfeldii performed more efficiently than carboy cultures in terms of growth rate (1.6-fold higher), growth maximum (15-fold higher) and desmethyl C spirolide (SPX-desMe-C) yield (5-fold higher [μg L^?1], though the yield [pg cell^?1basis] was lower). When bioreactor cultures of K. selliformis were operated in continuous mode, the yield of gymnodimine was substantially higher than a carboy or the bioreactor run in batch mode to growth max (793?μg day^?1 over 58?days in continuous culture was achieved versus an average of 60?μg day^?1 [carboy over 40?days] or 249?μg day^?1 [batch mode] over 26?days). Likewise in continuous bioreactor cultures of A. ostenfeldii run over 25?days, the yield of SPX-desMe-C (29?μg day^?1) was substantially higher than in same cultures run in batch mode or carboys (10.2 day^?1 and 7.7?μg day^?1 respectively). Similarly 5.4?L bioreactor batch cultures of K. brevisulcata reached 3.8-fold higher cell densities than carboy cultures, and when operated in continuous mode, the brevisulcatic acids were more efficiently produced than in batch culture (12?μg day^?1 versus 7?μg day^?1). When the bioreactor system was upscaled to 52?L, the maximum cell densities and toxin yields of K. brevisulcata cultures were somewhat less than those achieved in the smaller reactor, which was attributed to reduced light penetration.     

Production of Maltase by Wild-Type and a Constitutive Mutant of Saccharomyces italicus

The production of maltase, an inducible and repressible catabolic enzyme in Saccharomyces italicus, was studied and compared in batch, fed-batch, and continuous fermentations. Tight genetic controls on maltase synthesis limited the effect of environmental manipulations such as fed-batch or continuous culture in enhancement of maltase synthesis, and neither approach was able to improve the performance above the batch process for maltase production. S. italicus was mutated, and a constitutive producer of maltase was isolated. The mutant was detected by its ability to grow on sucrose, which is a noninducing substrate that is hydrolyzed by maltase; S. italicus does not possess invertase and will not normally grow on sucrose. Maltase production by this mutant was studied during growth on sucrose in batch and continuous cultures and marked improvement in enzyme productivity was observed. The specific activity of maltase produced by this mutant was more than twice that of the parent wild type: 2,210 and 1,370 U/g of cells for the mutant versus 890 and 510 U/g of cells for the wild type in batch and continuous cultures, respectively. Maltase specific productivity was increased from 74 to 288 U/g of cells per h by switching from batch growth of the wild type to continuous cultivation of the mutant.     

Selection of a Highly Monensin-Resistant Prevotella bryantii Subpopulation with Altered Outer Membrane Characteristics

Prevotella bryantii cultures treated with monensin grew more slowly than untreated cultures, but only if the monensin concentration was greater than 1 μM. Cultures that were repeatedly transferred (eight transfers or 25 doublings) with monensin always grew rapidly, even at a 10 μM concentration. The amount of monensin needed to facilitate half-maximal potassium depletion (K_d) from monensin-selected cells was 16-fold greater than “unadapted” wild-type cultures (3,200 versus 200 nM). Cells taken from continuous culture had a K_d of 100 nM, and these inocula could not grow in batch culture when the monensin concentration was greater than 300 nM. Continuous cultures treated with monensin nearly washed out, but the surviving cells had a K_d of 1,300 nM. When wild-type cells were transferred in batch culture with 10 μM monensin, the K_d did not reach its maximum value (3,200 nM) until after eight transfers (25 doublings). K_d declined when monensin was removed, and it took eight transfers to reach the control value (200 nM). The most probable number of wild-type cells was 1,000-fold lower than of the monensin-selected cells, but calculations based on relative growth advantage and K_d indicated that the wild-type culture had 1 to 10% highly monensin-resistant cells. Cell pellets of wild-type cultures were more difficult to disperse than were monensin-selected cells, and water-soluble phenol extracts of monensin-selected cells had 1.8-fold more anthrone-reactive material than did the wild type. Wild-type cultures that were washed in Tris buffer (pH 8.0) released little alkaline phosphatase and were agglutinated by lysozyme. Monensin-selected cultures leaked ninefold more alkaline phosphatase and were not agglutinated by lysozyme. Wild-type colonies taken from high-dilution agar roll tubes retained the lysozyme agglutination phenotype even if transferred with monensin, and monensin-selected colonies were never agglutinated. These observations indicated that wild-type P. bryantii cultures had a subpopulation with different outer membrane characteristics and increased monensin resistance.     

Predominant growth of α-amylase regulation mutant in continuous culture of Bacillus caldolyticus

The production of α-amylase in batch and continuous cultures of the strain SP of Bacillus caldolyticus was studied using a maltose-casitone medium. The three quantitative parameters of α-amylase production (maximum values of the specific production rate, volumetric productivity and concentration of α-amylase) increased in continuous culture by 2.5, 4.6 and 3.8 times respectively in comparison with a batch culture. It was found that a mutant strain M1 of α-amylase production was predominant in every run of the continuous cultures. The strain M1 differs from the strain SP in that it can produce α-amylase constitutively in a batch culture while the cells of strain SP require maltose for α-amylase production. On the contrary, α-amylase production by the strain M1 was repressed partly by maltose. Moreover, glucose repression on α-amylase production was not observed for the M1 strain while it was remarkable for the strain SP. The above-mentioned properties of the mutant M1 concerning the regulation of α-amylase production are not only advantageous for industrial use but also interesting from the viewpoint of basic microbiology.     

On-line growth measurements in bioreactors by titrating metabolic proton exchange

Recording the amount of titrant required to maintain constant pH in a bioreactor where cell metabolism causes acidity changes allows on-line determinations of growth kinetics in computer-controlled batch cultures. A system for making such measurements is described and its performance is investigated. Transient bicarbonate accumulation occurs if the culture produces CO₂ at high pH values and low gas transfer rates. We have developed a mathematical model for the titrant requirement as a function of the cell growth rate, the gas transfer properties of the bioreactor and the culture pH. According to this model, bicarbonate accumulation affects the stoichiometry between titrant and biomass but does not prevent determination of growth rate constants. These predictions are confirmed using model experiments and measurements during batch growth of microbial cultures.     

Modeling 1,2-dichloroethane biodegradation by <Emphasis Type="Italic">Klebsiella oxytoca va 8391</Emphasis> immobilized on granulated activated carbon

A mathematical model of the biodegradation of xenobiotics by microbial cells attached to particles of granulated activated carbon was developed. The model allowed the quantitative evaluation of different characteristics of the biofilm behavior: retarded microbial growth, increased concentration of immobilized cells compared to suspended cultures, potential cell detachment from the solid support and consequent independent growth of free cells. The applicability of the model was demonstrated for our own experimental data for 1,2- dichloroethane (DCA) biodegradation by Klebsiella oxytoca VA 8391 cells attached to granulated activated carbon. Two types of reactors, recirculated batch and continuous flow bioreactor, were studied. It was shown that in all investigated cases, the major contribution to DCA biodegradation was provided by the immobilized cells. Furthermore, immobilized cells were found to tolerate much higher substrate concentration and dilution rates in continuous culture than the free cells.     

Fermentation of Cellulosic Substrates in Batch and Continuous Culture by Clostridium thermocellum

Fermentation of dilute-acid-pretreated mixed hardwood and Avicel by Clostridium thermocellum was compared in batch and continuous cultures. Maximum specific growth rates per hour obtained on cellulosic substrates were 0.1 in batch culture and >0.13 in continuous culture. Cell yields (grams of cells per gram of substrate) in batch culture were 0.17 for pretreated wood and 0.15 for Avicel. Ethanol and acetate were the main products observed under all conditions. Ethanol:acetate ratios (in grams) were approximately 1.8:1 in batch culture and generally slightly less than 1:1 in continuous culture. Utilization of cellulosic substrates was essentially complete in batch culture. A prolonged lag phase was initially observed in batch culture on pretreated wood; the length of the lag phase could be shortened by addition of cell-free spent medium. In continuous culture with ~5 g of glucose equivalent per liter in the feed, substrate conversion relative to theoretical ranged from 0.86 at a dilution rate (D) of 0.05/h to 0.48 at a D of 0.167/h for Avicel and from 0.75 at a D of 0.05/h to 0.43 at a D of 0.11/h for pretreated wood. At feed concentrations of <4.5 g of glucose equivalent per liter, conversion of pretreated wood was 80 to 90% at D = 0.083/h. Lower conversion was obtained at higher feed substrate concentrations, consistent with a limiting factor other than cellulose. Free Avicelase activities of 12 to 84 mU/ml were observed, with activity increasing in this order: batch cellobiose, batch pretreated wood < batch Avicel, continuous pretreated wood < continuous Avicel. Free cellulase activity was higher at increasing extents of substrate utilization for both pretreated wood and Avicel under all conditions tested. The results indicate that fermentation parameters, with the exception of free cellulase activity, are essentially the same for pretreated mixed hardwood and Avicel under a variety of conditions. Hydrolysis yields obtained with C. thermocellum cellulase acting either in vitro or in vivo were comparable to those previously reported for Trichoderma reesei on the same substrates.     

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures was examined, using on-line off-gas analyses to measure the oxygen and carbon dioxide consumption rates continuously. A cell suspension from continuous cultures at steady state was used as the inoculum. It was observed that a dynamic phase occurred in the initial phase of the experiment. In this phase the bacterial ferrous iron oxidation and growth were uncoupled. After about 16 h the bacteria were adapted and achieved a pseudo-steady state, in which the specific growth rate and oxygen consumption rate were coupled and their relationship was described by the Pirt equation. In pseudo-steady state, the growth and oxidation kinetics were accurately described by the rate equation for competitive product inhibition. Bacterial substrate consumption is regarded as the primary process, which is described by the equation for competitive product inhibition. Subsequently the kinetic equation for the specific growth rate, μ, is derived by applying the Pirt equation for bacterial substrate consumption and growth. The maximum specific growth rate, μ _max, measured in the batch culture agrees with the dilution rate at which washout occurs in continuous cultures. The maximum oxygen consumption rate, q _O2,max, of the cell suspension in the batch culture was determined by respiration measurements in a biological oxygen monitor at excess ferrous iron, and showed changes of up to 20% during the course of the experiment. The kinetic constants determined in the batch culture slightly differ from those in continuous cultures, such that, at equal ferric to ferrous iron concentration ratios, biomass-specific rates are up to 1.3 times higher in continuous cultures. Received: 8 February 1999 / Accepted: 17 February 1999     

Production of an extracellular proteinase and α-amylase byBacillus subtilis in chemostat

The formation of two extracellular enzymes (α-amylase and a proteinase) ofB. subtilis was investigated in batch and continuous cultures. Differences were observed in the production of both enzymes in batch culture when studied in flasks and in a fermentor. The values of proteinase activity from continuous cultivation never reached those obtained during batch cultivation. The optimal dilution rate for proteinase synthesis was 0.1 – 0.2/h. The optimal dilution rate for the production of α-amylase was 0.2 –0.3/h.     

Biofilter performance and characterization of a biocatalyst degrading alkylbenzene gases

A biofilter treating alkylbenzene vapors was characterized for its optimal running conditions and kinetic parame-ters. Kinetics of the continuous biofilter were compared to batch kinetic data obtained with biofilm samples as well as with defined microbial consortia and with pure culture isolates from the biofilter. Both bacteria and fungi were present in the bioreactor. Five strains were isolated. Two bacteria, Bacillus and Pseudomonas, were shown to be dominant, as well as a Trichosporon strain which could, however, hardly grow on alkylbenzenes in pure culture. The remaining two strains were most often overgrown by the other three organisms in liquid phase batch cultures μ _max, K_S, K_I values and biodegradation rates were calculated and compared for the difterent mixed and pure cultures. Since filter bed acidification was observed during biofiltration studies reaching a pH of about 4, experiments were also undertaken to study the influence of pH on performance of the different cultures. Biodegradation and growth were possible in all cases, over the pH range 3.5–7.0 at appreciable rates, both with mixed cultures and with pure bacterial cultures. Under certain conditions, microbial activity was even observed in the presence of alkylbenzenes down to pH 2.5 with mixed cultures, which is quite unusual and explains the ability of the present biocatalyst to remove alkylbenzenes with high efficiency in biofilters under acidic conditions.     

Transient kinetics of yeast growth in batch and continous culture with an inhibitory carbon and energy source

Growth and acetate metabolism by Candida utilis (ATCC 9226) is reported for both acetate- and zinc-limited cultures in defined media. Acetate concentrations were varied from suboptimal to inhibitory levels in both types of media in differential shake flask culture and in batch and continuous cultures in stirred fermentors. Transient responses of steady-state cultures to small or large additions of concentrated sodium acetate, or to shifts in dilution rate or inlet acetate concentration are compared with one another and with simple mathematical models of growth and acetate metabolism. Exponential growth was observed during unrestricted growth (differential shake flask and batch cultures) with both types of media. Addition of acetate during unrestricted growth always caused lags and for larger pulses, lower specific growth rates were observed after exponential growth resumed. Inhibition by high acetate concentrations was much greater in acetate–limited than in zinc–limited cultures. During restricted growth (steady-state, continuous cultures), high acetate concentrations again consistently caused growth lags but stimulated, inhibited, or temporarily stopped acetage uptake. Qualitative agreement between the predictions of a simple mathematical model of acetate inhibition fitted to differential shake flask data and the observed transient data was surprisingly good.     

Effects of Culture Conditions on Poly(β-Hydroxybutyric Acid) Production by Haloferax mediterranei

The halobacterium Haloferax mediterranei accumulates poly(β-hydroxybutyrate) (PHB) as intracellular granules. The conditions for PHB production in batch and continuous cultures have been studied and optimized. Phosphate limitation is essential for PHB accumulation in large quantities. Glucose and starch are the best carbon sources. With 2% starch, 0.00375% KH₂PO₄, and 0.2% NH₄Cl in batch culture, a production of ca. 6 g of PHB per liter was reached, being 60% of the total biomass dry weight, and giving a yield over the carbon source of 0.33 g/g. The PHB production in continuous cultures was stable over a 3-month period. Our results demonstrate that H. mediterranei is an interesting candidate for industrial production of biological polyesters.     

Two-stage continuous operation of recombinant Escherichia coli using the bacteriophage λ Q − vector

A two-stage continuous culture of Escherichia coli in combination with a bacteriophage λ system was performed in order to overcome the intrinsic plasmid instability that is frequently observed in recombinant fermentation. A phage λ vector with a Q ⁻ mutation was used to enhance the expression of the λ system. The optimal values of the important operational variables such as the substrate concentration, the dilution rate, and the mean residence time on the expression of the cloned gene were determined in both batch and continuous cultures. For all culturing modes, the full induction of the cloned gene was observed 4 h after the temperature shift. In the two stage continuous culture, the overproduction reached their maxima at D=0.25 h⁻¹ with 1.5 S ₀ of the medium supply. The maximum productivity of the total β-galactosidase was 16.3×10⁶ U l⁻¹ h⁻¹, which was approximately seven times higher than that in the single-copy lysogenic stage. The recombinant cells were stable in the lysogenic state for more than 260 h, while they were stable for 40 h in the lytic state. The instability that developed rapidly in the second tank is believed to be due to the accumulation of lysis proteins as a result of vector leakage during the operation.