Continuous ethanol production by a flocculating strain of Kluyveromyces marxianus: bioreactor performance

A flocculating strain of Kluyveromyces marxianus was used for alcoholic fermentation in a continuous bioreactor working with zero residual concentration in effluent. Specific kinetic parameters were improved by increasing dilution rate, which is similar to results obtained with ultrafiltration systems. Specific biomass accumulation rate had always a value greater than 92.5% of specific biomass growth rate and was independent of the dilution rate. Productivity is shown to be 12.5 times greater than in conventional continuous operation and is directly proportional to dilution rate. Maximum biomass concentration also presents a linear relationship with dilution rate. The largest obtained biomass concentration is 8 times greater than in a conventional continuous fermentor.     

Dilution rate as a determinant of mycelial morphology in continuous culture

The morphology of mycelial fungi in liquid culture effects culture rheology and this in turn may affect product yield. It is therefore important to understand how environmental factors influence mycelial morphology and this paper describes the effect of dilution rate on two strains of Fusarium graminearum, the relatively sparsely branched parental strain (A3/5) and a relatively highly branched "colonial" variant (C106). At any given dilution rate, the concentration of mycelial fragments present at steady state of both strains remained approximately constant with time, suggesting that mycelial fragmentation occurred in a regular manner. However, for both strains fragment concentration decreased with increasing dilution rate. The strains had a similar morphology at a dilution rate of 0.07 h(-1). The length of the hyphal growth unit of A3/5 increased with increase in dilution rate, while that of C106 decreased with increase in dilution rate. At all dilution rates, C106 produced up to ten times more macroconidia than A3/5.     

Growth characteristics and expression of iron-regulated outer-membrane proteins of chemostat-grown biofilm cells of Pseudomonas aeruginosa.

An in vitro chemostat system was used to study the growth and the expression of iron-regulated outer-membrane proteins (IROMPs) by biofilm cells of Pseudomonas aeruginosa cultivated under conditions of iron limitation. The population of the planktonic cells decreased when the dilution rate was increased. At a dilution rate of 0.05 h-1, the populations of planktonic cells of both mucoid and nonmucoid P. aeruginosa were 3 x 10(9) cells/mL. This value dropped to 5 x 10(6) cells/mL when the dilution rate was increased to 1.0 h-1. The reverse was observed for the biofilm cells. The number of biofilm cells colonising the silicone tubing increased when the dilution rate was increased. The number of biofilm cells of the mucoid strain at steady state was 2 x 10(8) cells/cm (length) when the dilution rate was fixed at 0.05 h-1. The figure increased to 8 x 10(9) cells/cm when the dilution rate was increased to 1.0 h-1. The population of biofilm cells of the nonmucoid strain was 9 x 10(7) cells/cm (length) when the dilution rate was 0.05 h-1. It increased to 2 x 10(9) cells/cm when the dilution rate was set at 1.0 h-1. The expression of IROMPs was induced in the biofilm cells of both mucoid and nonmucoid strains when the dilution rates were 0.05 and 0.2 h-1. IROMPs were reduced but still detectable at the dilution rate of 0.5 h-1. However, the expression of IROMPs was repressed when the dilution rate was increased to 1.0 h-1. The data suggest that the biofilm cells of P. aeruginosa switch on the expression of IROMPs to assist iron acquisition when the dilution rate used for the chemostat run is below 0.5 h-1. The high affinity iron uptake system is not required by the biofilm cells when the dilution rate is increased because the trace amount of iron present in the chemostat is sufficient for the growth of adherent biofilm cells.     

Growth and phosphate uptake of a high phosphate accumulating bacterium, Arthrobacter globiformis PAB-6 in continuous culture

A high phosphate accumulating bacterium, Arthrobacter globiformis PAB-6, was grown in a chemostat under glucose-limitation. Two different growth patterns at steady state with various dilution rates were obtained. In one case, cells having a coccus shape tended to washout at a low dilution rate, 0.2 (h(-1)). In another, cells with a rod shape grew faster and gave a good steady-state growth at a dilution rate of 0.4. Such a close relationship between growth rate and cell morphology was found both in continuous and batch cultures. The amount of phosphate uptake per cell mass was almost constant irrespective of the dilution rate, but the rate of the uptake was maximum at about the dilution rate of 0.4. A clone of PAB-6 was isolated from the continuous culture with high dilution rate and had maximum specific growth rate of 0.7 in a simple glucosesalt medium.     

Predicting Production in Light-Limited Continuous Cultures of Algae

Equations relating productivity, growth rate, cell concentration, and light absorption lead to the prediction that, when incident light is below saturating intensity, maximal productivity will occur at half the maximal growth rate. The freshwater alga Chlorella pyrenoidosa TX71105 and the marine alga Dunaliella tertiolecta were grown in a small continuous culture apparatus with turbidostatic control. With both cultures, the cell concentration showed a linear decrease with dilution rate. Productivity was maximal at about one-half the maximal dilution rate. Average mass per cell increased near the maximal dilution rate, causing some asymmetry in the productivity versus dilution rate curve. The chlorophyll content per unit mass decreased in this region, but the chlorophyll content per cell remained constant. Best production rate in a light-limited algal culture was obtained when the growth rate at very low cell concentration was determined in the apparatus and the dilution rate was set at one-half that value.     

Do spatial effects appear at low dilution rate in chemostat?

The chemostat theory on two species competition has shown that the dilution rate where transition of dominance occurs – transition-dilution rate – is independent of limiting-nutrient concentration. However, we obtained the experimental data indicating that the transition-dilution rate changed with variations in limiting-ammonium concentrations, using the chemostat mixed-culture of the cyanobacterium Microcystis novacekii and the green alga Scenedesmus quadricauda. The transition-dilution rate was dependent on the concentration of limiting ammonium in the influx culture medium. We tried to simulate the experimental results. Though the dilution rate has been considered independent of nutrient concentration, we introduce the effective dilution rate that depends on nutrient concentration (ammonium concentration in this study). A hyperbolic Monod-type function is used to represent the effective dilution rate for each species. The maximum dilution rate of the function is set to be the mechanical dilution rate (nominal dilution rate) of the chemostat culture. The calculation shows that the nominal transition-dilution rate where transition of dominance occur decreases with increased concentration. This simulation is well consistent with our experimental data. These results may suggest that the species-specificity of limiting nutrients, here nitrogen. Or they may imply that the depreciation of nitrogen becomes critical when both dilution rate and concentration are very low, especially for the green algae. In the latter case, spatial effects are induced internally in the ecosystem.     

Continuous growth kinetics of Candida utilis in pineapple cannery effluent

Candida utilis was grown on a pineapple cannery effluent as the sole carbon and energy source in a chemostat at dilution rates between 0.10 and 0.62 h(-1) to determine the growth kinetics. The principal sugars in the effluent were sucrose, glucose, and fructose. The cell yield coefficient on carbohydrate varied with dilution rate and a maximum value of 0.63 was observed at a dilution rate of 0.33 h(-1). The steady-state concentrations of carbohydrate, reducing sugar, and chemical oxygen demand (COD) appeared to follow Monod saturation kinetics with increasing dilution rate, although none of the measured parameters represented a pure substrate. The maximum specific growth rate and reducing sugar saturation constant were 0.64 h(-1) and 0.060 g/L, respectively. A maximum cell mass productivity of 2.3 g/L h was observed at a dilution rate of 0.51 h(-1). At this dilution rate, only 68% of the COD was removed. A 95% COD removal was attained at a dilution rate of 0.10 h(-1). Optimal yeast productivity and COD reduction occurred at a dilution rate of 0.33 h(-1).     

Effect of specific growth rates on productivity in continuous open and partial cell retention animal cell bioreactors.

A clonal derivative of a transfectant of the SP2/0 myeloma cell line producing a chimeric monoclonal antibody was cultivated in both continuous open and continuous partially-closed bioreactors. Using an open system for the determination of kinetic parameters, we showed that the production of this chimeric mAb was growth associated. As such, the volumetric productivity increased linearly with increasing dilution rate up to the maximum dilution rate. Three continuous cultivations employing partial cell retention were conducted. In agreement with mathematical predictions, the product titer and volumetric productivity were independent of the degree of cell retention when the total dilution was held constant. When cells were maintained at a low specific growth rate, the product titer was independent of dilution rate and the volumetric productivity increased with increasing dilution rate, again in agreement with mathematical predictions. Since the partially-closed bioreactor could be operated at dilution rates in excess of the maximum specific cellular growth rate, volumetric productivities were greater than those achievable in the open bioreactor. However, when cells were maintained at a high specific growth rate, cell accumulation was limited and product titers decreased at high dilution rates. Therefore, the volumetric productivity in this latter case did not increase at higher dilution rates.     

Optimal operating policy of the ultrafiltration membrane bioreactor for enzymatic hydrolysis of cellulose

The dilution rate of an ultrafiltration membrane bioreactor in the enzymatic hydrolysis of cellulose was optimized using the kinetic model developed by Fan and Lee.(4) The sequence of optimal dilution rates was found to generally consist of an initial period of a minimal value (batch period), a subsequent period of maximum dilution rate, a period of a second batch, and a final period of a singular dilution rate. The effects of operating conditions, such as beta-glucosidase activity, operating time, maximum dilution rate, substrate feeding rate, and enzyme-to-substrate ratio on both the conversion yield and the sequence of optimal dilution rates were investigated. To evaluate the validity of kinetic model employed in this work, enzymatic hydrolysis was carried out using alpha-cellulose as a substrate in the ultrafiltration membrane bioreactor. The experimental data were well consistent with the simulation results. (c) 1993 John Wiley & Sons, Inc.     

Influence of dilution rate and dissolved oxygen concentration on continuous keto acid production by Gluconobacter oxydans subsp. melanogenum

The influence of dilution rate and dissolved oxygen concentration on continuous production of 2,5-diketogluconic acid (2,5-DKGA) by Gluconobacter oxydans subsp. melanogenum is described. Under suitable dissolved oxygen conditions, the glucose oxidation rate is independent of variations in dilution rates over a wide range (D < 0.33 h⁻¹). The higher dilution rate, however, leads to a partial oxidation of the substrate and therefore accumulation of more gluconic acid. Contrary to this, a longer residence time (lower dilution rate) was more suitable for better ketogenic activity, thereby facilitating accumulation of 2,5-DKGA in higher yields. A high dissolved oxygen concentration is further shown to positively influence the overall oxidation process through an organized induction of membrane-bound NAD(P)⁺-independent dehydrogenases.     

Effect of growth conditions on heat resistance of Arizona bacteria grown in a chemostat.

The effects of various growth conditions on the heat resistance of Arizona bacteria grown in a continuous-culture device (chemostat) were studied. Using either glucose, NH4Cl, NaH2PO4, or MgCl2 as the rate-limiting nutrient, it was found that the heat resistance, in all cases depended on the dilution rate and, hence, growth rate of the culture. Cells grown at high dilution rates were less heat resistant than those grown at low dilution rates. If, however, the dilution rate was maintained at a constant rate, the higher the growth temperature, the more heat resistant were the cells. Also at any given dilution rate, the cells were most heat resistant when grown at a near neutral pH. Most survival curves were biphasic in shape, indicating the presence in the population of two fractions of cells, one fraction being more resistant than the other. The size of the more heat-resistant fraction varied from almost 100% in very slow-growing cultures to practically 0% in cultures grown at a dilution rate of 0.67 h-1.     

Production of extracellular protease and glucose uptake in Bacillus clausii in steady-state and transient continuous cultures

The production of the extracellular alkaline protease Savinase (EC 3.4.21.62) and glucose uptake in a non-sporulating strain of Bacillus clausii were investigated by analysing steady-state and transients during continuous cultivations. The specific production rate was found to have an optimum at a dilution rate between 0.14 and 0.17 h(-1), whereas the yield of Savinase on glucose was found to increase with decreasing specific growth rate. A linear relationship between the ribosomal RNA content and the specific production rate was found, indicating that the translational capacity may be limiting for product formation. The dynamics of the production of Savinase were studied during step changes in the dilution rate. During a step down in the dilution rate the specific production rate decreased immediately until it reached a new steady value. During a step-up an initial cease in the production rate was observed, but when glucose stopped to accumulate the production rate was regained. The glucose uptake was further investigated when chemostat cultures growing at different dilution rates were exposed to glucose pulses. The maximal glucose uptake capacity was found to be dependent on the initial specific growth rate. Furthermore, the adaptation to high glucose concentrations was faster at high dilution rates than at low dilution rates.     

Kinetics of recombinant immunoglobulin production by mammalian cells in continuous culture

A clonal derivative of a transfectant of the SP2/O myeloma cell line producing a chimeric monoclonal antibody was maintained in steady-state, continuous culture at dilution rates ranging from 0.21 to 1.04 day(-1). The steady-state values for nonviable and total cell concentrations increased as the dilution rate decreased, while the viable cell concentration was roughly independent of the dilution rate. At steady state, the specific growth rate increased and the specific death rate decreased as the dilution rate increased. The maximum specific growth rate was 1.15 day(-1). Antibody production was growth associated and the specific rate of antibody production increased linearly as the specific growth rate increased.     

Continuous cultivation of the yeastSaccharomyces cerevisiae at different dilution rates and glucose concentrations in nutrient media

The influence of glucose concentration in nutrient media on the specific growth rate and biomass yield in the course of continuous fermentation ofSaccharomyces cerevisiae was investigated. An increase of glucose content in media decreased the specific growth rate and the biomass yield. Glucose concentration had significant effects on protein and phosphate contents of cells. However, an increased glucose concentration increased the fermentative power ofS. cerevisiae (SJA-method). An increase of the dilution rate decreased the cell concentration in the fermentor. Specific growth rate approached the values of the dilution rate. The best agreement has been obtained at a dilution rate of 0.20/h. This dilution rate proved to be most convenient for the investigated microorganism and cultivation conditions (media composition, pH, aeration intensity and temperature). Biomass yield proved to be decreased by an increase of the dilution rate.     

Calorimetric analysis of Escherichia coli in continuous culture

The heat evolution rate was measured in continuous culture of Escherichia coli k-12 on glucose-limiting medium. The specific heat production rate based on the dry cell mass increased in proportion to increase of dilution rate. The specific heat production rate based on a viable cell increased nonlinearly because the cell size varied in each dilution rate. Several thermochemical data were calculated for each dilution rate from the stoichiometric equation which was derived from elementary analysis of the cells. Growth yields based on energy measured directly was found to be less influenced by dilution rate than the growth yield based on consumed glucose. This suggested that the energy efficiency of utilization of catabolic energy for anabolism was almost constant.     

Production of the antitumoral retamycin during continuous fermentations of Streptomyces olindensis

Continuous fermentations were performed in order to correlate the production of retamycin, an anthracycline antibiotic produced by Streptomyces olindensis in submerged cultures, with the dilution rate. Maximum retamycin production was achieved at a dilution rate of 0.05 h⁻¹ (D=μ_x=0.05 h⁻¹), while higher dilution rates caused a decrease in antibiotic production, which ceased completely at a dilution rate of 0.30 h⁻¹. Otherwise, biomass productivity was favoured by high dilution rates, achieving a maximum at D=0.25 h⁻¹, whereas retamycin productivity reached a maximum at D=0.05 h⁻¹. Dilution rate influenced morphology, which was assessed by image analysis. The percentage of clumps decreased with an increase in dilution rate, with a correspondent increase in pellet percentage.     

Plasmid stability and kinetics of continuous production of glucoamylase by recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in an airlift bioreactor

Production of glucoamylase by recombinant Saccharomyces cerevisiae C468/pGAC9 (ATCC 20690) in a continuous stirred tank bioreactor was studied at different dilution rates. Plasmid stability was found to be growth (dilution rate) dependent; it increased with the dilution rate. Bioreactor productivity and specific productivity also increased with the dilution rate. A kinetic equation was used to model the plasmid stability kinetics. The growth rate ratio between plasmid-carrying and plasmid-free cells decreased from 1.397 to 1.215, and segregational instability or probability of plasmid loss from each cell division decreased from 0.059 to 0.020 as the dilution rate increased from 0.10 to 0.37 1/h. The specific growth rates increased with dilution rate, while the growth rate difference between plasmid-carrying and plasmid-free cell populations was negligible. This was attributed to the low copy number of the hybrid plasmid pGAC9. Thus, the growth rate had no significant effect on plasmid instability. The proposed kinetics was consistent with experimental results, and the model simulated the experimental data well.     

Cell yields and fermentation responses of a <Emphasis Type="Italic">Salmonella</Emphasis> Typhimurium poultry isolate at different dilution rates in an anaerobic steady state continuous culture

The objectives of these studies were to determine cell yield and fermentation responses of a Salmonella enterica serovar Typhimurium poultry isolate using various dilution rates in steady state continuous culture incubations. S. enterica Typhimurium cells were propagated in continuous cultures with a total volume of 0.50 l of Luria Bertani medium containing 0.1% glucose. Dilution rates from 0.0125 to 1.44/h were used. Cell protein concentration generally increased linearly with increased dilution rate up to a rate of 0.54/h and declined at the higher dilution rates. Glucose consumption gave a similar pattern to cell protein concentration by declining at the three highest dilution rates. Short chain fatty acid production was inconsistently influenced by dilution rate. Acetate, the most predominant fatty acid produced, declined at the higher dilution rates, as did propionate. Ammonia production remained stable at the lowest dilution rates, but increased significantly at a dilution rates above 0.27/h.     

Effect of dilution rates on the amount of wasted substrate during alcohol fermentation

Continuous ethanol production in a one-stage continuous stirred tank fermentor without recycle was carried out using a yeast strain Saccharomyces cerevisiae. Different dilution rates were used. Cell and ethanol concentrations in the culture medium decreased with increasing dilution rates, and the maximum value of 3.0 g l⁻¹h⁻¹was found at a dilution rate of 0.340 h⁻¹. Specific ethanol productivities increased as dilution rates were increased, and the highest value appeared at about the same dilution rate as that for the maximum fermentor productivity. A material balance equation, which relates total amount of spent medium to cell synsthesis, ethanol production, and overall maintenance, was introduced. The cellular yield and overall maintenance coefficients increased with increasing dilution rates. The fraction of limiting substrate utilized for overall maintenance, which includes the limiting substrate spent for purposes other than cell synthesis and ethanol production, decreased with increasing dilution rates. The non-product associated substrate utilization can be minimized if correct dilution rate is chosen.     

Continuous ethanol production from Jerusalem artichoke tubers. I. Use of free cells of Kluyveromyces marxianus

The Continuous fermentation of Jerusalem artichoke juice to ethanol by free cells of Kluyveromyces marxianus UCD (FST) 55-82 has been studied in a continuous-stirred-tank bioreactor at 35 degrees C and pH 4.6. A maximum yield of 90% of the theoretical was obtained at a dilution rate of 0.05 h(-1). About 95% of the sugars were utilized at dilution rates lower than 0.15 h(-1). Volumetric ethanol productivity and volumetric biomass productivity reached maximum values of 7 g ETOH/L/h and 0.6 g dry wt/L/h, respectively, at a dilution rate of 0.2 h(-1). The maintenance energy coefficient for K. marxianus culture was found to be 0.46 g sugar/g biomass/h/ Oscillatory behavior was following a change in dilution rate from a previous steady state and from batch to continuous culture. Values of specific ethanol production rate and specific sugar uptake were found to increase almost linearly with the increase of the dilution rate. The maximum specific ethanol production rate and maximum specific sugar uptake rate were found to be 2.6 g ethanol/g/ cell/h and 7.9 sugars/g cell/h, respectively. Washout occurred at a dilution rate of 0.41 h(-1).