Ethanol production from sucrose by immobilizedZymomonas mobilis cells in polyurethane foam

Summary The possibility of using polyurethane foam as a support for the immobilization ofZymomonas mobilis cells to carry out sucrose conversion to ethanol was investigated. Sucrose hydrolysis efficiencies of 90% and higher, volumetric reactor productivity of 20 gL^–1h^–1 and final ethanol concentration of 6.3% (v/v) at a dilution rate of 0.4 h^–1 show the good performance of polyurethane foams for whole cell immobilization.     

A continuous thermophilic cellulose fermentation in an upflow reactor by aClostridium thermocellum-containing mixed culture

Summary A continuous thermophilic cellulose fermentation by aCl. thermocellum-containing mixed culture was carried out in an upflow reactor for a period of 100 days. The cellulose conversion rate was finally 0.35 g.1^–1.h^–1. Evidence that the fermentation process was influenced by both pH and dilution rate was given by the changes of concentration of the main fermentation products, acetic acid and ethanol. The role of cellodextrins and glucose as reactive intermediates in the process of cellulose breakdown was established.     

Growth ofCatharanthus roseus cell suspensions in bioreactors: On-line analysis of oxygen and carbon dioxide levels in inlet and outlet gas streams

Summary Catharanthus roseus cells (C87N) grown in a 30 litre airlift vessel achieved a growth rate of 0.366 day^–1. The maximum biomass yield (9.13 gl^–1) was recorded after 168 hours (7 days). On-line analysis of the composition of inlet and outlet gas streams during the growth cycle allowed calculation of the metabolic activity of the cultures. Oxygen uptake on a dry weight basis reached a maximum of 4.5×10^–4 Moles O₂ g dry weight^–1 h^–1 after 96 hours (during the mid-logarithmic phase of growth) and a maximum of 2.7×10^–3 Moles O₂ l^–1 h^–1 on a volume basis (towards the end of the logarithmic phase). Carbon dioxide production ran in parallel with oxygen use with maxima at 4.2×10^–4 Moles CO₂ g dry weight^–1 h^–1 and 3.4×10^–3 Moles g l^–1 h^–1 respectively.     

Influence of dilution rate on the morphology and technological properties ofLactobacillus delbrueckii subsp.bulgaricus

Lactobacillus delbrueckii subsp.bulgaricus ATCC 11842 was grown in a chemostat at 45°C and pH 5.5 using glucose as the carbon source, with the aim of optimizing biomass production. Cells were grown in a complex medium under nitrogen. At dilution rates lower than 0.18h^–1, it was difficult to keep steady-state conditions and pleomorphic forms were observed. The addition of 30mM Ca²⁺ and Mn²⁺ reverted the cells to normal shape: 30mM Mg²⁺ had no effect. Increasing the dilution rate resulted in normal morphology without the addition of any cations. Under these conditions, a maximum productivity of 1.24g dry biomass 1^–1 h^–1 was obtained. The maximum growth yield, corrected for maintenance, was 30g biomass mol^–1 glucose and the maintenance energy was 0.26g glucose g^–1 biomass h^–1. Lactate was the main fermentation product at all glucose concentrations used in the fed medium. Cells grown at high dilution rates had normal technological properties (acid production and proteolysis) when tested in milk.     

Effect of the dilution rate on the biomass yield ofBacillus thuringiensis and determination of its rate coefficients under steady-state conditions

Depending on the biomass yield on glucose and the cell morphology ofBacillus thuringiensis, three different metabolic states were observed in continuous culture. At dilution rates between 0.18 h^–1 and 0.31 h^–1 vegetative cells, sporulating bacteria and spores coexisted, while glucose and amino acids were consumed. Only vegetative cells were observed at dilution rates between 0.42 h^–1 and 0.47 h^–1 and glucose was used as the main carbon and energy source. AtD = 0.50 h^–1 the biomass yield on glucose decreases sharply. To define better the specific growth rate range in which the microorganism uses mainly glucose, a dilution rate of 0.25–0.45 h^–1 was studied. The experimental data could be adjusted to a Monod model and the following rate coefficients and growth yields were determined: maximum specific growth rate 0.54 h^–1, saturation constant 0.56 mg glucose ml^–1, biomass growth yields 0.43 g cells (g glucose)^–1, and 0.76 g cells (g oxygen)^–1, and maintenance coefficients 0.065 g glucose (g cells)^–1 h^–1 and 0.039 g oxygen (g cells)^–1 h^–1.     

Continuous culture of Candida boidinii variant 60 growing on methanol

Summary A new variant, Candida boidinii variant 60, which is less sensitive to methanol and formaldehyde shocks was grown in continuous cultures with methanol as sole carbon source. The substrate concentration in the feeding medium was either 1% methanol or 3% methanol. Biomass production, methanol consumption, the formation of formaldehyde and gas exchange were measured at different dilution rates. With low methanol feeding (10 g/l) maximal productivity of 0.44 g biomass/l·h is obtained at a dilution rate of 0.14 h^–1. Maximal specific growth rate is 0.18 h^–1. A yield of 0.32 g biomass/g methanol was obtained and the respiration quotient was determined as 0.55. Independently of initial substrate concentration, biomass decreases if methanol and formaldehyde are accumulating in the culture broth.In the culture with high methanol feeding (30 g/l) cell concentratioon increases up to 9 g/l at D=0.04 h^–1. At higher dilution rates methanol and form-aldehyde appear in the medium. Formaldehyde is then preferably oxidized without energy advantages for the cells. It seems that this enables the cells to overcome toxic effects caused by methanol and formaldehyde.     

Cellulase production by Trichoderma reesei

Summary A model is proposed for the enzyme production by Trichoderma reesei (QM 9414), which assumes control of the active enzyme transport through the cell membrane as a key parameter for the enzyme activity change in the culture filtrate. In a stirred tank reactor, continuous cultivation of the fungus was carried out in the dilution rate range of D=0.01–0.032 h^–1. After changing the dilution rate it took 3–4 weeks to attain a steady state in enzyme activity. Reducing sugars, dissolved protein, enzyme activity (filter-paper and glucosidase activities), cellulose and nitrogen content of the sediment, the elementary analysis of the cell and the composition of the outlet gas were all determined during cultivation. At a dilution rate of D=0.025 h^–1 all of these properties change due to derepression (for D<0.025 h^–1) or repression (for D>0.025 h^–1) of the enzymes which are responsible for the active transport of cellulases from the cell into the medium. The cellulase excretion causes a decrease of the yield coefficient of growth and a reduction of the nitrogen content of the cells.In a two-stage system the time to attain a steady state increases to 4–6 weeks. At low dilution rates the enzyme activity is only slightly higher in the second stage than in the first. At high dilution rates, at which the enzyme is not excreted into the medium in the first stage, enzyme activity can be increased considerably in the second stage.     

Biofilm build-up of Pseudomonas putida in a chemostat at different dilution rates

Summary A test system was set up where the build-up of a biofilm on a defined surface could be studied in a carbon source limited chemostat.The attachment of P. putida ATCC 11172 to glass when growing on L-asparagine was studied at different dilution rates (specific growth rates) from 0.1 to 1.5 h^–1 The number of attached colony forming units (cfu) increased with dilution rate from 1×10⁶ cfu/cm² at 0.1 h^–1 to 4×10⁷ cfu/cm² at 1.0 h^–1 and then the attachment decreased to about 6×10⁶ cfu/cm² at higher dilution rates (1.1–1.5 h^–1). The number of attached cfu was measured after 24 h exposure. The value of the maximum specific growth rate in batch culture was 0.6 h^–1.The total amount of attached cell-mass followed roughly the same pattern as the viable count.The viable count of the cells suspended in the growth medium showed its lowest value at the same dilution rate as resulted in maximum adhesion.It was shown that the effect of growth rate on the biofilm build-up of P. putida is significant, and ought to be borne in mind when continuous culture systems are set up and results evaluated.     

Lactic acid productivity of a continuous culture of Lactobacillus delbreuckii

Summary Maximum volumetric productivities of biomass (1.40 gl^–1h^–1) and lactic acid (8.93 gl^–1h^–1) for a continuous culture ofLactobacillus delbreuckii occurred between dilution rates 0.35h^–1 and 0.40h^–1. All major nutrients were in excess in these cultures. Glucose utilisation was complete at dilution rates of 0.1h^–1 and lower. Product and biomass yields were constant in the dilution rate range studied (0.05h^–1 to 0.50h^–1).     

Continuous production of poly(3-hydroxybutyrate-co-3-hyhroxyvalerate) byAlcaligenes eutrophus

Summary Copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) were produced in a continuous culture ofAlcaligenes eutrophus with 17.5 gl ^–1 of fructose and 2.5gl ^–1 of pentanoic acid in the feed. The P(3HB-co-3HV) productivity was maximal at a dilution rate of 0.17h^–1 and yielded 0.31gl ^–1h^–1 under an ammonium-limited condition. The 3HV content in copolymers increased from 11 to 79 mol% as the dilution rate of cells was increased from 0.06 to 0.32h^–1.     

Degradation of sodium anthraquinone sulphonate by free and immobilized bacterial cultures

Aerobic biodegradation of a xenobiotic recalcitrant compound sodium anthraquinone-2-sulphonate (SAS), was investigated using as an inoculum a mixed microbial culture, which was activated sludge from industrial and domestic waste-water treatment plants. The difference in SAS degradation was examined using two main systems: (1) suspended cells and (2) immobilized cells, both in batch and in continuous culture. In the suspended cell system, under continuous culture conditions using SAS as a unique source of carbon and energy, it was possible to degrade about 95% of this substrate after 6 days. Maximal SAS removal rates in the suspended-cell system were 593 mg SAS l^–1 h^–1 and 88.7 mg SAS l^–1 h^–1 for dilution rates (D) of 0.05 h^–1 and 0.075 h^–1, respectively. In the immobilized-cell system, almost all SAS was degraded in 6 days and the maximal removal rate reached 88.7 mg SAS l^–1 h^–1 at D=0.05 h^–1. Application of a continuous-flow enrichment procedure resulted in selection of several kinds of micro-organisms and led to a progressive elimination of some species of Aeromonas. A stable microbial community of 11 strains has been established and characterized at D=0.075 h^–1. Most of them were Gram-negative and belonged to the genus Pseudomonas.     

Determination of cell lysis and death kinetics in continuous hybridoma cultures from the measurement of lactate dehydrogenase release

The death of the hybridoma VO 208 in a continuous culture at pH 7 and 6.8 was investigated by measuring both the appearance of visible dead cells which do not exclude the trypan blue dye and the release of lactate dehydrogenase (LDH) in the culture medium. The intracellular LDH was found to be completely released either when live cells lysed or when they were transformed into visible dead cells. No significant lysis of blue dead cells could be observed at the two different pH. Using a LDH balance over the culture system, cell lysis was found negligible at pH 7, but accounted for 20% of the total cell death at pH 6.8. A methodology is proposed to evaluate the rate constants of hybridoma lysis and total death. For the investigated cell line in continuous culture, the calculated total cell death rate constant was found to increase from 0.002 h^–1 to 0.01 h^–1 when decreasing the pH from 7 to 6.8.Abbreviations D dilution rate (h^–1) - k_b specific trypan-blue dead cells appearance rate (h^–1) - k_L specific lysis rate of viable cells (h^–1) - k_d specific death rate (h-1) - LDH₀ lactate dehydrogenase activity in the feed culture medium (IU.l^–1) - LDH lactate dehydrogenase activity in the outlet culture medium (IU.l^–1) - LDH_i intracellular lactate dehydrogenase activity of viable cells (IU.10^–9 cells) - r_LDH total rate of LDH release (IU.h^–1.L^–1) - r_b transformation rate of viable cells into blue dead cells (10⁹ cells.h^–1.L^–1) - x_v viable cell concentration (10⁹ cells.l^–1) - x_b trypan-blue dead cell concentration (10⁹ cells.l^–1)     

The isolation and characterisation of a salicylate-hydroxylase-producing strain of Pseudomonas putida

Summary A salicylate-hydroxylase-producing strain of Pseudomonas putida with an unusual capability to grow at toxic levels of salicylate up to 10 g l^–1 has been isolated. It grew well under continuous culture conditions, with optimum growth at pH 6.5 and a temperature of 25° C. The use of an ammonium salt as a nitrogen source, instead of nitrate, resulted in a 30–40% increase in its biomass yield coefficient. Optimum growth under continuous culture conditions was achieved using 4 g l^–1 salicylate at 25° C, pH 6.5 and 0.2 h^–1 dilution rate. High salicylate hydroxylase enzyme activity [236 units (U) l^–1] and productivity (424.8 U h^–1) were obtained at a dilution rate of 0.45 h^–1 using a mineral medium containing 4 g l^–1 of salicylate. Operating under continuous culture conditions with oxygen limitation and a slight accumulation of residual salicylate (0.2 g l^–1) resulted in a decrease in culture performance and enzyme productivity. Correspondence to: R. Marchant     

An analysis of the growth of Gluconobacter oxydans in chemostat cultures

Gluconobacter oxydans was grown successively in glucose and nitrogen-limited chemostat cultures. Construction of mass balances of organisms growing at increasing dilution rates in glucose-limited cultures, at pH 5.5, revealed a major shift from extensive glucose metabolism via the pentose phosphate pathway to the direct pathway of glucose oxidation yielding gluconic acid. Thus, whereas carbon dioxide production from glucose accounted for 49.4% of the carbon input at a dilution rate (D)=0.05 h^-1, it accounted for only 1.3% at D=0.26 h^-1. This decline in pentose phosphate pathway activity resulted in decreasing molar growth yields on glucose. At dilution rates of 0.05 h^-1 and 0.26 h^-1 molar growth yields of 19.5 g/mol and 3.2 g/mol, respectively, were obtained. Increase of the steady state glucose concentration in nitrogen-limited chemostat cultures maintained at a constant dilution rate also resulted in a decreased flow of carbon through the pentose phosphate pathway. Above a threshold value of 15–20 mM glucose in the culture, pentose phosphate pathway activity almost completely inhibited. In G. oxydans the coupling between energy generation and growth was very inefficient; yield values obtained at various dilution rates varied between 0.8–3.4 g/cells synthesized per 0.5 mol of oxygen consumed.     

L-Sorbose production in a continuous fermenter with and without cell recycle

The kinetics of continuous l-sorbose fermentation using Acetobacter suboxydans with and without cell recycle (100%) were investigated at dilution rates (D) of 0.05, 0.10, 0.15 and 0.3 h^–1. The biomass and sorbose concentrations for continuous fermentation without recycle increased as the dilution rate was increased from 0.05 to 0.10 h^–1. A maximum biomass concentration of 8.44 g l^–1 and sorbose concentration of 176.90 g l^–1 were obtained at D=0.10 h^–1. The specific rate of sorbose production and volumetric sorbose productivity at this dilution rate were 2.09 g g^–1 h^–1 and 17.69 g l^–1 h^–1. However, on further increasing the dilution rate to 0.3 h^–1, both biomass and sorbose concentrations decreased to 2.93 and 73.20 g l^–1 respectively, mainly due to washout of the reactor contents. However, the specific rate of sorbose formation and volumetric sorbose productivity at this dilution rate increased to 7.49 g g^–1 h^–1 and 21.96 g l^–1 h^–1 respectively. Continuous fermentation with 100% cell recycle served to further enhance the concentration of biomass and sorbose to 28.27 and 184.32 g l^–1 respectively (in the reactor at a dilution rate of 0.05 h^–1). Even though, there was a decline in the biomass and sorbose concentrations to 6.8 and 83.40 g l^–1 at a dilution rate of 0.3 h^–1, the specific rates of sorbose formation and volumetric sorbose productivity increased to 3.67 g g^–1h^–1 and 25.02 g l^–1 h^–1.     

Continuous production of acetone and butanol by Clostridium acetobutylicum in a two-stage phosphate limited chemostat

Summary When Clostridium acetobutylicum was grown in continuous culture under phosphate limitation (0.74 mM) at a pH of 4.3, glucose was fermented to butanol, acetone and ethanol as the major products. At a dilution rate of D=0.025 h^–1 and a glucose concentration of 300 mM, the maximal butanol and acetone concentrations were 130 mM and 74 mM, respectively. 20% of the glucose remained in the medium. On the basis of these results a two-stage continuous process was developed in which 87.5% of the glucose was converted into butanol, acetone and ethanol. The cells and minor amounts of acetate and butyrate accounted for the remaining 12.5% of the substrate. The first stage was run at D=0.125 h^–1 and 37° C and the second stage at D=0.04 h^–1 and 33° C. High yields of butanol and acetone were also obtained in batch culture under phosphate limitation.     

Metabolic and kinetic studies of hybridomas in exponentially fed-batch cultures using T-flasks

Exponentially fed-batch cultures (EFBC) of a murine hybridoma in T-flasks were explored as a simple alternative experimental tool to chemostats for the study of metabolism, growth and monoclonal antibody (MAb) production kinetics. EFBC were operated in the variable volume mode using an exponentially increasing and predetermined stepwise feeding profile of fresh complete medium. The dynamic and steady-state behaviors of the EFBC coincided with those reported for chemostats at dilution rates below the maximum growth rate. In particular, steady-state for growth rate and concentration of viable cells, glucose, and lactate was attained at different dilution rates between 0.005 and 0.05 h^–1. For such a range, the glucose and lactate metabolic quotients and the steady-state glucose concentration increased, whereas total MAb, volumetric, and specific MAb production rates decreased 65-, 6-, and 3-fold, respectively, with increasing dilution rates. The lactate from glucose yield remained relatively constant for dilution rates up to 0.03 h^–1, where it started to decrease. In contrast, viability remained above 80% at high dilution rates but rapidly decreased at dilution rates below 0.02 h^–1. No true washout occurred during operation above the maximum growth, as concluded from the constant viable cell number. However, growth rate decreased to as low as 0.01 h^–1, suggesting the requirement of a minimum cell density, and concomitant autocrine growth factors, for growth. Chemostat operation drawbacks were avoided by EFBC in T-flasks. Namely, simple and stable operation was obtained at dilution rates ranging from very low to above the maximum growth rate. Furthermore, simultaneous operation of multiple experiments in reduced size was possible, minimizing start-up time, media and equipment costs.Abbreviations EFBC exponentially-fed batch culture - CSC continuous suspended culture - MAb monoclonal antibody - D dilution rate - q _i metabolic quotient or specific rate of consumption or production of i     

Exopolysaccharide and extracellular metabolite production by Lactobacillus delbrueckii subsp. bulgaricus, grown on lactose in continuous culture

Lactobacillus delbrueckii subsp. bulgaricus NCFB 2483, when grown on lactose in continuous culture, showed increasing specific yields and volumetric productivities of exopolysaccharide (EPS) with increasing dilution rate. Specific and volumetric productivities of lactate and galactose, as extracellular metabolites, increased in response to the incremental changes in the dilution rate up to 0.4 h^–1. Elevated Y_p/s values determined for EPS (0.025 g EPSg lactose^–1) at the dilution rates of 0.3 h^–1–0.4 h^–1, relative to those determined at lower dilution rates, suggest a diversion of carbon flux towards EPS being associated with the higher rates of growth.     

A flow cytometry analysis of batch and continuous culture transient diauxic growth in Hansenula polymorpha

A flow cytometry analysis and in vitro enzyme activity study is carried out on the methylotrophic yeast, Hansenula polymorpha, during both (a) batch growth and (b) continuous cultures subjected to single perturbations in either system dilution rate or influent carbon substrate composition. Flow cytometry of yeasts growing diauxically on a glucose: methanol mixture during exponential growth, exhibit DNA and RNA distributions indicative of the S-synthesis-phase of the cell cycle. Cells at the stationary growth stage exhibit DNA and RNA distributions that indicate one portion of the population in the G ₀/G₁ resting phase and another in the M-mitosis-phase.Yeast cells grown at a steady-state of D=0.2 h¹, then shifted to D=0.35 h^–1, at a constant influent substrate mixture, are also examined with both flow cytometry and in vitro enzyme assays. Distributions of DNA, RNA, and total protein at either steady state and during the shift between dilution rates did not resemble any observed in batch culture. Flow cytometry indicates significant changes in cell composition within 20 min of the imposed dilution rate shift. In vitro enzyme assays show a response time in decreasing methanol oxidase activity of 2.5–3 h upon a dilution rate shift-up, while hexokinase activity increases to its steady-state level in less than 3 h. Similar cell compositional changes are reported for shifts in influent substrate methanol: glucose ratio at a constant dilution rate of D=0.35 h ^–1. Results suggest that an unsteady-state regime, oscillating between conditions that promote maximum enzyme activity of either glucose- or methanol-metabolizing enzymes, may allow simultaneous enhanced time-averaged production of both sets of enzymes.     

Corynebacterium glutamicum: morphological and ultrastructural changes of l-lysine producing cells in continuous culture

Summary Corynebacterium glutamicum was observed to undergo several morphological and ultrastructural changes during a shift in dilution rate when grown in phosphate-limiting continuous culture. At 0.1 h^–1 the cell culture appeared homogeneous and the average diameter of cells and the cell length was approximately 0.7 m and 1–1.5 m respectively. At 0.04 h^–1 there was essentially no change in these readings, but at this dilution rate there was a significant proportion of cells that measured three times the original length. At six residence times the elongated cells were increasing in number, but these changes occurred without diminishing the system's performance. Some changes in cell ultrastructure were observed during the shift in dilution rate. Thus, the presence of polyphosphate granules and of glycogen appeared in the cytoplasm of producing cells (0.04 h^–1). We have confirmed by mass spectrometry the absence of poly--hydroxybutyrate (PHB) in C. glutamicum.Correspondence to: N. Coello