Growth and cell wall composition of glucose-limited bean cells (Phaseolus vulgaris L.)

Glucose-limited bean cells (Phaseolus vulgaris L.) were grown in a modified bacterial fermentor at a constant pH of 4.8. The cultures were kept in steady state at different specific growth rates varying from 0.00216 h^–1 to 0.0106 h^–1. Culture conditions are described that are needed to start a continuous culture. First, it was essential to use log-phase cells as starting material. Second, it was important to increase the dilution rate gradually, otherwise cells in the culture aggregated. Cells grown at the highest dilution rate employed contained twice as much protein per gram dry weight as cells grown at the lowest dilution rate. The composition of the cell walls also varied with the dilution rate in contrast to their relatively constant composition when grown in batch culture.     

Morphological behaviour ofSaccharomyces cerevisiae during continuous fermentation

Summary Saccharomyces cerevisiae were observed to undergo drastic morphological changes when grown in continuous culture. Peak elongations and minimum cell volumes were found at intermediate dilution rates when it was believed the insitu glucose concentration was at its lowest. The shapes and sizes were reproducible and have been quantified at two different glucose feed concentrations.     

Acetone-butanol production by Clostridium acetobutylicum in an ammonium-limited chemostat at low pH values

Clostridium acetobutylicum was grown in continuous culture under ammonium limitation (15.15 mM NH₄ ⁺). At a pH of 6.0 and at various dilution rates only acetate, butyrate and ethanol were formed as non-gaseous products. A decrease of the pH to values between 5.2 and 4.3 resulted in a shift of the fermentation towards acetone-butanol formation.     

Effect of dilution rate on the biological nitrogen fixation in thePhaseolus vulgaris-Rhizobium phaseoli symbiosis

Summary Continuous culture was used to produceRhizobium phaseoli cells at different dilution rates.Phaseolus vulgaris seeds in sterile Leonard jars were then inoculated with the cells produced, and the system was left to interact for six weeks. Dry weight of the plants and number and weight of the nodules were measured. These data were compared to the response obtained with cells produced in batch culture. An increase in plant biomass and nodule weight and number were observed at higher dilution rates.     

Effects of organism type and growth conditions on cell disruption by impingement

Data for disruption of C. utilis, S. cerevisiae and B. subtilis cells by impingement of a high velocity jet of suspended cells against a stationary surface are compared. Differences between organisms were observed, but there were no general differences found between yeast and bacteria. In addition, growth conditions were found to have an effect on disruption with cells grown at a high specific growth rate easier to disrupt than cells grown at a low rate.Nomenclature a exponent of pressure (dimensionless) - D dilution rate (h^\s-1) - K dimensional rate constant (Pa ^\s-) - N number of passes (dimensionless) - P operating pressure (Pa) - R fraction of cells disrupted (dimensionless) - u_m maximum specific growth rate (h^\s-1)     

Lactase production in continuous culture by Trichoderma reesei Rut-C30

Summary Trichoderma reesei Rut-C30 was found to produce extracellular lactase when grown on lactose medium. Maximum enzyme levels in continuous culture were observed at dilution rates (D) between 0.02 and 0.027 hr^-1. The enzyme productivity reached 27.3 U/L hr at D = 0.027 hr^-1. Lactase synthesis appears to be inducible and subject to catabolite repression. Optimal growth temperature and pH for enzyme production were 28°C and pH 5. Maximum enzyme activity was observed at 63°C and pH 4.6. The apparent K_m, based on the nitrophenyl-galactopyranoside assay was estimated as 0.4 mM. The enzyme is suitable for lactose hydrolysis in acid whey.     

Continuous high rate production of ethanol by Zymomonas mobilis in an attached film expanded bed fermentor

Summary Experiments were conducted with Zymomonas mobilis in an attached film expanded bed (AFEB) fermentor at different dilution rates, using a feed glucose concentration of 100 gm/l. Vermiculite was used as the bed material for attachment of the bacterial film. Ethanol volumetric productivities were maximum at a dilution rate of 3.6hr^-1. The productivities were 105 gm/l-hr and 210 gm/l-hr based on total fermentor volume and bed volume, respectively.     

A new method for quantifying the probability of segregative plasmid loss inEscherichia coli B/r

Summary A new method for quantifying rates of segregative plasmid loss is described. The probability of plasmid loss is found from the decrease in the fraction of plasmid containing cells, following a synchronous division. Stability inEscherichia coli B/r increased with pH and dilution rate, but decreased with temperature.     

Growth requirements and the establishment of a chemically defined minimal medium inZymomonas mobilis

Summary A chemically defined minimal medium which fulfils the growth requirements of differentZymomonas mobilis strains has been established. The kinetics of ethanol production of the strains ATCC 10988, CU1, CP4 and 11163 grown on the minimal medium at different glucose concentrations were measured. All strains produced ethanol at rates similar to those on complete medium. The minimal medium described is suitable to study spontaneous metabolic deficiciencies and regulation of enzyme activities inZ.mobilis.     

Enhanced plasmid maintenance in a CSTR upon square-wave oscillations in the dilution rate

Summary The effects of forced square-wave perturbations in the dilution rate on plasmid maintenance and gene expression of a population ofEscherichia coli K12 strain carrying the vector plasmid pBR322 grown in a chemostat with a non-selective medium were studied. It was observed that in the control experiments, where the dilution rates were kept constant, the percentage of plasmid-containing cells decreased after a period of time. Eventually, the culture was displaced by the plasmid-free cells. However, when the cells were exposed to forced oscillations in the dilution rate, the reactor culture was able to maintain a mixed population of plasmid-free and plasmid-containing cells for a longer period of time. The above observation seems to be independent of the source of the host cells. That is, the same results were obtained when the plasmid-free cells were generated from the culture itself due to defective partitioning of the plasmids or introduced externally.     

Production of ethanol by adsorbed yeast cells

Summary Various ion exchange resins were tested for their ability to adsorb cells of Saccharomyces cerivisiae with the ultimate intention of developing a packed bed immobilized cell reactor for the continuous production of ethanol. The resins varied greatly in their ability to adsorb cells - the least effective resins retained less than 1 mg S. cerivisiae cells (dry weight)/g of resin (dry weight), and the most effective, 130–140 mg cells/g of resin. A column reactor packed with adsorbed yeast cells was operated continuously for over 200 hours using a 12% (w/v) glucose medium at dilution rates of 1.1 h^-1 and 1.44 h^-1 (based on void volume). High ethanol productivities of 53.1 and 62.0 g ethanol/l-h were obtained.     

Growth of Dioscorea deltoidea at high sugar concentrations

Dioscorea deltoidea cell suspension cultures were grown at initial sucrose concentrations of 35 to 200 g/L. The growth rates were similar (about 0.50 day^–1) with all of the initial sugar concentrations examined. The ratio of fresh weight to dry weight of cells was dependent on the initial sugar concentration, however, it remained fairly constant as long as the sugar was present in the growth medium. These results are different from results recently published, claiming that the growth rate of D. deltoidea cells is dependent on sugar concentration and the fresh weight to dry weight ratio increases throughout growth.     

Osmotic Stress Response: Quantification of Cell Maintenance and Metabolic Fluxes in a Lysine-Overproducing Strain of Corynebacterium glutamicum

Osmotic stress diminishes cell productivity and may cause cell inactivation in industrial fermentations. The quantification of metabolic changes under such conditions is fundamental for understanding and describing microbial behavior during bioprocesses. We quantified the gradual changes that take place when a lysine-overproducing strain of Corynebacterium glutamicum is grown in continuous culture with saline gradients at different dilution rates. The use of compatible solutes depended on environmental conditions; certain osmolites predominated at different dilution rates and extracellular osmolalities. A metabolic flux analysis showed that at high dilution rates C. glutamicum redistributed its metabolic fluxes, favoring energy formation over growth. At low dilution rates, cell metabolism accelerated as the osmolality was steadily increased. Flexibility in the oxaloacetate node proved to be key for the energetic redistribution that occurred when cells were grown at high dilution rates. Substrate and ATP maintenance coefficients increased 30- and 5-fold, respectively, when the osmolality increased, which demonstrates that energy pool management is fundamental for sustaining viability.     

Effect of glucose flow on the acetone butanol fermentation in fed batch culture

Summary Clostridium acetobutylicum was grown in fed-batch cultures at different feeding rates of glucose. The sugar converted to butanol and acetone increased with increasing the glucose flow, on the contrary the conversion to butyric acid was highest at slow glucose feeding rate. The acetic acid concentration was constant at the different flows of glucose. The solventogenesis was not inhibited at high flow of sugar.     

Effects of medium composition and nutrient limitation on loss of the recombinant plasmid pLG669-z and β -galactosidase expression by Saccharomyces cerevisiae

The effects of medium composition, nutrient limitation and dilution rate on the loss of the recombinant plasmid pLG669-z and plasmid-borne β -galactosidase expression were studied in batch and chemostat cultures of Saccharomyces cerevisiae strain CGpLG. The difference in growth rates between plasmid-free and plasmid-containing cells (Δμ) and the rate of segregation (R) were determined and some common factors resulting from the effect of medium composition on plasmid loss were identified. Glucose-limited chemostat cultures of CGpLG grown on defined medium were more stable at higher dilution rates and exhibited Δμ -dominated plasmid loss kinetics. Similar cultures grown on complex medium were more stable at lower dilution rates and exhibited R-dominated plasmid loss kinetics. Overall plasmid stability was greatest in phosphate-limited chemostat cultures grown on defined medium and was least stable in magnesium-limited cultures grown on defined medium. Δμ decreased and R increased with increased dilution rate, irrespective of medium composition. Increased plasmid loss rates at high or low dilution rates would appear to be characteristic of loss kinetics dominated by R or Δμ, respectively. Growth of glucose-limited chemostat cultures on complex medium decreased Δμ values but increased R values, in comparison to those cultures grown on defined medium. Any increased stability that a complex medium-induced reduction of Δμ may have conferred was counteracted by an increased R value. Increased β-galactosidase productivity was correlated with increased plasmid stability only in glucose-limited chemostat cultures grown on defined medium and not in those grown on complex medium. Previous studies have yielded contrasting responses with regard to the effect of dilution rate on recombinant plasmid loss from S. cerevisiae. Our findings can account for these differences and may be generally valid for the stability of similar yeast plasmid constructs. This information would facilitate the design of bioprocesses, where recombinant plasmid instability results in reduced culture productivity. Received 08 July 1996/ Accepted in revised form 14 January 1997     

Establishment and characterization of photoautotrophic protoplast-derived cultures ofNicotiana plumbaginifolia

A procedure is described for the rapid establishment of photoautotrophic protoplast-derived cultures ofNicotiana plumbaginifolia. Photoautotrophic growth was induced by lowering the glucose concentration to 2.5 g.l^–1 in the protoplast culture medium and by omitting glucose from the subsequent dilution medium. Four week-old highly viable suspensions were plated on an agar-medium without glucose in unsealed Petri dishes and kept in illuminated chambers flushed with 0.05 % or 2 % CO₂. Air-grown calli had net photosynthesis rates of 1.8 and 17 moles CO₂.g^–1 fresh wt.h^–1 in air at 0.034 % CO₂ and in air enriched with 1 % CO₂, respectively. Calli grown in 2 % CO₂ exhibited lower rates of net photosynthesis at the two CO₂ concentrations tested (0 and 7.5 moles CO₂.g^–1 fresh wt.h^–1, respectively). The contribution of photosynthesis to growth was estimated to be 80 % in air-grown calli and more than 90 % in calli grown in 2 % CO₂. The suitability of this photoautotrophic culture procedure is discussed with regard to the screening of photosynthetic mutants or transformants from protoplasts.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - IAA indoleacetic acid - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase     

Photoproduction of H2 and NADPH2 by polyurethane-immobilized cyanobacteria

Summary Cyanobacterial cells were grown in polyurethane foams (polyester or polyvinyl types). Chlorogloea fritschii, Nostoc muscorum and Mastigocladus laminosus remained immobilized in the foams and were used for continuous photoproduction of H₂ from ascorbate with methyl viologen (MV) and hydrogenase or Pt catalysts, for periods in excess of 9 days. Foam-immobilized N. muscorum continuously photoreduced exogenous NADP for at least 24 h in the presence of Fd-NADP reductase with ascorbate as electron donor.     

Amino acid consumption by Chloroflexus aurantiacus in batch and continuous cultures

Amino acid consumption was studied with batch and continuous chemostat cultures of Chloroflexus aurantiacus grown phototrophically in complex medium with casamino acids (Pierson and Castenholz 1974). Amino acids like Arg, Asx, Thr, Ala, Tyr, which were utilized during the early exponential phase by cells grown in batch cultures were consumed in chemostat cultures essentially at any of the dilution rates employed (0.018–0.104 h^-1). Those amino acids which were taken up during subsequent phases of growth were consumed in chemostat cultures preferentially at low dilution rates. For example, the consumption of Glx was enhanced during the late exponential phase and at low dilution rates. At high dilution rates Glx was not consumed at all. Since Glx utilization largely paralleled bacteriochlorophyll formation, it is discussed that formation of the photopigment depends on the intracellular availability of Glu as the exclusive precursor for tetrapyrrole synthesis.     

The effect of streptomycin and hypotonicity on survival of Pseudomonas aeruginosa

Summary P. aeruginosa proliferates well in a water environment; however, when subjected to high doses of streptomycin or gentamicin, the residual viable bacteria are killed by moderate water dilution of their media. These results lead to the suggestion that the mechanism of lethal action of aminoglycosides may operate through interference with the water balance system of the P. aeruginosa.     

Yeast cells entrapped in low-gelling temperature agarose for the continuous production of ethanol

Summary Continuous ethanol production byS. uvarum immobilized in a low-gelling temperature agarose namely SeaPlaque agarose was studied in a packed bed reactor at 30°C using sugarcane molasses containing 13.5% fermentable sugars as feed. The productivity at 95% conversion was 23 g/l.h (on reactor volume basis). The bioreactor was run continuously at a fixed dilution rate and it retained 60% of its initial activity upto 80 days.