Kinetic characterization and fed-batch fermentation for maximal simultaneous production of esterase and protease from Lysinibacillus fusiformis AU01

The simultaneous production of intracellular esterase and extracellular protease from the strain Lysinibacillus fusiformis AU01 was studied in detail. The production was performed both under batch and fed-batch modes. The maximum yield of intracellular esterase and protease was obtained under full oxygen saturation at the beginning of the fermentation. The data were fitted to the Luedeking–Piret model and it was shown that the enzyme (both esterase and protease) production was growth associated. A decrease in intracellular esterase and increase in the extracellular esterase were observed during late stationary phase. The appearance of intracellular proteins in extracellular media and decrease in viable cell count and biomass during late stationary phase confirmed that the presence of extracellular esterase is due to cell lysis. Even though the fed-batch fermentation with different feeding strategies showed improved productivity, feeding yeast extract under DO-stat fermentation conditions showed highest intracellular esterase and protease production. Under DO-stat fed-batch cultivation, maximum intracellular esterase activity of 820?×?10³ U/L and extracellular protease activity of 172?×?10³ U/L were obtained at the 16th?hr. Intracellular esterase and extracellular protease production were increased fivefold and fourfold, respectively, when compared to batch fermentation performed under shake flask conditions.     

Monitoring growth of Escherichia coli using a two-channel optical sensor

Summary The growth of Escherichia coli strain TG 1 was monitored, measuring simultaneously the culture fluorescence and the 360° reflection at 578 nm with a two-channel optical sensor. It was observed that the culture fluorescence at 366 nm excitation was approximately three times higher than the NADH fluorescence of washed E. coli cells whereas the 360° reflection at 578 nm was comparable. The reason for this effect was found to be the accumulation of riboflavin in the cultivation liquid of the E. coli cells being equal to approximately 0.05 mg/g biomass. In shaken batch cultivations of the same strain the amount of riboflavin in the cell-free cultivation liquid correlated with the biomass being a very sensitive indicator of E. coli growth.Correspondence to: W. S. Kunz     

Physiological characterisation of recombinant Aspergillus nidulans strains with different creA genotypes expressing A. oryzae alpha-amylase.

The physiology of three strains of Aspergillus nidulans was examined--a creA deletion strain, a wild type creA genotype and a strain containing extra copies of the creA gene, all producing Aspergillus oryzae alpha-amylase. The strains were cultured in batch and continuous cultivations and the biomass formation and alpha-amylase production was characterised. Overexpression of the creA gene resulted in a lower maximum specific growth rate and a slightly higher repression of the alpha-amylase production during conditions with high glucose concentration. No expression of creA also resulted in a decreased maximum specific growth rate, but also in drastic changes in morphology. Furthermore, the expression of alpha-amylase was completely derepressed and creA thus seems to be the only regulatory protein responsible for glucose repression of alpha-amylase expression. The effect of different carbon sources on the alpha-amylase production in the creA deletion strain was investigated and it was found that starch was the best inducer. The degree of induction by starch increased almost linearly with the concentration of starch in starch/glucose mixtures. High-density batch cultivation was performed with the creA deletion strain and a final titre of 6.0 g l(-1) of alpha-amylase was reached after 162 h of cultivation.     

Physiology of the growth and supersynthesis of DNA-polymerase in Escherichia coli during 2-stage continuous cultivation

The physiology of growth under the conditions of batch and continuous cultivation was studied with the recombinant strain of Escherichia coli CM 5199 capable of DNA polymerase I superproduction. The specific growth rate of the strain is 0.8 h-1 under the conditions of continuous cultivation which is almost 2.5 times greater than that in the exponential phase of batch cultivation. When the strain was cultivated at a flow rate above 0.3 h-1, the biomass concentration in the fermenter decreased and the culture was no more limited by the carbon source in the absence of other growth limiting components of the medium. Apparently, the metabolic product ceased to inhibit high growth rates of the culture under the conditions of continuous cultivation. The rate of DNA polymerase synthesis correlated with the specific growth rate and the respiration activity of the culture when the lambda pol A prophage was induced in the cells. The authors discuss the effectiveness of ribosome operation in the cells at a growth rate of 0.05 to 0.3 h-1 and the content of ribosomes at a higher growth rate in relation to DNA polymerase I synthesis.     

Chlorella pyrenoidosa 7-11-05 in batch and in homogeneous continuous culture under autotrophic conditions

The growth ofChlorella pyrenoidosa 7-11-05 in batch and in continuous culture using two types of cultivation vessels was compared. The high values of specific growth rate obtained in batch deep stirred tank culture containing small amount of inoculum are not easily attainable in continuous culture with regard to the high number of autospores formed throughout the ontogenetic cycle ofChlorella. The obtained surface and volume productivities prove that in both types of cultivation vessels a high growth intensity can be achieved. The deep tank perfectly mixed cultivation may become of significance in the future.     

Continuous culture with complete cell recycle to obtain high cell densities in product inhibited cultures; cultivation ofStreptococcus lactis for production of superoxide dismutase

Summary A cultivation system is described for cultivatingStreptococcus lactis in continuous culture with complete cell recycle. The aim was to obtain high cell densities for the production of su-peroxide dismutase (SOD) while avoiding the growth inhibiting effects of the lactic acid produced. This type of cultivation was performed both at constant and at increasing dilution rates. Comparisons made include those between cell mass productivity and SOD productivity in recycling cultivations and batch cultivations. In the recycling cultivation at increasing dilution rates a cell mass of 19 g/1 was obtained after 22 h of cultivation and the SOD productivity was 43.10³ U/1.h which is four times higher than for batch cultivations. The effect of recyclingS. lactis was also considered and no damage of the microorganisms was observed.     

Semi‐continuous in situ magnetic separation for enhanced extracellular protease production—modeling and experimental validation

In modern biotechnology proteases play a major role as detergent ingredients. Especially the production of extracellular protease by Bacillus species facilitates downstream processing because the protease can be directly harvested from the biosuspension. In situ magnetic separation (ISMS) constitutes an excellent adsorptive method for efficient extracellular protease removal during cultivation. In this work, the impact of semi‐continuous ISMS on the overall protease yield has been investigated. Results reveal significant removal of the protease from Bacillus licheniformis cultivations. Bacitracin‐functionalized magnetic particles were successfully applied, regenerated and reused up to 30 times. Immediate reproduction of the protease after ISMS proved the biocompatibility of this integrated approach. Six subsequent ISMS steps significantly increased the overall protease yield up to 98% because proteolytic degradation and potential inhibition of the protease in the medium could be minimized. Furthermore, integration of semi‐continuous ISMS increased the overall process efficiency due to reduction of the medium consumption. Process simulation revealed a deeper insight into protease production, and was used to optimize ISMS steps to obtain the maximum overall protease yield. Biotechnol. Bioeng. 2013; 110: 2161–2172. © 2013 Wiley Periodicals, Inc.     

Intracellular pH-based controlled cultivation of yeast cells: II. cultivation methodology

Intracellular pH (pH(i)) was measured on-line in a bioreactor using a fluorescent pH(i) indicator, 9-aminoacridine, and controlled fed-batch cultivations of yeast cells based on pH(i) (FB-pH(i)) were performed. In FB-pH(i) cultivations, automated glucose additions were made to the culture in response to culture pH(i). The average ethanol (an-aerobic product) yield was significantly lower [0.12 g g(-1) glucose in fed-batch pH(i) cultivations with 100 ppm glucose additions (FB-pH(i)-100 cultivation) vs. 0.48 g g(-1) glucose in batch] and cell yield was higher (0.54 g g(-1) glucose in FB-pH(i)-100 cultivation vs. 0.3 g g(-1) glucose in batch) compared to batch cultivation. An expression has been derived to calculate changes in pH(i) from measured fluorescence values when the cell concentration increases during growth. Cultivations based on pH(i), performed with different magnitudes of glucose addition (100, 50, and 10 ppm additions), showed that lower magnitudes of glucose addition resulted in lower ethanol yields while cell yield remained unaffected. The ratio of specific oxygen uptake rate to specific glucose uptake rate (OUR/GUR) increased with decreased in magnitude of glucose additions in FB-pH(i) cultivations, suggesting that the culture aerobic state was higher when the magnitude of glucose addition was lower. The average cell productivity in FB-pH(i) cultivations was 29% higher than in batch cultivation. Cells were also cultivated at high OUR conditions, and the results are compared with other cultivations. (c) 1993 John Wiley & Sons, Inc.     

Glucoamylase production in batch, chemostat and fed-batch cultivations by an industrial strain of Aspergillus niger

The Aspergillus niger strain BO-1 was grown in batch, continuous (chemostat) and fed-batch cultivations in order to study the production of the extracellular enzyme glucoamylase under different growth conditions. In the pH range 2.5–6.0, the specific glucoamylase productivity and the specific growth rate of the fungus were independent of pH when grown in batch cultivations. The specific glucoamylase producivity increased linearly with the specific growth rate in the range 0–0.1 h⁻¹ and was constant in the range 0.1–0.2 h⁻¹. Maltose and maltodextrin were non-inducing carbon sources compared to glucose, and the maximum specific growth rate was 0.19 ± 0.02 h⁻¹ irrespective of whether glucose or maltose was the carbon source. In fed-batch cultivations, glucoamylase titres of up to 6.5 g l⁻¹ were obtained even though the strain contained only one copy of the glaA gene. Received: 5 May 1999 / Received revision: 7 September 1999 / Accepted: 17 September 1999     

Continuous cultivation of recombinant Escherichia coli: Existence of an optimum dilution rate for maximum plasmid and gene product concentration

Growth yield factors, plasmid stability, cellular plasmid content, and cloned gene product activity for Escherichia coli HB101 containing plasmid pDM246 were measured at several dilution rates in continuous culture. Cell mass yield per mass of glucose consumed declined with increasing dilution rate. There was no evidence of plasmid segregational instability in any experiments, none of which employed selective medium. Plasmid content per cell varied with population-specific growth rate as observed in earlier batch experiments with the same strain. Plasmid content declined with increasing specific growth rate following indication of a maximum number of plasmids per cell at specific growth rates of ca. 0.3 h(-1). Cloned gene product (beta-lactamase) activity exhibited a sharp maximum with respect to dilution rate in continuous culture. Qualitatively different results were observed in previous experiments in batch cultivation in which specific growth rate changes were effected by altering medium composition.     

Cultivation of Thermoanaerobium brockii in continuous culture with complete cell recycling

The growth behaviour of the thermophilic anaerobic bacterium Thermoanaerobium brockii for the production of its intracellular secondary alcohol dehydrogenase (sADH) has been studied in batch cultures as well as in continuous cultivation with complete cell recycling. In batch culture the maximum specific growth rate, μ_MAX, was 0·5 h⁻¹, resulting in a cell density of 1·2 g l⁻¹ and an sADH activity of 1·3 units ml⁻¹. Higher glucose concentrations resulted in a decrease in ep cf7 max rs, enzyme productivity as well as biomass yield although an increase in total biomass was achieved. To improve cell density and productivity, continuous culture with complete cell recycling was used, resulting in an increase in cell density by 5 times and in productivity of the sADH by 3 times in comparison to those obtained in batch culture.     

Feather degradation by Kocuria rosea in submerged culture

A strain of Kocuria rosea with keratinolytic activity was studied. In batch culture, the optimum temperature for feather degradation, bacterial growth and protease secretion was at 40 °C. A specific growth rate of 0.17 h⁻¹ was attained in basal medium with feathers as fermentation substrate. Under these conditions, after 36 h of incubation, biomass and caseinolytic activity reached 3.2 g/l and 0.15 U/ml, respectively. Extracellular protease secretion was associated with the exponential growth phase. In batch fermentation, feather degradation up to 51% in 72 h was obtained with a conversion yield in biomass of 0.32 g/g. No organic acids were detected in the fermentation broth in significant amount. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production of cell-bound and vesicle-associated trypsin-like protease, alkaline phosphatase and N-acetyl-beta-glucosaminidase by Bacteroides gingivalis strain W50

Bacteroides gingivalis strain W50 was grown in batch and continuous culture on complex medium with haemin. In batch culture, cell-bound levels of trypsin-like protease (EC 3.4.21.4), alkaline phosphatase (EC 3.1.3.1) and N-acetyl-beta-glucosaminidase (EC 3.2.1.30) increased during the exponential phase of growth. These enzyme activities were also detected in extracellular vesicles and in extracellular soluble forms in the supernatant fluid, but in lower amounts per unit biomass compared to cell-bound levels. In continuous culture, at high relative growth rates (0.7-0.9 murel), the highest proportions of enzyme activities were cell-bound. In contrast, at low relative growth rates (0.1-0.2 murel), highest enzyme levels were detected in the extracellular vesicle fraction. Levels of extracellular soluble enzymes were always low compared to cell-bound or extracellular vesicle levels, but were highest at low relative growth rates. All three enzymes appeared to be relatively stable in their soluble forms. Vesicle production appeared to be associated with actively growing cells but was influenced by growth rate. The results are consistent with the hypothesis that cell-bound 'periplasmic' enzymes are encapsulated into vesicles which are subsequently released by the cells. Therefore, levels of total extracellular enzyme (extracellular vesicle plus extracellular soluble) may depend on the rate of vesicle formation superimposed on the rates of production of 'periplasmic' enzymes in the cell.     

Ethanol production from xylose by Thermoanaerobacter ethanolicus in batch and continuous culture

The fermentation of xylose by Thermoanaerobacter ethanolicus ATCC 31938 was studied in pH-controlled batch and continuous cultures. In batch culture, a dependency of growth rate, product yield, and product distribution upon xylose concentration was observed. With 27 mM xylose media, an ethanol yield of 1.3 mol ethanol/mol xylose (78% of maximum theoretical yield) was typically obtained. With the same media, xylose-limited growth in continuous culture could be achieved with a volumetric productivity of 0.50 g ethanol/liter h and a yield of 0.42 g ethanol/g xylose (1.37 mol ethanol/mol xylose). With extended operation of the chemostat, variation in xylose uptake and a decline in ethanol yield was seen. Instability with respect to fermentation performance was attributed to a selection for mutant populations with different metabolic characteristics. Ethanol production in these T. ethanolicus systems was compared with xylose-to-ethanol conversions of other organisms. Relative to the other systems, T. ethanolicus offers the advantages of a high ethanol yield at low xylose concentrations in batch culture and of a rapid growth rate. Its disadvantages include a lower ethanol yield at higher xylose concentrations in batch culture and an instability of fermentation characteristics in continuous culture.     

Establishing a Fed‐Batch Process for Protease Expression with Bacillus licheniformis in Polymer‐Based Controlled‐Release Microtiter Plates

Introducing fed‐batch mode in early stages of development projects is crucial for establishing comparable conditions to industrial fed‐batch fermentation processes. Therefore, cost efficient and easy to use small‐scale fed‐batch systems that can be integrated into existing laboratory equipment and workflows are required. Recently, a novel polymer‐based controlled‐release fed‐batch microtiter plate is described. In this work, the polymer‐based controlled‐release fed‐batch microtiter plate is used to investigate fed‐batch cultivations of a protease producing Bacillus licheniformis culture. Therefore, the oxygen transfer rate (OTR) is online‐monitored within each well of the polymer‐based controlled‐release fed‐batch microtiter plate using a µRAMOS device. Cultivations in five individual polymer‐based controlled‐release fed‐batch microtiter plates of two production lots show good reproducibility with a mean coefficient of variation of 9.2%. Decreasing initial biomass concentrations prolongs batch phase while simultaneously postponing the fed‐batch phase. The initial liquid filling volume affects the volumetric release rate, which is directly translated in different OTR levels of the fed‐batch phase. An increasing initial osmotic pressure within the mineral medium decreases both glucose release and protease yield. With the volumetric glucose release rate as scale‐up criterion, microtiter plate‐ and shake flask‐based fed‐batch cultivations are highly comparable. On basis of the small‐scale fed‐batch cultivations, a mechanistic model is established and validated. Model‐based simulations coincide well with the experimentally acquired data.     

Fluorene Cometabolism by Rhodococcus rhodochrous and Pseudomonas fluorescens Cultures

The transformation of fluorene by Rhodococcus rhodochrous strain 172 grown on sucrose and Pseudomonas fluorescens strain 26K grown on glycerol was studied as a function of the substrate concentration and the growth phase. Under certain cultivation conditions, fluorene was completely consumed from the medium. The specific transformation rate of fluorene was considerably higher when it was transformed in the presence of the cosubstrates than when it served as the sole carbon source. An approach to the evaluation of the specific transformation rate of fluorene during batch cultivations is proposed.     

Microalgae-mediated brewery wastewater treatment: effect of dilution rate on nutrient removal rates,biomass biochemical composition,and cell physiology

Microalgae have been used to remove nitrogen, phosphorus, and chemical oxygen demand (COD) from brewery wastewater (BWW). The microalga Scenedesmus obliquus was grown on BWW, using bubble column photobioreactors that operated under batch and continuous regimes. For the first time, the cell physiological status cell membrane integrity and enzymatic activity was monitored during the microalgae based BWW treatment, using flow cytometry. All the cultivations batch and continuous displayed a proportion of cells with intact membrane >?87%, although the continuous cultivations displayed a lower proportion of cells with enzymatic activity (20–40%) than the batch cultivations (97%). The dilution rate of 0.26 day^?1 was the most favorable condition, since the microalgae cultivation attained the maximum biomass productivity (0.2 g ash-free dry weight day^?1) and the total nitrogen and COD removal rates were the highest (97 and 74%, respectively), while the phosphorous removal rate was the third (23%).     

Experimental bioenergetics ofSaccharomyces cerevisiae in respiration and fermentation

Aerobic growth of Saccharomyces cerevisiae on glucose was investigated, focusing on the heat evolution as it relates to biomass and ethanol synthesis. “Aerobic fermentation” and “aerobic respiration” were established respectively in the experimental system by performing batch and fed-batch experiments. “Balanced growth” batch cultivations were carried out with initial sugar concentrations ranging from 10 to 70 g/L, resulting in different degrees of catabolite repression. The fermentative heat generation was continuously monitored in addition to the key culture parameters such as ethanol production rate, CO₂ evolution rate, O₂ uptake rate, specific growth rate, and sugar consumption rate. The respective variations of the above quantities reflecting the variations in the catabolic activity of the culture were studied. This was done in order to evaluate the microbial regulatory system, the energetics of microbial growth including the rate of heat evolution and the distribution of organic substrate between respiration and fermentation. This study was supported by closing C, energy, and electron balances on the system. The comparison of the fractions of substrate energy evolved as heat (δ_h) with the fraction of available electrons transferred to oxygen (?_O2) indicated equal values of the two (0.46) in the aerobic respiration (fed-batch cultivation). However, the glucose effect in batch cultivations resulted in smaller ?_O2 than δ_h, while both values decreased in their absolute values. The evaluation of the heat energetic yield coefficients, together with the fraction of the available electrons transferred to O, contributed to the estimation of the extent of heat production through oxidative phosphorylation.     

Oxygen enriched air supply in Escherichia coli processes: production of biomass and recombinant human growth hormone

In order to investigate the impact of high oxygen and carbon dioxide concentrations, Escherichia coli was grown in batch cultivations where the air supply was enriched with either oxygen or carbon dioxide. The effect of elevated concentrations of oxygen and carbon dioxide on stochiometric and kinetic constants was studied this way. The maximum growth rate was significantly reduced, the production of acetic acid and the biomass yield coefficient on glucose increased in cultures with carbon dioxide enriched air, compared to reference cultivations and cultivations with oxygen enriched air. The application of oxygen enriched air was studied in high cell density cultivations of Escherichia coli. Two production processes were chosen to investigate the impact of oxygen enrichment. Biomass concentration, specific growth rate, yield coefficient, respiration, mixed acid fermentation products and the product yield and quality for the recombinant product were investigated. First, a process for the production of biomass was investigated. Exponential growth could proceed for a longer time and higher growth rates could be maintained with oxygen enriched air supply. However, a higher specific oxygen consumption rate per glucose was measured after the start of the oxygen enrichment, indicating higher maintenance and consequently the growth rate and yield coefficient decreased drastically in the end of the process. Second, a process for the production of recombinant human growth hormone (rhGH) was investigated. Although the glucose feed rate and all medium components were doubled, the amount of produced biomass could only be increased by 77% when oxygen enriched air (40% oxygen) supply was applied. This was due to a decreased yield coefficient of biomass per glucose. The total amount of produced product was decreased by almost 50% compared to the control, although less proteolytically degraded variants were produced.     

Thermus 2S from Thai hot springs: isolation and immobilization

Three strains of thermophilic bacteria producing extracellular protease have been isolated from hot springs in Chiang Mai, Thailand. The bacterium producing the highest amount of protease has been selected and identified as belonging to the genusThermus, and is tentatively calledThermus 2S. The isolate is a Gram-negative, rod shaped bacterium. It exhibited maximum growth around 60°C at pH 7. Entrapment of the microbial cells in calcium alginate maintained the cell viability. Protease production from immobilized cells using 2 g wet cells per 10 ml 3% (w/v) sodium alginate was higher than that from a free-cell system using 2% inoculum.