Niels Bang Siemsen Jensen - Academia.edu (original) (raw)
Uploads
Papers by Niels Bang Siemsen Jensen
Applied and Environmental Microbiology, Jun 1, 2003
Lactococcus lactis subsp. lactis MG1363 was grown in batch cultures on a defined medium with gluc... more Lactococcus lactis subsp. lactis MG1363 was grown in batch cultures on a defined medium with glucose as the energy source under different aeration conditions, namely, anaerobic conditions, aerobic conditions, and microaerobic conditions with a dissolved oxygen tension of 5% (when saturation with air was used as the reference). The maximum specific growth rate was high (0.78 to 0.91 h ؊1) under all aeration conditions but decreased with increasing aeration, and more than 90% of the glucose was converted to lactate. However, a shift in by-product formation was observed. Increasing aeration resulted in acetate, CO 2 , and acetoin replacing formate and ethanol as end products. Under microaerobic conditions, growth came to a gradual halt, although more than 60% of the glucose was still left. A decline in growth was not observed during microaerobic cultivation when acetate was added to the medium. We hypothesize that the decline in growth was due to a lack of acetyl coenzyme A (acetyl-CoA) needed for fatty acid synthesis since acetyl-CoA can be synthesized from acetate by means of acetate kinase and phosphotransacetylase activities. MATERIALS AND METHODS Strain and growth conditions. The bacterium used throughout this study was the homofermentative laboratory strain L. lactis subsp. lactis MG1363 (14). This microorganism was grown in in-house bioreactors with a working volume of 1.0 liter under anaerobic and aerobic conditions or in an MBR bioreactor (MBR Bio Reactor AG, Wetzikon, Switzerland) with a working volume of 1.5 liters under microaerobic conditions. All cultures were incubated at 30°C, and the baffled bioreactors were fitted with four-blade Rushton turbines rotating at 350 rpm. A constant pH of 6.6 was maintained by automatic addition of 10 M KOH. Unless stated otherwise, the cells were grown in defined MS10 medium (7) supplemented with the following components to allow growth under aerobic conditions: MnSO 4 (1.25 ϫ 10 Ϫ5 g ⅐ liter Ϫ1), thiamine (1 mg ⅐ liter Ϫ1), and DL-6,8-thioctic acid (2.5 mg ⅐ liter Ϫ1). The glucose concentration was 10 g ⅐
Biotechnology and Bioengineering, May 5, 1999
An experimental procedure for the determination of intracellular concentrations of the phosphoryl... more An experimental procedure for the determination of intracellular concentrations of the phosphorylated sugars in the lactic acid bacterium Lactococcus lactis is presented. The first step of the procedure is a rapid sampling of a small volume of the growth medium into 60% (v/v) methanol precooled to -35 degrees C, bringing about a fast and complete stop of all metabolic activity. In contrast to yeast the metabolites leak out of the cells when these are brought into contact with methanol and are present in the medium and in the biomass after the quenching. A liquid-liquid extraction with chloroform at -25 degrees C ensures a total permeability of the cellular membrane towards the metabolites of interest as well as the inactivation of enzymes liable to alter their levels. The final step of the procedure consists in a solid phase extraction using columns with a high affinity for phosphorylated components. The internal standard was recovered to an extent of 85-95%.
Biotechnology and Bioengineering, Feb 12, 2002
Uniformly 13 C labeled glucose was fed to a lactic acid bacterium growing on a de®ned medium supp... more Uniformly 13 C labeled glucose was fed to a lactic acid bacterium growing on a de®ned medium supplemented with all proteinogenic amino acids except glutamate. Aspartate stemming from the protein pool and from the extracellular medium was enriched with 13 C disclosing a substantial de novo biosynthesis of this amino acid simultaneous to its uptake from the growth medium and a rapid exchange¯ux of aspartate over the cellular membrane. Phenylalanine, alanine, and threonine were also synthesized de novo in spite of their presence in the growth medium.
Applied and Environmental Microbiology, Jun 1, 2001
Minute amounts of oxygen were supplied to a continuous cultivation of Lactococcus lactis subsp. c... more Minute amounts of oxygen were supplied to a continuous cultivation of Lactococcus lactis subsp. cremoris MG1363 grown on a defined glucose-limited medium at a dilution rate of 0.1 h ؊1. More than 80% of the carbon supplied with glucose ended up in fermentation products other than lactate. Addition of even minute amounts of oxygen increased the yield of biomass on glucose by more than 10% compared to that obtained under anaerobic conditions and had a dramatic impact on catabolic enzyme activities and hence on the distribution of carbon at the pyruvate branch point. Increasing aeration caused carbon dioxide and acetate to replace formate and ethanol as catabolic end products while hardly affecting the production of either acetoin or lactate. The negative impact of oxygen on the synthesis of pyruvate formate lyase was confirmed. Moreover, oxygen was shown to down regulate the protein level of alcohol dehydrogenase while increasing the enzyme activity levels of the pyruvate dehydrogenase complex, ␣-acetolactate synthase, and the NADH oxidases. Lactate dehydrogenase and glyceraldehyde dehydrogenase enzyme activity levels were unaffected by aeration.
Biotechnology and Bioengineering, 2001
Applied and Environmental Microbiology, 2001
Minute amounts of oxygen were supplied to a continuous cultivation of Lactococcus lactis subsp. c... more Minute amounts of oxygen were supplied to a continuous cultivation of Lactococcus lactis subsp. cremoris MG1363 grown on a defined glucose-limited medium at a dilution rate of 0.1 h−1. More than 80% of the carbon supplied with glucose ended up in fermentation products other than lactate. Addition of even minute amounts of oxygen increased the yield of biomass on glucose by more than 10% compared to that obtained under anaerobic conditions and had a dramatic impact on catabolic enzyme activities and hence on the distribution of carbon at the pyruvate branch point. Increasing aeration caused carbon dioxide and acetate to replace formate and ethanol as catabolic end products while hardly affecting the production of either acetoin or lactate. The negative impact of oxygen on the synthesis of pyruvate formate lyase was confirmed. Moreover, oxygen was shown to down regulate the protein level of alcohol dehydrogenase while increasing the enzyme activity levels of the pyruvate dehydrogenase...
Biotechnology and Bioengineering, 2002
Uniformly 13 C labeled glucose was fed to a lactic acid bacterium growing on a de®ned medium supp... more Uniformly 13 C labeled glucose was fed to a lactic acid bacterium growing on a de®ned medium supplemented with all proteinogenic amino acids except glutamate. Aspartate stemming from the protein pool and from the extracellular medium was enriched with 13 C disclosing a substantial de novo biosynthesis of this amino acid simultaneous to its uptake from the growth medium and a rapid exchange¯ux of aspartate over the cellular membrane. Phenylalanine, alanine, and threonine were also synthesized de novo in spite of their presence in the growth medium. ã
Applied and Environmental Microbiology, 2003
Lactococcus lactis subsp. lactis MG1363 was grown in batch cultures on a defined medium with gluc... more Lactococcus lactis subsp. lactis MG1363 was grown in batch cultures on a defined medium with glucose as the energy source under different aeration conditions, namely, anaerobic conditions, aerobic conditions, and microaerobic conditions with a dissolved oxygen tension of 5% (when saturation with air was used as the reference). The maximum specific growth rate was high (0.78 to 0.91 h−1) under all aeration conditions but decreased with increasing aeration, and more than 90% of the glucose was converted to lactate. However, a shift in by-product formation was observed. Increasing aeration resulted in acetate, CO2, and acetoin replacing formate and ethanol as end products. Under microaerobic conditions, growth came to a gradual halt, although more than 60% of the glucose was still left. A decline in growth was not observed during microaerobic cultivation when acetate was added to the medium. We hypothesize that the decline in growth was due to a lack of acetyl coenzyme A (acetyl-CoA) n...
Applied and Environmental Microbiology, 2003
Lactococcus lactis subsp. lactis MG1363 was grown in batch cultures on a defined medium with gluc... more Lactococcus lactis subsp. lactis MG1363 was grown in batch cultures on a defined medium with glucose as the energy source under different aeration conditions, namely, anaerobic conditions, aerobic conditions, and microaerobic conditions with a dissolved oxygen tension of 5% (when saturation with air was used as the reference). The maximum specific growth rate was high (0.78 to 0.91 h−1) under all aeration conditions but decreased with increasing aeration, and more than 90% of the glucose was converted to lactate. However, a shift in by-product formation was observed. Increasing aeration resulted in acetate, CO2, and acetoin replacing formate and ethanol as end products. Under microaerobic conditions, growth came to a gradual halt, although more than 60% of the glucose was still left. A decline in growth was not observed during microaerobic cultivation when acetate was added to the medium. We hypothesize that the decline in growth was due to a lack of acetyl coenzyme A (acetyl-CoA) n...
Biotechnology and Bioengineering, 2001
The pyruvate metabolism in the lactic acid bacterium Lactococcus lactis was studied in anaerobic ... more The pyruvate metabolism in the lactic acid bacterium Lactococcus lactis was studied in anaerobic cultures under transient conditions. During growth of L. lactis in continuous culture at high dilution rate, homolactic product formation was observed, i.e., lactate was produced as the major end product. At a lower dilution rate, the pyruvate metabolism shifted towards mixed acidproduct formation where formate, acetate, and ethanol were produced in addition to lactate. The regulation of the shift in pyruvate metabolism was investigated by monitoring the dynamic behavior of L. lactis in continuous cultures subjected to step changes in dilution rate. Both shift-up and shift-down experiments were carried out, and these experiments showed that the enzyme pyruvate formate-lyase (PFL) plays a key role in the regulation of the shift. Pyruvate formate-lyase in vivo activity was regulated both at the level of gene expression and by allosteric modulation of the enzyme. A simple mathematical model was proposed to estimate the relative significance of the regulatory mechanisms involved.
Applied and Environmental Microbiology, Jun 1, 2003
Lactococcus lactis subsp. lactis MG1363 was grown in batch cultures on a defined medium with gluc... more Lactococcus lactis subsp. lactis MG1363 was grown in batch cultures on a defined medium with glucose as the energy source under different aeration conditions, namely, anaerobic conditions, aerobic conditions, and microaerobic conditions with a dissolved oxygen tension of 5% (when saturation with air was used as the reference). The maximum specific growth rate was high (0.78 to 0.91 h ؊1) under all aeration conditions but decreased with increasing aeration, and more than 90% of the glucose was converted to lactate. However, a shift in by-product formation was observed. Increasing aeration resulted in acetate, CO 2 , and acetoin replacing formate and ethanol as end products. Under microaerobic conditions, growth came to a gradual halt, although more than 60% of the glucose was still left. A decline in growth was not observed during microaerobic cultivation when acetate was added to the medium. We hypothesize that the decline in growth was due to a lack of acetyl coenzyme A (acetyl-CoA) needed for fatty acid synthesis since acetyl-CoA can be synthesized from acetate by means of acetate kinase and phosphotransacetylase activities. MATERIALS AND METHODS Strain and growth conditions. The bacterium used throughout this study was the homofermentative laboratory strain L. lactis subsp. lactis MG1363 (14). This microorganism was grown in in-house bioreactors with a working volume of 1.0 liter under anaerobic and aerobic conditions or in an MBR bioreactor (MBR Bio Reactor AG, Wetzikon, Switzerland) with a working volume of 1.5 liters under microaerobic conditions. All cultures were incubated at 30°C, and the baffled bioreactors were fitted with four-blade Rushton turbines rotating at 350 rpm. A constant pH of 6.6 was maintained by automatic addition of 10 M KOH. Unless stated otherwise, the cells were grown in defined MS10 medium (7) supplemented with the following components to allow growth under aerobic conditions: MnSO 4 (1.25 ϫ 10 Ϫ5 g ⅐ liter Ϫ1), thiamine (1 mg ⅐ liter Ϫ1), and DL-6,8-thioctic acid (2.5 mg ⅐ liter Ϫ1). The glucose concentration was 10 g ⅐
Biotechnology and Bioengineering, May 5, 1999
An experimental procedure for the determination of intracellular concentrations of the phosphoryl... more An experimental procedure for the determination of intracellular concentrations of the phosphorylated sugars in the lactic acid bacterium Lactococcus lactis is presented. The first step of the procedure is a rapid sampling of a small volume of the growth medium into 60% (v/v) methanol precooled to -35 degrees C, bringing about a fast and complete stop of all metabolic activity. In contrast to yeast the metabolites leak out of the cells when these are brought into contact with methanol and are present in the medium and in the biomass after the quenching. A liquid-liquid extraction with chloroform at -25 degrees C ensures a total permeability of the cellular membrane towards the metabolites of interest as well as the inactivation of enzymes liable to alter their levels. The final step of the procedure consists in a solid phase extraction using columns with a high affinity for phosphorylated components. The internal standard was recovered to an extent of 85-95%.
Biotechnology and Bioengineering, Feb 12, 2002
Uniformly 13 C labeled glucose was fed to a lactic acid bacterium growing on a de®ned medium supp... more Uniformly 13 C labeled glucose was fed to a lactic acid bacterium growing on a de®ned medium supplemented with all proteinogenic amino acids except glutamate. Aspartate stemming from the protein pool and from the extracellular medium was enriched with 13 C disclosing a substantial de novo biosynthesis of this amino acid simultaneous to its uptake from the growth medium and a rapid exchange¯ux of aspartate over the cellular membrane. Phenylalanine, alanine, and threonine were also synthesized de novo in spite of their presence in the growth medium.
Applied and Environmental Microbiology, Jun 1, 2001
Minute amounts of oxygen were supplied to a continuous cultivation of Lactococcus lactis subsp. c... more Minute amounts of oxygen were supplied to a continuous cultivation of Lactococcus lactis subsp. cremoris MG1363 grown on a defined glucose-limited medium at a dilution rate of 0.1 h ؊1. More than 80% of the carbon supplied with glucose ended up in fermentation products other than lactate. Addition of even minute amounts of oxygen increased the yield of biomass on glucose by more than 10% compared to that obtained under anaerobic conditions and had a dramatic impact on catabolic enzyme activities and hence on the distribution of carbon at the pyruvate branch point. Increasing aeration caused carbon dioxide and acetate to replace formate and ethanol as catabolic end products while hardly affecting the production of either acetoin or lactate. The negative impact of oxygen on the synthesis of pyruvate formate lyase was confirmed. Moreover, oxygen was shown to down regulate the protein level of alcohol dehydrogenase while increasing the enzyme activity levels of the pyruvate dehydrogenase complex, ␣-acetolactate synthase, and the NADH oxidases. Lactate dehydrogenase and glyceraldehyde dehydrogenase enzyme activity levels were unaffected by aeration.
Biotechnology and Bioengineering, 2001
Applied and Environmental Microbiology, 2001
Minute amounts of oxygen were supplied to a continuous cultivation of Lactococcus lactis subsp. c... more Minute amounts of oxygen were supplied to a continuous cultivation of Lactococcus lactis subsp. cremoris MG1363 grown on a defined glucose-limited medium at a dilution rate of 0.1 h−1. More than 80% of the carbon supplied with glucose ended up in fermentation products other than lactate. Addition of even minute amounts of oxygen increased the yield of biomass on glucose by more than 10% compared to that obtained under anaerobic conditions and had a dramatic impact on catabolic enzyme activities and hence on the distribution of carbon at the pyruvate branch point. Increasing aeration caused carbon dioxide and acetate to replace formate and ethanol as catabolic end products while hardly affecting the production of either acetoin or lactate. The negative impact of oxygen on the synthesis of pyruvate formate lyase was confirmed. Moreover, oxygen was shown to down regulate the protein level of alcohol dehydrogenase while increasing the enzyme activity levels of the pyruvate dehydrogenase...
Biotechnology and Bioengineering, 2002
Uniformly 13 C labeled glucose was fed to a lactic acid bacterium growing on a de®ned medium supp... more Uniformly 13 C labeled glucose was fed to a lactic acid bacterium growing on a de®ned medium supplemented with all proteinogenic amino acids except glutamate. Aspartate stemming from the protein pool and from the extracellular medium was enriched with 13 C disclosing a substantial de novo biosynthesis of this amino acid simultaneous to its uptake from the growth medium and a rapid exchange¯ux of aspartate over the cellular membrane. Phenylalanine, alanine, and threonine were also synthesized de novo in spite of their presence in the growth medium. ã
Applied and Environmental Microbiology, 2003
Lactococcus lactis subsp. lactis MG1363 was grown in batch cultures on a defined medium with gluc... more Lactococcus lactis subsp. lactis MG1363 was grown in batch cultures on a defined medium with glucose as the energy source under different aeration conditions, namely, anaerobic conditions, aerobic conditions, and microaerobic conditions with a dissolved oxygen tension of 5% (when saturation with air was used as the reference). The maximum specific growth rate was high (0.78 to 0.91 h−1) under all aeration conditions but decreased with increasing aeration, and more than 90% of the glucose was converted to lactate. However, a shift in by-product formation was observed. Increasing aeration resulted in acetate, CO2, and acetoin replacing formate and ethanol as end products. Under microaerobic conditions, growth came to a gradual halt, although more than 60% of the glucose was still left. A decline in growth was not observed during microaerobic cultivation when acetate was added to the medium. We hypothesize that the decline in growth was due to a lack of acetyl coenzyme A (acetyl-CoA) n...
Applied and Environmental Microbiology, 2003
Lactococcus lactis subsp. lactis MG1363 was grown in batch cultures on a defined medium with gluc... more Lactococcus lactis subsp. lactis MG1363 was grown in batch cultures on a defined medium with glucose as the energy source under different aeration conditions, namely, anaerobic conditions, aerobic conditions, and microaerobic conditions with a dissolved oxygen tension of 5% (when saturation with air was used as the reference). The maximum specific growth rate was high (0.78 to 0.91 h−1) under all aeration conditions but decreased with increasing aeration, and more than 90% of the glucose was converted to lactate. However, a shift in by-product formation was observed. Increasing aeration resulted in acetate, CO2, and acetoin replacing formate and ethanol as end products. Under microaerobic conditions, growth came to a gradual halt, although more than 60% of the glucose was still left. A decline in growth was not observed during microaerobic cultivation when acetate was added to the medium. We hypothesize that the decline in growth was due to a lack of acetyl coenzyme A (acetyl-CoA) n...
Biotechnology and Bioengineering, 2001
The pyruvate metabolism in the lactic acid bacterium Lactococcus lactis was studied in anaerobic ... more The pyruvate metabolism in the lactic acid bacterium Lactococcus lactis was studied in anaerobic cultures under transient conditions. During growth of L. lactis in continuous culture at high dilution rate, homolactic product formation was observed, i.e., lactate was produced as the major end product. At a lower dilution rate, the pyruvate metabolism shifted towards mixed acidproduct formation where formate, acetate, and ethanol were produced in addition to lactate. The regulation of the shift in pyruvate metabolism was investigated by monitoring the dynamic behavior of L. lactis in continuous cultures subjected to step changes in dilution rate. Both shift-up and shift-down experiments were carried out, and these experiments showed that the enzyme pyruvate formate-lyase (PFL) plays a key role in the regulation of the shift. Pyruvate formate-lyase in vivo activity was regulated both at the level of gene expression and by allosteric modulation of the enzyme. A simple mathematical model was proposed to estimate the relative significance of the regulatory mechanisms involved.