Influence of the Carbon: Nitrogen Ratio of the Growth Medium on the Cellular Composition and the Ability of the Methylotrophic Yeast Hansenula polymorpha to Utilize Mixed Carbon Sources

Egli, Th.; Quayle, J. R.

doi:10.1099/00221287-132-7-1779

Cited by 42 publications

(49 citation statements)

References 22 publications

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“…Second, elemental yield coefficients obtained under singlenutrient-limited growth conditions were used to calculate the boundaries of the dual-nutrient-limited growth zone according to the method of Egli and Quayle (13). For a particular dilution rate, the growth yield coefficients for nitrogen (Y X/N ) and carbon (Y X/C ) were constant under single-nutrient-limited conditions and changed within the dual-nutrient-limited growth zone: Y X/C decreased, whereas Y X/N increased (12).…”

Section: Resultsmentioning

confidence: 99%

Accumulation of Poly[( R )-3-Hydroxyalkanoates] in Pseudomonas oleovorans during Growth with Octanoate in Continuous Culture at Different Dilution Rates

Durner

Witholt

Egli

2000

Appl Environ Microbiol

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Pseudomonas oleovorans ATCC 29347 was grown in chemostat culture at different dilution rates with mineral media varying in their ratios of octanoate to ammonia (C 0 /N 0 ratio). At all dilution rates tested, three distinct growth regimes were observed: (i) carbon limitation with NH 4 ؉ in excess at low C 0 /N 0 ratios, (ii) purely nitrogen-limited growth conditions at high C 0 /N 0 ratios with residual octanoate in the culture supernatant, and (iii) an intermediate zone of dual-nutrient-limited growth conditions where both the concentration of octanoate and that of ammonia were very low. The dual-nutrient-limited growth zone shifted to higher C 0 /N 0 ratios with decreasing dilution rates, and the extension of the dual-nutrient-limited growth zone was inversely proportional to the growth rate. The cells accumulated the storage compound medium-chain-length poly[(R)-3-hydroxyalkanoate] (mcl-PHA) during dual (C and N)-nutrient-limited and N-limited growth conditions. Within the dual-nutrient-limited growth zone, the cellular mcl-PHA contents increased when the C 0 /N 0 ratio in the feed was increased, whereas the cellular mcl-PHA level was independent from the feed C 0 /N 0 ratio during N-limited growth. The monomeric composition of the accumulated mcl-PHA was independent of both the dilution rate and the feed C 0 /N 0 ratio and consisted of 12 mol% 3-hydroxyhexanoic acid and 88 mol% 3-hydroxyoctanoic acid. Accumulation of mcl-PHA led to an increase in the cellular C/N ratio and to changes in elemental growth yields for nitrogen and carbon.Pseudomonas oleovorans is able to accumulate mediumchain-length poly[(R)-3-hydroxyalkanoates] (mcl-PHAs) as intracellular carbon and energy storage compounds when grown with alkanes (9, 24), mcl-alkanoates (3, 21), n-alkanols (16), or many derivatives thereof (27,38). mcl-PHAs are hydrophobic polyesters and are attracting increasing interest for their possible application as biologically produced and inherently biodegradable substitutes for conventional plastic materials (6,8,10,20,32). Particularly, when subjected to nitrogen limitation in batch (22, 24) and chemostat culture (17,33,34), P. oleovorans has been reported to accumulate high cellular mcl-PHA contents.The nature and availability of essential nutrients are important parameters in determining the extent of storage compound accumulation in P. oleovorans (23,24). Generally, the production of microbial cell mass is limited by the restricted availability of a particular nutrient. In batch culture, the exhaustion of a specific nutrient terminates the exponentialgrowth phase, whereas in chemostat culture the biomass concentration is usually controlled by the permanent limitation of a single defined nutrient, typically the carbon and energy source. However, several authors have shown that two or more nutrients can simultaneously limit the production of biomass (for a summary, see reference 14). This phenomenon has also been observed for P. oleovorans, where we have found that under defined chemostat steady-state cultivation c...

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Section: Resultsmentioning

confidence: 99%

Accumulation of Poly[( R )-3-Hydroxyalkanoates] in Pseudomonas oleovorans during Growth with Octanoate in Continuous Culture at Different Dilution Rates

Durner

Witholt

Egli

2000

Appl Environ Microbiol

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“…Even after 70 generations no further increases in biomass occurred in response to a pulse of excess phosphate, so phosphatelimited growth had not been established at this low dilution rate. A possible explanation for this is that the culture had been growth-limited by more than one substrate at the lower dilution rate of 0.15 h-l, but not at 0-3 h-l, as described by Egli & Quayle (1986) and by Egli & Schmidt (1989).…”

Section: Plasmid Instability and Periodic Selection In A De$ned M Inmentioning

confidence: 99%

Effect of growth rate on plasmid maintenance by Escherichia coli HB101(pAT153)

Brownlie

Stephenson²,

Cole³

1990

Journal of General Microbiology

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~~~ ~The effects of changing the composition of the growth medium, the dilution rate and the source of the bacterial host on maintenance of the plasmid pAT153 in Escherichia coli HBlOl have been studied. In a medium supplemented with Casamino acids, the plasmid was maintained longer during phosphate-limited growth at a dilution rate of 0.3 h-l than at 0.15 h-l. In contrast, phosphate-limited growth was not achieved when the Casamino acids were replaced by proline, leucine and thiamin to satisfy the auxotrophic requirements of the host. Although 100% of the bacteria were still ampicillin resistant after 72 generations of growth at a dilution rate of 0.15 h-l, the original plasmid had almost totally been replaced by a structurally modified plasmid which lacked a functional tet gene.Further experiments confirmed that neither the host nor the plasmid was retained unchanged in the minimal medium. The changes were highly reproducible and reflected periodic selection of sub-populations which were either plasmid-free or carried a structurally modified plasmid, which had reverted to Leu+ or Pro+, or had acquired other chromosomal mutations which gave them a selective advantage. We conclude that ,in complex media the plasmid is maintained longer by E. coZi HBlOl at a high than at a low growth rate and that different results reported from different laboratories are largely due to differences in analytical techniques and the growth medium rather than to differences in the bacterial host or the plasmid used. A fermenter-adapted strain was isolated which reproducibly maintained the plasmid longer during phosphate-limited continuous growth than the original strain which had been cultured on laboratory media.

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“…An influence of C/N ratio in the medium on biomass composition has previously been reported for the bacteria Hyphomicrobium X (5) and Hyphomicrobium ZV620 (9) as well as for the yeasts Hansenula polymorpha (6) and Candida valida (15 principles of the calorimetric measurement procedure have been described previously (32).…”

mentioning

confidence: 99%

“…Consequently, a dual limitation, in which both the carbon as well as the nitrogen substrate is limiting, may occur (3,5,6,15). A limiting substrate is in this case defined by von Stockar and Auberson (31) as a substrate which is consumed at the maximal possible rate, i.e., the feed rate, and this rate is not determined by the concentration or feed rate of any other substance in the medium.…”

mentioning

confidence: 99%

Growth and metabolism of Saccharomyces cerevisiae in chemostat cultures under carbon-, nitrogen-, or carbon- and nitrogen-limiting conditions

et al. 1993

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Aerobic chemostat cultures of Saccharomyces cerevisiae were performed under carbon-, nitrogen-, and dual carbon-and nitrogen-limiting conditions. The glucose concentration was kept constant, whereas the ammonium concentration was varied among different experiments and different dilution rates. It was found that both glucose and ammonium were consumed at the maximal possible rate, i.e., the feed rate, over a range of medium C/N ratios and dilution rates. To a small extent, this was due to a changing biomass composition, but much more important was the ability of uncoupling between anabolic biomass formation and catabolic energy substrate consumption. When ammonium started to limit the amount of biomass formed and hence the anabolic flow of glucose, this was totally or at least partly compensated for by an increased catabolic glucose consumption. The primary response when glucose was present in excess of the minimum requirements for biomass production was an increased rate of respiration. The calculated specific oxygen consumption rate, at D = 0.07 h-', was more than doubled when an additional nitrogen limitation was imposed on the cells compared with that during single glucose limitation. However, the maximum respiratory capacity decreased with decreasing nitrogen concentration. The saturation level of the specific oxygen consumption rate decreased from 5.5 to 6.0 mmol/g/h under single glucose limitation to about 4.0 mmol/g/h at the lowest nitrogen concentration tested. The combined result of this was that the critical dilution rate, i.e., onset of fermentation, was as low as 0.10 h-1 during growth in a medium with a low nitrogen concentration compared with 0.20 hobtained under single glucose limitation.During growth of Saccharomyces cerevisiae in a chemostat with glucose as the sole limiting substrate, energy rather than carbon limits biomass formation (7,22,29), i.e., it can be regarded as a catabolic limitation. If, on the other hand, a nitrogen source such as ammonium is used as the limiting substrate, this can instead be viewed as an anabolic limitation. Several studies, concerning bacteria, have shown that anabolism and catabolism do not seem to be tightly coupled (3,5,12,(16)(17)(18)(19)(20)25). The catabolic activity is often higher than what can be explained by the anabolic requirements for ATP when the energy source is present in excess. When glucose-grown chemostat cultures of the bacterium Klebsiella aerogenes were subjected to a limitation other than a glucose limitation, it was found that the specific respiration rate increased compared with that of glucose-limited cells. In addition, when the energy source was in excess, the cells responded by excreting a number of products more oxidized than the carbon source, i.e., not a fermentative metabolism, a phenomenon named overflow metabolism (3,12,(17)(18)(19)(20)25). K aerogenes is the most extensively studied in this respect, but several other bacteria seem to respond in a similar way (16,20). In regards to the yeast S. cerevisiae, uncoupling between c...

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Influence of the Carbon: Nitrogen Ratio of the Growth Medium on the Cellular Composition and the Ability of the Methylotrophic Yeast Hansenula polymorpha to Utilize Mixed Carbon Sources

Cited by 42 publications

References 22 publications

Accumulation of Poly[( R )-3-Hydroxyalkanoates] in Pseudomonas oleovorans during Growth with Octanoate in Continuous Culture at Different Dilution Rates

Accumulation of Poly[( R )-3-Hydroxyalkanoates] in Pseudomonas oleovorans during Growth with Octanoate in Continuous Culture at Different Dilution Rates

Effect of growth rate on plasmid maintenance by Escherichia coli HB101(pAT153)

Growth and metabolism of Saccharomyces cerevisiae in chemostat cultures under carbon-, nitrogen-, or carbon- and nitrogen-limiting conditions

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