Methylobacterium rhodesianum cells tend to double the DNA content under growth limitations and accumulate PHB

Ackermann, Jörg‐Uwe; Müller, Susann; Lösche, Andreas; Bley, Thomas; Babel, Wolfgang

doi:10.1016/0168-1656(94)00138-3

Cited by 63 publications

(44 citation statements)

References 26 publications

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“…5c). The increasing average cell size indicated by the forward scattering of the Nile red stained cells during this growth phase can thus be attributed to PHB accumulation in the cells, a finding confirming those reported elsewhere (Ackermann et al, 1995;Müller et al, 1999). Flow cytometry allowed distinguishing two subpopulations with different DNA sets (Fig.…”

Section: Phb Production From a Non-toxic Substratesupporting

confidence: 69%

“…After 20 h, the cells began to adapt to repetitive phenol pulses and the C1 subpopulation dropped. The main part of the population obviously contained two chromosomes, a physiological state which seems typical for many bacteria under carbon limited conditions (see Ackermann et al, 1995;Müller and Babel, 2003). This pattern changed when the phenol was added once more (60 h).…”

Section: Influence Of Phenol Accumulation On Proliferation Activity Omentioning

confidence: 98%

“…A parameter of great importance for continuous processes is the proliferation activity that is reflected by the DNA content (Fouchet et al, 1993;Müller et al, 1995b;Scheper et al, 1987). Flow cytometry can also monitor a variety of metabolic activities (Ackermann et al, 1995;Chung et al, 1995;Müller et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

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Control of continuous polyhydroxybutyrate synthesis using calorimetry and flow cytometry

Maskow

Müller

Lösche

et al. 2006

Biotech & Bioengineering

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The substrate-carbon flow can be controlled in continuous bioreactor cultures by the medium composition, for example, by the C/N ratio. The carbon distribution is optimal when a maximum fraction flows into the desired product and the residual is just sufficient to compensate for the dilution of the microbial catalyst. Undershooting of the latter condition is reflected immediately by changes in the Gibbs energy dissipation and cellular states. Two calorimetric measurement principles were applied to optimize the continuous synthesis of polyhydroxybutyrate (PHB) by Variovorax paradoxus DSM4065 during growth with constantly increasing supply rates of fructose or toxic phenol. Firstly, the changed slope of the heat production rate in a complete heat balanced bioreactor (CHB) indicated optimum carbon channeling into PHB. The extent of the alteration depended directly on the toxic properties of the substrate. Secondly, a flow through calorimeter was connected with the bioreactor as a "measurement loop." The optimum substrate carbon distribution was indicated by a sudden change in the heat production rate independent of substrate toxicity. The sudden change was explained mathematically and exploited for the long-term control of phenol conversion into PHB. LASER flow cytometry measurements distinguished between subpopulations with completely different PHB-content. Populations grown on fructose preserved a constant ratio of two subpopulations with double and quadruple sets of DNA. Cells grown on phenol comprised a third subpopulation with a single DNA set. Rising phenol concentrations caused this subpopulation to increase. It may thus be considered as an indicator of chemostress.

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Section: Phb Production From a Non-toxic Substratesupporting

confidence: 69%

Section: Influence Of Phenol Accumulation On Proliferation Activity Omentioning

confidence: 98%

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Control of continuous polyhydroxybutyrate synthesis using calorimetry and flow cytometry

Maskow

Müller

Lösche

et al. 2006

Biotech & Bioengineering

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“…Escherichia coli and the non-polyphosphate-producing Micropruina glycogenica (37) were grown aerobically in 300-ml shake flasks in 50 ml peptone medium containing (per liter) 5 g peptone from meat (pancreatic), 3 g NaCl, 2 g K 2 HPO 4 , 10 g meat extract, 10 g yeast extract, and 5 g glucose (for E. coli) and in DSM 776 for M. glycogenica. Methylobacterium rhodesianum was grown aerobically in 100-ml shake flasks in 50 ml of the standard medium described by Ackermann et al (1). This strain was reported to produce poly-␤-hydroxybutyrate (PHB) to up to 95% of its dry weight.…”

Section: Methodsmentioning

confidence: 99%

Dynamics of Polyphosphate-Accumulating Bacteria in Wastewater Treatment Plant Microbial Communities Detected via DAPI (4′,6′-Diamidino-2-Phenylindole) and Tetracycline Labeling

Günther

Trutnau

Kleinsteuber

et al. 2009

Appl Environ Microbiol

107

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Wastewater treatment plants with enhanced biological phosphorus removal represent a state-of-the-art technology. Nevertheless, the process of phosphate removal is prone to occasional failure. One reason is the lack of knowledge about the structure and function of the bacterial communities involved. Most of the bacteria are still not cultivable, and their functions during the wastewater treatment process are therefore unknown or subject of speculation. Here, flow cytometry was used to identify bacteria capable of polyphosphate accumulation within highly diverse communities. A novel fluorescent staining technique for the quantitative detection of polyphosphate granules on the cellular level was developed. It uses the bright green fluorescence of the antibiotic tetracycline when it complexes the divalent cations acting as a countercharge in polyphosphate granules. The dynamics of cellular DNA contents and cell sizes as growth indicators were determined in parallel to detect the most active polyphosphate-accumulating individuals/subcommunities and to determine their phylogenetic affiliation upon cell sorting. Phylotypes known as polyphosphate-accumulating organisms, such as a "Candidatus Accumulibacter"-like phylotype, were found, as well as members of the genera Pseudomonas and Tetrasphaera. The new method allows fast and convenient monitoring of the growth and polyphosphate accumulation dynamics of not-yet-cultivated bacteria in wastewater bacterial communities.

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“…Members of the genus Methylobacterium are able to use reduced one-carbon compounds, such as methanol, as sole sources of carbon and energy. M. rhodesianum MB 126 accumulates PHB under conditions of excess methanol and limiting concentrations of nitrogen, phosphorus, or oxygen (1,7). Upon provision of sufficient nutrients and in the absence of a carbon source, PHB is remobilized and used as a source of carbon and energy.…”

mentioning

confidence: 99%

Production of the Chiral Compound ( R )-3-Hydroxybutyrate by a Genetically Engineered Methylotrophic Bacterium

Hölscher

Breuer

Adrian

et al. 2010

Appl Environ Microbiol

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In this study, a methylotrophic bacterium, Methylobacterium rhodesianum MB 126, was used for the production of the chiral compound (R)-3-hydroxybutyrate (R-3HB) from methanol. R-3HB is formed during intracellular degradation of the storage polymer (R)-3-polyhydroxybutyrate (PHB). Since the monomer R-3HB does not accumulate under natural conditions, M. rhodesianum was genetically modified. The gene (hbd) encoding the R-3HB-degrading enzyme, R-3HB dehydrogenase, was inactivated in M. rhodesianum. The resulting hbd mutant still exhibited low growth rates on R-3HB as the sole source of carbon and energy, indicating the presence of alternative pathways for R-3HB utilization. Therefore, transposon mutagenesis was carried out with the hbd mutant, and a double mutant unable to grow on R-3HB was obtained. This mutant was shown to be defective in lipoic acid synthase (LipA), resulting in an incomplete citric acid cycle. Using the hbd lipA mutant, we produced 3.2 to 3.5 mM R-3HB in batch and 27 mM (2,800 mg liter ؊1 ) in fed-batch cultures. This was achieved by sequences of cultivation conditions initially favoring growth, then PHB accumulation, and finally PHB degradation.Enantiomeric purity of a product or building block is often a prerequisite for its application in the health care field. Due to their chirality and the presence of two functional groups (i.e., hydroxyl and carbonic acid), (R)-3-hydroxyalkanoates are valuable building blocks for the synthesis of pharmaceutical products, such as carbapenem or macrolide antibiotics (36; for a review, see reference 9). (R)-3-hydroxyalkanoates can be obtained by hydrolysis of poly-(R)-3-hydroxyalkanoates (PHAs), which are synthesized as carbon storage polymers under conditions of nutrient limitation by many bacterial species (3). Poly-(R)-3-hydroxybutyrate (PHB), a homopolymer of (R)-3-hydroxybutyrate (R-3HB), is the most common naturally occurring PHA. For the recovery of the chiral monomers, chemical hydrolysis and a variety of biotechnological processes have been tested. The biotechnological processes include the in vitro or in vivo depolymerization of PHA using wild-type or genetically engineered microorganisms (23,24,30,34,37). Also, direct pathways for (R)-3-hydroxyalkanoate synthesis in non-PHA-producing strains have been established (15,25). For cultivation of the (R)-3-hydroxyalkanoate-producing bacteria, sugars and alkanoates have typically been used as carbon sources.In this study, production of R-3HB from methanol using a facultative methylotrophic bacterium, Methylobacterium rhodesianum MB 126, was assessed. Methanol, a cheap bulk chemical, is usually synthesized from natural gas or coal via syngas. In the near future, it will be possible to synthesize methanol in huge amounts directly from methane, a main component of biogas and natural gas, or from the greenhouse gas carbon dioxide (31). Thus, methanol is a promising substrate for new biotechnological processes. Members of the genus Methylobacterium are able to use reduced one-carbon compounds, such as methanol...

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Methylobacterium rhodesianum cells tend to double the DNA content under growth limitations and accumulate PHB

Cited by 63 publications

References 26 publications

Control of continuous polyhydroxybutyrate synthesis using calorimetry and flow cytometry

Control of continuous polyhydroxybutyrate synthesis using calorimetry and flow cytometry

Dynamics of Polyphosphate-Accumulating Bacteria in Wastewater Treatment Plant Microbial Communities Detected via DAPI (4′,6′-Diamidino-2-Phenylindole) and Tetracycline Labeling

Production of the Chiral Compound ( R )-3-Hydroxybutyrate by a Genetically Engineered Methylotrophic Bacterium

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