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1

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Detection and Isolation of Methanotrophic Bacteria Possessing Soluble Methane Monooxygenase (sMMO) Genes Using the Polymerase Chain Reaction (PCR) (1997)

Miguez, C.B. ; Bourque, D. ; Sealy, J.A. ; [et al.]

Springer

Microbial ecology 33 (1997), S. 21 -31

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ISSN: 1432-184X

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract Methanotrophic bacteria possessing sMMO activity have gained notoriety in recent years due to their ability to oxidize a wide variety of halogenated aliphatic compounds, including trichloroethylene (TCE), and are being used as the basis for developing new bioremediation processes. PCR primers were designed from DNA sequences of the alpha and beta subunits of the hydroxylase component of the sMMO from Methylococcus capsulatus (Bath). The mmoY1-mmoY2 primer set was derived from the beta subunit and was specific for the mmoY gene from M. capsulatus, but failed to produce the expected 395-nucleotide (nt) fragment from Methylosinus sporium (ATCC 35069) or from Methylosinus trichosporium (ATCC 36070), even at low stringency. A second primer set, primers mmoX1-mmoX2, was derived from the alpha subunit and produced the expected 369-nt fragment from all three methanotrophic cultures tested at the highest stringency used (72°C). Soil and groundwater samples were tested for the presence of sMMO-containing bacteria using these two primer sets. One diesel-contaminated soil sample and one TCE-contaminated groundwater sample gave positive results after amplification of total extracted DNA using the mmoX1-mmoX2 primers. Culture enrichment in small chemostats inoculated with the same positive samples led to the isolation of 13 cultures possessing sMMO activity and containing DNA amplifiable by the mmoX1-mmoX2 primers. Our results indicated that attempts to directly cultivate sMMO-positive bacteria may give false negative results with some environmental samples. We recommend that primers and/or gene probes based on the sMMO be used in parallel with the naphthalene oxidation test for any environmental assessment of the methanotrophic population. RAPD-PCR analysis revealed that half of these isolates appeared to be different from each other and from M. capsulatus, M. sporium, or M. trichosporium.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/s002489900004

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2

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Production of heterologous protein by Methylobacterium extorquens in high cell density fermentation (2004)

Bélanger, L ; Figueira, M.M ; Bourque, D ; [et al.]

Oxford, UK : Blackwell Publishing Ltd

FEMS microbiology letters 231 (2004), S. 0

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ISSN: 1574-6968

Source: Blackwell Publishing Journal Backfiles 1879-2005

Topics: Biology

Notes: The green fluorescent protein (GFP) was used as a model protein to study the recombinant protein production by the strain Methylobacterium extorquens ATCC 55366. Scale-up from shake flasks to 20 l fed-batch fermentation was achieved using methanol as a sole carbon and energy source and a completely minimal culture medium. Two different expression vectors were used to express GFP. Clone PCM-GFP containing the vector pCM110 with native promoter of the methanol dehydrogenase PmxaF produced approximately 100-fold more GFP than the clone PRK-GFP containing the vector pRK310 with the heterogeneous promoter Plac. Several fed-batch fermentations with and without selective pressure (tetracycline) were run in a 20 l stirred tank fermenter using the two different clones of M. extorquens. The methanol concentration was monitored with an on-line semiconductor gas sensor in the culture broth. It was maintained at a non-toxic level of 1.4 g l−1 with an adaptative control which regulates the methanol feed rate. The same growth profile was achieved in all fermentations. The maximum growth rate (μmax) was 0.18 h−1 with an overall yield (YX/S) of 0.3 g g−1 methanol. With this high cell density fermentation process, we obtained high levels (up to 4 g l−1) of GFP with the clone PCM-GFP. The maximum specific GFP production (YGFP/X) with this clone was 80 mg g−1 representing approximately 16% of the total cell protein. Additional feeding of pure oxygen to the fermenter permitted a longer phase of exponential growth but had no effect on the total yields of biomass and GFP. The specific GFP production of clone PCM-GFP remained unaffected in the presence or absence of selective pressure (tetracycline), within the initial 50 h of the fermentation culture. These results suggest that M. extorquens ATCC 55366 could be an interesting candidate for overexpression of recombinant proteins.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1016/S0378-1097(03)00956-X

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3

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Quantitative thin-layer chromatographic assay for the β-neoagarotetraose hydrolase of Pseudomonas atlantica

Groleau, D. ; Yaphe, W.

Amsterdam : Elsevier

Journal of Chromatography A 137 (1977), S. 234-239

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ISSN: 0021-9673

Source: Elsevier Journal Backfiles on ScienceDirect 1907 - 2002

Topics: Chemistry and Pharmacology

Type of Medium: Electronic Resource

URL: http://linkinghub.elsevier.com/retrieve/pii/S0021-9673(00)89265-1

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4

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Influence of oxygenation and carbon dioxide on the growth ofRhizobium meliloti in a fermenter (1990)

Bourque, D. ; Groleau, D.

Springer

World journal of microbiology and biotechnology 6 (1990), S. 273-280

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ISSN: 1573-0972

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Description / Table of Contents: Résumé La diminution de l'O2 dissous de 90 à 50% de la saturation dans un fermenteur, a affecté de manière négative tant la production de biomasse deRhizobium meliloti ATCC 9930 que le nombre de cellules revivifiables, bien que l'oxygène ne fut jamais limitant. Des quantités diminuées d'oxygène dissous ou une diminution accidentelle de la concentration en oxygène dissous, ont également causé une acidification appréciable du milieu de culture résultant de l'accumulation de CO2 dans le milieu. L'ajout de CO2 au gaz d'aération a affecté tant la production de biomasse que le temps moyen de génération, proportionnellement à la concentration de CO2; l'effet sur le nombre de cellules revivifiables était moins prononcé.R. meliloti peut être considéré comme un microorganisme modérément sensible au CO2. Une bonne croissance deR. meliloti requiert, entre autres, non seulement une aération (oxygénation) suffisante, mais aussi une bonne ventilation du CO2 engendré pendant la fermentation.

Notes: Abstract A decrease in dissolved O2 from 90% to 50% saturation in a fermenter adversely affected both blomass production ofRhizobium meliloti ATCC 9930 and viable cell number, although oxygen was never limiting. Lower amounts of dissolved oxygen, or accidental decreases in dissolved oxygen concentration, also caused appreciable acidification of the culture broth, which was the result of CO2 accumulation in the medium. Adding CO2 to the aeration gas mixture affected both biomass production and mean generation time in proportion to the CO2 concentration; the effect on viable cell number was less pronounced.R. meliloti may be considered as a microorganism moderately sensitive to CO2. GoodR. meliloti growth requires, among other things, not only sufficient aeration (oxygenation) but also good ventilation of the CO2 evolved during the fermentation.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF01201296

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5

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Fermentation study for the production of hepatitis B virus pre-S2 antigen by the methylotrophic yeastHansenula polymorpha (1991)

Roubin, M. R. ; Bastien, L. ; Shen, S. -H. ; [et al.]

Springer

Journal of industrial microbiology and biotechnology 8 (1991), S. 147-156

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ISSN: 1476-5535

Keywords: Methanol ; Yeast extract ; Two-phase process ; Periplasmic antigen ; Intracellular antigen

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: Summary Various physico-chemical parameters have been studied in order to improve the production of hepatitis B virus pre-S2 antigen (middle surface antigen) by the methylotrophic yeastHansenula polymorpha. Antigen production was done in two steps: first, production of cells on glycerol (Phase 1), followed by induction of antigen expression with methanol (Phase 2). Dense cultures ofH. polymorpha, equivalent to 35–40 g/l (dry weight), were readily obtained in small fermenters using minimal medium containing glycerol as carbon source. Antigen expression in this minimal medium, after induction with methanol, was however low and never exceeded 1.6 mg/l of culture. Antigen production was greatly enhanced by adding complex organic nitrogen sources along with methanol at induction time; yeast extract was the best of all the sources tested. In shake flasks, antigen production was proportional to yeast extract concentration up to 7% (w/v) yeast extract. it became clear that the nutritional conditions for good antigen expression were different from those for good biomass production. The effects of yeast extract were reproduced in small fermenters: antigen levels reached 8–9 mg/l in medium containing 6% (w/v) yeast extract during induction with methanol. The mechanisms of yeast extract's effects are still unknown but are probably nutritional. The recombinantH. polymorpha strain produced both periplasmic and intracellular antigen. The periplasmic antigen was shown to be present as 20–22-nm particles and was therefore immunogenic. Immunoblotting indicated that part of the pre-S2 antigen was present as a 24-kDa degradation product. These studies have led to a 140-fold increase in volumetric productivity of antigen and to a 4.6-fold increase in specific production.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF01575847

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6

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High-cell-density production of poly-β-hydroxybutyrate (PHB) from methanol by Methylobacterium extorquens: production of high-molecular-mass PHB (1995)

Bourque, D. ; Pomerleau, Y. ; Groleau, D.

Springer

Applied microbiology and biotechnology 44 (1995), S. 367-376

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ISSN: 1432-0614

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: Abstract Methylobacterium extorquens ATCC 55366 was successfully cultivated at very high cell densities in a fed-batch fermentation system using methanol as a sole carbon and energy source and a completely minimal culture medium for the production of polyβ-hydroxybutyrate (PHB). Cell biomass levels were between 100 g/l and 115 g/l (dry weight) and cells contained between 40% and 46% PHB on a dry-weight basis. PHB with higher molecular mass values than previously reported for methylotrophic bacteria was obtained under certain conditions. Shake-flask and fermentor experiments showed the importance of adjusting the mineral composition of the medium for improved biomass production and higher growth rates. High-cell-density cultures were obtained without the need for oxygen-enriched air; once the oxygen transfer capacity of the fermentor was reached, methanol was thereafter added in proportion to the amount of available dissolved oxygen, thus preventing oxygen limitation. Controlling the methanol concentration at a very low level (less than 0.01 g/l), during the PHB production phase, led not only to prevention of oxygen limitation but also to the production of very high-molecular-mass PHB, in the 900–1800 kDa range. Biomass yields relative to the total methanol consumed were in the range 0.29–0.33 g/g, whereas PHB yields were in the range 0.09–0.12 g/g. During the active period of PHB synthesis, PHB yields relative to the total methanol consumed were between 0.2 g/g and 0.22 g/g. M. extorquens ATCC 55366 appears to be a promising organism for industrial PHB production.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/s002530050569

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7

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High-cell-density production of poly-β-hydroxybutyrate (PHB) from methanol by Methylobacterium extorquens: production of high-molecular-mass PHB (1995)

Bourque, D. ; Pomerleau, Y. ; Groleau, D.

Springer

Applied microbiology and biotechnology 44 (1995), S. 367-376

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ISSN: 1432-0614

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: Abstract Methylobacterium extorquens ATCC 55366 was successfully cultivated at very high cell densities in a fed-batch fermentation system using methanol as a sole carbon and energy source and a completely minimal culture medium for the production of poly-β-hydroxybutyrate (PHB). Cell biomass levels were between 100 g/l and 115 g/l (dry weight) and cells contained between 40% and 46% PHB on a dry-weight basis. PHB with higher molecular mass values than previously reported for methylotrophic bacteria was obtained under certain conditions. Shake-flask and fermentor experiments showed the importance of adjusting the mineral composition of the medium for improved biomass production and higher growth rates. High-cell-density cultures were obtained without the need for oxygen-enriched air; once the oxygen transfer capacity of the fermentor was reached, methanol was thereafter added in proportion to the amount of available dissolved oxygen, thus preventing oxygen limitation. Controlling the methanol concentration at a very low level (less than 0.01 g/l), during the PHB production phase, led not only to prevention of oxygen limitation but also to the production of very high-molecular-mass PHB, in the 900–1800 kDa range. Biomass yields relative to the total methanol consumed were in the range 0.29–0.33 g/g, whereas PHB yields were in the range 0.09–0.12 g/g. During the active period of PHB synthesis, PHB yields relative to the total methanol consumed were between 0.2 g/g and 0.22 g/g. M. extorquens ATCC 55366 appears to be a promising organism for industrial PHB production.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00169931

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8

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Production of poly-β-hydroxybutyrate from methanol: characterization of a new isolate of Methylobacterium extorquens (1992)

Bourque, D. ; Ouellette, B. ; André, G. ; [et al.]

Springer

Applied microbiology and biotechnology 37 (1992), S. 7-12

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ISSN: 1432-0614

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology

Notes: Summary Poly-β-hydroxybutyric acid (PHB) and similar bacterial polyesters are promising candidates for the development of environment-friendly, totally biodegradable plastics. The use of methanol, one of the cheapest noble substrates available, may help to reduce the cost of producing such bioplastics. As a first step, a culture collection of 118 putative methylotrophic microorganisms was obtained from various soil samples without any laboratory enrichment step to favour culture diversity. The most promising culture was selected based on rapidity of growth and PHB accumulation and later identified as Methylobacterium extorquens. This isolate was obtained from soml contaminated regularly with used oil products for some 40 years. Concentrations of methanol greater than 8 g/l affected growth significantly and the methanol concentration was optimal at 1.7 g/l. PHB concentrations averaged 25–30% (w/v) of dry weight under non-optimized conditions. Controlling methanol concentration, using an open-loop configuration, led to biomass levels of 9–10 g/l containing 30–33% PHB while preventing methanol accumulation. The new isolate was also able to produce the co-polymer PHB/poly-β-hydroxyvalerate (PHV) using the mixture methanol + valerate. The PHV-to-PHB ratio was about 0.2 at the end of the fermentation. An average molecular mass varying between 2 and 3 × 105 Da was obtained for three PHB samples using two different measurement methods.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00174194

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