Physiological characteristics of four methylotrophic bacteria and their potential use in single-cell protein production (original) (raw)
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Food Technology and Biotechnology
Research background. This study aims to monitor the growth of the methylotrophic bacteria Methylobacterium organophilum in a culture medium with methanol as a carbon source and to verify the production of unicellular proteins and other biomolecules, such as carotenoids, exopolysaccharides and polyhydroxyalkanoates, making them more attractive as animal feed. Experimental approach. Bacterial growth was studied in shake flasks using different carbon/nitrogen (C:N) ratios to determine their best ratio for achieving the highest volumetric productivity of cells and substrate consumption rate. This optimal parameter was further used in a fed-batch operating bioreactor system to define the kinetic profile of cell growth. Methanol consumption was measured by HPLC analysis and the extracted pigments were analyzed by liquid chromatography/mass spectrometry. Chemical composition and rheological properties of the produced exopolysaccharides were also determined. Results and conclusions. The bes...
Growth and Polysaccharide Production by Methylocystis parvus OBBP on Methanol
Applied and Environmental Microbiology, 1979
Methylocystis parvus OBBP, an obligate methylotroph originally isolated as a methane-utilizing bacterium, was cultivated on methanol as a sole source of carbon. After adaptation to high methanol levels, this organism grew on methanol with a maximum specific growth rate of 0.65 h-1. The pH optimum for growth was between 7 and 9, and the temperature optimum was between 30 and 37°C. Methanol concentrations higher than 5% (by weight) were toxic. Formaldehyde, at a concentration greater than 1 mM, inhibited growth. Formate was neither a substrate nor an inhibitor. An extracellular viscous heteropolysaccharide was produced during growth. The maximum production of the total biomass was 14.5 g (dry weight) per liter of broth. The dried biomass contained 22% (wt/wt) crude protein and 62% (wt/wt) polysaccharide. The main components of the polysaccharide were D-glucose (82%) and L-rhamnose (14%).
Production of heterologous protein by Methylobacterium extorquens in high cell density fermentation
FEMS Microbiology Letters, 2004
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 P mxaF produced approximately 100-fold more GFP than the clone PRK-GFP containing the vector pRK310 with the heterogeneous promoter P lac . 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 31 with an adaptative control which regulates the methanol feed rate. The same growth profile was achieved in all fermentations. The maximum growth rate (W max ) was 0.18 h 31 with an overall yield (Y X =S ) of 0.3 g g 31 methanol. With this high cell density fermentation process, we obtained high levels (up to 4 g l 31 ) of GFP with the clone PCM-GFP. The maximum specific GFP production (Y GFP=X ) with this clone was 80 mg g 31 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. (C.B. M| ¤guez).
Journal of Applied Microbiology - J APPL MICROBIOL, 1995
continuous cultures of Methylobacterium sp. RXM to the addition of methanol pulses was studied. The increase of methanol concentration in the medium did not result in cell death under any of the conditions tested. When the growth rate of the steady-state cultures was low (D = 0.046 h-'), the specific growth rate increased. When the concentration of methanol in the pulse was increased from 36 mmol 1-' to 280 mmol 1-', uncoupled growth occurred and the molar cell yield decreased. Conversely, steady-state cultures at high growth rate (D = 0.2 h-') showed a decrease in both specific growth rate and molar cell yield after the addition of the methanol pulses (32 and 164 mmol 1-'). For all conditions, formaldehyde and formate were excreted into the medium but the levels did not exceed 1.13 mmol 1-'. Slow-growing cultures were characterized by cells with high derepressed specific activities of methanol dehydrogenase and low specific activities of formaldehyde and formate dehydrogenases, fast-growing cells had lower specific activity for methanol dehydrogenase and higher activities of formaldehyde and formate dehydrogenases, resulting in the excretion of lower concentrations of formaldehyde and formate. It is concluded that slow-growing cultures are more stable than fast-growing cultures for low methanol concentration fluctuations, and it is expected that maximum growth yields throughout the fermentation time are better achieved under the former conditions. However, for large fluctuations in the substrate concentration, the bacterial metabolic responses were identical both for slow-growing and fast-growing cultures.
Thermophilic mixed culture of bacteria utilizing methanol for growth
Applied microbiology, 1974
A thermophilic mixed population of bacteria, capable of utilizing methanol as its sole carbon-energy source at temperatures up to 65 C, was selected by enrichment and studied. A maximal cellular yield of 0.42 g per g of methanol was observed at 50 to 56 C. The maximal specific growth rate of the mixed population in continuous culture at 56 C was greater than 0.32 per h. The amino acid profile of the mixed culture indicated that a high quality protein was produced and the protein content was 71%. The properties of this culture and its ability to grow at elevated temperatures are discussed in terms of single-cell protein production and the treatment of industrial waste.
Chemostat optimization of biomass production of a mixed bacterial culture utilizing methanol
Applied Microbiology and Biotechnology, 1990
A continuous culture technique was used to optimize the medium composition and growth conditions of a mixed bacterial culture utilizing methanol. The improved medium resuited in satisfactory growth, high-yield coefficients and gave a product containing reduced polysaccharide concentrations. Optimal growth and biomass yields occurred at pH 6.8 a temperature of 37°C and dissolved oxygen at >20% saturation. The maximum growth rate was 0.58 h-1 and maximum biomass yield 0.48 g g-1. The protein content of the product ranged between 81%-83%, and nucleic acid content between 10%-12%, increasing with growth rate. The amino acid profile of the mixed culture product m e t and, in some cases, exceeded the UN Food and Agricultural Organization standard, indicating a good source of feed protein.
Methanogenesis mediated by methylotrophic mixed culture
Applied Biochemistry and Biotechnology, 1994
Enrichment of methanogenic cultures on methanol from the microbial population in the anaerobic digesters operated on agricultural wastes revealed a high rate of biomethanation efficiency. Routine maintenance of this enrichment in a minimal basal medium at room temperature resulted in maximal growth in 40-50 d, and indicated pigment production toward the end of the growth phase. The cultures grown in three different media, with different substrates under light and dark conditions, were analyzed for protein, pigment, and gaseous products, and morphological studies were carried out by light, phase-contrast, fluorescence, and electron microscopy. In different media with methanol as substrate, growth and pigment production were maximal for the light-grown cells, decreasing in the order: phototrophic (PS(m)) > mineral > basal medium. Methanation and phototrophic growth were inversely correlated under lightgrown conditions. In contrast, growth in the dark was predominently methanogenic in the decreasing order: mineral > basal > PS (m). Among other growth conditions tested, utilization of phototrophic substrates under light and dark conditions indicated the following: 1. Basal and mineral media were supportive of methanogenic growth under both light and dark conditions, although methane yields under light-grown conditions were low; *Author to whom all correspondence and reprint requests should be addressed.