Medium Composition Suitable for L-Lysine Production by Methylophilus methylotrophus in Fed-Batch Cultivation
ABSTRACT L-Lysine production was investigated in fed-batch fermentation using L-lysine producer of Methylophilus methylotrophus. By the addition of nutrient composition, containing L-methionine, K(2)HPO(4), NaH(2)PO(4), CuSO(4).5aq, MnSO(4).5aq, ZnSO(4).7aq, FeCl(3), MgSO(4).7aq and CaCl(2).2aq, in the feed medium, cell growth could be maintained through the cultivation, and L-lysine production reached to 7.86 g. In addition, the effect of counter ion for NH(4)(+) (Cl(-), SO(4)(2-), glutamate, succinate and citrate) was examined. The result showed that the cell growth in the medium using Cl(-) and glutamate were improved compared with that using SO(4)(2-), succinate and citrate, and L-lysine production in the medium using Cl(-) and glutamate reached to more than 9.0 g. In this experiment, there was a clear correlation between ionic strength and growth rate in the cultivation. In order to examine the influence of ionic strength on growth rate, the activity of enzymes in central metabolic pathway from methanol to pyruvate were assayed using samples at the log-phase and the stationary phase in fed-batch cultivation using (NH(4))(2)SO(4) and (NH(4))Cl as ammonium source. It was found that the higher ionic strength inhibited methanol oxidation activity, which linked to cell growth. In this report, it was revealed that maintaining a relatively low ionic strength had a positive effect on L-lysine production using L-lysine producer of M. methylotrophus.
SourceAvailable from: Irina L Tokmakova[Show abstract] [Hide abstract]
ABSTRACT: The isolation of auxotrophic mutants, which is a prerequisite for a substantial genetic analysis and metabolic engineering of obligate methylotrophs, remains a rather complicated task. We describe a novel method of constructing mutants of the bacterium Methylophilus methylotrophus AS1 that are auxotrophic for aromatic amino acids. The procedure begins with the Mu-driven integration of the Escherichia coli gene aroP, which encodes the common aromatic amino acid transporter, into the genome of M. methylotrophus. The resulting recombinant strain, with improved permeability to certain amino acids and their analogues, was used for mutagenesis. Mutagenesis was carried out by recombinant substitution of the target genes in the chromosome by linear DNA using the FLP-excisable marker flanked with cloned homologous arms longer than 1,000 bp. M. methylotrophus AS1 genes trpE, tyrA, pheA, and aroG were cloned in E. coli, sequenced, disrupted in vitro using a Kmr marker, and electroporated into an aroP carrier recipient strain. This approach led to the construction of a set of marker-less M. methylotrophus AS1 mutants auxotrophic for aromatic amino acids. Thus, introduction of foreign amino acid transporter genes appeared promising for the following isolation of desired auxotrophs on the basis of different methylotrophic bacteria.Applied and Environmental Microbiology 10/2009; 76(1):75-83. DOI:10.1128/AEM.02217-09 · 3.95 Impact Factor
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ABSTRACT: Common reed (Phragmites australis) and narrow-leaved cattail (Typha angustifolia L.) are two plant species used widely in artificial wetlands constructed to treat wastewater. In this study, the community structure and diversity of root-associated bacteria of common reed and narrow-leaved cattail growing in the Beijing Cuihu Wetland, China, were investigated using 16S rDNA library and PCR-denaturing gradient gel electrophoresis methods. Root-associated bacterial diversity was higher in common reed than in narrow-leaved cattail. In both plant species, the dominant root-associated bacterial species were Alpha, Beta and Gamma Proteobacteria, including the genera Aeromonas, Hydrogenophaga, Ideonella, Uliginosibacterium and Vogesella. Acidobacteria, Actinobacteria, Nitrospirae and Spirochaetes were only found in the roots of common reed. Comparing the root-associated bacterial communities of reed and cattail in our system, many more species of bacteria related involved in the total nitrogen cycle were observed in reed versus cattail, while species involved in total phosphorus and organic matter removal were mainly found in cattail. Although we cannot determine their nutrient removal capacity separately, differences in the root-associated bacterial communities may be an important factor contributing to the differing water purification effects mediated by T. angustifolia and P. australis wetlands. Thus, further work describing the ecosystem functions of these bacterial species is needed, in order to fully understand how effective common reed- and narrow-leaved cattail-dominated wetlands are for phytoremediation.World Journal of Microbiology and Biotechnology (Formerly MIRCEN Journal of Applied Microbiology and Biotechnology) 03/2013; DOI:10.1007/s11274-013-1316-2 · 1.35 Impact Factor
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ABSTRACT: We investigated the community structure of endophytic bacteria in narrowleaf cattail (Typha angustifolia L.) roots growing in the Beijing Cuihu Wetland, China, using the 16S rDNA library technique. In total, 184 individual sequences were used to assess the diversity of endophytic bacteria. Phylogenetic analysis revealed that 161 clones (87.5%) were affiliated with Proteobacteria, other clones grouped into Cytophaga/Flexibacter/Bacteroids (3.3%), Fusobacteria (3.8%), and nearly 5% were uncultured bacteria. In Proteobacteria, the beta and gamma subgroups were the most abundant, accounting for approximately 46% and 36.6% of all Proteobacteria, respectively. The dominant genera included Rhodoferax, Pelomonas, Uliginosibacterium, Pseudomonas, Aeromonas, Rhizobium, Sulfurospirillum, Ilyobacter and Bacteroides. While some of these endophytic bacteria are capable of fixing nitrogen and can therefore improve plant growth, other endophytes may play important biological roles by removing nitrogen, phosphorus and/or organic matter from the water body and thus have the potential to enhance the phytoremediation of eutrophic water bodies. These bacteria have the potential to degrade xenobiota such as methane, methanol, methylated amines, catechol, oxochlorate, urea, cyanide, and 2,4-dichlorophenol. Hence, the use of certain endophytic bacteria in the process of phytoremediation could be a powerful approach for the restoration of eutrophic systems.Research in Microbiology 11/2010; 162(2):124-31. DOI:10.1016/j.resmic.2010.09.021 · 2.83 Impact Factor