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Current Microbiology Dec 2022The dynamic microflora associated within, and in the surrounding aquatic environment, has been found to be responsible for the functional properties of many aquatic...
The dynamic microflora associated within, and in the surrounding aquatic environment, has been found to be responsible for the functional properties of many aquatic plants. The aim of the current work was to evaluate the effectiveness of Lemnaceae-based wastewater treatment system under tropical conditions and investigate the changes in the aquatic microflora upon plant growth. A biological wastewater treatment system was designed and investigated using mixed Lemnaceae culture comprising Lemna minor and Spirodela polyrhiza in a batch mode. A significant reduction in total solids (31.8%), biochemical oxygen demand (93.5%), and chemical oxygen demand (73.2%) was observed after seven days of duckweed growth using a low inoculum. A preliminary study on the change in the microbial population diversity and functionality, in the wastewater before and after treatment, revealed an increase in the denitrifying microflora in wastewater post-Lemnaceae treatment. Dominance of 10 bacterial phyla, contributing for 98.3% of the total bacterial communities, was recorded, and ~ 50.6% loss of diversity post-treatment of wastewater was revealed by the Shannon Index. Among 16 bacterial families showing relative abundance of ≥ 1% in untreated wastewater, Methylobacteriaceae, Pseudomonadaceae, Brucellaceae, Rhodobacteraceae, and Acetobacteraceae prevailed in the water post-treatment by duckweeds. This is a novel work done on the dynamics of aquatic microflora associated with Lemnaceae under tropical Indian conditions. It confirms the application of Lemnaceae-based wastewater treatment system as effective biofilter and calls for further studies on the active involvement of the endophytic and aquatic microflora in the functions of these plant.
Topics: Humans; Wastewater; Araceae; Plants; Bacteria; Population Dynamics; Water Purification
PubMed: 36585971
DOI: 10.1007/s00284-022-03149-0 -
Biotechnology Journal Feb 2023Methylobacterium extorquens AM1 (AM1), a model strain of methylotrophic cell factories (MeCFs) could be used to produce fine chemicals from methanol. Synthesis of...
Methylobacterium extorquens AM1 (AM1), a model strain of methylotrophic cell factories (MeCFs) could be used to produce fine chemicals from methanol. Synthesis of heterologous products usually needs reducing cofactors, but AM1 growing on methanol lack reducing power. Formate could be used as a reducing agent. In this study, mevalonic acid (MEV) yield of 0.067 gMEV/g methanol was reached by adding 10 mmol L sodium formate in MEV accumulating stage (at 72 h). The yield was improved by 64.57%, and represented the highest yield reported to date. C-labeling experiments revealed global effects of sodium formate on metabolic pathways in engineered Methylobacterium extorquens AM1. Sodium formate significantly increased the ratios of reducing equivalents, enhanced the metabolic rate of pathways demanding reducing cofactors and redirected the carbon flux to MEV synthesis. As a result, coupling formate to methanol-based production provide a promising way for converting C1 substances to useful chemical products.
Topics: Mevalonic Acid; Methylobacterium extorquens; Metabolic Engineering; Methanol; Formates; Carbon Cycle
PubMed: 36424513
DOI: 10.1002/biot.202200402 -
Bioscience, Biotechnology, and... Dec 2022C1-microorganisms that can utilize C1-compounds, such as methane and methanol, are ubiquitous in nature, and contribute to drive the global carbon cycle between two... (Review)
Review
C1-microorganisms that can utilize C1-compounds, such as methane and methanol, are ubiquitous in nature, and contribute to drive the global carbon cycle between two major greenhouse gases, CO2 and methane. Plants emit C1-compounds from their leaves and provide habitats for C1-microorganisms. Among C1-microorganisms, Methylobacterium spp., representative of methanol-utilizing methylotrophic bacteria, predominantly colonize the phyllosphere and are known to promote plant growth. This review summarizes the interactions between C1-mircroorganisms and plants that affect not only the fixation of C1-compounds produced by plants but also CO2 fixation by plants. We also describe our recent understanding of the survival strategy of C1-microorganisms in the phyllosphere and the application of Methylobacterium spp. to improve rice crop yield.
Topics: Methanol; Carbon Dioxide; Plants; Methane; Methylobacterium; Plant Leaves; Carbon
PubMed: 36367545
DOI: 10.1093/bbb/zbac176 -
Metabolic Engineering Nov 2022Formate is a promising, water-soluble C1 feedstock for biotechnology that can be efficiently produced from CO-but formatotrophy has been engineered in only a few...
Formate is a promising, water-soluble C1 feedstock for biotechnology that can be efficiently produced from CO-but formatotrophy has been engineered in only a few industrially-relevant microbial hosts. We addressed the challenge of expanding the feedstock range of bacterial hosts by adopting Pseudomonas putida as a robust platform for synthetic formate assimilation. Here, the metabolism of a genome-reduced variant of P. putida was radically rewired to establish synthetic auxotrophies that could be functionally complemented by expressing components of the reductive glycine (rGly) pathway. We adopted a modular engineering approach, dividing C1 assimilation in segments composed of both heterologous activities (sourced from Methylobacterium extorquens) and native biochemical reactions. Modular expression of rGly pathway elements enabled growth on formate as carbon source and acetate (predominantly for energy supply), and adaptive laboratory evolution of two lineages of engineered P. putida formatotrophs lead to doubling times of ca. 15 h. We likewise identified emergent metabolic features for assimilation of C1 units in these evolved P. putida populations. Taken together, our results consolidate the landscape of useful microbial platforms that can be implemented for C1-based biotechnological production towards a formate bioeconomy.
Topics: Pseudomonas putida; Metabolic Engineering; Formates; Methylobacterium extorquens; Glycine
PubMed: 36328297
DOI: 10.1016/j.ymben.2022.10.008 -
International Journal of Molecular... Sep 2022(Ca)-dependent pyrroloquinolinequinone (PQQ)-dependent methanol dehydrogenase (MDH) (EC: 1.1.2.7) is one of the key enzymes of primary C1-compound metabolism in...
(Ca)-dependent pyrroloquinolinequinone (PQQ)-dependent methanol dehydrogenase (MDH) (EC: 1.1.2.7) is one of the key enzymes of primary C1-compound metabolism in methylotrophy. PQQ-MDH is a promising catalyst for electrochemical biosensors and biofuel cells. However, the large-scale use of PQQ-MDH in bioelectrocatalysis is not possible due to the low yield of the native enzyme. Homologously overexpressed MDH was obtained from methylotrophic bacterium AM1 by cloning the gene of only one subunit, . The His-tagged enzyme was easily purified by immobilized metal ion affinity chromatography (36% yield). A multimeric form (α6β6) of recombinant PQQ-MDH possessing enzymatic activity (0.54 U/mg) and high stability was demonstrated for the first time. pH-optimum of the purified protein was about 9-10; the enzyme was activated by ammonium ions. It had the highest affinity toward methanol (K = 0.36 mM). The recombinant MDH was used for the fabrication of an amperometric biosensor. Its linear range for methanol concentrations was 0.002-0.1 mM, the detection limit was 0.7 µM. The properties of the invented biosensor are competitive to the analogs, meaning that this enzyme is a promising catalyst for industrial methanol biosensors. The developed simplified technology for PQQ-MDH production opens up new opportunities for the development of bioelectrocatalytic systems.
Topics: Alcohol Oxidoreductases; Ammonium Compounds; Ions; Methanol; Methylobacterium extorquens
PubMed: 36142248
DOI: 10.3390/ijms231810337 -
Applied Microbiology and Biotechnology Oct 2022The methylotrophic bacterium Methylorubrum extorquens AM1 has the potential to become a platform organism for methanol-driven biotechnology. Its ethylmalonyl-CoA pathway...
The methylotrophic bacterium Methylorubrum extorquens AM1 has the potential to become a platform organism for methanol-driven biotechnology. Its ethylmalonyl-CoA pathway (EMCP) is essential during growth on C1 compounds and harbors several CoA-activated dicarboxylic acids. Those acids could serve as precursor molecules for various polymers. In the past, two dicarboxylic acid products, namely mesaconic acid and 2-methylsuccinic acid, were successfully produced with heterologous thioesterase YciA from Escherichia coli, but the yield was reduced by product reuptake. In our study, we conducted extensive research on the uptake mechanism of those dicarboxylic acid products. By using 2,2-difluorosuccinic acid as a selection agent, we isolated a dicarboxylic acid import mutant. Analysis of the genome of this strain revealed a deletion in gene dctA2, which probably encodes an acid transporter. By testing additional single, double, and triple deletions, we were able to rule out the involvement of the two other DctA transporter homologs and the ketoglutarate transporter KgtP. Uptake of 2-methylsuccinic acid was significantly reduced in dctA2 mutants, while the uptake of mesaconic acid was completely prevented. Moreover, we demonstrated M. extorquens-based synthesis of citramalic acid and a further 1.4-fold increase in product yield using a transport-deficient strain. This work represents an important step towards the development of robust M. extorquens AM1 production strains for dicarboxylic acids. KEY POINTS: • 2,2-Difluorosuccinic acid is used to select for dicarboxylic acid uptake mutations. • Deletion of dctA2 leads to reduction of dicarboxylic acid uptake. • Transporter-deficient strains show improved production of citramalic acid.
Topics: Dicarboxylic Acids; Escherichia coli; Fumarates; Malates; Maleates; Methanol; Methylobacterium extorquens; Polymers; Succinates
PubMed: 36104545
DOI: 10.1007/s00253-022-12161-0 -
International Journal of Molecular... Aug 2022Herein, a novel laccase gene, , was amplified from and successfully expressed in with a molecular weight of approximately 50 kDa. The purified Melac13220 had no...
Herein, a novel laccase gene, , was amplified from and successfully expressed in with a molecular weight of approximately 50 kDa. The purified Melac13220 had no absorption peak at 610 nm and remained silent within electron paramagnetic resonance spectra, suggesting that Melac13220 belongs to the non-blue laccase group. Both inductively coupled plasma spectroscopy/optical emission spectrometry (ICP-OES) and isothermal titration calorimetry (ITC) indicated that one molecule of Melac13220 can interact with two iron ions. Furthermore, the optimal temperature of Melac13220 was 65 °C. It also showed a high thermolability, and its half-life at 65 °C was 80 min. Melac13220 showed a very good acid environment tolerance; its optimal pH was 1.5. Cu and Co can slightly increase enzyme activity, whereas Fe could increase Melac13220's activity five-fold. Differential scanning calorimetry (DSC) indicated that Fe could also stabilize Melac13220. Unlike most laccases, Melac13220 can efficiently decolorize Congo Red and Indigo Carmine dyes even in the absence of a redox mediator. Thus, the non-blue laccase from shows potential application value and may be valuable for environmental protection, especially in the degradation of dyes at low pH.
Topics: Coloring Agents; Escherichia coli; Hydrogen-Ion Concentration; Indigo Carmine; Laccase; Methylobacterium extorquens; Temperature
PubMed: 36077196
DOI: 10.3390/ijms23179804 -
Carbohydrate Polymers Nov 2022Methylobacterium extorquens is a facultative methylotrophic Gram-negative bacterium, often associated with plants, that exhibits a unique ability to grow in the presence...
Methylobacterium extorquens is a facultative methylotrophic Gram-negative bacterium, often associated with plants, that exhibits a unique ability to grow in the presence of high methanol concentrations, which serves as a single carbon energy source. We found that M. extorquens strain PA1 secretes a mixture of different exopolysaccharides (EPSs) when grown in reference medium or in presence of methanol, that induces the secretion of a peculiar and heterogenous mixture of EPSs, with different structure, composition, repeating units, bulk and a variable degree of methylation. These factors influenced 3D structure and supramolecular assets, diffusion properties and hydrodynamic radius, and likely contribute to increase methanol tolerance and cell stability. No direct methanol involvement in the EPSs solvation shell was detected, indicating that the polymer exposure to methanol is water mediated. The presence of methanol induces no changes in size and shape of the polymer chains, highlighting how water-methanol mixtures are a good solvent for refEPS and metEPS.
Topics: Methanol; Methylobacterium extorquens; Polymers; Stress, Physiological; Water
PubMed: 35989007
DOI: 10.1016/j.carbpol.2022.119863 -
Genome Biology and Evolution Aug 2022Methylobacterium is a group of methylotrophic microbes associated with soil, fresh water, and particularly the phyllosphere, the aerial part of plants that has been well...
Methylobacterium is a group of methylotrophic microbes associated with soil, fresh water, and particularly the phyllosphere, the aerial part of plants that has been well studied in terms of physiology but whose evolutionary history and taxonomy are unclear. Recent work has suggested that Methylobacterium is much more diverse than thought previously, questioning its status as an ecologically and phylogenetically coherent taxonomic genus. However, taxonomic and evolutionary studies of Methylobacterium have mostly been restricted to model species, often isolated from habitats other than the phyllosphere and have yet to utilize comprehensive phylogenomic methods to examine gene trees, gene content, or synteny. By analyzing 189 Methylobacterium genomes from a wide range of habitats, including the phyllosphere, we inferred a robust phylogenetic tree while explicitly accounting for the impact of horizontal gene transfer (HGT). We showed that Methylobacterium contains four evolutionarily distinct groups of bacteria (namely A, B, C, D), characterized by different genome size, GC content, gene content, and genome architecture, revealing the dynamic nature of Methylobacterium genomes. In addition to recovering 59 described species, we identified 45 candidate species, mostly phyllosphere-associated, stressing the significance of plants as a reservoir of Methylobacterium diversity. We inferred an ancient transition from a free-living lifestyle to association with plant roots in Methylobacteriaceae ancestor, followed by phyllosphere association of three of the major groups (A, B, D), whose early branching in Methylobacterium history has been heavily obscured by HGT. Together, our work lays the foundations for a thorough redefinition of Methylobacterium taxonomy, beginning with the abandonment of Methylorubrum.
Topics: Ecosystem; Methylobacterium; Phylogeny; Plant Leaves; Plants; RNA, Ribosomal, 16S
PubMed: 35906926
DOI: 10.1093/gbe/evac123 -
Archives of Microbiology Jul 2022Two novel Gram-stain-negative, aerobic, rod shaped bacterial strains BT290 and BT689 were isolated from soil collected in South Korea. Colony morphologies of both...
Two novel Gram-stain-negative, aerobic, rod shaped bacterial strains BT290 and BT689 were isolated from soil collected in South Korea. Colony morphologies of both strains were circular and convex while the colors of BT290 and BT689 were light-pink and white, respectively. Phylogenetic analysis based on 16S rRNA gene sequences revealed that BT290 and BT689 belong to a distinct lineage within the genus Microvirga (family Methylobacteriaceae, order Rhizobiales, class Alphaproteobacteria, phylum Proteobacteria, kingdom Bacteria). The 16S rR NA gene sequence similarity between two strains was 97.9%. Both strains had the similar quinone system, with ubiquinone 10 (Q-10) as the major respiratory quinone. The major polar lipids of strains BT290 and BT689 were phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG), phosphatidylcholine (PC), and phosphatidylglycerol (PG). The major cellular fatty acids of strain BT290 were C ω7c (58.2%) and C (17.7%), while those of strain BT689 were C ω7c (61.8%) and C (10.8%). On the bases of polyphasic analysis (phylogenetic, chemotaxonomic, and biochemical), strains BT290 and BT689 can be suggested as novel bacterial species within the genus Microvirga and the proposed names are Microvirga terrestris and Microvirga arvi, respectively. The type strain of Microvirga terrestris is BT290 (= KCTC 72367 = NBRC 114844) and the type strain of Microvirga arvi is BT689 (= KACC 22016 = NBRC 114858), respectively.
Topics: Alphaproteobacteria; Bacterial Typing Techniques; Base Composition; Bradyrhizobiaceae; DNA, Bacterial; Fatty Acids; Methylobacteriaceae; Phylogeny; Quinones; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Soil; Soil Microbiology
PubMed: 35895136
DOI: 10.1007/s00203-022-03109-z