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Water Research Apr 2019The aim of this study was to develop and test a non-diffusion limited, high cell density bioreactor for biodegradation of various phenol derivatives. The bioreactor was...
The aim of this study was to develop and test a non-diffusion limited, high cell density bioreactor for biodegradation of various phenol derivatives. The bioreactor was obtained using a straightforward one-step preparation method using cryostructuration and direct cross-linking of bacteria into a 3D structured (sponge-like) macroporous cryogel composite material consisting of 11.6% (by mass) cells and 1.2-1.7% polymer, with approximately 87% water (in the material pores). The macroporous cryogel composite material, composed of live bacteria, has pore sizes in the range of 20-150 μm (confirmed by SEM and Laser Scanning Confocal Microscopy). The enzymatic activity of bacteria within the cryogel structure and the effect of freezing on the viability of the cross-linked cells was estimated by MTT assay. Cryogels based on Pseudomonas mendocina, Rhodococcus koreensis and Acinetobacter radioresistens were exploited for the effective bioremediation of phenol and m-cresol, and to a lesser extent 2-chlorophenol and 4-chlorophenol, utilising these phenolic contaminants in water as their only source of carbon. For evaluation of treatment scalability the bioreactors were prepared in plastic "Kaldnes" carriers to improve their mechanical properties and allow application in batch or fluidised bed water treatment modes.
Topics: Biodegradation, Environmental; Bioreactors; Cryogels; Phenol; Water Purification
PubMed: 30739074
DOI: 10.1016/j.watres.2019.01.028 -
MSystems Apr 2024Plant-associated diazotrophs strongly relate to plant nitrogen (N) supply and growth. However, our knowledge of diazotrophic community assembly and microbial N...
Plant-associated diazotrophs strongly relate to plant nitrogen (N) supply and growth. However, our knowledge of diazotrophic community assembly and microbial N metabolism in plant microbiomes is largely limited. Here we examined the assembly and temporal dynamics of diazotrophic communities across multiple compartments (soils, epiphytic and endophytic niches of root and leaf, and grain) of three cereal crops (maize, wheat, and barley) and identified the potential N-cycling pathways in phylloplane microbiomes. Our results demonstrated that the microbial species pool, influenced by site-specific environmental factors (e.g., edaphic factors), had a stronger effect than host selection (i.e., plant species and developmental stage) in shaping diazotrophic communities across the soil-plant continuum. Crop diazotrophic communities were dominated by a few taxa (~0.7% of diazotrophic phylotypes) which were mainly affiliated with , , , and . Furthermore, eight dominant taxa belonging to and were identified as keystone diazotrophic taxa for three crops and were potentially associated with microbial network stability and crop yields. Metagenomic binning recovered 58 metagenome-assembled genomes (MAGs) from the phylloplane, and the majority of them were identified as novel species (37 MAGs) and harbored genes potentially related to multiple N metabolism processes (e.g., nitrate reduction). Notably, for the first time, a high-quality MAG harboring genes involved in the complete denitrification process was recovered in the phylloplane and showed high identity to . Overall, these findings significantly expand our understanding of ecological drivers of crop diazotrophs and provide new insights into the potential microbial N metabolism in the phyllosphere.IMPORTANCEPlants harbor diverse nitrogen-fixing microorganisms (i.e., diazotrophic communities) in both belowground and aboveground tissues, which play a vital role in plant nitrogen supply and growth promotion. Understanding the assembly and temporal dynamics of crop diazotrophic communities is a prerequisite for harnessing them to promote plant growth. In this study, we show that the site-specific microbial species pool largely shapes the structure of diazotrophic communities in the leaves and roots of three cereal crops. We further identify keystone diazotrophic taxa in crop microbiomes and characterize potential microbial N metabolism pathways in the phyllosphere, which provides essential information for developing microbiome-based tools in future sustainable agricultural production.
Topics: Microbiota; Agriculture; Soil; Nitrogen; Crops, Agricultural; Plant Development
PubMed: 38501864
DOI: 10.1128/msystems.01055-23 -
Journal of Applied Microbiology Apr 2012Metagenomic analysis of milk samples collected from Kankrej, Gir (Bos indicus) and crossbred (Bos taurus × B. indicus) cattle harbouring subclinical mastitis was...
AIMS
Metagenomic analysis of milk samples collected from Kankrej, Gir (Bos indicus) and crossbred (Bos taurus × B. indicus) cattle harbouring subclinical mastitis was carried out by next-generation sequencing 454 GS-FLX technology to elucidate the microbial community structure of cattle milk.
METHODS AND RESULTS
Milk samples from Kankrej, Gir and crossbred cattle were subjected to metagenomic profiling by pyrosequencing. The Metagenomic analysis produced 63·07, 11·09 and 7·87 million base pairs (Mb) of sequence data, assembled in 264 798, 56 114 and 36 762 sequences with an average read length of 238, 197 and 214 nucleotides in Kankrej, Gir and crossbred cattle, respectively. Phylogenetic and metabolic profiles by the web-based tool MG-RAST revealed that the members of Enterobacteriales were predominant in mastitic milk followed by Pseudomonadales, Bacillales and Lactobacillales. Around 56 different species with varying abundance were detected in the subclinically infected milk. Escherichia coli was found to be the most predominant species in Kankrej and Gir cattle followed by Pseudomonas aeruginosa, Pseudomonas mendocina, Shigella flexneri and Bacillus cereus. In crossbred cattle, Staphylococcus aureus followed by Klebsiella pneumoniae, Staphylococcus epidermidis and E. coli were detected in descending order. Metabolic profiling indicated fluoroquinolones, methicillin, copper, cobalt-zinc-cadmium as the groups of antibiotics and toxic compounds to which the organisms showed resistance. Sequences indicating potential of organisms exhibiting multidrug resistance against antibiotics and resistance to toxic compounds were also present. Interestingly, presence of bacteriophages against Staph. aureus, E. coli, Enterobacter and Yersinia species was also observed.
CONCLUSIONS
The analysis identified potential infectious organisms in mastitis, resistance of organisms to antibiotics and chemical compounds and the natural resistance potential of dairy cows.
SIGNIFICANCE AND IMPACT OF THE STUDY
The findings of this study may help in formulating strategies for the prevention and treatment of mastitis in dairy animals and consequently in reducing economic losses incurred because of it.
Topics: Animals; Anti-Bacterial Agents; Bacteria; Cattle; Crosses, Genetic; Female; High-Throughput Nucleotide Sequencing; Mastitis, Bovine; Milk
PubMed: 22277077
DOI: 10.1111/j.1365-2672.2012.05244.x -
The Biochemical Journal Jun 1999A pimeloyl-CoA synthetase from Pseudomonas mendocina 35 was purified and characterized, the DNA sequence determined, and the gene cloned into Escherichia coli to yield...
A pimeloyl-CoA synthetase from Pseudomonas mendocina 35 was purified and characterized, the DNA sequence determined, and the gene cloned into Escherichia coli to yield an active enzyme. The purified enzyme had a pH optimum of approximately 8.0, Km values of 0.49 mM for pimelic acid, 0.18 mM for CoA and 0.72 mM for ATP, a subunit Mr of approximately 80000 as determined by SDS/PAGE, and was found to be a tetramer by gel-filtration chromatography. The specific activity of the purified enzyme was 77.3 units/mg of protein. The enzyme was not absolutely specific for pimelic acid. The relative activity for adipic acid (C6) was 72% and for azaleic acid (C9) was 18% of that for pimelic acid (C7). The N-terminal amino acid was blocked to amino acid sequencing, but controlled proteolysis resulted in three peptide fragments for which amino acid sequences were obtained. An oligonucleotide gene probe corresponding to one of the amino acid sequences was synthesized and used to isolate the gene (pauA, pimelic acid-utilizing A) coding for pimeloyl-CoA synthetase. The pauA gene, which codes for a protein with a theoretical Mr of 74643, was then sequenced. The deduced amino acid sequence of the enzyme showed similarity to hypothetical proteins from Archaeoglobus fulgidus, Methanococcus jannaschii, Pyrococcus horikoshii, E. coli and Streptomyces coelicolor, and some limited similarity to microbial succinyl-CoA synthetases. The similarity with the protein from A. fulgidus was especially strong, thus indicating a function for this unidentified protein. The pauA gene was cloned into E. coli, where it was expressed and resulted in an active enzyme.
Topics: Acyl Coenzyme A; Adenosine Triphosphate; Amino Acid Sequence; Base Sequence; Chromatography, High Pressure Liquid; Cloning, Molecular; Coenzyme A Ligases; Enzyme Stability; Hydrogen-Ion Concentration; Kinetics; Molecular Sequence Data; Molecular Weight; Pimelic Acids; Pseudomonas; Recombinant Proteins; Restriction Mapping; Sequence Analysis; Sequence Homology, Amino Acid; Spectrum Analysis; Substrate Specificity
PubMed: 10359666
DOI: No ID Found -
Applied and Environmental Microbiology Dec 2003Pseudomonas mendocina KR-1 grew well on toluene, n-alkanes (C5 to C8), and 1 degrees alcohols (C2 to C8) but not on other aromatics, gaseous n-alkanes (C1 to C4),...
Pseudomonas mendocina KR-1 grew well on toluene, n-alkanes (C5 to C8), and 1 degrees alcohols (C2 to C8) but not on other aromatics, gaseous n-alkanes (C1 to C4), isoalkanes (C4 to C6), 2 degrees alcohols (C3 to C8), methyl tertiary butyl ether (MTBE), or tertiary butyl alcohol (TBA). Cells grown under carbon-limited conditions on n-alkanes in the presence of MTBE (42 micromoles) oxidized up to 94% of the added MTBE to TBA. Less than 3% of the added MTBE was oxidized to TBA when cells were grown on either 1 degrees alcohols, toluene, or dextrose in the presence of MTBE. Concentrated n-pentane-grown cells oxidized MTBE to TBA without a lag phase and without generating tertiary butyl formate (TBF) as an intermediate. Neither TBF nor TBA was consumed by n-pentane-grown cells, while formaldehyde, the expected C1 product of MTBE dealkylation, was rapidly consumed. Similar Ks values for MTBE were observed for cells grown on C5 to C8 n-alkanes (12.95 +/- 2.04 mM), suggesting that the same enzyme oxidizes MTBE in cells grown on each n-alkane. All growth-supporting n-alkanes (C5 to C8) inhibited MTBE oxidation by resting n-pentane-grown cells. Propane (Ki = 53 micromoles) and n-butane (Ki = 16 micromoles) also inhibited MTBE oxidation, and both gases were also consumed by cells during growth on n-pentane. Cultures grown on C5 to C8 n-alkanes also exhibited up to twofold-higher levels of growth in the presence of propane or n-butane, whereas no growth stimulation was observed with methane, ethane, MTBE, TBA, or formaldehyde. The results are discussed in terms of their impacts on our understanding of MTBE biodegradation and cometabolism.
Topics: Alkanes; Culture Media; Kinetics; Methyl Ethers; Oxidation-Reduction; Pseudomonas mendocina; Time Factors
PubMed: 14660389
DOI: 10.1128/AEM.69.12.7385-7394.2003 -
Applied and Environmental Microbiology May 2007Growth of the Pseudomonas mendocina ymp strain on insoluble ferrihydrite is enhanced by exogenous reductants with concurrent increase in soluble iron concentrations....
Growth of the Pseudomonas mendocina ymp strain on insoluble ferrihydrite is enhanced by exogenous reductants with concurrent increase in soluble iron concentrations. This shows that exogenous reductants play a substantial role in the overall microbial iron bioavailability. The exogenous reductants may work together with the siderophores, Fe-scavenging agents, to facilitate ferrihydrite dissolution.
Topics: Ferric Compounds; Iron; Pseudomonas mendocina; Reducing Agents; Solubility
PubMed: 17384310
DOI: 10.1128/AEM.02586-06 -
Applied and Environmental Microbiology Jul 2011Choline is abundant in association with eukaryotes and plays roles in osmoprotection, thermoprotection, and membrane biosynthesis in many bacteria. Aerobic catabolism of...
Choline is abundant in association with eukaryotes and plays roles in osmoprotection, thermoprotection, and membrane biosynthesis in many bacteria. Aerobic catabolism of choline is widespread among soil proteobacteria, particularly those associated with eukaryotes. Catabolism of choline as a carbon, nitrogen, and/or energy source may play important roles in association with eukaryotes, including pathogenesis, symbioses, and nutrient cycling. We sought to generate choline analogues to study bacterial choline catabolism in vitro and in situ. Here we report the characterization of a choline analogue, propargylcholine, which inhibits choline catabolism at the level of Dgc enzyme-catalyzed dimethylglycine demethylation in Pseudomonas aeruginosa. We used genetic analyses and 13C nuclear magnetic resonance to demonstrate that propargylcholine is catabolized to its inhibitory form, propargylmethylglycine. Chemically synthesized propargylmethylglycine was also an inhibitor of growth on choline. Bioinformatic analysis suggests that there are genes encoding DgcA homologues in a variety of proteobacteria. We examined the broader utility of propargylcholine and propargylmethylglycine by assessing growth of other members of the proteobacteria that are known to grow on choline and possess putative DgcA homologues. Propargylcholine showed utility as a growth inhibitor in P. aeruginosa but did not inhibit growth in other proteobacteria tested. In contrast, propargylmethylglycine was able to inhibit choline-dependent growth in all tested proteobacteria, including Pseudomonas mendocina, Pseudomonas fluorescens, Pseudomonas putida, Burkholderia cepacia, Burkholderia ambifaria, and Sinorhizobium meliloti. We predict that chemical inhibitors of choline catabolism will be useful for studying this pathway in clinical and environmental isolates and could be a useful tool to study proteobacterial choline catabolism in situ.
Topics: Bacteria, Aerobic; Burkholderia; Carbon; Choline; Energy Metabolism; Enzyme Inhibitors; Metabolic Networks and Pathways; Nitrogen; Pseudomonas; Sarcosine; Sinorhizobium meliloti
PubMed: 21602374
DOI: 10.1128/AEM.00504-11 -
Metabolic Engineering Sep 2018Liquid fuels sourced from fossil sources are the dominant energy form for mobile transport today. The consumption of fossil fuels is still increasing, resulting in a...
Liquid fuels sourced from fossil sources are the dominant energy form for mobile transport today. The consumption of fossil fuels is still increasing, resulting in a continued search for more sustainable methods to renew our supply of liquid fuel. Photosynthetic microorganisms naturally accumulate hydrocarbons that could serve as a replacement for fossil fuel, however productivities remain low. We report successful introduction of five synthetic metabolic pathways in two green cell factories, prokaryotic cyanobacteria and eukaryotic algae. Heterologous thioesterase expression enabled high-yield conversion of native fatty acyl-acyl carrier protein (ACP) into free fatty acids (FFA) in Synechocystis sp. PCC 6803 but not in Chlamydomonas reinhardtii where the polar lipid fraction instead was enhanced. Despite no increase in measurable FFA in Chlamydomonas, genetic recoding and over-production of the native fatty acid photodecarboxylase (FAP) resulted in increased accumulation of 7-heptadecene. Implementation of a carboxylic acid reductase (CAR) and aldehyde deformylating oxygenase (ADO) dependent synthetic pathway in Synechocystis resulted in the accumulation of fatty alcohols and a decrease in the native saturated alkanes. In contrast, the replacement of CAR and ADO with Pseudomonas mendocina UndB (so named as it is responsible for 1-undecene biosynthesis in Pseudomonas) or Chlorella variabilis FAP resulted in high-yield conversion of thioesterase-liberated FFAs into corresponding alkenes and alkanes, respectively. At best, the engineering resulted in an increase in hydrocarbon accumulation of 8- (from 1 to 8.5 mg/g cell dry weight) and 19-fold (from 4 to 77 mg/g cell dry weight) for Chlamydomonas and Synechocystis, respectively. In conclusion, reconstitution of the eukaryotic algae pathway in the prokaryotic cyanobacteria host generated the most effective system, highlighting opportunities for mix-and-match synthetic metabolism. These studies describe functioning synthetic metabolic pathways for hydrocarbon fuel synthesis in photosynthetic microorganisms for the first time, moving us closer to the commercial implementation of photobiocatalytic systems that directly convert CO into infrastructure-compatible fuels.
Topics: Biofuels; Carbon Dioxide; Chlamydomonas reinhardtii; Fatty Acids; Microorganisms, Genetically-Modified; Synechocystis
PubMed: 30144559
DOI: 10.1016/j.ymben.2018.08.008 -
Mussel Inspired Chemistry and Bacteria Derived Polymers for Oral Mucosal Adhesion and Drug Delivery.Frontiers in Bioengineering and... 2021Ulceration of the oral mucosa is common, can arise at any age and as a consequence of the pain lessens enjoyment and quality of life. Current treatment options often...
Ulceration of the oral mucosa is common, can arise at any age and as a consequence of the pain lessens enjoyment and quality of life. Current treatment options often involve the use of topical corticosteroids with poor drug delivery systems and inadequate contact time. In order to achieve local controlled delivery to the lesion with optimal adhesion, we utilized a simple polydopamine chemistry technique inspired by mussels to replicate their adhesive functionality. This was coupled with production of a group of naturally produced polymers, known as polyhydroxyalkanoates (PHA) as the delivery system. Initial work focused on the synthesis of PHA using CH50; once synthesized and extracted from the bacteria, the PHAs were solvent processed into films. Polydopamine coating was subsequently achieved by immersing the solvent cast film in a polymerized dopamine solution. Fourier Transform Infrared Spectroscopy (FTIR) spectroscopy confirmed functionalization of the PHA films via the presence of amine groups. Further characterization of the samples was carried out via surface energy measurements and Scanning Electron Microscopy (SEM) micrographs for surface topography. An adhesion test via reverse compression testing directly assessed adhesive properties and revealed an increase in polydopamine coated samples. To further identify the effect of surface coating, LIVE/DEAD imaging and Alamar Blue metabolic activity evaluated attachment and proliferation of fibroblasts on the biofilm surfaces, with higher cell growth in favor of the coated samples. Finally, biocompatibility was investigated in a rat model where the polydopamine coated PHA showed less inflammatory response over time compared to uncoated samples with sign of neovascularization. In conclusion, this simple mussel inspired polydopamine chemistry introduces a step change in bio-surface functionalization and holds great promise for the treatment of oral conditions.
PubMed: 34026742
DOI: 10.3389/fbioe.2021.663764 -
Scientific Reports Aug 2017Aerobic denitrification is a process reducing the nitrate into gaseous nitrogen forms in the presence of oxygen gas, which makes the nitrification and denitrification...
Aerobic denitrification is a process reducing the nitrate into gaseous nitrogen forms in the presence of oxygen gas, which makes the nitrification and denitrification performed simultaneously. However, little was known on the diversity of the culturable aerobic denitrifying bacteria in the surface water system. In this study, 116 strains of aerobic denitrifying bacteria were isolated from the sediment, water and biofilm samples in Liangshui River of Beijing. These bacteria were classified into 14 genera based on the 16 S rDNA, such as Pseudomonas, Rheinheimera, and Gemmobacter. The Pseudomonas sp., represented by the Pseudomonas stutzeri, Pseudomonas mendocina and Pseudomonas putida, composed the major culturable aerobic denitrifiers of the river, followed by Ochrobactrum sp. and Rheinheimera sp. The PCA plot showed the unclassified Pseudomonas sp. and Rheinheimera pacifica preferred to inhabit in biofilm phase while one unclassified Ochrobactrum sp. and Pseudomonas resinovorans had higher abundance in the sediment. In the overlying water, the Pseudomonas stutzeri and Ochrobactrum rhizosphaerae were found to have higher abundance, indicating these aerobic denitrifiers had different habitat-preferable characteristics among the 3 phases of river system. The findings may help select the niche to isolate the aerobic denitrifiers and facilitate the bioaugmentation-based purification of the nitrate polluted surface water.
Topics: Bacteria, Aerobic; Biofilms; Denitrification; Geologic Sediments; Microbiota; Nitrogen-Fixing Bacteria; Rivers
PubMed: 28855587
DOI: 10.1038/s41598-017-09556-9