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Xanthobacter sp. SoF1 (SoF1) is an autotrophic hydrogen‐oxidizing bacteria that produces protein‐rich biomass and has potential to be an alternative protein source that is more environmentally sustainable than animal and plant derived proteins. A protein‐rich powder derived from SoF1 was the test material in a 90‐day repeated‐dose oral toxicity study to explore major toxic effects, demonstrate target organs, and provide an estimate of a no‐observed‐adverse‐effect level (NOAEL). Daily doses of 0 (vehicle only), 375, 750, and 1500 mg/kg bw/day of the test material were administered by gavage to 10 Han:WIST rats/sex/group. An additional group was administered 1290 mg/kg bw/day whey protein concentrate as positive control. No treatment‐related adverse effects were observed, and no target organs were determined after 90/91 days of consecutive administration of the test item. A NOAEL of 1500 mg/kg bw/day was determined. A 90‐day repeated‐dose oral toxicity study was conducted to assess the subchronic toxicity of a microbial protein powder derived from Xanthobacter sp. SoF1. Daily doses of 0 (vehicle only), 375, 750, and 1500 mg/kg bw/day of the test material and 1290 mg/kg whey protein concentrate as a positive control were administered by gavage to 10 Han:WIST rats/sex/group. A no‐observed‐adverse‐effect‐level of 1500 mg/kg bw/day was determined. [ABSTRACT FROM AUTHOR]

One way of limiting the environmental impact of food production and improving food security is to replace part of the animal‐ or plant‐based protein in the human diet with protein sourced from microorganisms. The recently discovered bacterium Xanthobacter sp. SoF1 (VTT‐E‐193585) grows autotrophically using carbon dioxide gas as the only carbon source, yielding protein‐rich biomass that can be processed further into a powder and incorporated into various food products. Since the safety of this microbial protein powder for human consumption had not been previously assessed, its genotoxic potential was evaluated employing three internationally recognized and standardized studies: a bacterial reverse mutation test, an in vitro chromosomal aberration assay in human lymphocytes, and an in vitro micronucleus test in human lymphocytes. No biologically relevant evidence of genotoxicity or mutagenicity was found. The bacterium Xanthobacter sp. SoF1 (VTT‐E‐193585) grows autotrophically using carbon dioxide as the only carbon source, yielding protein‐rich biomass. Since the safety of this microbial protein for human consumption had not been assessed, its genotoxic potential was evaluated employing three internationally recognized and standardized studies: an in vitro chromosomal aberration assay in human lymphocytes, a bacterial reverse mutation test and an in vitro micronucleus test in human lymphocytes. No biologically relevant evidence of genotoxicity or mutagenicity was found. [ABSTRACT FROM AUTHOR]

The qualified presumption of safety (QPS) approach was developed to provide a regularly updated generic pre‐evaluation of the safety of microorganisms, intended for use in the food or feed chains, to support the work of EFSA's Scientific Panels. The QPS approach is based on an assessment of published data for each agent, with respect to its taxonomic identity, the body of relevant knowledge and safety concerns. Safety concerns identified for a taxonomic unit (TU) are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications'. In the period covered by this Statement, new information was found leading to the withdrawal of the qualification 'absence of aminoglycoside production ability' for Bacillus velezensis. The qualification for Bacillus paralicheniformis was changed to 'absence of bacitracin production ability'. For the other TUs, no new information was found that would change the status of previously recommended QPS TUs. Of 52 microorganisms notified to EFSA between April and September 2022 (inclusive), 48 were not evaluated because: 7 were filamentous fungi, 3 were Enterococcus faecium, 2 were Escherichia coli, 1 was Streptomyces spp., and 35 were taxonomic units (TUs) that already have a QPS status. The other four TUs notified within this period, and one notified previously as a different species, which was recently reclassified, were evaluated for the first time for a possible QPS status: Xanthobacter spp. could not be assessed because it was not identified to the species level; Geobacillus thermodenitrificans is recommended for QPS status with the qualification 'absence of toxigenic activity'. Streptoccus oralis is not recommended for QPS status. Ogataea polymorpha is proposed for QPS status with the qualification 'for production purposes only'. Lactiplantibacillus argentoratensis (new species) is included in the QPS list. [ABSTRACT FROM AUTHOR]

Rhamnose is a high-value carbohydrate used in flavorings, aromatics, and pharmaceuticals. Current demand for rhamnose is filled through plant-based sources; however, microbially originated rhamnolipids have been proposed as an alternative source. A mixed microbial biofilm, cultured from a wastewater sludge, was found to comprise > 8 dry weight% rhamnose when provided volatile fatty acids as carbon source, and 24 dry weight% when given glucose. The latter rhamnose concentration is a fourfold higher production mass than the current plant-based origin and is competitive with yields from pure microbial cultures. The biofilm was characterized based on total carbohydrate production at varying nutrient levels, individual carbohydrate monomer production from varying organic acid substrates, and microbial community composition—based on 16s rRNA. Biofilm carbohydrate production was maximized at a C:N ratio of 28 (mol:mol). The production of rhamnose varied significantly based on carbon substrate; glucose had the greatest yield of rhamnose, followed by propionic acid, lactic acid, acetic acid, valeric acid, and butyric acid. Microbial community analysis indicated an abundance of organisms within the Xanthobacter genus, which is known to produce rhamnose as zeaxanthin rhamnoside. Rhamnose production was heavily correlated with ribose production (R2 = 0.96). Results suggest that mixed microbial biofilms could be a competitive source of monomeric rhamnose that may be produced from mixed organic waste streams of variable composition via volatile fatty acids and glucose. [ABSTRACT FROM AUTHOR]

We demonstrate the synthesis of NH3 from N2 and H2O at ambient conditions in a single reactor by coupling hydrogen generation from catalytic water splitting to a H2-oxidizing bacterium Xanthobacter autotrophicus, which performs N2 and CO2 reduction to solid biomass. Living cells of X. autotrophicus may be directly applied as a biofertilizer to improve growth of radishes, a model crop plant, by up to ~1,440% in terms of storage root mass. The NH3 generated from nitrogenase (N2ase) in X. autotrophicus can be diverted from biomass formation to an extracellular ammonia production with the addition of a glutamate synthetase inhibitor. The N2 reduction reaction proceeds at a low driving force with a turnover number of 9 × 109 cell-1 and turnover frequency of 1.9 × 104 s-1·cell-1 without the use of sacrificial chemical reagents or carbon feedstocks other than CO2. This approach can be powered by renewable electricity, enabling the sustainable and selective production of ammonia and biofertilizers in a distributed manner. [ABSTRACT FROM AUTHOR]

A bacterial strain able to degrade dichloromethane (DCM) as the sole carbon source was isolated from a wastewater treatment plant receiving domestic and pharmaceutical effluent. 16S rDNA studies revealed the strain to be a Xanthobacter sp. (strain TM1). The new isolated strain when grown aerobically on DCM showed Luong type growth kinetics, with μmax of 0.094 h-1 and Sm of 1,435 mg l-1. Strain TM1 was able to degrade other aromatic and aliphatic halogenated compounds, such as halobenzoates, 2-chloroethanol and dichloroethane. The gene for DCM dehalogenase, which is the key enzyme in DCM degradation, was amplified through PCR reactions. Strain TM1 contains type A DCM dehalogenase (dcmAa), while no product could be obtained for type B dehalogense (dcmAb). The sequence was compared against 12 dcmAa from other DCM degrading strains and 98% or 99% similarity was observed with all other previously isolated DCM dehalogenase genes. This is the first time a Xanthobacter sp. is reported to degrade DCM. [ABSTRACT FROM AUTHOR]

The article focuses on the encoding of a putative gentisate 1,2-dioxygenase in the dibenzothiophene degradation gene cluster from Xanthobacter polyaromaticivorans 127W. The amino acid sequence showed high similarity in sequence with dioxygenases from Pseudomonas alcaligenes, Bradyrhizobium japonicum and Pseudomonas aeruginosa.

The coupling protein and ferredoxin from Xanthobacter autotrophicus Py2 alkene monooxygenase (Xamo) have been functionally expressed in both N-terminal affinity tagged fusion and native forms in Escherichia coli. However, attempts to express the NADH-oxidoreductase and oxygenase, always resulted in the production of inactive, insoluble proteins. Nevertheless, the recombinant reductase from the toluene 4-monooxygenase of Pseudomonas mendocina KR1 was found to functionally complement the Xamo system. In vitro reconstitution, using the recombinant coupling protein and other components purified from the wild type, showed that steady-state epoxidation rate and coupling efficiency were dependent on the relative concentration of Xamo components in the reaction. The optimal molar stoichiometric ratio of Xamo components was determined to be approximately 1:0.25–0.3:2:2 (oxygenase hexamer:reductase:ferredoxin:coupling protein), suggesting the formation of an efficient catalytic complex at the minimal stoichiometric ratio to saturate the probable two-fold symmetry binding sites on the oxygenase. [Copyright &y& Elsevier]

A highly efficient method of transposon mutagenesis was developed for genetic analysis of Xanthobacter autotrophicus Py2. The method makes use of a transposon delivery vector that encodes a hyperactive Tn5 transposase that is 1,000-fold more active than the wild-type transposase. In this construct, the transposase is expressed from the promoter of the tetA gene of plasmid RP4, which is functional in a wide variety of organisms. The transposon itself contains a kanamycin resistance gene as a selectable marker and the origin of replication from plasmid R6K to facilitate subsequent cloning of the resulting insertion site. To test the effectiveness of this method, mutants unable to produce the characteristic yellow pigment (zeaxanthin dirhamnoside) of X. autotrophicus Py2 were isolated and analyzed. Transposon insertions were obtained at high frequency: approximately 1×10–3 per recipient cell. Among these, pigment mutants were observed at a frequency of approximately 10–3. Such mutants were found to have transposon insertions in genes homologous to known carotenoid biosynthetic genes previously characterized in other pigmented bacteria. Mutants were also isolated in Pseudomonas stutzeri and in an Alcaligenes faecalis, demonstrating the effectiveness of the method in diverse Proteobacteria. Preliminary results from other laboratories have confirmed the effectiveness of this method in additional phylogenetically diverse species. [ABSTRACT FROM AUTHOR]

Short-chain aliphatic epoxides and ketones are two classes of toxic organic compounds formed biogenically and anthropogenically. In spite of their toxicity, these compounds are utilized as primary carbon and energy sources or are generated as intermediate metabolites in the metabolism of other compounds (e.g., alkenes, alkanes, and secondary alcohols) by a number of diverse bacteria. One bacterium capable of using both classes of compounds is the gram-negative aerobe Xanthobacter strain Py2. Studies of epoxide and ketone (acetone) metabolism by Xanthobacter strain Py2 have revealed a central role for CO2 in these processes. Both classes of compounds are metabolized by carboxylation reactions that produce β-keto acids as products. The epoxide- and ketone-converting enzymes are distinct carboxylases with molecular properties and cofactor requirements unprecedented for other carboxylases. Epoxide carboxylase is a four-component multienzyme complex that requires NADPH and NAD+ as cofactors. In the course of epoxide carboxylation, a transhydrogenation reaction occurs wherein NADPH undergoes oxidation and NAD+ undergoes reduction. Acetone carboxylase is a multimeric (three-subunit) ATP-dependent enzyme that forms AMP and inorganic phosphate as ATP hydrolysis products in the course of acetone carboxylation. Recent studies have demonstrated that acetone metabolism in diverse anaerobic bacteria (sulfate reducers, denitrifiers, phototrophs, and fermenters) also proceeds by carboxylation reactions. ATP-dependent acetone carboxylase activity has been demonstrated in cell-free extracts of the anaerobic acetone-utilizers Rhodobacter capsulatus, Rhodomicrobium vannielii, and Thiosphaera pantotropha. These studies have identified new roles for CO2 as a cosubstrate in the metabolism of two classes of important xenobiotic compounds. In addition, two new classes of carboxylases have been identified, the investigation of which promises to reveal new insights into biological strategies for the fixation of CO2 to organic substrates. [ABSTRACT FROM AUTHOR]

The regulation of C-metabolism in Xanthobacter strain 25a was studied during growth of the organism on acetate, formate and methanol in chemostat cultures. No activity of methanol dehydrogenase (MDH), formate dehydrogenase (FDS) or ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisC/O) could be detected in cells grown on acetate alone over a range of dilution rates tested. Addition of methanol or formate to the feed resulted in the immediate induction of MDH and FDH and complete utilization ( D=0.10 h) of acetate and the C-substrates. The activities of these enzymes rapidly dropped at the higher growth rates, which suggests that their synthesis is further controlled via repression by 'heterotrophic' substrates such as acetate. Synthesis of RuBisC/O already occurred at low methanol concentrations in the feed, resulting in additive growth yields on acetate/methanol mixtures. The energy generated in the oxidation of formate initially allowed an increased assimilation of acetate (and a decreased dissimilation), resulting in enhanced growth yields on the mixture. RuBisC/O activity could only be detected at the higher formate/acetate ratios in the feed. The data suggest that synthesis of RuBisC/O and CO fixation via the Calvin cycle in Xanthobacter strain 25 a is controlled via a (de)repression mechanism, as is the case in other facultatively autotrophic bacteria. Autotrophic CO fixation only occurs under conditions with a diminished supply of 'heterotrophic' carbon sources and a sufficiently high availability of suitable energy sources. The latter point is further supported by the clearly more pronounced derepressing effect exerted by methanol compared to formate. [ABSTRACT FROM AUTHOR]

All Xanthobacter strains studied are versatile autotrophic bacteria, able to grow on methanol and other substrates. Strain 25a, a yellow-pigmented, pleomorphic, Gram-negative bacterium, capable of autotrophic growth on methanol, formate, thiosulfate, and molecular hydrogen, was isolated from an enrichment culture inoculated with soil from a subtropical greenhouse. Subsequent studies showed that the organism also grows on a wide range of multicarbon substrates. Ammonia, nitrate and molecular nitrogen were used as nitrogen sources. The taxonomic relationship of strains H4-14 and 25a with previously described Xanthobacter strains was studied by numerical classification. Strain H4-14 was identified as a X. flavus strain, but the precise position of strain 25a remained uncertain. It probably belongs to a new species of the genus Xanthobacter. The levels of various enzymes involved in autotrophic and heterotrophic metabolism were determined following growth of strains H4-14 and 25a in batch and continuous cultures. The mechanisms involved in controlling ribulose-1,5-bisphosphate carboxylase/oxygenase synthesis in Xanthobacter strains appear to be comparable to those observed for other autotrophic bacteria, namely repression by organic compounds and derepression by autotrophic energy sources, such as methanol and hydrogen. [ABSTRACT FROM AUTHOR]

Yellow-pigmented bacteria showing typical characteristics of Xanthobacter spp. were isolated from enrichments with propene and 1-butene, using classical techniques. The generation time for growth on propene and 1-butene of these bacteria ranged from 5 to 7h. A NADH-dependent mono-oxygenase was identified in cell-free extract of Xanthobacter Py2. This mono-oxygenase was not influenced by potential inhibitors tested indicating that propene mono-oxygenase is different from other hydrocarbon mono-oxygenases described until now. Nitrogenase activity could be measured using the acetylene reduction assay with propene as energy source, because acetylene did not inhibit the mono-oxygenase activity. [ABSTRACT FROM AUTHOR]

N-fixation was investigated in the chemoautotrophic hydrogen bacterium Xanthobacter H4-14. N-fixing batch cultures of this organism could only be grown at pO values of around 0.02 bar, and in continuous culture dissolved oxygen tensions above 16 μM were found to inhibit N-fixation. Xanthobacter H4-14 utilized a variety of amino acids, nitrate and ammonia as nitrogen sources. Cell-free extracts from steady-state continuous cultures of ammonia grown, nitrate grown and N-fixing Xanthobacter were assayed for the presence of ammonia assimilation enzymes. No alanine dehydrogenase or glutamate dehydrogenase activity was detected. Ammonia was assimilated exclusively via the glutamine synthetase/glutamate synthase pathway, irrespective of the extracellular concentration of ammonia. [ABSTRACT FROM AUTHOR]