Amalonaticus Y19 Research Articles

Co-culture-based biological carbon monoxide conversion by Citrobacter amalonaticus Y19 and Sporomusa ovata via a reducing-equivalent transfer mediator

The biological conversion of carbon monoxide (CO) has been highlighted for the development of a C1 gas biorefinery process. Despite this, the toxicity and low reducing equivalent of CO uptake make biological conversion difficult. The use of synthetic co-cultures is an alternative way of enhancing the performance of CO bioconversion. This study evaluated a synthetic co-culture consisting of Citrobacter amalonaticus Y19 and Sporomusa ovata for acetate production from CO. In this consortium, the CO2 and H2 produced by the water-gas shift reaction of C. amalonaticus Y19, were utilized further by S. ovata. Higher acetate production was achieved in the co-culture system compared to the monoculture counterparts. Furthermore, syntrophic cooperation via various reducing equivalent carriers provided new insights into the synergistic metabolic benefits with a toxic and refractory substrate, such as CO. This study also suggests an appropriate model for examining the syntrophic interaction between microbial species in a mixed community.

Citrobacter amalonaticus Y19 for constitutive expression of carbon monoxide-dependent hydrogen-production machinery

BackgroundCitrobacter amalonaticus Y19 is a good biocatalyst for production of hydrogen (H2) from oxidation of carbon monoxide (CO) via the so-called water–gas-shift reaction (WGSR). It has a high H2-production activity (23.83 mmol H2 g−1 cell h−1) from CO, and can grow well to a high density on various sugars. However, its H2-production activity is expressed only when CO is present as an inducer and in the absence of glucose.ResultsIn order to avoid dependency on CO and glucose, in the present study, the native CO-inducible promoters of WGSR operons (CO dehydrogenase, CODH, and CODH-dependent hydrogenase, CO-hyd) in Y19 were carefully analyzed and replaced with strong and constitutive promoters screened from Y19. One engineered strain (Y19-PR1), selected from three positive ones after screening ~10,000 colonies, showed a similar CO-dependent H2-production activity to that of wild-type Y19, without being affected by glucose and/or CO. Compared with wild-type Y19, transcription of the CODH operon in Y19-PR1 increased 1.5-fold, although that of the CO-hyd operon remained at a similar level. To enhance the activity of CO-Hyd in Y19-PR1, further modifications, including an increase in gene copy number and engineering of the 5′ untranslated region, were attempted, but without success.ConclusionsConvenient recombinant Y19-PR1 that expresses CO-dependent H2-production activity without being limited by CO and glucose was obtained.

Improvement of Trimethylamine Uptake by Euphorbia milii: Effect of Inoculated Bacteria

In the last few years, a great emphasis has been placed on phytoremediation of indoor air pollution studies. However, limited work has been addressed to observe the bacteria potential to assist the phytoremediation process of trimethylamine (TMA). In this work, the ability of 4 different bacteria for individual TMA removal and IAA production were observed. In addition, the enhancement of TMA removal efficiency by Euphorbia milii with various inoculating bacteria were investigated. Bacillus thuringiensis , Citrobacter amalonaticus Y19, Bacillus nealsonii , and white colony-soil bacteria (WCSB) were able to absorb TMA and produce IAA individually. B. thuringiensis and C. amalonaticus Y19 were the two most effective bacteria to improve TMA removal efficiency by the plant. Since concentrations of IAA production by individual bacterium were highly correlated with TMA removal efficiency by plants in early periods of fumigation and highly correlated with leaf IAA production of bacterially inoculated plants, two predicted mechanisms on improving TMA uptake by bacterially inoculated plants are presented: (1) bacteria migration from plant roots to leaves increases leaf IAA concentration and (2) increasing concentration of bacterially inoculated root IAA inhibits transportation of IAA from leaves to roots, resulting in higher leaf IAA concentration. The higher concentration of leaf IAA is suggested to be a factor to increase stomatal opening which improves TMA removal efficiency of the plant.

Effect of culture medium on fermentative and CO-dependent H2 production activity in Citrobacter amalonaticus Y19

Citrobacter amalonaticus Y19 is a unique chemoheterotrophic microorganism capable of carbon monoxide (CO)-dependent hydrogen (H2) production. To improve its biocatalytic potential, the effects of medium composition on cell growth and H2 production were investigated. Among several carbon sources tested, maltose resulted in the best CO consumption and H2 production. Meanwhile, l-cysteine supplementation improved cell growth and H2 production, suggesting that this amino acid stimulates synthesis of FeS clusters. When molybdenum and selenium, which are essential cofactors of formate-hydrogen lyase (FHL), were added, formate accumulation was reduced. Optimized medium composition improved the activities of CO-dependent H2 production (6.3-fold), CODH (22.3-fold) and hydrogenase (2.9-fold). The optimized medium composition also enhanced H2 production in recombinant C. amalonaticus Y19-cooA, which homologously overexpresses CooA, the transcriptional activator of the coo operons. Cells grown under this condition exhibited improved stability in the course of repeated-batch CO-dependent H2 production under non-growing conditions.

Complete genome sequence of novel carbon monoxide oxidizing bacteria Citrobacter amalonaticus Y19, assembled de novo

We report here the complete genome sequence of Citrobacter amalonaticus Y19 isolated from an anaerobic digester. PacBio single-molecule real-time (SMRT) sequencing was employed, resulting in a single scaffold of 5.58Mb. The sequence of a mega plasmid of 291Kb size is also presented.

Cloning and functional expression of Citrobacter amalonaticus Y19 carbon monoxide dehydrogenase in Escherichia coli

Carbon monoxide (CO) is highly toxic but is an abundant carbon source that can be utilized for the production of hydrogen (H2). CO-dependent H2 production is catalyzed by a unique enzyme complex composed of carbon monoxide dehydrogenase (CODH) and CO-dependent hydrogenase (CO–H2ase), both of which contain metal cluster(s). In this study, CODH and the required maturation proteins from the novel facultative anaerobic bacterium Citrobacter amalonaticus Y19 were cloned and heterologously expressed in Escherichia coli. For functional expression of CODH in E. coli, only CooF (ferredoxin-like protein) and CooS (CODH), not the maturation proteins, were needed. The recombinant E. coli BL21(DE3)-cooFS showed a 3.5-fold higher specific CODH activity (4.9 U mg protein−1) compared to C. amalonaticus Y19 (Y19) (1.4 U mg protein−1). Purified heterologous CODH from the soluble cell-free extract of the recombinant E. coli showed a specific activity of 170.6 U mg protein−1. Recombinant E. coli harboring Y19 CODH and maturation proteins did not produce H2 from CO, suggesting that the native hydrogenases present in E. coli could not substitute the Y19 CO–H2ase for CO-dependent H2 production.

Improvement of carbon monoxide-dependent hydrogen production activity in Citrobacter amalonaticus Y19 by over-expressing the CO-sensing transcriptional activator, CooA

Citrobacter amalonaticus Y19 (Y19) can produce hydrogen (H2) from oxidation of carbon monoxide (CO) via the water-gas shift reaction. The reaction is catalyzed by two enzymes, carbon monoxide dehydrogenase (CODH) and carbon monoxide-dependent hydrogenase (CO-Hyd). The contig genome sequencing of Y19 exhibited the presence of unique CO oxidizing gene clusters encoding CODH (cooFS), CO-Hyd (cooMKLXUH) and a putative CO-responsive transcriptional activator (cooA). To improve CO-dependent H2 production activity, we developed recombinant Y19 by homologously over-expressing cooA. The over-expression of cooA improved the whole-cell CO-dependent H2 production activity (3.4-fold), and enzyme activities of CODH (5.3-fold) and CO-Hyd (1.2-fold). Furthermore, quantitative PCR analysis revealed a significant increase in the transcription of the genes located in CODH and CO-Hyd operons of recombinant Y19. The high CO-dependent H2 production activity of the recombinant C. amalonaticus was stably maintained during repeated exposure to CO.

Glycerol assimilation and production of 1,3-propanediol by Citrobacter amalonaticus Y19

Citrobacter amalonaticus Y19 (Y19) was isolated because of its ability for carbon monoxide-dependent hydrogen production (water-gas shift reaction). This paper reports the assimilation of glycerol and the production of 1,3-propanediol (1,3-PDO) by Y19. Genome sequencing revealed that Y19 contained the genes for the utilization of glycerol and 1,2-propanediol (pdu operon) along with those for the synthesis of coenzyme B12 (cob operon). On the other hand, it did not possess the genes for the fermentative metabolism of glycerol of Klebsiella pneumoniae, which consists of both the oxidative (dhaD and dhaK) and reductive (dhaB and dhaT) pathways. In shake-flask cultivation under aerobic conditions, Y19 could grow well with glycerol as the sole carbon source and produced 1,3-PDO. The level of 1,3-PDO production was improved when vitamin B12 was added to the culture medium under aerobic conditions. Under anaerobic conditions, cell growth and 1,3-PDO production on glycerol was also possible, but only when an exogenous electron acceptor, such as nitrate or fumarate, was added. This is the first report of the glycerol metabolism and 1,3-PDO production by C. amalonaticus Y19.

Decolorization of synthetic dyes by Citrobacter amalonaticus Y19

Newly isolated Citrobacter amalonaticus Y19 could decolorize various synthetic dyes containing different chromogenic groups including azo bond (Crocein Orange G, New Coccine, Chromotrope FB, Congo Red, Remazol Black B), anthraquinone (Reactive blue 2) and indigo (Indigo Carmine). Y19's specific degradation of Black B, due to its industrial importance and high decolorization activity, was studied in detail. Y19's decolorization rate, under anaerobic conditions and with glucose as the carbon source, was enhanced as the temperature was increased from 25 to 40 °C or as the initial dye concentration was increased from 25 to 2000 mg/L. The highest decolorization rate was estimated to be 171 mg dye/g cell h for an initial dye concentration of 2000 mg/L. High-performance liquid chromatograph (HPLC) and mass spectrum analyses indicated that Black B was degraded by reductive cleavage of the azo bond. However, one of the degradation products exhibited spontaneous oxidation under the aerobic conditions, and this resulted in an approximately 10% re-development of Black B's original color intensity. This is the first report of newly isolated C. amalonaticus Y19's efficiency as an azo dye-degrading microbe.

Comparison of hydrogen-production capability of four different Enterobacteriaceae strains under growing and non-growing conditions

Non-growing cells can function as whole-cell biocatalysts for hydrogen (H 2) production, a process that has recently drawn much attention. In order to evaluate their potential as whole-cell biocatalysts, we compared the H 2-production capability of four Enterobacteriaceae strains ( Citrobacter amalonaticus Y19, Escherichia coli K-12 MG1655, Escherichia coli DJT135, and Enterobacter aerogenes) under growing and non-growing conditions. We evaluated their H 2-production activity at varying temperatures (25–45 °C) and pH conditions (6.0–8.0) using glucose or formate as the carbon source. Under growing conditions with 10 mM glucose as a substrate, E. aerogenes exhibited the highest H 2-production activity (17.0 ± 0.2 μmol H 2 mg cell −1 h −1) among the four strains, but the final H 2 yield was similar (1.7–1.8 mol H 2 mol −1 glucose) in all four strains. H 2 production in the four strains proceeded through a formate-dependent pathway that involved the formate hydrogen lyase (FHL) complex. Under non-growing conditions with 20 mM formate as a substrate, we obtained high H 2-production activities, in the range of 95.5–195.2 μmol H 2 mg cell −1 h −1, with E. coli DJT135 exhibiting the highest activity (195.2 μmol H 2 mg −1 h −1) at pH 6.0 and 45 °C. In contrast, using glucose as the carbon substrate in non-growing cell experiments greatly reduced the H 2-production activity to 6.1–7.7 μmol H 2 mg cell −1 h −1. This study indicated that formate is a better substrate than glucose for H 2 production by non-growing cells, and that the H 2-production performance among the strains did not vary significantly, with the exception of E. coli K-12 MG1655.

Metabolic-flux analysis of hydrogen production pathway in Citrobacter amalonaticus Y19

For the newly isolated chemoheterotrophic bacterium Citrobacter amalonaticus Y19, anaerobic glucose metabolism and hydrogen ( H 2 ) production pathway were studied using batch cultivation and an in silico metabolic-flux analysis. Batch cultivation was conducted under varying initial glucose concentration between 1.5 and 9.5 g/L with quantitative measurement of major metabolites to obtain accurate carbon material balance. The metabolic flux of Y19 was analyzed using a metabolic-pathway model which was constructed from 81 biochemical reactions. The linear optimization program MetaFluxNet was employed for the analysis. When the specific growth rate of cells was chosen as an objective function, the model described the batch culture characteristics of Ci. amalonaticus Y19 reasonably well. When the specific H 2 production rate was selected as an objective function, on the other hand, the achievable maximal H 2 production yield ( 8.7 mol H 2 / mol glucose) and the metabolic pathway enabling the high H 2 yield were identified. The pathway involved non-native NAD(P)-linked hydrogenase and H 2 production from NAD(P)H which were supplied at a high rate from glucose degradation through the pentose phosphate pathway.

Various hydrogenases and formate-dependent hydrogen production in Citrobacter amalonaticus Y19

An isolate Citrobacter amalonaticus Y19 showed a typical mixed-acid fermentation with lactate and acetate as major end products when grown anaerobically on glucose and pyruvate, respectively. Production of hydrogen ( H 2 ) from glucose, formate, and reduced methylviologen (MV) and benzylviologen (BV) by the resting cells of Y19 indicates the presence of formate hydrogen lyase (FHL) activity and other hydrogenases. Study with subcellular fractions of Y19 exhibited that the FHL activity, dependent on soluble formate dehydrogenase activity, was detected in the broken cell extract, but not in the soluble or particulate fraction which are separated by centrifugation at 35 , 000 × g . Hydrogen production in the presence of reduced MV or BV was observed in both the soluble and particulate fractions. Uptake hydrogenase activity was observed in both the fractions when the oxidized forms of MV and BV were supplied as electron acceptor. In the soluble fraction, when formate was coupled with oxidized form of MV or BV, hydrogen production activity was recovered. These results indicate that, similar to E. coli, the strain Y19 expresses two different hydrogenases, one as the FHL complex and another as membrane-associated enzyme.