Ralstonia Eutropha Research Articles

Comparative metabolomic profiling of Cupriavidus necator B-4383 revealed production of cupriachelin siderophores, one with activity against Cryptococcus neoformans.

Cupriavidus necator H16 is known to be a rich source of linear lipopeptide siderophores when grown under iron-depleted conditions; prior literature termed these compounds cupriachelins. These small molecules bear β-hydroxyaspartate moieties that contribute to a photoreduction of iron when bound as ferric cupriachelin. Here, we present structural assignment of cupriachelins from C. necator B-4383 grown under iron limitation. The characterization of B-4383 cupriachelins is based on MS/MS fragmentation analysis, which was confirmed by 1D- and 2D-NMR for the most abundant analog (1). The cupriachelin congeners distinguish these two strains with differences in the preferred lipid tail; however, our rigorous metabolomic investigation also revealed minor analogs with changes in the peptide core, hinting at a potential mechanism by which these siderophores may reduce biologically unavailable ferric iron (4-6). Antifungal screening of the C. necator B-4383 supernatant extract and the isolated cupriachelin analog (1) revealed inhibitory activity against Cryptococcus neoformans, with IC50 values of 16.6 and 3.2μg/mL, respectively. This antifungal activity could be explained by the critical role of the iron acquisition pathway in the growth and pathogenesis of the C. neoformans fungal pathogen.

Structural analysis of multi-functional enzyme FadB from Cupriavidus necator H16: non-formation of FadAB complex

Structural analysis of multi-functional enzyme FadB from <i>Cupriavidus necator</i> H16: non-formation of FadAB complex

Cupriavidus metallidurans bacteria enhance sodium uptake by plants

Here we report that Cupriavidus metallidurans bacteria increase sodium (Na+) accumulation in Arabidopsis thaliana and quinoa (Chenopodium quinoa). Given the metal mobilizing feature of the bacteria, we tested this hypothesis that the bacteria can increase the uptake of Na+, a metal ion, by plants. In agar plate assays under salinity conditions, the bacterial inoculation significantly decreased plant biomass by 280% and 20% in Arabidopsis and quinoa, respectively. In pot experiments under the salinity conditions, the bacterial inoculation resulted in about 60% loss of the quinoa plant biomass. We detected a significant high accumulation of Na+ in the leaf and stem tissues of the bacteria-inoculated quinoa plants, suggesting an association between increased Na+ accumulation and bacteria-mediated growth retardation in the plants. Altogether we report a novel phenomenon, in which bacteria increase Na+ uptake by plants.

Sustainable biorefining and bioprocessing of green seaweed (Ulva spp.) for the production of edible (ulvan) and non-edible (polyhydroxyalkanoate) biopolymeric films

A sustainable biorefining and bioprocessing strategy was developed to produce edible-ulvan films and non-edible polyhydroxybutyrate films. The preparation of edible-ulvan films by crosslinking and plasticisation of ulvan with citric acid and xylitol was investigated using Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC) analysis. The edible ulvan film was tested for its gut-friendliness using Lactobacillus and Bifidobacterium spp. (yoghurt) and was shown to improve these gut-friendly microbiome's growth and simultaneously retarding the activity of pathogens like Escherchia coli and Staphylococcus aureus. Green macroalgal biomass refused after the extraction of ulvan was biologically processed by dark fermentation to produce a maximum of 3.48 (± 0.14) g/L of volatile fatty acids (VFAs). Aerobic processing of these VFAs using Cupriavidus necator cells produced 1.59 (± 0.12) g/L of biomass with 18.2 wt% polyhydroxybutyrate. The present study demonstrated the possibility of producing edible and non-edible packaging films using green macroalgal biomass as the sustainable feedstock.Graphical

Single-cell multimodal imaging uncovers energy conversion pathways in biohybrids.

Microbe-semiconductor biohybrids, which integrate microbial enzymatic synthesis with the light-harvesting capabilities of inorganic semiconductors, have emerged as promising solar-to-chemical conversion systems. Improving the electron transport at the nano-bio interface and inside cells is important for boosting conversion efficiencies, yet the underlying mechanism is challenging to study by bulk measurements owing to the heterogeneities of both constituents. Here we develop a generalizable, quantitative multimodal microscopy platform that combines multi-channel optical imaging and photocurrent mapping to probe such biohybrids down to single- to sub-cell/particle levels. We uncover and differentiate the critical roles of different hydrogenases in the lithoautotrophic bacterium Ralstonia eutropha for bioplastic formation, discover this bacterium's surprisingly large nanoampere-level electron-uptake capability, and dissect the cross-membrane electron-transport pathways. This imaging platform, and the associated analytical framework, can uncover electron-transport mechanisms in various types of biohybrid, and potentially offers a means to use and engineer R. eutropha for efficient chemical production coupled with photocatalytic materials.

Expression of His-tagged NADPH-dependent acetoacetyl-CoA reductase in recombinant Escherichia coli BL-21(DE3)

Poly-3-hydroxybutyrate (P(3HB)), a member of the polyhydroxyalkanoate (PHA) family, is a biodegradable polyester with diverse industrial applications. NADPH-dependent acetoacetyl-CoA reductase (phaB) is the enzyme which plays an essential role in P(3HB) synthesis by catalyzing the conversion of the intermediates. The expression of phaB enzyme using the recombinant Escherichia coli BL-21(DE3) and the purification of the synthesized enzyme were studied. The pET-B3 plasmid harbouring the phaB gene derived from Ralstonia eutropha H16, was driven by the lac promoter in E.coli BL-21(DE3). The enzyme was expressed with different induction time, temperatures and cell age. Results showed that the cell age of 4h, induction time of 12h at 37°C were identified as the optimal conditions for the enzyme reductase expression. A specific activity of 0.151 Umg-1 protein and total protein concentration of 0.518mgmg-1 of dry cell weight (DCW) were attained. Affinity chromatography was performed to purify the His-tagged phaB enzyme, in which enhanced the specific activity (14.44 Umg-1) and purification fold (38-fold), despite relative low yield (44.6%) of the enzyme was obtained. The purified phaB showed an optimal enzyme activity at 30°C and pH 8.0. The findings provide an alternative for the synthesis of the reductase enzyme which can be used in the industrial-scale production of the biodegradable polymers.

Synthesis and Properties of Polyhydroxyalkanoates on Waste Fish Oil from the Production of Canned Sprats

The waste fish oil obtained from Baltic sprat waste in the production of canned sprats was studied as a sole carbon substrate for PHA synthesis by the wild-type strain Cupriavidus necator B-10646. Sprat oil contained a set of fatty acids with a chain length from C14 to C24, saturation factor 0.63, and provided bacterial growth and PHA synthesis. Bacteria metabolized fatty acids unevenly utilizing polyenoic acids and not using monoenoic and saturated acids. The bacterial biomass yield and the intracellular polymer concentration were 6.5 ± 0.5 g/L and 65 ± 5% by fed-batch culture in flasks. The synthesized PHAs were three-component copolymers with a predominance (97–98 mol.%) of 3-hydroxybutyrate monomers and small inclusions of 3-hydroxyvalerate and 3-hydroxyhexanoate; the ratio of monomers changed slightly depending on the sprat oil concentration. The series of samples had a temperature (Tmelt) of 158–165 °C, a molecular weight (Mw) of 540–760 kDa, and a degree of crystallinity (Cx) of 66–72%. For the first time, the waste fish oil from the production of sprats studied as a carbon substrate is a promising, affordable, and renewable substrate for PHA biosynthesis.

Genetic inactivation of the Carnitine/Acetyl-Carnitine mitochondrial carrier of Yarrowia lipolytica leads to enhanced odd-chain fatty acid production

BackgroundMitochondrial carriers (MCs) can deeply affect the intracellular flux distribution of metabolic pathways. The manipulation of their expression level, to redirect the flux toward the production of a molecule of interest, is an attractive target for the metabolic engineering of eukaryotic microorganisms. The non-conventional yeast Yarrowia lipolytica is able to use a wide range of substrates. As oleaginous yeast, it directs most of the acetyl-CoA therefrom generated towards the synthesis of lipids, which occurs in the cytoplasm. Among them, the odd-chain fatty acids (OCFAs) are promising microbial-based compounds with several applications in the medical, cosmetic, chemical and agricultural industries.ResultsIn this study, we have identified the MC involved in the Carnitine/Acetyl-Carnitine shuttle in Y. lipolytica, YlCrc1. The Y. lipolytica Ylcrc1 knock-out strain failed to grow on ethanol, acetate and oleic acid, demonstrating the fundamental role of this MC in the transport of acetyl-CoA from peroxisomes and cytoplasm into mitochondria. A metabolic engineering strategy involving the deletion of YlCRC1, and the recombinant expression of propionyl-CoA transferase from Ralstonia eutropha (RePCT), improved propionate utilization and its conversion into OCFAs. These genetic modifications and a lipogenic medium supplemented with glucose and propionate as the sole carbon sources, led to enhanced accumulation of OCFAs in Y. lipolytica.ConclusionsThe Carnitine/Acetyl-Carnitine shuttle of Y. lipolytica involving YlCrc1, is the sole pathway for transporting peroxisomal or cytosolic acetyl-CoA to mitochondria. Manipulation of this carrier can be a promising target for metabolic engineering approaches involving cytosolic acetyl-CoA, as demonstrated by the effect of YlCRC1 deletion on OCFAs synthesis.

Cytotoxic effects of endophyte origin nanosilver and its computational studies

The present study aimed in the investigation of anticancer potential of silver nanoparticles (AgNPs) from endophytic bacterium Cupriavidus metallidurans (Cm-AgNPs) and Pantoea anthophila (Pa-AgNPs) of W. indica reported in our earlier studies, against human oral epidermoid carcinoma (KB 3–1) and COLON 26 cell lines. The cytotoxicity response was assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). Measurement of intracellular reactive oxygen species (ROS), mitochondrial membrane potential (ɅΨm) and apoptotic morphological changes were studied in KB 3–1 cell line. Both the endophytes were found to produce periplasmic nitrate reductase (PNR) that converts the ionic silver into stable nano silver. Hence the structure and function of PNR was determined by computationl tools and evolutionary relationships were constructed using Clustal Omega. The inhibitive properties of AgNP against selected cancer targets were analyzed by molecular docking tool Autodock 4.0. KB 3–1 cell line showed good anticancer response than COLON 26 cell lines. On comparison of AgNPs, Cm-AgNPs exhibited higher cytotoxicity with increased intracellular ROS levels, altered ɅΨm and apoptotic cell death than Pa-AgNPs. The three-dimensional structure of the PNR was modeled by Swiss model and validated using PROCHECK and PROVES. Docking study showed that the AgNP bound exactly in the inhibition site or in their close proximity that may enable the modulation of proteins. Thus the study provides an insight on the anticancer activity of AgNPs fabricated from endophytic extract.

SUSTAINABLE BIOREMEDIATION OF ACETAMINOPHEN USING BACTERIA: A CRITICAL REVIEW

Acetaminophen is a common antipyretic and analgesic pharmaceutical contaminant and has become one of the most emerging contaminants in the environment. Bioremediation is the suitable method to degrade acetaminophen as it is sustainable, mimics nature and is low-cost. The bioremediation of acetaminophen is performed by identifying the bacterial characteristics and the source of the bacteria involved in the degradation of acetaminophen. Based on this review study, gram-negative bacteria showed the highest efficiency in degrading acetaminophen by Pseudomonas aeruginosa strain HJ1012 and Cupriavidus necator with an efficiency of 99% and 100%, respectively. In addition, synergistic or antagonistic interaction among bacteria in mixed culture is a gap of study. The findings from the previous study showed that the optimum conditions are pH 7.0, temperature ranges between 30–40°C, and a poor culture medium of minimal salt solution. Furthermore, the study sheds light on enzyme involvement and characterisation of acetaminophen degradation pathways toward less harmful intermediates are addressed in order to achieve sustainable development goals through environmental security and health safety. These identified research gaps offer fundamental knowledge and shed insight into upcoming mechanism and application studies.

Automatic Control of Chemolithotrophic Cultivation of Cupriavidus necator: Optimization of Oxygen Supply for Enhanced Bioplastic Production

Gas fermentation is an upcoming technology to convert gaseous substrates into value-added products using autotrophic microorganisms. The hydrogen-oxidizing bacteria Cupriavidus necator efficiently uses CO2 as its sole carbon source, H2 as electron donor and O2 as electron acceptor. Surplus CO2 is stored in microbial storage material poly-(R)-3-hydroxybutyrate. O2 supply is the most critical parameter for growth and poly-(R)-3-hydroxybutyrate formation. A narrow O2 optimum between ~0.2 and ~4 mg/L was previously reported. Here, a standard benchtop bioreactor was redesigned for autotrophic growth of C. necator on explosive mixtures of CO2, H2 and O2. The bioreactor was equipped with mass flow control units and O2 and CO2 sensors. A controller for automated gas dosage based on a mathematical model including gas mass transfer, gas consumption and sensor response time was developed. Dissolved O2 concentrations were adjusted with high precision to 1, 2 and 4% O2 saturation (0.4, 0.8 and 1.5 mg/L dissolved O2, respectively). In total, up to 15 g/L cell dry weight were produced. Residual biomass formation was 3.6 ± 0.2 g/L under all three O2 concentrations. However, poly-(R)-3-hydroxybutyrate content was 71, 77 and 58% of the cell dry weight with 1, 2 and 4% dissolved O2, respectively.

Effect of Monomers of 3-Hydroxyhexanoate on Properties of Copolymers Poly(3-Hydroxybutyrate-co 3-Hydroxyhexanoate)

The properties of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) P(3HB-co-3HHx) copolymers with different ratios of monomers synthesized by the wild-type strain Cupriavidus necator B-10646 on sugars, and an industrial sample from Kaneka synthesized by the recombinant strain C. necator NSDG-ΔfadB1 on soybean oil, were studied in a comparative aspect and in relation to poly(3-hydroxybutyrate) P(3HB). The copolymer samples, regardless of the synthesis conditions or the ratio of monomers, had reduced values of crystallinity degree (50–60%) and weight average molecular weight (415–520 kDa), and increased values of polydispersity (2.8–4.3) compared to P(3HB) (70–76%, 720 kDa, and 2.2). The industrial sample had differences in its thermal behavior, including a lower glass transition temperature (−2.4 °C), two peaks in its crystallization and melting regions, a lower melting point (Tmelt) (112/141 °C), and a more pronounced gap between Tmelt and the temperature of thermal degradation (Tdegr). The process, shape, and size of the spherulites formed during the isothermal crystallization of P(3HB) and P(3HB-co-3ННx) were generally similar, but differed in the maximum growth rate of the spherulites during exothermic crystallization, which was 3.5–3.7 μm/min for P(3HB), and 0.06–1.25 for the P(3HB-co-3HHx) samples. The results from studying the thermal properties and the crystallization mechanism of P(3HB-co-3HHx) copolymers are important for improving the technologies for processing polymer products from melts.

Construction of Cupriavidus necator displayed with superoxide dismutases for enhanced growth in bioelectrochemical systems

It is of great significance to utilize CO2 as feedstock to synthesize biobased products, particularly single cell protein (SCP) as the alternative food and feed. Bioelectrochemical system (BES) driven by clean electric energy has been regarded as a promising way for Cupriavidus necator to produce SCP from CO2 directly. At present, the key problem of culturing C. necator in BES is that reactive oxygen species (ROS) generated in cathode chamber are harmful to bacterial growth. Therefore, it is necessary to find a solution to mitigate the negative effect of ROS. In this study, we constructed a number of C. necator strains displayed with superoxide dismutase (SOD), which allowed the decomposition of superoxide anion radical. The effects of promoters and signal peptides on the cell surface displayed SOD were analyzed. The proteins displayed on the surface were further verified by the fluorescence experiment. Finally, the growth of C. necator CMS incorporating a pBAD-SOD-E-tag-IgAβ plasmid could achieve 4.9 ± 1.0 of OD600 by 7 days, equivalent to 1.7 ± 0.3 g/L dry cell weight (DCW), and the production rate was 0.24 ± 0.04 g/L/d DCW, around 2.7-fold increase than the original C. necator CMS (1.8 ± 0.3 of OD600). This study can provide an effective and novel strategy of cultivating strains for the production of CO2-derived SCP or other chemicals in BES.Graphical

Secondary metabolites of rhizospheric fungal isolate Aspergillus carneus ABRF4 regulate the antibacterial and anti-proliferative activity against cancer cells

Abstract. Sahu MK, Yeeravalli R, Das A, Jha H. 2023. Secondary metabolites of rhizospheric fungal isolate Aspergillus carneus ABRF4 regulate the antibacterial and anti-proliferative activity against cancer cells. Nusantara Bioscience 15: 137-142. The medicinal capabilities of plants are influenced by soil chemistry, genotype, and climate. Many biotic and abiotic factors affect soil composition. Microorganisms constituting the soil microflora indicate a mutualistic relationship with plant rhizospheric region, and they play an important role in plant secondary metabolite production, yield, and efficacy. They are the major resources for structurally unique bioactive natural metabolites. The aim of the present study was to evaluate the bioactivity of the secondary metabolite extracted from the rhizospheric fungal isolate Aspergillus carneus ABRF4) isolated from the Achanakmar Biosphere Reserve, Chhattisgarh, India. The fungal secondary metabolites were extracted using several solvents by soxhlet extraction techniques. The crude and partially purified column fractions of A. carneus ABRF4 were characterized by Gas Chromatography-Mass Spectroscopy (GC-MS), Fourier Transform Infra-Red (FTIR) spectrum, and Thin Layer Chromatography (TLC). Results showed that the acetonitrile fraction had an antimicrobial activity with the variable zone of inhibition against human pathogens such as Bacillus circulans (MTCC-7906), Bacillus subtilis (MTCC 441), Staphylococcus aureus (MTCC-96), and Ralstonia eutropha (MTCC-2487). The crude extracts and the identified secondary metabolite, trans- 1,3-dimethyl-Cyclohexane, possess different anti-proliferative activity against human tissue-specific cancer cell lines, including breast cancer (MDA-MB-231, MDA-MB-468, and MCF-7), liver cancer (HepG2), lung cancer (A-549), and prostate cancer (DU-145) suggesting a potential therapeutic application of the isolated rhizospheric fungi.

Bioremediation potential of consortium Pseudomonas Stutzeri LBR and Cupriavidus Metallidurans LBJ in soil polluted by lead.

Pollution by lead (Pb) is an environmental and health threat due to the severity of its toxicity. Microbial bioremediation is an eco-friendly technique used to remediate contaminated soils. This present study was used to evaluate the effect of two bacterial strains isolated and identified from Bizerte lagoon: Cupriavidus metallidurans LBJ (C. metallidurans LBJ) and Pseudomonas stutzeri LBR (P. stutzeri LBR) on the rate of depollution of soil contaminated with Pb from Tunisia. To determine this effect, sterile and non-sterile soil was bioaugmented by P. stutzeri LBR and C. metallidurans LBJ strains individually and in a mixture for 25 days at 30°C. Results showed that the bioaugmentation of the non-sterile soil by the mixture of P. stutzeri LBR and C. metallidurans LBJ strains gave the best rate of reduction of Pb of 71.02%, compared to a rate of 58.07% and 46.47% respectively for bioaugmentation by the bacterial strains individually. In the case of the sterile soil, results showed that the reduction rate of lead was in the order of 66.96% in the case of the mixture of the two bacterial strains compared with 55.66% and 41.86% respectively for the addition of the two strains individually. These results are confirmed by analysis of the leachate from the sterile and non-sterile soil which showed an increase in the mobility and bioavailability of Pb in soil. These promising results offer another perspective for a soil bioremediation bioprocess applying bacterial bioremediation.

Adsorption of Cd (II) by a novel living and non-living Cupriavidus necator GX_5: optimization, equilibrium and kinetic studies

Biosorbents have been extensively studied for heavy metal adsorption due to their advantages of low cost and high efficiency. In the study, the living and non-living biomass of Cupriavidus necator GX_5 previously isolated were evaluated for their adsorption capacity and/or removal efficiency for Cd (II) through batch experiments, SEM and FT-IR investigations. The maximum removal efficiency rates for the live and dead biomass were 60.51% and 78.53%, respectively, at an optimum pH of 6, a dosage of 1 g/L and an initial Cd (II) concentration of 5 mg/L. The pseudo-second-order kinetic model was more suitable for fitting the experimental data, indicating that the rate-limiting step might be chemisorption. The Freundlich isotherm model fit better than the Langmuir isotherm model, implying that the adsorption process of both biosorbents was heterogeneous. FT-IR observation reflected that various functional groups were involved in Cd (II) adsorption: –OH, –NH, C=O, C–O and C–C groups for the living biomass and –OH, –NH, C–H, C = O, C–N and N–H groups for the dead biomass. Our results imply that non-living biosorbents have a higher capacity and stronger strength for absorbing Cd (II) than living biomass. Therefore, we suggest that dead GX_5 is a promising adsorbent and can be used in Cd (II)-contaminated environments.

Ability of Cupriavidus necator H16 to resist, bioremove, and accumulate some hazardous metal ions in water.

Bacterial biomasses are suitable and inexpensive biosorbents for the removal of metal ions. The Gram-negative betaproteobacterium Cupriavidus necator H16 is found in soil and freshwater environments. In this study, C. necator H16 was used to remove chromium (Cr), arsenic (As), aluminum (Al), and cadmium (Cd) ions from water. Minimum inhibition concentration (MIC) values of C. necator to Cr, As, Al, and Cd were found as 76, 69, 341, and 275 mg/L, respectively. The highest rates of Cr, As, Al, and Cd bioremoval were 45, 60, 54, and 78%, respectively. pH levels between 6.0 and 8.0 and an average temperature of 30 °C were optimum for the most efficient bioremoval. Scanning electron microscopy (SEM) images of Cd-treated cells showed that the morphology of the cells was significantly impaired compared to the control. Shifts in the Fourier transform infrared spectroscopy analysis (FTIR) spectra of the Cd-treated cell walls also confirmed the presence of active groups. As a result, it can be said that C. necator H16 has a moderate bioremoval efficiency for Cr, As, and Al and a high bioremoval efficiency for Cd.

Systematic Part Transfer by Extending a Modular Toolkit to Diverse Bacteria.

It is impractical to develop a new parts collection for every potential host organism. It is well-established that gene expression parts, like genes, are qualitatively transferable, but there is little quantitative information defining transferability. Here, we systematically quantified the behavior of a parts set across multiple hosts. To do this, we developed a broad host range (BHR) plasmid system compatible with the large, modular CIDAR parts collection for E.coli, which we named openCIDAR. This enabled testing of a library of DNA constructs across the Pseudomonadota─Escherichia coli, Pseudomonas putida, Cupriavidus necator, and Komagataeibacter nataicola. Part performance was evaluated with a standardized characterization procedure that quantified expression in terms of molecules of equivalent fluorescein (MEFL), an objective unit of measure. The results showed that the CIDAR parts enable graded gene expression across all organisms─meaning that the same parts can be used to program E.coli, P.putida, C.necator, and K.nataicola. Most parts had a similar expression trend across hosts, although each organism had a different average gene expression level. The variability is enough that to achieve the same MEFL in a different organism, a lookup table is required to translate a design from one host to another. To identify truly divergent parts, we applied linear regression to a combinatorial set of promoters and ribosome binding sites, finding that the promoter J23100 behaves very differently in K.nataicola than in the other hosts. Thus, it is now possible to evaluate any CIDAR compatible part in three other hosts of interest, and the diversity of these hosts implies that the collection will also be compatible with many other Proteobacteria (Pseudomonadota). Furthermore, this work defines an approach to generalize modular synthetic biology parts sets beyond a single host, implying that only a few parts sets may be needed to span the tree of life. This will accelerate current efforts to engineer diverse species for environmental, biotechnological, and health applications.

Valorization of whey-based side streams for microbial biomass, molecular hydrogen, and hydrogenase production.

Side streams of the dairy industry are a suitable nutrient source for cultivating microorganisms, producing enzymes, and high-value chemical compounds. The heterotrophic Escherichia coli and chemolithoautotroph Ralstonia eutropha are of major biotechnological interest. R. eutropha is a model organism for producing O2-tolerant [NiFe]-hydrogenases (Hyds) (biocatalysts), and E. coli has found widespread use as an expression platform for producing recombinant proteins, molecular hydrogen (H2), and other valuable products. Aiming at developing suitable cultivation media from side streams of the dairy industry, the pre-treatment (filtration, dilution, and pH adjustment) of cheese (sweet) whey (SW) and curd (acid) whey (AW), with and without the use of ß-glucosidase, has been performed. Growth parameters (oxidation-reduction potential (ORP), pH changes, specific growth rate, biomass formation) of E. coli BW25113 and R. eutropha H16 type strains were monitored during cultivation on filtered and non-filtered SW and AW at 37°C, pH 7.5 and 30°C, pH 7.0, respectively. Along with microbial growth, measurements of pH and ORP indicated good fermentative growth. Compared to growth on fructose-nitrogen minimal salt medium (control), a maximum cell yield (OD600 4.0) and H2-oxidizing Hyd activity were achieved in the stationary growth phase for R. eutropha. Hyd-3-dependent H2 production by E. coli utilizing whey as a growth substrate was demonstrated. Moreover, good biomass production and prolonged H2 yields of ~ 5mmol/L and cumulative H2 ~ 94mLg/L dry whey (DW) (ß-glucosidase-treated) were observed during the cultivation of the engineered E. coli strain. These results open new avenues for effective whey treatment using thermostable β-glucosidase and confirm whey as an economically viable commodity for biomass and biocatalyst production. KEY POINTS: • Archaeal thermostable β-glucosidase isolated from the metagenome of a hydrothermal spring was used for lactose hydrolysis in whey. • Hydrogenase enzyme activity was induced during the growth of Ralstonia eutropha H16 on whey. • Enhanced biomass and H2 production was shown in a genetically modified strain of Escherichia coli.

Whole Genome Sequence Analysis of Cupriavidus necator C39, a Multiple Heavy Metal(loid) and Antibiotic Resistant Bacterium Isolated from a Gold/Copper Mine.

Here a multiple heavy metal and antibiotic resistant bacterium Cupriavidus necator C39 (C. necator C39) was isolated from a Gold-Copper mine in Zijin, Fujian, China. C. necator C39 was able to tolerate intermediate concentrations of heavy metal(loid)s in Tris Minimal (TMM) Medium (Cu(II) 2 mM, Zn(II) 2 mM, Ni(II) 0.2 mM, Au(III) 70 μM and As(III) 2.5 mM). In addition, high resistance to multiple antibiotics was experimentally observed. Moreover, strain C39 was able to grow on TMM medium containing aromatic compounds such as benzoate, phenol, indole, p-hydroxybenzoic acid or phloroglucinol anhydrous as the sole carbon sources. The complete genome of this strain revealed 2 circular chromosomes and 1 plasmid, and showed the closest type strain is C. necator N-1T based on Genome BLAST Distance Phylogeny. The arsenic-resistance (ars) cluster GST-arsR-arsICBR-yciI and a scattered gene encoding the putative arsenite efflux pump ArsB were identified on the genome of strain C39, which thereby may provide the bacterium a robust capability for arsenic resistance. Genes encoding multidrug resistance efflux pump may confer high antibiotic resistance to strain C39. Key genes encoding functions in degradation pathways of benzene compounds, including benzoate, phenol, benzamide, catechol, 3- or 4-fluorobenzoate, 3- or 4-hydroxybenzoate and 3,4-dihydroxybenzoate, indicated its potential for degrading those benzene compounds.