Characterization of newly isolated thermotolerant bacterium Cupriavidus sp. CB15 from composting and its ability to produce polyhydroxyalkanoate from glycerol.

BACKGROUND: This study aimed to isolate a novel thermotolerant bacterium that is capable of synthesizing polyhydroxyalkanoate from glycerol under high temperature conditions. RESULTS: A newly thermotolerant polyhydroxyalkanoate (PHA) producing bacterium, Cupriavidus sp. strain CB15, was isolated from corncob compost. The potential ability to synthesize PHA was confirmed by detection of PHA synthase (phaC) gene in the genome. This strain could produce poly(3-hydroxybutyrate) [P(3HB)] with 0.95 g/L (PHA content 75.3 wt% of dry cell weight 1.24 g/L) using glycerol as a carbon source. The concentration of PHA was enhanced and optimized based on one-factor-at-a-time (OFAT) experiments and response surface methodology (RSM). The optimum conditions for growth and PHA biosynthesis were 10 g/L glycerol, 0.78 g/L NH4Cl, shaking speed at 175 rpm, temperature at 45 °C, and cultivation time at 72 h. Under the optimized conditions, PHA production was enhanced to 2.09 g/L (PHA content of 74.4 wt% and dry cell weight of 2.81 g/L), which is 2.12-fold compared with non-optimized conditions. Nuclear magnetic resonance (NMR) analysis confirmed that the extracted PHA was a homopolyester of 3-hydyoxybutyrate. CONCLUSION: Cupriavidus sp. strain CB15 exhibited potential for cost-effective production of PHA from glycerol.

Carboxymethyl Bacterial Cellulose from Nata de Coco: Effects of NaOH.

Bacterial cellulose from nata de coco was prepared from the fermentation of coconut juice with Acetobacter xylinum for 10 days at room temperature under sterile conditions. Carboxymethyl cellulose (CMC) was transformed from the bacterial cellulose from the nata de coco by carboxymethylation using different concentrations of sodium hydroxide (NaOH) and monochloroacetic acid (MCA) in an isopropyl (IPA) medium. The effects of various NaOH concentrations on the degree of substitution (DS), chemical structure, viscosity, color, crystallinity, morphology and the thermal properties of carboxymethyl bacterial cellulose powder from nata de coco (CMCn) were evaluated. In the carboxymethylation process, the optimal condition resulted from NaOH amount of 30 g/100 mL, as this provided the highest DS value (0.92). The crystallinity of CMCn declined after synthesis but seemed to be the same in each condition. The mechanical properties (tensile strength and percentage of elongation at break), water vapor permeability (WVP) and morphology of CMCn films obtained from CMCn synthesis using different NaOH concentrations were investigated. The tensile strength of CMCn film synthesized with a NaOH concentration of 30 g/100 mL increased, however it declined when the amount of NaOH concentration was too high. This result correlated with the DS value. The highest percent elongation at break was obtained from CMCn films synthesized with 50 g/100 mL NaOH, whereas the elongation at break decreased when NaOH concentration increased to 60 g/100 mL.

Synthesis, Characterization, and Application of Carboxymethyl Cellulose from Asparagus Stalk End.

Cellulose from Asparagus officinalis stalk end was extracted and synthesized to carboxymethyl cellulose (CMCas) using monochloroacetic acid (MCA) via carboxymethylation reaction with various sodium hydroxide (NaOH) concentrations starting from 20% to 60%. The cellulose and CMCas were characterized by the physical properties, Fourier Transform Infrared spectroscopy (FTIR), Differential scanning calorimetry (DSC), Scanning electron microscopy (SEM) and X-ray diffraction (XRD). In addition, mechanical properties of CMCas films were also investigated. The optimum condition for producing CMCas was found to be 30% of NaOH concentration for the carboxymethylation reaction, which provided the highest percent yield of CMCas at 44.04% with the highest degree of substitution (DS) at 0.98. The melting point of CMCas decreased with increasing NaOH concentrations. Crystallinity of CMCas was significantly deformed (p < 0.05) after synthesis at a high concentration. The L* value of the CMCas was significantly lower at a high NaOH concentration compared to the cellulose. The highest tensile strength (44.59 MPa) was found in CMCas film synthesized with 40% of NaOH concentration and the highest percent elongation at break (24.99%) was obtained in CMCas film treated with 30% of NaOH concentration. The applications of asparagus stalk end are as biomaterials in drug delivery system, tissue engineering, coating, and food packaging.

Physical Properties of Carboxymethyl Cellulose from Palm Bunch and Bagasse Agricultural Wastes: Effect of Delignification with Hydrogen Peroxide.

The aim of this work was to synthesize carboxymethyl cellulose (CMC) and produce CMC films from the cellulose of palm bunch and bagasse agricultural waste. The effect of various amounts of H2O2 (0-40% v/v) during delignification on the properties of cellulose, CMC, and CMC films was studied. As the H2O2 content increased, yield and the lignin content of the cellulose from palm bunch and bagasse decreased, whereas lightness (L*) and whiteness index (WI) increased. FTIR confirmed the substitution of a carboxymethyl group on the cellulose structure. A higher degree of substitution of CMC from both sources was found when 20%-30% H2O2 was employed. The trend in the L* and WI values of each CMC and CMC film was related to those values in their respective cellulose. Bleaching each cellulose with 20% H2O2 provided the cellulose with the highest viscosity and the CMC films with the greatest mechanical (higher tensile strength and elongation at break) and soluble attributes, but the lowest water vapor barrier. This evidence indicates that cellulose delignification with H2O2 has a strong effect on the appearance and physical properties of both CMCs.

Effect of sodium benzoate and chlorhexidine gluconate on a bio-thermoplastic elastomer made from thermoplastic starch-chitosan blended with epoxidized natural rubber.

Thermoplastic elastomer (TPE) was developed by blending thermoplastic starch (TPS) with rubber. Thermoplastic starch-chitosan (TPSC) was prepared by the solution mixing of cassava starch, chitosan (CTS) and glycerol in acidified water (lactic acid 1 wt%) at 80 °C follow by melt mixing at 130 °C. Sodium benzoate (BEN) and chlorhexidine gluconate (Cl) were added during the solution mixing as additives for antimicrobial properties. TPSC was melt-mixed with epoxidized natural rubber (ENR) (70/30 wt/wt). The tensile strength and elongation at break of the TPSC/ENR increased with the additive content. Elastic recovery was improved by the addition of Cl. A new peak in the FTIR data confirmed the reaction between the reactive functional groups of the CTS and the additives with the epoxy groups of ENR. These reactions and miscibility of the TPSC/ENR/additives blends improved the mechanical properties, elasticity, morphology, and antimicrobial properties of the blends.

Influence of Hydroxyl Groups on the Cell Viability of Polyhydroxyalkanoate (PHA) Scaffolds for Tissue Engineering.

Polyhydroxyalkanoates (PHAs) are biopolyesters that have been studied as tissue engineering materials because of their biodegradability, biocompatibility, and low cytotoxicity. In this study, poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-2,3-dihydroxybutyrate) [PHBVDB] containing hydroxyl groups was produced by recombinant Ralstonia eutropha. R. eutropha were constructed to express the propionate-coenzymeA transferase (pct) gene from Megasphaera elsdenii, and glycolate was used as the carbon source. Disruption of phaA encoding ß-ketothiolase in the phaCAB operon increased 2,3-dihydroxybutyrate (2,3-DHBA) compositions to 3 mol %. The PHBVDB film showed a lower water contact angle compared with other PHA films, indicating increased hydrophilicity due to the hydroxyl groups. The mechanical properties of the PHBVDB scaffold met the requirements for a soft tissue matrix. The effect of hydroxyl groups on cytotoxicity was evaluated with human mesenchymal stem cells. Results of cell proliferation and live/dead assays showed that the PHBVDB scaffold did not exhibit significant cytotoxicity toward the cells. These results indicate that PBVDB containing hydroxyl groups could be applied as a hydrophilicity-controlled scaffold for soft tissue engineering.

Biosynthesis of polyhydroxyalkanoates containing hydroxyl group from glycolate in Escherichia coli.

Polyhydroxyalkanoates (PHAs) containing hydroxyl groups in a side chain were produced in recombinant Escherichia coli JM109 using glycolate as the sole carbon source. The propionate-CoA transferase (pct) gene from Megasphaera elsdenii and the ß-ketothiolase (bktB) gene and phaCAB operon from Ralstonia eutropha H16 were introduced into E. coli JM109. A novel monomer containing a hydroxyl group, dihydroxybutyrate (DHBA), was the expected product of the condensation of glycolyl-CoA and acetyl-CoA by BktB. The recombinant strain produced a PHA containing 1 mol% DHBA. The incorporation of DHBA may have been restricted because the expression of phaAB1 competes for acetyl-CoA. The PHA containing DHBA units were evaluated regarding thermal properties, such as melting temperature, glass transition temperature and thermal degradation temperature. The current study demonstrates a potential use of PHA containing hydroxyl groups as renewable resources in biological materials.

Improved artificial pathway for biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with high C6-monomer composition from fructose in Ralstonia eutropha.

Poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)], a flexible and practical kind of polyhydroxyalkanoates, is generally produced from plant oils and fatty acids by several wild and recombinant bacteria. This study established an improved artificial pathway for the biosynthesis of P(3HB-co-3HHx) with high 3HHx composition from structurally unrelated fructose in Ralstonia eutropha. Depression of (R)-specific reduction of acetoacetyl-CoA by the deletion of phaB1 was an effective modification for formation of the C6-monomer unit from fructose driven by crotonyl-CoA carboxylase/reductase (Ccr). Co-overexpression of phaJ4a, which encodes medium-chain-length (R)-enoyl-CoA hydratase, with ccr promoted the incorporation of both 3HB and 3HHx units. Further introduction of emdMm, a synthetic gene encoding ethylmalonyl-CoA decarboxylase derived from mouse, was remarkably effective for P(3HB-co-3HHx) biosynthesis, probably by converting ethylmalonyl-CoA generated by the reductive carboxylase activity of Ccr back into butyryl-CoA. A high cellular content of P(3HB-co-3HHx) composed of 22mol% 3HHx could be produced from fructose by the engineered strain of R. eutropha with ΔphaB1 genotype expressing ccr, phaJ4a, and emd.

Overexpression of a type II 3-dehydroquinate dehydratase enhances the biotransformation of quinate to 3-dehydroshikimate in Gluconobacter oxydans.

Shikimate and 3-dehydroshikimate are useful chemical intermediates for the synthesis of various compounds, including the antiviral drug oseltamivir. Here, we show an almost stoichiometric biotransformation of quinate to 3-dehydroshikimate by an engineered Gluconobacter oxydans strain. Even under pH control, 3-dehydroshikimate was barely detected during the growth of the wild-type G. oxydans strain NBRC3244 on the medium containing quinate, suggesting that the activity of 3-dehydroquinate dehydratase (DHQase) is the rate-limiting step. To identify the gene encoding G. oxydans DHQase, we overexpressed the gox0437 gene from the G. oxydans strain ATCC621H, which is homologous to the aroQ gene for type II DHQase, in Escherichia coli and detected high DHQase activity in cell-free extracts. We identified the aroQ gene in a draft genome sequence of G. oxydans NBRC3244 and constructed G. oxydans NBRC3244 strains harboring plasmids containing aroQ and different types of promoters. All recombinant G. oxydans strains produced a significant amount of 3-dehydroshikimate from quinate, and differences between promoters affected 3-dehydroshikimate production levels with little statistical significance. By using the recombinant NBRC3244 strain harboring aroQ driven by the lac promoter, a sequential pH adjustment for each step of the biotransformation was determined to be crucial because 3-dehydroshikimate production was enhanced. Under optimal conditions with a shift in pH, the strain could efficiently produce a nearly equimolar amount of 3-dehydroshikimate from quinate. In the present study, one of the important steps to convert quinate to shikimate by fermenting G. oxydans cells was investigated.

Modification of ß-oxidation pathway in Ralstonia eutropha for production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from soybean oil.

Ralstonia eutropha H16 is a useful platform for metabolic engineering aiming at efficient production of polyhydroxyalkanaotes being attracted as practical bioplastics. This study focused on bifunctional (S)-specific 2-enoyl-CoA hydratase/(S)-3-hydroxyacyl-CoA dehydrogenase encoded by fadB to obtain information regarding ß-oxidation in this bacterium and to achieve compositional regulation of poly((R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate) [P(3HB-co-3HHx)] synthesized from soybean oil. In addition to two FadB homologs (FadB1 and FadB') encoded within the previously identified ß-oxidation gene clusters on the chromosome 1, a gene of third homolog (FadB2) was found on chromosome 2 of R. eutropha. The fadB homologs were disrupted in R. eutropha strain NSDG expressing a mutant gene of PHA synthase from Aeromonas caviae. The gene disruptions affected neither growth nor PHA production on fructose. On soybean oil, fadB' deletion led to reduction of PHA quantity attributed to decrease of 3HB unit, while fadB1 deletion slightly increased 3HHx composition without serious negative impact on both cell growth and PHA biosynthesis. Double deletion of fadB1 and fadB' significantly impaired the cell growth and PHA biosynthesis, indicating the major roles of fadB1 and fadB' in ß-oxidation. When fadB1 was deleted in several engineered strains of R. eutropha possessing additional (R)-enoyl-CoA hydratase gene(s), the net amounts of 3HHx unit in the PHA fractions showed 6-21% increase probably due to slightly enhanced supply of medium-chain-length 2-enoyl-CoAs through the partially impaired ß-oxidation. These results demonstrated that modification of ß-oxidation by fadB1 deletion was effective for increasing 3HHx composition in the copolyesters produced from soybean oil.