Oleovorans NRRL Research Articles

Selective Microbial Degradation of Saturated Methyl Branched‐Chain Fatty Acid Isomers

AbstractThree strains of Pseudomonas (P.) bacteria were screened for their capabilities of degrading chemically synthesized saturated branched‐chain fatty acids (sbc–FA). Mixtures of sbc–FA with the methyl‐branch located at various locales along the fatty acid were used as a carbon feedstock in shake‐flask culture. Utilization (and hence degradability) of the sbc–FA was monitored based on positive bacterial growth, fatty acid recovery rates and chromatographic (gas chromatography (GC) and GC‐mass spectroscopy (MS)) analysis of the recovered carbon source. P. putida KT2442 and P. oleovorans NRRL B‐14683 were both able to grow on sbc–FA utilizing 35 wt% and 27 wt% of the carbon source, respectively after 144 h. In contrast, P. resinovorans NRRL B‐2649 exhibited the most efficient use of the carbon source by utilizing 89 % of the starting material after 96 h resulting in a cell dry weight (CDW) of 3.1 g/L. GC and GC–MS analysis of the recovered carbon source revealed that the bacterial strains selectively utilized the isostearic acid in the sbc–FA mixture, and a new group of C10, C12, C14 and C16‐linear and/or branched‐chain fatty acids (approximately 4–29 wt%) were formed during degradation.

Glycerine and levulinic acid: Renewable co-substrates for the fermentative synthesis of short-chain poly(hydroxyalkanoate) biopolymers

Glycerine (a biodiesel co-product) and levulinic acid (a pulp and paper co-product) were used as co-substrates for the fermentative synthesis of short-chain polyhydroxyalkanoate (sc-PHA) biopolymers with tunable monomer and molecular weight characteristics. Pseudomonas oleovorans NRRL B-14682 utilized glycerine alone to produce poly(3-hydroxybutyrate) (PHB). When levulinic acid was added to the media at shake-flask scale in concentrations ⩽0.6wt.%, poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB/V) copolymers were produced with 3-HV contents ranging from 37 to 97mol%; a glycerine:levulinic acid ratio of 0.2%:0.8% (w/v) resulted in poly(3-hydroxyvalerate) (PHV). Ten-liter batch fermentations using glycerine:levulinic acid ratios of 1%:0, 0.75%:0.25%, 0.5%:0.5% and 0.25%:0.75% (w/v) resulted in PHB, P(73%-3HB-co-27%-3HV), P(30%-3HB-co-70%-3HV) and PHV with increasing number average molecular weights (×103g/mol) of 328, 511, 728 and 1330, respectively, owing to glycerine-based chain termination. These results provide a novel means by which glycerine and levulinic acid can be used collectively to produce an array of distinct sc-PHA biopolymers.

Efficient Utilization of Crude Glycerol as Fermentation Substrate in the Synthesis of Poly(3‐hydroxybutyrate) Biopolymers

AbstractOne refined and two crude glycerol (from biodiesel production) samples were utilized to produce poly(3‐hydroxybutyrate) (PHB) by Pseudomonas oleovorans NRRL B‐14682. A batch culture fermentation protocol including 1% glycerol and an aeration rate of 3 standard liters per minute proved best for PHB synthesis (av. yield = 1.0 ± 0.2 g/L at 48 h) and efficient glycerol utilization. PHB molecular weights decreased as MeOH concentration increased. Refined glycerol resulted in PHB polymers with number average molecular weights (Mn) of 314,000 g/mol which decreased by 17 and 90% as MeOH media concentrations increased to <0.005 and 0.85%, respectively. Proton (1H) NMR demonstrated the presence of glycerol‐ and methoxy‐based end‐capping, which was confirmed by 1H diffusion experiments (DOSY analyses). NMR diffusion analyses of the PHB polymers established their diffusivities, and confirmed that their relative molecular sizes were dependent on the impurities in the glycerol. In addition, DOSY analyses indicated that each end‐capped PHB polymer and the glycerol or methoxy groups bound to it had the same diffusion constants, demonstrating that they migrated together as covalent complexes. Non‐covalent complexation was eliminated by physically mixing free glycerol with PHB synthesized from oleic acid; their respective diffusivities were notably faster.

Characterization of biodegradable films from the extracellular polysaccharide produced by Pseudomonas oleovorans grown on glycerol byproduct

In this work, the film-forming capacity of the new microbial exopolysaccharide (EPS) composed by sugars and acyl groups, produced by Pseudomonas oleovorans NRRL B-14682, was studied. The films were transparent and quite flexible and tough when handled, but showed to be stiff under tensile and puncture tests. They presented a high water vapour permeability but a quite low permeability to carbon dioxide, which are typical of hydrophilic polysaccharide films. Furthermore, they showed good stability in contact with liquid water, after auto-crosslinking reactions at low pH, upon or after drying. Preliminary biodegradability tests indicated an easy biological degradation when exposed to soil microorganisms. The results obtained are rather promising regarding the film-forming capacity of the new EPS, as they were obtained only with the biopolymer itself. The films formulation may be complemented with additives (e.g. plasticizers, emulsifiers, nanocomposites), to design films for specific applications.

Effect of temperature on the dynamic and steady-shear rheology of a new microbial extracellular polysaccharide produced from glycerol byproduct

The effect of temperature on the dynamic and steady-shear rheology of aqueous solutions of an extracellular polysaccharide produced by microbial fermentation using Pseudomonas oleovorans NRRL B-14682 and glycerol byproduct, was studied. The biopolymer solutions have shown a pseudoplastic fluid behaviour and no gel formation was detected for all temperatures studied. As expected, the steady shear and dynamic parameters decreased with increasing temperature, due to the increase of polymer chain mobility. The EPS solutions were thermorheologically simple and stable, since the rheological parameters were successfully superimposed to and arbitrary reference temperature using the Time–Temperature Superposition principle, and the Cox-Merz rule was satisfactorily applied. In addition, the rheological properties were maintained under consecutive oscillatory and steady-state tests at 25 °C, after exposing the sample to increasing temperatures up to 80 °C. This polymer is a good candidate to be used in industrial processes involving temperature variations, at least in the range from 15 to 80 °C.

Production of a new exopolysaccharide (EPS) by Pseudomonas oleovorans NRRL B-14682 grown on glycerol

Abstract Pure glycerol and glycerol-rich product (GRP) obtained from the biodiesel industries were used as carbon source for the production of a new extracellular polysaccharide (EPS) by Pseudomonas oleovorans NRRL B-14682. The influence of temperature (20–40 °C) and pH (6.0–8.0) was studied. A temperature of 30 °C and pH control at 6.8 gave the maximum cell growth and EPS production. The culture attained a maximum cell dry weight (CDW) of 9.55 g l −1 and an EPS concentration of 11.82 g l −1 when cultivated with pure glycerol. GRP was a suitable carbon source, as shown by the slightly higher EPS concentration (12.18 g l −1 ). The EPS productivity obtained with GRP (3.85 g l −1 d −1 ) was almost twice that obtained with pure glycerol (2.00 g l −1 d −1 ). Also, the yield on glycerol was higher for the cultivation with GRP (0.36 g g −1 ) than for pure glycerol (0.28 g g −1 ). The EPS was a high molecular weight heteropolysaccharide, composed by neutral sugars (37–80 wt% galactose, 2–30 wt% glucose, 0.5–25 wt% mannose and 0.5–20 wt% rhamnose) and containing acyl group substituents (pyruvil, acetyl and succinyl were identified). The EPS forms highly viscous aqueous dispersions with many potential commercial applications.

Poly(hydroxyalkanoate) Biosynthesis from Crude Alaskan Pollock (Theragra chalcogramma) Oil

Six strains of Pseudomonas were tested for their abilities to synthesize poly(hydroxyalkanoate) (PHA) polymers from crude Pollock oil, a large volume byproduct of the Alaskan fishing industry. All six strains were found to produce PHA polymers from hydrolyzed Pollock oil with productivities (P; the percent of the cell mass that is polymer) ranging from 6 to 53% of the cell dry weight (CDW). Two strains, P. oleovorans NRRL B-778 (P = 27%) and P. oleovorans NRRL B-14682 (P = 6%), synthesized poly(3-hydroxybutyrate) (PHB) with number average molecular weights (Mn) of 206,000 g/mol and 195,000 g/mol, respectively. Four strains, P. oleovorans NRRL B-14683 (P = 52%), P. resinovorans NRRL B-2649 (P = 53%), P. corrugata 388 (P = 43%), and P. putida KT2442 (P = 39%), synthesized medium-chain-length PHA (mcl-PHA) polymers with Mn values ranging from 84,000 g/mol to 153,000 g/mol. All mcl-PHA polymers were primarily composed of 3-hydroxyoctanoic acid (C8:0) and 3-hydroxydecanoic acid (C10:0) amounting to at least 75% of the total monomers present. Unsaturated monomers were also present in the mcl-PHA polymers at concentrations between 13% and 16%, providing loci for polymer derivatization and/or crosslinking.

Characterization of an extracellular polysaccharide produced by a Pseudomonas strain grown on glycerol

A new extracellular charged polysaccharide composed mainly by galactose, with lower amounts of mannose, glucose and rhamnose, was produced by the cultivation of Pseudomonas oleovorans NRRL B-14682 using glycerol as the sole carbon source. Thermal and solid-state NMR analysis showed that this polymer is essentially amorphous, with a glass transition temperature of 155.7°C. The exopolysaccharide aqueous solutions have viscoelastic properties similar to that of Guar gum, but with affinity to salts as a result of its polyelectrolyte character. In addition, the exopolysaccharide has demonstrated good flocculating and emulsifying properties and film-forming capacity. These properties make this polymer a good alternative to more expensive natural polysaccharides, such as Guar gum, in several applications in the food, pharmaceutical, cosmetic, textile, paper and petroleum industries.

Genetic Characterization of the Poly(hydroxyalkanoate) Synthases of Various Pseudomonas oleovorans Strains

We identified the poly(hydroxyalkanoate) synthase (PHAS) genes of three strains of Pseudomonas oleovorans by using polymerase chain reaction (PCR)-based detection methods. P. oleovorans NRRL B-14682 contains Class I PHA synthase gene (phaC), NRRL B-14683 harbors Class II phaC1 and phaC2 genes, and NRRL B-778 contain both the Class I and II PHA synthase genes. Inverse-PCR and chromosomal walking techniques were employed to obtain the complete sequences of the Class I phaCs of NRRL B-778 (phbC778; 1698 bps) and B-14682 (phbC14682; 1899 bps). BLAST search indicated that these genes are new and had not been previously cloned. The gene product of phbC778 (i.e., PhbC778; 566 amino acid residues) is homologous to the Class I PHA synthases of Pseudomonas sp. HJ-2 and Pseudomonas sp. strain 61-3, and that of phbC14682 (PhbC14682; 632 amino acids) is homologous to PHAS of Delftia acidovorans. The PhbC14682 contains an extra sequence of 33 amino acids in its conserved alpha/beta-hydrolase domain, making it only the second Class I PHA synthase found to contain this cellular proteolytic sequence. Consistent with their Pseudomonas origin, the codon-usage profiles of PhbC778 and PhbC14682 are similar to those of Pseudomonas Class II PHASs. These new Pseudomonas Class I phbC genes provide valuable addition to the gene pool for the construction of novel PHASs through gene shuffling.

Synthesis of Short-/Medium-Chain-Length Poly(hydroxyalkanoate) Blends by Mixed Culture Fermentation of Glycerol

Glycerol was used as a substrate in the bio-production of poly(hydroxyalkanoates) (PHAs) in an effort to establish an alternative outlet for glycerol and produce value-added products. Pseudomonas oleovorans NRRL B-14682 and Pseudomonas corrugata 388 grew and synthesized poly(3-hydroxybutyrate) (P3HB) and medium-chain-length PHA (mcl-PHA) consisting primarily of 3-hydroxydecanoic acid (C(10:0); 44 +/- 2 mol %) and 3-hydroxydodecenoic acid (C(12:1); 31 +/- 2 mol %), respectively, from glycerol at concentrations up to 5% (v/v). Cellular productivity maximized at 40% for P. oleovorans in 5% (v/v) glycerol and 20% for P. corrugata in 2% (v/v) glycerol after 72 h. Increasing the glycerol media concentration from 1% to 5% (v/v) caused a 61% and 72% reduction in the molar mass (M(n)) of the P3HB and mcl-PHA polymers, respectively. Proton-NMR analysis of the glycerol-derived P3HB revealed that the M(n) decrease was the result of esterification of glycerol onto the polymer in a chain terminating position. However, no evidence of glycerol-based chain termination was present in the mcl-PHA. The growth patterns of P. oleovorans and P. corrugata on glycerol permitted their use as mixed cultures to produce natural blends of P3HB and mcl-PHA. By incorporating a staggered inoculation pattern and varying the duration of the fermentations, P3HB/mcl-PHA ratios were achieved that varied from 34:66 to 96:4.

Bacterial Poly(Hydroxyalkanoate) Polymer Production from the Biodiesel Co-product Stream

A co-product stream from soy-based biodiesel production (CSBP) containing glycerol, fatty acid soaps, and residual fatty acid methyl esters (FAME) was utilized as a fermentation feedstock for the bacterial synthesis of poly(3-hydroxybutyrate) (PHB) and medium-chain-length poly(hydroxyalkanoate) (mcl-PHA) polymers. Pseudomonas oleovorans NRRL B-14682 and P. corrugata 388 grew and synthesized PHB and mcl-PHA, respectively, when cultivated in up to 5% (w/v) CSBP. In shake flask culture, P. oleovorans grew to 1.3 ± 0.1 g/L (PHA cellular productivity = 13-27% of the bacterial cell dry weight; CDW) regardless of the initial CSBP concentration, whereas P. corrugata reached maximum cell yields of 2.1 g/L at 1% CSBP, which tapered off to 1.7 g/L as the CSBP media concentration was increased to 5% (maximum PHA cellular productivity = 42% of the CDW at 3% CSBP). While P. oleovorans synthesized PHB from CSBP, P. corrugata produced mcl-PHA consisting primarily of 3-hydroxyoctanoic acid (C8:0; 39 ± 2 mol%), 3-hydroxydecanoic acid (C10:0; 26 ± 2 mol%) and 3-hydroxytetradecadienoic acid (C14:2; 15 ± 1 mol%). The molar mass (Mn) of the PHB polymer decreased by 53% as the initial CSBP culture concentration was increased from 1% to 5% (w/v). In contrast, the Mn of the mcl-PHA polymer produced by P. corrugata remained constant over the range of CSBP concentrations used.

The synthesis of short- and medium-chain-length poly(hydroxyalkanoate) mixtures from glucose- or alkanoic acid-grown Pseudomonas oleovorans.

Pseudomonas oleovorans NRRL B-778 accumulated mixtures of poly-3-hydroxybutyrate (PHB) and medium-chain-length poly(hydroxyalkanoates) (mcl-PHAs) when grown on glucose, octanoic acid or oleic acid, whereas growth on nonanoic acid or undecanoic acid resulted in copolymers of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-co-HV). Acetone fractionation verified the presence of PHB/mcl-PHA mixtures. The acetone-insoluble (AIS) fractions of the polymers derived from glucose (PHA-glucose), octanoic acid (PHA-octanoic) and oleic acid (PHA-oleic) were exclusively PHB while the acetone-soluble (AS) fractions contained mcl-PHA composed of differing ratios of 3-hydroxy-acid monomer units, which ranged in chain length from 6 to 14 carbon atoms. In contrast, both the AIS and AS fractions from the polymers derived from nonanoic acid (PHA-nonanoic) and undecanoic acid (PHA-undecanoic) were composed of comparable ratios of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). The unfractionated PHA-glucose, PHA-octanoic and PHA-oleic polymers had melting temperatures (Tm) between 177 and 179 degrees C, enthalpies of fusion (AHf) of 20 cal/g and glasstransition temperatures (Tg) of 3-4 degrees C. This was due to the large PHB content in the polymer mixtures. On the other hand, the PHA-nonanoic and PHA-undecanoic polymers had thermal properties that supported their copolymer nature. In both cases, the Tm values were 161 degrees C, deltaHf values were 7 cal/g and Tg values were - 3 degrees C.

Microbial oxidation of cumene

A total of 1229 cultures, including 230 actinomycetes, 508 other bacteria, 12 fungi and 479 yeasts were screened for their ability to oxidize the isopropyl side chain of 2-phenyl propane (cumene). Four strains of actinomycetes and six strains of bacteria but no yeasts were found positive in converting 2-phenyl propane to its oxygenated products. Eight strains oxidized cumene through the alkyl side chain producing 2-phenyl-1-propanol. TwoBacillus strains oxidized cumene to an oxygenated product.Pseudomonas oleovorans NRRL B-3429 exhibited the highest alkyl side chain oxidation activity. The optimum reaction conditions for strain B-3429 are: 25 °C, pH 6.5 and 48 h of reaction. Octane-grown cells of strain B-3429 produced higher product yields (about 7.2-fold) than the glucose-grown cells. Prolonged incubation resulted in an increase in 2-phenyl-1-propionic acid production at the expense of 2-phenyl-1-propanol. The yield of 2-phenyl-1-propanol plus 2-phenyl-1-propionic acid was 5.1%. Reaction in the presence of methanol favored the accumulation of 2-phenyl-1-propionic acid and also increased the total yield. (The yield of 2-phenyl-1-propanol plus 2-phenyl-1-propionic acid was 14.9%.) Structures of the reaction products were confirmed by GC/MS and GC/IR analyses. Products contained 92% R(−) isomer.