Molecular Structure and Crystallization Dynamics of Poly(3-hydroxyl alkanoate) with Medium-Chain Length Biosynthesized from <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

The molecular structure of poly(3-hydroxyalkanoate) (P3HA) with medium-chain length, biosynthesized from Psuedomonas putida (P. putida), was investigated by thermal analysis, X-ray diffraction, and infrared spectrum. Cultivation in a medium of nonanoic acid and a mixed substrate of nonanoic acid and glycerin as carbon sources provided P3HA with monomer units of 7 and 9 carbons (P3HA_n) and 5, 6, 7, 8, 9, and 10 carbons (P3HA_mix). Since these P3HA has comparatively long side chains, the crystallinity was as low as about 10%. It was suggested that the hydrogen bonding plays an important role in constructing the crystal. The lamellar thickness was 1.20 nm, estimated from the melting temperature depression. This lamellar thickness corresponds to two repeating units. Crystallinity depending on time was observed by the CO stretching mode in the infrared spectrum, and then Avrami’s theory was applied to analyze the crystallization mechanism. The crystallization rate of P3HA_n was very low, on the order of a few hours. An Avrami exponent of 1.45 was estimated from the slope of the Avrami plot. This shows that the conformational arrangement is presumably promoted along the chain axis. The slow crystallization is attributed to the long side chain, which prevents aggregation of the polymer chains.     

Utilization of Cellulosic Wastes in Textile and Garment Industries: 2. Synthesis and Characterization of Cellulose Acetate from Knitted Rag

Cellulose acetate (CA) was synthesized from knitted rag, a cellulosic waste of Textile and Garment industries, in the glacial acetic acid, and subsequently acetic anhydride (Ac₂O) in presence of concentrated H₂SO₄ reaction medium. A low to high substitution products were obtained from single step up to seven steps acetylation of cellulose. In this way, it was possible to produce low cost and different grades or substituted acetylation derivatives of cellulose. The synthesized CA was characterized and investigation of its physical characteristics was done. Solubility, acetyl content, acetic acid content, degree of substitution, and molecular weight of CA increased gradually with the increase of the number of reaction steps attaining optimum value at the fourth step. The acetyl and acetic acid content of CA were increased from 39.95 % to 44.25 %, and from 55.73 % to 61.73 % respectively. Similarly, degree of substitution and molecular weight of CA were increased from 2.47 to 2.94, and from 74,249 to 121,437 respectively.     

Isolation and Characterization of a <Emphasis Type="Italic">Burkholderia</Emphasis> sp. USM (JCM15050) Capable of Producing Polyhydroxyalkanoate (PHA) from Triglycerides,Fatty Acids and Glycerols

A consortium of microorganisms from oil polluted wastewater sample was cultivated to promote polyhydroxyalkanoate (PHA) accumulation before subjecting the mixed cultures to sucrose density gradient ultracentrifugation. This resulted in the fractionation of the bacterial cells according to their physical features such as size, morphology and/or densities. An isolate was identified as Burkholderia sp. USM (JCM15050), which was capable of converting palm oil products [crude palm kernel oil (CPKO), palm olein (PO), palm kernel acid oil (PKAO), palm stearin (PS), crude palm oil (CPO), palm acid oil (PAO) and palm fatty acid distillate (PFAD)], fatty acids and various glycerol by-products into poly(3-hydroxybutyrate) [P(3HB)]. Up to 70 and 60 wt% of P(3HB) could be obtained when 0.5%(v/v) CPKO and glycerol was fed, respectively. Among the various fatty acids tested, lauric acid followed by oleic acid and myristic acid gave the best cell growth and PHA accumulation. Compared to Cupriavidus necator H16, the present isolate showed better ability to grow on and produce PHA from various glycerol by-products generated by the palm oil industry. This study demonstrated for the first time an isolate that has the potential to utilize palm oil and glycerol derivatives for the biosynthesis of PHA.     

Bacterial Cellulose from Simple and Low Cost Production Media by Gluconacetobacter xylinus

Bacterial cellulose pellicles were produced by Gluconacetobacter xylinus using non conventional low-cost carbon sources, such as glycerol remaining from biodiesel production and grape bagasse, a residue of wine production. The carbon sources assayed showed their suitability for microbial cellulose production, with relatively high production values such as 10.0 g/l for the culture medium with glycerol from biodiesel as carbon source and corn steep liquor as nitrogen source; and 8.0 g/l for the culture medium containing grape bagasse and corn steep liquor. Glucose, commercial glycerol and cane molasses were also assayed as carbon sources for comparison. The bacterial celluloses produced were characterized by means of scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy and thermogravimetric analysis. Morphological analysis showed that bacterial cellulose microfibrils produced from the non-conventional media used were several micrometers long and had rectangular cross-sections with widths and thicknesses in the range of 35–70 and 13–24 nm, respectively. X-ray patterns showed crystallinity levels in the range of 74–79 % (area method), whereas both X-ray patterns and infrared spectroscopy evidenced the presence of peaks characteristic of Cellulose I polymorph. Besides thermal properties were similar to those found for the pellicle obtained from glucose. The study performed showed the suitability of using wine residues or glycerol remaining from increasing biodiesel production as cheap carbon sources for production of bacterial cellulose microfibrils, with similar characteristics as those obtained by use of more expensive carbon sources such as glucose or commercial glycerol. On the other hand, the low cost nitrogen sources used (corn steep liquor or diammonium phosphate) also contributed to the economy of the bioprocess.     

Biodegradation of Polycyclic Aromatic Hydrocarbons by Exopolymers Synthesized by Moderately Halophilic Bacteria: Chemical Composition and Functional Properties

Three strains of halophilic bioemulsifier-producing bacteria; Bacillus sp. 2BSG-PDA-16, Bacillus sp. DV2-37 and Bacillus licheniformis ABRII6 were isolated from crude oil polluted water samples. Characteristics of exopolymers produced by these strains in media supplemented with various hydrocarbons instead of glucose were studied. Yield production, chemical composition, emulsifying, rheological and flocculating properties of exopolymers varied according to the strain and the carbon source. The highest amount of exopolymers synthesized by Bacillus sp. 2BSG-PDA-16, Bacillus sp. DV2-37 and B. licheniformis ABRII6 was 11, 18.5 and 12.4 g/l, respectively from media amended with glucose, while the most active emulsifiers were those obtained from media added with crude oil. Furthermore, all exopolymers produced were capable of emulsifying crude oil more efficiently than the three chemical surfactants tested as control (Tween 20, Tween 80 and Triton X-100). Respect to chemical composition, exopolymers produced on hydrocarbons always have lower content of carbohydrates and proteins than exopolymers produced in medium amended with glucose, however they showed higher amounts of uronic acids, sulfates and acetyl residues. The rheological study suggested that the exopolymers have characteristics of the pseudoplastic fluids. Efficiency of bacterial strains to remove PAH seems to agree with their potential applicability in oil bioremediation technology.     

Addition of Saturated Fatty Acids to Biodegradable Films: Effect on the Crystallinity and Viscoelastic Characteristics

Biodegradable films were produced by extrusion from blends of starch, glycerol and poly(butylene adipate co-terephthalate), a biodegradable polyester, with the addition of 1.5 % (wt/wt) saturated fatty acids (caproic, lauric or stearic). Films containing fatty acids had higher crystallinity than control films and the longer the carbon chain of the acid was, the more crystalline the film. Fatty acids with up to 12 carbon atoms did not alter the viscoelastic characteristic of the films. Further, the incorporation of fatty acids did not decrease the hydrophilicity of the films in comparison with the control film without a fatty acid.     

Compositional Limitations in Poly–3–Hydroxyalkanoates Produced by Pseudomonas oleovorans

It is well known that Pseudomonas oleovorans can utilize sodium octanoate for both cell growth and the synthesis of polyhydroxyalkanoates (PHAs), but it can utilize sodium butyrate only for limited cell growth and not for the polyester formation when this substrate is the sole carbon source. Therefore, these two substrates were evaluated as cofeeds for the possible incorporation of 3-hydroxybutyryl groups in the resulting PHA. When sodium butyrate and sodium octanoate were fed to P. oleovorans as cosubstrates in various proportions, the resultant cell density and polymer content were proportional to the amount of sodium octanoate in the feed. The PHA extracted from cells grown in all combinations of these cosubstrates had similar unit compositions of approximately 8 mole % 3-hydroxyhexanoate, 91 mole % 3-hydroxyoctanoate and 1 mole % 3-hydroxydecanoate. 3-Hydroxybutyrate units were not detected in any of the PHAs isolated, indicating that these units could not be incorporated in the copolymer synthesized by P. oleovorans either because the cell did not synthesize that monomer or, if it did, the PHA synthase could not copolymerize it with the longer chain monomers.     

Utilization of Porous Carbons Derived from Coconut Shell and Wood in Natural Rubber

The porous carbons derived from cellulose are renewable and environmentally friendly. Coconut shell and wood derived porous carbons were characterized with elemental analysis, ash content, X-ray diffraction, infrared absorbance, particle size, surface area, and pore volume. The results were compared with carbon black. Uniaxial deformation of natural rubber (NR) composites indicate the composites reinforced with the porous carbon from coconut shell have higher tensile moduli at the same elongation ratio than the composites reinforced with wood carbon. 40 % coconut shell composite showed a fivefold increase in tensile modulus compared to NR. Polymer–filler interactions were studied with frequency dependent shear modulus, swelling experiments and dynamic strain sweep experiments. Both linear and non-linear viscoelastic properties indicate the polymer–filler interactions are similar between coconut shell carbon and wood carbon reinforced composites. The swelling experiments, however, showed that the polymer–filler interaction is greater in the composites reinforced with coconut shell instead of wood carbon.     

Biosynthesis and Characterization of Poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) from Bacillus cereus S10

The biodegradable and biocompatible copolymer poly-(3-hydroxybutyrate-co-5 mol% 3-hydroxyvalerate), poly-(3HB-co-5 mol% 3HV), was synthesized by Bacillus cereus S10 and the highest yield was determined as 69.91 % at pH 7 and 30 °C after 48 h of incubation using a glucose as the sole carbon source. Poly-(3HB-co-5 mol% 3HV) was purified from bacterial biomass using chloroform. FTIR analysis showed absorption bands at 1,723, 1,274, 1,373, 1,453, 2,932 cm^?1 corresponding to C=O, C–O stretching, CH₃, –CH₂ and –CH groups, respectively. ¹H-NMR and ¹³C-NMR analysis confirmed that the copolymer was composed of 95 mol% of 3-hydroxybutrate and 5 mol% of 3-HV monomeric units. Poly-(3-HB-co-5 mol% 3HV) was used for nanoparticles preparation. The diameter of nanoparticles was 202 nm.     

Degradation of L- and DL-lactic acid oligomers in the presence ofFusarium moniliforme andPseudomonas putida

Poly(-alkanoates) derived from lactic acid enantiomers are known to degrade easily hydrolytically in aqueous media. The ability of two microorganisms, a filamentous fungus,Fusarium moniliforme, and a bacterium,Pseudomonas putida, to assimilate the degradation by-products of poly(lactic acid) (PLA), namely, lactic acid, lactyllactic acid dimers, and higher oligomers, was investigated in liquid culture. To distinguish the influence of chirality on bioassimilation, two series of substrates were considered which derived from the racemic and the L-form of lactic acid, respectively. The fate of these compounds was monitored by HPLC. Under the selected conditions,DL- andL-lactic acids were totally used by the two microorganisms regardless of the enantiomeric composition. Both microorganisms degraded the LL-dimer rather rapidly. However,F. moniliforme acted more rapidly thanP. putida. It is likely that the DD-dimer also biodegraded but at a slower rate, especially in the case of the fungi. Higher racemic oligomers were slowly assimilated by the two microorganisms, whereas higher L-oligomers appeared biostable probably because of their crystallinity. A synergistic effect was observed when both microorganisms were present in the same culture medium containing racemic oligomers.Presented at the 4th International Workshop on Biodegradable Plastics and Polymers, October 11–14, 1995. Durham, New Hampshire.     

Polyhydroxybutyrate Accumulation in Bacillus megaterium and Optimization of Process Parameters Using Response Surface Methodology

The accumulation of polyhydroxybutyrate of Bacillus megaterium is growth associated and significantly dependent on carbon sources. In the present investigation B. megaterium strain isolated from soil was studied for PHB production in fructose minimal media. The PHB production was found to be growth associated. The polymer production by the strain was found to vary from 24 to 48 % content (w/w) of the dry cell weight. Box Bohn design was used to study the interactive effect of four variables on cell growth and PHB production. The optimized medium conditions with the constrain to maximize cell growth and PHB content were glucose 4.32 g/L, Mannitol 4.52 g/L and Na succinate 3.45 g/L and PHB yield 1.38 g/L amounting to 49 % of dry cell weight which is more than 1.8 folds the basal medium. The polymer production by the strain was found to vary from 12.18 to 57.2 % content (w/w) of the dry cell weight.     

Production and degradation of polyhydroxyalkanoates in waste environment

Polyhydroxyalkanoates (PHAs) are energy/carbon storage materials accumulated under unfavorable growth condition in the presence of excess carbon source. PHAs are attracting much attention as substitute for non-degradable petrochemically derived plastics because of their similar material properties to conventional plastics and complete biodegradability under natural environment upon disposal. In this paper, PHA production and degradation in waste environment as well as its role in biological phosphorus removal are reviewed. In biological phosphorus removal process, bacteria accumulating polyphosphate (poly P) uptake carbon substrates and accumulate these as PHA by utilizing energy from breaking down poly P under anaerobic condition. In the following aerobic condition, accumulated PHA is utilized for energy generation and for the regeneration of poly P. PHA production from waste has been investigated in order to utilize abundant organic compounds in waste water. Since PHA content and PHA productivity that can be obtained are rather low, PHA production from waste product should be considered as a coupled process for reducing the amount of organic waste. PHAs can be rapidly degraded to completion in municipal anaerobic sludge by various microorganisms. ©     

Adsorption of Crystal Violet Dye from Aqueous Solution by Poly(Acrylamide-<Emphasis Type="Italic">co</Emphasis>-Maleic Acid)/Montmorillonite Nanocomposite

Poly(acrylamide-co-maleic acid)/montmorillonite nanocomposites, were synthesized via in situ polymerization with different maleic acid and MMT content. The capability of the hydrogel for adsorption of crystal violet (CV) was investigated in aqueous solutions at different pH values and temperatures. The pseudo-second-order kinetics model could fit successfully the adsorption kinetic data. The effects of maleic acid to acrylamide molar ratio (MA_R), weight percent of MMT (MMT%), the pH of medium and the solution temperature (T) on the CV adsorption capacity (q _e ) of adsorbents were studied by Taguchi experimental design approach. The results indicated that increasing the MMT% leads to a greater q _e . The q _e value of adsorbents increased also with increasing both MA_R and pH, while reduced when the temperature of medium increased. The relatively optimum conditions to achieve a maximum CV adsorption capacity for P(AAm/MA)/MMT adsorbents were found as: 0.06 for MA_R and 5 % of MMT%, medium pH = 7 and T = 20 °C.     

Tween 20 and Its Major Free Fatty Acids as Carbon Substrates for the Production of Polyhydroxyalkanoates in Pseudomonas aeruginosa ATCC 27853

The ability of Pseudomonas aeruginosa ATCC 27853 to grow and synthesize polyhydroxyalkanoates (PHAs) using Tween 20 as the sole carbon source was investigated. Tween 20 could support cell growth and PHA production. The polymer produced from Tween 20 was compared with those produced from its major free fatty acids components: lauric (C₁₂), myristic (C₁₄), and palmitic (C₁₆) acids. Gas-chromatographic analysis of methanolyzed samples and ¹³C-Nuclear Magnetic Resonance (NMR) showed that the PHAs obtained are composed of even carbon atoms 3-hydroxyalkanoates ranging from C₆ to C₁₄, with C₈ and C₁₀ as the predominant components. The nature of the carbon sources used had little influence on the composition, but was found to be important in determining the average molecular weight, shorter chain fatty acids yielding higher molecular weight products. Fast Atom Bombardment-Mass Spectrometry (FAB-MS) of partially pyrolyzed samples, coupled to statistical analysis, showed that these PHAs are random copolymers.     

Municipal Wastes Treatment and Production of Polyhydroxyalkanoate by Modified Two-Stage Batch Reactor

The municipal wastes were utilized as substrate for polyhydroxyalkanoate (PHA) using two strains of Bacillus licheniformis (PHAs-007, wild type and M2-12, mutant). Municipal wastes were subjected to separate wastewater and biosolid. Municipal biosolid was digested by anaerobic bacteria thereafter only the supernatant with soluble organic compounds was subjected into the PHA-producing reactor containing municipal wastewater. The mutant strain M2-12 gave the highest value of biomass (42.0 ± 2.0 g/L) and PHA concentration (37.4 ± 1.0 g/L with 88.9 % of dry cell weight, DCW) and reduced 76.5 % of soluble chemical oxygen demand after 60 h of cultivation. The value of pH, biochemical oxygen demand and total solid of the reclaimed wastewater after PHA recovery was 7.1, 20 and 97 mg/L, respectively. Moreover, the polymers produced by both strains of B. licheniformis were characterized. The resultant polymer from B. licheniformis PHAs-007 and M2-12 cultivated in the PHA-producing reactor was identified as poly-3-hydroxybutyrate-co-3-hydroxyvalerate [P(3HB-co-3HV)] and poly-3-hydroxybutyrate-co-4-hydroxybutyrate [P(3HB-co-4HB)], respectively. The results suggesting that the production of PHA by municipal wastes is feasible thus the PHA production stage can be integrated in waste treatment to produce PHA and treated municipal wastes at the same time.     

Biocompatible Polyurethane Scaffolds Prepared from Glycerol Monostearate-Derived Polyester Polyol

Biodegradable polyester polyol was synthesized from oleochemical glycerol monostearate (GMS) and glutaric acid under a non-catalyzed and solvent-free polycondensation method. The chemical structure of GMS-derived polyester polyol (GPP) was elucidated by FTIR, ¹H and ¹³C NMR, and molecular weight of GPP was characterized by GPC. The synthesized GPP with acid value of 3.03 mg KOH/g sample, hydroxyl value of 115.72 mg KOH/g sample and M_n of 1345 g/mol was incorporated with polyethylene glycol (PEG) and polycaprolactone diol (PCL diol) to produce a water-blown porous polyurethane system via one-shot foaming method. The polyurethanes were optimized by evaluating glycerol as a crosslinker, silicone surfactant and water blowing agent on tensile properties of polyurethanes. All polyurethanes underwent structural change, and crystalline hard segments of polyurethanes were shifted to higher temperature suggested that hard segments undergone re-ordering process during enzymatic treatment. In terms of biocompatibility, polyurethane scaffold produced by reacting 100% w/w of GPP with isophorone diisocyanate and additives showed the highest cells viability of 3T3 mouse fibroblast (94%, day 1), and MG63 human osteosarcoma (107%, day 1) and better cell adhesion as compared to reference polyurethane produced by only PEG and PCL diol (3T3 cell viability: 8%; MG63 cell viability: 2%). The current work demonstrated GPP synthesized from renewable and environmental friendly resources produced polyurethanes that allows improvement in physico-chemical, mechanical and biocompatibility properties. By blending with increasing content of GPP, the water-blown porous polyurethane scaffold has shown great potential as biomaterial for soft and hard tissue engineering.     

Application of recombinant gene technology for production of polyhydroxyalkanoic acids: Biosynthesis of poly(4-hydroxybutyric acid) homopolyester

Screening of a large number of bacteria revealed several strains, which utilize 1,4-butanediol and/or 4-hydroxybutyric acid (4HB) as a carbon source for growth and for synthesis of polyhydroxyalkanoic acids (PHA) containing 4HB as one constituent among others (mostly 3-hydroxybutyric acid). However, none of the wild-type strains investigated in this study was able to produce a homopolyester consisting solely of 4HB. Only several poly(3-hydroxybutyric acid)-leaky mutants ofAlcaligenes eutrophus strain JMP222 synthesized poly(4HB) homopolyester, which amounted to approximately 10% (w/w) of the cellular dry matter. If the PHA synthase structural gene ofA. eutrophus strain H16 was expressed in these mutants, the amount of poly(4HB) was increased to approximately 30% (w/w). The occurrence of poly(4HB) was demonstrated by gas chromatographic as well as¹H and¹³C nuclear magnetic resonance spectroscopic analysis.Paper presented at the Bio/Environmentally Degradable Polymer Society—Second National Meeting, August 19–21, 1993, Chicago, Illinois.     

Cell-recycle fed-batch production of a highly unsaturated polyhydroxyalkanoate from 1,3-butanediol byPseudomonas sp. A33

The recently isolatedPseudomonas sp. A33 was investigated for the production of a highly unsaturated polyhydroxyalkanoate (PHA) containing various alkyl and alkenyl pendent groups from 1,3-butanediol in a cell-recycle fed-batch production mode. The monomer composition and degree of unsaturation in PHA were dependent on the environmental conditions. The production temperature markedly influenced the content, composition, and degree of unsaturation of PHA. As the production temperature decreased from 30 to 10°C, the degree of unsaturation and content of PHA were increased, while the mole percentage of 3-hydroxybutyrate (3HB) was decreased. These temperature effects on the composition of PHA imply that the production can be used as a control variable for the biosynthesis of a highly unsaturated PHA and for the specific regulation of the composition of PHA. The biosynthetic pathway for a highly unsaturated PHA which is based on de novo fatty acid biosynthetic pathway is proposed. For the enhanced production of this functional PHA, a high cell density was achieved by cell-recycle continuous culture at 30°C, and then a large amount of PHA was accumulated at 15°C by fed-batch addition of the feeding solution containing excess 1,3-butanediol. The structures of monomer constituents of polymer were confirmed by gas chromatography—mass spectrometric analysis of trimethylsiyl derivatives of 3-hydroxyalkanoic acids methyl esters.     

Selective organic compounds degradation under controlling composting conditions

Organic matter stabilization resulted from the decrease of cellulose, xylan, arabinan, acetyl groups, glucuronic acids, galacturonic acids (easily biodegradable fractions) and the increase of lignin (resistant compound) and humic substances coming from the initial wastes have been studied. A central composite experimental design was used to investigate the influence of environmental composting parameters (moisture, aeration and particle size) on organic matter evolution. The organic matter evolution was clearly influenced by the studied composting parameters. All results were concordant, with an increase of humic substances and lignin and a decrease of the rest of the cellulose and hemicellulose compounds. Lower cellulose, xylan, acetyl groups and glucuronic acids contents (higher degradation) have been observed under low particle size (1 cm) and higher moisture content (70%). However lower lignin and higher humic substances under medium (3 cm) to low particle size and low moisture content (40%) have been found.     

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 (M_n) 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 M_n values ranging from 84,000 g/mol to 153,000 g/mol. All mcl-PHA polymers were primarily composed of 3-hydroxyoctanoic acid (C_8:0) and 3-hydroxydecanoic acid (C_10:0) 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. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.