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 (C_8:0; 39 ± 2 mol%), 3-hydroxydecanoic acid (C_10:0; 26 ± 2 mol%) and 3-hydroxytetradecadienoic acid (C_14:2; 15 ± 1 mol%). The molar mass (M_n) of the PHB polymer decreased by 53% as the initial CSBP culture concentration was increased from 1% to 5% (w/v). In contrast, the M_n of the mcl-PHA polymer produced by P. corrugata remained constant over the range of CSBP concentrations used.     

Extracellular Biopolymer Production by Aureobasidium pullulans MTCC 2195 Using Jackfruit Seed Powder

In this present paper, statistical screening and optimization of jackfruit seed powder based medium components were investigated for pullulan production from Aureobasidium pullulans. Seven medium variables jackfruit seed powder, K₂HPO₄, yeast extract, (NH₄)₂SO₄, NaCl, MgSO₄·7H₂O and ZnSO₄·7H₂O were screened by employing Plackett–Burman (PB) method. PB method showed jackfruit seed powder, ZnSO₄·7H₂O, K₂HPO₄ and yeast extract were significant. Central composite design of response surface method applied to optimize the significant variables identified from the PB experiment. Statistical analysis of the experimental results showed optimal values were found to be jackfruit seed powder 2 % (w/v), K₂HPO₄ 0.55 % (w/v), yeast extract 0.30 % (w/v) and ZnSO₄·7H₂O 0.006 % (w/v) with maximum pullulan concentration of 18.76 (g/L). Maximum pullulan concentration of 17.95 (g/L) was observed in the validation experiment. This experimental result explained the model was fitted 96 % as compare with the result predicted by response surface method.     

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.     

Optimization of Carbon Dioxide and Valeric Acid Utilization for Polyhydroxyalkanoates Synthesis by Cupriavidus necator

The utilization of captured CO₂ as a part of the CO₂ capture and storage system to produce biopolymers could address current environmental issues such as global warming and depletion of resources. In this study, the effect of feeding strategies of CO₂ and valeric acid on cell growth and synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] in Cupriavidus necator was investigated to determine the optimal conditions for microbial growth and biopolymer accumulation. Among the studied CO₂ concentrations (1–20 %), microbial growth and poly(3-hydroxybutyrate) accumulation were optimal at 1 % CO₂ using a gas mixture at H₂:O₂:N₂ = 7:1:91 % (v/v). When valeric acid was fed together with 1 % CO₂, (R)-3-hydroxyvalerate synthesis increased with increasing valeric acid concentration up to 0.1 %, but (R)-3-hydroxybutyrate synthesis was inhibited at >0.05 % valeric acid. Sequential addition of valeric acid (0.05 % at Day 0 followed by 0.025 % at Day 2) showed an increase in 3HV fraction without inhibitory effects on 3HB synthesis during 4 d accumulation period. The resulting P(3HB-co-3HV) with 17–32 mol  % of 3HV is likely to be biocompatible. The optimal concentrations and feeding strategies of CO₂ and valeric acid determined in this study for microbial P(3HB-co-3HV) synthesis can be used to produce biocompatible P(3HB-co-3HV).     

Utilization of Broken Rice for the Production of Poly(3-hydroxybutyrate)

The feasibility of utilizing non edible rice (broken rice) for production of fine materials such as poly(3-hydroxybutyrate) (PHB) was considered as one of the alternative ways of keeping the environment clean for sustainable development. Thus, production of PHB from broken rice by simultaneous saccharification and fermentation (SSF) was investigated. During the SSF process, the rice (15% w/v) material was hydrolyzed to glucose, which was utilized by Cupriavidus necator for growth and production of PHB. The PHB content reached 38% at 58 h fermentation. The PHB had weight average molar mass (Mw) and polydipersity index of 3.82 × 10⁵ (g/mol) and 4.15, respectively. Differential calorimetric scan of the PHB showed a melting temperature (Tm) of 176 °C. Given that the PHB was a homopolymer (which consisted of (R)-3-hydroxybutyric acid monomers), it was thought that broken rice could be a raw material for production of both PHB and (R)-3-hydroxybutyric acid. This SSF process would not only help in the utilization of broken rice or non edible rice, but would also serve as a model for utilization of other raw materials that contain starch for production of PHB.     

Effects of Poly(Ethylene Glycol) on the Production of Poly(β-Hydroxybutyrate) by Azotobacter vinelandii UWD

Azotobacter vinelandii UWD, ATCC 53799, an engineered strain derived from Azotobacter vinelandii UW was used in the poly(ethylene glycol) (PEG)-modulated synthesis of poly(-hydroxybutyrate) (PHB). To the best of our knowledge, this is the first report on modulating the production of PHB by amending the fermentation broth with PEG using A. vinelandii UWD. It was determined that A. vinelandii UWD is prone to back-mutation to the parent strain; hence fermentation experiments require the use of the antibiotic rifampicin. Diethylene glycol (DEG) and PEGs with molecular weights of 400, 2000, and 3400 Da and pentaerythritol ethoxylate (PEE) were used in the modulated fermentation experiments in a concentration of 2% (w/v). The molecular weight of the resulting polymers was reduced by up to 78%. No impact on the productivity of the strain was observed. Spectroscopic evidence showed that PEG-modulated synthesis resulted in the covalent attachment of the ethylene glycol moiety only when a small molecule, DEG, was used. PEGs had the same effects on the polymer formation in terms of molecular weight reduction as DEG, but no spectroscopic evidence was found for the formation of a covalent linkage between PHB and higher molecular weight PEGs.     

Degradation of Poly(3-hydroxybutyrate) and Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Some Soil Aspergillus spp.

Systematic screening of 45 soil fungi for degradation polyhydroxyalkanoic acids (PHAs) has led to the selection of 6 potent Aspergillus isolates belonging to A. flavus, A. oryzae, A. parasiticus, and A. racemosus. Degradation of PHAs as determined by tube assay method revealed that these Aspergillus spp. were more efficient in degrading poly(3-hydroxybutyrate) [P(3HB)] compared to copolymer of 3-hydroxybutyric acid and 3-hydroxyvaleric acid (P3HB-co-16% 3HV). Moreover, the extent of degradation in mineral base medium was much better than those in complex organic medium. For all the Aspergillus spp. tested, maximum degradation was recorded at a temperature of 37°C with significant inhibition of growth. The optimum pH range for degradation was 6.5–7.0 with degradation being maximum at pH 6.8. The extent of polymer degradation increased with increase in substrate concentration, the optimum concentration for most of the cultures being 0.4% and 0.2% (w/v) for P(3HB) and P(3HB-co-16%3HV) respectively. Supplementation of the degradation medium with additional carbon sources exerted significant inhibitory effect on both P(3HB) and P(3HB-co-16%3HV) degradation.     

Purification of Aspergillus fumigatus (Pdf1) Poly(β-hydroxybutyrate) (PHB) Depolymerase Using a New, Single-Step Substrate Affinity Chromatography Method: Characterization of the PHB Depolymerase Exhibiting Novel Self-Aggregation Behavior

The extracellular poly(-hydroxybutyrate) (PHB) depolymerase of Aspergillus fumigatus Pdf1 was purified by a new, simple, one-step affinity chromatography method using the substrate PHB. The purified enzyme was glycosylated, with the molecular mass of 40 KD, and exhibited a novel self-aggregation behavior by means of hydrophobic interaction that was resolved by Triton X-100 (TX-100) pretreatment of enzyme and also TX-100 incorporation in the native gel. The apparent K _m value of purified enzyme for PHB was 119 g/mL and 3-hydroxybutyrate was detected as the main endproduct of PHB hydrolysis. The depolymerase was insensitive to phenylmethyl sulfonyl fluoride (PMSF), sodium azide, ethylenediaminetetraacetic acid (EDTA), and para-chloromercuric benzoic acid (PCMB), but was inactivated by dithioerythritol (DTT) and showed specificity for short chain-length poly(-hydroxyalkanoates) (PHAs) such as PHB, poly(hydroxyvalerate) (PHV), and copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). Medium-chain-length PHA failed to get hydrolyzed. The enzyme, however, exhibited strong cross reactivity with the Comamonas sp. PHB depolymerase antibodies, but not with PHV depolymerase antibodies of Pseudomonas lemoignei. Southern hybridization and dot blot analysis of A. fumigatus Pdf1 genomic DNA with alkaline phosphatase labeled probes of P. lemoignei PHB and PHV depolymerase genes revealed no homology, although the enzyme hydrolyzed both PHB and PHV.     

Influence of Carbon Sources on The Production and Characterization of The Exopolysaccharide (EPS) by Bacillus sphaericus 7055 Strain

In this study, EPS produced by five Bacillus spp. strains was determined. The only one strain (B. sphaericus 7055) was selected due to its high EPS production and it was investigated by growing this strain in LB broth medium containing various carbon sources. The highest EPS production of this strain was found in medium containing fructose. However, the effect of different concentrations of fructose and molasses on EPS production by the strain was studied. The maximum EPS yield of the strain 7055 was recorded with 2.5% (w/v) fructose, also the highest EPS production was found in 2.5% (w/v) molasses. The strain 7055 was found to contain (98.6%) galactose and (1.4%) glucuronic acid in control medium whereas the composition of the strain 7055 (2.5% (w/v) fructose) was found to be (99.9%) neutral sugar and (0.1%) Glucuronic acid while the strain 7055 (2.5% (w/v) molasses) was found to contain (65.9%), neutral sugars and (34.1%) glucuronic acid.     

Co-digestion of sewage sludge with glycerol to boost biogas production

The feasibility of adding crude glycerol from the biodiesel industry to the anaerobic digesters treating sewage sludge in wastewater treatment plants was studied in both batch and continuous experiments at 35 °C. Glycerol addition can boost biogas yields, if it does not exceed a limiting 1% (v/v) concentration in the feed. Any further increase of glycerol causes a high imbalance in the anaerobic digestion process. The reactor treating the sewage sludge produced 1106 ± 36 ml CH₄/d before the addition of glycerol and 2353 ± 94 ml CH₄/d after the addition of glycerol (1% v/v in the feed). The extra glycerol-COD added to the feed did not have a negative effect on reactor performance, but seemed to increase the active biomass (volatile solids) concentration in the system. Batch kinetic experiments showed that the maximum specific utilization rate (μ_max) and the saturation constant (K_S) of glycerol were 0.149 ± 0.015 h^?1 and 0.276 ± 0.095 g/l, respectively. Comparing the estimated values with the kinetics constants for propionate reported in the literature, it can be concluded that glycerol uptake is not the rate-limiting step during the process.     

Environmentally-Friendly Synthesis of Ag Nanoparticles by Fusarium sporotrichioides for the Production of PVA/Bentonite/Ag Composite Nanofibers

Membranes and filters made of nanofibers can have many medicines and water treatment applications. The use of silver nanoparticles (AgNPs) with antibacterial activity in these structures improve their efficiency. However, due to the toxicity of the compounds used in the chemical synthesis of AgNPs, in this study, AgNPs were obtained through a biological process using Fusarium sporotrichioides. AgNPs preparation conditions were optimized, including F. sporotrichioides medium and AgNO₃ concentration. Next, a PVA nanofiber membrane with bentonite and AgNPs (Bio-AgNPs or Chem-AgNPs) was prepared using electrospinning. The optimal conditions for the production of Bio-AgNPs were the culture of F. sporotrichioides in the MGYP culture medium and 12 M of AgNO₃. The Bio-AgNPs particle size and zeta potential were 58 nm and ??16.8 mV, respectively, with antibacterial activity. The PVA/NB/AgNPs nanofibers operation conditions included 7.5% w/w PVA, 3% w/w bentonite, and AgNPs 5% w/w at a voltage of 11 kV, feed rate of 0.5 mL/h, and 15 cm distance between the needle and the collector. The average diameter of the PVA/NB/Bio-AgNPs nanofibers was 230 nm. Also, the presence of silver in the nanofibers was confirmed through EDX and XRD methods. The antibacterial assay of the nanofibers showed that the inhibition zone of PVA/NB/Bio-AgNPs against E. coli and S. aureus was 0.62 and 0.36 mm, which is better than PVA/NB/Chem-AgNPs and comparable with chloramphenicol. The produced membrane is suitable for water treatment, food packaging, and wound dressing because of its good thermal, mechanical, and antibacterial properties.

     

Microbial degradation of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in compost

The microbial degradation of tensile test pieces made of poly(3-hydroxybutyrate) [P(3HB)] or copolymers with 10% [P(3HB-co-10%3HV)] and 20% [P(3HB-co-20%3HV)] 3-hydroxyvaleric acid was studied in small household compost heaps. Degradation was measured through loss of weight (surface erosion) and changes in molecular weight and mechanical strength. It was concluded, on the basis of weight loss and loss of mechanical properties, that P(3HB) and P(3HB-co-3HV) plastics were degraded in compost by the action of microorganisms. No decrease inM _w could be detected during the degradation process. The P(3HB-co-20%3HV) copolymer was degraded much faster than the homopolymer and P(3HB-co-10%3HV). One hundred nine microbial strains capable of degrading the polymersin vitro were isolated from the samples used in the biodegradation studies, as well as from two other composts, and identified. They consisted of 61 Gram-negative bacteria (e.g.,Acidovorax facilis), 10 Gram-positive bacteria (mainlyBacillus megaterium), 35Streptomyces strains, and 3 molds.     

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.     

Crystallization behavior of predominantly syndiotactic poly(β-hydroxybutyrate)

Predominantly syndiotactic poly(-hydroxybutyrate), syn-PHB, of variable syndioregularity (syndyad fractions 0.59, 0.62, 0.64, and 0.71) and molecular weight was prepared by the dibutyltin dimethoxide catalyzed ring opening of racemic-butyrolactone (BL). The crystallization behavior of the syn-PHB polymers was investigated by DSC and X-ray diffraction analyses. DSC of films after melting and annealing showed at least one, and often two distinct melting transitions occuring over a broad (often 40°C) temperature range. These results indicate that syn-PHB chain segments of variable syndioregularity form crystalline regions with very different thermodynamic stabilities. Maximum degrees of crystallinity for melt annealed 0.64- and 0.71-syn-PHB was observed at an annealing temperature (T _c ) of 30°C. AtT _c values at 45°C and higher, crystallization of relatively lower syndioregular chain segments was apparently excluded to variable degrees dependent onT _c and sample syndiotactic dyad content. After crystallization of syn-PHB samples at elevated temperatures, ambient temperature annealing resulted in an observed lower temperature melting transition at 50°C. This result showed little to no dependence on syn-PHB syndio-regularity andT _c . Both solution precipitated 0.62-syn-PHB and 0.71-syn-PHB have WAXS patterns with poorly resolved crystalline reflections superimposed on amorphous haloes indicating low levels of crystallinity (17% and 25%, respectively) and poorly formed crystals. Isothermal crystallization monitored by DSC showed that the syn- and natural origin PHB showed fastest crystallization rates at temperatures between 50°C and 70°C and 60°C and 90°C, respectively. From the dependence of the higher melting transition onT _c it was determined that the equilibrium melting temperatures for 0.62-syn-PHB (M _n =83,700 g/mol) and a 0.64-syn-PHB (M _n =11,900 g/mol) were 157 and 154°C, respectively. An Avrami analysis of syn-PHB yielded results similar to that found for natural origin PHB indicating that crystal growth occurs by a two-dimensional mechanism.Guest Editor: Dr. Graham Swift, Rohm & Haas.     

Synthesis of a block copolymer of poly(3-hydroxybutyrate) and poly(ethylene glycol) and its application to biodegradable polymer blends

A block copolymer {P[(R,S)-HB-b-EG]} of atactic poly[(R,S)-3-hydroxybutyrate] {P[(R,S)-HB]} and poly(ethylene glycol) (PEG) was prepared by the ring-opening polymerization of -butyrolactone in the presence of a macroinitiator (PEG/ZnEt₂/H₂O) which had been produced by the reaction of ,-dihydroxy PEG ( _n=3000) with ZnEt₂/H₂O (1/0.6) catalyst. The block copolymer ( _n=10,500, _w/ _n=1.2) was an A-B-A triblock copolymer comprising atactic P[(R,S)-HB] (A) and PEG (B) segments. The miscibility, physical properties, and biodegradability of binary blends of microbial poly[(R)-3-hydroxybutyrate] {P[(R)-HB]} with the block copolymer P[(R,S)-HB-b-EG] has been studied. The glass-transition temperature (T _g) data showed that the P[(R)-HB]/P[(R,S)-HB-b-EG] blend was miscible in the amorphous state. The P[(R)-HB] film became flexible and tough by means of blending with P[(R,S)-HB-b-EG] block copolymer. The enzymatic degradation of blend films was carried out at 37°C and pH 7.4 in a 0.1M phosphate solution of an extracellular PHB depolymerase fromAlcaligenes faecalis. The enzymatic degradation took place solely on the surface of the blend films.     

Kinetics of Aerobic Polymer Degradation in Soil by Means of the ASTM D 5988-96 Standard Method

The standard test method ASTM D 5988-96 for determining the degree and rate of aerobic biodegradation of plastic materials in contact with soil was applied to poly(3-hydroxybutyrate) and poly(-caprolactone). The method proved to be reliable and supplied reproducible measurements of CO₂ production, provided potassium (instead of barium) hydroxide was used as a trapping solution. The trends of CO₂ evolution, as a function of time, observed for the degradation of polymer powders in soil are similar to those predicted by simple first order kinetics in solution. The experimental data are described by a Michaelis–Menten type model, which accounts for the heterogeneity of the polymer-soil system. The kinetic equation deduced predicts the degradation rate to the proportional to the exposed polymer surface area.     

Degradation of poly(3-hydroxybutyrate), PHB,by bacteria and purification of a novel PHB depolymerase fromComamonas sp.

Bacteria capable of growing on poly(3-hydroxybutyrate), PHB, as the sole source of carbon and energy were isolated from various soils, lake water, activated sludge, and air. Although all bacteria utilized a wide variety of monomeric substrates for growth, most of the strains were restricted to degrade PHB and copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate, P(3HB-co-3HV). Five strains were also able to decompose a homopolymer of 3-hydroxyvalerate, PHV. Poly(3-hydroxyoctanoate), PHO, was not degraded by any of the isolates. One strain, which was identified asComamonas sp., was selected, and the extracellular depolymerase of this strain was purified from the medium by ammonium sulfate precipitation and by chromatography on DEAE-Sephacel and Butyl-Sepharose 4B. The purified PHB depolymerase was not a glycoprotein. The relative molecular masses of the native enzyme and of the subunits were 45,000 or 44,000, respectively. The purified enzyme hydrolyzed PHB, P(3HB-co-3HV), and—at a very low rate—also PHV. Polyhydroxyalkanoates, PHA, with six or more carbon atoms per monomer or characteristic substrates for lipases were not hydrolyzed. In contrast to the PHB depolymerases ofPseudomonas lemoignei andAlcaligenes faecalis T₁, which are sensitive toward phenylmethylsulfonyl fluoride (PMSF) and which hydrolyze PHB mainly to the dimeric and trimeric esters of 3-hydroxybutyrate, the depolymerase ofComamonas sp. was insensitive toward PMSF and hydrolyzed PHB to monomeric 3-hydroxybutyrate indicating a different mechanism of PHB hydrolysis. Furthermore, the pH optimum of the reaction catalyzed by the depolymerase ofComamonas sp. was in the alkaline range at 9.4.     

Distribution of Poly(β-propiolactone) Aerobic Degrading Microorganisms in Different Environments

The distribution of degading microorganisms of high molecular weight poly(-propiolactone) (PPL), whose individual structural units are similar to those of poly(-hydroxybutyrate) (PHB) and poly(€-caprolactone) (PCL), was examined. Despite the fact that PPL is a chemosynthetic polymer, many kinds of PPL-degrading microorganisms were found to be distributed as resident populations widely in natural environments. A total of 77 strains of PPL-degrading microorganisms was isolated. From standard physiological and biochemical tests, at least 41 strains were referred to as Bacillus species. Microbial degradation of fibrous PPL proceeded rapidly in some enrichment cultures but was not as complete as that of PHB. Most of the isolated PPL-degrading microorganisms were determined to be PCL degraders and/or PHB degraders. Therefore, it can be assumed that mostly PPL is recognized by the microorganisms as PHB or another natural substrate of the same type as which PCL is regarded. Microbial degradation of PPL was confirmed by some Bacillus strains from type culture collections. The similarity of microbial degradation between PPL and PCL was found to be very close.     

Pseudomonas lemoignei has five poly(hydroxyalkanoic acid) (PHA) depolymerase genes: A comparative study of bacterial and eukaryotic PHA depolymerases

Four polyhydroxyalkanoate (PHA) depolymerases were purified from the culture fluid ofPseudomonas lemoignei: poly(3-hydroxybutyrate) (PHB), depolymerase A (M _r , 55,000), and PHB depolymerase B (M _r , 67,000) were specific for PHB and copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) as substrates. The third depolymerase additionally hydrolyzed poly(3-hydroxyvalerate) (PHV) at high rates (PHV depolymerase;M _r , 54,000). The N-terminal amino acid sequences of the three purified proteins, of a fourth partially purified depolymerase (PHB depolymerase C), and of the PHB depolymerases ofComamonas sp. were determined. Four PHA depolymerase genes ofP. lemoignei (phaZ1,phaZ2,phaZ3, andphaZ4) have been cloned inEscherichia coli, and the nucleotide sequence ofphaZ1 has been determined recently (D. Jendrossek, B. Müller, and H. G. Schlegel,Eur. J. Biochem. 218, 701–710, 1993). In this study the nucleotide sequences ofphaZ2 andphaZ3 were determined.PhaZ1,phaZ2, andphaZ4 were identified to encode PHB depolymerase C, PHB depolymerase B, and PHV depolymerase, respectively.PhaZ3 coded for a novel PHB depolymerase ofP. lemoignei, named PHB depolymerase D. None of the four genes harbored the PHB depolymerase A gene, which is predicted to be encoded by a fifth depolymerase gene ofP. lemoignei (phaZ5) and which has not been cloned yet. The deduced amino acid sequences ofphaZ1–phaZ3 revealed high homologies to each other (68–72%) and medium homologies to the PHB depolymerase gene ofAlcaligenes faecalis T₁ (25–34%). Typical leader peptide amino acid sequences, lipase consensus sequences (Gly-Xaa-Ser-Xaa-Gly), and unusually high proportions of threonine near the C terminus were found in PhaZ1, PhaZ2, and PhaZ3. Considering the biochemical data of the purified proteins and the amino acid sequences, PHA depolymerases ofP. lemoignei are most probably serine hydrolases containing a catalytical triad of Asp, His, and Ser similar to that of lipases. A comparison of biochemical and genetic data of various eubacterial and one eukaryotic PHA depolymerases is provided also.Paper presented at the Bio/Environmentally Degradable Polymer Society—Second National Meeting, August 19–21, 1993, Chicago, Illinois.     

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.