Kinetic Study of Biopolymer (PHB) Synthesis in <Emphasis Type="Italic">Alcaligenes</Emphasis> sp. in Submerged Fermentation Process Using TEM

Poly-β-hydroxybuyrate (PHB) is a carbon—energy storage material which is accumulated as intracellular granule in variety of microorganism under nutrient starved conditions. Solid PHB is a biodegradable thermoplastic polymer and is utilizable in various ways similar to many conventional plastics. Ralstonia eutropha (Alcaligenes sp.), a gram negative bacteria accumulates PHB as insoluble granules inside the cells when nutrients other than carbon are limited. In this report effort has been made to analyze PHB granule synthesis inside Alcaligenes sp. NCIM 5085 by transmission electron microscopy and qualitative estimation of PHB was carried out by fourier transform infrared spectroscopy which provide better precision compared to other conventional techniques previously applied for PHB determination. Maximum PHB concentration of 2.20 ± 0.40 g/L and cell biomass of 3.42 ± 0.20 g/L was obtained after 48.0 h of fermentation. Leudking-Piret equation deduced mixed growth associated product formation which varies from earlier reports.     

Utilization of molasses spentwash for production of bioplastics by waste activated sludge

Present study describes the treatment of molasses spentwash and its use as a potential low cost substrate for production of biopolymer polyhydroxybutyrate (PHB) by waste activated sludge. Fluorescence microscopy revealed the presence of PHB granules in sludge biomass which was further confirmed by fourier transform-infra-red spectroscopy (FT-IR) and ¹³C nuclear magnetic resonance (NMR). The processing of molasses spentwash was carried out for attaining different ratios of carbon and nitrogen (C:N). Highest chemical oxygen demand (COD) removal and PHB accumulation of 60% and 31% respectively was achieved with raw molasses spentwash containing inorganic nitrogen (C:N ratio = 28) followed by COD removal of 52% and PHB accumulation of 28% for filtered molasses containing inorganic nitrogen (C:N ratio = 29). PHB production yield (Y_p/s) was highest (0.184 g g^?1 COD consumed) for deproteinized spentwash supplemented with nitrogen. In contrast, the substrate consumption and product formation were higher in case of raw spentwash. Though COD removal was lowest from deproteinized spentwash, evaluation of kinetic parameters suggested higher rates of conversion of available carbon to biomass and PHB. Thus the process provided dual benefit of conversion of two wastes viz. waste activated sludge and molasses spentwash into value-added product-PHB.     

Ethanol production from banana peels using statistically optimized simultaneous saccharification and fermentation process

Dried and ground banana peel biomass (BP) after hydrothermal sterilization pretreatment was used for ethanol production using simultaneous saccharification and fermentation (SSF). Central composite design (CCD) was used to optimize concentrations of cellulase and pectinase, temperature and time for ethanol production from BP using SSF. Analysis of variance showed a high coefficient of determination (R²) value of 0.92 for ethanol production. On the basis of model graphs and numerical optimization, the validation was done in a laboratory batch fermenter with cellulase, pectinase, temperature and time of nine cellulase filter paper unit/gram cellulose (FPU/g-cellulose), 72 international units/gram pectin (IU/g-pectin), 37 °C and 15 h, respectively. The experiment using optimized parameters in batch fermenter not only resulted in higher ethanol concentration than the one predicted by the model equation, but also saved fermentation time. This study demonstrated that both hydrothermal pretreatment and SSF could be successfully carried out in a single vessel, and use of optimized process parameters helped achieve significant ethanol productivity, indicating commercial potential for the process. To the best of our knowledge, ethanol concentration and ethanol productivity of 28.2 g/l and 2.3 g/l/h, respectively from banana peels have not been reported to date.     

Enhanced production of poly(3-hydroxybutyrate) by filamentation-suppressed recombinantEscherichia coli in a defined medium

Fed-batch cultures of recombinantEscherichia coli strains were carried out for the production of poly(3-hydroxybutyric acid) (PHB) in a chemically defined medium. TheE. coli strains used were XL1-Blue, harboring pSYL105, a stable high-copy number plasmid containing theAlcaligenes eutrophus polyhydroxyalkanoate (PHA) genes, and XL1-Blue, harboring pSYL107, which is pSYL105 containing theE. coli ftsZ gene to suppress filamentation. With XL1-Blue(pSYL105) the final cell mass and PHB concentration obtained in 62 h were 102 and 22.5 g/L, respectively. Fed-batch culture of XL1-Blue(pSYL107) under identical conditions resulted in a final cell mass and PHB concentration of 127.5 and 48.2 g/L, respectively. The PHB contents obtained with XL1-Blue(pSYL105) and XL1-Blue(pSYL107) were 22.1 and 37.8%, respectively. Therefore, PHB was more efficiently produced in a defined medium by employing filamentation-suppressed recombinantE. coli.     

Purification and Properties of Extracellular Poly(3-hydroxybutyrate) Depolymerase Produced by <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> 202

An extracellular poly(3-hydroxybutyrate) (PHB) depolymerase produced by a thermotolerant fungal soil isolate, Aspergillus fumigatus 202, was purified and characterized. Maximum PHB depolymerase production was obtained at the end of 48 h with initial medium pH 7.0 and 45 °C in Bushnell Haas Minerals medium containing PHB as sole source of carbon. The PHB depolymerase was purified using size exclusion chromatography to a fold purification of 20.62 and 61.62% yield. SDS-PAGE and isoelectric focusing revealed the molecular weight and pI of the purified enzyme as 63,744 Da and 4.2, respectively. N-terminal amino acid sequence of purified enzyme was HAXDAYLVK. This non-glycosylated enzyme was most active at pH 9.0 and 45 °C. Purified enzyme was inactivated by N-bromosuccinimide and dithiothreitol suggesting the involvement of tryptophan residues and disulfide bonds at its active site. Nonionic detergents like Tween 20, Tween 80 and Triton X-100 inhibited the enzyme activity. Ions like Ca⁺² and Mg⁺² (5 mM) increased the enzyme activity 1.5 times. Fe⁺² effectively inhibited the enzyme activity to 88% whereas Hg⁺² completely inhibited the enzyme.     

Bioethanol from sea lettuce with the use of crude enzymes derived from waste

The mid-gut gland of the scallop contains saccharification enzymes such as cellulase and amylase, and these enzymes have been disposed of together with the mid-gut gland after the removal of the adductor muscle, an edible part of the scallop. We used a drip from the mid-gut gland of the scallop, obtained by squeezing the gland, as an inexpensive enzyme mixture and tried to produce bioethanol from the glucans present in sea lettuce by the method of simultaneous saccharification and fermentation (SSF) with the use of baker’s yeast. The ethanol concentration attained was as high as 7.2 g/L, which corresponded to ~37% of the conversion of glucans in sea lettuce in the solid-state SSF. Furthermore, we ascertained that the drip and sea lettuce contain nutrients that are indispensable for maintaining the yeast activity, and, thus, the SSF did not require any additional nutrients, such as yeast extract or peptone, the use of which increases the cost of fermentation to a high level.     

1,3-Propanediol Production Potential by a Locally Isolated Strain of Klebsiella Pneumoniae in Comparison to Clostridium Beijerinckii NRRL B593 from Waste Glycerol

The fermentative production of 1,3-propanediol (1,3-PDO) by Klebsiella pneumoniae under different initial substrate concentrations (between 5 and 110 g/L) was investigated. It was found that glycerol was almost 100% utilized and 1,3-PDO production increased up to 20 g/L of influent substrate concentration, but there was a significant decrease in both glycerol consumption and 1,3-PDO production at substrate concentrations exceeding 20 g/L. Furthermore, pH control was essential, and a lack of pH control negatively effects of 1,3-PDO production. In the second part of the study, two microorganisms, namely Clostridium beijerinckii NRRL B593 and K. pneumoniae were comparatively studied in terms of their 1,3-PDO productivity under pH controlled conditions. Higher 1,3-PDO production was achieved under pH controlled fermentation conditions (pH = 7) for both microorganisms. Even though the two microorganisms had almost the same 1,3-PDO yield (0.60 mol/mol for C. beijerinckii, 0.61 mol/mol for K. pneumoniae) at the end of fermentation period, K. pneumoniae completed the 1,3-PDO production in one-third of the time (t = 8 h with a productivity of 1.34 g/L/h) than C. beijerinckii (t = 24 h). The results of this study clearly indicated that a substrate inhibition is a challenge that needs to be studied further for higher productivities.     

Accumulation of Polyhydroxyalkanoates by Rhizobacteria Underneath Nickel-Hyperaccumulators from Serpentine Ecosystem

Nickel-resistant bacteria isolated from underneath Ni-hyperaccumulators growing on serpentine soils were screened for production of polyhydroxyalkanoates. These rhizobacteria accumulated poly-3-hydroxybutyric acid [P(3HB)] accounting 3.9–67.7% of cell dry weight during growth in gluconate and/or glucose. Cupriavidus pauculus KPS 201 utilized only gluconate and accumulated about 67.7% P(3HB) while, Bacillus firmus AND 408 utilized both carbon sources for polymer synthesis. The isolates being resistant to Ni also accumulated substantial amount of P(3HB) when grown in presence of the heavy metal and this was revealed by transmission electron microscopic studies. Although B. firmus AND 408 produced only P(3HB) at higher concentrations of gluconate, C. pauculus KPS 201 synthesized copolymer of 3-hydroxybutyric acid (3HB) and 3-hydroxyvaleric acid (3HV) [P(3HB-co-3HV)]. In presence of 0.8% gluconate and 4 mM Ni, KPS 201 cells produced PHA amounting 81% CDW, which contained 76 and 24 mol% 3HB and 3HV monomers, respectively.     

Purification and characterization of extracellular poly(3-hydroxybutyrate) depolymerases

Five extracellular PHB depolymerases of bacteria isolated from various sources were purified to electrophoretic homogeneity and compared with known extracellular PHB depolymerase fromAlcaligenes faecalis T1. The molecular mass of these enzymes were all around 40–50 kDa. Nonionic detergent, diisopropylfluorophosphate and dithiothreitol inhibited the PHB depolymerase activity of all these enzymes. Trypsin abolished PHB depolymerase activity, but not theD-3-hydroxybutyric acid dimer hydrolase activity of all the enzymes. These results showed that the basic properties of these PHB depolymerases resemble those of theA. faecalis T1 enzyme. Analysis ofN-terminal amino acid sequence of the purified enzymes revealed that these enzymes includingA. faecalis T1 enzyme fall into three groups.     

Fungal Degradation of the Bioplastic PHB (Poly-3-hydroxy- butyric acid)

PHB (poly-3-hydroxybutyric acid) is a thermoplastic polyester synthesized by Ralstonia eutropha and other bacteria as a form of intracellular carbon and energy storage and accumulated as inclusions in the cytoplasm of these bacteria. The degradation of PHB by fungi from samples collected from various environments was studied. PHB depolymerization was tested in vials containing a PHB-containing medium which were inoculated with isolates from the samples. The degradation activity was detected by the formation of a clear zone below and around the fungal colony. In total, 105 fungi were isolated from 15 natural habitats and 8 lichens, among which 41 strains showed PHB degradation. Most of these were deuteromycetes (fungi imperfecti) resembling species of Penicillium and Aspergillus and were isolated mostly from soils, compost, hay, and lichens. Soil-containing environments were the habitats from which the largest number of fungal PHB degraders were found. Other organisms involved in PHB degradation were observed. A total number of 31 bacterial strains out of 67 isolates showed clear zones on assay medium. Protozoa, possible PHB degraders, were also found in several samples such as pond, soil, hay, horse dung, and lichen. Lichen, a fungi and algae symbiosis, was an unexpected sample from which fungal and bacterial PHB degraders were isolated.     

Value addition of vegetable wastes by solid-state fermentation using Aspergillus niger for use in aquafeed industry

Vegetable waste typically has high moisture content and high levels of protein, vitamins and minerals. Its value as an agricultural feed can be enhanced through solid-state fermentation (SSF). Two experiments were conducted to evaluate the nutritional status of the products derived by SSF of a mixture of dried vegetable waste powder and oil cake mixture (soybean flour, wheat flour, groundnut oil cake and sesame oil cake at 4:3:2:1 ratio) using fungi Aspergillus niger S₁4, a mangrove isolate, and A. niger NCIM 616. Fermentation was carried out for 9 days at 35% moisture level and neutral pH. Significant (p < 0.05) increase in crude protein and amino acids were obtained in both the trials. The crude fat and crude fibre content showed significant reduction at the end of fermentation. Nitrogen free extract (NFE) showed a gradual decrease during the fermentation process. The results of the study suggest that the fermented product obtained on days 6 and 9 in case of A. niger S₁4 and A. niger NCIM 616 respectively contained the highest levels of crude protein.     

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.     

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.     

Appraisal of methane production and anaerobic fermentation kinetics of livestock manures using artificial neural networks and sinusoidal growth functions

This study aimed to perform a comparative analysis of the performance of five models (Gompertz, logistic, Richards, the first-order, artificial neural networks) in predicting methane production rate from anaerobic digestion of livestock manures. The input variables were fermentation time, digestion temperature, biogas temperature, ambient temperature, pH, and specific biogas production rate. The physicochemical compositions of cow manure and sheep manure showed that volatile solid (VS) contents were close to each other in manure compositions (77.6% and 64.7%, respectively), while the potential of methane production from cow manure (673.44 mL CH₄/g VS) was greater than that from sheep manure (320.32 mL CH₄/g VS). The determination coefficients (R²) for logistic function, Gompertz, Richards, the first-order, and ANN models were obtained as 0.968, 0.967, 0.975, 0.825, and 0.995 for the cow manure, respectively. In case of the sheep manure, the R² values obtained from these models were 0.976, 0.979, 0.981, 0.968 and 0.991, respectively. Although the determination coefficients of all models were in satisfactory agreement with the experimental data, the ANN model showed competitive lower RMSE values of 0.111 and 0.164 for cow and sheep manure data sets, respectively, indicating its superior performance than other models.

     

Effect of complex nitrogen source on the synthesis and accumulation of poly(3-hydroxybutyric acid) by recombinantEscherichia coli in flask and fed-batch cultures

When a recombinantEscherichia coli XL1-Blue harboring pSYL105 was cultured in a complex medium, a poly(3-hydroxybutyric acid) (PHB) concentration of 7.16 g/L was obtained in 48 h. However, a PHB concentration of only 0.91 g/L was obtained in 60 h by culturing in a defined medium. Also, fed-batch culture in a defined medium resulted in considerably lower PHB accumulation than in a complex medium. With the aim to produce a high concentration of PHB at a reduced medium cost, we examined 10 complex nitrogen sources for their ability to promote PHB synthesis in a defined medium. Tryptone, casamino acids, and casein hydrolysate promoted PHB synthesis to a higher extent than the others tested. PHB synthesis was also enhanced during fedbatch cultures when a defined medium was supplemented with various complex nitrogen sources. With tryptone supplementation a PHB concentration of 66.7 g/L could be obtained in 44 h. Yeast extract was less effective for promoting PHB synthesis than tryptone. Corn steep liquor, which did not enhance PHB synthesis significantly, could promote PHB synthesis considerably when supplemented together with yeast extract in both flask and fed-batch cultures.     

A Processing,Characterization and Marine Biodegradation Study of Melt-Extruded Polyhydroxyalkanoate (PHA) Films

A series of polyhydroxyalkanoates (PHA), all containing 1% nucleating agent but varying in structure, were melt-processed into films through single screw extrusion techniques. This series consisted of three polyhydroxybutyrate (PHB) and three polyhydroxybutyrate-valerate (PHBV) resins with varying valerate content. Processing parameters of temperature in the barrel (165–173 °C) and chill rolls (60 °C) were optimized to obtain cast films. The gel-permeation chromatography (GPC) results showed a loss of 8–19% of the polymer’s initial molecular weight due to extrusion processing. Modulated differential scanning calorimetry (MDSC) displayed glass transition temperatures of the films ranging from −4.6 to 6.7 °C depending on the amount of crystallinity in the film. DSC data were also used to calculate the percent crystallinity of each sample and slightly higher crystallinity was observed in the PHBV series of samples. X-ray diffraction patterns did not vary significantly for any of the samples and crystallinity was confirmed with X-ray data. Dynamic mechanical analysis (DMA) verified the glass transition trends for the films from DSC while loss modulus (E′) reported at 20 °C showed that the PHBV (3,950–3,600 MPa) had the higher E′ values than the PHB (3,500–2,698 MPa) samples. The Young’s modulus values of the PHB and PHBV samples ranged from 700 to 900 MPa and 900 to 1,500 MPa, respectively. Polarized light microscopy images revealed gel particles in the films processed through single-screw extrusion, which may have caused diminished Young’s modulus and tensile strength of these films. The PHBV film samples exhibited the greatest barrier properties to oxygen and water vapor when compared to the PHB film samples. The average oxygen transmission rate (OTR) and water vapor transmission rate (WVTR) for the PHBV samples was 247 (cc-mil/m²-day) and 118 (g-mil/m²-day), respectively; while the average OTR and WVTR for the PHB samples was 350 (cc-mil/m²-day) and 178 (g-mil/m²-day), respectively. Biodegradation data of the films in the marine environment demonstrated that all PHA film samples achieved a minimum of 70% mineralization in 40 days when run in accordance with ASTM 6691. For static and dynamic incubation experiments in seawater, microbial action resulting in weight loss as a function of time showed all samples to be highly biodegradable and correlated with the ASTM 6691 biodegradation data.     

A Novel Affinity Chromatographic Material for the Purification of Extracellular Polyhydroxybutyrate Depolymerases

A novel affinity chromatographic material, which is composed of silica matrix, coated with polyhydroxybutyrate (PHB) powder, suitable for the purification of PHB depolymerases, was developed. The surface morphology of the PHB-silica coated particles (silica-PHB composite particles) was examined by scanning electron microscopy and revealed a successful uniform coating of silica particles with PHB. Moreover, the complex of these materials retained its homogeneity even after incubation at 80 °C for 6 h, whereas the strong binding of PHB on silica surface was further verified by thermal gravimetric analysis and by PHB extraction- from silica surface- experiments. This novel material was demonstrated to be suitable for both, the one-step on-batch and on-column purification of Thermus thermophilus extracellular PHB depolymerase. The enzyme exhibited higher affinity against the composite of silica-PHB particles than PHB powder, since the one-step purification-fold and the overall recovery of the enzyme were 2.8 and 4 times higher respectively, in the first case. Reusability of the silica-PHB composites particles was examined by determining the recoveries of PHB depolymerase. The enzyme recoveries were ranged from 30 to 35% for the first five uses, whereas for further uses recoveries gradually dropped to 15–18% indicating that the particles could be used repeatedly for five times. This material could be also a suitable support for lipases or other proteins that exhibit strong affinity to hydrophobic materials.     

Evaluation of buckwheat and barley tea wastes as ethanol fermentation substrates

Buckwheat tea waste (BWTW) and barley tea waste (BTW), by-products of the beverage industry, are alternative carbohydrate sources for ethanol production. In this study, optimal enzyme loading for enzymatic saccharification of BWTW and BTW was determined, and simultaneous saccharification and fermentation (SSF) was performed by Saccharomyces cerevisiae and Mucor indicus to produce ethanol. Optimal enzyme loading for enzymatic saccharification of 2?% w/v BWTW and BTW was 0.5?% (weight of enzyme/weight of tea wastes) for BWTW and 0.1?% for BTW. Ethanol production from BWTW by S. cerevisiae and M. indicus after 48?h of SSF was 49.9/100?g of BWTW and 47.9/100?g of BWTW, respectively, with 0.5?% enzyme loading. Ethanol production from BTW by S. cerevisiae and M. indicus after 48?h of SSF was 20.5/100?g of BTW and 21.6/100?g of BTW, corresponding to 62 and 66?% of the theoretical yield based on starch content, respectively, with 0.1?% of enzyme loading. Furthermore, S. cerevisiae produced 76?% of the theoretical yield based on the total glucose from starch in BWTW and BTW when a mixture of BWTW and BTW was used as a substrate, with 0.2?% enzyme loading and no additional nitrogen or mineral sources.     

Poly[(R)-3-hydroxy butyrate] Melt Processing: Strategy for Prevention of Degradation Reactions

Poly[(R)-3-hydroxy butyrate] (PHB) as well as all the components of the poly[(R)-3-hydroxy alkanoate]s (PHAs) family tend to degrade during processing at temperatures above their melting point via a hydrolytic mechanism induced by moisture attack and a concerted reaction mechanism induced by temperature. Therefore, the PHAs stabilization in the molten state is particularly important for production of biodegradable ecocompatible plastic items of commercial value. In order to refrain the indicated negative degradation effects, PHB was melt processed in a torque rheometer in the presence of a polymeric carbodiimide agent. Processed specimens were characterized by using FTIR, GPC, DSC, TGA and tensile properties. GPC analysis showed that the increase of the additive content in the formulation resulted in an increase of PHB molecular weight. However, decreasing in PHB thermal stability, glass transition temperature and mechanical properties, was observed with the increase of the additive amount. This behaviour most likely arises from the formation of new chemical structures promoted by the presence of the additive aimed at preventing the onset of hydrolitic processes.     

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.