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.     

Biochemical and Genetic Characterization of an Extracellular Poly(3-Hydroxybutyrate) Depolymerase from Acidovorax Sp. Strain TP4

To determine the properties of enzymes from bacteria that degrade polypropiolactone (PPL), we isolated 13 PPL-degrading bacteria from pond water, river water, and soil. Nine of these strains were identified as Acidovorax sp., three as Variovorax paradoxus, and one as Sphingomonas paucimobilis. All the isolates also degraded poly(3-hydroxybutyrate) (PHB). A PPL-degrading enzyme was purified to electrophoretical homogeneity from one of these bacteria, designated Acidovorax sp. TP4. The purified enzyme also degraded PHB. The molecular weight of the enzyme was estimated as about 50,000. The enzyme activity was inhibited by diisopropylfluorophosphate, dithiothreitol, and Triton X-100. The structural gene of the depolymerase was cloned in Escherichia coli. The nucleotide sequence of the cloned DNA fragment contained an open reading frame (1476 bp) specifying a protein with a deduced molecular weight of 50,961 (491 amino acids). The deduced overall sequence was very similar to that of a PHB depolymerase of Comamonas acidovorans YM1609. From these results it was concluded that the isolated PPL-degrading enzyme belongs to the class of PHB depolymerases. A conserved amino acid sequence, Gly-X₁-Ser-X₂-Gly (lipase box), was found at the N-terminal side of the amino acid sequence. Site-directed mutagenesis of the TP4 enzyme confirmed that ²⁰Ser in the lipase box was essential for the enzyme activity. This is the first report of the isolation a PHB depolymerase from Acidovorax.     

Natural and Artificial Functionalized Biopolyesters I. Chemoenzymatic Mediated Synthesis of Chiral Precursors

Design of optically active polymeric materials for temporary therapeutic and environmental applications requires the working-out of functionalized polymers with structures and properties adjusted to the considered applications. Biocompatibility of selected polymers is a required property in regard to the interactions between living organisms and macromolecular systems. It is therefore important to prepare highly optically active monomers and their corresponding hydrolyzable and biocompatible polymers. Bacterium Clostridium tetanomorphum is an useful source of enzymes for bioconversion and particularly in the chemoenzyrnatic route to optically active alkylmalolactonic acid esters and their optically pure stereoisomers. 3-Methylaspartase, involved in the glutamate fermentation pathway, is a very interesting enzyme, which can provide chiral precursors with high optically purity. This chemoenzymatic strategy can be used to prepare the four stereoisomers of 3-methylmalolaconic acid esters, by enzymatic resolution of natural and artificial stereoisomers of 3-methylaspartic acid. A series of 3-alkymalolactonic acid esters with alkyl equal to ethyl or isopropyl, have been also synthesized and transformed into corresponding optically active polyesters.     

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.     

Picocyanobacteria – Sensitive Bioindicators of Contaminant Stress in an Alpine Lake (Traunsee Austria)

The abundance and photosynthetic activity ofpicocyanobacteria in the oligotrophic alpine lake Traunseewere measured at a station located close to the outlet ofindustrial soda waste and at a mid-lake reference stationduring spring, 1999 through to autumn, 2000.Picocyanobacterial numbers measured by flow cytometry inTraunsee (0.7–13.2 × 10⁴ ml^-1) were comparable tothose of other oligotrophic lakes, and there was nosignificant difference between the contaminated and thereference sampling location. Picoplankton (<2 m)photosynthetic rates measured in vitro by the ¹⁴C-technique were significantly reduced at the contaminated siterelative to the reference station at low photosyntheticallyavailable radiation (10 E m^-2 s^-1), while nodifference between these two stations was found at moderatelyhigh light intensity (100 E m^-2 s^-1). Theinvestigation was complemented by laboratory experiments withcultured picocyanobacteria. Three Synechococcus spp.strains were exposed to water taken from either of the twoTraunsee stations and from a control station located inneighbouring Attersee. Cell-specific photosynthetic activitymeasured by 4-h in vitro incubations revealed no significantdifference among the three stations investigated. Growthrates of the same three Synechococcus spp. strains weremeasured by flow cytometry over several days in thelaboratory. One strain, in particular, was sensitive to watertaken from the contaminated site; growth rate of this strainwas significantly reduced, relative to when exposed to watertaken from the reference station. Taken together, our resultsdemonstrate that picocyanobacteria are highly sensitivebioindicators of contaminant stress. The overall impact ofthe emissions from the industrial outlet on thepicocyanobacteria was, however, relatively minor.     

Accumulation of Heavy Metals by in vitro cultures of plants

The aim of the project is to study heavy metals accumulation by the selected plants in both laboratory and field conditions. Within the experiments the aspen (Populus tremula × tremuloides), sunflower (Helianthus annuus) and corn (Zea mays) plants were studied. The reasons for this selection were: a fast growth of these plants, an accumulation capacity and an ability to survive in different types of soils. The study was carried out on the aspen plantlets grown in vitro. The plants were exposed to the aqueous solutions having concentrations 0.1 mM, 0.5 mM of Pb²⁺ or Ni²⁺, respectively. The accumulation capacityfor aspen, was about 70% of Pb²⁺ originally present in the solution. The starting concentration of Pb²⁺ (0.5 mM) exhibited no negative impact on the growth. Besides in vitro expositions, a pilot-scale phytoremediation experiment was carried out at the polluted industrial area (Zn – 75000 mg/kg), (Pb – 16000 mg/kg), (Cr – 590 mg/kg), (Cd – 90 mg/kg) and (Cu – 1700 mg/kg).     

Sorption of strontium on bentonite

Sorption of Sr on bentonite was studied using the batch technique. Distribution coefficients (K_d) were determined as a function of contact time, pH, sorbent and sorbate concentration and temperature. The data were interpreted in terms of Freundlich, Langmuir and Dubinin-Radushkevich isotherms. Thermodynamic parameters for the sorption system were determined at three different temperatures. The positive value of the heat of sorption, ΔH° = 30.62 kJ/mol at 298 K, shows that the sorption of strontium on bentonite is endothermic. The negative value of the free energy of sorption, ΔG° = −10.69 kJ/mol at 298 K, shows the spontaneity of the reaction. ΔG° becomes more negative with increasing temperature, which shows that the sorption process is more favorable at higher temperatures. The mean free energy for sorption, E 9 kJ/mol, suggests that ion exchange is the predominant mode of sorption in the Sr concentration range studied, i.e. 0.01 – 0.3 mol/dm³. The presence of complementary cations depresses the sorption of strontium on bentonite in the order Ca²⁺>Mg²⁺>K⁺>Na⁺. Some organic complexing agents and natural ligands also affect the sorption of strontium. The desorption studies with ground water at low strontium loadings on bentonite show that about 90% of Sr is irreversibly sorbed on the bentonite.     

Bacterial degradation of a new polyester,polyethylene glycol-phthalate polyester

Microorganisms which can assimilate a new polyester synthesized from polyethylene glycol (PEG) as a dihydroxyl compound and phthalic acid as a dicarboxyl compound were isolated from soils by enrichment culture techniques. Two cultures, K and N, were obtained: Culture K grew on PEG 4000 polyester and culture N assimilated PEG 6000 polyester. Each culture included two bacteria indispensable for the degradation of polyesters: bacteria K1 and K2 for PEG 4000 polyester-utilizing culture K and bacteria N1 and N2 for PEG 6000 polyester-utilizing culture N. Bacteria K2 and N2 were responsible for the hydrolysis of ester bonds in a polyester and both were identified as the same species,Comamonas acidovorans. Bacteria K1 and N6 could assimilate PEG as a sole carbon and energy source. Both are Gram-negative, non-spore-forming rods and resembled each other on their colony characteristics, although strain K1 could not grow on PEG 6000.C. acidovorans N2 (K2) grew on dialkyl phthalates (C₂–C₄) and phthalate and tributyrin, but not on PEG, diphthalic PEG, and PEG phthalate polyesters. Their culture supernatant and washed cells hydrolyzed PEG (400–20,000) phthalate and sebacate polyesters.C. acidovorans had higher esterase activity toward PEG phthalate, isophthalate, and terephthalate polyesters than known esterase and lipases. The esterase seemed to be an extracellular one and attached to the cell surface.     

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.     

Biodegradable Polymers from 5-Hydroxylevulinic Acid: 1. Synthesis and Characterization of Poly(5-hydroxylevulinic acid)

As an attempt to synthesize new biodegradable polymers from renewable cellulose resources, melt polycondensation of 5-hydroxylevulinic acid (5-HLA) was reported for the first time. The resulting product, poly(5-hydroxylevulinic acid) (PHLA), was synthesized and characterized with GPC, FTIR, ¹H NMR and DSC. The in vitro degradation behaviors in phosphate-buffered saline (PBS) and in deionized water (DW) were also examined. The molecular weight of PHLA is not high (several 1,000s), but it possesses unordinary high glass transition temperature (as high as 120 °C). This is very different from existing aliphatic polyesters that usually have T _gs lower than 60 °C. The high T _g is attributed to the formation of inter- and/or intramolecular hydrogen bonds due to a characteristic keto–enol tautomerism equilibrium in the polymer structure. PHLA readily degraded hydrolytically in aqueous media.     

Uptake of chromium by Aspergillus foetidus

Aspergillus foetidus has the ability to take up chromium during the stationary phase of growth and under growth-nonsupportive conditions. We observed a 97% decrease in hexavalent chromium (initial concentration 5 µg/g) at the end of 92 h of growth, which may be due to its reduction to Cr (III) and/or complexation with organic compounds released due to the metabolic activity of the fungus. Replacement culture studies under growth-nonsupportive conditions revealed that the maximum uptake of Cr (VI) at pH 7.0 is 2 mg/g of dry biomass. At low or high pH values, Cr (VI) uptake is significantly reduced. In addition, the initial rate of total chromium uptake is also enhanced by higher biomass concentrations and the presence of glucose. The results obtained through this investigation indicate the possibility of treating waste effluents containing hexavalent chromium using Aspergillus foetidus.     

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.     

Landfill Leachate Ecotoxicity Experiments using Lemna minor

The effects of varying concentrations of landfill leachate on the growth, frond area, chlorophyll content and fluorescence of four strains of Lemna minor were assessed. Growth fluorescence and frond chlorophyll content decreased after seven days exposure to leachate, although responses differed between the strains and end parameters. A L. minor bioassay was used to assess leachate toxicity and the effectiveness of a constructed wetland treatment system and pre-treatment aeration and settlement in reducing toxicity. Pre-treatments were found to significantly reduce toxicity, so their incorporation in any treatment system may increase pollutant stripping.     

Effects of Nitrogen Supply on the Sensitivity to O3 of Growth and Photosynthesis of Japanese Beech (Fagus crenata) Seedlings

To obtain basic information for evaluating critical levels of O₃ under different nitrogen loads for protecting Japanese beech forests, two-year-old seedlings of Fagus crenata Blume were grown in potted andisol supplied with N as NH₄NO₃ solution at 0, 20 or 50 kg ha⁻¹ year⁻¹ and exposed to charcoal-filtered air or O₃ at 1.0, 1.5 and 2.0 times the ambient concentration from 16 April to 22 September 2004. The O₃ induced significant reductions in the whole-plant dry mass, net photosynthetic rate at 380 μmol mol⁻¹ CO₂ (A ₃₈₀), carboxylation efficiency (CE) and concentrations of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and total soluble protein (TSP) in the leaves. The concentrations of Rubisco and TSP were negatively correlated with the concentration of leaf acidic amino acid, suggesting that O₃ enhanced the degradation of protein such as Rubisco. The N supply to the soil did not significantly change the whole-plant dry mass and A ₃₈₀, whereas it significantly increased the CE and concentrations of Rubisco and total amino acid. No significant interactive effects of O₃ and N supply to the soil were detected on the growth, photosynthetic parameters and concentrations of protein and amino acid in the leaves. In conclusion, N supply to the soil at ≤50 kg ha⁻¹ year⁻¹ does not significantly change the sensitivity to O₃ of growth and net photosynthesis of Fagus crenata seedlings.     

Properties and biodegradability of chain-extended copoly(succinic anhydride/ethylene oxide)

Chain-extension reactions were carried out using titanium-iso-propoxide (TIP) as a catalyst for a series of polyesters or copolyesterethers with low molecular weights (M _n =1500–10,000) synthesized by the ring-opening copolymerization of succinic anhydride (SA) with ethylene oxide (EO). The copolymers having aM _n from 25,000 to 50,000 of different properties were obtained. Both the melting point (T _m ) and the fusion heat (H), which indicate the crystallinity of the copolymers, rose with an increase in SA content in the copolymers. Semitransparent films were prepared by compression molding of the copolymers. The biodegradation of the copolymer films was evaluated by enzymatic hydrolysis by lipases and by an aerobic gas evolution test in standard activated sludge. The hydrolyzability of these copolymers by three kinds of lipases was affected by their copolymer composition SA/EO, form, andM _n . The copolyesterether (SA/EO=43/57,M _n =48,900) was more easily biodegraded by standard activated sludge compared to the polyester (SA/EO=47/53,M _n =36,300).Presented at the Pacifichem-95, December 17–22, 1995, Honolulu, Hawaii.     

Production of Polyhydroxyalkanoates from Fatty Wastes

The production of polyesters from triglyceride containing substrates was investigated. A first filter step based on lipase activity was followed and those bacteria potentially able to degrade oils or animal fats were further tested for their polymer accumulation properties, selected and kept for further studies. In a second step, bacteria were directly grown on animal fats and/or vegetable oils, and polyhydroxyalkanoates (PHAs) accumulation was verified under appropriate incubation conditions. Each substrate, whether of animal or vegetable derivation, supported the growth of a number of the newly isolated strains and among those, some strains were also found to produce reasonably high amounts of PHA. The repeat-unit composition of the polyesters was determined by gas chromatography (GC) analysis of the ?-hydroxyalkanoate methyl esters from the hydrolyzed polymer and some class of co-polymers were also detected. These properties, coupled with the ability of some of the selected isolates to grow and produce lipases on a minimal medium, could be considered as promising in view of possible industrial applications. The overall results indicate that PHAs could be produced from waste containing considerable amounts of fat, oil and grease (FOG), that generally need to be treated for their disposal.     

Miscibility and Biodegradability of Poly(Butylene Succinate)/Poly(Butylene Terephthalate) Blends

As one of the biodegradable polymers, the blend of poly(butylene succinate) and poly(butylene terephthalate) is dealt with in this study. In our previous work, it was demonstrated that PBS and PBT are immiscible not only from the changes of T _g but also from logG–log G plots. It is expected that the biodegradability of the blends could be improved by enhancing the miscibility. We tried to induce the transesterification reaction between two polyesters with various time intervals to enhance the miscibility of the blends. The extent of transesterification reaction was examined by ¹H-NMR. We utilized a dynamic mechanical thermal analyzer and a rotational rheometer to investigate the changes in miscibility. We also verified the biodegradability of PBS/PBT blends after the transesterification reaction by the composting method.     

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.     

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.     

Distribution of Cd,Ck, Pb and Zn in Soil and Vegetation Compartments in Stands of Five Boreal Tree Species in N.E. Sweden

Concentrations and total quantity of cadmium (Cd), cupper (Cu),lead (Pb) and zink (Zn) were determined in biomass and soil compartments in a replicated tree species experiment with 27-yr-old stands growing on former farmland in N.E. Sweden. Sequentialextractions of soil samples were performed in order to estimate the exchangeable and an organically bound fraction of each element. The tree species included were Picea abies (L.)H. Karst., Pinus sylvestris L., Pinus contorta Dougl., Larix sibirica Ledeb., and Betula pendula Roth.Tree species influenced the rate of removal of Cu, Pb and Zn incase of stemwood harvesting, and of Cd, Cu and Zn in the case ofwhole-tree harvesting. B. pendula and P. abies had higher quantities and average concentrations of Zn in the biomass. For all species, >50% of the Zn in the stems was found in the bark. P. abies and L. sibirica had higher quantities of Cu in the biomass than the other species.P. abies and P. contorta had high quantities of Cd inthe biomass in relation to the other species. Branches and stembark contained high concentrations of Cd and Pb in relation to foliage and stemwood. Dead branches had especially high concentrations of Pb. The high accumulation rate of Zn in thebiomass of B. pendula was related to a low exchangeable amount of Zn in the A horizon. In the superficial centimeters ofthe A horizon, a depletion similar to that found for Zn was detected for Cu, whereas for Cd and Pb, no correlations were found between quantities of elements in the trees and element pools in the soil.