Degradation of polyhydroxyalkanoates and the composition of microbial destructors under natural conditions

The degradation dynamics of polyhydroxyalkanoates of different composition has been studied in an eutrophic storage reservoir for two seasons. It has been shown that the biodegradation of polymers under natural conditions depends not only on their structure and physicochemical properties but also, to a great extent, on a complex of weather-climatic conditions affecting the state of the reservoir ecosystem. The molecular genetic analysis of 16S rRNA has revealed bacterial species (clones) probably involved in the degradation of polyhydroxyalkanoates in a model storage reservoir.     

Experimental assessment of combined effect of nitrates and acute gamma-irradiation on green algae Scenedesmus quadricauda growth

The combined effect of acute gamma-irradiation at doses of 0, 50, 100, 150 and 200 Gy and nitrates in concentrations of 0.04 g/dm3 (that corresponds to maximum permissible concentrations for fishery waters), 0.1, 0.25, 0.5, 1.0, 2.5 g/dm3 (that is close to NO3(-) level in water of a reservoir R-17 used as radioactive waste storage of the "Mayak" Production Association) and 5.0 g/dm3 (that is close to NO3(-) level in the water of radioactive waste storage reservoir R-9) on the unicellular green algae Scenedesmus quadricauda growth has been studied in laboratory conditions. It was shown that the joint effects of nitrates and y-radiation had an antagonistic character. Thus, it may be concluded that chemical pollution is the factor limiting the development of green algae in reservoir R-17; probably, both factors, chemical and radiating, are essential to the algocenosis degradation in reservoir R-9.     

Fungal degradation of polyhydroxyalkanoates and a semiquantitative assay for screening their degradation by terrestrial fungi.

The current problems with decreasing fossile resources and increasing environmental pollution by petrochemical-based plastics have stimulated investigations to find biosynthetic materials which are also biodegradable. Bacterial reserve materials such as polyhydroxyalkanoates (PHA) have been discovered to possess thermoplastic properties and can be synthesized from renewable resources. Poly-beta-hydroxybutyric acid (PHB) is at present the most promising PHA; and BIOPOL, its copolymer with poly-beta-hydroxy-valerate (PHV), is already industrially produced (ICI, UK), and used as packaging material (WELLA, FRG). According to the literature, PHA degradation has so far mainly been observed in bacteria; only under certain environmental conditions has fungal degradation of PHAs been indicated. Since fungi constitute an important part of microbial populations participating in degradation processes, a simple screening method for fungal degradation of BIOPOL, a PHA-based plastic, was developed. Several media with about 150 fungal strains from different terrestrial environments and belonging to different systematic and ecological groups were used. PHA depolymerization was tested on three PHB-based media, each with 0.1% BIOPOL or PHB homopolymer causing turbidity of the medium. The media contained either a comparatively low or high content of organic carbon (beside PHA) or were based on mineral medium with PHA as the principal source of carbon. The degradation activity was detectable due to formation of a clear halo around the colony (Petri plates) or a clear zone under the colony (test tubes).(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of growth stage on activities of polyhydroxyalkanoate (PHA) polymerase and PHA depolymerase in <Emphasis Type="Italic">Pseudomonas putida</Emphasis> U

Background  

Medium chain length (mcl-) polyhydroxyalkanoates (PHA) are synthesized by many bacteria in the cytoplasm as storage compounds for energy and carbon. The key enzymes for PHA metabolism are PHA polymerase (PhaC) and depolymerase (PhaZ). Little is known of how mcl-PHA accumulation and degradation are controlled. It has been suggested that overall PHA metabolism is regulated by the β-oxidation pathway of which the flux is governed by intracellular ratios of [NADH]/[NAD] and [acetyl-CoA]/[CoA]. Another level of control could relate to modulation of the activities of PhaC and PhaZ. In order to investigate the latter, assays for in vitro activity measurements of PhaC and PhaZ in crude cell extracts are necessary.     

Biodegradation of polyhydroxyalkanoates by soil microbiocoenoses of different structures and detection of microorganisms-destructors

Biodegradation of microbial linear polymers of hydroxyalkanoic acids (polyhydroxyalkanoates, PHAs) by soil microbiocoenoses of different structures has been studied during two field seasons in different weather conditions. This process was shown to be influenced by the polymer chemical composition, temperature, humidity, and the microbial soil component. The PHA degradation was accompanied by a decrease in the polymer molecular weight and an increase in the degree of crystallinity, indicating the preferential destruction of the amorphous phase compared to the crystalline one. The quantity of the true PHA destructors developing at the surface of the polymer samples was lower than the quantity of accompanying bacteria. The dominant PHA destructors under the test conditions were identified as bacteria of the genera Variovorax, Stenotrophomonas, Acinetobacter, Pseudomonas, Bacillus, and Xanthomonas and as micromycetes from Penicillium, Paecilomyces, Acremonium, Verticillium. and Zygosporium.     

Microbial degradation of polyhydroxyalkanoates in tropical soils

The integrated study addressing biodegradation of microbial linear polyesters of hydroxyalkanoic acids (polyhydroxyalkanoates, PHAs) in tropical conditions by microbial communities of Vietnamese soils was performed in locations close to Hanoi and Nha Trang, which differed in their weather conditions and microbial communities. It shows that PHA degradation in tropical soils is influenced by polymer chemical composition, specimen shape, and microbial characteristics. The homopolymer of 3-hydroxybutyric acid is degraded at higher rates than the copolymer of 3-hydroxybutyric and 3-hydroxyvaleric acids. The average rates of mass loss were 0.04–0.33% per day for films and 0.02–0.18% for compact pellets. PHA degradation was accompanied by a decrease in the polymer molecular mass and, usually, an increase in the degree of crystallinity, suggesting preferential degradation of the amorphous phase. Under the study conditions, representatives of the bacterial genera Burkholderia, Bacillus, Cupriavidus, Mycobacterium, and Nocardiopsis and such micromycetes as Acremonium, Gongronella, Paecilomyces, and Penicillium, Trichoderma have been identified as major PHA degraders.     

Genome sequence of the polyhydroxybutyrate producer Pseudomonas extremaustralis, a highly stress-resistant Antarctic bacterium

Pseudomonas extremaustralis 14-3b presents genes involved in the synthesis of different polyhydroxyalkanoates, in tolerance and degradation of pollutants, and in microaerobic metabolism. Several genomic islands were detected. Genetic machinery could contribute to the adaptability to stressful conditions. This is the first genome sequence reported from a Pseudomonas isolated from cold environments.     

Glycogenformation by Rhodococcus species and the effect of inhibition of lipid biosynthesis on glycogen accumulation in Rhodococcus opacus PD630

Members of the genus Rhodococcus were investigated for their ability to produce glycogen during cultivation on gluconate or glucose. Strains belonging to Rhodococcus ruber, Rhodococcus opacus, Rhodococcus fascians, Rhodococcus erythropolis and Rhodococcus equi were able to produce glycogen up to 0.2–5.6% of cellular dry weight (CDW). The glycogen content varied from 0.8% to 3.2% of CDW in cells of R. opacus PD630, which is a well-known oleaginous bacterium, during the exponential growth phase, when cultivated on diverse carbon sources. Maltose and pyruvate promoted glycogen accumulation by cells of strain PD630 to a greater extent than glucose, gluconate, lactose, sucrose or acetate. This strain was able to produce triacylglycerols, polyhydroxyalkanoates and glycogen as storage compounds during growth on gluconate, although triacylglycerols were always the main product under the conditions of this study. Cerulenin, an inhibitor of de novo fatty acid synthesis, inhibited the accumulation of triacylglycerols from gluconate and increased the content of polyhydroxyalkanoates (from 2.0% to 4.2%, CDW) and glycogen (from 0.1% to 3.0%, CDW). An increase of the polyhydroxyalkanoates and glycogen content was also observed in two mutants of R. opacus PD630, which produced reduced amounts of triacylglycerols during cultivation of cells on gluconate.     

Degradation of proteins upon storage at near-neutral pH: Indications of a proteolytic/gelatinolytic activity associated with aggregates

Background

The twin phenomena of aggregation and degradation are classically associated with protein storage. However, although aggregation has been thought to be a possible consequence of protein degradation, it has never before been proposed to be a cause of degradation.

Methods

Proteins stored under physiological conditions and electrophoresed on SDS-PAGE were examined zymographically for the presence of detergent-resistant high molecular weight (HMW) forms, and association of such HMW forms with time-correlated, seeding-dependent gelatinolytic activity, under various conditions.

Results

Eight different proteins aggregate naturally during storage at near-neutral pH, with concomitant development of ‘gelatinolytic’ activity diminished greatly by storage at low temperatures, extremes of pH, arginine, imidazole, BSA, azide, EDTA, DTT, PMSF (but not AEBSF), and diisopropyl fluorophosphate (DFP), suggesting involvement of surface serine residues in a novel aggregate-borne proteolytic activity.

Conclusions

Naturally-formed aggregates of proteins appear to use surface serines to perform peptide bond hydrolysis, explaining degradation of proteins during storage, and indicating why aggregates are cytotoxic.

General significance

The study suggests that a bi-directional cause–effect relationship operates between protein aggregation, and protein degradation, providing clues to the design of better conditions for long-term protein storage.     

Polyhydroxyalkanoate (PHA)/inorganic phase composites for tissue engineering applications

Polyhydroxyalkanoates are emerging as a class of biodegradable polymers for applications in tissue engineering. Members of the polyhydroxyalkanoates family encompass a wide variety of materials, from hard and brittle materials to soft and elastomeric. Over the years, efforts have been made to extend the group of polyhydroxyalkanoates and to investigate their use in numerous biomedical applications, such as sutures, cardiovascular patches, wound dressings, guided tissue repair/regeneration devices, and tissue engineering scaffolds. Along with the development of polyhydroxyalkanoates, researchers have looked into the possibility of designing composites in combination with inorganic phases to further improve the mechanical properties, rate of degradation, and also impart bioactivity. Poly(3-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) are some of the polymers which have been studied extensively to fabricate composites in combination with hydroxyapatite, bioactive glass, and glass-ceramic fillers or coatings. This paper reviews international research carried out toward development of polyhydroxyalkanoates/inorganic phase composites in terms of systems investigated, microstructures, properties achieved, and applications, with special focus on tissue engineering scaffolds. A comparison between different composite systems developed in the past few years is presented. The paper also addresses the prospect of potential further development of polyhydroxyalkanoates/inorganic phase composites with optimized microstructure and properties for improved tissue engineering scaffolds.     

聚羟基脂肪酸酯的应用展望

聚羟基脂肪酸酯(Polyhydroxyalkanoates,简称PHA)是由微生物合成的天然高分子基材料,作为微生物碳源和能源的储备物质。目前,PHA的单体种类有150多种,致使PHA的品种繁多、材料学性质各不相同。PHA具有材料多变性、非线性光学性能、压电性能、气体阻隔性能、热塑性、生物可降解性、良好的生物相容性等特点,使其在塑料包装、化工、医药、农业、生物能源等诸多领域的具有很大的应用前景。文中系统介绍了目前PHA的应用和未来的发展。     

Methods for detection and visualization of intracellular polymers stored by polyphosphate-accumulating microorganisms

Polyphosphate-accumulating microorganisms (PAOs) are important in enhanced biological phosphorus (P) removal. Considerable effort has been devoted to understanding the biochemical nature of enhanced biological phosphorus removal (EBPR) and it has been shown that intracellular polymer storage plays an important role in PAO's metabolism. The storage capacity of PAOs gives them a competitive advantage over other microorganisms present that are not able to accumulate internal reserves. Intracellular polymers stored by PAOs include polyphosphate (poly-P), polyhydroxyalkanoates (PHAs) and glycogen. Staining procedures for qualitative visualization of polymers by optical microscopy and combinations of these procedures with molecular tools for in situ identification are described here. The strengths and weaknesses of widely used polymer quantification methods that require destruction of samples, are also discussed. Finally, the potential of in vivo nuclear magnetic resonance (NMR) spectroscopy for on-line measurement of intracellular reserves is reported.     

The production of polyhydroxyalkanoates in recombinant Escherichia coli

Polyhydroxyalkanoates, the natural polyester that many microorganisms accumulate to store carbon and reducing equivalents, have been considered as a future alternative of traditional plastic due to their special properties. In Escherichia coli, a previous non-polyhydroxyalkanoates producer, pathway engineering has been developed as a very powerful approach to set up microbial production process through the introduction of direct genetic changes by recombinant DNA technology. Various metabolic pathways leading to the polyhydroxyalkanoates accumulation with desirable properties at low-cost and high-productivity have been developed. At the same time, high density fermentation technology of E. coli provides an efficient polyhydroxyalkanoates production strategy. This review focused on metabolic engineering, fermentation and downstream process aiming to polyhydroxyalkanoates production in E. coli.     

Biodegradation of polyhydroxyalkanoates by soil microbial communities of different structures and detection of PHA degrading microorganisms

Biodegradation of microbial linear polymers of hydroxyalkanoic acids (polyhydroxyalkanoates, PHAs) by soil microbial communities of different structures has been studied during two field seasons in different weather conditions. This process was shown to be influenced by the polymer chemical composition, temperature, humidity, and the microbial soil component. The PHA degradation was accompanied by a decrease in the polymer molecular weight and an increase in the degree of crystallinity, indicating the preferential destruction of the amorphous phase compared to the crystalline one. The quantity of the true PHA destructors developing at the surface of the polymer samples was lower than the quantity of accompanying bacteria. The dominant PHA degrading microorganisms under the test conditions were identified as bacteria of the genera Variovorax, Stenotrophomonas, Acinetobacter, Pseudomonas, Bacillus, and Xanthomonas and as micromycetes from Penicillium, Paecilomyces, Acremonium, Verticillium, and Zygosporium.     

Occurrence, production, and export of lipophilic compounds by hydrocarbonoclastic marine bacteria and their potential use to produce bulk chemicals from hydrocarbons

Petroleum (or crude oil) is a complex mixture of hydrocarbons. Annually, millions of tons of crude petroleum oil enter the marine environment from either natural or anthropogenic sources. Hydrocarbon-degrading bacteria (HDB) are able to assimilate and metabolize hydrocarbons present in petroleum. Crude oil pollution constitutes a temporary condition of carbon excess coupled to a limited availability of nitrogen that prompts marine oil-degrading bacteria to accumulate storage compounds. Storage lipid compounds such as polyhydroxyalkanoates (PHAs), triacylglycerols (TAGs), or wax esters (WEs) constitute the main accumulated lipophilic substances by bacteria under such unbalanced growth conditions. The importance of these compounds as end-products or precursors to produce interesting biotechnologically relevant chemicals has already been recognized. In this review, we analyze the occurrence and accumulation of lipid storage in marine hydrocarbonoclastic bacteria. We further discuss briefly the production and export of lipophilic compounds by bacteria belonging to the Alcanivorax genus, which became a model strain of an unusual group of obligate hydrocarbonoclastic bacteria (OHCB) and discuss the possibility to produce neutral lipids using A. borkumensis SK2.     

Acute impact of tetracycline and erythromycin on the storage mechanism of polyhydroxyalkanoates

The study investigated acute impact of tetracycline and erythromycin on substrate storage under aerobic conditions. A fill and draw reactor fed with peptone mixture was maintained at steady-state at a sludge age of 10 days; the acclimated biomass was used in a series of batch runs. The first run served as control reactor with organic substrate alone and the others were started with antibiotic doses of 50 mg/L and 200 mg/L for assessing intracellular storage. Parallel batch reactors were also conducted for recording oxygen uptake rate profiles. Both antibiotics enhanced substrate storage, leading to higher levels of polyhydroxyalkanoates incorporated into biomass, but they impaired its internal utilization for microbial growth. The observed decrease in oxygen consumption under the acute effect of antibiotics could partially be related to substrate storage – except for 50 mg/L of erythromycin dosing – suggesting an additional substrate binding mechanism by antibiotics, leading to residual biodegradable substrate.     

Influence of sludge retention time on tolerance of copper toxicity for polyphosphate accumulating organisms linked to polyhydroxyalkanoates metabolism and phosphate removal

This study explored the influence of sludge retention time (SRT) on tolerance of copper invasion for polyphosphate accumulating organisms (PAOs) in an enhanced biological phosphorus removal (EBPR). The experimental data showed the anaerobic polyhydroxyalkanoates (PHA) storage for the sludge at 10d SRT was less influenced by copper invasion than those at 5d and 15d SRTs. The reaction of PAOs aerobically taking up phosphate for the sludge at 5d or 15d SRT almost ceased at 2 mg Cu L⁻¹, whereas PAOs in the sludge at 10d SRT retained half of the ability to take up phosphate. Both the PHAs degradation and synthesis rates decreased with increasing copper concentration, regardless of the SRTs. However, the copper inhibition of the former was greater than that of the later.