Comparative characteristics of biosynthesis of polyhydroxybutyrate from methanol by Methylobacteria extorquens G10 and Methyloligella halotolerans C2

The biosynthesis of polyhydroxybutyrate by Methylobacteria extorquens G10 and Methyloligella halotolerans C2 via the serine pathway of C1 metabolism was comparatively studied. Nitrogen limitation stimulated synthesis of the biopolymer in both cultures. It was shown that, despite the similarity of the pathways of methanol metabolism and those of polyhydroxybutyrate biosynthesis, the methylobacteria synthesized polymers of different molecular weights. In the case of M. extorquens G10, an increase in the content of the residual nitrogen in the culture medium was found to result in a reduction of the molecular weight of the polymer from 250 to 85 kDa, whereas M. halotolerans C2 synthesized a polymer of high molecular weight (approximately 3000 kDa) regardless of the residual content of the nitrogen source. It was established that the examined methylobacteria can utilize not only pure methanol but also a crude one, a feature that made it possible to significantly reduce the cost of the resulting polyhydroxybutyrate.     

Proteomic phenotyping of Novosphingobium nitrogenifigens reveals a robust capacity for simultaneous nitrogen fixation, polyhydroxyalkanoate production, and resistance to reactive oxygen species

Novosphingobium nitrogenifigens Y88(T) (Y88) is a free-living, diazotrophic Alphaproteobacterium, capable of producing 80% of its biomass as the biopolymer polyhydroxybutyrate (PHB). We explored the potential utility of this species as a polyhydroxybutyrate production strain, correlating the effects of glucose, nitrogen availability, dissolved oxygen concentration, and extracellular pH with polyhydroxybutyrate production and changes in the Y88 proteomic profile. Using two-dimensional differential in-gel electrophoresis and tandem mass spectrometry, we identified 217 unique proteins from six growth conditions. We observed reproducible, characteristic proteomic signatures for each of the physiological states we examined. We identified proteins that changed in abundance in correlation with either nitrogen fixation, dissolved oxygen concentration, or acidification of the growth medium. The proteins that correlated with nitrogen fixation were identified either as known nitrogen fixation proteins or as novel proteins that we predict play roles in aspects of nitrogen fixation based on their proteomic profiles. In contrast, the proteins involved in central carbon and polyhydroxybutyrate metabolism were constitutively abundant, consistent with the constitutive polyhydroxybutyrate production that we observed in this species. Three proteins with roles in detoxification of reactive oxygen species were identified in this obligate aerobe. The most abundant protein in all experiments was a polyhydroxyalkanoate granule-associated protein, phasin. The full-length isoform of this protein has a long, intrinsically disordered Ala/Pro/Lys-rich N-terminal segment, a feature that appears to be unique to sphingomonad phasins. The data suggest that Y88 has potential as a PHB production strain due to its aerobic tolerance and metabolic orientation toward polyhydroxybutyrate accumulation, even in low-nitrogen growth medium.     

Kinetic parameters of a culture of the hydrogen-oxidizing Ralstonia eutropha,grown under the regimen of biosynthesis of polyhydroxybutyrate

Kinetic parameters of a culture of the hydrogen-oxidizing bacterium Ralstonia eutropha, grown on a gas substrate under the conditions favoring autotrophic biosynthesis of polyhydroxybutyrate, were studied. The following parameters, making it possible to control and optimize the process in industrial situations, were determined: specific rate of substrate consumption, physical properties of culture medium, and coefficients of heat emission and mass transfer.     

Influence of cultivation conditions on spatial structure and functional activity of OmpF-like porin from outer membrane of <Emphasis Type="Italic">Yersinia pseudotuberculosis</Emphasis>

The influence of cultivation conditions of pseudotuberculosis bacteria on the spatial structure and the functional activity of nonspecific OmpF-like porin was studied by means of optical spectroscopy, scanning microcalorimetry, and bilayer lipid membrane technique. With this goal, porin samples isolated from microbial masses grown at different temperatures, nutrient medium densities, and growth phases were characterized. According to CD data, the porin samples under investigation represent beta-sheet proteins. It was found that the protein isolated from the colonial culture of pseudotuberculosis bacteria grown at low temperature has the most compact structure. Using intrinsic protein fluorescence, it was shown that different conditions of pseudotuberculosis bacteria cultivation (temperature, medium, growth phase) led to the changes in spectral properties of porin fluorescence due to the redistribution of the contributions of tyrosine and different classes of tryptophan residues to the total protein emission. Heat inactivation of porin samples was studied using CD spectroscopy, intrinsic protein fluorescence, and scanning microcalorimetry. Spatial features of the porin samples were found to affect their functional activities. Considering all these data, it is possible to correlate the spatial structure and functional activity of porin samples isolated under different cultivation conditions of bacteria and the composition of the outer membrane lipid matrix.     

Kinetic Parameters of a Culture of the Hydrogen-Oxidizing Bacterium Ralstonia eutrophaGrown under Conditions Favoring Polyhydroxybutyrate Biosynthesis

Kinetic parameters of a culture of the hydrogen-oxidizing bacterium Ralstonia eutrophagrown on a gas substrate under conditions favoring autotrophic biosynthesis of polyhydroxybutyrate were studied. The following parameters, making it possible to control and optimize the process in industrial situations, were determined: specific rate of substrate consumption, physical properties of the culture medium, and coefficients of heat emission and mass transfer.     

Physicochemical properties of multicomponent polyhydroxyalkanoates

X-ray structure analysis, IR spectrometry, differential thermal analysis, and viscosimetry have been used to investigate the properties of novel five-component polyhydroxyalkanoates formed by short- and medium-chain-length monomers synthesized by the bacterium Wautersia eutropha B5786. As the molar fraction of hydroxyhexanoate contained in polyhydroxyalkanoates samples increased from 2.5 to 18.0 mol%, their degree of crystallinity decreased from 72 to 57%. The melting temperature of multicomponent polyhydroxyalkanoates (Tm) and their temperature for the onset of decomposition (Td) are lower than those of polyhydroxybutyrate, whose Tm is 168-170 degrees C and Td 260-265 degrees C. In multicomponent polymers (PHA(SC+MC)), both parameters decrease as the molar fraction of hydroxyhexanoate grows to 156 and 252 degrees C, respectively, in the range of hydroxyhexanoate content studied. Hydroxyhexanoate influences the physicochemical properties of polyhydroxyalkanoates similarly to hydroxyvalerate; as the fraction of either of these medium-chain-length monomers in polyhydroxyalkanoates increases, the crystallinity of the polymer decreases, but its thermostability remains unchanged.     

Unfolding and refolding studies of frutalin, a tetrameric D-galactose binding lectin.

Protein refolding is currently a fundamental problem in biophysics and molecular biology. We have studied the refolding process of frutalin, a tetrameric lectin that presents structural homology with jacalin but shows a more marked biological activity. The initial state in our refolding puzzle was that proteins were unfolded after thermal denaturation or denaturation induced by guanidine hydrochloride, and under both conditions, frutalin was refolded. The denaturation curves, measured by fluorescence emission, gave values of conformational stability of 17.12 kJ.mol-1 and 12.34 kJ.mol-1, in the presence and absence of d-galactose, respectively. Native, unfolded, refolded frutalin and a distinct molecular form denoted misfolded, were separated by size-exclusion chromatography (SEC) on Superdex 75. The native and unfolded samples together with the fractions separated by SEC were also analyzed for heamagglutination activity by CD and fluorescence spectroscopy. The secondary structure content of refolded frutalin estimated from the CD spectra was found to be close to that of the native molecule. All the results obtained confirmed the successful refolding of the protein and suggested a nucleation-condensation mechanism, whereby the sugar-binding site acts as a nucleus to initiate the refolding process. The refolded monomers, after adopting their native three-dimensional structures, spontaneously assemble to form tetramers.     

Composite biodegradable materials based on polyhydroxyalkanoate

Conditions for the processing and mixing of biodegradable polymers at temperatures less than their thermal destruction (130–150°C) using standard equipment have been identified. The structure of the polyhydroxybutyrate/valerate (PHB/V) copolymer has been revealed and peculiarities of the crystal phase formation at different monomer ratios have been investigated. It was shown that pure PHB with molecular mass 180–270 kDa has elastic module approximately 1.2 GPa, strength approximately 25 MPa, and elongation at break approximately 10%. The most active biodestructors of PHB, PHB/V, and their composites have been selected (Aspergillus caespitosus), and the ability of basidiomycete Panus tigrinus to biodegrade polyalkanoates was demonstrated for the first time. It was shown that A. caespitosus degraded PHB/V and Biopol films along with the PHB with the destruction rate depending on the technology of the film production, on the molecular mass, and on the extend of the polymer crystallinity.     

Unfolding and refolding studies of frutalin, a tetrameric D-galactose binding lectin.

Protein refolding is currently a fundamental problem in biophysics and molecular biology. We have studied the refolding process of frutalin, a tetrameric lectin that presents structural homology with jacalin but shows a more marked biological activity. The initial state in our refolding puzzle was that proteins were unfolded after thermal denaturation or denaturation induced by guanidine hydrochloride, and under both conditions, frutalin was refolded. The denaturation curves, measured by fluorescence emission, gave values of conformational stability of 17.12 kJ x mol(-1) and 12.34 kJ x mol(-1), in the presence and absence of d-galactose, respectively. Native, unfolded, refolded frutalin and a distinct molecular form denoted misfolded, were separated by size-exclusion chromatography (SEC) on Superdex 75. The native and unfolded samples together with the fractions separated by SEC were also analyzed for heamagglutination activity by CD and fluorescence spectroscopy. The secondary structure content of refolded frutalin estimated from the CD spectra was found to be close to that of the native molecule. All the results obtained confirmed the successful refolding of the protein and suggested a nucleation-condensation mechanism, whereby the sugar-binding site acts as a nucleus to initiate the refolding process. The refolded monomers, after adopting their native three-dimensional structures, spontaneously assemble to form tetramers.     

羟基脂肪酸聚酯制备与热性质研究

不同的碳源条件下,真养产碱杆菌可在胞内积累聚羟丁酸(PHB)或含羟基戊酸单体(HV)比例不等的聚羟丁戊酸共聚物(PHBv)。利用次氯酸钠,氯仿混合液体系提取上述羟基脂肪酸聚酯(PHA),提取率为85％,纯度达97．O％。以差示扫描热分析法对PHB和PHBV材料进行热性质研究,发现材料中的HV组分逐渐增加．材料的熔点Tm,熔化焓Hm逐渐下降。热分解峰值逐渐向低温区偏移。但含HV为6lmol％的PHBV材料有关热性质出现回升现象。     

Application of two component biodegradable carriers in a particle-fixed biofilm airlift suspension reactor: development and structure of biofilms

Two component biodegradable carriers for biofilm airlift suspension (BAS) reactors were investigated with respect to development of biofilm structure and oxygen transport inside the biofilm. The carriers were composed of PHB (polyhydroxybutyrate), which is easily degradable and PCL (caprolactone), which is less easily degradable by heterotrophic microorganisms. Cryosectioning combined with classical light microscopy and CLSM was used to identify the surface structure of the carrier material over a period of 250 days of biofilm cultivation in an airlift reactor. Pores of 50 to several hundred micrometers depth are formed due to the preferred degradation of PHB. Furthermore, microelectrode studies show the transport mechanism for different types of biofilm structures, which were generated under different substrate conditions. At high loading rates, the growth of a rather loosely structured biofilm with high penetration depths of oxygen was found. Strong changes of substrate concentration during fed-batch mode operation of the reactor enhance the growth of filamentous biofilms on the carriers. Mass transport in the outer regions of such biofilms was mainly driven by advection.     

Enzymatic degradation of monolayer for poly(lactide) revealed by real-time atomic force microscopy: effects of stereochemical structure, molecular weight, and molecular branches on hydrolysis rates

The influences of the stereochemical structure, the molecular weight, and the number of molecular branches for poly(lactide) (PLA) on enzymatic hydrolysis rates of PLA monolayers were studied by atomic force microscopy (AFM) and the Langmuir-Blodgett (LB) technique. Monolayers of six kinds of PLA with different molecular weights, stereochemical structure, and numbers of molecular branches were prepared by LB techniques and then characterized by AFM in air. The PLA molecules covered homogeneously with a silicon substrate and did not form lamellar crystals in the monolayer. We determined the initial hydrolysis rate of PLA monolayers in presence of proteinase K by volumetric analysis from the continuous AFM height images. The presence of D-lactyl unit reduced the hydrolysis rate of the monolayer. The hydrolysis rate for the linear PLLA samples increased with a decrease in the molecular weight. In contrast, the rates of erosion for branched PLLA monolayers were independent of the molecular weight of samples. The erosion rate of branched PLLA monolayers was found to be dependent on the average molecular weight of PLLA segment in branched molecules, not on the overall molecular weight of samples. From these results, furthermore, the hydrolysis mode of PLAs by proteinase K is discussed.     

Enzymatic and non-enzymatic degradation of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) copolyesters produced by Alcaligenes sp. MT-16

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3HB-co-3HV), copolyesters with a variety of 3HV contents (ranging from 17 to 60 mol%) were produced by Alcaligenes sp. MT-16 grown on a medium containing glucose and levulinic acid in various ratios, and the effects of hydrophilicity and crystallinity on the degradability of the copolyesters were evaluated. Measurements of thermo-mechanical properties and Fourier-transform infrared spectroscopy in the attenuated total reflectance revealed that the hydrophilicity and crystallinity of poly(3HB-co-3HV) copolyesters decreased as 3HV content in the copolyester increased. When the prepared copolyester film samples were non-enzymatically hydrolysed in 0.01 N NaOH solution, the weights of all samples were found to have undergone no changes over a period of 20 weeks. In contrast, the copolyester film samples were degraded by the action of extracellular polyhydroxybutyrate depolymerase from Emericellopsis minima W2. The overall rate of weight loss was higher in the films containing higher amounts of 3HV, suggesting that the enzymatic degradation of the copolyester is more dependent on the crystallinity of the copolyester than on its hydrophilicity. Our results suggest that the degradability characteristics of poly(3HB-co-3HV) copolyesters, as well as their thermo-mechanical properties, are greatly influenced by the 3HV content in the copolyesters.     

Evolutionary relationships among prehistoric human populations: an evaluation of relatedness patterns based on facial morphometric data using molecular data

The study of evolutionary relationships among human populations is fundamental to inferring processes that determine their structure and history. Among the different data types used to infer such relationships, molecular data, particularly nuclear and mitochondrial DNA, are preferred because of their high heritability and the low probability of changes during development. However, although the reliability of relatedness patterns based on other traits is discussed, except in unusual circumstances most prehistoric populations remain within the domain of morphological study. Therefore the primary goal of this study is to test the reliability of relatedness patterns constructed on the basis of craniometric data on a regional scale. In particular, we analyze samples from populations belonging to the Chaco, Pampa, and Patagonia regions of South America for which craniometric and molecular data are available. We compare a strongly supported relatedness pattern based on molecular data with the results obtained through landmark-based and semilandmark-based facial data. The matrices based on Euclidean distance for morphometric data and DA distances for molecular data were used to perform principal coordinates analyses and to obtain reticulograms. Finally, a principal components analysis of all individuals was performed with morphometric data. The results indicate that ordination analyses yield slightly different results depending on the morphometric data used. However, the reticulograms obtained with both landmark-based and semilandmark-based data allow the separation of the Chubut samples from the Chaco samples, with the Pampa sample in between the others; this pattern is congruent with molecular-based analyses. As a consequence, our results indicate that facial morphometric data allow the inference of the structure and history of the prehistoric populations for the studied region.     

Microbial production of polyhydroxybutyrate with tailor-made properties: an integrated modelling approach and experimental validation

The microbial production of polyhydroxybutyrate (PHB) is a complex process in which the final quantity and quality of the PHB depend on a large number of process operating variables. Consequently, the design and optimal dynamic operation of a microbial process for the efficient production of PHB with tailor-made molecular properties is an extremely interesting problem. The present study investigates how key process operating variables (i.e., nutritional and aeration conditions) affect the biomass production rate and the PHB accumulation in the cells and its associated molecular weight distribution. A combined metabolic/polymerization/macroscopic modelling approach, relating the process performance and product quality with the process variables, was developed and validated using an extensive series of experiments and measurements. The model predicts the dynamic evolution of the biomass growth, the polymer accumulation, the consumption of carbon and nitrogen sources and the average molecular weights of the PHB in a bioreactor, under batch and fed-batch operating conditions. The proposed integrated model was used for the model-based optimization of the production of PHB with tailor-made molecular properties in Azohydromonas lata bacteria. The process optimization led to a high intracellular PHB accumulation (up to 95% g of PHB per g of DCW) and the production of different grades (i.e., different molecular weight distributions) of PHB.     

Construction of a stress-induced system in Escherichia coli for efficient polyhydroxyalkanoates production

In the application of engineered Escherichia coli in industrial polyhydroxybutyrate production process, one of the major concerns is the induction of the metabolic pathway. In this study, we developed a stress-induced system by which the PHB biosynthesis pathways can be induced under stress conditions. Fermentation results showed that recombinant E. coli DH5α (pQKZ103) harboring this system was able to accumulate polyhydroxybutyrate up to 85.8% of cell dry weight in minimal glucose medium without adding any inducer. Growth experiment with GFP as a reporter indicated that the induction of this system happened at the late exponential phase and was sensitive to stressed environment. This system can also be applied in many other biotechnological processes.     

Model-based intensification of a fed-batch microbial process for the maximization of polyhydroxybutyrate (PHB) production rate

An integrated metabolic–polymerization–macroscopic model, describing the microbial production of polyhydroxybutyrate (PHB) in Azohydromonas lata bacteria, was developed and validated using a comprehensive series of experimental measurements. The model accounted for biomass growth, biopolymer accumulation, carbon and nitrogen sources utilization, oxygen mass transfer and uptake rates and average molecular weights of the accumulated PHB, produced under batch and fed-batch cultivation conditions. Model predictions were in excellent agreement with experimental measurements. The validated model was subsequently utilized to calculate optimal operating conditions and feeding policies for maximizing PHB productivity for desired PHB molecular properties. More specifically, two optimal fed-batch strategies were calculated and experimentally tested: (1) a nitrogen-limited fed-batch policy and (2) a nitrogen sufficient one. The calculated optimal operating policies resulted in a maximum PHB content (94% g/g) in the cultivated bacteria and a biopolymer productivity of 4.2 g/(l h), respectively. Moreover, it was demonstrated that different PHB grades with weight average molecular weights of up to 1513 kg/mol could be produced via the optimal selection of bioprocess operating conditions.

     

Improvement of the enzymatic synthesis of ethyl valerate by esterification reaction in a solvent system

The present study reports the improved enzymatic synthesis of ethyl valerate (green apple flavor) by esterification reaction of ethanol and valeric acid in heptane medium. Lipase from Thermomyces lanuginosus (TLL) was immobilized by physical adsorption on polyhydroxybutyrate (PHB) particles and used as a potential biocatalyst. The effect of certain parameters that influence the ester synthesis was evaluated by factorial design. The experimental conditions that maximized the synthesis of ethyl valerate were 30.5°C, 18% m/v of biocatalyst (TLL–PHB), absence of molecular sieves, agitation of 234?rpm, and 1,000?mM of each reactant (ethanol and valeric acid). Under these conditions, conversion percentage ≈92% after 105?min of reaction was observed. Soluble TLL was also used as biocatalyst and the highest conversion was of 82% after 120?min of reaction. Esterification reaction performed in a solvent-free system exhibited conversion of 13% after 45?min of reaction catalyzed by immobilized lipase, while the soluble lipase did not exhibit catalytic activity. The synthesis of the ester was confirmed by Fourier transform infrared spectroscopy and gas chromatography–mass spectrometry analyses. After six consecutive cycles of ethyl valerate synthesis, the prepared biocatalyst retained ≈86% of its original activity.     

Biosynthesis,properties and potential of natural–synthetic hybrids of polyhydroxyalkanoates and polyethylene glycols

Chemical conjugation with poly(ethylene glycols) (PEGs) are established procedures to facilitate solubilisation of hydrophobic compounds. Such techniques for PEGylation have been applied to polyhydroxybutyrate. ‘BioPEGylation’ of such polyhydroxyalkanoates (PHAs) to form natural–synthetic hybrids has been demonstrated through the addition of PEGs to microbial cultivation systems. The strategic addition of certain PEGs not only supports hybrid synthesis but may also provide a technique for control of PHA composition and molecular mass, and by extension, their physico-mechanical properties. PHA composition and molecular mass control by PEGs is dependent upon the polyethers’ molecular mass, loading in the cultivation system, time of introduction and microbial species. Hybrid characterisation studies are in their infancy, but results to date suggest that PHA–PEG hybrids have subtle, but significant, differences in their physiochemical and material properties as a consequence of the PEGylation.