PHA-rubber blends: synthesis, characterization and biodegradation

Medium chain length polyhydroxyalkanoates (mcl-PHA) and different rubbers; namely natural rubber, nitrile rubber and butadiene rubber were blended at room temperature using solution blending technique. Blends constituted 5%, 10% and 15% of mcl-PHA in different rubbers. Thermogravimetric analysis of mcl-PHA showed the melting temperature of the polymer around 50 degrees C. Thermal properties of the synthesized blend were studied by Differential Scanning Calorimetry which confirmed effective blending between the polymers. Blending of mcl-PHA with natural rubber led to the synthesis of a different polymer having the melting point of 90 degrees C. Degradation studies of the blends were carried out using a soil isolate, Pseudomonas sp. 202 for 30 days. Extracellular protein concentration as well as OD660 due to the growth of Pseudomonas sp. 202 was studied. The degradation of blended plastic material, as evidenced by % weight loss after degradation and increase in the growth of organism correlated with the amount of mcl-PHA present in the sample. Growth of Pseudomonas sp. 202 resulted in 14.63%, 16.12% and 3.84% weight loss of PHA:rubber blends (natural, nitrile and butadiene rubber). Scanning electron microscopic studies after 30 days of incubation further confirmed biodegradation of the films.     

Effects of crosslinking on the thermal stability of hemoglobins. II. The stabilization of met-, cyanomet-, and carbonmonoxyhemoglobins A and S with bis(3,5-dibromosalicyl) fumarate

Hemoglobins A and S were crosslinked between Lys 82 beta 1 and Lys 82 beta 2 using bis (3,5-dibromosalicyl) fumarate (J. A. Walder et al. (1979) Biochemistry 18, 4265). Thermal denaturation experiments were used to compare the stabilities of the met, cyanomet, and carbonmonoxy forms of these crosslinked hemoglobins to the corresponding uncrosslinked proteins. Uncrosslinked carbonmonoxy- and cyanomethemoglobins had transition temperatures about 11 degrees C higher than the corresponding met samples. The increase in denaturation temperature (Tm) due to crosslinking was 15 degrees C for the methemoglobins, 10 degrees C for the cyanomethemoglobins, and 4 degrees C for the carbonmonoxy ones. There was no significant difference in stability between the met and carbonmonoxy crosslinked proteins. In order of increasing stability the samples were: met Hb S less than met Hb A less than CO Hb S less than CO Hb A = CN-met Hb A less than met XL-Hb S = CO XL-Hb S less than met XL-Hb A = CO XL-Hb A less than CN-met XL-Hb A. The slight decrease in the stability of Hb S (beta 6 Glu----Val) compared to Hb A can be explained by the replacement of an external ionic group by a hydrophobic residue in Hb S. In mixtures of crosslinked and normal Hb A, the Tm of the uncrosslinked material was slightly increased by the presence of the more stable crosslinked hemoglobin. The effects of both crosslinking and cyanide or carbon monoxide binding can be explained by Le Chatelier's principle since both would favor the native form of the protein.     

Strain-Induced Alignment in Collagen Gels

Collagen is the most abundant extracellular-network-forming protein in animal biology and is important in both natural and artificial tissues, where it serves as a material of great mechanical versatility. This versatility arises from its almost unique ability to remodel under applied loads into anisotropic and inhomogeneous structures. To explore the origins of this property, we develop a set of analysis tools and a novel experimental setup that probes the mechanical response of fibrous networks in a geometry that mimics a typical deformation profile imposed by cells in vivo. We observe strong fiber alignment and densification as a function of applied strain for both uncrosslinked and crosslinked collagenous networks. This alignment is found to be irreversibly imprinted in uncrosslinked collagen networks, suggesting a simple mechanism for tissue organization at the microscale. However, crosslinked networks display similar fiber alignment and the same geometrical properties as uncrosslinked gels, but with full reversibility. Plasticity is therefore not required to align fibers. On the contrary, our data show that this effect is part of the fundamental non-linear properties of fibrous biological networks.     

Crosslinked fibrous collagen for use as a dermal implant: control of the cytotoxic effects of glutaraldehyde and dimethylsuberimidate

Collagen intended for use as a dermal implant may be crosslinked to increase its strength and persistence in vivo. Sheets of rat fibrous dermal collagen were crosslinked with either glutaraldehyde or dimethylsuberimidate and the cytotoxicity to human dermal fibroblasts resulting from these treatments was measured by following the inhibition of [3H]leucine incorporation into protein. Both agents were cytotoxic at the concentrations required to effect adequate crosslinking (0.005% and 25 mM, respectively). This cytotoxicity could be limited by extensive washing and by incubation with 5 mM L-lysine, with 66 mM (0.25% w/v) sodium borohydride, or with 71.3 mM (1% w/v) dimedone. However, cytotoxicity was most efficiently controlled by treatment with a combination of 66 mM sodium borohydride and 5 mM L-lysine or 66 mM sodium borohydride and 71.3 mM dimedone. [3H]Leucine incorporation by cells exposed to crosslinked collagen treated with these combinations approached 100% of the values recorded with cells exposed to uncrosslinked collagen.     

Studies on the characteristics of drug-loaded gelatin nanoparticles prepared by nanoprecipitation

The morphology of gelatin nanoparticles loaded with three different drugs (Tizanidine hydrochloride, Gatifloxacin and Fluconazole) and their characteristics of entrapment and release from gelatin nanoparticles were investigated by the analysis on nanoparticle size distribution, SEM and FT-IR in this study. The particles were prepared by nanoprecipitation using water and ethanol as a solvent and a nonsolvent, respectively. The exclusion of a crosslinking agent from the procedure led the system to have an irregularly-shaped morphology. Nonetheless, the uncrosslinked case of Gatifloxacin loading generally led to a more homogeneous population of nanoparticles than the uncrosslinked case of Tizanidine hydrochloride loading. No loading was achieved in the case of Fluconazole, whereas both Tizanidine hydrochloride and Gatifloxacin are observed of being capable of being loaded by nanoprecipitation. Tizanidine hydrochloride-loaded, blank and Gatifloxacin-loaded nanoparticles yielded, under crosslinked condition, 59.3, 23.1 and 10.6% of the used dried mass. The crosslinked Tizanidine hydrochloride-loaded particles showed the loading efficiency of 13.8%, which was decreased to 1.1% without crosslinking. A crosslinker such as glutaraldehyde is indispensable to enhance the Tizanidine hydrochloride-loading efficiency. To the contrary, the Gatifloxacin-loading efficiency for crosslinked ones was lower by a factor of 2-3 times than that for uncrosslinked ones. This is due to the carboxylic groups of Gatifloxacin and the aldehyde groups of glutaraldehyde competing with each other during the crosslinking process, to react with the amino groups of gelatin molecules. The loading efficiency of gelatin nanoparticles reported by other investigators greatly varies. Nevertheless, the loading efficiency reported by us is in good agreement with the drug-loading data of gelatin nanoparticles reported by other investigators. The 80% of loaded Tizanidine hydrochloride was released around 15 h after start-up of the release experiment, while the 20% of loaded Gatifloxacin was released more rapidly, as free Gatifloxacin, than the loaded Tizanidine hydrochloride and it showed the trend of sustained slow release during the remaining period of its release experiment. Furthermore, the result of comparative FT-IR analysis is consistent to that of the corresponding drug release study.     

Estimation of the yield coefficient of Pseudomonas sp. strain DP-4 with a low substrate (2,4-dichlorophenol [DCP]) concentration in a mineral medium from which uncharacterized organic compounds were eliminated by a non-DCP-degrading organism

The yield coefficient (YC) of Pseudomonas sp. strain DP-4, a 2, 4-dichlorophenol (DCP)-degrading organism, was estimated from the number of CFU produced at the expense of 1 unit amount of DCP at low concentrations. At a low concentration of DCP, the YC can be overestimated in pure culture, because DP-4 assimilated not only DCP but also uncharacterized organic compounds contaminating a mineral salt medium. The concentration of these uncharacterized organic compounds was nutritionally equivalent to 0.7 microg of DCP-C ml(-1). A mixed culture with non-DCP-degrading organisms resulted in elimination of ca. 99.9% of the uncharacterized organic compounds, and then DP-4 assimilated only DCP as a substrate. In a mixed culture, DP-4 degraded an initial concentration of 0.1 to 10 microg of C ml of DCP(-1) and the number of CFU of DP-4 increased. In the mixed culture, DCP at an initial concentration of 0.07 microg of C ml(-1) was degraded. However, the number of CFU of DP-4 did not increase. DCP at an extremely low initial concentration of 0.01 microg of C ml(-1) was not degraded in mixed culture even by a high density, 10(5) CFU ml(-1), of DP-4. When glucose was added to this mixed culture to a final concentration of 1 microg of C ml(-1), the initial concentration of 0.01 microg of C ml of DCP(-1) was degraded. These results suggested that DP-4 required cosubstrates to degrade DCP at an extremely low initial concentration of 0.01 microg of C ml(-1). The YCs of DP-4 at the expense of DCP alone decreased discontinuously with the decrease of the initial concentration of DCP, i.e., 1.5, 0.19, or 0 CFU per pg of DCP-C when 0.7 to 10, 0.1 to 0.5, or 0.07 microg of C ml of DCP(-1) was degraded, respectively. In this study, we developed a new method to eliminate uncharacterized organic compounds, and we estimated the YC of DP-4 at the expense of DCP as a sole source of carbon.     

Lux-biosensor assessment of pH effects on microbial sorption and toxicity of chlorophenols

Lux-marked bacterial biosensors and a commercial toxicity testing bacterial strain (Microtox) were exposed to 2,4-dichlorophenol (DCP) and the light output response measured. Increasing DCP concentrations caused a decrease in light output in all three biosensors with an order of sensitivity (in terms of luminescence decrease over the DCP concentration range) of Pseudomonas fluorescens < Escherichia coli < Microtox. Adsorption of DCP to E. coli was measured using uniformly ring labelled [14C]DCP and found to be very rapid. The effect of pH on toxicity and adsorption was also investigated. Low pH values increased the amount of DCP adsorbed to the cell and increased the toxicity of DCP.     

松材线虫对其携带的细菌繁殖的影响

1.引言松材线虫病（Bursaphelenchus xylophilus）是松树的一种毁灭性病害,在日本、中国、韩国和北美、尼日利亚和葡萄牙等国家蔓延,造成了巨大经济损失,其中以日本和中国受害最重.一直认为松材线虫是引起该病的唯一病原,但近十几年来的研究发现,细菌在致病过程中可能起着重要作用,相继从病木和松材线虫体上分离到能对黑松苗有致萎活性的细菌.赵博光等首次根据实验提出松材线虫病是线虫和细菌共同侵染引起的复合侵染病害的假说,并在以后的试验中得到了验证.关于松材线虫对其细菌繁殖的影响研究鲜有报道.本试验采用从感病松树上分离并鉴定了的细菌菌株中选取假单胞属7株、其它属的细菌菌株3株,     

Comparison of Tryptophan-nicotinamide Metabolism between Male and Female Rats

A dipeptidyl carboxypeptidase (DCP) activity was detected in cell-free extracts of Pseudomonas sp. WO24. After purification and characterization the enzyme was found to be homogeneous by SDS-PAGE, and had a molecular mass of 74,000 Da by SDS–PAGE and 72,000 Da by gel filtration, indicating that it is monomeric. The isoelectric point was 5.2 and optimum pH was 6.5–7.0. It showed a specific activity of 780 μmol/min/mg, which is the highest of the values shown by known enzymes. The enzyme hydrolyzed angiotensin I to angiotensin II and sequentially released Phe-Arg and Ser-Pro from the C-terminus bradykinin. The DCP could not cleave imido-bonds, Gly-Gly bonds, or tripeptides. The enzymatic activity was completely inhibited by 0.001 mm EDTA and 0.1 mm o-phenanthroline, but it was not affected by general serine and cysteine protease inhibitors. Addition of Zn^{2 +} completely restored the original activity of the inactivated DCP treated with EDTA. These results suggest that this enzyme is a zinc metalloprotease. The characteristics of the purified enzyme are slightly different from those of the DCPs from Escherichia coli, Pseudomonas maltophilia, and Corynebacterium equi, and considerably from those of the DCP from Bacillus pumilus.     

Effects of Ca crosslinking on structure and properties of waterborne polyurethane-carboxymethylated guar gum films

Films from waterborne polyurethane (WPU) and carboxymethylated guar gum (CMGG) with different contents (20–80 wt%) were prepared through solution casting method, and then were crosslinked with calcium chloride. The effect of CMGG content on the miscibility, morphology and physical properties of the blend films is investigated by Fourier transform infrared spectroscopy, scanning electron microscopy, density measurements, differential scanning calorimetry, dynamic mechanical thermal analysis, thermogravimetric analysis, water sensitivity measurements, solvent-swelling and tensile tests. The results reveal that the uncrosslinked films exhibit good miscibility when CMGG content is lower than 60 wt%, whereas typical “sea-island” structure occurs when the CMGG content further increases. After crosslinking with calcium ion, the blend films form a relatively dense architecture, which leads to better miscibility, higher storage modulus and thermal stability. The crosslinked films also exhibit better tensile strength (11.6–56.5 MPa) and solvent-resistance than that of the uncrosslinked films over the entire composition range. A model describing the configuration of Ca²⁺-chelating structure was proposed to illustrate the different structures of the two series of the blend films.     

Effect of pretreatment of rubber material on its biodegradability by various rubber degrading bacteria

The effect of pretreatment of several cis-1,4-polyisoprene containing rubbers on their biodegradability was examined. Tests were carried out with six recently isolated and characterized rubber degrading bacteria belonging to the genera Gordonia (strains Kb2, Kd2 and VH2), Mycobacterium, Micromonospora and Pseudomonas. All strains were able to use natural rubber (NR) as well as NR latex gloves as sole carbon source. Extraction of NR latex gloves by organic solvents resulted in an enhancement of growth for three of the selected strains. On the other hand, growth of Gordonia sp. (strain Kb2 and Kd2), Mycobacterium fortuitum NF4 and Micromonospora aurantiaca W2b on synthetic cis-1,4-polyisoprene did only occur after removal of the antioxidants, that are usually added during manufacture to prevent aging of the materials. Detailed degradation studies performed with Gordonia sp. Kb2 revealed an enhanced mineralization of pretreated NR latex gloves and mineralization of purified natural rubber (NR), indicating the actual mineralization of cis-1,4-polyisoprene rubber constituent even after removal of non-rubber constituent that may act as co-metabolic substrate and support microbial growth. Further analysis by scanning electron microscopy (SEM) clearly demonstrated the enhanced colonization efficiency of these bacteria towards pretreated NR latex gloves. Colonization was additionally visualized by staining of overgrown NR latex gloves with Schiff's reagent, and the purple color produced in the area of degradation was an evidence for the accumulation of aldehydes containing oligomers. Further enhancement of latex gloves degradation could be achieved after successive replacement of mineral salts medium during cultivation. Thereby, a rapid disintegration of untreated NR latex gloves material was accomplished by Gordonia sp. strain VH2.     

Removal of copper ion by Pseudomonas spp.

Copper-resistant Pseudomonas sp. 41Y, Pseudomonas pseudomallei 13-1 and Pseudomonas aeruginosa 7 were used in the present study. When the latter two organisms were added to copper-containing 1/3 strength Tryptic Soy Broth, more than 99.5% of the copper ion was removed from the medium within 24 h. If copper solution was added to hog waste slurry, a reduction in the copper ion concentration could be detected only when the added bacteria started to grow in it, whereas in a mineral medium supplemented with glycerol-2-phosphate, both bacteria could remove about 50% of the copper ion from the medium within 24 h. When cell suspension of Pseudomonas sp. 41Y was autoclaved, no copper ion removal was observed. Different incubation temperatures, including 30 degrees C, 37 degrees C and 45 degrees C, had no effect on the percent of copper ion removed by both Pseudomonas sp. 41Y and P. pseudomallei 13-1. On the other hand, if the pH value of the solution was lowered from 8.2 to 6.0, there was a drastic decrease in copper removal. A similar reduction of copper ion removal ability was also observed with the addition of lead ion. When cells of Pseudomonas sp. 41Y were embedded in sodium alginate, there was a decrease in its ability to remove copper ion as compared to the free-living cells.     

Anaerobic desulfurization of ground rubber with the thermophilic archaeon Pyrococcus furiosus – a new method for rubber recycling

The anaerobic sulfur-reducing archaeon Pyrococcus furiosus was investigated regarding its capacity to desulfurize rubber material. The microorganism's sensitivity towards common rubber elastomers and additives was tested and several were shown to be toxic to P. furiosus. The microorganism was shown to utilize sulfur in vulcanized natural rubber and an increase in cell density was obtained when cultivated in the presence of spent tire rubber. Ethanol-leached cryo-ground tire rubber treated with P. furiosus for 10 days was vulcanized together with virgin rubber material (15% w/w) and the mechanical properties of the resulting material were determined. The increase in the stress at break value and the decrease in swell ratio and stress relaxation rate obtained for material containing microbially treated rubber (compared to untreated material) show the positive effects of microbial desulfurization on rubber.     

Scanning electron microscopy of dairy equipment surfaces contaminated by two milk-borne micro-organisms

Ethanol dehydration followed by argon replacement induced drying (ARID) was found to be a suitable method for the preparation of glass, stainless steel and rubber surfaces which had been in contact with inoculated milk and which were to be examined using scanning electron microscopy (SEM). This technique was used to examine samples of all three materials which had been subjected to both single and repeated inoculation with whole milk containing a Pseudomonas sp. or a Micrococcus sp. and incubated for various periods. Some samples were also prepared for SEM using a cryofixation technique. The Pseudomonas sp. was found to proliferate on glass and stainless steel surfaces but not on rubber. Due to the clumping tendency of the Micrococcus sp. proliferation of this organism was more difficult to assess accurately. In general there was no difference in results obtained between single and repeated inoculation. Various factors which may have aided attachment of micro-organisms to surfaces were identified viz. , surface channels present in stainless steel, milk deposits and the production of extracellular material. The value of using both the cryofixation and chemical preparatory techniques for the identification of artifacts is discussed.     

Diverse responses to UV-B radiation and repair mechanisms of bacteria isolated from high-altitude aquatic environments

Acinetobacter johnsonii A2 isolated from the natural community of Laguna Azul (Andean Mountains at 4,560 m above sea level), Serratia marcescens MF42, Pseudomonas sp. strain MF8 isolated from the planktonic community, and Cytophaga sp. strain MF7 isolated from the benthic community from Laguna Pozuelos (Andean Puna at 3,600 m above sea level) were subjected to UV-B (3,931 J m-2) irradiation. In addition, a marine Pseudomonas putida strain, 2IDINH, and a second Acinetobacter johnsonii strain, ATCC 17909, were used as external controls. Resistance to UV-B and kinetic rates of light-dependent (UV-A [315 to 400 nm] and cool white light [400 to 700 nm]) and -independent reactivation following exposure were determined by measuring the survival (expressed as CFU) and accumulation of cyclobutane pyrimidine dimers (CPD). Significant differences in survival after UV-B irradiation were observed: Acinetobacter johnsonii A2, 48%; Acinetobacter johnsonii ATCC 17909, 20%; Pseudomonas sp. strain MF8, 40%; marine Pseudomonas putida strain 2IDINH, 12%; Cytophaga sp. strain MF7, 20%; and Serratia marcescens, 21%. Most bacteria exhibited little DNA damage (between 40 and 80 CPD/Mb), except for the benthic isolate Cytophaga sp. strain MF7 (400 CPD/Mb) and Acinetobacter johnsonii ATCC 17909 (160 CPD/Mb). The recovery strategies through dark and light repair were different in all strains. The most efficient in recovering were both Acinetobacter johnsonii A2 and Cytophaga sp. strain MF7; Serratia marcescens MF42 showed intermediate recovery, and in both Pseudomonas strains, recovery was essentially zero. The UV-B responses and recovery abilities of the different bacteria were consistent with the irradiation levels in their native environment.     

Histone-DNA interactions within chromatin. Isolation of histones from DNA-histone adducts induced in nuclei by UV light.

We have developed a method by which to isolate histones that have been crosslinked to DNA following irradiation of calf thymus nuclei by UV light. The procedure involves separation of protein-DNA adducts from uncrosslinked protein by Sepharose 4B chromatography under dissociating conditions. Histones which are crosslinked to DNA are released by chemical hydrolysis of the DNA and identified by SDS gel electrophoresis. The results indicate that, of the histones, H1 and H3 become crosslinked to the DNA most readily under our irradiation conditions.     

Novel Dehalogenase Mechanism for 2,3-Dichloro-1-Propanol Utilization in Pseudomonas putida Strain MC4

A Pseudomonas putida strain (MC4) that can utilize 2,3-dichloro-1-propanol (DCP) and several aliphatic haloacids and haloalcohols as sole carbon and energy source for growth was isolated from contaminated soil. Degradation of DCP was found to start with oxidation and concomitant dehalogenation catalyzed by a 72-kDa monomeric protein (DppA) that was isolated from cell lysate. The dppA gene was cloned from a cosmid library and appeared to encode a protein equipped with a signal peptide and that possessed high similarity to quinohemoprotein alcohol dehydrogenases (ADHs), particularly ADH IIB and ADH IIG from Pseudomonas putida HK. This novel dehalogenating dehydrogenase has a broad substrate range, encompassing a number of nonhalogenated alcohols and haloalcohols. With DCP, DppA exhibited a k(cat) of 17 s(-1). (1)H nuclear magnetic resonance experiments indicated that DCP oxidation by DppA in the presence of 2,6-dichlorophenolindophenol (DCPIP) and potassium ferricyanide [K(3)Fe(CN)(6)] yielded 2-chloroacrolein, which was oxidized to 2-chloroacrylic acid.     

2,4-Dichlorophenol degradation by the soil fungus Mortierella sp

We investigated the degradation pathways and kinetics of 2,4-dichlorophenol (DCP) by an endemic soil fungus, Mortierella sp. (Zygomycetes). Mortierella sp. degraded 32% of added DCP (final concentration, 250 microM) within 1 h. We identified four aromatic metabolites and found two DCP degradation pathways (a hydroxylation pathway and a dechlorination pathway). This is the first report of a dechlorination pathway in Zygomycetes.     

Identification and characterization of genes from Streptomyces sp. strain K30 responsible for clear zone formation on natural rubber latex and poly(cis-1,4-isoprene) rubber degradation

Streptomyces sp. strain K30 was isolated from soil next to a city high way in Münster (Germany) according to its ability to degrade natural and synthetic poly(cis-1,4-isoprene) rubber and to form clear zones on natural rubber latex agar plates. The clear zone forming phenotype was used to clone the responsible gene by phenotypic complementation of a clear zone negative mutant. An open reading frame (lcp) of 1,191 bp was identified, which was preceded by a putative signal sequence and restored the capability to form clear zones on natural rubber latex in the mutant. The putative translation product exhibited strong homologies (50% aa identity) to a putative secreted protein from Streptomyces coelicolor strain A3(2), another clear zone forming strain. Heterologous expression of lcp of Streptomyces sp. strain K30 in Streptomyces lividans strain TK23 enabled the latter to form clear zones on latex-overlay agar plates and to accumulate a degradation product of about 12 kDa containing aldehyde groups. Two ORFs putatively encoding a heterodimeric molybdenum hydroxylase (oxiAB) were identified downstream of lcp in Streptomyces sp. strain K30 strain which exerted a positive effect on clear zone formation and enabled the strain to oxidize the resulting aldehydes. Heterologous expression of a fragment harboring lcp plus oxiAB in S. lividans TK23 resulted in accumulation of aldehydes only in the presence of 10 mM tungstate. Determination of protein content during cultivation on poly(cis-1,4-isoprene) revealed an increase of the cellular protein, and gel permeation chromatography analysis indicated a shift of the molecular weight distribution of the rubber to lower values in the transgenic S. lividans strains and in the wild type, thus confirming utilization and degradation of rubber. Therefore, for the first time, genes responsible for clear zone formation on natural rubber latex and synthetic cis-1,4-polyisoprene degradation in Gram-positive bacteria were identified and characterized.     

Historical and recent achievements in the field of microbial degradation of natural and synthetic rubber

This review intends to provide an overview of historical and recent achievements in studies of microbial degradation of natural and synthetic rubber. The main scientific focus is on the key enzymes latex-clearing protein (Lcp) from the Gram-positive Streptomyces sp. strain K30 and rubber oxygenase A (RoxA) from the Gram-negative Xanthomonas sp. strain 35Y, which has been hitherto the only known rubber-degrading bacterium that does not belong to the actinomycetes. We also emphasize the importance of knowledge of biodegradation in industrial and environmental biotechnology for waste natural rubber disposal.