Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) depolymerase from Aspergillus sp. NA-25

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) degrading thermophilic fungus was isolated from soil sample collected from waste disposal site, Islamabad, Pakistan. It was able to grow efficiently on a medium containing PHBV as a sole source of carbon and has been identified as Aspergillus sp. NA-25 by 18S rRNA. Using 9% of inoculum maximum production of PHBV depolymerase was observed at 45°C, pH 7.0 in the presence of 0.2% lactose as an additional carbon source. PHBV depolymerase was purified by precipitation with 80% ammonium sulfate and gel filtration chromatography on Sephadex G-75. The four enzyme forms obtained after gel filtration were analyzed on SDS-PAGE and their molecular weights (36, 68, 72 and 90 kDa) were determined. They were characterized on the basis of effect of different temperatures, pH, metal ions and different reagents on the PHBV activity and stability. It is obvious that the fungal strain Aspergillus sp. NA-25 is capable of degrading PHBV with the help of different types of depolymerases.     

Biosynthesis and Thermal Properties of PHBV Produced from Levulinic Acid by Ralstonia eutropha

Levulinic acid (LA) can be cost-effectively produced from a vast array of renewable carbohydrate-containing biomaterials. LA could facilitate the commercialization of the polymer poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) and PHBV-based products as carbon substrates. Therefore, this paper focused on the production of PHBV by Ralstonia eutropha with LA for hydroxyvalerate (HV) production, which plays an important role in enhancing the thermal properties of PHBV. Accordingly, the HV content of PHBV varied from 0–40.9% at different concentrations of LA. Stimulation of cell growth and PHBV accumulation were observed when 2–6 g L⁻¹ LA was supplied to the culture. The optimal nitrogen sources were determined to be 0.5 g L⁻¹ ammonium chloride and 2 g L⁻¹ casein peptone. It was determined that the optimal pH for cell growth and PHBV accumulation was 7.0. When the cultivation was performed in large scale (2 L fermenter) with a low DO concentration of 30% and a pH of 7.0, a high maximum dry cell weight of 15.53 g L⁻¹ with a PHBV concentration of 12.61 g L⁻¹ (53.9% HV), up to 81.2% of the dry cell weight, was obtained. The melting point of PHBV found to be decreased as the fraction of HV present in the polymer increased, which resulted in an improvement in the ductility and flexibility of the polymer. The results of this study will improve the understanding of the PHBV accumulation and production by R. eutropha and will be valuable for the industrial production of biosynthesized polymers.     

Improving culture conditions for poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production by Bacillus sp. ND153, a bacterium isolated from a mangrove forest in Vietnam

The production of polyhydroxyalkanoate (PHA) by Bacillus sp. ND153, a bacterium strain isolated from a mangrove forest in Vietnam, was studied. Bacillus sp. ND153 was grown on HM-1 medium with different carbon sources (e.g. glucose, sucrose, maltose, dextrin, and starch). Glucose was found to be the most suitable carbon source for PHA accumulation, whereas starch and dextrin favored cell growth over PHA accumulation. Optimization of the culture medium for PHA production was investigated by applying factorial design, and a maximum PHA content of 79 % (w/w) was obtained with low concentrations of NH₄Cl and MgSO₄ and a high concentration of KH₂PO₄ in the medium. Propionate was used as the precursor for the production of copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and the amount of 3-hydroxyvalerate (3HV) in the polymer showed an increasing linear trend with the increase in propionate concentration from 0.2 g l^?1 to 1.0 g l^?1. Thus, the production of PHBV by Bacillus sp. ND153, with 3HV fraction ranging from 1 mol% to 30 mol%, was noted to be high, and the characteristics of fast cell growth and accumulation of PHA exhibited by Bacillus sp. ND153 make it a promising choice for biopolyester production.     

Effects of pH on Lignin and Cellulose Degradation by Streptomyces viridosporus

Lignocellulose degradation by Streptomyces viridosporus results in the oxidative depolymerization of lignin and the production of a water-soluble lignin polymer, acid-precipitable polymeric lignin (APPL). The effects of the culture pH on lignin and cellulose metabolism and APPL production by S. viridosporus are reported. Dry, ground, hot-water-extracted corn (Zea mays) lignocellulose was autoclaved in 1-liter reagent bottles (5 g per bottle) and inoculated with 50-ml volumes of S. viridosporus cells suspended in buffers of specific pH (pH 6.0 to 9.2 at 0.4 pH unit intervals). Four replicates of inoculated cultures and of uninoculated controls at each pH were incubated as solid-state fermentations at 37°C. After 6 weeks of incubation the percent loss of lignocellulose, lignin, and carbohydrate and the amount of APPL produced were determined for each replicate. Optimal lignocellulose degradation, as shown by substrate weight loss, was observed in the pH range of 8.4 to 8.8. Only minor differences were seen in the Klason lignin, carbohydrate, protein, and ash contents of the APPLS produced by cultures at each pH. The effects of pH on the degradation of a spruce (Picea pungens) [¹⁴C-lignin]lignocellulose and a Douglas fir (Pseudotsuga menziesii) [¹⁴C-glucan]-lignocellulose were also determined at pH values between 6.5 and 9.5 (0.5 pH unit intervals). The incubations were carried out for 3 weeks at 37°C with bubbler-tube cultures. The percentage of initial ¹⁴C recovered as ¹⁴CO₂, ¹⁴C-labeled water-soluble products, and [¹⁴C]APPL was then determined. The mineralization of lignin and cellulose to CO₂ was optimal at pHs 6.5 and 7.0, respectively. However, the optimum for lignin and cellulose solubilization was pH 8.5, which correlated with the pH 8.5 optimum for APPL production. Overall, the data show that, whereas lignin mineralization is optimal at neutral to slightly acidic pHs, lignocellulose degradation with lignin solubilization and APPL production is promoted by alkaline pHs. These findings indicate that lignin-solubilizing actinomycetes may play an important role in the metabolism of lignin in neutral to alkaline soils in which ligninolytic fungi are not highly competitive.     

Novel Extracellular PHB Depolymerase from Streptomyces ascomycinicus: PHB Copolymers Degradation in Acidic Conditions

The ascomycin-producer strain Streptomyces ascomycinicus has been proven to be an extracellular poly(R)-3-hydroxybutyrate (PHB) degrader. The fkbU gene, encoding a PHB depolymerase (PhaZ_Sa), has been cloned in E. coli and Rhodococcus sp. T104 strains for gene expression. Gram-positive host Rhodococcus sp. T104 was able to produce and secrete to the extracellular medium an active protein form. PhaZ_Sa was purified by two hydrophobic interaction chromatographic steps, and afterwards was biochemically as well as structurally characterized. The enzyme was found to be a monomer with a molecular mass of 48.4 kDa, and displayed highest activity at 45°C and pH 6, thus being the first PHB depolymerase from a gram-positive bacterium presenting an acidic pH optimum. The PHB depolymerase activity of PhaZ_Sa was increased in the presence of divalent cations due to non-essential activation, and also in the presence of methyl-β-cyclodextrin and PEG 3350. Protein structure was analyzed, revealing a globular shape with an alpha-beta hydrolase fold. The amino acids comprising the catalytic triad, Ser¹³¹-Asp²⁰⁹-His²⁶⁹, were identified by multiple sequence alignment, chemical modification of amino acids and site-directed mutagenesis. These structural results supported the proposal of a three-dimensional model for this depolymerase. PhaZ_Sa was able to degrade PHB, but also demonstrated its ability to degrade films made of PHB, PHBV copolymers and a blend of PHB and starch (7∶3 proportion wt/wt). The features shown by PhaZ_Sa make it an interesting candidate for industrial applications involving PHB degradation.     

Enhanced production and partial characterization of an extracellular polysaccharide from newly isolated Azotobacter sp. SSB81

A strain was selected by its highest extracellular polysaccharide (EPS) production ability compare to other isolates from the same rhizospheric soil. The selected strain was identified by 16S rDNA sequencing and designated as SSB81. Phylogenetic analysis of the gene sequence showed its close relatedness with Azotobacter vinelandii and Azotobacter salinestris. Maximum EPS (2.52 g l⁻¹) was recovered when the basal medium was supplemented with glucose (2.0%), riboflavin (1 mg l⁻¹) and casamino acid (0.2%). The EPS showed a stable viscosity level at acidic pH (3.0–6.5) and the pyrolysis temperature was found to be at 116.73 °C with an enthalpy (ΔH) of 1330.72 Jg⁻¹. MALDI TOF mass spectrometric result suggests that polymer contained Hex₅Pent₃ as oligomeric building subunit. SEM studies revealed that the polymer had a porous structure with small pore size distribution indicating the compactness of the polymer. This novel EPS may find possible application as a polymer for environmental bioremediation and biotechnological processes.     

Members of the Family Comamonadaceae as Primary Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate)-Degrading Denitrifiers in Activated Sludge as Revealed by a Polyphasic Approach

The distribution and phylogenetic affiliations of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)-degrading denitrifying bacteria in activated sludge were studied by a polyphasic approach including culture-independent biomarker and molecular analyses as well as cultivation methods. A total of 23 strains of PHBV-degrading denitrifiers were isolated from activated sludges from different sewage treatment plants. 16S ribosomal DNA (rDNA) sequence comparisons showed that 20 of the isolates were identified as members of the family Comamonadaceae, a major group of β-Proteobacteria. When the sludges from different plants were acclimated with PHBV under denitrifying conditions in laboratory scale reactors, the nitrate removal rate increased linearly during the first 4 weeks and reached 20 mg NO₃⁻-N h⁻¹ g of dry sludge⁻¹ at the steady state. The bacterial-community change in the laboratory scale sludges during the acclimation was monitored by rRNA-targeted fluorescence in situ hybridization and quinone profiling. Both approaches showed that the population of β-Proteobacteria in the laboratory sludges increased sharply during acclimation regardless of their origins. 16S rDNA clone libraries were constructed from two different acclimated sludges, and a total of 37 clones from the libraries were phylogenetically analyzed. Most of the 16S rDNA clones were grouped with members of the family Comamonadaceae. The results of our polyphasic approach indicate that β-Proteobacteria, especially members of the family Comamonadaceae, are primary PHBV-degrading denitrifiers in activated sludge. Our data provide useful information for the development of a new nitrogen removal system with solid biopolymer as an electron donor.     

Metabolic Engineering of a Novel Propionate-Independent Pathway for the Production of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) in Recombinant Salmonella enterica Serovar Typhimurium

A pathway was metabolically engineered to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biodegradable thermoplastic with proven commercial applications, from a single, unrelated carbon source. An expression system was developed in which a prpC strain of Salmonella enterica serovar Typhimurium, with a mutation in the ability to metabolize propionyl coenzyme A (propionyl-CoA), served as the host for a plasmid harboring the Acinetobacter polyhydroxyalkanoate synthesis operon (phaBCA) and a second plasmid with the Escherichia coli sbm and ygfG genes under an independent promoter. The sbm and ygfG genes encode a novel (2R)-methylmalonyl-CoA mutase and a (2R)-methylmalonyl-CoA decarboxylase, respectively, which convert succinyl-CoA, derived from the tricarboxylic acid cycle, to propionyl-CoA, an essential precursor of 3-hydroxyvalerate (HV). The S. enterica system accumulated PHBV with significant HV incorporation when the organism was grown aerobically with glycerol as the sole carbon source. It was possible to vary the average HV fraction in the copolymer by adjusting the arabinose or cyanocobalamin (precursor of coenzyme B₁₂) concentration in the medium.     

Production of poly-3-(hydroxybutyrate-co-hydroxyvalerate) by Haloferax mediterranei using rice-based ethanol stillage with simultaneous recovery and re-use of medium salts

Haloferax mediterranei holds promise for competitive industrial-scale production of polyhydroxyalkanoate (PHA) because cheap carbon sources can be used thus lowering production costs. Although high salt concentration in production medium permits a non-sterile, low-cost process, salt disposal after process completion is a problem as current environmental standards do not allow total dissolved solids (TDS) above 2000 mg/l in discharge water. As the first objective of this work, the waste product of rice-based ethanol industry, stillage, was used for the production of PHA by H. mediterranei in shake flasks. Utilization of raw stillage led to 71 ± 2 % (of dry cell weight) PHA accumulation and 16.42 ± 0.02 g/l PHA production. The product yield coefficient was 0.35 while 0.17 g/l h volumetric productivity was attained. Simultaneous reduction of BOD₅ and COD values of stillage by 83 % was accomplished. The PHA was isolated by osmotic lysis of cells, purification by sodium dodecyl sulfate and organic solvents. The biopolymer was identified as poly-3-(hydroxybutyrate-co-15.4 mol%-hydroxyvalerate) (PHBV). This first report on utilization of rice-based ethanol stillage for PHBV production by H. mediterranei is currently the most cost effective. As the second objective, directional properties of decanoic acid together with temperature dependence of water solubility in decanoic acid were applied for two-stage desalination of the spent stillage medium. We report for the first time, recovery and re-use of 96 % of the medium salts for PHA production thus removing the major bottleneck in the potential application of H. mediterranei for industrial production of PHBV. Final discharge water had TDS content of 670 mg/l.     

Anaerobic degradation of poly-3-hydroxybutyrate and poly-3-hydroxybutyrate-co-3-hydroxyvalerate

The anaerobic degradation of the polyesters poly-3-hydroxybutyrate (PHB) and poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) was investigated with special regard to intermediate products, kinetics, and yields. During the degradation of PHBV acetate, propionate, n-butyrate, and n-valerate were detected. Additionally, 3-hydroxybutyrate and 3-hydroxyvalerate and four dimeric esters of these two molecules were identified by GC-MS measurements. Three different test systems for the anaerobic degradation of polyesters were studied. It was not possible to get reproducible results by means of the Anaerobic Sturm-test, a simple system based on carbon dioxide measurement. Secondly, a system based on the GC measurement of accumulated organic acids was investigated. A degradation of 90% in two days was calculated by a carbon balance. Best results were reached with the third test system based on the measurement of methane with a gas meter. A degradation of 99% was observed within 30 days.     

Development of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) fibers for skin tissue engineering: effects of topography, mechanical, and chemical stimuli

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), a biodegradable polyester, was electrospun to form defect-free fibers with high surface-area-to-volume ratio for skin regeneration. Several parameters such as solvent ratio, polymer concentration, applied voltage, flow rate, and tip-to-target distance were optimized to achieve defect-free morphology. The average diameter of the PHBV fibers was 724 ± 91 nm. PHBV was also solvent-cast to form 2-D films, and its mechanical properties, porosity, and degradation rates were compared with PHBV fibers. Our results demonstrate that PHBV fibers exhibited higher porosity, increased ductility, and faster degradation rate when compared with PHBV 2-D films (p < 0.05). In vitro studies with PHBV fibers and 2-D films were carried out to evaluate the adhesion, viability, proliferation, and gene expression of human skin fibroblasts. Cells adhered and proliferated on both PHBV fibers and 2-D films. However, the proliferation of cells on the surface of PHBV fibers was comparable to tissue culture polystyrene (TCPS, control) (p > 0.05). The gene expression of collagen I and elastin was significantly up-regulated when compared with TCPS control, whereas collagen III was down-regulated on PHBV fibers and 2-D film after 14 days in culture. The less ductile PHBV 2-D films showed higher levels of elastin expression. Furthermore, the PHBV fibers in the presence and absence of an angiogenesis factor (R-Spondin 1) were evaluated for their wound healing capacity in a rat model. The wound contracture in R-Spondin-1-loaded PHBV fibers was found to be significantly higher when compared with PHBV fibers alone after 7 days (p < 0.05). Furthermore, the presence of fibers promoted an increase in collagen and aided re-epithelialization. Thus our results demonstrate that the topography and mechanical and chemical stimuli have a pronounced influence on the cell proliferation, gene expression, and wound healing.     

Stenotrophomonas maltophilia Gd2: A potential and novel isolate for fibrinolytic enzyme production

The bacterium with an ability to produce extracellular fibrinolytic protease was isolated and identified as Stenotrophomonas maltophilia Gd2 based on ribotyping. The in-vitro fibrinolytic profile of this enzyme depicted 73% of fibrin clot dissolution within 4 h. Fibrinolytic enzyme yield influenced by different physiological (incubation time, temperature, agitation and pH), nutritional (macronutrients such as carbon and nitrogen sources) and biological (inoculums age and inoculums concentration) parameters of fermentation which were optimized based on one-factor-at-a-time (OFAT) approach. The enzyme yield improved from 886 to 1795 FU ml⁻¹ upon OFAT; optimized conditions include temperature – 33 °C, pH – 8.0, incubation time – 36 h, agitation – 150 RPM, 3% v/v inoculums and age of inoculum – 18 h. Further optimization of enzyme production was achieved with implementation of Plackett-Burman media designing where the production levels increased to 3411 FU ml⁻¹ and noticed that peptone, pH, dextrose and K₂HPO₄ was found to be significant factor. This ms reports the highest fibrinolytic enzyme yield with S. maltophilia to that of literature reports.     

Immobilization and characterisation of a lipase from a new source, Bacillus sp. ITP-001

A new source of lipase from Bacillus sp. ITP-001 was immobilized by physical adsorption on the polymer poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) in aqueous solution. The support and immobilized lipase were characterised, compared to the lyophilised lipase, with regard to the specific surface area, adsorption–desorption isotherms, pore volume (Vp) and size (dp) by nitrogen adsorption, differential scanning calorimetry, thermogravimetric analysis, chemical composition analysis, Fourier transform infrared spectroscopy and biochemical properties. The immobilized enzyme displayed a shift in optimum pH towards the acidic side with an optimum at pH 4.0, whereas the optimum pH for the free enzyme was at pH 7.0; the optimum temperature of activity was 80 and 37 °C for the free and immobilized enzyme, respectively. The inactivation rate constant for the immobilized enzyme at 37 °C was 0.0038 h^?1 and the half-life was 182.41 h. The kinetic parameters obtained for the immobilized enzyme gave a Michaelis–Menten constant (K _m) of 49.10 mM and a maximum reaction velocity (V _max) of 205.03 U/g. Furthermore, the reuse of the lipase immobilized by adsorption allowed us to observe that it could be reused for 10 successive cycles, duration of each cycle (1 h), maintaining 33 % of the initial activity.     

Degradation of 3-Phenoxybenzoic Acid by a Bacillus sp

3-Phenoxybenzoic acid (3-PBA) is of great environmental concern with regards to endocrine disrupting activity and widespread occurrence in water and soil, yet little is known about microbial degradation in contaminated regions. We report here that a new bacterial strain isolated from soil, designated DG-02, was shown to degrade 95.6% of 50 mg·L⁻¹ 3-PBA within 72 h in mineral salt medium (MSM). Strain DG-02 was identified as Bacillus sp. based on the morphology, physio-biochemical tests and 16S rRNA sequence. The optimum conditions for 3-PBA degradation were determined to be 30.9°C and pH 7.7 using response surface methodology (RSM). The isolate converted 3-PBA to produce 3-(2-methoxyphenoxy) benzoic acid, protocatechuate, phenol, and 3,4-dihydroxy phenol, and subsequently transformed these compounds with a q _max, K _s and K _i of 0.8615 h⁻¹, 626.7842 mg·L⁻¹ and 6.7586 mg·L⁻¹, respectively. A novel microbial metabolic pathway for 3-PBA was proposed on the basis of these metabolites. Inoculation of strain DG-02 resulted in a higher degradation rate on 3-PBA than that observed in the non-inoculated soil. Moreover, the degradation process followed the first-order kinetics, and the half-life (t _1/2) for 3-PBA was greatly reduced as compared to the non-inoculated control. This study highlights an important potential application of strain DG-02 for the in situ bioremediation of 3-PBA contaminated environments.     

Anaerobic degradation of poly-3-hydroxybutyrate and poly-3-hydroxybutyrate-co-3-hydroxyvalerate

The anaerobic degradation of the polyesterspoly-3-hydroxybutyrate (PHB) andpoly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) wasinvestigated with special regard to intermediateproducts, kinetics, and yields. During the degradationof PHBV acetate, propionate, n-butyrate, andn-valerate were detected. Additionally,3-hydroxybutyrate and 3-hydroxyvalerate and fourdimeric esters of these two molecules were identifiedby GC-MS measurements. Three different test systemsfor the anaerobic degradation of polyesters werestudied. It was not possible to get reproducibleresults by means of the Anaerobic Sturm-test, a simplesystem based on carbon dioxide measurement. Secondly,a system based on the GC measurement of accumulatedorganic acids was investigated. A degradation of 90%in two days was calculated by a carbon balance. Bestresults were reached with the third test system basedon the measurement of methane with a gas meter. Adegradation of 99% was observed within 30 days.     

Studies on intracellular degradation of polyhydroxyalkanoic acid-polyethylene glycol copolymer accumulated by Azotobacter chroococcum MAL-201

Azotobacter chroococcum MAL-201 accumulates poly(3-hydroxybutyric acid) [PHB] when grown in glucose containing nitrogen-free Stockdale medium. The same medium supplemented with valerate alone and valerate plus polyethylene glycol (PEG) leads to the accumulation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [PHBV] and PEG containing PHBV-PEG polymers, respectively. The intracellular degradation of these polymers as studied in carbon-free Stockdale medium showed a rapid degradation of PHB followed by PHBV, while it was least in case of PHBV-PEG. The rate of such degradation was 44.16, 26.4 and 17.0 mg h(-1)l(-1) for PHB, PHBV and PHBV-PEG, respectively. During the course of such of PHBV and PHBV-PEG degradation the 3HB mol% of polymers decreased significantly with increase of 3HV mol fraction, the EG mol% in PHBV-PEG, however, remained constant. After 50h of degradation the decrease in intrinsic viscosity and molecular mass of PHBV-PEG were 37.5 and 43.6%, respectively. These values appeared low compared to PHB and PHBV. Moreover, the increasing EG content of polymer retarded their extent of degradation. Presence of PEG, particularly of low molecular weight PEG was inhibitory to intracellular PHA depolymerise (i-PHA depolymerase) activity and the relative substrate specificity of the i-PHA depolymerase of MAL-201 appeared to be PHB > PHBV > PHBV-PEG.     

Recent advances in studies of the mechanisms of microbial degradation of lignins

Major advances in our understanding of the biochemical and enzymological mechanisms of lignin biodegradation have been made in the past three years. Research has principally involved two ligninolytic microorganisms, the white rot fungus Phanerochaete chrysosporium and the actinomycete Streptomyces viridosporus. Research has been centred on attempts to identify the microbial catalysts that mediate lignin decay in these two microbes. Emphasis has been on studies concerned with isolating specific lignin catabolic enzymes and/or reduced forms of oxygen involved in attacking the lignin polymer. The possibility that lignin degradation might be non-enzymatic and mediated by extracellular reduced oxygen species such as hydrogen peroxide (H₂O₂), superoxide (O₂∪c-|_.), hydroxyl radical (·OH) or singlet oxygen (¹O₂) has been investigated with both microorganisms. Using methods which have not always been unequivocal, the question of involvement of reduced oxygen species in lignin degradation by P. chrysosporium has been examined exhaustively. Evidence for the involvement of H₂O₂ is conclusive. However, there is little evidence to support the involvement of other extracellular reduced oxygen species, including ·OH, directly in the process of lignin degradation. Scavenger studies have been inconclusive because of questions of their specificity. If activated oxygen species are involved, the activated oxygen is probably held within the active site of an enzyme molecule. With S. viridosporus, scavenger studies also strongly indicate that extracellular reduced oxygen species are not involved in lignin degradation since scavengers generally do not significantly affect the ligninolytic system. The involvement of specific enzymes in lignin degradation by both P. chrysosporium and S. viridosporus has now been confirmed. With P. chrysosporium, ligninolytic enzymes recently discovered include extracellular non-specific peroxidases and oxygenases. They show numerous activities including dehydrogenative, peroxidatic, oxygenative and C_α?C_β cleavages of lignin side chains. At least one P. chrysosporium enzyme, a unique H₂O₂-requiring oxygenase, has been purified to homogeneity. Evidence has been presented to show that S. viridosporus also produces a ligninolytic enzyme complex involved in demethylation of lignin's aromatic rings and in the oxidation of lignin side chains and cleavage of β-tether linkages within the polymer. The combined activites of these enzymes generate water-soluble polymeric modified lignin fragments, which are then slowly degraded further by S. viridosporus. The β-ether cleaving enzyme complex is probably membrane associated, but it is not extracellular. These first isolations of ligninolytic enzymes have changed the course of basic research on lignin biodegradation. New research priorities are already emerging and include enzyme purifications, kinetic studies, enzyme reaction mechanism studies and screenings for more enzymes. In addition, genetic studies are being carried out with both P. chrysosporium and S. viridosporus. Genetic manipulations include not only classical mutagenesis techniques, but also recombinant DNA techniques such as protoplast fusion. This latter technique has already been used to generate overproducers of the ligninolytic enzyme complex in S. viridosporus and it has been successfully used to recombine mutant strains of P. chrysosporium.     

In vitro effect of pH and ethanol on biofilm formation by clinicalica-positiveStaphylococcus epidermidis strains

Biofilm production is an important step in the pathogenesis ofStaphylococcus epidermidis associated biomaterial infections.Staphylococcus epidermidis strains isolated from dialysis fluid (n=9) and needle cultures (n=14) were phenotyped and genotyped for extracellular polysaccharide production and were examined for their ability to produce slime in a medium at various pH levels (3, 5, 7, 9 and 12) and with ethanol supplementation (0, 2, 5, 10 and 15%) using a semi-quantitative adherence assay. A total of 23 clinicalicaADBC positiveS. epidermidis, one reference strain (S. epidermidis CIP 106510) used as positive control, and oneicaADBC negative strain (E21) were investigated. Qualitative biofilm production analysis revealed that 15 of the 23icaADBC positive strains (65.21%) produced slime on Congo Red agar plates. Quantitative biofilm was determined by measuring the optical density at 570 nm (OD₅₇₀). The results show that the slime production depended on the pH value of the medium and the ethanol concentration. At highly acidic (pH 3) and alkaline (pH 12) levels, the OD₅₇₀ was lower, while at pH 7 the adhesion was moderate. In addition the cells adhered strongly with 2% ethanol than with the other concentrations. Our results suggest that pH and ethanol were stress factors that led toS. epidermidis biofilm formation and also play a possible role in the pathogenesis of biomaterial-related infections.     

An organic solvent-, detergent-, and thermostable alkaline protease from the mesophilic, organic solvent-tolerant Bacillus licheniformis 3C5

Bacillus licheniformis 3C5, isolated as mesophilic bacterium, exhibited tolerance towards a wide range of non-polar and polar organic solvents at 45°C. It produced an extracellular organic solvent-stable protease with an apparent molecular mass of approximately 32 kDa. The inhibitory effect of PMSF and EDTA suggested it is likely to be an alkaline serine protease. The protease was active over a broad range of temperatures (45–70°C) and pH (8–10) range with an optimum activity at pH 10 and 65°C. It was comparatively stable in the presence of a relatively high concentration (35% (v/v)) of organic solvents and various types of detergents even at a relatively high temperature (45°C). The protease production by B. licheniformis 3C5 was growth-dependent. The optimization of carbon and nitrogen sources for cell growth and protease production revealed that yeast extract was an important medium component to support both cell growth and the protease production. The overall properties of the protease produced by B. licheniformis 3C5 suggested that this thermo-stable, solvent-stable, detergent-stable alkaline protease is a promising potential biocatalyst for industrial and environmental applications.     

Hydrogenase-3 Contributes to Anaerobic Acid Resistance of Escherichia coli

Background

Hydrogen production by fermenting bacteria such as Escherichia coli offers a potential source of hydrogen biofuel. Because H₂ production involves consumption of 2H⁺, hydrogenase expression is likely to involve pH response and regulation. Hydrogenase consumption of protons in E. coli has been implicated in acid resistance, the ability to survive exposure to acid levels (pH 2–2.5) that are three pH units lower than the pH limit of growth (pH 5–6). Enhanced survival in acid enables a larger infective inoculum to pass through the stomach and colonize the intestine. Most acid resistance mechanisms have been defined using aerobic cultures, but the use of anaerobic cultures will reveal novel acid resistance mechanisms.

Methods and Principal Findings

We analyzed the pH regulation of bacterial hydrogenases in live cultures of E. coli K-12 W3110. During anaerobic growth in the range of pH 5 to 6.5, E. coli expresses three hydrogenase isoenzymes that reversibly oxidize H₂ to 2H⁺. Anoxic conditions were used to determine which of the hydrogenase complexes contribute to acid resistance, measured as the survival of cultures grown at pH 5.5 without aeration and exposed for 2 hours at pH 2 or at pH 2.5. Survival of all strains in extreme acid was significantly lower in low oxygen than for aerated cultures. Deletion of hyc (Hyd-3) decreased anoxic acid survival 3-fold at pH 2.5, and 20-fold at pH 2, but had no effect on acid survival with aeration. Deletion of hyb (Hyd-2) did not significantly affect acid survival. The pH-dependence of H₂ production and consumption was tested using a H₂-specific Clark-type electrode. Hyd-3-dependent H₂ production was increased 70-fold from pH 6.5 to 5.5, whereas Hyd-2-dependent H₂ consumption was maximal at alkaline pH. H₂ production, was unaffected by a shift in external or internal pH. H₂ production was associated with hycE expression levels as a function of external pH.

Conclusions

Anaerobic growing cultures of E. coli generate H₂ via Hyd-3 at low external pH, and consume H₂ via Hyd-2 at high external pH. Hyd-3 proton conversion to H₂ is required for acid resistance in anaerobic cultures of E. coli.