Gene Cloning and Characterization of a Poly(<Emphasis Type="SmallCaps">l</Emphasis>-Lactic Acid) Depolymerase from <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. Strain DS04-T

A gene encoding a poly(l-lactic acid) (PLA) depolymerase from Pseudomonas sp. strain DS04-T was cloned and overexpressed in Escherichia coli. The recombinant PLA depolymerase with a molecular weight of 19.2 kDa was purified to homogeneity. The optimum pH and temperature of the PLA depolymerase are 8.5 and 60 °C, respectively. K⁺, Ca²⁺ and Ni²⁺ enhance the enzyme activity, while Na⁺, Zn²⁺, Mg²⁺, Cu²⁺, Fe²⁺, Mn²⁺ and Co²⁺ inhibit it. The inhibition of different chemicals on the PLA depolymerase activity were examined, in which EDTA was found to have a significantly inhibitory effect. The main degradation product of the depolymerase is identified as lactic acid monomer by mass spectrometric analysis. Physicochemical properties, substrate specificity and sequence analysis indicated that PME is a new type of PLA depolymerase.     

Purification and Properties of Extracellular Poly(3-hydroxybutyrate) Depolymerase Produced by <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> 202

An extracellular poly(3-hydroxybutyrate) (PHB) depolymerase produced by a thermotolerant fungal soil isolate, Aspergillus fumigatus 202, was purified and characterized. Maximum PHB depolymerase production was obtained at the end of 48 h with initial medium pH 7.0 and 45 °C in Bushnell Haas Minerals medium containing PHB as sole source of carbon. The PHB depolymerase was purified using size exclusion chromatography to a fold purification of 20.62 and 61.62% yield. SDS-PAGE and isoelectric focusing revealed the molecular weight and pI of the purified enzyme as 63,744 Da and 4.2, respectively. N-terminal amino acid sequence of purified enzyme was HAXDAYLVK. This non-glycosylated enzyme was most active at pH 9.0 and 45 °C. Purified enzyme was inactivated by N-bromosuccinimide and dithiothreitol suggesting the involvement of tryptophan residues and disulfide bonds at its active site. Nonionic detergents like Tween 20, Tween 80 and Triton X-100 inhibited the enzyme activity. Ions like Ca⁺² and Mg⁺² (5 mM) increased the enzyme activity 1.5 times. Fe⁺² effectively inhibited the enzyme activity to 88% whereas Hg⁺² completely inhibited the enzyme.     

The Properties of Poly(<Emphasis Type="SmallCaps">l</Emphasis>-Lactide) Prepared by Different Synthesis Procedure

Different synthesis methods were applied to determine optimal conditions for polymerization of (3S)-cis-3,6-dimethyl-1,4-dioxane-2,5-dione (l-lactide), in order to obtain poly(l-lactide) (PLLA). Bulk polymerizations (in vacuum sealed vessel, high pressure reactor and in microwave field) were performed with tin(II) 2-ethylhexanoate as the initiator. Synthesis in the vacuum sealed vessel was carried out at the temperature of 150 °C. To reduce the reaction time second polymerization process was carried out in the high pressure reactor at 100 °C and at the pressure of 138 kPa. The third type of rapid synthesis was done in the microwave reactor at 100 °C, using frequency of 2.45 GHz and power of 150 W at the temperature of 100 °C. The temperature in this method was controlled via infrared system for in-bulk measuring. The solution polymerization (with trifluoromethanesulfonic acid as initiator) was possible even at the temperature of 40 °C, yielding PLLA with narrow molecular weight distribution in a very short period of time (less than 6 h). The obtained polymers had the number-average molecular weights ranging from 43,000 to 178,000 g mol⁻¹ (polydispersity index ranging from 1 to 3) according to the gel permeation chromatography measurements. The polymer structure was characterized by Fourier transform infrared and NMR spectroscopy. Thermal properties of the obtained polymers were investigated using thermogravimetry and differential scanning calorimetry.     

Hydrolysis of <Emphasis Type="Italic">Sasa senanensis</Emphasis> culm with dilute sulfuric acid for production of a fermentable substrate

To prepare a substrate for microbial conversion of xylose into xylitol, the culm of Sasa senanensis was hydrolyzed with dilute sulfuric acid. When the reaction temperature was fixed at 121°C, an optimum yield of xylose was obtained by treatment with 2% sulfuric acid for 1 h. An increase in the sulfuric acid concentration or a prolonged reaction time resulted in a decrease in the xylose yield. A fermentable substrate with a relatively high xylose concentration (36.7 g l⁻¹) was obtained by hydrolysis with 2% sulfuric acid with a liquid-to-solid ratio of 5 g g⁻¹. During hydrolysis at elevated temperatures, certain undesired byproducts were also generated, such as degradation products of solubilized sugars and lignin, which are potential inhibitors of microbial metabolism. These compounds were, however, successfully removed from the hydrolysate by treatment with activated char.     

Enzyme-Assisted Extraction and Pb<Superscript>2+</Superscript> Biosorption of Polysaccharide from <Emphasis Type="Italic">Cordyceps militaris</Emphasis>

The enzyme assisted extraction conditions of polysaccharide from Cordyceps militaris mycelia were firstly investigated by kinetics analysis and the optimal operating was found to be: extraction temperature 40 °C; solid-solvent ratio 1:20; extraction pH 4.0; cellulase concentration 2.0%. The polysaccharide extraction yield was 5.99% under these optimized conditions. Furthermore, a fundamental investigation of the biosorption of Pb²⁺ from aqueous solution by the C. militaris polysaccharide was performed under batch conditions. The suitable pH (5.0), polysaccharide concentration (0.20 g L^?1), initial Pb²⁺ concentration (300 mg L^?1) and contact time (40 min) were outlined to enhance Pb²⁺ biosorption from aqueous medium. The Langmuir isotherm model and pseudo first order kinetic model fitted well to the data of Pb²⁺ biosorption, suggesting the biosorption of Pb²⁺ onto C. militaris polysaccharide was monolayer biosorption and physical adsorption might be the rate-limiting step that controlled the adsorption process. FTIR analysis showed that the main functional groups of C. militaris polysaccharide involved in adsorption process were carbonyl, carboxyl, and hydroxyl groups.     

Kinetic Analysis of the Temperature Effect on Polyhydroxyalkanoate Production by <Emphasis Type="Italic">Haloferax mediterranei</Emphasis> in Synthetic Molasses Wastewater

Haloferax mediterranei is an extremely halophilic archaeon that is able to synthesize polyhydroxyalkanoate (PHA) in high salt environment with low sterility demand. In this study, a mathematical model was validated and calibrated for describing the kinetic behavior of H. mediterranei at 15, 20, 25, and 35 °C in synthetic molasses wastewater. Results showed that the production of PHA by H. mediterranei, ranging from 390 to 620 mg h^?1 L^?1, was strongly dependent on the temperature. The specific growth rate (µ _max), specific substrate utilization rate (q _max), and specific decay rate (k _d) of H. mediterranei increased with temperature following Arrhenius equation prediction. The estimated activation energy was 58.31, 25.59, and 22.38 kJ mol^?1 for the process of cell growth, substrate utilization, and cell decay of H. mediterranei, respectively. The high temperature triggered the increased PHA storage even without nitrogen limitation. Thus, working at high temperatures seems a good strategy for improving the PHA productivity of H. mediterranei.     

An Original Halo-Alkaline Protease from <Emphasis Type="Italic">Bacillus halodurans</Emphasis> Strain US193: Biochemical Characterization and Potential Use as Bio-Additive in Detergents

Over a hundred of halophilic/halotolerant microorganisms were screened for alkaline protease production. The bacterium showing the highest enzyme production was characterized and identified as Bacillus halodurans US193 on the basis of 16S rRNA gene analysis. It was alkalophilic, thermophilic and halotolerant since it grew optimally at pH 9.7 and 50?°C with tolerance of up to 125 g NaCl l^?1. The alkaline protease was purified 4.9 times with about 40186.1 U/mg as specific activity. It exhibited optimal activity at pH 10, 70?°C and 0.25 M NaCl with perfect stability at wide ranges of pH (6–12), temperatures (30–60?°C) and NaCl concentrations (0–2 M). The serine alkaline protease maintained high stability in the presence of Cu²⁺, Mg²⁺, Ba²⁺ and Ca²⁺ ions, various organic solvents [50% (v/v)] and ionic and non ionic detergent additives. In addition, it was more compatible with various commercialized detergents than other reported detergent proteases, and was very efficient in blood stain removal. These findings let B. halodurans US193 alkaline protease be an ideal candidate for many industrial processes at harsh conditions, especially as a bio-additive in detergent industry.     

<Emphasis Type="Italic">Mesua ferrea</Emphasis> L. Seed Oil Based Highly Branched Environment Friendly Polyester Resin/Clay Nanocomposites

To develop a high performance environment friendly material, highly branched polyester/clay nanocomposites have been prepared from Mesua ferrea Linn seed oil-based polyester resin and hydrophilic bentonite nanoclay. The prepared nanocomposites were characterized by Fourier transform infra-red spectroscopy, X-ray diffractometer, scanning electron microscope, transmission electron microscope and rheological studies. Partial exfoliation of clay layers by the polymer chains with good interfacial interactions was observed in the nanocomposites. The formation of delaminated nanocomposites was manifested through the enhancement of tensile strength, scratch hardness, chemical resistance, impact resistance, thermostability, etc. The results show enhancement of three times in tensile strength and 18 °C in thermostability by inclusion of 5 wt% nanoclay as compared to the pristine polymer. By the influence of 5 wt% nanoclay four times enhancement in elongation at break as compared to the pristine polymer was noticed. Thus these nanocomposites have the potential to be used in many advanced applications.     

Effect of Solvent Composition on Porosity,Surface Morphology and Thermal Behavior of Metal Alginate Prepared from Algae (<Emphasis Type="Italic">Undaria pinnatifida</Emphasis>)

Alginates, extracted from algae are linear unbranched polymers containing β-(1→4)-linked d-mannuronic acid (M) and α-(1→4)-linked l-guluronic acid (G) residues. The conversion of alginic acid into the metal alginate is confirmed using FTIR spectroscopy. Asymmetric and symmetric stretching of free carboxyl group present in metal alginate occurs almost at the same position in various solvent compositions. Total intrusion volume of metal alginate prepared in propanol (0.0742 mL/g) is greater compared to those in ethanol (0.0648 mL/g) and methanol (0.0393 mL/g) as solvent. Surface morphology as well as porosity and pore size distribution of metal alginate are greatly influenced by solvent. It can be seen from thermal analysis results that calcium alginate prepared using different solvent compositions started decomposing at 100 °C, but rapid degradation started around 200 °C. The results showed a stepwise weight loss during thermal sweep, indicating different types of reactions during degradation. First and second step of rapid degradation was situated around 200–300 and 300–550 °C, respectively; whereas the final step is situated around 550–650 °C. The trend of degradation was similar for all the solvents, although the amount of final residue varied from one solvent to another. At the same time, lower thermal stability was also observed with higher heating rates. Additionally, a kinetic analysis was performed to fit with TGA data, where the entire degradation process has been considered as three consecutive first order reactions.     

Synthesis and Properties of <Emphasis Type="Italic">N</Emphasis>-Maleyl Chitosan-Cross-Linked Poly(Acrylic Acid-co-Acrylamide) Superabsorbents

A high-swelling superabsorbent was synthesized with biodegradable N-maleyl chitosan as cross-linker, acrylic acid (AA) and acrylamide (AM) as the monomers, ammonium peroxodisulfate–sodium bisulfite (NaHSO₃) as redox initiation system, by means of aqueous solution polymerization. The best reaction condition was based on the orthogonal experiment design. The optimal conditions on distilled water absorbency and on 0.9 wt% NaCl solution absorbency were monomer concentration 20 wt%, mole ratio of AA to (AA + AM) 60%, the neutralization degree of AA 40%, cross-linker concentration 2% and monomer concentration 25 wt%, mole ratio of AA to (AA + AM) 60%, neutralization degree of AA 50% and cross-linker concentration 1%, respectively. Factors influencing the water absorbency of superabsorbent also were investigated, by single factor experiment method. The absorbency of superabsorbents in distilled water and 0.9 wt% NaCl solution increased and then decreased with the increasing of monomer concentration, mole ratio of AA to (AA + AM) and degree of neutralization of AA. With the increasing of cross-linker concentration, the absorbency in distilled water increased and then decreased, but it decreased all the time in 0.9 wt% NaCl solution. In enzymatic degradation tests, the weight loss of superabsorbent was related to the content of cross-linker.     

Investigation of Biosurfactant Activity and Asphaltene Biodegradation by <Emphasis Type="Italic">Bacillus cereus</Emphasis>

In this research, a biosurfactant-producing bacterium with capability of asphaltene degradation was isolated from oil-contaminated soil samples, and identified as Bacillus cereus. This strain produced an effective biosurfactant in the presence of molasses and the surface tension was reduced to the level of 36.4 mN/m after 48 h under optimum conditions. The optimum values of carbon-to-nitrogen ratio (C:N), pH, and temperature for biosurfactant production were determined as 30:1, 7.3 and 29 °C, respectively, using response surface methodology. The maximum emulsification activity in the culture broth was 53 % after 48 h using kerosene at 25 °C. The goodness of fit of four growth kinetic models including Tessier, Contois, Logistic and Westerhoff was compared for the bacterial growth and molasses utilization of B. cereus in 5-L batch bioreactor during 120 h. Conducted kinetic study showed that biosurfactant production had a good fit with the Contois growth kinetic model (R² = 0.962) and the maximum specific growth rate (µ _max ), saturation constant (K _s ) and the yield of biomass per substrate (Y _x/s ) were determined to be 0.145 h^?1, 1.83 g/L and 0.428 g/g, respectively. The asphaltene biodegradation in flask was evaluated by FTIR analysis and quantified by a spectrophotometer. This bacterium was able to degrade up to 40 % of asphaltene as a sole carbon and energy source after 60 days at 28 °C. The resulting surface tension of 30.2 mN/m with the critical micelle concentration of 23.4 mg/L indicated good efficiency of the biosurfactant.     

Kinetic Study of Biopolymer (PHB) Synthesis in <Emphasis Type="Italic">Alcaligenes</Emphasis> sp. in Submerged Fermentation Process Using TEM

Poly-β-hydroxybuyrate (PHB) is a carbon—energy storage material which is accumulated as intracellular granule in variety of microorganism under nutrient starved conditions. Solid PHB is a biodegradable thermoplastic polymer and is utilizable in various ways similar to many conventional plastics. Ralstonia eutropha (Alcaligenes sp.), a gram negative bacteria accumulates PHB as insoluble granules inside the cells when nutrients other than carbon are limited. In this report effort has been made to analyze PHB granule synthesis inside Alcaligenes sp. NCIM 5085 by transmission electron microscopy and qualitative estimation of PHB was carried out by fourier transform infrared spectroscopy which provide better precision compared to other conventional techniques previously applied for PHB determination. Maximum PHB concentration of 2.20 ± 0.40 g/L and cell biomass of 3.42 ± 0.20 g/L was obtained after 48.0 h of fermentation. Leudking-Piret equation deduced mixed growth associated product formation which varies from earlier reports.     

Degradation of a Terephthalate-containing Polyester by Thermophilic Actinomycetes and <Emphasis Type="Italic">Bacillus</Emphasis> Species Derived from Composts

We intended to find thermophilic degraders of terephthalate-containing Biomax^® films. Films in mesh bags were buried in composts (inside temperature: approximately 55–60 °C), resulting in the degradation of them in 2 weeks. Fluorescent microscopy of films recovered from composts showed that microorganisms gradually covered the surface of a film during composting. DGGE analysis of microorganisms on the composted film indicated the presence of Bacillus species as main species (approximately 80% of microbial flora) and actinomycetes (approximately 10–20%) as the second major flora. Isolation of Biomax^®-utilizing bacteria was focused on these two genera: two actinomycetes and one Bacillus species were isolated as pure best degraders from the composted polymer films, which were fragmented into small pieces. All the strains were thermophilic and identified, based on their 16S rDNA analyses. Degradation of polymer films was confirmed by (1) accelerated fragmentation of films in composts, compared with a control (no inoculum) and resultant decrease in molecular weights, (2) growth in a powdered Biomax^® medium, compared with a control without powdered Biomax^®, and (3) production of terephthalate in a powdered Biomax^® medium. In this way, we concluded that these bacteria were useful for degradation of thermostable Biomax^® products.     

A Microscopic Study of Paper Decayed by <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> and <Emphasis Type="Italic">Paecilomyces variotii</Emphasis>

The current study is interested in evaluating the decay of cotton, Whatman and chemical pulp caused by Trichoderma harzianum and Paecilomyces variotii. The structural changes of the paper were evaluated by Infrared Spectroscopy (FTIR) and Scanning Electron Microscope (SEM). The SEM results show differences in hyphae colonization and paper decay patterns between studied species under the current study; P. variotii caused an eroded structure in the cotton (cavity forming), whereas the initial T. harzianum colonization produced rupture and erosion (soft-rot decay type II) for the three types of paper ,the gaps were elongated with sharp pointed ends, which consisted either of individual cavities or in chains. Moreover, FTIR results confirmed that there a relationship could be observed between fungal decay and crystalline cellulose content because the intensity of peaks at 1335 and 1111 cm^?1 significantly decreased due to the fungal decay. Furthermore, the intensity of O–H stretching absorption slightly decreased, and this may be attributed to hydrolysis of cellulose molecules.     

Optimization of Poly(<Emphasis Type="SmallCaps">dl</Emphasis>-Lactic Acid) Degradation and Evaluation of Biological Re-polymerization

Poly(dl-lactic acid) or PLA is a biodegradable polymer. It has received much attention since it plays an important role in resolving the global warming problem. The protease produced by Actinomadura keratinilytica strain T16-1 was previously reported as having PLA depolymerase potential and being applicable to PLA biodegradation, which was used in this work. Therefore, this research demonstrates the important basic knowledge on the biological degradation process by the crude PLA-degrading enzyme from strain T16-1. Its re-polymerization was evaluated. The optimization of PLA degradation by statistical methods based on central composite design was determined. Approximately 6700 mg/l PLA powder was degraded by the crude enzyme under optimized conditions: an initial enzyme activity of 200 U/ml, incubated at 60 °C for 24 h released 6843 mg/l lactic acid with 82% conversion, which was similar to the commercial enzyme proteinase K (81%). The degradable products were re-polymerized repeatedly by using commercial lipase as a catalyst under a nitrogen atmosphere for 6 h. A PLA oligomer was achieved with a molecular weight of 378 Da (n = 5). This is the first report to demonstrate the high efficiency of the enzyme to degrade 100% of PLA powder and to show the biological recycling process of PLA, which is promising for the treatment and utilization of biodegradable plastic wastes in the future.     

Preparation and Characterization of Collagens from the Skin of Northern Snakehead (<Emphasis Type="Italic">Channa argus)</Emphasis>

Channa argus, a type of snakehead fish native to China, is a popular food fish in certain Asian countries but is a known destructive invasive species in the US. In this study, the two collagens, i.e. acid-soluble collagen (ASC) and pepsin-solubilized collagen (PSC), were obtained from C. argus skin. The yield of ASC was 28.0% and that of PSC was 16.8% on the dry bases. The collagens were identified as the collagen of type I by SDS–PAGE patterns. The Tds were approximately 27.0?°C. Similar ultraviolet spectra of both collagens were observed. Fourier Transform infrared spectra indicated PSC structure had a little change due to the loss of terminal domains by pepsin digestion. The results of XRD proved that the two collagens retained their helical structures. The results suggest that the collagens isolated from C. argus can potentially be alternative sources of vertebrate collagens for use in the food and other industries.     

Biodegradation of Poly(ε-caprolactone) (PCL) Film by <Emphasis Type="Italic">Alcaligenes faecalis</Emphasis>

The biodegradation behavior of PCL film with high molecular weight (80,000 Da) in presence of bacterium Alcaligenes faecalis and the analysis of degraded polymer film have been carried out. Thin Films of PCL were prepared by means of solution casting method and the bacterial degradation behavior was carried in basal medium, in presence of bacteria with time variation after UV treatment. It was observed that after UV treatment the degradation of polymer film was increased and the degradation rate followed a three steps degradation mechanism. The degraded polymer film was analyzed by means of Differential Scanning Calorimeter (DSC), Thermo Gravimetric Analyzer (TGA) and Fourier Transform Infrared Spectroscope (FTIR). DSC results revealed that at the initial stages of the degradation up to 15–20 days, the bacterium preferentially degrades the amorphous parts of the polymer film over the crystalline zone. Thermo gravimetric analysis highlighted the low temperature stability of degraded films with extent of degradation. FTIR results showed the chain scission mechanism of the polymer chains and also supported the preferential degradation of amorphous phase over crystalline phase in the initial stages of the degradation.     

Selective catalytic reduction of NO<Subscript><Emphasis Type="Italic">x</Emphasis></Subscript> using RDF char and municipal solid waste char based catalyst

Experiments were performed in order to investigate the possibility for the development of catalysts for low-temperature selective catalytic reduction (SCR) using municipal waste char and RDF byproduct. Physical and chemical activations, using water, and HCl and KOH, were employed to increase the catalytic activities. The characteristics of the activated catalysts were investigated using N₂ adsorption–desorption and FT-IR. The catalysts activated chemically using basic treatment showed higher NO _x removal efficiencies than those activated physically or chemically using acidic treatment. The de-NO _x performance of the activated catalysts was dependent on the chemical properties, such as oxygen functional groups as well as physical properties, such as specific surface area and pore volume. In order to investigate the effect of MnO _x , which has been reported to be efficient for the removal of NO _x in low-temperature SCR processes, the chemically activated catalyst was impregnated with manganese. The Mn-impregnated catalyst had the highest NO _x conversion at all of the temperatures tested in this study.     

Water Retention Capacity of Polysaccharides from Prickly Pear Nopals of <Emphasis Type="Italic">Opuntia Ficus Indica</Emphasis> and <Emphasis Type="Italic">Opuntia Litoralis</Emphasis>: Physical–Chemical Approach

Polysaccharides were isolated from nopals mucilage pulp and peel of Opuntia Ficus Indica (OFI) and Opuntia litoralis (OL) by aqueous extraction and purified by ultrafiltration. Studying the glycosyl residue composition, these polysaccharides were assumed to be rhamnogalacturonan I (RG-I). The macromolecular features of these compounds have been characterized by SEC/MALLS and by low shear viscosimetry. In the present work, we have undertaken a comparative study about different polysaccharides resulting from OFI and OL growing in different area. This comparison is to see the influence of the geographical area in which these two plants push on the mechanism of retention of water by the different polysaccharides extract. The polysaccharides resulting from the nopal peels of the two plants are highly methylated (>70%), thus they are much more hydrophobic especially for peels of OFI growing in the desert area than those resulting from pulps. Consequently, they probably prevent the evaporation of water in nopals by increasing their water retention capacity. Prickly pear nopals of OFI and OL contain a significant amount of water (>80%), carbohydrates (75% compared to the soluble matter), proteins (8% compared to the soluble matter) and salt (17% compared to the soluble matter). Thus, they represent an important source of water and alimentation especially in the arid and semi-arid areas.     

Adsorption of Crystal Violet Dye from Aqueous Solution by Poly(Acrylamide-<Emphasis Type="Italic">co</Emphasis>-Maleic Acid)/Montmorillonite Nanocomposite

Poly(acrylamide-co-maleic acid)/montmorillonite nanocomposites, were synthesized via in situ polymerization with different maleic acid and MMT content. The capability of the hydrogel for adsorption of crystal violet (CV) was investigated in aqueous solutions at different pH values and temperatures. The pseudo-second-order kinetics model could fit successfully the adsorption kinetic data. The effects of maleic acid to acrylamide molar ratio (MA_R), weight percent of MMT (MMT%), the pH of medium and the solution temperature (T) on the CV adsorption capacity (q _e ) of adsorbents were studied by Taguchi experimental design approach. The results indicated that increasing the MMT% leads to a greater q _e . The q _e value of adsorbents increased also with increasing both MA_R and pH, while reduced when the temperature of medium increased. The relatively optimum conditions to achieve a maximum CV adsorption capacity for P(AAm/MA)/MMT adsorbents were found as: 0.06 for MA_R and 5 % of MMT%, medium pH = 7 and T = 20 °C.