Elena Ionata

Application of a new xylanase activity from Bacillus amyloliquefaciens XR44A in brewer's spent grain saccharification

Background Cellulases and xylanases are the key enzymes involved in the conversion of lignocelluloses into fermentable sugars. Western Ghat region (India) has been recognized as an active hot spot for the isolation of new microorganisms. The aim of this work was to isolate new microorganisms producing cellulases and xylanases to be applied in brewers spent grain saccharification. Results 93 microorganisms were isolated from Western Ghat and screened for the production of cellulase and xylanase activities. Fourteen cellulolytic and seven xylanolytic microorganisms were further screened in liquid culture. Particular attention was focused on the new isolate Bacillus amyloliquefaciens XR44A, producing xylanase activity up to 10.5 U mL−1. A novel endo-1,4-beta xylanase was identified combining zymography and proteomics and recognized as the main enzyme responsible for B. amyloliquefaciens XR44A xylanase activity. The new xylanase activity was partially characterized and its application in saccharification of brewers spent grain, pretreated by aqueous ammonia soaking, was investigated. Conclusion The culture supernatant of B. amyloliquefaciens XR44A with xylanase activity allowed a recovery of around 43% xylose during brewers spent grain saccharification, similar to the value obtained with a commercial xylanase from Trichoderma viride, and a maximum arabinose yield of 92%, around 2-fold higher than that achieved with the commercial xylanase.

A Novel Thermoacidophilic Cellulase from Alicyclobacillus acidocaldarius

A novel cellulase was isolated from the thermoacidophilic bacterium Alicyclobacillus acidocaldarius ATCC27009 grown in medium containing carboxymethylcellulose. The enzyme is a glycosylated monomer of 56.2 kDa, relatively thermostable, with optimal pH and temperature of 4.0 and 65 degrees C, respectively. Enzymatic assays on several polysaccharides demonstrated that CelG was specific for carboxymethylcellulose.

The effect of Pleurotus ostreatus arabinofuranosidase and its evolved variant in lignocellulosic biomasses conversion.

The fungal arabinofuranosidase from Pleurotus ostreatus PoAbf recombinantly expressed in Pichia pastoris rPoAbf and its evolved variant rPoAbf F435Y/Y446F were tested for their effectiveness to enhance the enzymatic saccharification of three lignocellulosic biomasses, namely Arundo donax, corn cobs and brewers spent grains (BSG), after chemical or chemical-physical pretreatment. All the raw materials were subjected to an alkaline pretreatment by soaking in aqueous ammonia solution whilst the biomass from A. donax was also pretreated by steam explosion. The capability of the wild-type and mutant rPoAbf to increase the fermentable sugars recovery was assessed by using these enzymes in combination with different (hemi)cellulolytic activities. These enzymatic mixtures were either entirely of commercial origin or contained the cellulase from Streptomyces sp. G12 CelStrep recombinantly expressed in Escherichia coli in substitution to the commercial counterparts. The addition of the arabinofuranosidases from P. ostreatus improved the hydrolytic efficiency of the commercial enzymatic cocktails on all the pretreated biomasses. The best results were obtained using the rPoAbf evolved variant and are represented by increases of the xylose recovery up to 56.4%. These data clearly highlight the important role of the accessory hemicellulolytic activities to optimize the xylan bioconversion yields.

Biodegradation of poly-ε-caprolactone/poly-β-hydroxybutyrate blend

Poly-e-caprolactone (PCL), poly-β-hydroxybutyrate (PHB) and one of their blends have been incubated in presence of some microbial consortia isolated from plastic-contaminated environments. In the conditions used in this work, both pure PCL and PHB samples were degraded with strong erosion of the amorphous zones. The PCL/PHB 70/30 blend showed, after only 20 days of incubation, that spheres of PCL were bordering with spherulites of PHB indicating complete degradation. The crystallinity content of homopolymers and blend were investigated at different degradation times: whilst PCL crystallinity remains constant, both PHB and the blend PHB-phase crystallinity increased. Data from differential scanning calorimetry are well fitted with those obtained by scanning electron microscopy, gel permeation chromatography and weight loss analysis.

Lignocellulose-Adapted Endo-Cellulase Producing Streptomyces Strains for Bioconversion of Cellulose-Based Materials

Twenty-four Actinobacteria strains, isolated from Arundo donax, Eucalyptus camaldulensis and Populus nigra biomass during natural biodegradation and with potential enzymatic activities specific for the degradation of lignocellulosic materials, were identified by a polyphasic approach. All strains belonged to the genus Streptomyces (S.) and in particular, the most highly represented species was Streptomyces argenteolus representing 50% of strains, while 8 strains were identified as Streptomyces flavogriseus (synonym S. flavovirens) and Streptomyces fimicarius (synonyms Streptomyces acrimycini, Streptomyces baarnensis, Streptomyces caviscabies, and Streptomyces flavofuscus), and the other four strains belonged to the species Streptomyces drozdowiczii, Streptomyces rubrogriseus, Streptomyces albolongus, and Streptomyces ambofaciens. Moreover, all Streptomyces strains, tested for endo and exo-cellulase, cellobiase, xylanase, pectinase, ligninase, peroxidase, and laccase activities using qualitative and semi-quantitative methods on solid growth medium, exhibited multiple enzymatic activities (from three to six). The 24 strains were further screened for endo-cellulase activity in liquid growth medium and the four best endo-cellulase producers (S. argenteolus AE58P, S. argenteolus AE710A, S. argenteolus AE82P, and S. argenteolus AP51A) were subjected to partial characterization and their enzymatic crude extracts adopted to perform saccharification experiments on A. donax pretreated biomass. The degree of cellulose and xylan hydrolysis was evaluated by determining the kinetics of glucose and xylose release during 72 h incubation at 50°C from the pretreated biomass in the presence of cellulose degrading enzymes (cellulase and β-glucosidase) and xylan related activities (xylanase and β-xylosidase). The experiments were carried out utilizing the endo-cellulase activities from the selected S. argenteolus strains supplemented with commercial β-gucosidase and xylanase preparations from Genencore (Accellerase BG and Accellerase XY). Cellulose and xylan conversion, when conducted using commercial (hemi)cellulases, gave glucose and xylose yields of 30.17 and 68.9%, respectively. The replacement of the cellulolytic preparation from Genencor (Accellerase 1500), with the endo-cellulase from S. argenteolus AE58P resulted in almost 76% of the glucose yield obtained in the presence of the commercial counterpart. Due to the promising results obtained by using the enzymatic crude extracts from S. argenteolus AE58P in the pretreated A. donax saccharification experiments, the proteins putatively responsible for endo-cellulase activity in this strain were identified by proteomics. Several proteins were confidently identified in different Streptomyces spp., eight of which belong to the class of Carbohydrate active enzymes. Overall results highlighted the biotechnological potential of S. argenteolus AE58P being an interesting candidate biocatalyst-producing bacterium for lignocellulose conversion and production of biochemicals and bioenergy.

Olive mill wastewater anaerobically digested : phenolic compounds with antiradical activity

The recovery of phenolic compounds, present in the olive fruits and its by-products, has been intensively studied by the antioxidant properties. Olive mill wastewater (OMW) is a phenolic-rich industrial effluent that can be advantageously valorized by the anaerobic digestion to the methane and agricultural fertilizer productions. The objective of this work was to evaluate the antiradical activity of OMW after anaerobic digestion in order to maximize the valorization of this type of effluents. The digested flow was obtained from an anaerobic hybrid reactor treating OMW at different organic loading rates (OLR), from 3.3 to 7.1 kg COD m 3 d -1 . OLR rise was applied by increasing progressively the OMW volume fraction from 8 % to 83 % in the feed mixture. The input and output streams, obtained at different OMW volume fractions, were characterized in terms of antiradical activity and phenolic compounds identification and quantification. Despite of the fraction decrease on total phenolic compounds provided by OMW anaerobic digestion, the antiradical activity is still significantly high (EC50 = 3.24) in the digested effluent. Oleuropein was the main phenolic compound present in the substrate before and after anaerobic digestion (about 15 % of the initial value). Others phenolic compounds present are: gallic acid, hydroxytyrosol, tyrosol, and quercetin. These data confirmed that, after the OMW anaerobic treatment to produce biomethane, the remaining flow yet contain useful compounds with antiradical activity.

Microbiological degradation of pentane by immobilized cells of Arthrobacter sp.

The increasing production of several plastics such as expanded polystyrene, widely used as packaging and building materials, has caused the release of considerable amounts of pentane employed as an expanding agent. Today many microorganisms are used to degrade hydrocarbons in order to minimize contamination caused by several industrial activities. The aim of our work was to identify a suitable microorganism to degrade pentane. We focused our attention on a strain of Arthrobacter sp. which in a shake-flask culture produced 95% degradation of a 10% mixture of pentane in a minimal medium after 42 days of incubation at 20 °C. Arthrobacter sp. cells were immobilized on a macroporous polystyrene particle matrix that provides a promising novel support for cell immobilization. The method involved culturing cells with the expanded polystyrene in shake-flasks, followed by in situ growth within the column. Scanning electron microscopy analysis showed extensive growth of Arthrobacter sp. on the polymeric surface. The immobilized microorganism was able to actively degrade a 10% mixture of pentane, allowing us to obtain a bioconversion yield of 90% after 36 h. Moreover, in repeated-batch operations, immobilized Arthrobacter sp. cells were able to maintain 85–95% pentane degradation during a 2 month period. Our results suggest that this type of bioreactor could be used in pentane environmental decontamination.

Extremophilic (Hemi)cellulolytic Microorganisms and Enzymes

The second generation bioethanol represents a main challenge in global efforts to utilize renewable resources rather than fossil fuels. However, the close association of cellulose and hemicelluloses to lignin in the plant cell wall makes it difficult to degrade lignocellulose into fermentable sugars. Consequently, pretreatments are necessary to make the polysaccharides more accessible to the enzymes, but the high temperature and extreme pH conditions required give rise to problems when using conventional enzymes in the saccharification step (Galbe and Zacchi 2002). Microorganisms thriving in habitats characterized by harsh conditions, and the enzymes derived therein, represent a helpful tool in the development of bioethanol production processes. In fact, they allow bioconversions at non-conventional conditions under which common biocatalysts are denatured. The use of high operational temperatures allows energy savings by reducing the cooling cost after high temperature pretreatments, and, in ethanol production, thermophilic conditions permit ethanol evaporation allowing harvest during fermentation.

Biodegradability Study on Films for Packaging Based on Isotactic Polypropylene Modified with Natural Terpene Resins

This chapter discusses the influence of a natural terpene resin, poly (α-pinene) on tensile behaviour, gas permeability and biodegradability of isotactic polypropylene (iPP)-based films. The presence of poly(α-pinene) confers improvement of physical and mechanical properties of films, compared to those of plain iPP.

Optimization of Arundo donax Saccharification by (Hemi)cellulolytic Enzymes from Pleurotus ostreatus

An enzymatic mixture of cellulases and xylanases was produced by Pleurotus ostreatus using microcrystalline cellulose as inducer, partially characterized and tested in the statistical analysis of Arundo donax bioconversion. The Plackett-Burman screening design was applied to identify the most significant parameters for the enzymatic hydrolysis of pretreated A. donax. As the most significant influence during the enzymatic hydrolysis of A. donax was exercised by the temperature (°C), pH, and time, the combined effect of these factors in the bioconversion by P. ostreatus cellulase and xylanase was analyzed by a 33 factorial experimental design. It is worth noting that the best result of 480.10 mg of sugars/gds, obtained at 45°C, pH 3.5, and 96 hours of incubation, was significant also when compared with the results previously reached by process optimization with commercial enzymes.