In this work, miniemulsion polymerization was used as a technique for cellulase immobilization on poly(methyl methacrylate) (PMMA) polymeric nanoparticles. Polymerizations with non-ionic surfactant, Lutensol AT50, concomitantly with a buffering agent, NaHCO 3 , led to high enzymatic activity values. From the study of the effect of surfactant concentration, 2.7 wt.%of Lutensol AT50 was sufficient to obtain stable nanoparticles with a high enzyme activity value. The increase in enzyme concentration in the polymerization reactions led to higher relative activity values at the end of the reactions. A cellulaseconcentration study indicated that the maximum immobilization yield was 60%, obtained when 6 wt.% of cellulase was added. Both free and immobilized enzymes presented the same behavior in relation to thermal stability, having their relative activity values reduced to 50% after 60 min of hydrolysis at 55 ºC and pH 6.0. In relation to storage stability, it was verified that immobilized enzyme keeps its activity throughout seven days when stored at 4 ºC in phosphate buffer, pH 6.0. Based on the results presented in this work, the immobilization of cellulase on PMMA polymeric nanoparticles by miniemulsion polymerization can be seen as a promising, feasible and innovative technique, which aims to cooperate with improvement, in both an economic and environmental sense, of several productive industrial processes.
organism used generally genetically modified strains of Hypocrea jecorina (anamorph Trichoderma reesei). Although the high cost of production in SmF systems due to increased fermentation time with low productivity, has resulted in a shift towards the solid state fermentation (SSF) systems but the advantage of better monitoring and handling are still associated with the submerged cultures (86). Though there are reports on cellulases production by SSF, the large scale commercial processes are still using the proven technology of SmF because the SSF is still non competitive. The appropriate technology, operation controls and improved bioreactor design may make it viable e.g. the enzyme in SSF crude product after concentration can be directly use in agro-biotechnological applications viz. silage or feed additive, ligno-cellulosic hydrolysis, and for processing of natural fiber. Solid state fermentation (SSF) may become a competitive method for the production of cellulases as it offers numerous advantages such as high productivity, relatively high concentrations of the products and less effluent generation. Tengerdy (92) compared cellulase production in SmF and SSF systems and indicated that there was a 10 fold reduction in the production cost in SSF than SmF. Pandey et al.
Physiological studies were conducted to determine the optimum cultural conditions for maximal carboxymethyl cellulase (CMCase) formation by Aspergillus terreus DSM 826. Shaking condition at 150 rpm is favorable for the production of CMCase from rice straw and sugar cane bagasse. The highest enzyme yield was obtained at the third day of incubation at 30 °C for both cases; however CMCase formation occurred at a broad range of pH values, with maximal formation of A. terreus DSM 826 CMCase at pH 4.5 and 5.0 when rice straw and sugar cane bagasse were used as sole carbon source, respectively. Carboxymethyl cellulose (CMC) was found to be a good inducer for CMCase formation in both agricultural wastes with CMC concentrations of 0.5 and 1.0 % (w/v) in case of rice straw and sugar cane bagasse, respectively. High level of enzyme formation was obtained with the addition of ammonium chloride as nitrogen source in both cases and at a concentration of 0.4 % (v/v Tween-80) as an addition to medium containing rice straw. However this addition did not influence the production of CMCase in case of using sugar cane bagasse as carbon source.
On each sampling day (days 10, 20, 30, 60, 90, and 120), three chambers of each plant and trophic condition were ﬁl- tered ( Ф = 0.45 m) and fractioned into POC and DOC. The DOC was ﬁltered successively ( Ф = 0.45 m and 0.2 m) and the carbon concentration was measured using a carbon ana- lyzer (Shimadzu TOC-L CPH, Japan). The DOC daily rate was calculated according to Eq. (1) . The DOC daily rate was con- sidered as mineralization or immobilization. The difference between the concentrations of ﬁltered dissolved carbon (0.45 and 0.2 m) was assumed as formation of organic compounds including microbial biomass (immobilization). Cellulose con- tent of remaining POC was measured by gravimetric analysis with a prior acid digestion (H2SO4 − 72%, 3 h). 20
Experiments were carried out based on VDI 4630 guidelines for the “Fermentation of organic materials” methodology (VDI, 2006). The tests were performed in 500 mL reactors containing 300 g of the ellulose sludge, with a concentration of 64% VS. The batch tests were conducted in triplicates at 38 ºC (100.4 °F) for a time period of 35 days using Eudiometer methods (DIN 38414- 8, VDI 4630). Inoculum from a full-scale biogas plant (KTG Biogas AG, Germany), fed with maize silage, was used for the batch experiments. The seeding sludge was ﬁltered to eliminate particles larger than 4mm. The TS was 3% and the VS accounted for 64%. The substrate/ ellulose ratio based on the initial VS was 0.4. The cumulative volume of produced biogas from each eudiometer was measured daily as the equivalent volume of acidified water displaced. Results are presented at standard temperature and pressure (STP).
The CelB is a halophilic cellulase. Its activities increase 10 fold in the presence of 2.5M NaCl . Such property has been observed with other endoglucanases, but to a much less extent [56, 57]. No significant structure change was observed when salt concentration was increased, which rules out the possibility of refolding of the protein under high salt. The surface of CelB is highly negatively charged (Fig 5). However, there are several small positive charged patches present. It is possible that under low salt conditions, these positively charged patches serve as an attaching point for the neighboring highly negatively charged CelB molecules. And these molecules may form oligomer through electrostatic interactions. In fact, as observed by dynamic light scattering, the diameters of particles in CelB solution increase significantly under the low salt conditions (data not shown). The formation of such oligomers would pre- vent the substrate binding and, therefore, hinder the catalytic activities.
Cellulase activity was measured as described previously with slight modification [14, 26]. Briefly, colonies of cellulase-expressing transformants were transferred into 500 μl of SD medium without leucine, tryptophan, and uracil supplemented with 1% casamino acids in a deep 96-well plate and grown at 30°C with shaking at 1800 rpm for 24 h in a specialized shaker for deep-well plates (MBR-022UP; Taitec, Aichi, Japan). Next, a 100-μl aliquot of each culture was inoculated into 500 μl of SD medium without leucine, tryptophan, and uracil supple- mented with 1% casamino acids and again grown at 30°C with shaking at 1800 rpm for 24 h. After centrifugation at 3000 rpm for 10 min, the cellulase activity of 5-μL aliquots of superna- tant was determined by measuring the concentration of reducing sugar with 2% (w/v) Avicel buffered in 50 mM acetate buffer (pH 5.0) as the substrate, as described previously.[14, 26] Cellulase reactions were conducted at 50°C for 4 h. All measurements were performed in triplicate.
duced by filamentous fungi, because hyphae have the natu- ral ability to cover the solid nutritive surface of the substrate and even to enter its pores, and thus become strongly attached to the substrate (Raimbault, 1998). The easy growth of filamentous fungi on solid media relies on the high capacity of hydrolytic enzyme synthesis in the me- dia along with a high content of polymerized sugars, which are the inducers of gene expression of these enzymes (Sachslehner et al., 1998; Sternberg and Mandels, 1979). The ordinary content of cellulose in abundant natural crop residues, such as sugarcane bagasse and rice straw, is about 40% w/w (Cen and Xia, 1999). When these residues are used as substrates for solid state cultures (SSC), the cellu- lose concentration in the medium is around 6-28%, consid- ering that moisture levels vary between 30 and 85% (Krishna, 2005), while for SmC, the maximum cellulose concentration is about 0.5 to 6% (Chahal, 1985). Therefore, the induction of cellulase synthesis has reduced power in SmC relative to SSC.
For the biochemical characterization of CelDZ1, the cellulolytic activity of the enzyme was determined by quantification of the amount of reducing sugar released from the substrate using the 3,5–dinitrosalicylic acid (DNS) method . One unit (U) of activity was defined as the quantity of enzyme required to release 1 μmol of reducing sugar per min. The standard reaction consisted of 50 mM phosphate buffer at pH 5 and 1% (w/v) CMC as the substrate, and 3 μg/mL enzyme. Enzyme reactions were carried out on a MJ Research thermal cycler at 70°C for 5 min unless stated otherwise. The reactions were terminated by the addition of equal vol- ume of DNS and the mixture was boiled for 5 min to develop the colour occurring due to the reaction with reducing sugars. Enzymic activity was recorded by measuring the absorbance at 540 nm. For the determination of the enzyme’s optimal pH, the reactions were carried out at 40°C in 50 mM acetate, phosphate, Tris-HCl and glycine buffers for pH values 4–6, 7, 8–9 and 10, respectively. The temperature profiling of CelDZ1 was performed by incubating the stan- dard reaction at temperatures ranging from 40 to 90°C. Kinetic parameters were determined by using the standard reaction format with CMC concentrations ranging from 0.3 to 3%. Data analysis and curve fitting was performed using the Graphpad Prism 5 software. For the sub- strate specificity experiments, CMC was replaced in the standard reaction by other soluble polysaccharides. For the insoluble substrates such as Avicel and filter paper the reaction time was 24 h, and the enzyme concentration was increased 10 fold. In the thermostability studies, CelDZ1 was replaced in the standard assay by the pre-incubated enzyme in various tempera- tures and for different time intervals. Halostability and halotolerance studies were also executed in the standard reaction with the only difference being the addition of salts. The same applies for the metals and denaturing agents studies. All measurements were obtained from at least three independent experiments carried out in triplicates.
Correlating the evolution of acidity with pH, the low production of propionic and acetic acids and variation in cell concentration may be directly related to the low pH values throughout the fermentation, especially after 96 hours. Analyzing the results, it can be observed that the fermentation time was around 120 hours, the same result obtained by Kośmider et al. (2010), that investigated propionic acid production by fermentation at 30 o C
Highest coefficient α rate for pasture with RY + WC (5.2), which is even higher than that of the proposed model (4.8), shows that ryegrass has higher N contents in the vegetal tissue when intercropped with white clover. Further, sometime in its development N absorption by plant was higher than its requirements due to a higher availability of the mineral in the soil (Figure 1). White clover’s greater participation, featuring 24.75% of the biomass of pasture after 35 days exclusion, may have cooperated towards N fixation in the soil and its reception in the pasture during the initial evaluation stage. Higher N concentration in the vegetal tissue during this period and intense reduction of N rates in ryegrass due to DM accumulation (β = -0.94) underpins the above hypothesis (Figure 1).
Figure 1. Concentrations of chemokines in sputum (left side) and plasma (right side) of healthy control (HC, N = 9) and patients with asthma (AS, N = 9) or chronic obstructive pulmo- nary disease (COPD, N = 11). The concentration of CCL11 (A, B), CXCL8 (C, D) or CCL2 (E, F) in sputum or plasma was meas- ured by ELISA. Data are re- ported as the log mean value. The horizontal lines indicate the median value for each group. *P < 0.05 compared to HC and + P < 0.05 compared to COPD (Spearman’s rank correlation test).
In 1995, the EUCLIDES system started to be designed; it was under development for 12 years. The system was developed by Instituto de Energía Solar–Universidad Politécnica de Madrid (IES‐UPM), BP Solar and with assistance by ZSW (Zentrum fur Sonnenenergieund Wasserstoff-Forschung, Germany). The system consisted of a reflective parabolic trough working at low-to-medium geometric concentration (32X), silicon solar cells (the SATURN cells, high efficiency solar cells with buried front contacts, as discussed in detail in Section 2.2.3), and single north–south‐orientated horizontal axis . The system achieved an efficiency of 14% (Marta Vivar et al. 2011; G. Sala et al. 1996; A. Luque & Andreev 2007). Its success lead to EUCLIDES II, a demonstration plant of 480 kWp installed in Tenerife in 1988. The project revealed several issues regarding the reliability of the industrialization of less commercially ready components such as the mirrors and some receiver elements. It was also identified the need for qualification standards which would have avoided several subsystem defects that were ignored. The investment of the European commission (IDEOCONTE project) on a third-generation of the EUCLIDES system, see Fig.1.21, (Marta Vivar 2009; G. Sala et al. 2005) has enhanced the activity in CPV field. The overall objective of this project was to identify the most appropriate CPV system configuration, based on silicon concentrator cells. With the third generation of EUCLIDES, M. Vivar arrived to the conclusion that at that time PV market, it would be necessary to increase the efficiency or reduce component costs or even both approaches in order to achieve the required cost targets (Marta Vivar et al. 2011).
In Table 1 are shown the values of PM 10 concentrations at 1.5 m high considering all the emissions (traffic + background) for the actual street configuration (option A) compared with PM 10 concentrations measurements made on seven strategic points along the street, on both sides of the road. Additionally the table shows the mean concentration value for the 1.5 m plane and also for the mean concentration weighted by the wind frequency, considered the air quality index (AQ index), for the same plane.
Figure 2. Morphological variables of hydroponic lettuce in function of the nitrate concentration in the nutrient solution: SFM (shoot fresh mass); RFM (root fresh mass); SDM (shoot dry mass); RDM (root dry mass); PH (plant height); SL (stem length); RL (root length); LA (leaf area); LN (number of leaves) (variáveis morfológicas da alface em cultivo hidropônico em função dos teores de nitrato na solução nutritiva. SFM (massa fresca da parte aérea); RFM (massa fresca da raiz); SDM (massa seca da parte aérea); RDM (massa seca da raiz); AP (altura da planta); SL (comprimento do caule); RL (comprimento de raiz); LA (área foliar); LN (número de folhas)). Alegre, UFES, 2009.
Carboxymethyl cellulose is a soluble substrate and easily hydrolyzed. In this work, isolate 380 showed an enzyme activity of 0.12 UEA after 6 hours of incubation. A study involving the influence of temperature on enzyme activity showed variations in the production, with 0.001 mg mL -1 of the enzyme at 50°C for a production of 344 mg mL -1 at 45°C (Shabeb et al. 2010). This result leads us to infer that isolate 380 may show an excellent production of cellulase at low and high temperatures, a crucial factor for its inoculation along the composting process, degrading cellulose sources in mesophilic and thermophilic phases. Furthermore, the hydrolysis of microcrystalline cellulose, for example, requires the synergistic action of different cellulases, which leads to the production of various enzymes.
aﬀect the interaction between duplication and concentration. Two important simplifying assumptions in the literature with far-reaching implications are (1) equal treatment of all Þrms, i.e., the restriction to symmetric equilibria and (2) constant returns to scale in the output sector. We build on an insight by Salant and Shaﬀer (1998, 1999), who have shown that for a wide class of two-stage models, interior and asymmetric, yet superior, equilibria may exist. In essence, con- stant returns to scale create a non-convexity which places the social optimum at an equilibrium that is not only asymmetric but is in fact a corner solution. It follows that the combination of symmetry and constant returns to scale assumptions deÞnes a suboptimal benchmark against which to gauge performance. In this paper, we shall keep the constant returns to scale assumption and focus on relaxing the sym- metry requirement.
Nevertheless, the value of such a simplified relationship as that proposed by Dusek et al. (2006), with its implied simple relationship between aerosols and CDNC, is tantalizing, not only from the standpoint of model usage but from that of re- mote sensing. Such parameters as, for example, the accumu- lation mode number concentration, are readily available from existing remote retrieval algorithms such as that for MODIS (Remer et al., 2005) and have a substantial validation history. In contrast, algorithms to retrieve CCN activity, while avail- able, have been little used and then with very modest success (e.g., Gasso and Hegg, 2003). Indeed, a recent assessment by Kapustin et al. (2006) suggests that effective remote retrieval of CCN activity will be very challenging. It therefore seems worthwhile to pursue a simple empirical CDNC-aerosol re- lationship, such as that implied by the Dusek et al. (2006) study, a bit further. In this study, we examine data on cloud microphysics and aerosol properties for selected venues of most importance to indirect aerosol forcing of climate, seek- ing such a relationship.
Heterologous expression of CelC2 cellulase in E. meliloti elicits Hole on the Tip (HoT) phenotype in T. repens. Previous studies showed that the purified cellulase CelC2 isozyme from wild-type ANU843 degrades the cell wall at the apex of the root hair tip (“HoT” phenotype) when incubated with intact seedling roots of its compatible host, white clover 10 . E. meliloti 1021 is capable of infecting and nodulating Medicago but not Trifolium and its genome does not encode CelC homologs 16 . To assess CelC2 effects on primary infection of a non-cognate legume host, T. repens seedlings were inoculated with E. meliloti wild-type or celC-overexpress- ing (1021C2 + ) strains. Similar symbiotic tests were performed with the clover-compatible ANU843 strain or its overexpressing CelC2 derivative (CelC2 + ). Canonical symbiotic infection phenotypes occurring at early stages of rhizobia-root hair recognition, namely Hac (Hair curling) and noi (nodule initiation), together with Hole on the Tip (HoT) were monitored and documented with a digital microscope (Fig. 1 , Table 1 ).