Production of pectate lyase by penicillium viridicatum RFC3 in solid-state and submerged fermentation

Pectate lyase (PL) was produced by the filamentous fungus Penicillium viridicatum RFC3 in solid-state cultures of a mixture of orange bagasse and wheat bran (1 : 1 w/w), or orange bagasse, wheat bran and sugarcane bagasse (1 : 1 : 0.5 w/w), and in a submerged liquid culture with orange bagasse and wheat bran (3%) as the carbon source. PL production was highest (1,500U mL -1 or 300Ug -1 of substrate) in solid-state fermentation (SSF) on wheat bran and orange bagasse at 96 hours. PL production in submerged fermentation (SmF) was influenced by the initial pH of the medium. With the initial pH adjusted to 4.5, 5.0, and 5.5, the peak activity was observed after 72, 48, and 24 hours of fermentation, respectively, when the pH of the medium reached the value 5.0. PL from SSF and SmF were loaded on Sephadex-G75 columns and six activity peaks were obtained from crude enzyme fromSSF and designated PL I, II, III, IV, V, andVI, while five peaks were obtained fromcrude enzyme fromSmF and labeled PL I', II', III', IV', and VII'. Crude enzyme and fraction III from each fermentative process were tested further. The optimum pH for crude PL from either process was 5.5, while that for PL III was 8.0. The maximum activity of enzymes from SSF was observed at 35°C, but crude enzyme was more thermotolerant than PL III, maintaining its maximum activity up to 45°C. Crude enzyme from SmF and PL III' showed thermophilic profiles of activity, with maximum activity at 60 and 55°C, respectively. In the absence of substrate, the crude enzyme from SSF was stable over the pH range 3.0-10.0 and PL III was most stable in the pH range 4.0-7.0. Crude enzyme from SmF retained 70%-80% of its maximum activity in the acid-neutral pH range (4.0-7.0), but PIII showed high stability at alkaline pH (7.5-9.5). PL from SSF was more thermolabile than that from SmF. The latter maintained 60% of its initial activity after 1 h at 55°C. The differing behavior of the enzymes with respect to pH and temperature suggests that they are different isozymes. Copyright © 2010 Viviani Ferreira et al.

Ultra-structural mapping of sugarcane bagasse after oxalic acid fiber expansion (OAFEX) and ethanol production by Candida shehatae and Saccharomyces cerevisiae

Background: Diminishing supplies of fossil fuels and oil spills are rousing to explore the alternative sources of energy that can be produced from non-food/feed-based substrates. Due to its abundance, sugarcane bagasse (SB) could be a model substrate for the second-generation biofuel cellulosic ethanol. However, the efficient bioconversion of SB remains a challenge for the commercial production of cellulosic ethanol. We hypothesized that oxalic-acid-mediated thermochemical pretreatment (OAFEX) would overcome the native recalcitrance of SB by enhancing the cellulase amenability toward the embedded cellulosic microfibrils. Results: OAFEX treatment revealed the solubilization of hemicellulose releasing sugars (12.56 g/l xylose and 1.85 g/l glucose), leaving cellulignin in an accessible form for enzymatic hydrolysis. The highest hydrolytic efficiency (66.51%) of cellulignin was achieved by enzymatic hydrolysis (Celluclast 1.5 L and Novozym 188). The ultrastructure characterization of SB using scanning electron microscopy (SEM), atomic force microscopy (AFM), Raman spectroscopy, Fourier transform-near infrared spectroscopy (FT-NIR), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) revealed structural differences before and after OAFEX treatment with enzymatic hydrolysis. Furthermore, fermentation mediated by C. shehatae UFMG HM52.2 and S. cerevisiae 174 showed fuel ethanol production from detoxified acid (3.2 g/l, yield 0.353 g/g; 0.52 g/l, yield, 0.246 g/g) and enzymatic hydrolysates (4.83 g/l, yield, 0.28 g/g; 6.6 g/l, yield 0.46 g/g). Conclusions: OAFEX treatment revealed marked hemicellulose degradation, improving the cellulases ability to access the cellulignin and release fermentable sugars from the pretreated substrate. The ultrastructure of SB after OAFEX and enzymatic hydrolysis of cellulignin established thorough insights at the molecular level. © 2013 Chandel et al; licensee BioMed Central Ltd.

The effect of silver nanoparticles and nystatin on mixed biofilms of Candida glabrata and Candida albicans on acrylic

The aim of this study was to compare biofi lm formation by Candida glabrata and Candida albicans on acrylic, either individually or when combined (single and dual species) and then examine the antimicrobial effects of silver nanoparticles and nystatin on these biofi lms. Candidal adhesion and biofi lm assays were performed on acrylic surface in the presence of artifi cial saliva (AS) for 2 h and 48 h, respectively. Candida glabrata and C. albicans adherence was determined by the number of colony forming units (CFUs) recovered from the biofi lms on CHROMagar ® Candida . In addition, crystal violet (CV) staining was used as an indicator of biofi lm biomass and to quantify biofi lm formation ability. Pre-formed biofi lms were treated either with silver nanoparticles or nystatin and the effect of these agents on the biofi lms was evaluated after 24 h. Results showed that both species adhered to and formed biofi lms on acrylic surfaces. A signifi cantly ( P < 0.05) higher number of CFUs was evident in C. glabrata biofi lms compared with those formed by C. albicans . Comparing single and dual species biofi lms, equivalent CFU numbers were evident for the individual species. Both silver nanoparticles and nystatin reduced biofi lm biomass and the CFUs of single and dual species biofi lms ( P < 0.05). Silver nanoparticles had a signifi cantly ( P < 0.05) greater effect on reducing C. glabrata biofi lm biomass compared with C. albicans . Similarly, nystatin was more effective in reducing the number of CFUs of dual species biofi lms compared with those of single species ( P < 0.05). In summary, C. glabrata and C. albicans can co-exist in biofi lms without apparent antagonism, and both silver nanoparticles and nystatin exhibit inhibitory effects on biofi lms of these species. © 2013 ISHAM.

Digestibility, fermentation and rumen microbiota of crossbred heifers fed diets with different soybean oil availabilities in the rumen

The goal of this study was to evaluate the effects of different soybean oil availabilities on the intake and partial and total digestibility of dry matter (DM) and nutrients, rumen fermentation parameters, efficiency of microbial synthesis, and the rumen microbiota of crossbred beef heifers. Nine crossbred heifers fitted with rumen and duodenal cannulae were evaluated in a triple 3 × 3 Latin square design with three treatments and three periods in three simultaneous repetitions. Heifers approximately 18 months old, with mean initial and final body weights of 316.3±28.8 and 362.6±34.4 kg, respectively, were fed a diet containing 600. g/kg of corn silage and 400. g/kg concentrate with a 58.0. g/kg fat content in the total diet. The sources of lipids included soybean grain, rumen-protected fat, and soybean oil. The statistical analyses were conducted using PROC MIXED from SAS, and the means were compared using Tukey's test (P<0.05). Dietary lipid sources did not affect nutrient intake (P>0.05). Except the apparent digestibility of organic matter (P=0.024), the apparent digestibility of the other nutrients did not differ among the treatment groups. Regarding body nitrogen retention, the soybean grain treatment was more effective than the rumen-protected fat treatment (P=0.045); however, the soybean oil treatment did not differ from the other two treatments. In relation to the efficiency of microbial protein synthesis (g. N/kg of organic matter apparently digested in the rumen corrected for microbial organic matter), the soybean oil and soybean grain treatments were more efficient than the rumen-protected fat treatment (P=0.001). Animals fed rumen-protected fat had larger numbers of protozoa (P<0.001) and fungi (P<0.001) than those supplemented with soybean grain and soybean oil. The dietary lipid sources did not affect pH, the molar concentration of propionate and total volatile fatty acids (P>0.05), whereas the concentrations of ammonia nitrogen and acetate were higher in animals fed with rumen-protected fat than in those submitted to the other treatments. The use of different soybean oil availabilities did not affect nutrient intake; however, treatments with soybean oil and soybean grain were more efficient regarding nutrient intake than rumen-protected fat because they reduced the numbers of fungi and protozoa and consequently improved the efficiency of microbial protein synthesis. © 2013 Elsevier B.V.

Methane emissions and estimates of ruminal fermentation parameters in beef cattle fed different dietary concentrate levels

Using sorghum silage, the effect of roughage/concentrate ratios was evaluated on nutrient intake, digestibility, ruminal parameters and methane production by beef cattle. Three treatments (0, 30 and 60% of concentrate in DM of the diet) were distributed in three Latin squares, with nine animals and three periods. Dry matter intake increased as the grain concentration in diet increased; pH showed opposite behavior. Methane emissions were lower for animals fed the diet exclusively with sorghum silage as compared with those fed 30% of concentrate, but was similar to that of animals receiving 60% of concentrate. Losses of ingested gross energy as methane were reduced by 33% when grain concentration was increased in the diet. Concentrations of propionic and butyric acids were greater in diets with grain concentrate; acetic acid concentration was not affected. Concentrate in diet increases available energy for the metabolism, measured by lower losses of ingested gross energy as ruminal methane. © 2013 Sociedade Brasileira de Zootecnia.

Influence of artificial aging in marginal adaptation of mixed class V cavities

The aim of this study was to investigate whether the artificial aging by thermal cycling had influenced the marginal adaptation of class V restorations with/without chlorhexidine application in the bond process. Twelve intact human third molars were used. Class V cavity preparations were performed on the buccal surface and the teeth received 35% phosphoric acid-etching procedure (Ultradent Products Inc., South Jordan, Utah, USA). Subsequently, the samples were divided in two groups: Untreated acid-etched dentin and chlorhexidine application as an adjunct in the bond process. The adhesive Single Bond 2 (3M ESPE, St. Paul, MN, USA) was used after 2% chlorhexidine application, and the restorations were performed with FiltekTM Z350 XT (3M ESPE) composite resin. The specimens were submitted to artificial aging by thermal cycling with 3,000 cycles. Analyzes were performed on scanning electron microscopy using replicas of marginal adaptation in percentage of continuous margin before and after the artificial aging. The data were analyzed by paired test and the results showed statistically significant differences in the percentage of continuous margin with/without chlorhexidine treatment before and after thermal cycling. This study concluded that the artificial aging by thermal cycling influenced the marginal adaptation of mixed class V composite restorations.

Estudo do pré-tratamento do bagaço de cana-de-açúcar e caracterização físico-química

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo do composto derivado do óleo de Ricinus communis L. (mamona) sobre a bactéria e biopolímero da fermantação etanólica

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Ação biocida do Poliquilgerm® derivado dom óleo de Ricinus Communis L. (mamona) sobre bactérias contaminantes da fermentação etanólica

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Produção de poligalacturonase termoestável pelo fungo Rhizomucor pusillus A 13.36 em fermentação em estado sólido, purificação e caracterização da enzima

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Purificação e caracterização bioquímica de poligalacturonases produzidas pelo fungo Penicillium viridicatum RFC3 em fermentação em estado sólido e submersa

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Chemical inhibition of the contaminant Lactobacillus fermentum from distilleries producing fuel bioethanol

The purpose of this study was to determine the Minimum Inhibitory Concentration (MIC) of pure or mixed chemicals for Saccharomyces cerevisiae and Lactobacillus fermentum in the samples isolated from distilleries with serious bacterial contamination problems. The biocides, which showed the best results were: 3,4,4' trichlorocarbanilide (TCC), tested at pH 4.0 (MIC = 3.12 mg/l), TCC with benzethonium chloride (CBe) at pH 6.0 (MIC = 3.12 mg/l) and TCC mixed with benzalkonium chloride (CBa) at pH 6.0 (MIC = 1.53 mg /l). If CBa was used in sugar cane milling in 1:1 ratio with TCC, a 8 times reduction of CBa was possible. This formulation also should be tested in fermentation steps since it was more difficult for the bacterium to develop resistance to biocide. There was no inhibition of S. cerevisiae and there were only antibiotics as an option to bacterial control of fuel ethanol fermentation by S. cerevisiae.

Influence of the combination of probiotic cultures during fermentation and storage of fermented milk

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The effect of exogenous fibrolytic enzymes and a ferulic acid esterase-producing inoculant on the fibre degradability, chemical composition and conservation characteristics of alfalfa silage

The objective of this study was to determine the effect of applying fibrolytic enzymes at ensiling, either alone or in combination with a ferulic acid esterase-producing bacterial silage inoculant, on the silage conservation characteristics and nutritive value of alfalfa (Medicago sativa L). Second-cut alfalfa (340 g DM/kg fresh crop) was harvested, wilted, chopped and sub-sampled into 24 batches. Samples were randomly allocated in triplicate to one of four enzyme product treatments supplying endoglucanases and xylanases: none (control), EN1, EN2, EN3; applied alone or in combination with a ferulic acid esterase-producing silage inoculant (FAEI). Treatments were arranged in a 4 x 2 factorial design. All enzyme treatments were applied at 2 ml enzyme product/kg herbage DM, and inoculant was applied at 1 x 10(5) cfu/g fresh herbage. Samples were packed into laboratory-scale silos and stored for 7, 27 or 70 days, and analysed for dry matter (DM) losses, aerobic stability, chemical composition and in vitro ruminal degradability. The use of enzymes did not affect (P>0.05) ensilage DM losses or lactic or acetic acid concentrations after 70 days of ensilage, compared to the control silage. Silage produced using EN1 had lesser neutral detergent fibre (aNDF, P=0.046) and acid detergent fibre (ADF; P=0.006) concentrations than control silage. However, no difference (P>0.05) was observed between the control silage and silage produced with EN1 for aNDF or ADF degradability (NDFD, ADFD). Silages produced with FAEI had greater DM losses (P=0.017) and pH (P<0.001) and lesser NDFD (P=0.019), ADFD (P=0.010) and proportion of lactic acid in the total fermentation products (P=0.006) after 70 days of ensilage, compared to uninoculated silages. The use of fibrolytic enzymes did not have a major effect on the ensilage fermentation of alfalfa, either ensiled alone or with an inoculant. No advantage in ruminal DM or fibre degradability was observed for silages produced with fibrolytic enzymes. The use of a ferulic acid esterase-producing inoculant alone did not improve the nutritive value of alfalfa silage, and did not promote any incremental effects when applied in combination with fibrolytic enzyme products. Crown Copyright (C) 2014 Published by Elsevier B.V. All rights reserved.

Chemical Composition, Rumen Fermentation Kinetics, Digestibility and Energy Value of Cassava Leaves Hay at Different Storage Times

Cassava leaves have been widely used as a protein source for ruminants in the tropics. However, these leaves contain high level of hydro-cyanic acid (HCN) and condensed tannins (CT). There are evidences that making hay can eliminate more than 90% of HCN and that long-term storage can reduce CT levels. A complete randomized design with four replicates was conducted to determine the effect of different storage times (0-control, 60, 90 and 120 days) on chemical composition, in vitro rumen fermentation kinetics, digestibility and energy value of cassava leaves hay. Treatments were compared by analyzing variables using the GLM procedure (SAS 9.1, SAS Institute, Inc., Cary, NC). Crude protein (CP) and ether extract (EE) of the cassava hay were not affected (P > 0.05) by storage time (17.7% and 3.0%, respectively). Neutral detergent fiber, acid detergent fiber, total carbohydrate and non-fiber carbohydrate were not affected either (P>0.05) by storage time (47.5, 32.6, 72.3 and 25.8% respectively). However, other parameters were influenced. CT was lower (P<0.05) in hay after 120 days of storage compared with control (1.75% versus 3.75%, respectively). Lignin and insoluble nitrogen in neutral detergent, analyzed without sodium sulfite, were higher (P<0.01) after 120 days of storage, compared with the control (11.22 versus 13.57 and 1.65 versus 3.81% respectively). This suggests that the CT has bound to the fiber or CP and became inactive. Consequently, the in vitro digestibility of organic matter (50.36%), total digestible nutrients (44.79%) and energy (1.61 Mcal/KgMS), obtained from gas production data at 72 h of incubation, has increased (P<0.05) with storage times (56.83%, 51.53% and 1.86 Mcal/KgMS, respectively). The chemical composition and fermentative characteristics of cassava hay suffered variations during the storage period. The best values were obtained after 90 days of storage. This is probably due to the reduction in condensed tannins.