Síntese, extração e caracterização de biopolímeros de origem microalgal para desenvolvimento de nanofibras

Pesquisas com microalgas estão crescendo devido aos possíveis bioprodutos oriundos de sua biomassa, bem como as suas diferentes aplicabilidades. Microalgas podem ser cultivadas para a produção de biopolímeros com características de biocompatibilidade e biodegradabilidade. Nanofibras produzidas por electrospinning a partir de poli-β-hidroxibutirato (PHB) geram produtos com aplicabilidade na área de alimentos e médica. O objetivo deste trabalho foi selecionar microalgas com maior potencial para síntese de biopolímeros, em diferentes meios de cultivo, bem como purificar poli-β-hidroxibutirato e desenvolver nanofibras. Este trabalho foi dividido em cinco artigos: (1) Seleção de microalgas produtoras de biopolímeros; (2) Produção de biopolímeros pela microalga Spirulina sp. LEB 18 em cultivo com diferentes fontes de carbono e redução de nitrogênio; (3) Síntese de biopolímeros pela microalga Spirulina sp. LEB 18 em cultivos autotróficos e mixotróficos; (4) Purificação de poli-β- hidroxibutirato extraído da microalga Spirulina sp. LEB 18; e (5) Produção de nanofibras a partir de poli-β-hidroxibutirato de origem microalgal. Foram estudadas as microalgas Cyanobium sp., Nostoc ellipsosporum, Spirulina sp. LEB 18 e Synechococcus nidulans. Os biopolímeros foram extraídos nos tempos de 5, 10, 15, 20 e 25 d de cultivo a partir de digestão diferencial. Para os experimentos com diferentes nutrientes, foi utilizado como fonte de carbono, bicarbonato de sódio, acetato de sódio, glicose e glicerina modificando-se as concentrações de nitrogênio e fósforo. Os cultivos foram realizados em fotobiorreatores fechados de 2 L. A concentração inicial de inóculo foi 0,15 g.L-1 e os ensaios foram mantidos em estufa termostatizada a 30 ºC com iluminância de 41,6 µmolfótons.m -2 .s -1 e fotoperíodo 12 h claro/escuro. Para a purificação de PHB, foi utilizada a biomassa da cianobactéria Spirulina sp. LEB 18, cultivada em meio Zarrouk. Após a extração do biopolímero bruto, a amostra foi desengordurada com hexano e purificada com 1,2-carbonato de propileno. Foram determinadas as purezas e as propriedades térmicas no PHB purificado. O biopolímero utilizado para produzir as nanofibras apresentava 70 % de pureza. A técnica para produção de nanofibras foi o electrospinning. As microalgas que apresentaram máxima produtividade foram Nostoc ellipsosporum e Spirulina sp. LEB 18 com rendimento de biopolímero 19,27 e 20,62 % em 10 e 15 d, respectivamente, na fase de máximo crescimento celular. O maior rendimento de biopolímeros (54,48 %) foi obtido quando se utilizou 8,4 g.L-1 de NaHCO3, 0,05 g.L-1 de NaNO3 e 0,1 g.L-1 de K2HPO4. A condição que proporcionou maior pureza do PHB foi a 130 ºC e 5 min de contato entre o solvente (1,2-carbonato de propileno) e o PHB. As análises térmicas para todas as amostras foram semelhantes em relação ao PHB padrão (Sigma-Aldrich). A purificação com 1,2-carbonato de propileno foi eficiente para o PHB extraído de microalga, alcançando pureza acima de 90 %. A condição que apresentou menores diâmetros de nanofibras foi ao utilizar solução contendo 20 % de biopolímero solubilizado em clorofórmio. As condições do electrospinning que apresentou nanofibras com diâmetros de 470 e 537 nm foram, vazão 150 µL.h-1 , diâmetro do capilar 0,45 mm e voltagens entre 24,1 e 29,6 kV, respectivamente. A microalga Spirulina sp. LEB 18 produz PHB ao utilizar menores concentrações de nutrientes no meio de cultivo, que pode ser purificado com 1,2-carbonato de propileno. Este biopolímero possui aplicabilidade para produção de nanofibras.

POTENCIAL DA ASSOCIAÇÃO Pisolithus microcarpus COM MUDAS DE Corymbia citriodora EM SOLO CONTAMINADO COM ZINCO

O uso de plantas com potencial de associação com microrganismos é uma prática frequente em solos contaminados por metais pesados, considerada de baixo custo e ambientalmente correta. O trabalho objetivou avaliar o crescimento do Corymbia citriodora (Hook.) K.D. Hill & L.A.S. Johnson e o efeito da inoculação com Pisolithus microcarpus UFSC-Pt116 em solo contaminado com Zn. O delineamento experimental foi inteiramente casualizado em arranjo fatorial (2 x 6), sendo com e sem inóculo e seis doses de Zn (0, 300, 600, 900, 1200 e 1500 mg kg-1 de solo), com seis repetições. As mudas foram inoculadas e cultivadas durante 90 dias em viveiro. Após 67 dias do transplante definitivo foi avaliado o percentual de colonização ectomicorrízica, a altura de planta, diâmetro do colo, número de folhas, índice de clorofila total, volume radicular, massa seca das folhas, da haste caulinar, radicular e total, relação massa seca aérea/massa seca radicular e a relação altura/diâmetro do colo. O percentual de colonização ectomicorrízica em Corymbia citriodora é estimulado pelo acréscimo de até 1412,21 mg kg-1 de Zn no solo. O Corymbia citriodora é tolerante a adição de até 1500 mg kg-1 de zinco em solo com 81% de argila, mesmo sem a inoculação com Pisolithus microcarpus. A análise de correlação canônica evidencia que a inoculação com P. microcarpus favorece a massa seca total, radicular e da parte aérea de Corymbia citriodora cultivado em solo com 81% de argila contaminada com 600 mg kg-1 de Zn.

In vivo and in vitro disappearance of energy and nutrients in novel carbohydrates and cereal grains by pigs

In vivo and in vitro experiments were conducted to determine digestibility of GE and nutrients, as well as DE and ME of carbohydrates fed to growing pigs. The objective of Exp. 1 was to determine the DE and ME of 4 novel carbohydrates fed to pigs. The 4 novel carbohydrates were 2 sources of resistant starch (RS 60 and RS 70), soluble corn fiber (SCF), and pullulan. These carbohydrates were produced to increase total dietary fiber (TDF) intake by humans. Maltodextrin (MD) was used as a highly digestible control carbohydrate. The DE and ME for RS 60 (1,779 and 1,903 kcal/kg, respectively), RS 75(1,784 and 1,677 kcal/kg, respectively), and SCF (1,936 and 1,712 kcal/kg, respectively) were less (P < 0.05) than for MD (3,465 and 3,344 kcal/kg, respectively) and pullulan (2,755 and 2,766 kcal/kg, respectively), and pullulan contained less (P < 0.05) DE and ME than MD. However, there was no difference in the DE and ME for RS 60, RS 75, and SCF. The varying degrees of small intestinal digestibility and differences in fermentability among these novel carbohydrates may explain the differences in the DE and ME among carbohydrates. Therefore, the objectives of Exp. 2 were to determine the effect of these 4 novel carbohydrates and cellulose on apparent ileal (AID) and apparent total tract (ATTD) disappearance, and hindgut disappearance (HGD) of GE, TDF, and nutrients when added to diets fed to ileal-cannulated pigs. The second objective was to measure the endogenous flow of TDF to be able to calculate the standardized ileal disappearance (SID) and standardized total tract (STTD) disappearance of TDF in the 4 novel fibers fed to pigs. Results of the experiment indicated that the AID of GE and DM in diets containing cellulose or the novel fibers was less (P < 0.05) than of the maltodextrin diet, but the ATTD of GE and DM was not different among diets. The addition of RS 60, RS 75, and SCF did not affect the AID of acid hydrolysed ether extract (AEE), CP, or ash, but the addition of cellulose and pullulan reduced (P < 0.01) the AID of CP. The average ileal and total tract endogenous losses of TDF were calculated to be 25.25 and 42.87 g/kg DMI, respectively. The SID of TDF in diets containing RS 60, SCF, and pullulan were greater (P < 0.01) than the SID of TDF in the cellulose diet, but the STTD of the SCF diet was greater (P < 0.05) than for the cellulose and pullulan diets. Results of this experiment indicate that the presence of TDF reduces small intestinal disappearance of total carbohydrates and energy which may reduce the DE and ME of diets and ingredients. Therefore, the objective of Exp. 3 was to determine the DE and ME in yellow dent corn, Nutridense corn, dehulled barley, dehulled oats, polished rice, rye, sorghum, and wheat fed to growing pigs and to determine the AID and ATTD of GE, OM, CP, AEE, starch, total carbohydrates, and TDF in these cereal grains fed to pigs. Results indicated that the AID of GE, OM, and total carbohydrates was greater (P < 0.001) in rice than in all other cereal grains. The AID of starch was also greater (P < 0.001) in rice than in yellow dent corn, dehulled barley, rye, and wheat. The ATTD of GE was greater (P < 0.001) in rice than in yellow dent corn, rye, sorghum, and wheat. With a few exceptions, the AID and ATTD of GE and nutrients in Nutridense corn was not different from the values for dehulled oats. Likewise, with a few exceptions, the AID, ATTD, and HGD of GE, OM, total carbohydrates, and TDF in yellow corn, sorghum, and wheat were not different from each other. The AID of GE and AEE in dehulled barley was greater (P < 0.001) than in rye. The ATTD of GE and most nutrients was greater (P < 0.001) in dehulled barley than in rye. Dehulled oats had the greatest (P < 0.001) ME (kcal/kg DM) whereas rye had the least ME (kcal/kg DM) among the cereal grains. Results of the experiment indicate that the presence of TDF and RS may reduce small intestinal digestibility of starch in cereal grains resulting in reduced DE and ME in these grains. Digestibility experiments involving animals are time consuming and expensive. Therefore, the objective of Exp. 4 was to correlate DM and OM digestibility obtained from 3 in vitro procedures with ATTD of GE and with the concentration of DE in 50 corn samples that were fed to growing pigs. The second objective was to develop a regression model that can predict the ATTD of GE or the concentration of DE in corn. The third objective was to evaluate the suitability of using the DaisyII incubator as an alternative to the traditional water bath when determining in vitro DM and OM digestibility. Results indicated that corn samples incubated with Viscozyme for 48 h in the DaisyII incubator improved (P < 0.001) the ability of the procedure to detect small differences in the ATTD of GE or to detect small differences in the concentration of DE in corn. Likewise, compared with using cellulase or fecal inoculum, the variability in the ATTD of GE and the variability in the DE in corn was better (R2 = 0.56; P < 0.05 and R2 = 0.53; P < 0.06, respectively) explained if Viscozyme was used than if cellulase or fecal inoculum was used. A validated regression model that predicted the DE in corn was developed using Viscozyme and with the corn samples incubated in the DaisyII incubator for a 48 h. In conclusion, this present work used the pig as a model for human gastrointestinal function and evaluates carbohydrates from 2 different nutritional perspectives – humans and animals. The addition of novel carbohydrates reduced the digestibility of energy in the diets without necessarily reducing the digestibility of other nutrients. Thus, supplementation of novel carbohydrates in the diets may be beneficial for the management of diabetes. Aside from diabetic management, cereal grains such as rye and sorghum, may also help in BW management because of there low caloric value, but for undernourished individuals, dehulled oats, dehulled barley, and rice are the ideal grains. From an animal nutrition standpoint, high concentration of dietary fiber is undesirable because it reduces feed efficiency. Therefore, the inclusion of feed ingredients that have a high concentration of dietary fiber is often limited in animal diets. Although in vivo determination is ideal, in vitro procedures are useful tools to determine caloric value of food and feed ingredients.

Eliminação/valorização de resíduos de frutas (kiwi) por digestão anaeróbia

Este estudo tem como objetivo a valorização económica de resíduos agroindustriais, nomeadamente resíduos de kiwi, por digestão anaeróbia de forma a otimizar a produção de biogás e a sua qualidade (% CH4). Trata-se de uma pesquisa experimental que consistiu na avaliação da produção de biogás usando diferentes proporções de substrato e inóculo, quatro valores distintos para a razão C:N, inóculo de diferentes digestores e colheita do inóculo em épocas distintas do ano. Os ensaios foram desenvolvidos num reator batch em condições mesofílicas, sendo o processo acompanhado por monitorização dos parâmetros: pH, Alcalinidade, Ácidos Gordos Voláteis (AGV), Sólidos Totais (ST), Sólidos Voláteis (SV), Carência Química de Oxigénio (CQO) e Carbono. Para a totalidade dos ensaios, o valor de pH no reator praticamente não apresentou variação, mantendo-se em torno de 7,0; a alcalinidade do meio, 1500 mg CaCO3/L, revelou-se adequada uma vez que no final do processo de DA a concentração de AGV (400-600 mg/L), nunca excede os valores considerados críticos. Os resultados obtidos apresentam valores interessantes para um número considerável de ensaios. Das 10 experiências realizadas, em duas foram obtidos resultados muito significativos em relação à literatura; o ensaio com 1% de substrato (experiência 2) registou uma produção de biogás de 1628 L/kg SV com uma %CH4 de 57% e o biogás de maior qualidade, 85% de metano, foi obtido no ensaio com 5% de resíduo de kiwi (experiência 10). As experiências em que se avaliou o efeito da razão C:N foram as menos produtivas, possivelmente devido à inibição da atividade da população microbiana pelo KNO3. A qualidade do inóculo revelou-se determinante num conjunto de ensaios, nomeadamente quando a sua colheita foi realizada no inverno, com o digestor a apresentar temperaturas bastante baixas. De acordo com os valores mais favoráveis para a produção de biogás, por tonelada de resíduo de kiwi poderá ser obtido um valor monetário bruto de 102 €, resolvendo-se um problema de eliminação deste resíduo, com valorização energética simultaneamente.

Prácticas culturales para el manejo de la Sigatoka negra en bananos y plátanos

Black sigatoka (Mycosphaerella fijiensis) control consists in the continuous use of fungicide sprays and cultural practices. Cultural control reduces inoculum sources of the pathogen and favorable environmental conditions for its development, as well as it increases the vigor of the plants. In order to manage the disease it is necessary to know its behavior through the time, its relationship with the weather and the crop management. The most important cultural practice to reduce the inoculum source is the removal of the whole affected leaves or only the diseased portions of the leaves. The removed tissue is placed over the ground and the use of urea is feasible to accelerate its decomposition. An alternative practice is the "mini-composting" that consists in forming small heaps with the dead leaves and harvested plants for its quick degradation, which allows to reduce the pathogen inoculum and to incorporate nutrients and organic matters into the soil. The early pruning of the tips of the Young leaves before they show symptoms of the disease and the quick elimination of harvested plants reduce the inoculum. Also, the agronomic management of the crop helps to reduce the favorable conditions and to increase the vigor of the plants: this management considers plantation density, drainage systems, irrigation methods, control of weeds, chemical-biological fertilization, and nematode control.

Biotecnologia de Dunaliella salina para produção de B caroteno

Dissertação de mest. em Aquacultura, Unidade de Ciências e Tecnologia dos Recursos Aquáticos, Univ. do Algarve, 1997

Aspectos da interacção entre Phytophthora cinnamomi e a doença do declínio em Q. suber e Q. rotundifolia

Faculdade de Engenharia de Recursos Naturais, Univ. do Algarve, 2001

Especificidad del hongo micorrizico (Rhizoctonia sp.) en Phalaenopsis sp., Cymbidium sp., Trichoceros antenifer, Oncidium excavatum, y Cyrtochilum sp.

Las orquídeas producen abundantes semillas pequeñas, careciendo de endospermo, cotiledones y sustancias de reserva para llevar a cabo su germinación. Es por esto que estratégicamente las semillas establecen una relación simbiótica con un hongo micorrízico que favorece a su germinación y desarrollo. El objetivo de este estudio fue determinar la especificidad del hongo micorrízico (Rhizoctonia sp.) en la germinación de cinco géneros de orquídeas. Se usaron dos medios de cultivo: 1) PhytamaxTM y 2) avena-agar+Rhizoctonia sp. Los resultados evaluados a los 45 y 75 días demuestran que no existe especificidad entre el hongo Rhizoctonia sp. y Trichoceros antenifer, especie de la cual se aisló el hongo. La germinación en tres de los cinco géneros evaluados fue mayor en el tratamiento avena-agar+Rhizoctonia sp., evidenciando también mayor tamaño y vigor las plantas que se desarrollaron en este tratamiento, aunque estos datos no fueron evaluados. Los resultados sugieren también que la planta hospedera del hongo inoculado podría tener una ventaja en cuanto al tiempo requerido para su germinación; sin embargo otras, especies también se ven favorecidas con el inóculo, aunque éste sea obtenido de otra fuente.

Improved production of chlorogenic acid from cell suspension cultures of Lonicera macranthoids

Purpose: To evaluate the potential of Lonicera macranthoids Hand. -Mazz. Yulei1 suspension culture system for enhanced production of the main secondary metabolite, chlorogenic acid. Methods: The callus of L. macranthoides Hand.-Mazz. “Yulei1” was suspension cultured in B5 liquid medium supplemented with different plant growth regulators. Biomass accumulation was calculated by weight method and chlorogenic acid production was measured using high performance liquid chromatography (HPLC). HPLC was carried out on C18 analytical column at 35 °C and the detection wavelength was set at 324 nm. Results: The results showed that maximum accumulation of biomass and chlorogenic acid were achieved 15 days after culture growth. The optimized conditions for biomass accumulation and chlorogenic acid production were 50 g/L of inoculum on fresh weight basis, B5 medium supplemented with plant growth regulators, 30 - 40 g/L sucrose and initial medium pH of 5.5. Maximum accumulation of chlorogenic acid and biomass were observed when the culture medium was supplemented with 2.0 mg/L6-BA. Optimal accumulation of chlorogenic acid was observed using combination of hormones 2.0 mg/L 6-Benzyladenine (BA) + 0.5 mg/L2, 4-Dichlorophenoxyacetic acid (2,4-D), while optimal accumulation of biomass was observed with 2.0 mg/L 6-BA + 2.0 mg/L2, 4-D. In addition, phenylalanine also contributed to the synthesis of chlorogenic acid at a concentration > 50 mg/L. Conclusion: Cell suspension cultures of L. macranthoides Hand.-Mazz. “Yulei1” have successfully been established. The findings provide a potential basis for large scale production of chlorogenic acid using cell suspension cultures of L. macranthoides.

Asian soybean rust in Brazil: past, present, and future.

ABSTRACT: Asian soybean rust, caused by the fungus Phakopsora pachyrhizi, is the most severe disease of the crop and can cause yield losses of up to 90%. The disease was first reported in Brazil in 2001. Epidemics of the disease are common in the country, where the fungus can survive year?round. Regulatory measures to reduce the inoculum between seasons and avoid late-season soybean have been adopted to manage the disease. Disease control has relied mainly on chemical control, but a lower sensibility of the fungus to fungicides has been reported in Brazil. Major?resistance genes have been mapped and incorporated into the cultivars. With the reduced efficacy of the fungicides, the adoption of integrated measures to control the disease will be important for the sustainability of the crop. This review presents the main changes in the soybean crop system caused by the introduction of the fungus in Brazil, the current management strategies adopted to avoid losses, and the new trends that, together with biotechnological strategies, can improve management in the future. RESUMO: A ferrugem?asiática da soja, causada pelo fungo Phakopsora pachyrhizi, é a doença mais severa da cultura e pode causar perdas de produtividade de até 90%. A doença foi relatada pela primeira vez no Brasil em 2001. Epidemias da doença são comuns no País, onde o fungo pode sobreviver durante todo o ano. Medidas regulatórias para reduzir o inóculo entre safras e evitar a semeadura tardia de soja têm sido adotadas para manejar a doença. O controle da doença tem se baseado principalmente no controle químico, mas uma menor sensibilidade do fungo aos fungicidas tem sido relatada no Brasil. Genes de resistência têm sido mapeados e incorporados às cultivares. Por causa da redução da eficiência dos fungicidas, a adoção de medidas integradas para o controle da doença será importante para a sustentabilidade da cultura. Este artigo de revisão apresenta as principais mudanças no sistema de produção da soja causadas pela introdução do fungo no Brasil, as medidas de controle atualmente usadas para evitar perdas, e as novas tendências que, juntas com estratégias biotecnológicas, podem melhorar o manejo da doença no futuro.

Molecular identification and characterization of phosphate solubilizing pseudomonas sp. isolated from rhizosphere of mash bean (Vigna mungo L.) for growth promotion in wheat

Bio-inoculants have potential role in plant growth promotion. The present study evaluated the potential of Pseudomonas strains as bio-inoculants in wheat on the basis of plant growth promotion and physiological characterization. The 16S rRNA gene sequencing and phylogenetic analysis revealed that four isolated strains belonged to genus Pseudomonas. These strains were positive for phosphorus solubilization and indole acetic acid production, whereas only two strains were positive candidate for their nitrogen fixing ability as determined by presence or absence of nifH gene through amplification from polymerase chain reaction. The pot experiment showed that the integrated use of Pseudomonas strains as co-inoculant and 50% applied mineral fertilizers enhanced the maximum wheat growth and development from 58 to 140% for different shoot and root growth parameters. The strain NCCP-45 and NCCP-237 were closely related to Pseudomonas beteli and Pseudomonas lini, respectively. These isolated strains can be used to increase crop productivity by using as a bio-fertilizer inoculum.

Exploring the potential of novel biomixtures and Lentinula edodes fungus for the degradation of selected pesticides. Evaluation for use in biobed systems

An approach to reduce the contamination of water sourceswith pesticides is the use of biopurificaction systems. The active core of these systems is the biomixture. The composition of biomixtures depends on the availability of local agro-industrial wastes and design should be adapted to every region. In Portugal, cork processing is generally regarded as environmentally friendly and would be interesting to find applications for its industry residues. In this work the potential use of different substrates in biomixtures, as cork (CBX); cork and straw, coat pine and LECA (Light Expanded Clay Aggregates), was tested on the degradation of terbuthylazine, difenoconazole, diflufenican and pendimethalin pesticides. Bioaugmentation strategies using the white-rot fungus Lentinula edodes inoculated into the CBX, was also assessed. The results obtained from this study clearly demonstrated the relevance of using natural biosorbents as cork residues to increase the capacity of pesticide dissipation in biomixtures for establishing biobeds. Furthermore, higher degradation of all the pesticides was achieved by use of bioaugmented biomixtures. Indeed, the biomixtures inoculated with L. edodes EL1were able to mineralize the selected xenobiotics, revelling that these white-rot fungi might be a suitable fungus for being used as inoculum sources in on-farm sustainable biopurification system, in order to increase its degradation efficiency. After 120 days, maximum degradation of terbuthylazine, difenoconazole, diflufenican and pendimethalin, of bioaugmented CBX, was 89.9%, 75.0%, 65.0% and 99.4%, respectively. The dominant metabolic route of terbuthylazine in biomixtures inoculated with L. edodes EL1 proceeded mainly via hydroxylation, towards production of terbuthylazine-hydroxy-2 metabolite. Finally, sorption process to cork by pesticides proved to be a reversible process,working cork as a mitigating factor reducing the toxicity to microorganisms in the biomixture, especially in the early stages.