Produção de ácido indol-3-acético e fitases por fermentação em estado sólido e aplicação em Eucalyptus grandis x E. urophylla
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Data
2019-02-01
Autores
Prado, Débora Zanoni do [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O Eucalyptus é um importante gênero florestal plantado mundialmente para fins comerciais. O Brasil é líder global em produtividade de Eucalyptus, devido às condições climáticas favoráveis e a eficientes programas de melhoramento baseados em plantações clonais, porém alguns clones de Eucalyptus possuem dificuldades de propagação, principalmente relacionadas ao enraizamento. A inoculação fúngica e bacteriana pode auxiliar o enraizamento e desenvolvimento vegetal pela produção de metabólitos tais como ácido indol-3-acético (AIA) e fitases. O objetivo deste trabalho foi produzir AIA e fitases por fermentação em estado sólido (FES) utilizando Aspergillus spp., Trichoderma spp. e Bacillus spp. e a inoculação das cepas de maior produtividade em mudas de Eucalyptus grandis x E. urophylla (clone IPB2) visando otimizar o desenvolvimento vegetal. A FES ocorreu utilizando bagaço de mandioca, farelos de soja e trigo e grãos secos de destilaria com solúveis (DDGS) de sorgo e milho como substratos. Nos filtrados da FES foram determinados a concentração de AIA e a atividade de fitase. As características físicas e químicas dos substratos [macroporosidade (%), microporosidade (%), retenção de água (mL/cm3), condutividade elétrica (mS/cm3), pH, proteína bruta (%), lipídeos (%), hemicelulose (%), celulose (%) e lignina (%)] foram determinadas e correlacionadas à produtividade das biomoléculas, a fim de esclarecer a influência na alteração do metabolismo microbiano para maior ou menor produtividade de AIA e fitases. Os micro-organismos com maior produtividade de AIA e fitases foram selecionados para a continuidade do estudo. Foram implantados dois experimentos, o primeiro testando a produção de auxina pela fermentação de A. flavipes em farelo de soja (0,5 mm) com adição de água (15 mL) e triptofano (1,5 %, m/m), descrito na patente BR 10 2018 007927 1 e o segundo testando a cepa 01 de A. niger cultivada em farelo de trigo com adição de água (10 mL), produtor de fitase. Os tratamentos de ambos experimentos foram aplicados em formas sólida e líquida ao substrato Carolina I® nas doses 0, 40, 80, 120 e 160 mg de AIA kg-1 e 0, 85, 170, 255 e 340 U de fitase por kg-1 de substrato, respectivamente. O plantio das estacas foi realizado nos substratos tratados e no controle. O experimento com AIA, cujo objetivo foi avaliar o enraizamento, teve duração de 40 dias; enquanto o experimento com fitase, cujo objetivo foi avaliar o desenvolvimento vegetal de forma geral, se prolongou por 5 meses. Foram realizadas avaliações de sobrevivência (%), enraizamento (%), comprimento da parte aérea e das raízes e as massas fresca e seca das raízes. Para o experimento com fitase, também foram determinados o comprimento da parte aérea, diâmetro médio do colo, a massa seca total, a intensidade de coloração verde das folhas e o número de folhas. Nossos resultados confirmaram a capacidade de produção de AIA e fitases pela maioria dos micro-organismos utilizados neste estudo. Houve influência negativa de lignina na produtividade de AIA por Bacillus e Trichoderma e influência positiva do pH, hemicelulose e microporos para a produção de AIA por Bacillus. O produto descrito na patente BR 10 2018 007927 1, na forma sólida e na faixa de dosagem de 40 a 120 mg kg-1, incrementou a porcentagem de enraizamento, o comprimento radicular e as massas fresca e seca das raízes, enquanto o tratamento com 120 mg kg-1 do produto na forma líquida induziu o aumento do comprimento das raízes e da massa seca radicular, além dos efeitos positivos no enraizamento o produto não apresentou citotoxicidade em fibroblastos (NIH/3t3). Já a inoculação A. niger, em geral, prejudicou o desenvolvimento do clone IBP2, principalmente nos tratamentos com a forma líquida, indicando a produção de metabólitos fitotóxicos pelo micro-organismo. Assim, em estudos futuros, sugerimos que a fitase produzida por A. niger seja testada em Eucalyptus grandis x E. urophylla na forma purificada.
Eucalyptus is an important forest genus planted worldwide for commercial purposes. Brazil is the global leader in Eucalyptus productivity due to favorable climatic conditions and efficient breeding programs based on clonal plantations, but some Eucalyptus clones have difficulties of propagation, mainly related to rooting. Fungal and bacterial inoculation can positively influence rooting and plant development by producing metabolites such as indole-3-acetic acid (IAA) and phytases. This work aimed to produce IAA and phytases under solid state fermentation (SSF) using Aspergillus spp., Trichoderma spp. and Bacillus spp. and inoculate the best producers in cuttings of Eucalyptus grandis x E. urophylla (clone IPB2), aiming to optimize plant development. The SSF technique was performed using cassava bagasse, soybean and wheat bran and distillers dried grains with solubles (DDGS) of sorghum and maize as substrates. The concentration of IAA and phytase activity were determined in SSF filtrates. The physical and chemical characteristics of the substrates [macroporosity (%), microporosity (%), water retention (mL/cm3 ), electrical conductivity (mS/cm3 ), pH, brute protein (%), lipids (%), hemicellulose %), cellulose (%) and lignin (%)] were determined and correlated to the biomolecules productivity in order to clarify their influence on the alteration of microbial metabolism for higher or lower productivity of IAA and phytases. The microorganisms with higher productivity of IAA and phytases were selected for the study continuation. Two experiments were carried out, the first one tested the fermentation of A. flavipes in soybean bran (0.5 mm) with addition of water (15 mL) and tryptophan (1.5%, m/m), described in patent BR 10 2018 0079271, source of IAA and the second tested the strain 01 of A. niger, grown on wheat bran with addition of water (10 mL), source of phytase. The treatments of both experiments were applied in solid and liquid forms to the substrate Carolina I® at 0, 40, 80, 120 and 160 mg of IAA kg-1 and 0, 85, 170, 255 and 340 U of phytase kg-1 of substrate, respectively. The planting of cuttings was carried out on treated substrates and on control. The experiment with IAA, which aimed rooting improvement, lasted 40 days, while the experiment with phytase, whose objective was to evaluate plant development, lasted 5 months. Plants survival (%), rooting (%), shoot and root length, and fresh and dry mass of the roots were evaluated. For the phytase experiment, shoot length, colon diameter, total dry mass, green intensity in leaves and number of leaves were also determined. Our results confirmed the production of IAA and phytases by most of the microorganisms tested in this study. There was a negative influence of 8 lignin on the productivity of IAA by Bacillus and Trichoderma and positive influence of pH, hemicellulose and micropores for the production of IAA by Bacillus. The product described in the patent BR 10 2018 007927 1, in solid form, and in the range from 40 to 120 mg kg-1 , increased rooting percentage, root length and fresh and dry mass of the roots, while treatment with 120 mg kg-1 of the product in liquid form induced increases in root length, and root dry mass. In addition to the positive rooting effects, the product did not show cytotoxicity in fibroblasts (NIH/3t3). However, A. niger inoculation generally impaired the development of the IBP2 clone, mainly in treatments with the liquid form, indicating the production of phytotoxic metabolites by the microorganism. Thus, in future studies, we suggest testing purified A. niger phytases in Eucalyptus grandis x E. urophylla
Eucalyptus is an important forest genus planted worldwide for commercial purposes. Brazil is the global leader in Eucalyptus productivity due to favorable climatic conditions and efficient breeding programs based on clonal plantations, but some Eucalyptus clones have difficulties of propagation, mainly related to rooting. Fungal and bacterial inoculation can positively influence rooting and plant development by producing metabolites such as indole-3-acetic acid (IAA) and phytases. This work aimed to produce IAA and phytases under solid state fermentation (SSF) using Aspergillus spp., Trichoderma spp. and Bacillus spp. and inoculate the best producers in cuttings of Eucalyptus grandis x E. urophylla (clone IPB2), aiming to optimize plant development. The SSF technique was performed using cassava bagasse, soybean and wheat bran and distillers dried grains with solubles (DDGS) of sorghum and maize as substrates. The concentration of IAA and phytase activity were determined in SSF filtrates. The physical and chemical characteristics of the substrates [macroporosity (%), microporosity (%), water retention (mL/cm3 ), electrical conductivity (mS/cm3 ), pH, brute protein (%), lipids (%), hemicellulose %), cellulose (%) and lignin (%)] were determined and correlated to the biomolecules productivity in order to clarify their influence on the alteration of microbial metabolism for higher or lower productivity of IAA and phytases. The microorganisms with higher productivity of IAA and phytases were selected for the study continuation. Two experiments were carried out, the first one tested the fermentation of A. flavipes in soybean bran (0.5 mm) with addition of water (15 mL) and tryptophan (1.5%, m/m), described in patent BR 10 2018 0079271, source of IAA and the second tested the strain 01 of A. niger, grown on wheat bran with addition of water (10 mL), source of phytase. The treatments of both experiments were applied in solid and liquid forms to the substrate Carolina I® at 0, 40, 80, 120 and 160 mg of IAA kg-1 and 0, 85, 170, 255 and 340 U of phytase kg-1 of substrate, respectively. The planting of cuttings was carried out on treated substrates and on control. The experiment with IAA, which aimed rooting improvement, lasted 40 days, while the experiment with phytase, whose objective was to evaluate plant development, lasted 5 months. Plants survival (%), rooting (%), shoot and root length, and fresh and dry mass of the roots were evaluated. For the phytase experiment, shoot length, colon diameter, total dry mass, green intensity in leaves and number of leaves were also determined. Our results confirmed the production of IAA and phytases by most of the microorganisms tested in this study. There was a negative influence of 8 lignin on the productivity of IAA by Bacillus and Trichoderma and positive influence of pH, hemicellulose and micropores for the production of IAA by Bacillus. The product described in the patent BR 10 2018 007927 1, in solid form, and in the range from 40 to 120 mg kg-1 , increased rooting percentage, root length and fresh and dry mass of the roots, while treatment with 120 mg kg-1 of the product in liquid form induced increases in root length, and root dry mass. In addition to the positive rooting effects, the product did not show cytotoxicity in fibroblasts (NIH/3t3). However, A. niger inoculation generally impaired the development of the IBP2 clone, mainly in treatments with the liquid form, indicating the production of phytotoxic metabolites by the microorganism. Thus, in future studies, we suggest testing purified A. niger phytases in Eucalyptus grandis x E. urophylla
Descrição
Palavras-chave
Aspergillus, Trichoderma, Bacillus, enraizamento, fitohormônio, enzima, rooting, phytohormone, enzyme