José Carlos Germani

Screening Brazilian Macrophomina phaseolina isolates for alkaline lipases and other extracellular hydrolases.

Macrophomina phaseolina, phylum Ascomycota, is a phytopathogenic fungus distributed worldwide in hot dry areas. There are few studies on its secreted lipases and none on its colony radial growth rate, an indicator of fungal ability to use nutrients for growth, on media other than potato-dextrose agar. In this study, 13 M. phaseolina isolates collected in different Brazilian regions were screened for fast-growth and the production of hydrolases of industrial interest, especially alkaline lipases. Hydrolase detection and growth rate determination were done on citric pectin, gelatin, casein, soluble starch, and olive oil as substrates. Ten isolates were found to be active on all substrates tested. The most commonly detected enzymes were pectinases, amylases, and lipases. The growth rate on pectin was significantly higher (P < 0.05), while the growth rates on the different media identified CMM 2105, CMM 1091, and PEL as the fastest-growing isolates. The lipase activity of four isolates grown on olive oil was followed for 4 days by measuring the activity in the cultivation broth. The specific lipolytic activity of isolate PEL was significantly higher at 96 h (130 mU mg protein(-1)). The broth was active at 37 °C, pH 8, indicating the potential utility of the lipases of this isolate in mild alkaline detergents. There was a strong and positive correlation (0.86) between radial growth rate and specific lipolytic activity.

Genetic variability of Bipolaris sorokiniana isolates using URP-PCR

Spot blotch disease, caused by Bipolaris sorokiniana, is one of the major diseases of wheat and is responsible for large losses of wheat crops worldwide. We used polymerase chain reaction (PCR) with universal rice primers (URP) for molecular characterization of 60 monosporic B. sorokiniana isolates from Brazil and other countries, and evaluated the diversity of the samples. PCR amplification generated 232 different DNA fragments ranging in size from 100 to 2018 bp. The primers URP-4R, URP-2R, and URP-1F generated greater numbers of amplified fragments (36, 30, and 25, respectively) from the single-spore isolates, and some diversity was observed among the isolates generated using these primers. The primers URP-2F, URP-6R, URP-17R, URP-30F, and URP-38F produced a pattern of monomorphic fragments and 73% of the isolates showed an average of 44 different DNA-amplified fragments. Primer URP-2F generated a 578-bp fragment that was common to 83.7% of the isolates; primer URP-6R generated a 548-bp fragment and primer URP-38F generated a 650bp product that was common to 89.1% and 80% of the isolates, respectively. The URP-PCR primers provided important information about the genetic profiles of the monosporic cultures, which showed intraspecific variability among the monoconidial isolates and among the monosporic cultures that originated from the same polysporic strain. Our results indicate that URPs are sensitive and give reproducible results for assaying the genetic variability of B. sorokiniana.

Recovery of Extracellular Lipolytic Enzymes from Macrophomina phaseolina by Foam Fractionation with Air

Macrophomina phaseolina was cultivated in complex and simple media for the production of extracellular lipolytic enzymes. Culture supernatants were batch foam fractionated for the recovery of these enzymes, and column design and operation included the use of P 2 frit (porosity 40 to 100 μm), air as sparging gas at variable flow rates, and Triton X-100 added at the beginning or gradually in aliquots. Samples taken at intervals showed the progress of the kinetic and the efficiency parameters. Best results were obtained with the simple medium supernatant by combining the stepwise addition of small amounts of the surfactant with the variation of the air flow rates along the separation. Inert proteins were foamed out first, and the subsequent foamate was enriched in the enzymes, showing estimated activity recovery (R), enrichment ratio (E), and purification factor (P) of 45%, 34.7, and 2.9, respectively. Lipases were present in the enriched foamate.

Production of lipolytic enzymes by bacteria isolated from biological effluent treatment systems

This work aimed to evaluate the production of lipolytic complexes, produced by microorganisms isolated from a biological treatment system of effluents from a hotel. To select the best lipolytic microorganism for use in biotechnological processes, we tested 45 bacterial isolates recovered from the raw effluent of the hotels restaurant waste tank. Lipase production was assayed in culture medium supplemented with olive oil and rhodamine B, incubated at 25 °C and 30 °C for 24 h - 48 h. Results showed 22 isolates lipase producers. All isolates were inoculated on medium without yeast extract to select the ones with highest enzyme yields. Out of these, nine isolates showed high lipase activity. The strain with the larger halo was assayed in submerged culture using an orbital shaker and a bioreactor, with three different substrates (olive oil, grape seed oil, and canola oil). Isolate G40 identified as Acinetobacter baylyi was selected to run the production assays because it showed the best result in the solid medium. In the bioreactor, maximum lipase production was obtained after 12 h of culture with the three substrates evaluated: 0,358 U/mL.min-1 in olive oil, 0,352 U/mL.min-1 with grapeseed oil, and 0,348 U/mL.min-1 with canola oil.

DIVERSIDADE DE BACTÉRIAS GRAM-POSITIVAS PRESENTES NO PESCADO E NO CONVÉS DE BARCOS PESQUEIROS APORTADOS NO MUNICÍPIO DE PASSO DE TORRES (SC)

O pescado e um alimento de excelente valor nutritivo; entretanto, e muito perecivel, necessitando de condicoes sanitarias adequadas desde a sua captura ate a comercializacao. Objetivou-se, neste estudo, determinar a presenca de populacoes bacterianas em pescado desde a sua captura ate a entrega ao distribuidor. As amostras foram coletadas em diferentes pontos: no conves do barco pesqueiro, no pescado e no peixe armazenado no momento do desembarque. Um total de 95 micro-organismos isolados foram identificados atraves de tecnicas tradicionais, provas bioquimicas e sequenciamento do 16S rDNA. As bacterias identificadas foram: Aerococcus viridans, Bacillus spp., Brochothrix thermosphacta, Leifsonia aquaticum, Exiguobacterium aurantiacum, Marinococcus halotolerans, Micrococcus luteus, Rhodococcus corynebacterioides, Staphylococcus aureus, S. epidermidis, Salinicoccus alkaliphilus, S. roseus, Streptococcus iniae e Vagococcus fluvialis. Os micro-organismos isolados apontaram uma diversidade bacteriana ligada a influencia de fatores de origem marinha, humana e de contaminacao ambiental, que podem ser