Luiz Antonio de Oliveira

Rhizobia amylase production using various starchy substances as carbon substrates

Six isolates of indigenous rhizobia of Central Amazonia were screened for the production of amylases in liquid media using various starchy substances as carbon sources. All rhizobia strains could produce more extracellular protein, biomass and amylases with the different kinds of carbon substrates. Among the carbon sources tested maltose was the best substrate for protein and amylase production. In general, peach palm flour and corn starch (maizena®) were also considered to be good carbon sources for rhizobia amylases. On the other hand, the biomass production by the rhizobia isolates was higher in the presence of oat flour. INPA strain R-926 was a good amylase producer in maltose (1.94 U) and corn starch (0.53 U) media. INPA strain R-991 was also a good amylase producer in maltose (1.66 U) and corn starch (1.59 U) yielding significant extracellular amylase. Correlation analysis showed significant and positive relationships between rhizobia amylases and final pH (r = 0.49, P < 0.05), extracellular protein (r = 0.47, P < 0.47) and biomass production (r = 0.69, P < 0.01) in the maltose medium. The results obtained in this study revealed several Central Amazonian rhizobia strains as promising sources of amylase for biotechnological applications, especially in starch industry.

Seasonal dynamics of arbuscular mycorrhizal fungi in plants of Theobroma grandiflorum Schum and Paullinia cupana Mart. of aN agroforestry system in Central Amazonia, Amazonas State, Brazil

The seasonal dynamics of Arbuscular Mycorrhizal Fungi (AMF) was investigated in the rhizosphere of two fruit species in a terra firme (upland) ecosystem in Central Amazonia. Two host species (Theobroma grandiflorum and Paullinia cupana) and nine sampling months (August, September and December/1998, February, April, May and December/1999, February and May/2000) were studied in a completely randomized design, with five replications, set in a 2 x 9 factorial experiment. Soil (0-20 cm depth) and root samples were collected between August 1998 and May 2000. The mean percent colonization of AMF for both species reached maximal values in February and May 2000 (rainy season). In April and May 1999, February and May 2000 (rainy season) the highest AMF spore numbers were registered. The pluvial precipitation was significantly positively correlated with AMF number spores for both fruit species, and significant positive correlation only with AMF colonization of P. cupana. Soil moisture content was positively correlated with colonization and spore numbers of AMF for both species evaluated. AMF colonization and AMF spore numbers of T. grandiflorum were positively correlated with soil Mg and K concentrations. AMF spore numbers of T. grandiflorum were also negatively correlated with effective cation exchange capacity (ECEC). AMF colonization and AMF spore numbers in the rhizosphere of P. cupana were positively correlated with pH and Mn concentrations. AMF colonization was also positively correlated with AMF spore numbers for both species evaluated. In conclusion, this study showed that AMF colonization and sporulation are seasonal and dependent on host plant species, pluvial precipitation, soil moisture content and soil chemistry in Central Amazonia conditions.

Características fisiológicas e ecológicas de isolados de rizóbios oriundos de solos ácidos de Iranduba, Amazonas

O aluminio e a acidez dos solos da Amazonia podem diminuir a populacao de rizobios que fixam o nitrogenio, solubilizam fosfatos pouco soluveis e aumentam a disponibilidade do fosforo em sistemas produtivos. O objetivo deste trabalho foi avaliar a capacidade nodulifera, tolerância a acidez e ao aluminio, bem como a capacidade de solubilizar fosfatos insoluveis de isolados de rizobios, obtidos de amostras de solos agricolas do Municipio de Iranduba, AM. As amostras de solos apresentavam isolados de rizobios com potencialidades para serem utilizados como fonte de inoculos para o caupi, de cujos nodulos foram extraidos para testes de tolerância e solubilizacao. O inoculo INPA-M10 tendeu a superar todos os demais quanto ao rendimento da biomassa seca da parte aerea das plantas. Houve correlacao significativa entre o numero e massa dos nodulos, o que indica que os isolados apresentaram nodulos de tamanho semelhante. A sensibilidade a acidez e ao aluminio foi apresentada por 43% dos isolados; os demais se comportaram como tolerantes. A solubilizacao de fosfatos de calcio foi observada na presenca de 57% dos isolados. Nessa mesma proporcao tambem foram encontrados isolados que solubilizaram fosfato de aluminio.

Características fisiológicas e ecológicas de isolados de rizóbios oriundos de solos ácidos e álicos de Presidente Figueiredo, Amazonas

Some isolated rhizobias, besides fixing N 2 , are also able to solubilize low soluble phosphates, making the P available both for plants and for themselves. Some factors like toxic Al and acidity, as in some Amazonian soils, may decrease the population of these microorganisms. The present study evaluated the nodulation ability, tolerance to acidity and toxic aluminum, as well as the ability to solubilize Ca and Al phosphates of rhizobia isolated from agricultural soils the municipality of from Presidente Figueiredo, AM. Samples of soil under agricultural cultivation were collected and used as a source of inoculum for the cowpea bean. Some rhizobia isolates were tested for tolerance to acidity and toxic Al, as well as to verify their abilities to solubilize phosphates. The soil samples contained rhizobia populations capable of inducing the nodulation and improving the aerial biomass of cowpea bean under acidity (pH 4,5) and alic (2 cmol c Al.L -1 ) conditions. The rhizobia population from the soil samples idnetified as INPA-PF2, INPA-PF3, INPA-PF4, INPA-PF5, INPA-PF13, INPA-PF15, INPA-PF22 and INPA-PF24 promoted increases of aerial biomass when compared to the control tratment (absence of inoculum). Twenty-five percent of the isolates were tolerant to acidity and 23% to Al. The Ca phosphate was solubilized by 39% of the isolated rhizobia. However, just one isolate presented high solubilization index. The Al phosphate solubilization ability was identified in 67% of the isolates. The majority of the isolates that solubilized Ca phosphate (76.5% of the lineages) also solubilized the Al phosphate.

Seleção de Basidiomycetes da Amazônia para produção de enzimas de interesse biotecnológico

Mushrooms, edible basidiomycetes, have been extensively used as producers of different substances of economical interest, such as enzymes, antibiotics, vitamins, amino acids, and steroids. The objective of this study is to detect the production of enzymes such as amylases, proteases, cellulases, phenoloxidases and pectinases for lineages of basidiomycetes originating from the Amazonian forest. For the production of enzymes, mushrooms were cultivated in a liquid medium with substratum (0,5%), pH adjusted for each enzyme, and incubated at 28 °C, under agitation and 140 rpm for 96 or 120 hours. After this period, the samples were filtered for the separation of the mycelial mass. The filtrates were inoculated in solid medium surface perforated cup-plates of 6 mm diameter, appropriate for the detection of enzymes in Petri dishes. The plates were incubated at 28 °C for 24 hours, for observation to reveal enzymatic halos. It was verified the activity of the amylase and protease produced by the mushrooms grown in the liquid medium, with different nutritive sources. It was possible to detect the cellulase and protease production for all the ones that were isolated, 40% produced amylases, 50% produced phenoloxidases, and 10% produced pectinases. Regarding the activity of the amylase, the wheat bran substrate presented the largest degradation halos revealing Daedalea sp.4E6 and Daedalea sp. 1A, Stereaceae 22B and Pycnoporus sanguineus 12B mushrooms. Considering the substrata tested for protease production, it was observed that the fish protein concentrate was the best. The P. sanguineus 12B, Stereaceae 22B and Cantharellus guyanensis 4Bl mushrooms were the best protease producers.

Partial characterization of amylases of two indigenous Central Amazonian rhizobia strains

A producao e parcial caracterizacao de extratos brutos de amilase de duas estirpes de rizobio (R-926 e R-991) foram avaliadas. Para ambas as estirpes, as maximas atividades amiloliticas foram obtidas no inicio/meio da fase exponencial de crescimento. As amilases rizobiais foram ativas numa variacao de pH de 4,5 a 8,5 e temperatura de 30 a 50 oC. Nenhum dos ions testados (K+, Na+, Ca2+, Hg2+, Mg2+, Mn2+, Cu2+ e Zn2+) foi exigido para a atividade catalitica da estirpe R-926. A amilase produzida pelo R991 foi estimulada na presenca de K+, Hg2+ e Zn2+. Os surfactantes SDS, Triton X-100 e Tween-80 nao exerceram um pronunciado efeito inibitorio sobre as atividades enzimaticas, e SDS e Tween-80 causaram os maiores efeitos estimulatorios. A atividade amilolitica rizobial foi reduzida em ate 30% na presenca de EDTA; a amilase produzida pela estirpe R-926 tambem foi inibida pelo HgCl2, sugerindo, ao menos em parte, a importância de Ca2+ e residuos de cisteina na atividade amilolitica dessa estirpe.

Lipase Activity among Bacteria Isolated from Amazonian Soils

The objective of this study was to select lipase-producing bacteria collected from different counties of the Amazon region. Of the 440 bacteria strains, 181 were selected for the lipase assay in qualitative tests at Petri dishes, being 75 (41%) lipase positive. The enzymatic index was determined during fifteen days at different temperatures (30°, 35°, 40°, and 45°C). The highest lipase activity was observed within 72 hours at 30°C. Twelve bacteria strains presented an index equal to or greater than the standard used like reference, demonstrating the potential of microbial resource. After the bioassay in Petri dishes, the selected bacteria strains were analyzed in quantitative tests on p-nitrophenyl palmitate (p-NPP). A group of the strains was selected for other phases of study with the use in oleaginous substrates of the Amazonian flora, aiming for the application in processes like oil biotransformation.

Atividade enzimática de isolados de rizóbia nativos da Amazônia Central crescendo em diferentes níveis de acidez

A importância das bacterias conhecidas como rizobia no estabelecimento de leguminosas tem sido amplamente reconhecida. Porem, poucas sao as informacoes referentes ao perfil enzimatico dessas bacterias beneficas. O estudo objetivou investigar a influencia do pH do meio solido sobre a atividade enzimatica de rizobios nativos da Amazonia Central. Essa triagem constitui o primeiro passo no processo de selecao de microorganismos beneficos, como produtores de enzimas de aplicacao biotecnologica. Nesse estudo, 64 isolados de rizobia foram testados para a producao extracelular de amilase, lipase, pectinase e protease, em meio YMA modificado. Excetuando a atividade pectinolitica, todas as outras enzimas (amilase, lipase e protease) foram detectadas nos isolados investigados. Dois isolados (INPA R-975 e INPA R-926) exibiram atividades amiloliticas, lipoliticas e proteoliticas. Os indices enzimaticos amiloliticos e proteoliticos variaram significativamente entre os isolados e as condicoes de pH do meio de cultura. De maneira geral, as maiores atividades amiloliticas e proteoliticas foram exibidas pelos isolados INPA R-957, INPA R-915B e INPA R-991 em pH 6,5. O isolado INPA R-957 tambem se mostrou amilolitico e proteolitico nos pHs 5,0 e 8,0. Esse estudo mostrou que alguns rizobios nativos da Amazonia representam fontes promissoras de amilase e protease de uso biotecnologico, sobretudo na tecnologia de alimentos.

Ocorrência de nodulação em leguminosas florestais de terra firme nativas da região de Manaus-AM.

Fez-se um levantamento da nodulacao em mudas de especies florestais nativas, em area proxima de Manaus na Amazonia, caracterizada por uma vegetacao do tipo tropical umida de terra firme, em Latossolo Amarelo de varias texturas. Este levantamento foi feito com a extracao de mudas com sistema radicular intacto, sendo registrada a presenca de nodulos e o tipo de solo em que se fez a coleta. Estas mudas foram transplantadas para recipientes plasticos com solo de floresta, com o objetivo de verificar se ocorria nodulacao por Rhizobium nativo em condicoes de viveiro. Apos um ano, sob estas condicoes, foram feitas observacoes da presenca de nodulos, atividade da nitrogenase, bem como algumas caracteristicas dos nodulos. Das 34 especies pesquisadas, 22 mostraram-se capazes de nodular. Destas, nove sao da subfamilia Mimosoideae, seis da Caesalpinioideae e quatro da Papilionoideae, sendo que doze foram coletadas em solo arenoso, seis em solo argiloso e uma nos dois tipos de solo. Baixas atividades da nitrogenase foram encontradas na maioria das especies. Foi observada a ocorrencia de nodulos em algumas especies (Cedrelinga catenaeformis, Diplotropis purpurea e Tachigalia sp.) que nao haviam sido mencionadas como capazes de nodular em recente revisao.

Fixação de nitrogênio (redução de acetileno) em cupins (Insecta: Isoptera) da Amazônia Central

Vinte e uma espécies de cupins foram coletndas de seus cupinzeiros em áreas de pastagem, floresta secundária e floresta primária, numa fazc:ndR na Amazônia Central no Brasil A atividade de nitrog_nase associ-a· da com os cupms foi testada imsdiatamznte pelo método de redução de acetileno. Todas as atividades maiores (acima de 100 nmoles C~H4 prcduz1dos/g de peso seco/hora) ocorreram em cupins do gónero Nasutiter· mos coletedos na pastagem. Nasutitermes é conheci· do como o gênero mais comum na área. As taxas de redução do acetileno detectadas em cupins da floresta primária e secundária foram mais baixas que na pasta· gcm; porém, cupins do gênero Nasutitermes não foram testados na floresta secundária A d ferença da taxa de redução de ccctileno entre os operários e soldados foi pequena. As taxas foram lineares dentro de pe· rlodos de três horas de incubação, exceto em dois de nove casos. A fixação de nllrogênio associada com cupins pode ser um fator ecológico Importante na Amazônia Central.