62ª Reunião Anual da SBPC

C. Ciências Biológicas - 10. Microbiologia - 2. Microbiologia Aplicada

FILAMENTOUS FUNGI ISOLATED FROM Neoteredo reynei: POTENTIAL PRODUCING CELLULASES

Nelson Rosa Ferreira 1 Ana Paula Bastos Ferreira 2 Alberdan Silva Santos 1

1. Laboratório de Investigação Sistemática em Biotecnologia e Química Fina, UFPa 2. Faculdade de Química, Universidade Federal do Pará

INTRODUÇÃO:

Fungi are lower eukariotes microorganisms that have been important in both ancient and modern biotechnological processes and are known as excellent metabolites producer agents of antibiotics, alcohols, enzymes, organic acids, pharmaceuticals and diverse organic compounds. Fungi present a different way of nutrition, secreting a wide range of secondary metabolites and powerful enzymes, such peroxidase, hydrolases among others, into the environment that oxidize and hydrolase lignins, cellulose, polysaccharides to micro molecules such phenyl alcane and glucose. The shipworms (Neoteredo reyney), also known in the Amazon region as "turus", are marine bivalve mollusks species belonging to the family Teredinidae, these are fixed in the wood at the end of their larval stage and as they grow build long galleries lined with limestone. They are important decomposers of wood (xylophagous), especially in mangroves. It is believed that the ability of these animals to survive in the logs of trees using cellulose as a carbon source is related to the interaction with saccharifying agents (bacteria and fungi) present in the intestinal tract. The microflora in the gut of xylophagous is an example of symbiosis between an animal and a variety of microorganisms. The characteristics of microorganisms present in the intestines of these animals have been commonly studied. An important benefit of these microorganisms is to enable carbon, nitrogen and energy nutrients to their hosts. This interaction has already been observed in several insects, including wasps (Sirex noctilio), bees (Apis mellifera), ant (Myrmecia greedy) and termites (Cryptotermes hilli, Neotermes insularis, Schedorhinotermes actuosus, Schedorhinotermes breinli). The objective of this work is to isolate naturally occurring microorganisms from shipworm and assess the capabilities of saccharification of these microorganisms in different types of cellulose.

METODOLOGIA:

The collection of Neoteredo reynei (Bivalvia, Teredinidae) was held in October of 2007 in the rural municipality of Bragança, distant 210 km from the city of Belem, state capital of Pará (Brazil) in the following positions: 1°'03'S and 46°'42 'W. The shipworms were collected directly in the mangrove (swamp) during low tide and removed from the interior of logs with the aid of ax. They were immediately placed in ice coolers for storage and subsequent selection. Three cryopreserved specimens were randomly selected for cutting parts of the tissue along its structure, totaling an amount of 10g tissue. The mass obtained was transferred aseptically to a conical flask containing 100 ml of sterile sodium chloride 1% (w/v). Aliquots of 200 ml were collected from the material under continuous agitation and dispensed in Petri dishes containing individual potato dextrose agar and Sabouraud agar. Aliquots were seeded evenly and incubated at 30.0 ± 0.5 ºC for 5 days. Then fungi colonies were isolated and transferred to GPY agar. The mycelial growth speed was evaluated by the biometric orthogonal axes method. We used three culture media for the assessment of mycelial growth: i) CMC agar (with sodium carboxymethylcellulose). ii) MCC gar (with microcrystalline cellulose), and Modified GPY (with glucose). The microorganisms were incubated at 30.0 ± 0.5 ºC for 72 hours and subsequently were evaluated every 24 h intervals by the measurement of mycelial growth diameters in the two directions of the orthogonal axes and statistical analysis was applied. After evaluation of mycelial growth, were added to 10 ml of congo red dye (2.5 g l-1) diluted in Tris-HCl 0.1 M pH 8.0 on each plate of different culture media (CMC agar, CCM agar and modified GPY agar) . After 30 minutes the solution was discarded and the colonies were washed with 5 ml of sodium chloride solution 0.5 M. Was considered positive test results showed that a clear halo around the mycelium. Based on the results of the speed of mycelial growth and the halo of degradation of cellulose were initially selected two strains of microorganisms for the submerged cultivation. Thus, standardized mycelium fragments of these two strains were inoculated in liquid MCC and CMC medium. The cultures were maintained at 30.0 ± 1.0 ° C, 150 rpm for 10 days. This period, aliquots were removed at intervals of 48 hours to evaluate the content of reducing sugars, proteins and pH.

RESULTADOS:

The collection of shipworms was held in end of summer Amazon and large quantities of mangrove woody biomass containing these molluscs are observed after high tide, was perceived a higher density of shipworms in small logs, although it has also been observed in the presence of larger trunks. The interior of the trunk showed a slightly red color, showing a contrast with the outside. Despite the firm consistency of wood colonized by shipworm, the formation of galleries in the trunks was observed, varying according to the size of the animal.The simultaneous primary cultures on PDA and Sabouraud agar allowed isolating microorganisms that apparently did not grow in these two culture media. This provided a larger number of filamentous fungi strains with different morphological characteristics, noting the growing conditions established. A total of twelve strains were selected and then isolated in GPY agar culture medium. By evaluating the speed of mycelial growth, it was possible to select two strains (NR02 and NR04) that showed the best growth profiles in both sources of cellulose (CMC and CMM). Thus, the agar plates with the remaining microorganism mycelial growth were covered with congo red solution. It was observed that the halos of saccharification of cellulose were formed in the ranged between 1-6 mm depending on the strain of microorganism. The two strains selected for the submerged cultivation showed different production profiles of reducing sugars and proteins. The strain NR02 showed the maximum production of reducing sugars equivalent to 1.1 mg ml-1 on the seventh day of culture and 2.5 mg ml-1 protein in the same period, when CMC was used as carbon source. Otherwise, for the same strain using MCC, the maximum amount of reducing sugars was 0.5 mg ml-1 on the fifth day of culture and 1.8 mg ml-1 protein in the same period. Strain NR04 presented the maximum production of 0.62 and 0.48 mg ml-1 of reducing sugars, when used CMC and MCC respectively. The production of protein equivalent to the same period of cultivation was 1.7 and 1.5 mg ml-1. The results show that the microorganism TN02 presented better adaptive capacity and greater production of endoglucanases in the conditions laid. In so much, we can infer that this strain can be used in future biotechnological processes, and will be necessary more studies concerning the cellulase activities.

CONCLUSÃO:

The results of this study indicate the possibility of using naturally occurring symbiotic microorganisms from marine xylophages originating in the Amazon region as a biotransformation agent capable of saccharified cellulose. This is the first work directed to research of microbial flora of "turus" with biotechnological purposes.

Instituição de Fomento: CNPQ, FAPESPA.

Palavras-chave: shipworms, Neoteredo reynei, cellulases.