Bioprospecção de bactérias xilanolíticas e celulolíticas a partir de conteúdo ruminal de bovinos leiteiros

Abstract

Ruminants are animals capable of utilizing the fiber contained in vegetables as feed due to the microrganisms present in their digestive tract. The prospection of organisms with potential ability to degrade cellulose and xylan in the rumen microbiota is of great importance in anthropogenic activities and industrial processes. Samples of ruminal content were collected weekly from fistulated bovines for later bacteria isolation by making use of the technique described by Hungate (1966) with modifications by Bryant and Burkey (1951). The isolates were evaluated regarding their morphotintorial characteristics by using Gram stain and regarding their motility by inoculation in supplemented SIM media. The isolates energetic metabolism was also evaluated by respiratory test, as well as their enzymatic profile by zymmograms. Once characterized, the isolates were stored in -20ºC freezer for molecular identification and later inclusion in the EMBRAPA microorganism collection. A total of 67 isolated were obtained, of which 52 were Gram positive cocci, 10 were Gram negative rods and 5 were Gram positive rods. The motility test revealed that only 12 out of the 67 isolates were motile and in the respiratory test it was possible to observe that 14 bacteria were obligate anaerobes. The zymmograms showed that 14 isolates were potencially cellulolytic and 53 were potencially xylanolytic. The commercial biochemical identification kits revealed that out of the 67 bacteria, 49,3% obtained species identification with acceptable (15,15%) or good identification (34,15%), while 50,7% were not identified by this technique. The isolates were grouped in biogroups, according to the similarities in their biochemical profiles, and up to three integrals were chosen for ribotyping. The reaction was only possible with the rods. One member of each ribotype and up to three members of each biogroup were selected for amplification of the DNA region that encodes the 16S rRNA and later sequencing. After analysis of the resulting sequences, the species Selenomonas ruminantium, Streptococcus sp., Pseudobutyrivibrio sp. e Lactobacillus vitulinus were identified among the isolates. One of the isolates did not present satisfactory identification. It was possible to evaluate that the two different diets did not cause significant changes in the bacterial community profile. The comparison between both identification methods showed that the biochemical kits utilized during this research should not be used for the identification of ruminal bacteria. The sequencing of the the DNA region that encodes the 16S rRNA is still the safest form of identification. The identification of potencially cellulolytic and xylanolytic isolates as Selenomonas ruminantium and Lactobacillus vitulinus presents new evidence of the fibrolytic activity in individuals from those species. More studies are needed so as to verify the phytase expression by those bacteria. The results in this study are important for better comprehension of the rumen ecosystem and, in the future, could contribute to improvements in the industrial hydrolysis of lignocellulosic material.