Isolamento, caracterização bioquímica e filogenia de uma bacteria ruminal degradante de celulose
DOI:
https://doi.org/10.17533/udea.rccp.v32n2a05Palavras-chave:
celulolítica, fermentação, monofilético, rúmen, ShigellaResumo
Antecedentes: As bactérias celulolíticas hidrolizam a celulosa em celobiose e glicose, e o isolamento desses microrganismos fornece informações sobre a diversidade do rúmen. Objetivo: Identificar e caracterizar um microorganismo isolada do rúmen de uma vaca, com capacidade para hidrolisar a celulose. Métodos: Técnicas de cultura anaeróbica foram utilizadas para isolar bactérias ruminais que degradam a celulose. A atividade β-D-glucanase foi mostrada utilizando mancha de vermelho Congo, e o padrão de fermentação de carbohidratos foi obtida com o kit API 50CHB/E. A extracção foi realizada de DNA e amplificou-se os genes 16S rDNA utilizando os iniciadores 8F (AGA GTT TGA 5'-TCC TGG CTC AG-3'), e 1492R (5' CTT GGT TAC GTT ACG TCA T 3'). A árvore filogenética foi reconstruída com o algoritmo de máxima parcimônia (réplicas 5000). A sequência de rDNA 16S foi depositada no banco de dados do NCBI (número de acesso: KM094184). Resultados: O isolado mostrou uma atividade celulolítica com coloração vermelho Congo; además esta bactéria fermentação de glicerol, ribose, xilose, sacarose, galactose e glicose. No entanto, com as provas bioquímicas não se identificou a bactérias isolada, já que não se encontrou na base de dados do software API WEB. A inferência filogenética indicou que esta bactéria pertence ao género Shigella, com 98% de identidade de máximo respeito para outras espécies taxonômicas. Conclusão: A análise filogenética do gene 16S rRNA mostrou as bactérias isoladas do ambiente ruminal como um membro do género Shigella, que condições mesofilicas é um candidato atraente para obter oligossacarídeos da biomassa lignocelulósica.
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Akintokun AK, Adeyosoye OI, Abiola-Olagunju O, Joel EO. Identification and occurrence of heterophilic rumen bacteria and fungi isolated from selected Nigerian breeds of cattle. Appl Environ Microbiol 2014; 2(6):303-308.
Artzi L, Bayer EA, Moraïs S. Cellulosomes: bacterial nanomachines for dismantling plant polysaccharides. Nature Reviews Microbiology 2016;15:83-95.
Baltaci MO, Adiguzel A. Isolation, identification and molecular characterization of cellulolytic bacteria from rumen samples collected from Erzurum slaughter house, Turkey. Res J Biotech 2016; 11(2):32-38.
Boots B, Lillis L, Clipson N, Petrie K, Kenny D, Boland T. Responses of anaerobic rumen fungal diversity (phylum Neocallimastigomycota) to changes in bovine diet. J Appl Microbiol 2013; 114:626–635.
Creevey CJ, Kelly WJ, Henderson G, Leahy SC. Determining the culturable accessibility of the rumen bacterial microbiome. Microb Biotechnol 2014; 7:467-479.
Cobos MA. Clostridium paraputrificum var. Ruminantium: Colonization and degradation of shrimp carapaces in vitro observed by scanning electron microscopy. In: Wallace RJ and Lahlou-Kassi A, editors. Rumen Ecology Research Planning. Procedings of a Workshop Held at ILRI. Addis Ababa, Ethiopia: International Livestock Research Institute; 1995.
Deng W, Xi D, Mao H, Wanapat M. The use of molecular techniques based on ribosomal RNA and DNA for rumen microbial ecosystem studies: a review. Mol Biol Rep 2008;35(2):265-274.
Denman SE, McSweeney CS. The early impact of genomics and metagenomics on ruminal microboilogy. Ann Rev Anim Biosci 2015; 3:447-465.
Dias J, Marcondes MI, Noronha MF, Resende RT, Machado FS, Mantovani HC, Suen G. Effect of pre-weaning diet on the ruminal archaeal, bacterial, and fungal communities of dairy calves. Front Microbiology 2017; 8:1553-1570.
Flint HJ, Bayer EA. Plant cell wall breakdown by anaerobic microorganisms from the mammalian digestive tract. Ann NY Acad Sci 2008; 1125:280-288.
Flint HJ, Bayer EA, Lamed R, White BA. Polysaccharide utilization by gut bacteria: potential for new insights from genomic analysis. Nature Rev Microbiol 2008; 6:121-131.
Fonty G, Joblin K, Chavarot M, Roux R, Naylor G, Michallon F. Establishment and development of ruminal hydrogenotrophs in methanogen-free lambs. Appl Environ Microbiol 2007; 73(20): 6391-6403. http://aem.asm.org/content/73/20/6391.full.pdf.
Forbes C, Hughes D, Fox J, Ryan P, Colleran E. High-rate anaerobic degradation of 5 and 6 carbon sugars under thermophilic and mesophilic conditions. Bioresour Technol 2010; 101(11):3925-3930.
Gharechahi J, Zahiri HS, Noghabi KA, Salekdeh GH. In-depth diversity analysis of the bacterial community resident in the camel rumen. Syst Appl Microbiol 2015; 38(1):67-76.
Gupta P, Samant K, Sahu A. Isolation of cellulose-degrading bacteria and determination of their cellulolytic potential. Int J Microbiol 2012; Article ID 578925:1-5. http://dx.doi.org/10.1155/2012/578925.
Guzman CE, Bereza-Malcolm LT, De Groef B, Franks AE. Uptake of milk with and without solid feed during the monogastric phase:effect on fi brolytic and methanogenic microorganisms in the gastrointestinal tract of calves. Anim Sci J 2016; 87:378–388.
Hatful GF, Jacobs Jr WR. Molecular Genetics of Mycobacteria. 2a ed. Washington, DC: American Society for Microbiology Press; 2014.
Kenters N, Henderson G, Jeyanathan J, Kittelmann S, Janssen PH. Isolation of previously uncultured rumen bacteria by dilution to extinction using a new liquid culture medium. J Microbiol Methods 2011;84:52-60.
Ley de-Coss A, Arce EC, Cobos PM, Hernández SD, Pinto RR. Estudio comparativo entre la cepa de Pediococcus acidilactici aislada del rumen de borregos y un consorcio de bacteria ruminales. Agrociencia 2013; 47:567-578.
Mateo-Sánchez JM, Cobos-Peralta MA, Trinidad-Santos A, Cetina-Alcalá V, Vargas-Hernández J. Aislamiento de bacterias ruminales degradadoras del aserrín. Agrociencia 2002; 36(5):523-530. http://www.redalyc.org/articulo.oa?id=30236503.
Mosoni P, Chaucheyras-Durand F, Béra-Maillet C, Forano E. Quantification by real-time PCR of cellulolytic bacteria in the rumen of sheep after supplementation of a forage diet with readily fermentable carbohydrates: effect of a yeast additive. J Appl Microbiol 2007; 103(6):2676-2685.
Newbold CJ, de la Fuente G, Belanche A, Ramos-Morales E, McEwan NR. The role of ciliate protozoa in the rumen. Front Microbiol 2015; 6:1313-1322. NOM-062-ZOO. 1999.
Norma Oficial Mexicana. Especificaciones técnicas para la producción, cuidado y uso de los animales de laboratorio. http://www.fmvz.unam.mx/fmvz/ principal/archivos/062ZOO.PDF.
Nyonyo T, Shinkai T, Tajima A, Mitsumori M. Effect of media composition, including gelling agents, on isolation of previously uncultured rumen bacteria. Lett Appl Microbiol 2013; 56:63-70.
Penatti MPA, Hollanda LM, Nakazato G, Campos TA, Lancellotti M, Angellini M, Brocchi M, Rocha MM, Dias da Silveira W. Epidemiological characterization of resistance and PCR typing of Shigella flexneri and Shigella sonnei strains isolated from bacillary dysentery cases in Southeast Brazil. Braz J Med Biol Res 2007; 40(2): 249-258. http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2007000200012.
Rakotoarivonina H, Larson MA, Morrison M, Girardeau JP, Gaillard-Martinie B, Forano E, Mosoni P. The Ruminococcus albuspilA1-pilA2 locus: expression and putative role of two adjacent pil genes in pilus formation and bacterial adhesion to cellulose. Microbiol 2005; 151:1291-1299.
Russell JB, Muck RE, Weimer PJ. Quantitative analysis of cellulose degradation and growth of cellulolytic bacteria in the rumen. FEMS Microbiol Ecol 2009; 67(2):183-197. http://mic.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.27735-0
Samsudin AA, Wright ADG, Jassim RA. Cellulolytic bacteria in the foregut of the dromedary camel (Camelus dromedarius). Appl Environ Microbiol 2012; 78(24):8836-8839. http://aem.asm.org/content/78/24/8836.full.
Singh KM, Jisha TK, Reddy B, Parmar N, Patel A, Patel AK, Joshi CG. Microbial profiles of liquid and solid fraction associated biomaterial in buffalo rumen fed green and dry roughage diets by tagged 16S rRNA gene pyrosequencing. Mol Biol Rep 2015; 42(1):95-103.
Slutzki M, Jobby MK, Chitayat S, Karpol A, Dassa B, Barak Y, Lamed R, Smith SP, Bayer EA. Intramolecular clasp of the cellulosomal Ruminococcus flavefaciens ScaA dockerin module confers structural stability. FEBS Open Bio 2013; 3:398-405.http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3821032/.
Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 2007; 24(8):1596-1599. http://mbe.oxfordjournals.org/content/24/8/1596.long.
Ten LN, Im WT, Kim MK, Kang MS, Lee ST. Development of a plate technique for screening of polysaccharide-degrading microorganisms by using a mixture of insoluble chromogenic substrates. J Microbiol Methods 2004; 56(3):375-382.
Valledor L, Escandón M, Meijón M, Nukarinen E, Cañal MJ, Weckwerth W. A universal protocol for the combined isolation of metabolites, DNA, long RNAs, small RNAs, and proteins from plants and microorganisms. Plant J 2014; 79(1):173-180. http://onlinelibrary.wiley.com/doi/10.1111/tpj.12546/pdf.
Venkatesh B. Current challenges in commercially producing biofuels from lignocellulosic biomass. ISRN Biotechnology 2014; Article ID 463074:31. http://dx.doi.org/ 10.1155/ 2014/463074.
Wanapat M, Cherdthong A. Use of real-time PCR technique in studying rumen cellulolytic bacteria population as affected by level of roughage in swamp buffalo. Curr Microbiol 2009; 58(4):294-299. http://link.springer.com/article/10.1007%2Fs00284-008-9322-6#/page-1.
Wang A, Gao L, Ren N, Xu J, Liu C, Cao G, Yu H, Liu W, Hemme CL, He Z, Zhou J. Isolation and characterization of Shigella flexneri G3, capable of effective cellulosic saccharification under mesophilic conditions. Appl Environ Microbiol 2011; 77(2):517-523.http://aem.asm.org/content/77/2/517.long.
Weimer PJ, Price NPJ, Kroukamp O, Joubert LM, Wolfaardt GM, Van Zyl WH. Studies of the extracellular glycocalyx of the anaerobic cellulolytic bacterium Ruminococcus albus 7. Appl Environ Microbiol 2006; 72(12):7559-7566. http://aem.asm.org/content/72/12/7559.full.
Wilson DB. Microbial diversity of cellulose hydrolysis. Curr Opin Microbiol 2011; 14:259–263.
Zhivin O, Dassa B, Moraïs S, Utturkar SM, Brown SD, Henrissat B, Lamed R, Bayer EA. Unique organization and unprecedented diversity of the Bacteroides (Pseudobacteroides) cellulosolvens cellulosome system. Biotechnol Biofuels 2017;10:211-218.
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