In Situ Identification of the Synthrophic Protein Fermentative

RESEARCH LETTER In situ identiﬁcation of the synthrophic protein fermentative Coprothermobacter spp. involved in the thermophilic anaerobic digestion process Maria Cristina Gagliano, Camilla Maria Braguglia & Simona Rossetti

Water Research Institute, IRSA-CNR, Monterotondo, RM, Italy

Correspondence: Simona Rossetti, Water Abstract Research Institute, CNR, Via Salaria km 29,300, 00015 Monterotondo, RM, Italy. Thermophilic bacteria have recently attracted great attention because of their Tel.: +39 06 90672697; potential application in improving different biochemical processes such as fax: +39 06 90672787; anaerobic digestion of various substrates, wastewater treatment or hydrogen e-mail: [email protected] production. In this study we report on the design of a specific 16S rRNA-tar- geted oligonucleotide probe for detecting members of Coprothermobacter genus Received 7 March 2014; revised 29 June characterized by a strong protease activity to degrade proteins and peptides. 2014; accepted 2 July 2014. Final version published online xxxx xxx xxx. The newly designed CTH485 probe and helper probes hCTH429 and hCTH439 were optimized for use in fluorescence in situ hybridization (FISH) on thermo- DOI: 10.1111/1574-6968.12528 philic anaerobic sludge samples. In situ probing revealed that thermo-adaptive mechanisms shaping the 16S rRNA gene may affect the identification of ther- Editor: Robert Gunsalus mophilic microorganisms. The novel developed FISH probe extends the possibility to study the widespread thermophilic syntrophic interaction of Keywords Coprothermobacter spp. with hydrogenotrophic methanogenic archaea, whose Coprothermobacter ; thermophilic anaerobic establishment is a great benefit for the whole anaerobic system. digestion; FISH; syntrophy; proteolytic bacteria.

rich wastewater (Cai et al., 2011). Coprothermobacter spp. Introduction were identified in several studies focused on the analysis of Anaerobic digestion (AD) is one of the few sustainable the microbial community structure from anaerobic thermo- technologies that both produce energy and treat waste philic reactors treating sewage sludge (Hatamoto et al., streams. Due to their advantages mainly related to the 2008; Kobayashi et al., 2008; Lee et al., 2009; Luo et al., higher organic matter removal, methane production and 2013; Pervin et al., 2013), other substrates such as slaugh- pathogens removal, anaerobic processes operating under terhouse waste (Palatsi et al., 2011), food waste and cattle thermophilic conditions have attracted great attention manure (Tandishabo et al., 2012). The growth of Coproth- (Ho et al., 2013). Our knowledge about the microbial ermobacter spp. on protein substrates is also closely associ- consortia involved in this process is limited because of a ated with hydrogen production (Kawagoshi et al., 2005; lack of phylogenetic and metabolic data on these predom- Tandishabo et al., 2012). In particular, Coprothermobacter inantly uncultivated microorganisms (Riviere et al., can accomplish protein degradation in syntrophic associa- 2009). Further understanding of the identity and function tion with hydrogenotrophic methanogenic archaea (Sasaki of the microbial components would therefore allow a bet- et al., 2011b). ter control of the biological processes. To study and monitor natural anaerobic ecosystems, as Coprothermobacter proteolyticus was originally isolated well as anaerobic engineering processes, in situ detection from a methanogenic enrichment culture inoculated from a methods such as fluorescence in situ hybridization (FISH) thermophilic digester (Ollivier et al., 1985). Coprothermob- are widely used for the identification, quantification and,

MICROBIOLOGY LETTERS MICROBIOLOGY acter is generally associated with the microbial population in combination with other techniques, characterization of anaerobic digesters because of its proteolytic properties. of phylogenetically deﬁned microbial populations. Only This makes it an ideal candidate for facilitating the treat- microscopy-based tools can indeed link isolated extracted ment and processing under high temperature of protein- DNA markers with intact, individual cells in their natural