New DNA Sequences from Bacteria Converting Phenol into Acetate under Strict Anaerobic Conditions J. E. Hernandez O. Chemical and Environmental Engineering Department, Bioinspired Engineering Research Group, The University of Nottingham, Malaysia. Chemical and Environmental Engineering Department, The University of Nottingham, UK. Email: cpp02jeh@yahoo.com.mx Abstract—This work reports new fragments of DNA sequences related to microbes able to degrade phenol into acetate under strict anaerobic conditions. For this purpose, anaerobic digesting sludge was acclimatised to degrade phenol, then heat treated and in turn used as fermentative sludge. The resulting microbial community was able to convert phenol into acetate under anaerobic conditions (kinetic constants: 0.396 ± 0.01 and 0.345 ± 0.04 mg of compound L -1 day -1 , respectively). Microscopic, chemical and molecular analyses revealed that only bacteria were present in the final sludge and thus methanogens were eliminated. The bacteria were mainly Gram-negative sporeforming rods, belonging to the Deltaproteobacteria class and had a tendency for aggregation. These are also phenotypically related to organisms thriving at extreme environments. Cloning, temperature gradient gel electrophoresis (TGGE) and probe matching of a short 16S DNA fragment revealed that these new microbes are evolutionary related to, and share 90% of similarities with, Desulfovibrio sp. Index Terms—anaerobic fermentation, bacteria phenol acetate, acetogenesis, SRB I. INTRODUCTION Phenol is an industrial commodity and a priority hazardous pollutant [1], [2]. For decades, there has been an interest in reducing the negative environmental impact of phenol bearing streams upon waste treatment processes and biogas production [3]. The fermentative step involved in the breakdown of the aromatic ring into acetate is energetically limited and is carried out by syntrophic microbes [4]. Nevertheless, the vast majority of the microorganisms involved and the pathways used for this process remain unknown. Historically, attempts to study acetate formation from anaerobic degradation of phenol by acetogens have been carried out with 2-broethane sulphonic acid (BESA), Manuscript received July 20, 2014; revised February 10, 2015. chloroform or heating at 85 ˚C. These techniques aimed at the elimination or inactivation of H2 and acetate consumers [5]–[8]. Sulphate reducing prokaryotes (SRP) have been suspected to be responsible for acetogenesis by aromatic ring degradation under non-methanogenic conditions [9], [10]. Some SRP are able to survive in extreme environments with low redox potential, hard energy conservation limits close to the equilibrium (ΔG = 0), freezing or boiling temperatures, heavy metal reduction and other slow growth conditions [11]–[17]. Until today, complete phylogeny of SRP is unknown and is difficult to decipher. SRP are widely distributed in both Bacteria and Archaea domain [18]. Besides, they are very closely related to non-SRP and therefore difficult to discriminate. This is complicated by the lack of data in public databases, lack of oligonucleotide probes targeting all SRP, evolutionary differences between the 16S rRNA and functional genes due to horizontal gene transfer [11], [19], [18]. Recently, unambiguous identification has been possible only among very close phylogenetically related SRP with the help of a probe database [20]. This work aims at reporting new bacterial sequences related to Desulfovibrio microbes that were able to convert phenol into acetate under strict anaerobic conditions. Optic light microscopy and SEM were employed to reveal the effect of experimental procedures on the morphology of the microbial community. Evolutionary relationship was inferred by applying a 16S rDNA based approach. Short gene fragments were amplified and studied by complementing cloning, TGGE and probe matching. II. MATERIALS AND METHODS A. Culture Setup Actively digesting sludge was collected from the Aldwarke waste water treatment plant, Rotherham, UK. Henceforward, all the procedures involving the sludge 1 Journal of Medical and Bioengineering Vol. 5, No. 1, February 2016 ©2016 Engineering and Technology Publishing doi: 10.12720/jomb.5.1.1-10 R. G. J. Edyvean Department of Chemical and Process Engineering, Kroto Institute, The University of Sheffield, North Campus, Broad Lane, Sheffield, S3 7HQ, U.K.