Methane Production from Oleate: Assessing the Bioaugmentation Potential of Syntrophomonas Zehnderi
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water research 44 (2010) 4940e4947 Available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/watres Methane production from oleate: Assessing the bioaugmentation potential of Syntrophomonas zehnderi A.J. Cavaleiro*, D.Z. Sousa, M.M. Alves Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal article info abstract Article history: The potential for improving long-chain fatty acids (LCFA) conversion to methane was Received 2 April 2010 evaluated by bioaugmenting a non-acclimated anaerobic granular sludge with Syntropho- Received in revised form monas zehnderi. Batch bioaugmentation assays were performed with and without the solid 2 July 2010 microcarrier sepiolite, using 1 mM oleate as sole carbon and energy source. When S. Accepted 13 July 2010 zehnderi was added to the anaerobic sludge methane production from oleate was faster. Available online 21 July 2010 High methane yields, i.e. 89 Æ 5% and 72 Æ 1%, were observed in bioaugmented assays in the absence and presence of sepiolite, respectively. Sepiolite stimulated a faster methane Keywords: production from oleate and prevented the accumulation of acetate. Acetoclastic activity Bioaugmentation was affected by oleate in the absence of sepiolite, where methane production rate was 26% Syntrophomonas zehnderi lower than in assays with microcarrier. Methane ª 2010 Elsevier Ltd. All rights reserved. Oleate Sepiolite 1. Introduction possible (Stams, 1994). Presently, there are 7 species of syn- trophic bacteria reported as capable of growing on LCFA with Long-chain fatty acids (LCFA) are produced during the hydrolysis more than 12 carbon atoms. Among these bacteria only 3 of oils and fats and are commonly present in fatty-wastewaters. mesophilic species can utilize unsaturated LCFA, namely These compounds can be efficiently converted to methane in Syntrophomonas sapovorans, Syntrophomonas curvata and Syn- anaerobic bioreactors, if the appropriate technology and feeding trophomonas zehnderi (Sousa et al., 2009). strategy are applied (Alves et al., 2009; Cavaleiro et al., 2009). Several authors reported the presence of syntrophic aceto- Nonetheless, LCFA accumulation during the degradation of genic bacteria belonging to the Syntrophomonadacea family in lipid-rich wastewaters has been frequently described. microbial communities degrading different LCFA, either in Broughton et al. (1998) reported the accumulation of stearate and mesophilic enrichment cultures (Hatamoto et al., 2007; Sousa palmitate during batch degradation of sheep tallow, and these et al., 2007b) or in continuous bioreactors (Sousa et al., 2007a). LCFA were also found in bioreactors fed with milk-oleate In the work from Sousa et al. (2007a) Syntrophomonas-related synthetic wastewaters (Cavaleiro et al., 2009; Pereira et al., 2002). microorganisms appear as predominant microorganisms in Complete LCFA degradation evolves through the coordi- sludges that were submitted to contact with saturated and nated activity of syntrophic bacteria, which convert LCFA to unsaturated LCFA, but not in the sludgeusedas inoculumfor the acetate and hydrogen, and methanogenic archaea that utilize bioreactors. Thus, extended contact with LCFA might stimulate these substrates, making the overall conversion energetically the occurrence of syntrophic bacteria (Sousa et al., 2007a). * Corresponding author. Tel.: þ351 253 604 400; fax: þ351 253 678 986. E-mail address: [email protected] (A.J. Cavaleiro). 0043-1354/$ e see front matter ª 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2010.07.039 water research 44 (2010) 4940e4947 4941 It is likely that LCFA degradation relies on the development In this work, the potential for improving methane production of syntrophic communities and, thus, the addition of LCFA- from LCFA was studied by bioaugmenting anaerobic sludge with degrading bacteria to anaerobic sludge can potentially be used S. zehnderi. A direct link between S. zehnderi and oleate degrada- to (1) accelerate bioreactors’ start-up phase, which is generally tion, either in continuous and fed-batch reactors, has been time-consuming, or (2) promote the recovery of disrupted previously reported (Sousa et al., 2008). This bacterium is able to treatment processes when LCFA accumulation/adsorption degrade a wide range of saturated and unsaturated fatty acids onto the biomass could not be prevented. (with 4e18 carbon atoms), which makes it suitable for using as Bioaugmentation has been frequently applied in polluted a bioaugmenting strain in LCFA degradation (Sousa et al., 2007c). site or bioreactors to fasten the removal of undesired compounds, to improve the performance of on-going bio- 2. Materials and methods logical processes, and to ease the establishment of specific populations in microbial communities (Bouchez et al., 2000; 2.1. Preparation of the bioaugmenting culture Jiang et al., 2007). Bioaugmentation of anaerobic waste and wastewater-treating processes, with pure or mixed cultures, S. zehnderi DSM 17840T was pre-grown in co-culture with has also been reported (Table 1). Methanobacterium formicicum DSM 1535T in bicarbonate-buff- Cirne et al. (2006) studied the effects of bioaugmentation as ered mineral salt medium. Anaerobic medium was prepared a means of improving the hydrolysis and solubilization of lipids. as described by Stams et al. (1993), dispensed in bottles and Batch assays with a model waste containing 10% triolein were sealed with butyl rubber septa and aluminum screw caps. bioaugmented with Clostridium lundense, a lipolytic strain iso- Bottles were pressurized with a mixture of N2/CO2 (80:20 vol/ lated from bovine rumen. The hydrolysis of the lipid fraction vol, 1.7 Â 105 Pa) and autoclaved for 20 min at 121 C. Before was improved, but LCFA degradation appeared to be the limiting inoculation, mineral medium was reduced with 0.8 mM step in the complete conversion of the substrate to methane. sodium sulfide and supplemented with bicarbonate and salts Bioaugmentation success depends on the survival of the plus vitamins solutions (Stams et al., 1993). Sodium oleate inoculated microbial culture, which is influenced by several (99%, Fluka) was added to the medium from a sterile stock parameters including phenotypic characteristics of the selected solution. Inoculation of S. zehnderi and M. formicicum active strains, complex microbial interactions and environmental cultures, as well as the addition of the solutions, was per- factors (Jiang et al., 2007). Therefore, the applicability and limits formed aseptically using sterile syringes and needles. S. of bioaugmentation can be initially tested in microcosms or zehnderi was incubated with 3Â 0.5 mM oleate (successive batch assays, but should be further evaluated in conditions substrate additions) at 37 C, statically and in the dark. Prior comparable to the real environment where bioaugmentation is the bioaugmentation assays, S. zehnderi co-culture was to be performed. Batch assays allow effective retention of the centrifuged (1600 g, 10 min, 4 C) and washed (2Â) with inoculated culture in the system, since washout is avoided, and anaerobic medium. Co-culture was distributed in two bottles provide more homogeneous environmental conditions. To under N2 atmosphere; one of this bottles was heat treated increase the success of bioaugmentation strategies, some (121 C, 40 min, 2Â) in order to inactivate the culture. authors used carrier materials such as alginate, agarose and polyurethane, to provide a temporary protectiveenvironment to 2.2. Microcarrier characterization the inoculum (Boon et al., 2002). The use of microcarriers, such as sepiolite and diabase, has also been reported as beneficial for Sepiolite, a clay mineral, was the solid microcarrier selected stimulating methanogenic activity (Sanchez et al., 1994). for this work based on the reports of Alves et al. (1999) and Table 1 e Examples of bioaugmentation studies in anaerobic bioreactors. Bioreactor Microorganism(s) Type of waste/wastewater Reference UASBR Desulfomonile tiedjei (as a pure culture 3-Chlorobenzoate Ahring et al., 1992 and in co-culture) UASBR Desulfitobacterium hafniense strain DCB-2 Pentachlorophenol Christiansen and Ahring, 1996 UASBR Dehalospirillum multivorans Tetrachloroethene Ho¨ rber et al., 1998 UASBR Phenol, o- and p-cresol degrading enriched Phenolic compounds Hajji et al., 2000 microbial consortium AnSBBR Enriched culture of sulphate reducing bacteria Sulphate-rich wastewater Mohan et al., 2005 (immobilized in alginate beds) Batch Clostridium lundense (DSM 17049T) Restaurant lipid-rich waste Cirne et al., 2006 Batch Azoarcus sp. strain DN11 Benzene contaminated groundwater Kasai et al., 2007 Batch Caldicellulosiruptor lactoaceticus or Dictyoglomus sp. Cattle manure Nielsen et al., 2007 Two stage CSTR Caldicellulosiruptor lactoaceticus Cattle manure Nielsen et al., 2007 Batch Ralstonia sp. HM-1 Cd and Zn contaminated sediment Park et al., 2008 UASBR Sulfurospirillum barnesii (immobilized in Selenate and nitrate or sulphate Lenz et al., 2009 polyacrylamide gels) AnSBBR e anaerobic sequencing batch biofilm reactor; UASBR e upflow anaerobic sludge blanket reactor; CSTR e continuous stirred tank reactor. 4942 water research 44 (2010) 4940e4947 Sanchez et al. (1994). Chemically sepiolite is a hydrated methane concentration measured in bottles’ headspace and magnesium silicate with general formula Si12Mg8O30(O- the theoretical stoichiometric value for complete conversion $ Æ À3