CORE Metadata, citation and similar papers at core.ac.uk Provided by Wageningen University & Research Publications Biodegradation (2008) 19:161–177 DOI 10.1007/s10532-007-9123-9 ORIGINAL PAPER Molecular characterization of mesophilic and thermophilic sulfate reducing microbial communities in expanded granular sludge bed (EGSB) reactors Stephanie A. Freeman Æ Reyes Sierra-Alvarez Æ Mahmut Altinbas Æ Jeremy Hollingsworth Æ Alfons J. M. Stams Æ Hauke Smidt Received: 28 November 2006 / Accepted: 10 April 2007 / Published online: 4 May 2007 Ó Springer Science+Business Media B.V. 2007 Abstract The microbial communities established in (thermophilic reactor). The addition of copper(II) to mesophilic and thermophilic expanded granular the influent of both reactors did not noticeably affect sludge bed reactors operated with sulfate as the the composition of the bacterial or archaeal commu- electron acceptor were analyzed using 16S rRNA nities, which is in agreement with the very low targeted molecular methods, including denaturing soluble copper concentrations (3–310 mglÀ1) present gradient gel electrophoresis, cloning, and phyloge- in the reactor contents as a consequence of extensive netic analysis. Bacterial and archaeal communities precipitation of copper with biogenic sulfides. Fur- were examined over 450 days of operation treating thermore, clone library analysis confirmed the phy- ethanol (thermophilic reactor) or ethanol and later a logenetic diversity of sulfate-reducing consortia in simulated semiconductor manufacturing wastewater mesophilic and thermophilic sulfidogenic reactors containing citrate, isopropanol, and polyethylene operated with simple substrates. glycol 300 (mesophilic reactor), with and without the addition of copper(II). Analysis, of PCR- Keywords Anaerobic wastewater treatment Á amplified 16S rRNA gene fragments using denaturing Copper Á Ethanol Á Methanogens Á Sulfate reducing gradient gel electrophoresis revealed a defined shift bacteria Á DGGE Á 16S rRNA gene clone library in microbial diversity in both reactors following a change in substrate composition (mesophilic reactor) and in temperature of operation from 308Cto558C Introduction Sulfate-reducing bacteria (SRB) have long been studied as an integral part of the global sulfur cycle, S. A. Freeman Á R. Sierra-Alvarez (&) Á J. Hollingsworth Department of Chemical and Environmental Engineering, which comprises important instances of electron University of Arizona, P.O. Box 21001, Tucson, AZ exchange in oceans, sediments, and water systems. 85721, USA SRB, as well as sulfate-reducing archaea, are able to e-mail: [email protected] use sulfate as a terminal electron acceptor for growth S. A. Freeman Á M. Altinbas Á A. J. M. Stams Á H. Smidt using an organic compound or hydrogen as an Laboratory of Microbiology, Wageningen University, electron donor. SRB are a polyphyletic group as they P.O. Box 8033, 6700 EJ Wageningen, The Netherlands are commonly found in the class of d-Proteobacteria, the low G+C Gram positive class Clostridia, the M. Altinbas Department of Environmental Engineering, Istanbul genus Thermodesulfovibrio within Nitrospira, and the Technical University, Istanbul, Turkey genus Thermodesulfobacterium (Stahl et al. 2002). 123 162 Biodegradation (2008) 19:161–177 The ability of dissimilatory sulfate reduction also the feed of TR, and from the shift in TR from ethanol occurs in a branch of hyperthermophilic archaea to a simulated semiconductor manufacturing waste- including several Archaeoglobus species (Burggraf water containing a mixture of isopropanol, citrate and et al. 1990; Stetter et al. 1993). polyethylene glycol (PEG, Mn = 300) with and Recent studies have shown the potential of sulfate without copper(II). A complementary set of methods reducing bioreactors for the removal of heavy metals targeting 16S rRNA and the encoding gene were from wastewater (Lens et al. 1998). Removal of used, including denaturing gradient gel electrophore- heavy metals by SRB is mainly due to the formation sis (DGGE) analysis of PCR-amplified 16S rRNA of highly insoluble precipitates with biogenic sulfide, gene fragments, restriction fragment length polymor- including entrapment of precipitated sulfide minerals phism (RFLP) fingerprinting, cloning and sequenc- by biolfilm exopolymers. The solubility product of ing. Phylogenetic analysis of partial sequences of most metal sulfides is extremely low (Stumm and clone libraries was performed to reveal the identity of Morgan 1996) and, therefore, this technology can the predominant groups of bacteria and archaea in the reduce metal concentrations in the wastewater to very sulfidogenic biofilms, and to gain insights into the low concentrations (in the ppb range). Studies of transformation processes occurring in the reactor mesophilic SRB applied to metal decontamination biofilms. have increased in recent years, but existing applica- tions of these environmental biotechnologies at the practical scale are still limited. Technological appli- Materials and methods cation of thermophilic SRB bacteria for metal removal has not yet been described. Thermophilic EGSB bioreactor operation, wastewater bioreactors could potentially accommodate hotter composition, and sample fixation process waters resulting from closed-cycle manufacturing. Two laboratory scale expanded granular sludge An interesting application of sulfate reducing blanket (EGSB) bioreactors (V = 2.9 l) were inoc- bioreactors is in the treatment of the high flows of ulated with granular sludge from an industrial copper-rich wastewaters generated from semiconduc- sulfidogenic reactor fed ethanol (10 g volatile tor manufacturing. Sulfidogenic bioreactors could suspended solids (VSS) lÀ1). The sludge was adapted also facilitate the removal of organic compounds in to a wastewater containing ethanol (493– these effluent streams. Anaerobic microorganisms 1,480 mg lÀ1) and sulfate (1,800–5,320 mg lÀ1) for can effectively remove a variety of organic com- 46 days prior to inoculation. A mesophilic reactor pounds often found in semiconductor manufacturing (MR) at 308C and a thermophilic reactor (TR) at effluents, including citric acid, polyethylene glycol 558C were operated for approximately 450 days with (average Mn 300), isopropanol, and oxalic acid using non-limiting concentrations of sulfate, at a hydraulic sulfate as the main electron acceptor (Hollingsworth retention time of 8 h. The influent of MR was mixed et al. 2005). with recycled effluent in a fluidized-bed crystalliza- Despite considerable interest shown by microbi- tion reactor (V = 0.4 l) placed upstream from the ologists and environmental engineers, the present bioreactor. The crystallizer was supplied with understanding of the microbial diversity in sulfate 150 grams of acid-washed quartz sand (50–70 mesh) reducing bioreactors is incomplete, especially in to promote heterogeneous nucleation of copper thermophilic processes. The aim of this study was minerals. The effluent recirculation in the bioreactor to analyze the microbial communities established in a was maintained at a recycle ratio of 15, in order to mesophilic reactor (MR) and a thermophilic reactor maintain fluidization of the sand in the crystallizer as (TR) operated at 308C and 558C, respectively, and well as maintain sludge bed fluidization in the supplied with sulfate as the main electron acceptor bioreactor. The TR operated as a stand-alone reactor and ethanol as carbon source and electron donating and heavy metal precipitation by biogenic sulfides substrate. A second objective was to evaluate the occurred within the reactor. Both reactors were impact on the microbiota present in the reactor initially fed with 1,480 mg lÀ1 ethanol and biofilms resulting from the addition of copper(II) to 5,320 mg lÀ1 sulfate. At day 199, the electron donor 123 Biodegradation (2008) 19:161–177 163 in the MR was switched from ethanol to a simulated bacterial amplification was 2 min of initial denatur- semiconductor manufacturing wastewater containing ation at 958C, 35 cycles of denaturation at 958C for 425 mg lÀ1 isopropanol, 1,370 mg lÀ1 citric acid, and 30 s, annealing at 568C for 45 s, and elongation at À1 615 mg l polyethylene glycol (average Mn approx. 728C for 1 min, with a final 5 min elongation at 728C. 300). Divalent copper (Cu(II)) was added to the The thermal sequence used for archaeal amplification influent of MR and TR at an average concentration of differed only in the annealing step, which was 528C 4.2 ± 0.4 mg lÀ1 on day 374 and further increased to for 40 s, and the elongation step, which was 90 s long. 24.6 ± 0.1 mg lÀ1 on day 407. The concentration of Cu(II) in the influent of the MR was increased further Cloning and RFLP analysis to 66.4 ± 0.4 mg lÀ1 on day 430. Cu(II) was added to the medium as CuCl2Á2H2O. Bacterial and archaeal 16S rRNA gene clone library The basal mineral medium contained (in mg lÀ1): analysis was performed with total DNA extracted NH4Cl (280), NaHCO3 (4,000), KCl (270), K2HPO4 from the biofilm samples, with the exception of the (169), CaCl2 Á 2H2O (10), MgCl2 Á 6H2O (150), yeast clone libraries of samples obtained on day 447 which extract (20), and 1 ml lÀ1 of trace element solution used cDNA. The bacterial primers 968f (no GC (Hollingsworth et al. 2005). The initial pH of the clamp) and 1401r and the archaeal primers A109(T)-f reactor medium ranged between 7.7 and 8.0, resulting and 515r (no GC clamp) were used in PCR ampli- in effluent pH values from 7.4 to 8.0. fication, as described above. Amplified 16S rRNA Sludge samples were taken from both bioreactors gene products were purified and cloned as described periodically and immediately frozen at –80 8C and previously (Roest et al. 2005b). Cell lysates were stored until DNA extraction was performed. The MR screened using RFLP and restricting with MspI, CfoI, was sampled for clone library analysis on day 280, and AluI (Promega Corp., Madison, WI, USA). 197 and 447, and for DGGE analysis on days 8, 16, Restriction fragments were analyzed on 12% 22, 30, 62, 84, 140, 197, 280, 367, 424, and 447. The Poly(NAT)1 gels (Elchrom Scientific, Cham, Swit- TR was sampled for clone library analysis on day 62, zerland).