Diversity and Activity of Sulfate- reducing bacteria in Sulfidogenic Wastewater Treatment Reactors If we knew what we are doing, it would not be called research would it? Albert Einstien ii Diversity and Activity of Sulfate- reducing bacteria in Sulfidogenic Wastewater Treatment Reactors Proefschrift ter verkrijging van de graad van doctor aan de Technische Universiteit Delft, op gezag van de Rector Magnificus Prof. dr. ir. J. T. Fokkema, voorzitter van het College voor Promoties, in het openbaar te verdedigen op vrijdag 19 oktober 2007 om 10.00 uur door Shabir Ahmad DAR Master in Science of Bioprocess Technology, Asian Institute of Technology (AIT), Thailand geboren te Srinagar, J&K, India iii Dit proefschrift is goedgekeurd door de promotor: Prof. dr. J. G. Kuenen Toegevoegd promotor Dr. G. Muyzer Samenstelling promotie commissie: Rector Magnificus Voorzitter Prof. dr. J.G. Kuenen Delft University of Technology, Promotor Dr. G. Muyzer Delft University of Technology, Toegevoegd promotor Prof. dr. F. Widdel Max-Planck-Institute for Marine Microbiology, Bremen, Germany Prof. dr. ir. M.C.M. van Loosdrecht Delft University of Technology Prof. dr. H.J. Laanbroek Utrecht University Prof. dr. ir.A.J.M. Stams Wageningen University Prof. dr. ir. P.N.L. Lens Wageningen University This study was carried out in the Environmental Biotechnology group of the Department of Biotechnology at Delft University of Technology, Delft, the Netherlands. This research was financially supported by The Netherlands Organization for Scientific Research – (NWO Earth and Life Sciences). ISBN 978-90-9022271-4 Printed by: PrintPartners IPSKAMP iv Contents Chapter 1 General Introduction 1 Chapter 2 Nested PCR-Denaturing gradient gel 37 electrophoresis approach to determine the diversity of sulfate-reducing bacteria in complex microbial communities Chapter 3 Analysis of diversity and activity of sulfate- 55 reducing bacterial communities in sulfidogenic bioreactors using 16S rRNA and dsrAB genes as molecular markers Chapter 4 Co-existence of physiologically similar 85 sulfate-reducing bacteria in a full-scale sulfidogenic bioreactor fed with a single electron donor Chapter 5 Competition and coexistence of sulfate 107 reducing bacteria, acetogens and methanogens in a lab-scale anaerobic bioreactor as affected by changing substrate to sulfate ratio Chapter 6 General Discussion 131 Summary 145 Samenvatting 151 List of Publications 157 Curriculum Vitae 159 Acknowledgements 161 v vi CHAPTER 1 General Introduction Chapter 1 1 Biological sulfur cycle Sulfur is an essential element for the growth of many life forms (microorganisms, plants, animals), and their sulfur content typically varies between 0.1 and 1.5% of dry weight. The element sulfur occurs in a large variety of oxidation states (Table 1), oxidation state -2 (completely reduced) to oxidation state +6 (completely oxidized) (148). However, only three oxidation states are abundantly present in nature, i.e., -2 (sulfhydryl, R-SH and sulfide, HS-), 0 (elemental sulfur, S0), and 2- +6 (sulfate, SO4 ). Most of the sulfur is found in sediments and rocks in the form of sulfate (primarily gypsum, CaSO4) and sulfide minerals (primarily pyrite, FeS2), although the oceans constitute the most significant reservoir of sulfur for the biosphere (in the form of dissolved inorganic sulfate). Another large part is incorporated into the biomass as sulfur-containing compounds, such as cysteine and methionine. Table 1 Oxidation states of sulphur in common compounds (166) Oxidation Compounds state - -2 Dihydrogen sulfide H2S, hydrogen sulfide ion HS , sulfide ion S2-, as in FeS; thiocyanate SCN- 2- -1 Disulfane H2S2, disulfide S2 as in pyrite FeS2; 1- – - thiosulfate sulfane S ; polysulfaides S(S)nS 0 Elemental sulfur Sn; organic polysulfanes R-Sn-R; - - polythionates O3S(S)nSO3 +1 Dichlorodisulfane Cl-S-S-Cl 2- +2 Sulfur dichloride SCl2; sulfoxylate SO2 2- +3 Dithionate S2O4 2- - +4 Sulfur dioxide SO2; sulfite SO3 ; bisulfite HSO3 2- - +5 Dithionate S2O6 ; sulfonate RSO3 ; thiosulfate sulfone - SO3 2- 2- +6 Sulfur trioxide SO3; sulfate SO4 ; peroxosulfate SO5 These compounds are continuously converted into each other by a combination of biological, chemical and geochemical processes. The conversions of the inorganic sulfur compounds and to a lesser extent also those of the organic sulfur compounds are dominated by microbiological transformations. The biochemical oxidations and reductions of sulfur compounds constitute the biological sulfur cycle, which is schematically shown in Fig. 1. Reduction of sulfur compounds can be either assimilatory for the synthesis of organic sulfur compounds or dissimilatory in order to dispose of excess reducing equivalents. The sulphide produced can be either deposited as metal sulfides, or it can be oxidized to elemental 2 Introduction sulfur or sulfate. These conversions involve the metabolism of several different specific groups of bacteria and archaea. Organic sulphur compounds Assimilatory mineralization Sulfidic sulfate reduction processes minerals Sulfate (e.g. reserves dissimilatory pyrites) (seawater) sulfate reduction 2- biological 2- SO4 oxidation with - S O2 or NO3 chemical dissimilatory oxidation sulfur reduction biological biological oxidation with O2 oxidation with O2 or - anaerobic NO3 anaerobic oxidation by oxidation by phototrophic bacteria 0 phototrophic S bacteria Sulfur deposits Fig. 1 The sulphur cycle (142) 1.1 Dissimilatory oxidation of reduced sulfur compounds Reduced sulfur compounds are used by many bacteria and some archaea to carry out dissimilatory sulfur oxidation. Winogradsky in 1887 suggested the name “Schwefelbacterien” (sulfur bacteria) for these bacteria. They include phototrophic green (Chlorobiaceae), and purple sulfur bacteria (Chromatiaceae and Ectothiorhodospiraceae) (54) and non-phototrophic colorless sulfur bacteria that belong either to the Proteobacteria (e.g. the genera Beggiatoa, Thiobacillus, Thiomicrospira, Thioploca, Thiospira, Thiothrix and Thiovulum) or to the archaea (Sulfolobus and Acidianus) (96, 97). The anaerobic sulfur and thiosulfate oxidizers are represented by photosynthetic green and purple sulfur bacteria. These bacteria oxidize H2S by using it as source of reducing power in CO2 fixation. Green sulfur bacteria are strict anaerobes that deposit the sulfur (So) they produce extracellularly. The purple sulfur bacteria, with the exception of Ectothiorhodospiraceae spp., deposit the sulfur 3 Chapter 1 intracellularly (127). Some of the latter can grow as chemolithoautotrophs under microaerophilic conditions. Under H2S limitation, they oxidize the sulfur further to sulfate. Non-phototrophic colorless sulfur bacteria comprise aerobes as well as anaerobes. Among the bacterial aerobes, the most important groups comprise Thiobacillaceae and Beggiatoacea. These groups include obligate and facultative autotrophs as well as mixotrophs and heterotrophs. Other H2S oxidizers found in aquatic environments include Thiovulum (autotrophic) (186), Achromatium, Thiothrix, Thiobacterium (98) and Thiomicrospira (97). Many of these groups produce intracellular and extracellular sulfur when oxidizing H2S. The archaea, Sulfolobus spp. and Acidianus spp. are able to oxidize sulfur to sulfuric acid at temperatures between 55 and 85°C (17). These organisms are facultative autotrophs. Two examples of facultative, anaerobic sulfur oxidizing bacteria are Thiobacillus denitrificans (83) and Thermothrix thiopara (15). The former a mesophile and the latter a thermophile, use nitrate as terminal electron acceptor and reduce it to oxides of nitrogen and dinitrogen. It should be noted that many other bacterial groups such as Paracoccus and Hydrogenobacter species posses the capability to oxidize sulfur compounds. 1.2 Dissimilatory sulfate reduction and the key enzymes involved. 2- The most oxidized form of sulfur is sulfate (SO4 ). A broad range of organisms, such as higher plants, algae, fungi and most prokaryotes can use sulfate as a sulfur source and carry out assimilatory sulfate- reduction. A variety of fermentative bacteria can use partially reduced sulfur. However, the ability to use sulfate as electron acceptor during the degradation of organic compounds is only restricted to the group of sulfate reducing prokaryotes. Even though an alternative microaerobic metabolism of some sulfate-reducing bacteria (SRB) was reported (43, 93), these bacteria can grow only under anoxic, reduced conditions (181). Dissimilatory sulfate reduction, the focus of this thesis, is the central metabolic pathway that drives the global sulfur cycle. The SRB reduce sulfate by oxidizing hydrogen and various organic compounds and directing the electrons arising from the oxidation to the sulfate reducing system. Compounds such as sulfur or thiosulfate are used as alternative external electron acceptor, whereas sulfide is the end product. Typical substrates (electron donors and/or carbon sources) for SRB are lactate, ethanol, propionate and H2. Three enzymes are generally involved in the dissimilatory reduction of sulfate. All of which are located inside the cytoplasm, to which sulfate is transported in an active process (66, 91). These include ATP sulfurylase, the iron-sulfur 4 Introduction flavo-protein adenosine-5’-phosphosulfate (APS) reductase and the dissimilatory sulfite reductases. 2- The free sulfate anion (SO4 ) is chemically inactive and not easily reduced and thus the initial reaction in the reduction of sulfate is an activation step where ATP and sulfate form adenylyl sulfate (APS),