Sulfate reducing processes at extreme salinity and temperature: extending its application window Marcus Vinícius Guerini Vallero Promotor Prof. dr. ir. G. Lettinga hoogleraar in de anaerobe zuiveringstechnologie en hergebruik van afvalstoffen Co-promotor Dr. ir. P. N. L. Lens universitair docent bij het sub-departement Milieutechnologie, Wageningen Universiteit Samenstelling promotiecommissie Prof. dr. E. Foresti University of São Paulo EESC-USP, Brazil Prof. dr. ir. A. J. M. Stams Wageningen Universiteit Prof. dr. ir. C. J. N. Buisman Wageningen Universiteit Dr. ir. R. T. van Houten TNO Milieu, Energie en Procesinnovatie Sulfate reducing processes at extreme salinity and temperature: extending its application window Marcus Vinícius Guerini Vallero Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, Prof. dr. ir. L. Speelman, in het openbaar te verdedigen op maandag 3 november 2003 des namiddags te vier uur in de Aula Cover: Two bioreactor concepts (UASB and SAMBaR) utilized in the investigations described in this thesis. The inserts show the respective view of the macro (sludge type) and micro (scanning electron and light microscopy) of the biomass present in each reactor concept. CIP-DATA KONINKLIKE BIBLIOTHEEK, DEN HAAG Author: Vallero, M. V. G. Title: Sulfate reducing processes at extreme salinity and temperature: extending its application window ISBN: 90-5808-909-6 Publication year: 2003 Subject headings: sulfate reduction, anaerobic, UASB, membrane bioreactor, extreme, halophilic, mesophilic, thermophilic. Thesis Wageningen University, Wageningen, the Netherlands – with references - with summary in Dutch. Aos meus pais Itamar e Maria, meu irmão Marcelo e a Marina ACKNOWLEDGEMENTS First of all I would like to thanks the Brazilian Government agency for the development of science and technology (CNPq) which sponsored the four years of my Ph.D. in Wageningen. My tenure in the Sub-department of Environmental Technology positively changed my life. I do hope that I will make a visible contribution to my country, Brazil, and possibly to the world beyond its boundaries. I would like to express my sincere gratitude to my supervisor Piet Lens for introducing me to the stinking and very interesting field of sulfate reduction. His creativity, optimism, enthusiasm and peculiar sense of humor were always inspiring. I would also like to thank to my promotor Gatze Lettinga for his goodwill and absolute commitment to ensure that I started and completed the Ph.D. program in due time. I extend my appreciation also to Dora Lettinga for her sincere hospitality. This Ph.D. period in the Netherlands would not start without the introduction to the sanitation field provided by my former research colleagues and professors in Brazil: Carlos Blundi (in memoriam), Eugênio Foresti, Marcelo Zaiat, Mario Kato, Bernardo Teixeira, Nemésio Salvador and Viviana Baldochi. For all my colleagues in Brazil I send my honest wishes of esteem and consideration. Many thanks to Paula Paulo for introducing me to the laboratories and analytical apparatus of our department. Her willingness to help and to have a delightful conversation will never be forgotten. Great part of this work would not be accomplished without the help of diligent students. I would like to show my appreciation to them for their excellent contribution: Rafa Herran, Eva Camarero, Juliana de Morais, Wenrui Xu, Yang Shuo and Alexia Gordillo. I am also grateful to the members of our dedicated technical team at the department: Geert Meijer, Ilse Gerrits, Sjoerd Hobma, Hillion Wegh and Vinnie de Wilde. The contribution of the research colleagues Bernd van Houten and Caroline Plugge from the Department of Microbiology is also highly appreciated. I would like also to extend my compliments to our industrious secretaries: Liesbeth Kesaulÿa, Anita van de Weerd and Heleen Vos. Their unreserved enthusiasm and readiness for handling the complex Dutch bureaucracy is greatly acknowledged. In order to come through this thesis, I had the pleasure to interact with many research colleagues and collaborators. Some of them have long left for greener pastures. Some of these research colleagues always came to my rescue whenever I was confronted with research pitfalls such as reactor set-up and analytical procedures. Others were always keen to invite me for unforgettable evenings savoring the excellent Belgian (ok, also Dutch) beers. It has been very pleasant to work in the delightful atmosphere with Jan Sipma, Marcel Zandvoort (thanks for the translation of Chapter 10 !), Bego Osuna, Giovanni Esposito, Francisco Cervantes (and Liz Levario), Agnieszka Popenda, Sonia Lopes, Johan Vermeulen, Joost van Buuren, Iemke Bisschops, Bas Buys, Jules van Lier, Jarno Gieteling, Sonja Parshina, Adrie Veeken, Look Hulshoff Pol, Lucas Seghezzo, Sjon Kortekaas, Robin van Leerdam, Peter van der Maas, Velu Muniandi, Nidal Mahmoud, Martijn Smit, Adriaan Mels, Jan Weijma, Loan Nguyen, Gabor Szanto, Maha Halasheh and Jaap Vogelaar. I also extend the compliments to the cycling enthusiasts of our department: Marjo Lexmond, Chiel Cuypers, Eric van Hullebusch and Marc Boncz. I do hope I will keep riding my ‘Jan Janssen’ in Brazilian soils. A warm hug I send to my Brazilian fellows Renato Leitão and André dos Santos. Thanks very much for your companionship and to make me stick to the ‘Brazilian way of life”. Os mais sinceros votos de reconhecimento e carinho eu envio aos meus queridos familiares. Considero-me uma pessoa privilegiada por poder ter trilhado todo este caminho que culmina na redação desta tese. Nada disso seria possível sem o vosso apoio irrestrito e paciência. Eu amo vocês !! If I may add just one more line, I would like to acknowledge how the companionship and love of Marina Marchioretto are important for me. Your enthusiasm and affection makes me realize how fortunate I was to have come across you. I am persuaded to believe that together we are stronger than ever. Marcus VG Vallero Wageningen, the Netherlands. November, 2003. ABSTRACT Vallero, M.V.G. (2003). Sulfate reducing processes at extreme salinity and temperature: extending its application window. Doctoral Thesis, Wageningen University, Wageningen, the Netherlands, pp.214. The characteristics of various sulfate-rich wastewaters, such as temperature, pH and salinity, are determined by the (industrial) process from which they originate, and can be far from the physiological optima of the sulfur cycle microorganisms. The main goal of the research described in this thesis was to investigate and develop high rate sulfate reducing wastewater treatment processes for the treatment of inorganic sulfate-rich wastewaters under extreme conditions, i.e. high temperature and high salinity. In this thesis, several simple organic bulk chemicals were tested as electron donor, viz. lower alcohols (methanol and ethanol) and volatile fatty acids (formate, acetate and propionate). With respect to the start-up of upflow anaerobic sludge bed (UASB) reactors at high salinity or high temperature, the results obtained in this investigation indicate that the appearance of a targeted metabolic property (sulfate reduction at high salinity or at high temperature) is independent of the strategy for biomass acclimation (direct exposure vs. stepwise exposure). The stepwise adaptation of thermophilic sulfidogenic methanol degrading biomass to a high osmolarity environment, both at 55°C or at 70°C, likely does not occur in UASB reactors, as probably no methanol halotolerant thermophilic sulfate reducing bacteria (SRB) were present in the thermophilic inoculum sludge used in the investigations described in this thesis. Exposing the sludge directly to a very high salinity (50 g NaCl.L-1) stimulated the growth of a mesophilic (30°C) propionate- and ethanol-utilizing 2- -1 -1 halotolerant SRB population, which supported high rate sulfate reduction (up to 3.6 g SO4 .L .day ) in a UASB reactor. The start-up of thermophilic (55 to 65°C) and extreme thermophilic (70°C or higher) anaerobic bioreactors inoculated with mesophilic sludges at the targeted temperature proceeded fast and stable, as it provoked the rapid selection of (extreme) thermophiles. Therefore, the key for the successful treatment of high salinity or hot wastewaters is to invest enough time for the growth of the targeted microorganism in the biomass. The results of this investigation show that the competition between SRB, methane producing archaea and acetogenic bacteria for substrate is highly dependent of the type of substrate and operational conditions imposed to the bioreactor. This thesis describes a situation where the production of acetate and methane was completely suppressed in methanol-fed sulfate reducing UASB reactors operated at 70°C. As a result, for the first time a fully sulfate reducing granular sludge has been cultivated in a methanol-fed thermophilic sulfate reducing reactor 2- -1 -1 (with sulfate reduction rates as high as 14.4 g SO4 .L .day ), provided that an operational temperature of 70°C is kept. The production of methane can be easily suppressed in thermophilic methanol fed reactors, either by running the reactor at temperatures equal or higher than 65°C or by exposing 55°C operated reactors to a short (2 days) temperature (65 – 70°C) shock. Methanogenesis can also be easily suppressed in mesophilic propionate- and ethanol-fed reactors, provided high salinity conditions prevail (e.g. above 50 mS.cm-1). It seems, however, that the production of acetate, with