View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Universidade do Minho: RepositoriUM 352 © IWA Publishing 2012 Water Science & Technology | 66.2 | 2012 Role of syntrophic microbial communities in high-rate methanogenic bioreactors Alfons J. M. Stams, Diana Z. Sousa, Robbert Kleerebezem and Caroline M. Plugge ABSTRACT Anaerobic purification is a cost-effective way to treat high strength industrial wastewater. Alfons J. M. Stams (corresponding author) Caroline M. Plugge Through anaerobic treatment of wastewaters energy is conserved as methane, and less Laboratory of Microbiology, Wageningen University, sludge is produced. For high-rate methanogenesis compact syntrophic communities of fatty Dreijenplein 10, 6703 HB, Wageningen, The Netherlands acid-degrading bacteria and methanogenic archaea are essential. Here, we describe the E-mail: [email protected] microbiology of syntrophic communities in methanogenic reactor sludges and provide information Alfons J. M. Stams on which microbiological factors are essential to obtain high volumetric methane production rates. Diana Z. Sousa IBB-Institute for Biotechnology and Bioengineering, Fatty-acid degrading bacteria have been isolated from bioreactor sludges, but also from other Centre for Biological Engineering, University of Minho, sources such as freshwater sediments. Despite the important role that fatty acid-degrading Campus de Gualtar, 4710-057 Braga, Portugal bacteria play in high-rate methanogenic bioreactors, their relative numbers are generally low. fi Robbert Kleerebezem This nding indicates that the microbial community composition can be further optimized to achieve Department of Biotechnology, even higher rates. Delft University of Technology, Julianalaan 67, 2628 BC, Delft, Key words | anaerobic treatment, granulation, methanogenesis, syntrophic communities, UASB The Netherlands reactor ANAEROBIC WASTEWATER TREATMENT Anaerobic treatment is one of the most cost-effective ways to transfer limitations around the biofilm. Anaerobic treatment treat industrial wastewater with high organic matter content systems can operate at different temperatures and are suited (Kosaric & Blaszczyk ; Lettinga ; van Lier et al. for different types of wastewater. Currently, anaerobic treat- ). In particular, the introduction of the Upflow Anaero- ment technologies are widely applied to treat wastewaters bic Sludge Blanket (UASB) reactor, over three decades ago, from food processing industries, pulp and paper industries has resulted in improved treatment of industrial waste- and chemical and petrochemical industries (Lettinga ; waters. In a UASB reactor the wastewater is pumped from Macarie ). the bottom into the reactor, and is purified while passing a bed of compact biomass (granular sludge), which is formed by self-immobilization of anaerobic bacteria and METHANOGENESIS methanogenic archaea (Lettinga et al. ; van Lier et al. ). The high density of these granules prevents microor- Physiologically and phylogenetically different microbial ganisms from being washed out, while the short groups are involved in the mineralization of complex intermicrobial distances are favourable for the transfer of organic matter to methane and carbon dioxide (Gujer & metabolites from the bacteria to the methanogens. A variety Zehnder ; de Bok et al. ; Stams et al. ). In gen- of additional anaerobic bioreactor designs has been devel- eral, biopolymers including proteins, carbohydrates, nucleic oped. Expanded Granular Sludge Blanket (EGSB) and acids and fats are first hydrolyzed to mono- and oligomers, Internal Circulation (IC) reactors have replaced the more and these are then fermented to products that can be used conventional UASB systems (Franklin ). EGSB systems by methanogens directly (acetate, hydrogen, formate) and have a comparable design to UASB reactors, but contain an to other fatty acids such as propionate, butyrate, branched- expanded granular sludge bed minimizing external mass chain fatty acids and long-chain fatty acids (Gujer & doi: 10.2166/wst.2012.192 353 A. J. M. Stams et al. | Syntrophic communities in bioreactors Water Science & Technology | 66.2 | 2012 Zehnder ). Acetogenic bacteria oxidize these fatty acids Table 1 | Standard Gibbs free energy changes of reactions involved in syntrophic propio- nate and butyrate degradation. Data from Thauer et al. (1977) anaerobically to acetate, CO2,H2 and formate (Gujer & Zehnder ; Stams & Plugge ). Generally, about 70% of the methane is formed from acetate, while the Fatty-acid oxidation by acetogens À À 00 Propionate þ3H2O acetate ΔG ¼þ76 kJ remainder is formed from H2/CO2 and formate (Gujer & À þ þHCO þH þ3H Zehnder ; Conrad ). However, acetate can also be 3 2 Àþ À Àþ þþ Δ 00¼þ degraded by syntrophic communities of bacteria and Propionate 2HCO3 acetate H 3 formate G 72 kJ À À þ 0 Butyrate þ2H O 2acetate þH þ2H ΔG0 ¼þ48 kJ archaea, resulting in a more prominent role of H2/CO2 2 2 Àþ À Àþ þ Δ 00¼þ and formate as methanogenic substrates (Hattori ). In Butyrate 2HCO3 2 acetate H G 46 kJ þ Àþ mesophilic digesters, high ammonia levels can cause syn- 2 formate 3H2O trophic acetate oxidation (Westerholm et al. ). The Hydrogen and formate utilization by methanogens þ Àþ þ þ Δ 00¼ ammonia tolerance of these syntrophic acetate oxidizers 4H2 HCO3 H CH4 3H2O G 136 kJ Àþ þ Àþ þ Δ 00¼ gives them a competitive advantage in ammonia-stressed 4 Formate H 3HCO3 2H2O CH4 G 130 kJ systems. These bacteria, in association with ammonia-toler- Acetate utilization by methanogens À À 00 ant hydrogenotrophic methanogens, may consequently Acetate þH2O HCO3 þCH4 ΔG ¼31 kJ adopt the role of dominant acetate consumers in environ- ments in which ammonia restrains aceticlastic methanogenic activity (Westerholm et al. ). energy for growth from these conversions when the con- Propionate and butyrate are key intermediates in the centration of the products (especially hydrogen and mineralization of complex organic matter. The complete formate) is kept low. This results in an obligate dependence oxidation of propionate and butyrate can account for 20– of acetogenic bacteria on methanogenic archaea. Obli- 43% of the total methane formation, depending on the gately syntrophic communities of propionate- and type of digester and the nature of the organic compounds butyrate-degrading acetogenic bacteria and methanogenic (Mackie & Bryant ). archaea have several unique features: they degrade fatty acids coupled to growth, while neither the bacterium nor the methanogen alone is able to degrade these compounds; SYNTROPHIC FATTY ACID DEGRADATION intermicrobial distances influence biodegradation rates and specific growth rates, which in methanogenic bioreactors Easily degradable compounds (soluble polysaccharides, pro- results in the formation of aggregates of bacteria and teins, sugars and amino acids) in industrial wastewater are archaea (granular sludge); the syntrophic communities rapidly degraded by primary fermenters during storage and grow in conditions that are close to thermodynamical equi- the passage to the anaerobic bioreactor. Therefore, the librium, and the communities have evolved biochemical main substrates that enter the anaerobic bioreactor are mix- mechanisms that allow sharing of chemical energy (Stams tures of fatty acids, mainly acetate, propionate and butyrate. & Plugge ). There is still discussion on whether hydro- Propionate and butyrate are important intermediates in gen and formate are the primary compounds for methanogenesis. They are formed in the anaerobic fermen- interspecies electron transfer, and it is still unclear what tation of polysaccharides and proteins (Stams ; Schink their relative importance is (Stams & Plugge ; Müller & Stams ). Typically these fatty acids are further et al. ; Worm et al. ). degraded by syntrophic associations of anaerobic bacteria The reported maximum specific growth rates of propio- and methanogenic archaea, which are dependent on each nate- and butyrate-degrading acetogenic bacteria in other for energy conservation and growth (McInerney suspended co-culture with methanogens were 0.10 and À et al. ; Sousa et al. ; Stams & Plugge ). For 0.19 day 1, respectively (McInerney et al. ; Boone & complete and high-rate methanogenesis such syntrophic Bryant ; Mountfort & Bryant ). It is evident that communities are indispensable. fatty acid-degrading bacteria grow slowly, but it is not Bacteria that degrade and grow on fatty acids have to clear fully how fast they can grow in tight physical aggrega- cope with the unfavourable energetics of the conversion tion with methanogens. In fact, specific growth rates of processes. Table 1 illustrates the conversion of propionate syntrophic communities are difficult to determine as the and butyrate to the methanogenic substrates acetate, hydro- conversion rate per cell is not constant, but is dependent gen and formate. It is evident that bacteria can only derive on cell density of the partner microorganism. 354 A. J. M. Stams et al. | Syntrophic communities in bioreactors Water Science & Technology | 66.2 | 2012 Syntrophic propionate-degrading bacteria HS-CoA-dependent decarboxylation to acetyl-CoA and finally to acetate. Boone & Bryant () described Syntrophobacter wolinii,a propionate-degrading bacterium that grows in syntrophic Syntrophic butyrate- and LCFA-degrading bacteria association with methanogens or sulfate-reducing bacteria (Table 2). Several other mesophilic and thermophilic bac- McInerney et al.() enriched and characterized Syntro- teria that grow in syntrophy with