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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 00 Propionate þ3H2O acetate ΔG ¼þ76 kJ remainder is formed from H2/CO2 and formate (Gujer & þ þHCO þH þ3H Zehnder ; Conrad ). However, acetate can also be 3 2 þ þ þþ Δ 00¼þ degraded by syntrophic communities of bacteria and Propionate 2HCO3 acetate H 3 formate G 72 kJ þ 0 Butyrate þ2H O 2acetate þH þ2H ΔG0 ¼þ48 kJ archaea, resulting in a more prominent role of H2/CO2 2 2 þ þ þ Δ 00¼þ and formate as methanogenic substrates (Hattori ). In Butyrate 2HCO3 2 acetate H G 46 kJ þ þ 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 þ þ þ Δ 00¼ ammonia tolerance of these syntrophic acetate oxidizers 4H2 HCO3 H CH4 3H2O G 136 kJ þ þ þ 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 00 ant hydrogenotrophic methanogens, may consequently Acetate þH2O HCO3 þCH4 ΔG ¼ 31 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 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 methanogens have been phomonas wolfei, a Gram-positive bacterium that described since then as reviewed by McInerney et al. degrades butyrate and some other short-chain fatty acids (). These include Gram-negative bacteria (Syntropho- in syntrophic association with methanogens (Table 2). Sev- bacter and Smithella) and Gram-positive bacteria eral other bacteria have been described that grow with (Pelotomaculum and Desulfotomaculum). Both groups are butyrate or higher fatty acids in syntrophy with methanogens phylogenetically related to sulfate-reducing bacteria and or sulfate reducers as hydrogen scavengers (McInerney et al. some species are also able to use sulfate as a terminal elec- ; Sousa et al. ). Thus far, Algorimarina butyrica is tron acceptor for growth. Syntrophobacter and the only psychrophilic and marine bacterium that is Desulfotomaculum species reduce sulfate, but Smithella known to degrade butyrate in syntrophy with methanogens and Pelotomaculum cannot reduce sulfate. Most syntrophic (Kendall et al. ). This bacterium is not able to grow propionate-degrading bacteria are able to grow in pure cul- with other short-chain fatty acids like valerate or caproate ture by fermentation of fumarate or pyruvate. Fermentative or longer chain fatty acids such as palmitate. Mesophilic growth or sulfate-dependent growth was used to obtain the bacteria capable of syntrophic fatty acid metabolism are bacteria in pure culture. The only exceptions are Pelotoma- mainly species of Syntrophomonas, though S. bryantii was culum schinkii (de Bok et al. ) and P. propionicicum previously named Syntrophospora bryantii and Clostridium (Imachi et al. ), which seem to be true propionate- bryantii (Stieb & Schink ; Zhao et al. ;Wu degrading syntrophs. P. thermopropionicum and Desulfoto- et al. 2006a; Sousa et al. ). The only exception is maculum thermobenzoicum (subsp. thermopropionicum) are moderately thermophilic and grow in syntrophy with thermophilic methanogens (Imachi et al. ; Plugge et al. ). A marine propionate-degrading syntrophic com- munity has been described, but the identity of the propionate-degrading bacterium is not known (Kendall et al. ). In sulfate-rich environments sulfate reduction is the main terminal process and several sulfate reducers that degrade propionate have been described. Therefore, in marine habitats syntrophic growth on propionate seems to be restricted to sulfate-depleted zones. Two pathways for propionate metabolism are known, the methylmalonyl-CoA pathway and a dismutation pathway. In the latter pathway two propionate molecules are converted to acetate and butyrate, the butyrate being degraded to acetate and hydrogen as described below. Thus far, this pathway is only found in Smithella propionica (Liu et al. ; de Bok et al. ). The methylmalonyl-CoA pathway is found in the other syntrophic propionate-oxidizing bacteria (McInerney et al. ). In the methylmalonyl-CoA pathway, propionate is first activated to propionyl-CoA and then car- boxylated to methylmalonyl-CoA. Methylmalonyl-CoA is rearranged to form succinyl-CoA, which is converted to suc- cinate. Succinate is oxidized to fumarate, which is then Figure 1 | Scanning electron microscopic (SEM) images that show embedded micro- hydrated to malate and oxidized to oxaloacetate. Pyruvate colonies in an anaerobic methanogenic granule. The bar represents 50 μM is formed by decarboxylation, and is further oxidized in a (upper panel) and 5 μM (lower panel). 355 A. J. M. Stams et al. | Syntrophic communities in bioreactors Water Science & Technology | 66.2 | 2012
Table 2 | Isolation source of fatty-acid degrading bacteria (in grey: bacteria isolated from anaerobic reactor sludge) and the respective methanogenic partner
Substrates used Syntrophic bacterium Syntrophic partner in co-culturea Isolated from References
Syntrophobacter Methanospirillum C3 Granular sludge from a full-scale Harmsen et al.() fumaroxidans hungatei UASB reactor treating sugar beet waste, Breda, The Netherlands
Syntrophobacter Methanospirillum C3 Anoxic sludge of a municipal Wallrabenstein et al. pfennigii hungatei sewage plant in Konstanz, () Germany
Syntrophobacter Methanospirillum C3 Granular sludge from a UASB Chen et al.() sulfatireducens hungatei reactor treating brewery wastewater in Beijing, China
Syntrophobacter Methanospirillum C3 Anaerobic sewage digester sludge in Boone & Bryant () wolinii hungatei Illinois, USA Desulfovibrio sp.
Pelotomaculum Methanospirillum C3 Granular sludge from a full-scale de Bok et al.() schinkii hungatei UASB reactor treating sugar beet waste, Breda, The Netherlands
Pelotomaculum Methanothermobacter C3 Granular sludge from a Imachi et al.() thermopropionicum themautotrophicus thermophilic laboratory-scale UASB reactor, Japan
Pelotomaculum Methanospirillum C3 Granular sludge from a mesophilic Imachi et al.() propionicicum hungatei laboratory-scale UASB reactor, Japan
Smithella propionica Methanospirillum C3,C4 Granular sludge from a Liu et al.() hungatei mesophilic laboratory-scale Methanogenium sp. UASB reactor, USA
Desulfotomaculum Methanothermobacter C3,C4 Granular sludge from a Plugge et al.() thermobenzoicum thermautotrophicus thermophilic laboratory-scale thermosyntrophicum UASB reactor, The Netherlands
Desulfotomaculum Methanococcus C3,C4 North Sea oil reservoir formation Nilsen et al.() thermocisternum thermolithotrophicus water, Norway
Algorimarina butyrica Methanogenium sp. C4 Permanently cold, shallow anoxic Kendall et al.() marine sediments in Skan Bay, Alaska
Syntrophomonas Methanospirillum C4–C11 Marine anoxic mud taken Stieb & Schink (); bryantii hungatei near Cuxhaven, mud of Zhao et al.(); Desulfovibrio sp. a creek near Konstanz, Wu et al.(a) anaerobic digester sludge, Germany
Syntrophomonas Methanobacterium C4–C8,C10 Sample from the walls of a distilled- Wu et al.(a) cellicola formicicum spirit fermenting cellar in Hebei Desulfovibrio sp. province, China
Syntrophomonas Methanobacterium C4–C18,C18:1 Granular sludge from a UASB Zhang et al.(; curvata formicicum reactor treating brewery ) wastewater in Beijing, China
Syntrophomonas erecta Methanospirillum C4–C8 Granular sludge of a UASB reactor Zhang et al.(); erecta hungatei treating bean-curd farm Wu et al.(b) wastewater in Beijing, China
Syntrophomonas erecta Methanobacterium C4–C8 River sediment in Beijing and rice Wu et al.(b) sporosyntropha formicicum field mud in Yunnan Province, China
(continued) 356 A. J. M. Stams et al. | Syntrophic communities in bioreactors Water Science & Technology | 66.2 | 2012
Table 2 | continued
Substrates used Syntrophic bacterium Syntrophic partner in co-culturea Isolated from References
Syntrophomonas Methanospirillum C4–C18 Granular sludge from a laboratory- Hatamoto et al.() palmitatica hungatei scale UASB reactor treating palm oil mill effluent, Japan
Syntrophomonas Methanospirillum C4–C18 Anaerobic sewage digester sludge in Lorowitz et al.(); saponavida hungatei Illinois, USA Wu et al.() Desulfovibrio sp.
Syntrophomonas Methanospirillum C4–C18,C16:1,C18:1, Sewage digester sludge in Roy et al.(); sapovorans hungatei C18:2 Marquette-Lez-Lille, France Zhang et al.()
Syntrophomonas Methanobacterium C4–C8 Rice field mud in Jangxi province, Wu et al.() wolfei formicicum China methylbutyratica
Syntrophomonas Methanospirillum C4–C8 Sewage digester sludge and rumen McInerney et al.(; wolfei wolfei hungatei digester, USA ); Desulfovibrio sp. Zhang et al.()
Syntrophomonas Methanobacterium C4–C18,C16:1,C18:1, Anaerobic sludge from an EGSB Sousa et al.() zehnderi formicicum C18:2 treating oleate-based effluent, Braga, Portugal
Syntrophus Methanospirillum C4–C8,C16,C18 Sewage sludge from a municipal Jackson et al.() aciditrophicus hungatei sewage treatment plant in Desulfovibrio sp. Norman, Oklahoma, USA
Syntrophothermus Methanothermobacter C4–C10 Granular sludge from a laboratory- Sekiguchi et al.() lipocalidus thermoautotrophicus scale thermophilic UASB reactor, Japan
Thermosyntropha Methanobacterium sp. C4–C18,C18:1,C18:2 Alkaline hot springs of Lake Svetlitshnyi et al. lipolytica Bogoria, Kenya ()
Thermosyntropha Methanothermobacter C4–C18,C18:1,C18:2 Hot spring in Yunnan province, Zhang et al.() tengcongensis thermautotrophicus China aThe number of carbon atoms of the fatty acids and the position of the double bond are indicated.
Syntrophus aciditrophicus, a Gram-negative benzoate- fatty acids, first activation to a HS-CoA derivative takes degrading bacterium that it is also able to degrade place. The HS-CoA-derivative is then dehydrogenated to medium- and long-chain fatty acids in co-culture with a form an enoyl-CoA. After water addition, a second dehydro- methanogen (Jackson et al. ). Thermophilic syntrophic genation takes place to form a ketoacylacetyl-CoA. After butyrate-degrading bacteria are Thermosyntropha lipolytica hydrolysis acetyl-CoA and an acyl-CoA are formed, which (Svetlitshnyi et al. ) and Syntrophothermus lipocalidus enters another cycle of dehydrogenation and the cleaving (Sekiguchi et al. ). None of the fatty acid-degrading off of acetyl-CoA. bacteria that grow syntrophically with methanogens has been described to reduce sulfate. Most fatty acid degraders are able to ferment crotonate, which was used to obtain EFFECT OF GRANULATION pure cultures. However, Syntrophomonas sapovorans and S. zehnderi are not able to ferment crotonate, and are The formation of granular sludge is one of the reasons for only available in syntrophic methanogenic co-cultures the success of anaerobic treatment of wastewaters (Kosaric (Roy et al. ; Sousa et al. ). & Blaszczyk ; Lettinga ). In particular, aggregation Butyrate and longer chain fatty acids are degraded via of syntrophic communities is essential to achieve high rates so-called β-oxidation (Schink & Stams ; McInerney of methanogenesis with propionate and butyrate. Figure 1 et al. ). In a series of reactions acetyl groups are cleaved illustrates the dense packing of microorganisms and micro- off yielding acetate, hydrogen and formate. To metabolize colonies in the granule structure. As pointed out by 357
Table 3 | Detection and quantification of syntrophic bacteria in anaerobic reactors .J .Stams M. J. A.
Reactor type Reactor feed and operational conditions Syntrophic bacteria abundance Detection/Quantification method Reference
Mesophilic laboratory-scale Industrial wastewater from a brewery Syntrophobacter spp. Férnandez et al. W UASB reactor OLR¼ 4g L 1 day 1 ; T¼ 30 C 5–10% FISH (probe SYN835) ()
COD al et
Thermophilic full-scale Municipal sewage sludge Pelotomaculum spp. Imachi et al. . 1 W | anaerobic reactor CODin¼ 59 g L ; T¼ 53 C 0.1% Real-time PCR (primers () bioreactors in communities Syntrophic DEM116f-Ih820) Thermophilic UASB laboratory- Synthetic wastewater containing sucrose, acetate, Pelotomaculum spp. Imachi et al. scale reactor propionate, and peptone or yeast extract 1.1% Real-time PCR (primers () 1 W CODin¼ 4gL ; T¼ 55 C DEM116f-Ih820) 0.5 ± 0.2% FISH (probe Ih820) Thermophilic laboratory-scale Wastewater from clear liquor manufacture Pelotomaculum spp. Imachi et al. 1 W anaerobic reactor CODin¼ 12 g L ; T¼ 55 C 3.9% Real-time PCR (primers () DEM116f-Ih820) 4.1 ± 1.1% FISH (probe Ih820) Mesophilic UASB laboratory- Synthetic wastewater Pelotomaculum spp. Imachi et al. 1 W scale reactor CODin¼ 2gL ; T¼ 35 C 0.1% Real-time PCR (primers () DEM116f-Ih820) <0.1% FISH (probe Ih820) Mesophilic full-scale anaerobic Municipal sewage sludge Pelotomaculum spp. Imachi et al. 1 W reactor CODin¼ 58 g L ; T¼ 35 C 0.3% Real-time PCR (primers () DEM116f-Ih820) Mesophilic laboratory-scale Synthetic wastewater made from powdered whole Ariesyady et al. reactor (inoculated with sludge milk Syntrophobacter, Smithella spp. (b) 1 1 W from an anaerobic digester OLR¼ 1.5 gCOD L day ; T¼ 37 C 2.7–7.3% MAR-FISH (probes from municipal wastewater SmiSR354, SmiLR150, treatment plant) Synbac824) Syntrophomonadaceae detected MAR-FISH (probe but not quantified Synm700) Mesophilic full-scale anaerobic Syntrophobacter fumaroxidans McMahon et al.
digester (two reactors) 0.27 ± 0.14% /0.37 ± 0.12% Membrane hybridization () Technology & Science Water Syntrophobacter pfennigii (probe S.fum464) 0.33 ± 0.08% /0.48 ± 0.08% Membrane hybridization Syntrophobacter wolinii (probe S.pfn460) 0.44 ± 0.12% /0.43 ± 0.11% Membrane hybridization Smithella propionica (probe S.wol223) <0.2% /0.28 ± 0.29% Membrane hybridization Syntrophomonadaceae (probe S.pro450) ± ± 0.42 0.16% /1.76 0.25% Membrane hybridization | (probe Synm700) 66.2 | (continued) 2012 358
Table 3 continued | Stams M. J. A.
Reactor type Reactor feed and operational conditions Syntrophic bacteria abundance Detection/Quantification method Reference
Mesophilic laboratory-scale Synthetic mixture of organic fraction of municipal Syntrophobacter fumaroxidans McMahon et al. anaerobic digester solid waste, primary sludge and waste activated 0.25 ± 0.05% Membrane hybridization () al et .
sludge Syntrophobacter pfennigii (probe S.fum464) |
3 1 W bioreactors in communities Syntrophic OLR¼ 9.4 kgVS m day ; T¼ 37 C 0.67 ± 0.07% Membrane hybridization Syntrophobacter wolinii (probe S.pfn460) 0.47 ± 0.06% Membrane hybridization Smithella propionica (probe S.wol223) 0.23 ± 0.04% Membrane hybridization Syntrophomonadaceae (probe S.pro450) 0.65 ± 0.03% Membrane hybridization (probe Synm700) Mesophilic full-scale anaerobic Excess sludge of domestic wastewater treatment Syntrophus, Smithella Ariesyady et al. digester plant facility propionica (a) W OLR¼ 2.5 kg m 3 day 1 ; T¼ 40 C 2.0 ± 0.7% FISH (probe SmiSR354) Syntrophobacter 0.5 ± 0.4% FISH (probe Synbac824) Syntrophomonas 1.5 ± 0.5% FISH (probe Synm700) Mesophilic full-scale anaerobic Mixture of swine and cattle manure and a variety of Syntrophomonadaceae Hansen et al. reactor industrial organic waste streams 0.2–1% Membrane hybridization () Syntrophomonas (probe Synm700) 0.2–1% Membrane hybridization (probe Syn126) Mesophilic full-scale anaerobic Edible tallow refinery wastewater Syntrophomonadaceae Menes & reactors (four reactors) Poultry slaughterhouse wastewater 3% FISH (probe Butox) Travers () Brewery wastewater <0.01% Potato processing wastewater <0.01% <0.01%
Anaerobic lagoon Woolscouring wastewater Syntrophomonadaceae Menes & Technology & Science Water <0.01% FISH (probe Butox) Travers () Mesophilic full-scale anaerobic Excess sludge of domestic wastewater treatment Syntrophus, Smithella Ariesyady et al. digester plant facility propionica (a) W OLR¼ 2.5 kg m 3 day 1 ; T¼ 40 C 2.0 ± 0.7% FISH (probe SmiSR354) Syntrophobacter 0.5 ± 0.4% FISH (probe Synbac824) Syntrophomonas ± 1.5 0.5% FISH (probe Synm700) | 66.2
OLR: Organic loading rate; COD: Chemical oxygen demand; FISH: Fluorescence in situ hybridization; MAR-FISH: Microautoradiography combined with FISH; Real-time PCR: Real-time polymerase chain reaction. | 2012 359 A. J. M. Stams et al. | Syntrophic communities in bioreactors Water Science & Technology | 66.2 | 2012
Schink & Thauer () by bringing hydrogen-producing quantification of different trophic groups in anaerobic bacteria into close proximity with hydrogen-consuming environments and combined with flux measurements will methanogens the distance that hydrogen has to diffuse allow for a detailed understanding of anaerobic conversion from the producing to the consuming microorganisms processes. Based on the improved understanding of electron becomes shorter, and this results in higher conversion transfer in anaerobic environments it can readily be envi- rates. Thus, the compact microbial structure in methano- sioned that the anaerobic digestion process can still be genic granular sludge is functional. It has been observed improved significantly. that when the structure is disrupted the specific methano- genic activity with propionate decreases considerably, while the specific methanogenic activity with acetate is CONCLUSIONS not affected (Grotenhuis et al. ; Schmidt & Ahring ). Another factor that affects the specific activity is the size High-rate methanogenesis is required to treat industrial of the granules. When granules are too large, the centre of wastewaters efficiently and conserve the chemical energy the granules will become substrate limited. Moreover, in the organic pollutants as biogas. In the complete conver- biogas may accumulate in the granules that are weakened sion of organic pollutants to methane and carbon dioxide, in structure inside. This may cause a disintegration of the syntrophic methanogenic communities of bacteria and mature granules into smaller particles that grow again. In archaea play an indispensable role. Both Gram-negative this way, the average size of the granules is determined by (deltaproteobacteria) and Gram-positive bacteria with the the wastewater composition. ability to grow with propionate and butyrate in syntrophy In the granules competition for space takes place as well. with methanogens have been described. Some of the To efficiently degrade fatty acids, the microorganisms in the described bacteria have been obtained in pure culture, granule should mainly consist of acetogenic fatty acid- while others are only maintained in syntrophic co-culture degrading bacteria and acetoclastic and hydrogenotrophic with a defined methanogen. In high-rate methanogenic bio- methanogens. Fluorescence in situ hybridization (FISH) reactors propionate-degrading and butyrate-degrading has been used to study the structural organization of anaero- acetogenic bacteria form microcolonies with methanogens. bic granular sludge (Harmsen et al. ; Sekiguchi et al. The short distances between the bacteria and methanogens ). In these studies, a close association between Syntro- are favourable for transfer of metabolites from bacteria to phobacter species and hydrogenotrophic methanogens archaea and result in high conversion rates. To further belonging to the Methanomicrobiales group has been improve the rate of methane formation with mixtures of observed. Nevertheless, microbial community analysis with fatty acids as substrates the underlying mechanisms of inter- molecular techniques has shown that granular sludge of species electron transfer in syntrophic communities need to full-scale reactors rarely harbours high numbers of acetogenic be better understood. In addition, insight is needed into how bacteria (Table 3). This suggests that fatty acid-degrading by process engineering the compact structure of syntrophic communities establish high specific conversion rates, and communities in granules can be obtained and maintained. that even higher conversion rates can be achieved by mini- mizing the numbers of primary fermenters in the granules. Still, due to the limitations in quantification of individual ACKNOWLEDGEMENT groups of bacteria in mixed anaerobic biomass, fluxes can hardly be attributed to concentrations of specific microorgan- Our research is funded by grants from the division of Chemi- isms. Consequently, conversion rates are expressed per cal Sciences (CW) and Earth and Life Sciences (ALW) of fi amount of lumped biomass in terms of volatile suspended The Netherlands Organisation for Scienti c Research solids (VSS). Herewith, it remains unclear if energy is (NWO) and by the Technology Foundation (STW), the shared equally between the partner microorganisms and applied science division of NWO. what the mutual effects of different acetogenic substrates are on their degradation. Detailed quantitative insight is REFERENCES required about the electron fluxes between the partner organ- isms in anaerobic sludges and the way they share the Ariesyady, H. 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First received 16 September 2011; accepted in revised form 5 March 2012