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Microbial communities in anaerobic digestion processes for waste and wastewater treatment: a microbiological update
Microbial communities in anaerobic digestion processes for waste and wastewater treatment: a microbiological update Takashi Narihiro and Yuji Sekiguchi Anaerobic digestion technology is the biological treatment of organic waste and wastewater without input of external electron acceptors (oxygen), offering the potential to reduce treatment cost and to produce energy as ‘biogas’ (methane) from organic waste. The technology has become enormously popular in the past two decades, and knowledge of microbiological aspects of the technology has also accumulated significantly. Major advances have been made in elucidating the diversity of yet-to-be cultured microbes in anaerobic digestion processes, and the cultivation of uncultured organisms is of great interest with regard to gaining insights into the function of these organisms. In addition, recent advances have been made in the development of microbial fuel cells as an alternative, direct energy-yielding treatment system. Addresses Bio-Measurement Research Group, Institute for Biological Resources and Functions, National Institute of Advanced Science and Technology (AIST), AIST Tsukuba Central 6, Ibaraki 305-8566, Japan Corresponding author: Sekiguchi, Yuji ([email protected])
Current Opinion in Biotechnology 2007, 18:273–278 This review comes from a themed issue on Environmental biotechnology Edited by Eliora Z Ron and Philip Hugenholtz Available online 25th April 2007 0958-1669/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2007.04.003
Introduction In theory, anaerobic digestion technology is an ideal biological means for the removal of organic pollutants in waste and wastewater. The technology has two significant advantages over the conventional aerobic biological treatment: firstly, it is cost-effective because aeration is not required and a small amount of excess sludge is produced, and secondly, it generally produces gaseous methane as an energy resource [1,2]. The largest application of this digestion technology is the stabilization of sludge, such as a sludge digester commonly used at municipal wastewater treatment plants. The technology has also become popular in dilute and concentrated wastestream treatment fields; anaerobic digestion technology for wastewater treatment can now also be considered a matured biotechnology. Owing to the development of sophisticated wastewater treatment technologies (such as upflow anaerobic sludge blanket [UASB] technology), more than 1500 anaerobic www.sciencedirect.com
wastestream treatment processes are now installed and operating worldwide [3,4]. So far, the most practical target of this technology is high-rate treatment of high-strength industrial organic wastewater [1,4]. The types of wastewater that can be treated by such technology have recently been expanded notably; for example, low-strength organic wastewater, complex wastewater containing persistent chemical compounds, and wastewater discharged at temperatures ranging from psychrophilic (4 8C to 20 8C) to thermophilic (above 45 8C) [2,5]. In addition, the technology has also been shown to offer interesting potentials for metal removal and recovery with sulfate reduction, removal of nitrates with nitrification, and bioremediation for breakdown of toxic substances. One of the most advanced fields associated with the technology in the past few years is the microbiology of anaerobic digestion processes. Because knowledge of the ecology and function of the microbial community in these processes is required to better control the biological processes, considerable effort has been made to understand the microbial community structure by using culture-dependent and culture-independent molecular approaches [2,6,7]. Through these analyses, particularly those targeting the 16S rRNA gene, detailed pictures of the community compositions are being documented. In addition, several functionally important anaerobes, playing key roles in the treatment process, have been cultivated and characterized. Furthermore, the ecophysiology of yet-to-be cultured organisms in the ecosystems is being elucidated using a variety of approaches. In this review, we focus on microbiological aspects of anaerobic (mainly methanogenic) microbial communities for high-rate organic waste and wastewater treatment, and update the recent findings in this field. We highlight a variety of recent approaches in microbiological fields, including microbial community analysis, the domestication of uncultured microorganisms, and ecophysiological analysis of yet-to-be cultured anaerobes that are relevant to the technology. In addition, we raise the topic of microbial fuel cells, an alternative, direct energyyielding wastewater treatment system, and describe recent findings on the microbiological aspects.
Community analysis of anaerobic digestion processes: the uncultured, yet-to-be characterized lineages In anaerobic treatment processes, there has been a relatively limited number of studies conducting 16S rRNA gene cloning-based analyses of the microbial community Current Opinion in Biotechnology 2007, 18:273–278
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(often known as ‘sludge’) [2,6,7]. However, constituents of more than 20 bacterial phyla have been detected in anaerobic (mostly methanogenic) waste and wastewater sludges [2,6,7]. For example, 16S rRNA gene clones that were frequently and commonly retrieved from these sludges were distributed in various prokaryotic taxa such as the phyla Proteobacteria (mainly in the class Deltaproteobacteria), Chloroflexi, Firmicutes, Spirochaetes, and Bacteroidetes in the domain Bacteria. Similarly, clones in the classes Methanomicrobia, Methanobacteria, and Thermoplasmata in the domain Archaea are those of typical phylotypes found in such sludges [6]. In addition to these relatively known taxa, phylotypes belonging to a variety of uncultured candidate phyla (or classes) (known as ‘clone cluster’) were often detected in these sludges [6,7,8, 9,10,11,12]. Within the domain Bacteria, diverse uncultivated taxonomic groups, such as OP10, BA024, OP8, TM6, EM3, OP3, and OS-K, were detected in these sludges [6] (note that most of the candidate taxa in this review are named according to the article by Hugenholtz [13]). Finding key (or dominant) populations that belong to such uncultured lineages at various taxonomic levels (from species to phylum levels) is one of the major advances in the microbiology of anaerobic digestion processes in the past few years. One recent finding in the predominant populations of the clone clusters is that of the candidate bacterial phylum WWE1. Chouari et al. [10] described that 81% of all the bacterial 16S rRNA gene clones retrieved from sludge from an anaerobic mesophilic digester were assigned with the group WWE1. Fluorescence in situ hybridization (FISH) analysis revealed that WWE1-type cells had rod and filamentous morphotypes, and the rRNA from WWE1 cells accounted for 12% of the total bacterial rRNA [10]. Although the ecophysiological function of WWE1-type bacteria remains unknown, their high abundance in the sludge suggests they might play a certain role in the digestion process. Another example of the predominant populations that belong to clone clusters is that of the bacterial phylum Deferribacteres. The phylum Deferribacteres (formerly recognized as the phylum ‘Synergistes’) contained phenotypically diverse genera such as Deferribacter, Denitrovorans, Geovibrio, Flexistipes and ‘Synergistes’ [14]. Within the phylum, several 16S rRNA gene clones that are distantly related to known cultivated species, forming distinct clone clusters at the subphylum (class) levels, have also been retrieved from anaerobic (methanogenic) sludges. The distribution of members of this phylum was thoroughly explored in a total of 93 anaerobic ecosystems, such as anaerobic digesters, soil and gut samples, indicating that Deferribacteres-type clones were widely distributed in such anaerobic ecosystems [11]. The clones retrieved from methanogenic digester sludges were assigned to four different subphyla of Deferribacteres, none Current Opinion in Biotechnology 2007, 18:273–278
of which contains cultured representatives. More recently, Diaz et al. [15] showed that 34% of all the bacterial clones detected from a full-scale methanogenic UASB process treating brewery wastewater were affiliated with uncultured clades of the phylum Deferribacteres. Given their high occurrence in such methanogenic ecosystems, they might play a role in part of the food web for the methanogenic degradation of organic compounds; however, their ecophysiology remains unknown. With respect to the uncultured archaeal lineages, archaeal 16S rRNA gene clones affiliated with the candidate taxon WSA2 were retrieved in abundance from a mesophilic methanogenic digester decomposing sewage sludge [9] (this candidate taxon is also named according to the review by Hugenholtz [13], although Chouari et al. [9] refer to the taxon as ‘ArcI’ group in their report). This group is a clone cluster at the subphylum (or class) level within the archaeal phylum Euryarchaeota, and phylotypes of WSA2 were sometimes detected in other anaerobic treatment processes [6]. Chouari et al. [9] also obtained highly enriched WSA2 cells using formate- or hydrogen-containing media, implying that WSA2-related microorganisms are methanogens. Another unique, uncultured archaeal taxon that is also often found in methanogenic sludges is subphylum C2 of the archaeal phylum Crenarchaeota. For example, 16% of the archaeal rRNA gene clones analyzed from a mesophilic methanogenic digester were found to belong to members of Crenarchaeota, particularly the subphylum C2 [9]. C2-type rRNA gene clones were also detected from a full-scale, methanogenic (partially sulfidogenic) UASB process treating paper mill wastewater [16] and a laboratory-scale UASB reactor treating methanethiol [17]. Collins et al. [8] recently surveyed C2-type phylotypes in anaerobic sludges from a variety of methanogenic wastewater treatment systems, detecting these phylotypes in abundance (14–78% of the total archaeal clones analyzed). From FISH observations in thin-sections of methanogenic sludge granules, cells of the C2-type phylotypes were found to be rods (1.5 mm in length and 0.7 mm in width), forming dense microcolonies within the sections. Interestingly, they were juxtaposed to Methanosaeta cells [8]. These findings may imply that they are active members of the sludge ecosystems, interacting with aceticlastic methanogens in situ. Further studies are needed to clearly describe the ecophysiological functions of these unique Archaea.
Community changes within anaerobic digestion processes Recent microbiological studies focus not only on the description of a microbial community at a particular operational time, but also on the community shift (population dynamics) of anaerobic sludge along with different operational periods (or conditions). www.sciencedirect.com
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Such population dynamics of resident microbes in anaerobic treatment processes have also been analyzed by using rRNA and rRNA-gene based methods. For example, Diaz et al. [15] and Zheng et al. [18] studied the community changes along with the maturation of sludge granules in UASB reactors using rRNA gene-based denaturing gradient gel electrophoresis (DGGE) analysis and FISH. Similarly, Hori et al. [19] analyzed changes in the microbial community succession in a thermophilic methanogenic reactor under deteriorative and stable conditions, which were induced by acidification and neutralization, using rRNA gene-based single-strand conformation polymorphism (SSCP), quantitative PCR, and FISH. The results indicated that the methanogenic community in the process was significantly affected by volatile fatty acid concentrations.
Newly isolated microorganisms from anaerobic treatment processes The domestication (cultivation) of uncultured organisms is of great interest in this field to gain insights into the function of these organisms. In the past few years, newly discovered microorganisms have been successfully isolated from anaerobic sludges, and the information regarding their physiology in conjugation with phylogeny is updated regularly: examples include carbohydrate degraders [20,21], a protein degrader [22], fatty acid oxidizers [23,24,25–32], terephthalate oxidizers [33], and methanogens [34–36]. One of the most significant advances in this field is the isolation of organisms from subphylum I of the bacterial phylum Chloroflexi. The subphylum I was recognized as a clone cluster, members of which were frequently detected in anaerobic environments in abundance [6,15,16,37,38]. Recently, four filamentous strains that belong to the Chloroflexi subphylum I were successfully isolated and cultured from anaerobic wastewater treatment sludges, and these strains were characterized in detail to give their taxonomic placements [20,21]. At present, the subphylum contains two thermophilic species of the genus Anaerolinea, one mesophilic species of the genus Levilinea, and one mesophilic species of the genus Leptolinea, and the subphylum was named as the new class Anaerolineae. These organisms are known to be one of the major populations in mesophilic and thermophilic sludge granules of UASB reactors [37,38]. One species (Anaerolinea thermophila) is associated with filamentous bulking of methanogenic granular sludge [37]. All strains possess filamentous morphotypes, growing fermentatively with a range of carbohydrates and yeast extract as substrates. These findings suggest that members of the class might play a key role in the primary degradation of carbohydrates and cellular materials (such as amino acids) in methanogenic digestion processes. Several important proton-reducing syntrophic bacteria affiliated with the group ‘Desulfotomaculum cluster I’ have www.sciencedirect.com
been isolated and cultured from anaerobic wastewater processes. Imachi et al. [23,39] first isolated Pelotomaculum thermopropionicum from a laboratory-scale UASB reactor operated under thermophilic conditions. This bacterium can degrade propionate in co-culture with hydrogenotrophic methanogens [23]. Later, the obligately syntrophic, propionate-oxidizing bacterium Pelotomaculum schinkii [24], a mesophilic, syntrophic propionate-oxidizing strain MGP [40], two mesophilic, syntrophic phthalatedegrading species Pelotomaculum terephthalicum and Pelotomaculum isophthalicum [33,41], and a mesophilic, syntrophic benzoate-oxidizing species Sporotomaculum syntrophicum [25] were also isolated from anaerobic wastewater treatment processes. Interestingly, all these syntrophs lack the ability to dissimilatory reduce sulfate, although other members of the ‘Desulfotomaculum cluster I’ are known to be sulfate reducers. From a set of various molecular and cultivation-based analyses for non-sulfate-reducing Desulfotomaculum-type organisms, it was hypothesized that these microorganisms have recently adopted a syntrophic lifestyle to thrive in low-sulfate, methanogenic environments and thus have lost their ancestral ability for dissimilatory sulfate/sulfite reduction [40]. Importantly, by using DNA-based stable isotope probing (SIP) analyses members of the genus Pelotomaculum were found to be a key contributor to the syntrophic propionate degradation in natural anaerobic ecosystems (e.g. flooded soil and freshwater marshes) [42,43]. Among the characterized species of these syntrophic organisms of the ‘Desulfotomaculum cluster I’, P. thermopropionicum has been intensively studied in recent years. Kosaka et al. [44] analyzed the genome of P. thermopropionicum and proposed a novel propionate-oxidizing pathway (methylmalomyl coenzyme A pathway), which differs from that of the previously characterized pathway in other bacteria. Together with proteomic data, they assumed that fumarase plays a central role in the regulation of syntrophic propionate degradation. Additionally, it was reported that the cells of P. thermopropionicum coaggregated when they were co-cultivated with Methanothermobacter thermautotrophicus cells in the presence of propionate [45,46]. Interestingly, the flagellum-like filaments of P. thermopropionicum participate in coaggregation of each cell [45], suggesting that these filaments might have a role in keeping the two organisms closely juxtaposed with each other for efficient interspecies hydrogen transfer. More recently, the flagellum-like filaments of P. thermopropionicum were found to be electrically conductive, suggesting the possibility of direct exocellular electron transfer through the flagellum-like filaments (this topic is further discussed below) [47,48] Although the number of descriptions of these new anaerobes is increasing, there is still much work to be done to domesticate the yet-to-be cultured microbes that are recalcitrant to artificial cultivation. To avoid difficulties Current Opinion in Biotechnology 2007, 18:273–278
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in cultivation, several techniques such as FISH combined with microautoradiography (MAR–FISH), DNA-based stable isotope probing (SIP) and RNA-SIP have been developed and applied to anaerobic sludges [49]. Visualizing substrate uptake pattern within the methanogenic granule sections with new techniques, such as radioactive tracer technique plus b imaging [8], SIP-Raman microscopy [50] and SIP-secondary ion mass spectrometry (SIMS) [51], might also help to point the way to the cultivation of uncultured organisms. Collins et al. [8] attempted to apply the radioactive tracer technique and b imaging to sludge granules to speculate the function of uncultured Crenarchaeota cells (mentioned above). Furthermore, metagenomic approaches for microbial consortia containing yet-to-be cultured microbial cells have recently been applied to a wide range of environments to gain new insight into the potential metabolic activities of predominant microbes based on gene information. For example, the report by Wexler et al. [52] is, to our knowledge, the first attempt at employing a metagenomic approach for anaerobic digester sludges.
Recent issue: microbial fuel cells revisited Microbial fuel cells (MFC) have recently received much attention for their potential to directly recover electricity as an energy resource from waste and wastewater [48, 53,54]. Interest in the MFC process originally began in the 1960s, when it was discovered that resident microorganisms in the process oxidize the organic substances in waste and wastewater as electron donors and transfer electrons to the anode electrode via soluble electron mediators such as ferricyanide. However, the addition of such mediators was considered infeasible for actual electron recovery because of their toxicity and instability in a prolonged operation of the MFC process [53,55,56]. Recently, Bond and Lovley [55] and Chaudhuri and Lovley [56] showed that Geobacter sulfurreducens and Rhodoferax ferrireducens, respectively, directly transfer electrons to the anode surface via components associated with their cell wall; these findings again shed light on the MFC process research. The electron transfer efficiency from acetate to electricity was found to be 96.8% with G. sulfurreducens cells, whereas the electron transfer efficiency from glucose was reported to be 81% with R. ferrireducens cells. Several attempts to produce electricity with a complex microbial community have also been reported [54,57,58]. For example, Rabaey et al. [57] reported that the MFC process enriched with an anaerobic granular sludge achieved 81% efficiency for electron transfer from glucose. Most recently, it is reported that electrically conductive pilus-like filaments (they called ‘bacterial nanowires’) of the metal-reducing bacterium Shewanella oneidensis mediated exocellular electron transfer to metal surface [47]. As mentioned above, a similar finding was made when the syntrophic propionate-oxidizer P. thermopropionicum was cultivated Current Opinion in Biotechnology 2007, 18:273–278
in pure culture with fumarate or in co-culture with M. thermautotrophicus with propionate [47]. This suggests the possibility of direct exocellular electron transfer from the syntrophic microbe through such electrically conductive, flagellum-like filaments [47,48].
Conclusions As described in this short update, significant advances have been made in elucidating the diversity of yet-to-be cultured organisms in anaerobic (methanogenic) digestion processes. Although some important anaerobes have been cultivated and characterized, there is still work to be done for a vast number of remaining anaerobes that have not yet been cultured. For the remainder, further characterization of their ecophysiological traits with cultivation or with much more elegant methods (such as SIP) is needed. The accumulation of such information will offer substantial information for more sophisticated management of the digestion technology. Recent attention to the potential of the MFC process to directly recover electricity as an energy resource from waste and wastewater could suggest a future direction of the technological development of the anaerobic digestion process. Further investigations of the MFC process will be necessary to improve the process, as well as to fully understand the role of the microbial community contributing the generation of electricity.
Acknowledgements This study was supported by the Project ‘Development of Technologies for Analyzing and Controlling the Mechanism of Biodegrading and Processing’, which was ensured to the New Energy and Industrial Technology Development Organization (NEDO).
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36. Jiang B, Parshina SN, van Doesburg W, Lomans BP, Stams AJM: Methanomethylovorans thermophila sp. nov., a thermophilic, methylotrophic methanogen from an anaerobic reactor fed with methanol.. Int J Syst Evol Microbiol 2005, 55:2465-2470. 37. Sekiguchi Y, Takahashi H, Kamagata Y, Ohashi A, Harada H: In situ detection, isolation, and physiological properties of a thin filamentous microorganism abundant in methanogenic granular sludges: a novel isolate affiliated with a clone cluster, the green non-sulfur bacteria, subdivision I. Appl Environ Microbiol 2001, 67:5740-5749. 38. Yamada T, Sekiguchi Y, Imachi H, Kamagata Y, Ohashi A, Harada H: Diversity, localization, and physiological properties of filamentous microbes belonging to Chloroflexi subphylum I in mesophilic and thermophilic methanogenic sludge granules. Appl Environ Microbiol 2005, 71:7493-7503. 39. Imachi H, Sekiguchi Y, Kamagata Y, Ohashi A, Harada H: Cultivation and in situ detection of a thermophilic bacterium capable of oxidizing propionate in syntrophic association with hydrogenotrophic methanogens in a thermophilic methanogenic granular sludge. Appl Environ Microbiol 2000, 66:3608-3615. 40. Imachi H, Sekiguchi Y, Kamagata Y, Loy A, Qiu YL, Hugenholtz P, Kimura N, Wagner M, Ohashi A: Harada H: Non-sulfatereducing, syntrophic bacteria affiliated with Desulfotomaculum cluster I are widely distributed in methanogenic environments. Appl Environ Microbiol 2006, 72:2080-2091. In this paper, the occurrence and abundance of ‘Desulfotomaculum cluster I’ in low-sulfate, methanogenic environments were analyzed using 16S rRNA-based molecular approaches, and the new strain MGP was successfully isolated in co-culture with a hydrogenotrophic methanogen. Interestingly, strain MGP could not dissimilatory reduce sulfur compounds, but the strain contained (and expressed) dsrAB, key genes in the sulfate respiration. 41. Qiu YL, Sekiguchi Y, Imachi H, Kamagata Y, Tseng IC, Cheng SS, Ohashi A, Harada H: Identification and isolation of anaerobic, syntrophic phthalate isomer-degrading microbes from methanogenic sludges treating wastewater from terephthalate manufacturing. Appl Environ Microbiol 2004, 70:1617-1626.
methanogen Methanothermobacter thermautotrophicus coaggregated via flagellum-like filaments when they grow in co-culture with propionate. 46. Ishii S, Kosaka T, Hotta Y, Watanabe K: Simulating the contribution of coaggregation to interspecies hydrogen fluxes in syntrophic methanogenic consortia. Appl Environ Microbiol 2006, 72:5093-5096. This paper describes a simple model for simulating the contribution of coaggregation to interspecies hydrogen transfer fluxes between syntrophic bacteria and methanogens. 47. Gorby YA, Yanina S, McLean JS, Rosso KM, Moyles D, Dohnalkova A, Beveridge TJ, Chang IS, Kim BH, Kim KS et al.: Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms. Proc Natl Acad Sci USA 2006, 103:11358-11363. Using scanning tunneling microscopy, the authors report that three bacterial species, Shewanella oneidensis, Synechocystis sp., and Pelotomaculum thermopropionicum, produced electrically conductive piluslike filaments. Because P. thermopropionicum uses these electrically conductive filaments to coaggregate with methanogens, it is suggested that P. thermopropionicum possibly mediates interspecies electron transfer with methanogens not only via protons but also via the filaments. 48. Logan BE, Regan JM: Electricity-producing bacterial communities in microbial fuel cells. Trends Microbiol 2006, 14:512-518. This paper reviews current knowledge of the microbial communities found in microbial fuel cells. 49. Hatamoto M, Imachi H, Ohashi A, Harada H: Identification and cultivation of anaerobic, syntrophic long-chain fatty acid degrading microbes from mesophilic and thermophilic methanogenic sludges. Appl Environ Microbiol 2007, 73:1332-1340. 50. Huang WE, Griffiths RI, Thompson IP, Bailey MJ, Whiteley AS: Raman microscopic analysis of single microbial cells. Anal Chem 2004, 76:4452-4458. 51. Derito CM, Pumphrey GM, Madsen EL: Use of field-based stable isotope probing to identify adapted populations and track carbon flow through a phenol-degrading soil microbial community. Appl Environ Microbiol 2005, 71:7858-7865.
42. Lueders T, Pommerenke B, Friedrich MW: Stable-isotope probing of microorganisms thriving at thermodynamic limits: Syntrophic propionate oxidation in flooded soil. Appl Environ Microbiol 2004, 70:5778-5786.
52. Wexler M, Bond PL, Richardson DJ, Johnston AWB: A wide hostrange metagenomic library from a waste water treatment plant yields a novel alcohol/aldehyde dehydrogenase. Environ Microbiol 2005, 7:1917-1926.
43. Chauhan A, Ogram A: Fatty acid-oxidizing consortia along a nutrient gradient in the Florida Everglades. Appl Environ Microbiol 2006, 72:2400-2406. DNA-based stable isotope proving was employed to investigate the fate of carbon from propionate and butyrate in freshwater marshes.
53. Logan BE, Hamelers B, Rozendal R, Schrorder U, Keller J, Freguia S, Aelterman P, Verstraete W, Rabaey K: Microbial fuel cells: methodology and technology. Environ Sci Technol 2006, 40:5181-5192.
44. Kosaka T, Uchiyama T, Ishii S, Enoki M, Imachi H, Kamagata Y, Ohashi A, Harada H, Ikenaga H, Watanabe K: Reconstruction and regulation of the central catabolic pathway in the thermophilic propionate-oxidizing syntroph Pelotomaculum thermopropionicum. J Bacteriol 2006, 188:202-210. This paper discusses the whole picture of the possible central catabolic pathway of Pelotomaculum thermopropionicum based on draft genome sequencing. 45. Ishii S, Kosaka T, Hori K, Hotta Y, Watanabe K: Coaggregation facilitates interspecies hydrogen transfer between Pelotomaculum thermopropionicum and Methanothermobacter thermautotrophicus. Appl Environ Microbiol 2005, 71:7838-7845. On the basis of beautiful scanning electron microscopic images, the authors suggested that the thermophilic propionate-oxidizing bacterium Pelotomaculum thermopropionicum and the hydrogenotrophic
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54. Lovley DR: Microbial fuel cells: novel microbial physiologies and engineering approaches. Curr Opin Biotechnol 2006, 17:327-332. 55. Bond DR, Lovley DR: Electricity production by Geobacter sulfurreducens attached to electrodes. Appl Environ Microbiol 2003, 69:1548-1555. 56. Chaudhuri SK, Lovley DR: Electricity generation by direct oxidation of glucose in mediatorless microbial fuel cells. Nat Biotechnol 2003, 21:1229-1232. 57. Rabaey K, Boon N, Siciliano SD, Verhaege M, Verstraete W: Biofuel cells select for microbial consortia that self-mediate electron transfer. Appl Environ Microbiol 2004, 70:5373-5382. 58. Logan BE: Simultaneous wastewater treatment and biological electricity generation. Water Sci Technol 2005, 52:31-37.
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Current Opinion in Biotechnology 2007, 18:273–278 This review comes from a themed issue on Environmental biotechnology Edited by Eliora Z Ron and Philip Hugenholtz Available online 25th April 2007 0958-1669/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2007.04.003
Introduction In theory, anaerobic digestion technology is an ideal biological means for the removal of organic pollutants in waste and wastewater. The technology has two significant advantages over the conventional aerobic biological treatment: firstly, it is cost-effective because aeration is not required and a small amount of excess sludge is produced, and secondly, it generally produces gaseous methane as an energy resource [1,2]. The largest application of this digestion technology is the stabilization of sludge, such as a sludge digester commonly used at municipal wastewater treatment plants. The technology has also become popular in dilute and concentrated wastestream treatment fields; anaerobic digestion technology for wastewater treatment can now also be considered a matured biotechnology. Owing to the development of sophisticated wastewater treatment technologies (such as upflow anaerobic sludge blanket [UASB] technology), more than 1500 anaerobic www.sciencedirect.com
wastestream treatment processes are now installed and operating worldwide [3,4]. So far, the most practical target of this technology is high-rate treatment of high-strength industrial organic wastewater [1,4]. The types of wastewater that can be treated by such technology have recently been expanded notably; for example, low-strength organic wastewater, complex wastewater containing persistent chemical compounds, and wastewater discharged at temperatures ranging from psychrophilic (4 8C to 20 8C) to thermophilic (above 45 8C) [2,5]. In addition, the technology has also been shown to offer interesting potentials for metal removal and recovery with sulfate reduction, removal of nitrates with nitrification, and bioremediation for breakdown of toxic substances. One of the most advanced fields associated with the technology in the past few years is the microbiology of anaerobic digestion processes. Because knowledge of the ecology and function of the microbial community in these processes is required to better control the biological processes, considerable effort has been made to understand the microbial community structure by using culture-dependent and culture-independent molecular approaches [2,6,7]. Through these analyses, particularly those targeting the 16S rRNA gene, detailed pictures of the community compositions are being documented. In addition, several functionally important anaerobes, playing key roles in the treatment process, have been cultivated and characterized. Furthermore, the ecophysiology of yet-to-be cultured organisms in the ecosystems is being elucidated using a variety of approaches. In this review, we focus on microbiological aspects of anaerobic (mainly methanogenic) microbial communities for high-rate organic waste and wastewater treatment, and update the recent findings in this field. We highlight a variety of recent approaches in microbiological fields, including microbial community analysis, the domestication of uncultured microorganisms, and ecophysiological analysis of yet-to-be cultured anaerobes that are relevant to the technology. In addition, we raise the topic of microbial fuel cells, an alternative, direct energyyielding wastewater treatment system, and describe recent findings on the microbiological aspects.
Community analysis of anaerobic digestion processes: the uncultured, yet-to-be characterized lineages In anaerobic treatment processes, there has been a relatively limited number of studies conducting 16S rRNA gene cloning-based analyses of the microbial community Current Opinion in Biotechnology 2007, 18:273–278
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(often known as ‘sludge’) [2,6,7]. However, constituents of more than 20 bacterial phyla have been detected in anaerobic (mostly methanogenic) waste and wastewater sludges [2,6,7]. For example, 16S rRNA gene clones that were frequently and commonly retrieved from these sludges were distributed in various prokaryotic taxa such as the phyla Proteobacteria (mainly in the class Deltaproteobacteria), Chloroflexi, Firmicutes, Spirochaetes, and Bacteroidetes in the domain Bacteria. Similarly, clones in the classes Methanomicrobia, Methanobacteria, and Thermoplasmata in the domain Archaea are those of typical phylotypes found in such sludges [6]. In addition to these relatively known taxa, phylotypes belonging to a variety of uncultured candidate phyla (or classes) (known as ‘clone cluster’) were often detected in these sludges [6,7,8, 9,10,11,12]. Within the domain Bacteria, diverse uncultivated taxonomic groups, such as OP10, BA024, OP8, TM6, EM3, OP3, and OS-K, were detected in these sludges [6] (note that most of the candidate taxa in this review are named according to the article by Hugenholtz [13]). Finding key (or dominant) populations that belong to such uncultured lineages at various taxonomic levels (from species to phylum levels) is one of the major advances in the microbiology of anaerobic digestion processes in the past few years. One recent finding in the predominant populations of the clone clusters is that of the candidate bacterial phylum WWE1. Chouari et al. [10] described that 81% of all the bacterial 16S rRNA gene clones retrieved from sludge from an anaerobic mesophilic digester were assigned with the group WWE1. Fluorescence in situ hybridization (FISH) analysis revealed that WWE1-type cells had rod and filamentous morphotypes, and the rRNA from WWE1 cells accounted for 12% of the total bacterial rRNA [10]. Although the ecophysiological function of WWE1-type bacteria remains unknown, their high abundance in the sludge suggests they might play a certain role in the digestion process. Another example of the predominant populations that belong to clone clusters is that of the bacterial phylum Deferribacteres. The phylum Deferribacteres (formerly recognized as the phylum ‘Synergistes’) contained phenotypically diverse genera such as Deferribacter, Denitrovorans, Geovibrio, Flexistipes and ‘Synergistes’ [14]. Within the phylum, several 16S rRNA gene clones that are distantly related to known cultivated species, forming distinct clone clusters at the subphylum (class) levels, have also been retrieved from anaerobic (methanogenic) sludges. The distribution of members of this phylum was thoroughly explored in a total of 93 anaerobic ecosystems, such as anaerobic digesters, soil and gut samples, indicating that Deferribacteres-type clones were widely distributed in such anaerobic ecosystems [11]. The clones retrieved from methanogenic digester sludges were assigned to four different subphyla of Deferribacteres, none Current Opinion in Biotechnology 2007, 18:273–278
of which contains cultured representatives. More recently, Diaz et al. [15] showed that 34% of all the bacterial clones detected from a full-scale methanogenic UASB process treating brewery wastewater were affiliated with uncultured clades of the phylum Deferribacteres. Given their high occurrence in such methanogenic ecosystems, they might play a role in part of the food web for the methanogenic degradation of organic compounds; however, their ecophysiology remains unknown. With respect to the uncultured archaeal lineages, archaeal 16S rRNA gene clones affiliated with the candidate taxon WSA2 were retrieved in abundance from a mesophilic methanogenic digester decomposing sewage sludge [9] (this candidate taxon is also named according to the review by Hugenholtz [13], although Chouari et al. [9] refer to the taxon as ‘ArcI’ group in their report). This group is a clone cluster at the subphylum (or class) level within the archaeal phylum Euryarchaeota, and phylotypes of WSA2 were sometimes detected in other anaerobic treatment processes [6]. Chouari et al. [9] also obtained highly enriched WSA2 cells using formate- or hydrogen-containing media, implying that WSA2-related microorganisms are methanogens. Another unique, uncultured archaeal taxon that is also often found in methanogenic sludges is subphylum C2 of the archaeal phylum Crenarchaeota. For example, 16% of the archaeal rRNA gene clones analyzed from a mesophilic methanogenic digester were found to belong to members of Crenarchaeota, particularly the subphylum C2 [9]. C2-type rRNA gene clones were also detected from a full-scale, methanogenic (partially sulfidogenic) UASB process treating paper mill wastewater [16] and a laboratory-scale UASB reactor treating methanethiol [17]. Collins et al. [8] recently surveyed C2-type phylotypes in anaerobic sludges from a variety of methanogenic wastewater treatment systems, detecting these phylotypes in abundance (14–78% of the total archaeal clones analyzed). From FISH observations in thin-sections of methanogenic sludge granules, cells of the C2-type phylotypes were found to be rods (1.5 mm in length and 0.7 mm in width), forming dense microcolonies within the sections. Interestingly, they were juxtaposed to Methanosaeta cells [8]. These findings may imply that they are active members of the sludge ecosystems, interacting with aceticlastic methanogens in situ. Further studies are needed to clearly describe the ecophysiological functions of these unique Archaea.
Community changes within anaerobic digestion processes Recent microbiological studies focus not only on the description of a microbial community at a particular operational time, but also on the community shift (population dynamics) of anaerobic sludge along with different operational periods (or conditions). www.sciencedirect.com
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Such population dynamics of resident microbes in anaerobic treatment processes have also been analyzed by using rRNA and rRNA-gene based methods. For example, Diaz et al. [15] and Zheng et al. [18] studied the community changes along with the maturation of sludge granules in UASB reactors using rRNA gene-based denaturing gradient gel electrophoresis (DGGE) analysis and FISH. Similarly, Hori et al. [19] analyzed changes in the microbial community succession in a thermophilic methanogenic reactor under deteriorative and stable conditions, which were induced by acidification and neutralization, using rRNA gene-based single-strand conformation polymorphism (SSCP), quantitative PCR, and FISH. The results indicated that the methanogenic community in the process was significantly affected by volatile fatty acid concentrations.
Newly isolated microorganisms from anaerobic treatment processes The domestication (cultivation) of uncultured organisms is of great interest in this field to gain insights into the function of these organisms. In the past few years, newly discovered microorganisms have been successfully isolated from anaerobic sludges, and the information regarding their physiology in conjugation with phylogeny is updated regularly: examples include carbohydrate degraders [20,21], a protein degrader [22], fatty acid oxidizers [23,24,25–32], terephthalate oxidizers [33], and methanogens [34–36]. One of the most significant advances in this field is the isolation of organisms from subphylum I of the bacterial phylum Chloroflexi. The subphylum I was recognized as a clone cluster, members of which were frequently detected in anaerobic environments in abundance [6,15,16,37,38]. Recently, four filamentous strains that belong to the Chloroflexi subphylum I were successfully isolated and cultured from anaerobic wastewater treatment sludges, and these strains were characterized in detail to give their taxonomic placements [20,21]. At present, the subphylum contains two thermophilic species of the genus Anaerolinea, one mesophilic species of the genus Levilinea, and one mesophilic species of the genus Leptolinea, and the subphylum was named as the new class Anaerolineae. These organisms are known to be one of the major populations in mesophilic and thermophilic sludge granules of UASB reactors [37,38]. One species (Anaerolinea thermophila) is associated with filamentous bulking of methanogenic granular sludge [37]. All strains possess filamentous morphotypes, growing fermentatively with a range of carbohydrates and yeast extract as substrates. These findings suggest that members of the class might play a key role in the primary degradation of carbohydrates and cellular materials (such as amino acids) in methanogenic digestion processes. Several important proton-reducing syntrophic bacteria affiliated with the group ‘Desulfotomaculum cluster I’ have www.sciencedirect.com
been isolated and cultured from anaerobic wastewater processes. Imachi et al. [23,39] first isolated Pelotomaculum thermopropionicum from a laboratory-scale UASB reactor operated under thermophilic conditions. This bacterium can degrade propionate in co-culture with hydrogenotrophic methanogens [23]. Later, the obligately syntrophic, propionate-oxidizing bacterium Pelotomaculum schinkii [24], a mesophilic, syntrophic propionate-oxidizing strain MGP [40], two mesophilic, syntrophic phthalatedegrading species Pelotomaculum terephthalicum and Pelotomaculum isophthalicum [33,41], and a mesophilic, syntrophic benzoate-oxidizing species Sporotomaculum syntrophicum [25] were also isolated from anaerobic wastewater treatment processes. Interestingly, all these syntrophs lack the ability to dissimilatory reduce sulfate, although other members of the ‘Desulfotomaculum cluster I’ are known to be sulfate reducers. From a set of various molecular and cultivation-based analyses for non-sulfate-reducing Desulfotomaculum-type organisms, it was hypothesized that these microorganisms have recently adopted a syntrophic lifestyle to thrive in low-sulfate, methanogenic environments and thus have lost their ancestral ability for dissimilatory sulfate/sulfite reduction [40]. Importantly, by using DNA-based stable isotope probing (SIP) analyses members of the genus Pelotomaculum were found to be a key contributor to the syntrophic propionate degradation in natural anaerobic ecosystems (e.g. flooded soil and freshwater marshes) [42,43]. Among the characterized species of these syntrophic organisms of the ‘Desulfotomaculum cluster I’, P. thermopropionicum has been intensively studied in recent years. Kosaka et al. [44] analyzed the genome of P. thermopropionicum and proposed a novel propionate-oxidizing pathway (methylmalomyl coenzyme A pathway), which differs from that of the previously characterized pathway in other bacteria. Together with proteomic data, they assumed that fumarase plays a central role in the regulation of syntrophic propionate degradation. Additionally, it was reported that the cells of P. thermopropionicum coaggregated when they were co-cultivated with Methanothermobacter thermautotrophicus cells in the presence of propionate [45,46]. Interestingly, the flagellum-like filaments of P. thermopropionicum participate in coaggregation of each cell [45], suggesting that these filaments might have a role in keeping the two organisms closely juxtaposed with each other for efficient interspecies hydrogen transfer. More recently, the flagellum-like filaments of P. thermopropionicum were found to be electrically conductive, suggesting the possibility of direct exocellular electron transfer through the flagellum-like filaments (this topic is further discussed below) [47,48] Although the number of descriptions of these new anaerobes is increasing, there is still much work to be done to domesticate the yet-to-be cultured microbes that are recalcitrant to artificial cultivation. To avoid difficulties Current Opinion in Biotechnology 2007, 18:273–278
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in cultivation, several techniques such as FISH combined with microautoradiography (MAR–FISH), DNA-based stable isotope probing (SIP) and RNA-SIP have been developed and applied to anaerobic sludges [49]. Visualizing substrate uptake pattern within the methanogenic granule sections with new techniques, such as radioactive tracer technique plus b imaging [8], SIP-Raman microscopy [50] and SIP-secondary ion mass spectrometry (SIMS) [51], might also help to point the way to the cultivation of uncultured organisms. Collins et al. [8] attempted to apply the radioactive tracer technique and b imaging to sludge granules to speculate the function of uncultured Crenarchaeota cells (mentioned above). Furthermore, metagenomic approaches for microbial consortia containing yet-to-be cultured microbial cells have recently been applied to a wide range of environments to gain new insight into the potential metabolic activities of predominant microbes based on gene information. For example, the report by Wexler et al. [52] is, to our knowledge, the first attempt at employing a metagenomic approach for anaerobic digester sludges.
Recent issue: microbial fuel cells revisited Microbial fuel cells (MFC) have recently received much attention for their potential to directly recover electricity as an energy resource from waste and wastewater [48, 53,54]. Interest in the MFC process originally began in the 1960s, when it was discovered that resident microorganisms in the process oxidize the organic substances in waste and wastewater as electron donors and transfer electrons to the anode electrode via soluble electron mediators such as ferricyanide. However, the addition of such mediators was considered infeasible for actual electron recovery because of their toxicity and instability in a prolonged operation of the MFC process [53,55,56]. Recently, Bond and Lovley [55] and Chaudhuri and Lovley [56] showed that Geobacter sulfurreducens and Rhodoferax ferrireducens, respectively, directly transfer electrons to the anode surface via components associated with their cell wall; these findings again shed light on the MFC process research. The electron transfer efficiency from acetate to electricity was found to be 96.8% with G. sulfurreducens cells, whereas the electron transfer efficiency from glucose was reported to be 81% with R. ferrireducens cells. Several attempts to produce electricity with a complex microbial community have also been reported [54,57,58]. For example, Rabaey et al. [57] reported that the MFC process enriched with an anaerobic granular sludge achieved 81% efficiency for electron transfer from glucose. Most recently, it is reported that electrically conductive pilus-like filaments (they called ‘bacterial nanowires’) of the metal-reducing bacterium Shewanella oneidensis mediated exocellular electron transfer to metal surface [47]. As mentioned above, a similar finding was made when the syntrophic propionate-oxidizer P. thermopropionicum was cultivated Current Opinion in Biotechnology 2007, 18:273–278
in pure culture with fumarate or in co-culture with M. thermautotrophicus with propionate [47]. This suggests the possibility of direct exocellular electron transfer from the syntrophic microbe through such electrically conductive, flagellum-like filaments [47,48].
Conclusions As described in this short update, significant advances have been made in elucidating the diversity of yet-to-be cultured organisms in anaerobic (methanogenic) digestion processes. Although some important anaerobes have been cultivated and characterized, there is still work to be done for a vast number of remaining anaerobes that have not yet been cultured. For the remainder, further characterization of their ecophysiological traits with cultivation or with much more elegant methods (such as SIP) is needed. The accumulation of such information will offer substantial information for more sophisticated management of the digestion technology. Recent attention to the potential of the MFC process to directly recover electricity as an energy resource from waste and wastewater could suggest a future direction of the technological development of the anaerobic digestion process. Further investigations of the MFC process will be necessary to improve the process, as well as to fully understand the role of the microbial community contributing the generation of electricity.
Acknowledgements This study was supported by the Project ‘Development of Technologies for Analyzing and Controlling the Mechanism of Biodegrading and Processing’, which was ensured to the New Energy and Industrial Technology Development Organization (NEDO).
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31. Sekiguchi Y, Imachi H, Susilorukmi A, Muramatsu M, Ohashi A, Harada H, Hanada S, Kamagata Y: Tepidanaerobacter syntrophicus gen. nov., sp. nov., an anaerobic, moderately thermophilic, syntrophic alcohol- and lactate-degrading bacterium isolated from thermophilic digested sludges. Int J Syst Evol Microbiol 2006, 56:1621-1629.
17. de Bok FA, van Leerdam RC, Lomans BP, Smidt H, Lens PN, Janssen AJ, Stams AJ: Degradation of methanethiol by methylotrophic methanogenic archaea in a lab-scale upflow anaerobic sludge-blanket reactor. Appl Environ Microbiol 2006, 72:7540-7547. 18. Zheng D, Angenent LT, Raskin L: Monitoring granule formation in anaerobic upflow bioreactors using oligonucleotide hybridization probes. Biotechnol Bioeng 2006, 94:458-472. 19. Hori T, Haruta S, Ueno Y, Ishii M, Igarashi Y: Dynamic transition of a methanogenic population in response to the concentration of volatile fatty acids in a thermophilic anaerobic digester. Appl Environ Microbiol 2006, 72:1623-1630. 20. Sekiguchi Y, Yamada T, Hanada S, Ohashi A, Harada H, Kamagata Y: Anaerolinea thermophila gen. nov., sp. nov and Caldilinea aerophila gen. nov., sp. nov., novel filamentous thermophiles that represent a previously uncultured lineage of the domain Bacteria at the subphylum level.. Int J Syst Evol Microbiol 2003, 53:1843-1851. www.sciencedirect.com
32. Kendall MM, Liu Y, Boone DR: Butyrate- and propionatedegrading syntrophs from permanently cold marine sediments in Skan Bay, Alaska, and description of Algorimarina butyrica gen. nov., sp. nov.. FEMS Microbiol Lett 2006, 262:107-114. 33. Qiu YL, Sekiguchi Y, Hanada S, Imachi H, Tseng IC, Cheng SS, Ohashi A, Harada H, Kamagata Y: Pelotomaculum terephthalicum sp. nov. and Pelotomaculum isophthalicum sp. nov.: two anaerobic bacteria that degrade phthalate isomers in syntrophic association with hydrogenotrophic methanogens. Arch Microbiol 2006, 185:172-182. 34. Ma K, Liu XL, Dong XZ: Methanobacterium beijingense sp. nov., a. novel methanogen isolated from anaerobic digesters.. Int J Syst Evol Microbiol 2005, 55:325-329. 35. Ma K, Liu XL, Dong XZ: Methanosaeta harundinacea sp. nov., a novel acetate-scavenging methanogen isolated from a UASB reactor.. Int J Syst Evol Microbiol 2006, 56:127-131. Current Opinion in Biotechnology 2007, 18:273–278
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36. Jiang B, Parshina SN, van Doesburg W, Lomans BP, Stams AJM: Methanomethylovorans thermophila sp. nov., a thermophilic, methylotrophic methanogen from an anaerobic reactor fed with methanol.. Int J Syst Evol Microbiol 2005, 55:2465-2470. 37. Sekiguchi Y, Takahashi H, Kamagata Y, Ohashi A, Harada H: In situ detection, isolation, and physiological properties of a thin filamentous microorganism abundant in methanogenic granular sludges: a novel isolate affiliated with a clone cluster, the green non-sulfur bacteria, subdivision I. Appl Environ Microbiol 2001, 67:5740-5749. 38. Yamada T, Sekiguchi Y, Imachi H, Kamagata Y, Ohashi A, Harada H: Diversity, localization, and physiological properties of filamentous microbes belonging to Chloroflexi subphylum I in mesophilic and thermophilic methanogenic sludge granules. Appl Environ Microbiol 2005, 71:7493-7503. 39. Imachi H, Sekiguchi Y, Kamagata Y, Ohashi A, Harada H: Cultivation and in situ detection of a thermophilic bacterium capable of oxidizing propionate in syntrophic association with hydrogenotrophic methanogens in a thermophilic methanogenic granular sludge. Appl Environ Microbiol 2000, 66:3608-3615. 40. Imachi H, Sekiguchi Y, Kamagata Y, Loy A, Qiu YL, Hugenholtz P, Kimura N, Wagner M, Ohashi A: Harada H: Non-sulfatereducing, syntrophic bacteria affiliated with Desulfotomaculum cluster I are widely distributed in methanogenic environments. Appl Environ Microbiol 2006, 72:2080-2091. In this paper, the occurrence and abundance of ‘Desulfotomaculum cluster I’ in low-sulfate, methanogenic environments were analyzed using 16S rRNA-based molecular approaches, and the new strain MGP was successfully isolated in co-culture with a hydrogenotrophic methanogen. Interestingly, strain MGP could not dissimilatory reduce sulfur compounds, but the strain contained (and expressed) dsrAB, key genes in the sulfate respiration. 41. Qiu YL, Sekiguchi Y, Imachi H, Kamagata Y, Tseng IC, Cheng SS, Ohashi A, Harada H: Identification and isolation of anaerobic, syntrophic phthalate isomer-degrading microbes from methanogenic sludges treating wastewater from terephthalate manufacturing. Appl Environ Microbiol 2004, 70:1617-1626.
methanogen Methanothermobacter thermautotrophicus coaggregated via flagellum-like filaments when they grow in co-culture with propionate. 46. Ishii S, Kosaka T, Hotta Y, Watanabe K: Simulating the contribution of coaggregation to interspecies hydrogen fluxes in syntrophic methanogenic consortia. Appl Environ Microbiol 2006, 72:5093-5096. This paper describes a simple model for simulating the contribution of coaggregation to interspecies hydrogen transfer fluxes between syntrophic bacteria and methanogens. 47. Gorby YA, Yanina S, McLean JS, Rosso KM, Moyles D, Dohnalkova A, Beveridge TJ, Chang IS, Kim BH, Kim KS et al.: Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms. Proc Natl Acad Sci USA 2006, 103:11358-11363. Using scanning tunneling microscopy, the authors report that three bacterial species, Shewanella oneidensis, Synechocystis sp., and Pelotomaculum thermopropionicum, produced electrically conductive piluslike filaments. Because P. thermopropionicum uses these electrically conductive filaments to coaggregate with methanogens, it is suggested that P. thermopropionicum possibly mediates interspecies electron transfer with methanogens not only via protons but also via the filaments. 48. Logan BE, Regan JM: Electricity-producing bacterial communities in microbial fuel cells. Trends Microbiol 2006, 14:512-518. This paper reviews current knowledge of the microbial communities found in microbial fuel cells. 49. Hatamoto M, Imachi H, Ohashi A, Harada H: Identification and cultivation of anaerobic, syntrophic long-chain fatty acid degrading microbes from mesophilic and thermophilic methanogenic sludges. Appl Environ Microbiol 2007, 73:1332-1340. 50. Huang WE, Griffiths RI, Thompson IP, Bailey MJ, Whiteley AS: Raman microscopic analysis of single microbial cells. Anal Chem 2004, 76:4452-4458. 51. Derito CM, Pumphrey GM, Madsen EL: Use of field-based stable isotope probing to identify adapted populations and track carbon flow through a phenol-degrading soil microbial community. Appl Environ Microbiol 2005, 71:7858-7865.
42. Lueders T, Pommerenke B, Friedrich MW: Stable-isotope probing of microorganisms thriving at thermodynamic limits: Syntrophic propionate oxidation in flooded soil. Appl Environ Microbiol 2004, 70:5778-5786.
52. Wexler M, Bond PL, Richardson DJ, Johnston AWB: A wide hostrange metagenomic library from a waste water treatment plant yields a novel alcohol/aldehyde dehydrogenase. Environ Microbiol 2005, 7:1917-1926.
43. Chauhan A, Ogram A: Fatty acid-oxidizing consortia along a nutrient gradient in the Florida Everglades. Appl Environ Microbiol 2006, 72:2400-2406. DNA-based stable isotope proving was employed to investigate the fate of carbon from propionate and butyrate in freshwater marshes.
53. Logan BE, Hamelers B, Rozendal R, Schrorder U, Keller J, Freguia S, Aelterman P, Verstraete W, Rabaey K: Microbial fuel cells: methodology and technology. Environ Sci Technol 2006, 40:5181-5192.
44. Kosaka T, Uchiyama T, Ishii S, Enoki M, Imachi H, Kamagata Y, Ohashi A, Harada H, Ikenaga H, Watanabe K: Reconstruction and regulation of the central catabolic pathway in the thermophilic propionate-oxidizing syntroph Pelotomaculum thermopropionicum. J Bacteriol 2006, 188:202-210. This paper discusses the whole picture of the possible central catabolic pathway of Pelotomaculum thermopropionicum based on draft genome sequencing. 45. Ishii S, Kosaka T, Hori K, Hotta Y, Watanabe K: Coaggregation facilitates interspecies hydrogen transfer between Pelotomaculum thermopropionicum and Methanothermobacter thermautotrophicus. Appl Environ Microbiol 2005, 71:7838-7845. On the basis of beautiful scanning electron microscopic images, the authors suggested that the thermophilic propionate-oxidizing bacterium Pelotomaculum thermopropionicum and the hydrogenotrophic
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54. Lovley DR: Microbial fuel cells: novel microbial physiologies and engineering approaches. Curr Opin Biotechnol 2006, 17:327-332. 55. Bond DR, Lovley DR: Electricity production by Geobacter sulfurreducens attached to electrodes. Appl Environ Microbiol 2003, 69:1548-1555. 56. Chaudhuri SK, Lovley DR: Electricity generation by direct oxidation of glucose in mediatorless microbial fuel cells. Nat Biotechnol 2003, 21:1229-1232. 57. Rabaey K, Boon N, Siciliano SD, Verhaege M, Verstraete W: Biofuel cells select for microbial consortia that self-mediate electron transfer. Appl Environ Microbiol 2004, 70:5373-5382. 58. Logan BE: Simultaneous wastewater treatment and biological electricity generation. Water Sci Technol 2005, 52:31-37.
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