Detection, phylogeny and population dynamics of syntrophic propionate - oxidizing bacteria in anaerobic granular sludge

The research described this thesis concerns the diversity and phylogeny of syntrophic propionate-oxidizing bacteria and their ecology in granular sludge, from which they were obtained. 16S rRNA was used as a molecular marker to study both the phylogeny and the ecology of these bacteria. Sequence analysis of the 16S rRNA gave information on the phylogeny of the syntrophic bacteria, while specific oligonucleotide probes based on these sequences enabled quantification and detection of these bacteria. In situ hybridization techniques were applied to study the architecture of granular sludge. Granular sludge was also enriched for propionate-oxidizing bacteria and studied with dot blot and in situ hybridization to combine their presence in the sludge to the physiological processes observed.In this chapter the results are summarized and discussed in a broader context, starting with the diversity of the syntrophic propionate-oxidizing bacteria. Subsequently, the application of the ribosomal approach for the detection of syntrophs in the sludge is discussed. Next, the ecology and the localization of the microbes in granular sludge fed with different substrates is presented in models of sludge architecture, and finally the main conclusions of the research are presented.Diversity of syntrophic propionate-oxidizing bacteria. For more than one decade Syntrophobacter wolinii was the only well-described syntrophic propionateoxidizing bacterium (Boone and Bryant, 1980). Although other syntrophic propionateoxidizing cocultures were enriched, no other bacterium could be isolated (Koch et al., 1983; Boone and Xun 1987; Mucha et al., 1988; Stains et al., 1992).It was initially assumed that S.wolinii was an obligately syntrophic bacterium, which only used propionate as carbon source, and only grew in coculture with H 2-and/or formate-utilizing microbes such as Desulfovibrio G11 or Methanospirillum hungatei (Boone and Bryant, 1980). However, at the beginning of the nineties, when the research of this thesis was initiated, this view changed. Stams et al. (1993) enriched a syntrophic propionate oxidizer on malate, which could grow in pure culture on fumarate. This bacterium, strain MPOB, later named Syntrophobacter fumaroxidans (Chapter 8), could oxidize propionate using fumarate as alternative electron acceptor. Meanwhile, Schink and coworkers, succeeded to grow S.wolinii in the absence of H 2-and/or formate-utilizing microbes on propionate and sulfate, and on pyruvate (Dörner, 1992; Wallrabenstein et al., 1994). These findings showed that these syntrophs were not obligately syntrophic, but could be grown in pure culture with other carbon sources as well. The phylogenetic analysis of the 16S rRNA of the syntrophs showed that S. wolinii, S. fumaroxidans and the later isolated Syntrophobacter pfennigii, are not only closely related to each other, but are related to Gram-negative sulfate-reducing bacteria (Chapters 2 and 3). Moreover, they are sulfate-reducing bacteria themselves, as all three Syntrophobacter spp. could oxidize propionate by the reduction of sulfate.The increasing knowledge of this group of syntrophs may lead to the isolation of more related syntrophic propionate-oxidizing species. However, there are presently arguments that syntrophy is not a physiological characteristic of this phylogenetic group. Two sulfate-reducing bacteria have been isolated that were related to the genus Syntrophobacter, but which did not use propionate syntrophically under the conditions tested. These include, Desulforhabdus amnigenus, isolated by the growth on acetate (Oude Elferink et al., 1995), and Desulfoacinicum infernum, a thermophilic bacterium isolated on lactate from a petroleum resevoir (Rees et al., 1995). 16S rRNA sequence analysis revealed a close and a moderate relation, respectively of D. amnigenus and D. infernum, with the genus Syntrophobacter, and both most likely share a common ancestor with the latter genus. Whether these bacteria lost the ability to grow syntrophically or that Syntrophobacter gained this ability, remains unclear.16S rRNA analysis of two other cultures enriched from granular sludge showed that syntrophic propionate-oxidation was not restricted to the genus Syntrophobacter. The SYN7 bacterium was not related to Syntrophobacter, but moderately related to the syntrophic benzoate oxidizing genus Syntrophus (Wallrabenstein et al., 1995a) (Chapter 6). Furthermore, the sporeforming syntrophic propionate-oxidizing bacteria SporeA and SporeB (Chapter 4), are related to Gram-positive sulfate-reducing genus Desulfotomaculum. Although widely spread throughout the domain of Bacteria, the syntrophic propionate oxidizers that are phylogenetically analyzed so far have one feature in common, they are related to sulfate reducers. It is even possible that they are all sulfate reducers. Such a linkage between syntrophic propionate oxidation and sulfate reduction may originate from common enzymatic pathways, such as the methyl-malonyl CoA pathway, present in the members of the genus Syntrophobacter (Houwen et al., 1990; Stams et al., 1993). Also Desulfobulbus possess such a pathway (Stams et al. 1984), but so far, has not been shown to grow syntrophically on propionate. It is interesting to find out why Desulfobulbus spp. can not oxidize propionate syntrophically, while Syntrophobacter spp. are able to do so. Furthermore, other sulfate-reducing bacteria need to be tested or tested more extensively for the capacity to grow syntrophically on propionate, for instance Desulforhabdus amnigenus or members of the genus Desulfotomaculum.The purification of strain MPOB, resulting in the description of Syntrophobacter fumaroxidans sp . nov., allowed for the testing of growth on many carbon sources in the presence or absence of sulfate. Preliminary experiments indicated that this S.fumaroxidans strain could also grow, although after a long lag-phase, on alcohols and amino acids by the reduction of sulfate. As a consequence it is quite a versatile sulfate reducer and resembles other sulfate reducers in their growth on many different substrates (Widdel and Bak, 1992). However, in methanogenic ecosystems such as granular sludge, propionate appears to be the only substrate available for S.fumaroxidans. This bacterium seems to be specialized in the syntrophic oxidation of propionate, and this feature may be of advantage over other sulfate-reducing bacteria present in methanogenic granular sludge.1 6S rRNA sequence analysis of cocultures and enrichment cultures. Phylogenetic analyses of syntrophic propionate-oxidizing bacteria were performed with mixed cultures. The cultures from which the 16S rRNA genes were obtained, had an increasing complexity. The first culture was a defined coculture consisting of Syntrophobacter wolinii and Desulfovibrio G11 (Boone and Bryant, 1980). Following the analysis of the 16S rRNA gene of Desulfovibrio G11, it was obvious that the other 16S rRNA gene obtained from the culture had to be derived from S.wolinii (Chapter 2). This was not the case for the phylogenetic analysis of the highly purified, but not defined cultures MPOB and KOPROP1, later named S. fumaroxidans and S.pfennigii, respectively (Chapter 8; Wallrabenstein et al., 1995b). Although no other 16S rRNA genes were cloned, it had to be shown that the sequences obtained really came from the dominant syntrophic bacteria. The required evidence was given by in situ hybridization with specific oligonucleotide probes (Chapter 3). Because of the higher complexity of the cultures, more elaborate work was needed for the phylogenetic analysis of the dominant syntrophic propionate oxidizers in the less defined enrichments described in the Chapters 4 and 6. In the case of SYN7, several 16S rRNA genes were cloned and characterized, but the SYN7 sequence was the most abundant one. In situ hybridization with SYN7-specific probes showed that this sequence was indeed abundantly present in the culture, next to sequences that hybridized with a Desulfobulbus- specific probe. However, these Desulfobulbus -like 16S rRNA sequences were not present in the clone library, probably caused by preferential amplification or cloning of the 16S rRNA-genes. This shows that this method of amplification and cloning of 16S rRNA genes may cause biases and should always be followed by feedback methods like in situ hybridization. In the case of the sporeforming syntrophs, only one bacterial morphology was observed, while at least two 16S rRNA sequences were derived from the culture, which were derived form bacteria indicated as SporeA and SporeB. In situ hybridizations with specific probes for the two sequences were performed to investigate from which bacteria these sequences were derived. Unfortunately, this did not result in unambiguous fluorescent signals, probably because the Gram-positive cell wall could not be made penetrable for the probes with our methods. Therefore, it still can not be ruled out that both sequences were obtained from the same organism. However, dot blot hybridizations showed that only the probe specific for the SporeB sequence hybridized with another enrichment of propionate oxidizers by pasteurization, indicating that the sequences are present in separate organisms.Despite of these problems, the 16S rRNA sequence-analysis done on these enrichment cultures gave valuable information on the phylogenetic status of the dominant bacteria in the cultures, which may help in the isolation of these and other syntrophic propionate-oxidizing bacteria in the future. Furthermore, specific oligonucleotide probes were designed on the basis of these sequences, which have helped to study their presence and their dynamics in granular sludge as described below.Detection of syntrophic propionate-oxidizing bacteria in granular sludge. Fluorescently-labeled specific 16S rRNA-based oligonucleotide probes were used to detect the syntrophic propionate-oxidizing bacteria in anaerobic granular sludge.For this, two types of granular sludge were studied, (i) granular sludge that originated from an upflow anaerobic sludge bed (UASB) reactor treating sugarbeet-processing wastewater (Chapter 5) and (ii) sludge from an UASB reactor treating potatoprocessing wastewater (Chapters 6 and 7). S. wolinii and S. pfennigii were never detected in these two types of granular sludge, not even after enrichment on propionate. S. fumaroxidans -like bacteria were present in large numbers in sludge that originated from the sugarbeet wastewater-treating UASB-reactor. SYN7 was present in the potato-wastewater sludge, but could only be detected significantly after enrichment on propionate. Both S.fumaroxidans and SYN7 were only detected in the sludge from which they were originally enriched. From the two sporeforming syntrophic bacteria, just the SporeB sequence was found in potato-processing wastewater treating sludge, but only after pasteurization and enrichment on propionate .It seems that each type of sludge possesses its specific syntrophic propionateoxidizing bacterium, depending on the wastewater to be treated. This could be due to selection caused by the wastewater itself, or due to the different sources of inoculum used to start up the reactors, such as activated sludge or manure. To test this, granular sludge could be transferred from one reactor to another reactor that treats a different type of wastewater, and then follow the population dynamics of the syntrophs. Furthermore, the abundance of S. wolinii and S.pfennigii could be determined in the sludge from which they were isolated, and more importantly, it could be investigated if also other syntrophs are present in that sludge.Architecture of granular sludge. The architecture of the three investigated sludge types appeared to be dependent on the influent of the reactor. The granules obtained from the UASB reactor treating sugarbeet wastewater, fed with either sucrose or volatile fatty acids (VFA), and the granules from the UASB reactor treating potato wastewater, all had a layered structure in which both syntrophic and methanogenic bacteria were observed. However, there are marked differences in the architecture and number of layers between the three sludge types. Moreover, the abundance of the different trophic groups varied, and the organization of the syntrophic consortia and the microbes present were quite distinct. To illustrate these differences, models are designed that represent active granules of a moderate size. However, the centre of large granules often consisted of large cavities, probably due to substrate transport limitation, which cause lysis of the microbes (Alphenaar et al., 1993).The sludge fed with sucrose and VFA that originated from the UASB- reactor treating sugarbeet wastewater, had been operated with a defined influent for several months. This presumably led to a reduction of the diversity of the organisms in the sludge. Based on the results obtained by in situ hybridization (Chapter 5), the following schematic architecture of the sucrose-fed sludge is proposed. This sludge probably contains only four trophic groups of microorganisms, that form granules of a very simple architecture. Fermentative bacteria are present in the loosely bound outer layer and form propionate and acetate from sucrose. Inside the granule syntrophic microcolonies are present in which the syntrophic acetogens utilize the propionate to form acetate and hydrogen, and carbon dioxide and/or formate. These are then used by the Methanobacter- like methanogens also present in the syntrophic microcolonies. The acetate is utilized by Methanosaeta sp. microcolonies present adjacent to the syntrophic microcolonies. The results obtained from the VFA-grown sludge (Chapter 5) showed an architecture different from that of the sucrose fed sludge, which leads to the following model. The outer layer contains bacteria that are more inside the granule, underneath this layer there is a thick layer of mainly aceticlastic Methanosaeta sp., but also Methanosarcina microcolonies are seen in this layer. These latter methanogens have a lower affinity for acetate than Methanosaeta and will only by present if the concentrations are sufficiently high (Jetten et al., 1990). Underneath this layer there is a layer of syntrophic microcolonies and microcolonies of Methanosaeta sp. , degrading propionate and acetate, respectively. The results obtained from the in situ hybridizations of granular sludge of the UASB-reactor treating potato-processing wastewater (Chapter 7) was quite different to the granules described above, which leads to the proposal of the following model. This sludge was fed with industrial potato wastewater containing at least starch, lactate, butyrate, propionate and acetate as substrates. Therefore, the microbial community in the sludge is more diverse. This is clearly shown in the many different morphologies present in the thick outer layer of the sludge. Underneath the outer layer there is a layer present with small syntrophic microcolonies and Methanosaeta microcolonies and maybe other kinds of microcolonies. Remarkably, these microcolonies were all concentrically orientated. In situ hybridizations indicated that the microcolonies in the centre were less active than those between the centre and the outer layer, probably due to substrate limitation (Chapter 7). This suggests a development of the granule from the centre towards the outer layer.The diversity of the potato-starch grown granules was also shown by the enrichment on propionate and on propionate with sulfate, which selected for syntrophic and sulfidogenic. propionate-oxidizing bacteria, respectively (Chapter 7). This research showed that both trophic groups of bacteria were present in the inoculum sludge. The sulfidogenic propionate oxidizing Desulfobulbus sp. colonized the outer layers of the granules, while the syntrophic SYN7 bacteria formed active microcolonies inside the granule. The morphological diversity in both types of granules decreased dramatically, due to cultivation on one substrate. This was seen by the disappearance of the diverse outer layer, and by the decrease of bacterial rRNA in the dot blot hybridizations.The most interesting difference between the sludge that originated from the sugarbeet- wastewater UASB-reactor and the potato-wastewater treating reactor was the organisation and the types of microbes in the syntrophic microcolonies. The sludge grown on sucrose and VFA contained syntrophic microcolonies consisting of thick MPOB-like rods juxta-positioned with rods that hybridized with a Methanobacteriales -specific probe, morphologically resembling Methanobrevibacter cells. A similar morphological organization of syntrophic microcolonies was previously observed (MacLeod et al, 1990; Grotenhuis et al., 1991; Prensier et al., 1988; Wu et al., 1992). Methanobrevibacter only uses hydrogen as substrate, which has a low solubility in the water, and because every MPOB-like cell is surrounded by methanogens, interspecies hydrogen transfer might the mechanism by which the reducing equivalents are transferred. However, MacLeod et al. (1990) measured a high formate utilizing activity in sludge with syntrophic microcolonies containing Methanobrevibacter -like cells, although they did not measure hydrogen utilizing activity of the sludge. In contrast to the granules mentioned above, the granules treating potato-wastewater contained syntrophic microcolonies with densely packed small SYN7 rods that were intertwined with chains of cells morphologically resembling Methanospirillum hungatei. These cells hybridized with a probe specific for the genus Methanogenium and related organisms, including M. hungatei. This type of microcolonies have not been reported before. In the SYN7 microcolonies not every syntrophic acetogen is surrounded by methanogens. Since Methanospirillum can also utilize formate, it is possible that formate transfer might occur in these microcolonies.A layered structure of granules was proposed and shown in models by others (MacLeod et al., 1990; Macario et al., 1991). Macleod et al. (1990) suggested for sucrose-fed granules also the presence of four trophic groups of microbes organized as follows; a central core of Methanosaeta (Methanothrix) surrounded by a layer of hydrogen-producing acetogens and hydrogen-consuming methanogens, and an outer layer of fermentative bacteria. In the model described here the two inner layers are always mixed, and in the case of VFA grown granules Methanosaeta even form a layer around the syntrophic microcolonies. The earlier observation that only Methanosaeta cells were present in the central core (MacLeod et al, 1990) could be due to the size of the granules, so that only acetate is able to reach the central core. A general model for the architecture of granules converting organic matter based on previous models and the observations presented here suggests the presence of two active layers and a substrate-transport limited central core. In the outer layers mainly microorganisms are present which degrade the substrates in the surrounding medium or influent, independently from other microorganisms. The inner layers consist of microbes that degrade substrates which are produced by the primary layer, e.g fatty acids, acetate and hydrogen and/or formate, and which are dependent on each other for growth, such as the syntrophic consortia. The presence and size of the central core depends on the size of the granule. In large granules there will be a central core consisting of large cavities occupied by Methanosaeta sp. which degrade acetate, produced by the second layer as the terminal intermediate in the breakdown of organic matter. The methane which is produced throughout the granule will disappear from the granule through the numerous cavities and channels in the granule.Conclusions. The main conclusions of the research presented in this thesis are:i There are at least three phylogenetically different groups of syntrophic propionate- oxidizing bacteria which are all related to sulfate-reducing bacteria. The first group is the genus Syntrophobacter , consisting of the species S.wolinii S. pfennigii and S. fumaroxidans, which are closely related to each other and belong to the delta subclass of Proteo-bacteria. These bacteria were shown to be sulfate reducers themselves. The second group is related to the genus Syntrophus also belonging to the delta subclass. The third group are sporeforming syntrophic bacteria related to the Gram-positive genus Desulfotomaculum.ii Granular sludge originating from different sources treating various wastewaters contained specific syntrophic propionate-oxidizing subpopulation. Sludge originating from the UASB reactor treating sugarbeet-processing wastewater, mainly contained S. fumaroxidans as propionate-oxidizer, which are juxta-positioned with Methanobrevibacter -like cells in syntrophic microcolonies. Sludge from the UASB reactor treating potato-processing wastewater, contains SYN7-like bacteria growing in microcolonies which are intertwined with chains of Methanospirillum -like methanogens.iii The architecture of granular sludge and the presence of specific microbial subpopulations in this sludge is dependent on the substrates and electron acceptors available in the influent. Fermentative bacteria and sulfate-reducing acetogens are located in the outer layers of the granule, while syntrophic consortia and aceticlastic methanogens are located in the inner layers.