1. INTRODUCTION
3.3. Results & discussion
3.3.3. Microbial community analyses
Archaeal and bacterial community structures were analyzed at a steady state in each operational phase by DGGE. The obtained molecular fingerprints are shown in Fig. 3-3. A total of four archaeal bands (CA1 to 4) and ten bacterial bands (CB1 to 10) of interest were excised for sequencing analysis and phylogenetic affiliation (Table 3-4). All archaeal sequences were classified into two methanogen orders, Methanomicrobiales (CA1 and 4) and Methanosarcinales (CA2 and 3), by the RDP Classifier (bootstrap cutoff, 80%). CA1 and 4 were closely related (≥97% sequence similarity) to hydrogenotrophic Methanospirillum and Methanolinea, respectively, while CA2 and 3 were affiliated with the obligate aceticlastic Methanosaetaceae family. This implies that both hydrogen- and acetate-utilizing methanogenic pathways likely developed in the reactors. Given that two Methanosaetaceae-related bands, CA2 and 3, appeared as the dominant bands across all lanes, aceticlastic methanogenesis was probably the major route for methane production throughout the experiment in both reactors. Methanosaetaceae species reportedly predominates in stable AD environments where the concentration of residual VFAs is low (below ca. 1 mM acetate) (Speece, 1996). The prevailing occurrence of Methanosaetaceae-related methanogens accords with the fact that the residual VFAs were generally maintained at low levels during the experimental phases except for Phases 1B and 2E (Fig. 3-2). Although R1 suffered from a serious build-up of VFAs
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during Phase 1B, its performance was restored after five turnovers and then sampled for molecular fingerprinting. This may be why CA2 and 3 still occur as the dominant bands in the DGGE lane for Phase 1B. Meanwhile, their intensity visibly declined in the lane for Phase 2E, where the reactor failed with a continuous build-up of VFAs.
The retrieved bacterial sequences were classified across five phyla, Actinobacteria (CB10), Bacteroidetes (CB2, 3 and 4), Chloroflexi (CB9), Firmicutes (CB5 and 8), and Proteobacteria (CB6 and 7), except for CB1, which was unclassified even at the phylum level. CB1 was closely related to an uncultured clone from an anaerobic fixed-bed reactor treating terephthalic acid wastewater (Li et al., 2014). This band appeared as an apparent band during the phases fed with substrate containing whey but was not visible in Phases 1E and 2A (100% Ulva). Although not clarified, this suggests that the corresponding population to CB1 might be involved in fermenting an easily degradable organic fraction of whey. Only two bacterial sequences (CB2 and 4) were closely related to known species. CB2 was assigned to the genus Parabacteroides and closely related to both Parabacteroides chartae and Macellibacteroides fermentans with 99.6% similarity. This is due to the high rRNA gene sequence homology between M. fermentans and P. chartae strains, which hinders their differentiation by 16S rRNA gene sequencing (Kitahara et al., 2013). Members of these species ferment various sugars to organic acids, such as acetate, butyrate, and lactate (Jabari et al., 2012a; Tan et al., 2012), and the population corresponding to CB2 was possibly responsible for degrading sugars and polysaccharides. CB4 was affiliated with the genus Meniscus originally isolated from AD sludge and closely related to Meniscus glaucopis (Irgens, 1977). This species is able to produce organic acids, including acetate and propionate, by fermenting sugars but is unable to utilize starch or cellulose. Therefore, the bacterium deduced from CB4 likely played a role in the acidogenesis of simple sugars rather than the hydrolysis of polysaccharides.
CB5, 6, and 7, although not closely related to any published species, were classified at the genus level by the RDP Classifier. CB5 was affiliated with the genus Saccharofermentans and showed a 96.4% similarity to Saccharofermentans acetigenes. Members of this fermentative genus utilize various saccharides to produce organic acids, primarily acetate and lactate (Chen et al., 2010).
The corresponding population to this band likely contributed to the acidogenic fermentation of substrate carbohydrates. CB6 and CB7 were assigned to Syntrophobacter and Smithella, respectively, which are both syntrophic propionate degraders that oxidize propionate to acetate and H2/CO2 in consortium with H2 scavengers like hydrogenotrophic methanogens (de Bok et al., 2004).
The bacteria corresponding to CB6 and 7 were probably involved in syntrophic propionate
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metabolism in the reactors. Interspecies hydrogen transfer between VFA degraders and hydrogenotrophic methanogens is essential for effective stabilization of VFAs. This syntrophy plays a key role in preventing process imbalance and restoring an imbalanced process in AD systems (Phase 1B). It seems that, in the experimental reactors, Syntrophobacter (CB6)- and Smithella (CB7)-related bacteria and Methanospirillum (CA1)- and Methanolinea (CA4)-related methanogens formed a syntrophic consortium responsible for the stabilization of VFAs, particularly propionate.
This corresponds to the sudden emergence of CA1 after the restoration of stable VFA levels in Phase 1B. Given the COD removal and YM, although hydrogen-utilizing SRBs could also contribute, hydrogenotrophic methanogenesis was likely the major hydrogen-scavenging process in both reactors (Table 3-3).
The remaining bacterial sequences (CB3, 8, 9, and 10) were classified only at the order or phylum level. Although the functions of their corresponding populations are unclear, the band sequences were all assigned to bacterial groups commonly abundant in AD environments and closely related to uncultured clones from anaerobic digesters (refer to their closest relatives in Table 3). CB9 and 10, which were assigned to the phylum Chloroflexi and the order Actinomycetales, respectively, appeared when the Ulva proportion in the mixed substrate was less than 75% (Phases 1A to 1C and 2C to 2E). The appearance of the putative, filamentous bacteria can be related to the low concentration of residual NH4+ in these phases (<90 mg/L; Table 3-3). Due to the high surface- to-volume ratio, filamentous bacteria can take advantage of high substrate affinity and better cope with nutrient-limited conditions (Liu & Liu, 2006). This is in line with the appearance of CB9 and 10 at a higher intensity in phases with lower NH4+ concentrations.
49 Figure 3-3. Archaeal (A) and bacterial (B) 16S rRNA gene DGGE
profiles. Each lane is labeled with the corresponding experimental phase.
Figure 3-4 Cluster dendrogram constructed based on the bacterial DGGE profiles. Each community profile is labeled with the corresponding experimental phase.
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Table 3-4. Phylogenetic affiliation of archaeal and bacterial 16S rRNA gene sequences retrieved from DGGE
Band Closest relative Accession no. Similarity (%) Classificationa
Archaea
CA1 Methanospirillum hungatei strain JF-1 NR074177 99.6 Methanospirillum
CA2 Methanosaeta concilii strain X16932 KM408635 100.0 Methanosaetaceae
Methanosaeta concilii GP-6 CP002565 100.0
CA3 Uncultured archaeon clone UPA1 KR081310 99.6 Methanosaetaceae
CA4 Methanolinea tarda strain NOBI-1 NR028163 97.4 Methanomicrobiales
Bacteria
CB1 Uncultured bacterium clone UAFB_TA_43_14 KJ476606 98.9 Bacteria
CB2 Bacteroides sp. BDNS3 FJ188412 99.8 Parabacteroides
Macellibacteroides fermentans strain LIND7H NR117913 99.6
CB3 Bacterium Oil-Tsu-11 gene AB081544 96.9 Bacteroidetes
Clostridium sp. 6-44 gene AB596885 94.3
CB4 Meniscus glaucopis strain ATCC 29398 NR104837 98.1 Meniscus
Uncultured bacterium gene LCFA-B02 AB244309 97.6
CB5 Saccharofermentans acetigenes strain P6 NR115340 96.4 Saccharofermentans
CB6 Uncultured Deltaproteobacteria bacterium clone QEDR2D804 CU922160 100.0 Syntrophobacter
Uncultured bacterium clone SGE34G GU390032 100.0
CB7 Syntrophus sp. clone B3 AJ133796 97.2 Smithella
Smithella propionica strain LYP NR024989 96.7
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CB8 Uncultured Firmicutes bacterium clone 2D2 HQ003638 100.0 Firmicutes
Uncultured Firmicutes bacterium gene B18 AB780905 100.0
CB9 Uncultured bacterium clone SSOTU85 HM346756 99.8 Chloroflexi
Uncultured bacterium gene Down11 AB795489 96.8
CB10 Uncultured actinobacterium gene RSg 13-9 AB603826 99.8 Actinomycetales
Uncultured bacterium clone Eb48 EF063623 99.8
a The lowest rank assigned by the RDP Classifier at a bootstrap confidence threshold of 80%.
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