Microbial Community Analysis of Anaerobic Reactors Treating Soft Drink Wastewater

<div><p>The anaerobic packed-bed (AP) and hybrid packed-bed (HP) reactors containing methanogenic microbial consortia were applied to treat synthetic soft drink wastewater, which contains polyethylene glycol (PEG) and fructose as the primary constituents. The AP and HP reactors achieved high COD removal efficiency (>95%) after 80 and 33 days of the operation, respectively, and operated stably over 2 years. 16S rRNA gene pyrotag analyses on a total of 25 biofilm samples generated 98,057 reads, which were clustered into 2,882 operational taxonomic units (OTUs). Both AP and HP communities were predominated by <i>Bacteroidetes</i>, <i>Chloroflexi</i>, <i>Firmicutes</i>, and candidate phylum KSB3 that may degrade organic compound in wastewater treatment processes. Other OTUs related to uncharacterized <i>Geobacter</i> and <i>Spirochaetes</i> clades and candidate phylum GN04 were also detected at high abundance; however, their relationship to wastewater treatment has remained unclear. In particular, KSB3, GN04, <i>Bacteroidetes</i>, and <i>Chloroflexi</i> are consistently associated with the organic loading rate (OLR) increase to 1.5 g COD/L-d. Interestingly, KSB3 and GN04 dramatically decrease in both reactors after further OLR increase to 2.0 g COD/L-d. These results indicate that OLR strongly influences microbial community composition. This suggests that specific uncultivated taxa may take central roles in COD removal from soft drink wastewater depending on OLR.</p></div

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<p>AP, anaerobic packed-bed reactor; HP, hybrid packed-bed reactor; COD: chemical oxygen demand; HRT, hydraulic retention time; OLR, organic loading rate; N.D. not determined.</p><p>Operational parameter of anaerobic packed-bed (AP) and hybrid packed-bed (HP) reactors.</p

Principal coordinate analysis based on the abundances of 16S rRNA gene OTUs (unweighted UniFrac).

<p>For this analysis, observed 16S rRNA gene OTUs were normalized to 1,400 reads per sample. A and H indicate the samples taken from the anaerobic packed-bed (AP) and hybrid packed-bed (HP) reactors. The numbers following A and H indicate days of the operation for biomass sampling.</p

Distance matrix tree of 16S rRNA gene sequences assigned to the <i>Geobacter</i> retrieved from anaerobic reactors based on the neighbor-joining method.

<p>Boldface indicates the sequences obtained in this study. The 16S rRNA gene sequences of <i>Thermodesulfobacterium commune</i> DSM 2178 (AF418169), <i>Thermodesulfobacterium hveragerdense</i> DSM 12571 (NR_029311), and <i>Thermodesulfobacterium hydrogeniphilum</i> DSM 14290 (NR_025146) were used as outgroup. The bar indicates 10% base substitution. Branching points supported probabilities >95%, >75%, and >50% by bootstrap analyses (based on 1,000 replicates) are indicated by solid circle, open circles, and open square, respectively.</p

Bubble plot indicates the relative abundances of major OTUs retrieved from anaerobic packed-bed (AP) and hybrid packed-bed (HP) reactors.

<p>The numbers below AP and HP on top row indicate days of the operation for biomass sampling.</p

Distance matrix tree of 16S rRNA gene sequences assigned to the candidate phyla GN04 and KSB3 retrieved from anaerobic reactors based on the neighbor-joining method.

<p>Boldface indicates the sequences obtained in this study. The 16S rRNA gene sequences of <i>Methanosaeta harundinacea</i> 8Ac (AY817738), <i>Methanosaeta pelagica</i> 03d30q (AB679167), <i>Methanosaeta concilii</i> opfikon (X51423) were used as outgroup. The bar indicates 10% base substitution. Branching points supported probabilities >95%, >75%, and >50% by bootstrap analyses (based on 1,000 replicates) are indicated by solid circle, open circles, and open square, respectively.</p