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We report, for the first time, extensive biologically mediated phosphate removal from wastewater during high-rate anaerobic digestion (AD). A hybrid sludge bed/fixed-film (packed pumice stone) reactor was employed for low-temperature (12°C) anaerobic treatment of synthetic sewage wastewater. Successful phosphate removal from the wastewater (up to 78% of influent phosphate) was observed, mediated by biofilms in the reactor. Scanning electron microscopy and energy dispersive X-ray analysis revealed the accumulation of elemental phosphorus (∼2%) within the sludge bed and fixed-film biofilms. 4′, 6-diamidino-2-phenylindole (DAPI) staining indicated phosphorus accumulation was biological in nature and mediated through the formation of intracellular inorganic polyphosphate (polyP) granules within these biofilms. DAPI staining further indicated that polyP accumulation was rarely associated with free cells. Efficient and consistent chemical oxygen demand (COD) removal was recorded, throughout the 732-day trial, at applied organic loading rates between 0.4 and 1.5 kg COD m-3 d-1 and hydraulic retention times of 8–24 h, while phosphate removal efficiency ranged from 28 to 78% on average per phase. Analysis of protein hydrolysis kinetics and the methanogenic activity profiles of the biomass revealed the development, at 12°C, of active hydrolytic and methanogenic populations. Temporal microbial changes were monitored using Illumina MiSeq analysis of bacterial and archaeal 16S rRNA gene sequences. The dominant bacterial phyla present in the biomass at the conclusion of the trial were the Proteobacteria and Firmicutes and the dominant archaeal genus was Methanosaeta. Trichococcus and Flavobacterium populations, previously associated with low temperature protein degradation, developed in the reactor biomass. The presence of previously characterized polyphosphate accumulating organisms (PAOs) such as Rhodocyclus, Chromatiales, Actinobacter, and Acinetobacter was recorded at low numbers. However, it is unknown as yet if these were responsible for the luxury polyP uptake observed in this system. The possibility of efficient phosphate removal and recovery from wastewater during AD would represent a major advance in the scope for widespread application of anaerobic wastewater treatment technologies.

The development and activity of a cold-adapting microbial community was monitored during low-temperature anaerobic digestion (LtAD) treatment of wastewater. Two replicate hybrid anaerobic sludge bed-fixed-film reactors treated a synthetic sewage wastewater at 12°C, at organic loading rates of 0.25–1.0 kg chemical oxygen demand (COD) m−3 d−1, over 889 days. The inoculum was obtained from a full-scale anaerobic digestion reactor, which was operated at 37°C. Both LtAD reactors readily degraded the influent with COD removal efficiencies regularly exceeding 78% for both the total and soluble COD fractions. The biomass from both reactors was sampled temporally and tested for activity against hydrolytic and methanogenic substrates at 12°C and 37°C. Data indicated that significantly enhanced low-temperature hydrolytic and methanogenic activity developed in both systems. For example, the hydrolysis rate constant (k) at 12°C had increased 20–30-fold by comparison to the inoculum by day 500. Substrate affinity also increased for hydrolytic substrates at low temperature. Next generation sequencing demonstrated that a shift in a community structure occurred over the trial, involving a 1-log-fold change in 25 SEQS (OTU-free approach) from the inoculum. Microbial community structure changes and process performance were replicable in the LtAD reactors.

No abstract

High-rate anaerobic digestion (AD) is a reliable, efficient process to treat wastewaters and is often operated at temperatures exceeding 30 • C, involving energy consumption of biogas in temperate regions, where wastewaters are often discharged at variable temperatures generally below 20 • C. High-rate ambient temperature AD, without temperature control, is an economically attractive alternative that has been proven to be feasible at laboratory-scale. In this study, an ambient temperature pilot scale anaerobic reactor (2 m 3 ) was employed to treat real dairy wastewater in situ at a milk processing plant, at organic loading rates of 1.3 ± 0.6 to 10.6 ± 3.7 kg COD/m 3 /day and hydraulic retention times (HRT) ranging from 36 to 6 h. Consistent high levels of COD removal efficiencies, ranging from 50 to 70% for total COD removal and 70 to 84% for soluble COD removal, were achieved during the trial. Within the reactor biomass, stable active archaeal populations were observed, consisting mainly of Methanothrix (previously Methanosaeta) species, which represented up to 47% of the relative abundant active species in the reactor. The decrease in HRT, combined with increases in the loading rate had a clear effect on shaping the structure and composition of the bacterial fraction of the microbial community, however, without affecting reactor performance. On the other hand, perturbances in influent pH had a strong impact, especially when pH went higher than 8.5, inducing shifts in the microbial community composition and, in some cases, affecting negatively the performance of the reactor in terms of COD removal and biogas methane content. For example, the main pH shock led to a drop in the methane content to 15%, COD removals decreased to 0%, while the archaeal population decreased to ∼11% both at DNA and cDNA levels. Functional redundancy in the microbial community underpinned stable reactor performance and rapid reactor recovery after perturbations.