Phosphorus removal from fermented dairy manure concurrent with polyhydroxybutyrate-co-valerate synthesis under aerobic conditions.

Wastewater phosphorus removal achieved biologically is associated with the process known as enhanced biological phosphorus removal (EBPR). In contrast with canonical EBPR operations that employ alternating anaerobic-aerobic conditions and achieve asynchronous carbon and phosphorus storage, research herein focused on phosphorus removal achieved under aerobic conditions synchronously with volatile fatty acid (VFA) storage as polyhydroxybutyrate-co-valerate (PHBV). 90.3 ± 3.4 % soluble phosphorus removal was achieved from dairy manure fermenter liquor; influent and effluent concentrations were 38.6 ± 9.5 and 3.7 ± 0.8 mgP/L, respectively. Concurrently, PHBV yield ranged from 0.17 to 0.64 mgCOD/mgCOD, yielding 147-535 mgCODPHBV/L. No evidence of EBPR mechanisms was observed, nor were canonical phosphorus accumulating organisms present; additionally, the polyphosphate kinase gene was not present in the microbial biomass. Phosphorus removal was primarily associated with biomass growth and secondarily with biomass complexation. Results demonstrate that concurrent PHBV synthesis and phosphorus recovery can be achieved microbially under aerobic dynamic feeding conditions when fed nutrient rich wastewater.

Interrogating the performance and microbial ecology of an enhanced biological phosphorus removal/post-anoxic denitrification process at bench and pilot scales.

Research focused on interrogating post-anoxic enhanced biological phosphorus removal (EBPR) at bench and pilot scales. Average bench-scale effluent ranged from 0.33 to 1.4 mgP/L, 0.35 to 3.7 mgNH3 -N/L, and 1.1 to 3.9 mgNOx -N/L. Comparatively, the pilot achieved effluent (50th percentile/average) of 0.13/0.2 mgP/L, 9.7/8.2 mgNH3 -N/L, and 0.38/3.3 mgNOx -N/L under dynamic influent and environmental conditions. For EBPR process monitoring, P:C ratio data indicated that 0.2-0.4 molP/molC will result in stable EBPR; relatedly, a target design influent volatile fatty acid (VFA):P ratio would exceed 15 mgCOD/mgP. Post-anoxic EBPR was enriched for Nitrobacter spp. at 1.70%-20.27%, with Parcubacteria also dominating; the former is putatively associated with nitritation and the latter is a putative fermenting heterotrophic organism. Post-anoxic specific denitrification rates (SDNRs) (20°C) ranged from 0.70 to 3.10 mgN/gVSS/h; there was a strong correlation (R2 = 0.94) between the SDNR and %Parcubacteria for systems operated at a 20-day solids residence time (SRT). These results suggest that carbon substrate potentially generated by this putative fermenter may enhance post-anoxic EBPR. PRACTITIONER POINTS: Post-anoxic EBPR can achieve effluent of <0.2 mgP/L and <12 mgN/L. The P:C and VFA:P ratios can be predictive for EBPR process monitoring. Post-anoxic EBPR was enriched for Nitrobacter spp. over Nitrospira spp. and also for Parcubacteria, which is a putative fermenting heterotrophic organism. Post-anoxic specific denitrification rates (20°C) ranged from 0.70 to 3.10 mgN/gVSS/h. BLASTn analysis of 16S rDNA PAO primer set was shown to be improved to 93.8% for Ca. Accumulibacter phosphatis and 73.2%-94.0% for all potential PAOs.

Interrogating nitritation at a molecular level: Understanding the potential influence of Nitrobacter spp.

Water resource recovery facilities (WRRFs) increasingly must maximize nitrogen and phosphorus removal, but concurrently face challenges to reduce their energy usage and environmental footprint. In particular, biological nutrient removal (BNR), which targets removal of phosphorus and nitrogen, exhibits a large energy demand. However, a BNR process achieving partial oxidation of NH3 to NO2 (nitritation) could reduce energy demands, with secondary environmental emission benefits. Research was conducted on bench-scale systems performing nitritation and nitrification to better understand how mixed microbial consortia, cultured on real wastewater, can sustain nitritation. BNR configurations achieved nitrite accumulation ratios of 64-82%, with excellent overall effluent quality. Applying phylogenetic, transcriptomic, and metabolomic methods, coupled with process monitoring, results indicate that partial nitritation may be induced through a combination of: (1) Employing ammonia-based aeration control, with an ammonia setpoint of 2, 3 mgN/L; (2) Maintaining an aerobic period DO of 1.0-2.0 mg/L; and (3) Operating BNR post-anoxically, integrated within enhanced biological phosphorus removal (EBPR). Significant nitritation was achieved despite the presence Nitrobacter spp., but nitrite oxidoreductase must be functionally impaired or structurally incomplete. Overall, this research demonstrated the value of interrogating a mixed microbial consortia at a macro and molecular level to explore unique metabolic responses such as nitritation.

Effects of anaerobic HRT and VFA loading on the kinetics and stoichiometry of enhanced biological phosphorus removal.

Enhanced biological phosphorus removal (EBPR) can recover significant quantities of wastewater phosphorus. However, this resource recovery process realizes limited use largely due to process stability concerns. The research evaluated the effects of anaerobic HRT (τAN ) and VFA concentration-critical operational parameters that can be externally controlled-on EBPR performance. Evaluated alone, τAN (1-4 h) exhibited no statistical effect on effluent phosphorus. However, PHA increased with VFA loading and biomass accumulated more phosphorus. Regarding resiliency, under increasing VFA loads PAOs hydrolyzed more phosphorus to uptake/catabolize VFAs; moreover, PHA synthesis normalized to VFA loading increased with τAN , suggesting fermentation. Kinetically, PAOs exhibited a Monod-like relationships for qPHAAN and qVFAAN as a function of anaerobic P release; additionally, qPAE exhibited a Monod-like relationship with end-anaerobic PHA concentration. A culminating analysis affirmed the relationship between enhanced aerobic P uptake, and net P removal, with a parameter (phosphorus removal propensity factor) that combines influent VFA concentration with τAN . PRACTITIONER POINTS: Evaluated alone τAN exhibits no statistical effect on effluent phosphorus in an EBPR configuration. Increased PHA synthesis, associated with increased VFAs and/or extended τAN, enhances aerobic phosphorus removal. PHA synthesis normalized to VFA loading increased with τAN , suggesting fermentation in the EBPR anaerobic zone. Aerobic phosphorus uptake increases linearly with anaerobic phosphorus release, with the slope exceeding unity. Increased VFAs can be substituted for shorter anaerobic HRTs, and vice versa, to enhance EBPR performance.

Assessing the Effects of RAS Fermentation on EBPR Performance and Associated Microbial Ecology.

Enhanced biological phosphorus removal (EBPR) is an engineered water resource recovery facility (WRRF) process configuration that can produce effluent P < 0.5 mg/L. To consistently achieve low effluent P concentrations, EBPR requires volatile fatty acids (VFAs) to induce requisite biochemical reactions. Moreover, returned activated sludge (RAS) nitrate concentrations must be minimized. Returned activated sludge fermentation can potentially address process needs. However, research detailed herein highlights concerns with RAS fermentation integrated with EBPR. Under 2 and 4 hours of RAS fermentation periods, no consequential VFA production was observed; similar results were observed in batch tests with RAS from a full-scale EBPR WRRF. More critically, EBPR performance was poor, with average effluent concentrations of 1.0 to 2.4 mg/L. Furthermore, the glycogen accumulating organism (GAO) fraction under RAS fermentation was 4.3 to 8.7 times higher than in a conventional EBPR mixed microbial consortium (MMC). Integrated RAS fermentation-EBPR only performed well under "high" RAS nitrate; thus, should RAS fermentation be implemented, careful control to prevent anaerobic conditions in the fermentation zone is required.

Characterizing and contrasting the microbial ecology of laboratory and full-scale EBPR systems cultured on synthetic and real wastewaters.

The anthropogenic discharge of phosphorus (P) into surface waters can induce the proliferation of cyanobacteria and algae, which can negatively impact water quality. Enhanced biological P removal (EBPR) is an engineered process that can be employed to efficiently remove significant quantities of P from wastewater. Within this engineered system, the mixed microbial consortium (MMC) becomes enriched with polyphosphate accumulating organisms (PAOs). To date much knowledge has been developed on PAOs, and the EBPR process is generally well understood; nonetheless, the engineered process remains underutilized. In this study, investigations were conducted using qPCR and Illumina MiSeq to assess the impacts of wastewater (synthetic vs. real) on EBPR microbial ecology. While a strong relationship was demonstrated between EBPR metrics (P:C; influent VFA:P) and excellent P removal across diverse EBPR systems and MMCs, no such correlations existed with the specific MMCs. Moreover, MMCs exhibited distinct clusters based on substrate, and qPCR results based on the putative PAO Accumulibacter did not correlate with BLASTN eubacterial results for either Accumulibacter or Rhodocyclaceae. More critically, PAO-based sequences aligned poorly with Accumulibacter for both eubacterial and PAO primer sets, which strongly suggests that the conventional PAO primers applied in FISH and qPCR analysis do not sufficiently target the putative PAO Accumulibacter. In particular, negligible alignment was observed for PAO amplicons obtained from a MMC performing excellent EBPR on crude glycerol (an atypical substrate). A synthetic wastewater-based MMC exhibited the best observed BLASTN match of the PAO amplicons, raising concerns about the potential relevance in using synthetic substrates in the study of EBPR.

Polyhydroxyalkanoate synthesis by mixed microbial consortia cultured on fermented dairy manure: Effect of aeration on process rates/yields and the associated microbial ecology.

Polyhydroxyalkanoates (PHAs) are biodegradable polymers that can substitute for petroleum-based plastics in a variety of applications. One avenue to commercial PHA production involves coupling waste-based synthesis with the use of mixed microbial consortia (MMC). In this regard, production requires maximizing the enrichment of a MMC capable of feast-famine PHA synthesis, with the metabolic response induced through imposition of aerobic-dynamic feeding (ADF) conditions. However, the concept of PHA production in complex matrices remains unrefined; process operational improvements are needed, along with an enhanced understanding of the MMC. Research presented herein investigated the effect of aeration on feast-famine PHA synthesis, with four independent aeration state systems studied; MMC were fed volatile fatty acid (VFA)-rich fermented dairy manure. Regardless of the aeration state, all MMC exhibited a feast-famine response based on observed carbon cycling. Moreover, there was no statistical difference in PHA synthesis rates, with qPHA ranging from 0.10 to 0.19 CmmolPHA gVSS-1 min-1; VFA uptake rates exhibited similar statistical indifferences. PHA production assessments on the enriched MMC resulted in maximum intracellular concentrations ranging from 22.5 to 90.7% (mgPHA mgVSS-1); at maximum concentration, the mean hydroxyvalerate mol content was 73 ± 0.6%. While a typical feast-famine dissolved oxygen (DO) pattern was observed at maximum aeration, less resolution was observed at decreasing aeration rates, suggesting that DO may not be an optimal process monitoring parameter. At lower aeration states, nitrogen cycling patterns, supported by molecular investigations targeting AOBs and NOBs, indicate that NO2 and NO3 sustained feast-famine PHA synthesis. Next-generation sequencing analysis of the respective MMC revealed numerous and diverse genera exhibiting the potential to achieve PHA synthesis, suggesting functional redundancy embedded in the diverse MMC. Ultimately, results demonstrate that aeration can be controlled in waste-based ADF systems to sustain PHA production potential, while enriching for a diverse MMC that exhibits potential functional redundancy. Reduced aeration could also enhance cost competitiveness of waste-based PHA production, with potential further benefits associated with nitrogen treatment.

Dairy manure resource recovery utilizing two-stage anaerobic digestion - Implications of solids fractionation.

Dairy manure management is increasingly becoming an environmental challenge. In this regard, manure anaerobic digestion (AD) can be applied to address environmental concerns; however, dairy manure AD remains economically uncompetitive. Ongoing research is focused on enhanced resource recovery from manure, including maximizing AD methane yield through a novel multi-stage AD configuration. Research presented herein centered on the hypothesis that separately digesting fine and coarse solids from fermented dairy manure would improve methane production; the hypothesis was disproven. While maximum methane concentration was realized on fine solids, combined solids AD yielded enhanced VS destruction. The diverse combined-solids substrate enriched for a more heterogeneous bacterial/archaeal consortium that balanced fermentation and methanogenesis to yield maximum product (methane). However, results suggest that targeted AD of the fat-rich fine solids could be a more optimal approach for processing manure; alternate (non-AD) methods could then be applied to extract value from the fibrous fraction.

EBPR using crude glycerol: assessing process resiliency and exploring metabolic anomalies.

Enhanced biological phosphorus removal (EBPR) is predicated on exposing bacteria to cyclical anaerobic/aerobic environments while providing volatile fatty acids (VFAs). Combined, this environment enriches for phosphorus accumulating organisms (PAOs) and induces metabolisms to ensure excess phosphorus removal. Crude glycerol (CG), a byproduct of biodiesel manufacturing, is an alternate waste stream that could be substituted to achieve excess phosphorus removal; research into the use of CG yielded unexpected findings. While CG was an excellent substrate to accomplish and/or help achieve excess phosphorus removal, CG-fed bacteria did not consistently exhibit theoretical EBPR metabolisms. Specifically, anaerobic phosphorus release was not required for successful EBPR; however, carbon cycling patterns were consistent with theory. Analysis of results suggests that PAOs will first leverage carbon to generate energy anaerobically; only as needed will the bacteria utilize polyphosphate reserves anaerobically. Results also demonstrated that excess phosphorus removal can be achieved with a small fraction of PAOs.

Methane production on thickened, pre-fermented manure.

Over 9 million dairy cows generate an estimated 226 billion kg of wet manure annually in the US. The purpose of this study was to demonstrate the viability of a novel two-stage anaerobic digestion (AD) process for producing methane-rich biogas on pre-fermented dairy manure. In summary, it was observed that AD of thickened pre-fermented manure can generate comparable biogas quantities to AD using raw manure, with enhanced methane content. Despite receiving a lower quality (i.e., partially biodegraded) substrate, biogas stoichiometry and overall process stability in the two-stage system was also comparable to AD receiving raw manure. Finally, the two-stage AD was more enriched with the acetoclastic methanogen Methanosarcinaceae (Msc; compared to AD of raw manure) and biogas production appeared closely linked with the Msc fraction. In fact, the enhanced enrichment of Msc likely contributed to the successful and stable operations.

Advancing post-anoxic denitrification for biological nutrient removal.

The objective of this research was to advance a fundamental understanding of a unique post-anoxic denitrification process for achieving biological nutrient removal (BNR), with an emphasis on elucidating the impacts of surface oxygen transfer (SOT), variable process loadings, and bioreactor operational conditions on nitrogen and phosphorus removal. Two sequencing batch reactors (SBRs) were operated in an anaerobic/aerobic/anoxic mode for over 250 days and fed real municipal wastewater. One SBR was operated with a headspace open to the atmosphere, while the other had a covered liquid surface to prevent surface oxygen transfer. Process performance was assessed for mixed volatile fatty acid (VFA) and acetate-dominated substrate, as well as daily/seasonal variance in influent phosphorus and ammonia loadings. Results demonstrated that post-anoxic BNR can achieve near-complete (>99%) inorganic nitrogen and phosphorus removal, with soluble effluent concentrations less than 1.0 mgN L(-1) and 0.14 mgP L(-1). Observed specific denitrification rates were in excess of typical endogenous values and exhibited a linear dependence on the glycogen concentration in the biomass. Preventing SOT improved nitrogen removal but had little impact on phosphorus removal under normal loading conditions. However, during periods of low influent ammonia, the covered reactor maintained phosphorus removal performance and showed a greater relative abundance of polyphosphate accumulating organisms (PAOs) as evidenced by quantitative real-time PCR (qPCR). While GAOs were detected in both reactors under all operational conditions, BNR performance was not adversely impacted. Finally, secondary phosphorus release during the post-anoxic period was minimal and only occurred if nitrate/nitrite were depleted post-anoxically.

Effect of anaerobic HRT on biological phosphorus removal and the enrichment of phosphorus accumulating organisms.

The purpose of this research was to develop a better understanding of the dynamic effects of anaerobic hydraulic retention time (HRT) on both enhanced biological phosphorus removal (EBPR) performance and enrichment of phosphorus accumulating organisms (PAOs). The research was conducted using laboratory-scale sequencing batch reactors inoculated with mixed microbial consortia and fed real wastewater. Exposing microorganisms to extended anaerobic HRTs is not recommended for EBPR configured systems. In this research, however, longer anaerobic exposure did not negatively affect performance even if volatile fatty acids were depleted. Further, extended anaerobic HRTs may positively affect phosphorus removal through enhanced aerobic uptake. The EBPR consortia also appear to maintain reserve energetic capacity in the form of polyphosphate that can be used to survive and grow under variable operational and environmental conditions. Finally, the tested EBPR systems yield mixed microbial consortia enriched with PAOs (specifically Candidatus Accumulibacter phosphatis) at approximately 7.1 to 21.6% of the total population.