Cynthia K. Brinkman

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.

Should we build "obese" or "lean" anaerobic digesters?

Conventional anaerobic digesters (ADs) treating dairy manure are fed with raw or fermented manure rich in volatile fatty acids (VFAs). In contrast, pre-fermented AD (PF-AD) is fed with the more recalcitrant, fiber-rich fraction of manure that has been pre-fermented and depleted of VFAs. Thus, the substrate of PF-AD may be likened to a lean diet rich in fibers while the pre-fermentation stage fermenter is fed a relatively rich diet containing labile organic substances. Previous results have shown that conventional and pre-fermented ADs fed with raw or pre-fermented manure, respectively, produced comparable methane yields. The primary objective of this study was to characterize, using next-generation DNA sequencing, the bacterial communities in various bioreactors (pre-fermentation stage fermenter; various operational arrangements PF-AD; conventional single-stage AD; and a full scale AD) and compare the Firmicutes to Bacteroidetes (F/B) ratios in these different systems. Firmicutes and Bacteroidetes constituted the two most abundant phyla in all AD samples analyzed, as well as most of the samples analyzed in the fermenters and manure samples. Higher relative abundance of Bacteroidetes, ranging from 26% to 51% of bacteria, tended to be associated with PF-AD samples, while the highest relative abundance of Firmicutes occurred in the fermenter (maximum of 76% of bacteria) and manure (maximum of 66% of bacteria) samples. On average, primary stage fermenters exhibited microbiological traits linked to obesity: higher F/B ratios and a ‘diet’ that is less fibrous and more labile compared to that fed to PF-AD. On the other hand, microbial characteristics associated with leanness (lower F/B ratios combined with fibrous substrate) were associated with PF-AD. We propose that bacterial communities in AD shift depending on the quality of substrate, which ultimately results in maintaining VFA yields in PF-AD, similar to the role of bacterial communities and a high fiber diet in lean mice.

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.

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.

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.

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.