Bioaugmentation and Correlating Anaerobic Digester Microbial Community to Process Function

This dissertation describes two research projects on anaerobic digestion (AD) that investigated the relationship between microbial community structure and digester function. Both archaeal and bacterial communities were characterized using high-throughput (Illumina) sequencing technology with universal 16S rRNA gene primers. In the first project, bioaugmentation using a methanogenic, aerotolerant propionate enrichment culture was investigated as a possible method to increase digester methane production. Nine anaerobic digesters, seeded with different biomass, were operated identically and their quasi steady state function was compared. Before bioaugmentation, different seed biomass resulted in different quasi steady state function, with digesters clustering into high, medium or low methane (CH4) production groups. High CH4 production correlated with neutral pH and high Methanosarcina abundance, whereas low CH4 production correlated to low pH and high Methanobacterium and DHVEG-6 family abundance. After bioaugmentation, CH4 production from the high CH4-producing digesters transiently increased by 11±3% relative to non-bioaugmented controls (p \u3c0.05, n=3), whereas no functional changes were observed for medium and low CH4 producing digesters. The CH4 production increase after bioaugmentation was correlated to increased relative abundance of Methanosaeta and Methanospirillum originating from the bioaugment culture. In conclusion, different anaerobic digester seed biomass can result in different quasi steady state function. The bioaugmentation employed can result in a period of increased methane production. In the second project, a quantitative structure activity relationship (QSAR) between anaerobic microorganism relative abundance values and digester methane production rate was developed using 150 lab-scale anaerobic digesters seeded with 50 biomass samples obtained from 49 US states. Although all digesters were operated identically for a minimum of 5 retention cycles, their quasi steady-state performance varied significantly, with the average daily methane production rate ranging from 0.09±0.004 to 0.98±0.05 L-CH4/LR-day (average ± standard deviation). Analysis of over 4.1 million-sequence reads revealed approximately 1300 operation taxonomical units (OTUs) at the genus level across all digesters, with each digester having 158±27 OTUs (mean ± standard deviation). Using Spearman’s rank correlation, 10 OTUs, which included one archaeal OTU, were found to significantly correlate to digester methane production rate. The relative abundance values of the 10 OTUs were used as descriptors to develop a MLR equation, with good statistical prediction of the digester methane production rates. The results are encouraging and provide an initial step for further research to develop more robust QSAR models to predict the function of anaerobic and other bioprocesses using microbial community descriptors

Anaerobic Digester Bioaugmentation Influences Quasi Steady State Performance and Microbial Community

Nine anaerobic digesters, each seeded with biomass from a different source, were operated identically and their quasi steady state function was compared. Subsequently, digesters were bioaugmented with a methanogenic culture previously shown to increase specific methanogenic activity. Before bioaugmentation, different seed biomass resulted in different quasi steady state function, with digesters clustering into three groups distinguished by methane (CH4) production. Digesters with similar functional performance contained similar archaeal communities based on clustering of Illumina sequence data of the V4V5 region of the 16S rRNA gene. High CH4 production correlated with neutral pH and high Methanosarcina abundance, whereas low CH4production correlated to low pH as well as high Methanobacterium and DHVEG 6 family abundance. After bioaugmentation, CH4 production from the high CH4 producing digesters transiently increased by 11 ± 3% relative to non-bioaugmented controls (p \u3c 0.05, n = 3), whereas no functional changes were observed for medium and low CH4producing digesters that all had pH higher than 6.7. The CH4 production increase after bioaugmentation was correlated to increased relative abundance of Methanosaeta and Methaospirillum originating from the bioaugment culture. In conclusion, different anaerobic digester seed biomass can result in different quasi steady state CH4production, SCOD removal, pH and effluent VFA concentration in the timeframe studied. The bioaugmentation employed can result in a period of increased methane production. Future research should address extending the period of increased CH4 production by employing pH and VFA control concomitant with bioaugmentation, developing improved bioaugments, or employing a membrane bioreactor to retain the bioaugment

Relating Anaerobic Digestion Microbial Community and Process Function

Anaerobic digestion (AD) involves a consortium of microorganisms that convert substrates into biogas containing methane for renewable energy. The technology has suffered from the perception of being periodically unstable due to limited understanding of the relationship between microbial community structure and function. The emphasis of this review is to describe microbial communities in digesters and quantitative and qualitative relationships between community structure and digester function. Progress has been made in the past few decades to identify key microorganisms influencing AD. Yet, more work is required to realize robust, quantitative relationships between microbial community structure and functions such as methane production rate and resilience after perturbations. Other promising areas of research for improved AD may include methods to increase/control (1) hydrolysis rate, (2) direct interspecies electron transfer to methanogens, (3) community structure–function relationships of methanogens, (4) methanogenesis via acetate oxidation, and (5) bioaugmentation to study community–activity relationships or improve engineered bioprocesses

Correlating Methane Production to Microbiota in Anaerobic Digesters Fed Synthetic Wastewater

A quantitative structure activity relationship (QSAR) between relative abundance values and digester methane production rate was developed. For this, 50 triplicate anaerobic digester sets (150 total digesters) were each seeded with different methanogenic biomass samples obtained from full-scale, engineered methanogenic systems. Although all digesters were operated identically for at least 5 solids retention times (SRTs), their quasi steady-state function varied significantly, with average daily methane production rates ranging from 0.09 ± 0.004 to 1 ± 0.05 L-CH4/LR-day (LR = Liter of reactor volume) (average ± standard deviation). Digester microbial community structure was analyzed using more than 4.1 million partial 16S rRNA gene sequences of Archaea and Bacteria. At the genus level, 1300 operational taxonomic units (OTUs) were observed across all digesters, whereas each digester contained 158 ± 27 OTUs. Digester function did not correlate with typical biomass descriptors such as volatile suspended solids (VSS) concentration, microbial richness, diversity or evenness indices. However, methane production rate did correlate notably with relative abundances of one Archaeal and nine Bacterial OTUs. These relative abundances were used as descriptors to develop a multiple linear regression (MLR) QSAR equation to predict methane production rates solely based on microbial community data. The model explained over 66% of the variance in the experimental data set based on 149 anaerobic digesters with a standard error of 0.12 L-CH4/LR-day. This study provides a framework to relate engineered process function and microbial community composition which can be further expanded to include different feed stocks and digester operating conditions in order to develop a more robust QSAR model

Meta-Analysis of Non-Reactive Phosphorus in Water, Wastewater, and Sludge, and Strategies to Convert it for Enhanced Phosphorus Removal and Recovery

Current and future trends indicate that mining of natural phosphorus (P) reserves is occurring faster than natural geologic replenishment. This mobilization has not only led to P supply concerns, but has also polluted many of the world\u27s freshwater bodies and oceans. Recovery and reuse of this nuisance P offers a long-term solution simultaneously addressing mineral P accessibility and P-based pollution. Available physical, chemical, and biological P removal/recovery processes can achieve low total P (TP) concentrations (≤100 μg/L) and some processes can also recover P for direct reuse as fertilizers (e.g., struvite). However, as shown by our meta-analysis of over 20,000 data points on P quantity and P form, the P in water matrices is not always present in the reactive P (RP) form that is most amenable to recovery for direct reuse. Thus, strategies for removing and recovering other P fractions in water/wastewater are essential to provide environmental protection via P removal and also advance the circular P economy via P recovery. Specifically, conversion of non-reactive P (NRP) to the more readily removable/recoverable RP form may offer a feasible approach; however, extremely limited data on such applications currently exist. This review investigates the role of NRP in various water matrices; identifies NRP conversion mechanisms; and evaluates biological, physical, thermal, and chemical processes with potential to enhance P removal and recovery by converting the NRP to RP. This information provides critical insights into future research needs and technology advancements to enhance P removal and recovery

Methane Yield and Lag Correlate with Bacterial Community Shift Following Bioplastic Anaerobic Co-Digestion

Past plastic management practices have resulted in pollution. An improved management scenario may involve adding used bioplastic to anaerobic digesters to increase methane for renewable energy. In this work, effects of polyhydroxybutyrate (PHB) bioplastic anaerobic co-digestion with synthetic primary sludge on operation and microbial communities were investigated. Co-digesters treating sludge were co-fed 20% untreated or pretreated (55 °C, pH 12) PHB. Pretreament resulted in shorter lag (5 d shorter) before methane production increased after co-digestion. At steady-state, co-digesters converted 86% and 91% of untreated and pretreated PHB to methane, respectively. Bacterial communities were different before and after bioplastic co-digestion, whereas no archaeal community change was observed. Relative abundance of 30 significant bacteria correlated with methane production and lag following PHB addition. No previously known PHB degraders were detected following PHB co-digestion. Microbial communities in anaerobic digesters treating synthetic primary sludge are sufficiently capable of co-digesting PHB to produce additional methane

Ion exchange nutrient recovery from anaerobic membrane bioreactor permeate

Nutrient recovery from municipal wastewater was evaluated using anion exchange media loaded with hydrated ferric oxide (HFO) and copper (Cu2+) (Dow‐HFO‐Cu resin) to selectively capture phosphate, followed by clinoptilolite for ammonium removal and recovery. Nutrients were concentrated in the regenerants and recovered as precipitated struvite. Media exchange capacity after multiple ion exchange cycles was determined using permeate from an anaerobic membrane bioreactor (AnMBR) treating synthetic or actual municipal wastewater from a full‐scale water reclamation facility. Regeneration through five ion exchange cycles using relatively low concentration regenerant solution (2% NaCl and 0.5% NaOH) resulted in the highest phosphate exchange capacity and phosphate recovery. This regenerant also provided the most consistent ammonium recovery. Column tests treating AnMBR permeate were performed over five ion exchange cycles; Dow‐HFO‐Cu resin exchange capacities ranged from 1.6 to 2.8 mg PO4‐P/g dry media. A maximum of 94% of the removed phosphate was recovered during regeneration. The rate and extent of regeneration was insensitive to regenerant salt concentrations in the range investigated. Precipitation using a mixture of the spent regeneration brines from the Dow‐HFO‐Cu resin and clinoptilolite columns produced low molar ratios of Mg:NH4:PO4, suggesting that the recovered product was not pure struvite

Toxicity of Various Pyrolysis Liquids From Biosolids on Methane Production Yield

Aqueous pyrolysis liquid (APL) is a high-COD byproduct of wastewater biosolids pyrolysis that is comprised of numerous complex organic compounds and ammonia nitrogen (NH3-N). One potential beneficial use of APL is as a co-digestate to produce more biogas in anaerobic digesters. However, some APL organics and NH3-N are known to inhibit methane-producing microbes. Autocatalytic pyrolysis which uses previously-produced biochar as a catalyst during biosolids pyrolysis, increases energy-rich py-gas while eliminating bio-oil production and reducing COD concentration in the APL (catalyzed APL). However, the catalyzed APL still has a high organic strength and no suitable treatment strategies have yet been identified. In this study, the methane production yields and methanogenic toxicity of non-catalyzed and catalyzed APLs were investigated. Both non-catalyzed and catalyzed APLs were produced at 800°C from a mix of digested primary and raw waste activated sludge from a municipal water resource reclamation facility. Using the anaerobic toxicity assay, APL digester loading rates higher than 0.5 gCOD/L for non-catalyzed and 0.10 gCOD/L for catalyzed APL were not sustainable due to toxicity. The IC50 values (APL concentration that inhibited methane production rate by 50%) for non-catalyzed and catalyzed APLs were 2.3 and 0.3 gCOD/L, respectively. Despite having significantly fewer identified organic compounds catalytic APL resulted in higher methanogenic toxicity than non-catalytic APL. NH3-N was not the main inhibitory constituent and other organics in APL, including 3,5-dimethoxy-4-hydroxybenzaldehyde, 2,5-dimethoxybenzyl alcohol, benzene, cresol, ethylbenzene, phenols, styrene, and xylenes as well as nitrogenated organics (e.g., benzonitrile, pyridine) ostensibly caused considerable methane production inhibition. Future research focused on pretreatment methods to overcome APL toxicity and the use of acclimated biomass to increase methane production rates during APL anaerobic digestion or co-digestion is warranted

An Inexpensive, Reproducible Method to Quantify Activated Sludge Foaming Potential: Validation Through Lab-Scale Studies and Year-Long Full-Scale Sampling Campaign

Activated sludge is a conventional treatment process for biochemical oxygen demand (BOD) and total suspended solids (TSS) removal at water resource recovery facilities (WRRFs). Foaming events are a common operational issue in activated sludge and can lead to decreased treatment efficiency, maintenance issues, and potential environmental health risks. Stable foaming events are caused by biological and chemical drivers (i.e., microbes and surfactants) during the aeration process. However, foaming events are difficult to predict and quantify. We present an inexpensive and easy-to-use method that can be applied at WRRFs to quantify foaming potential. Subsequently, the method was applied over a year-long full-scale study while data on microbial community composition and functional parameters associated with foaming potential were collected from activated sludge samples at South Shore Water Reclamation Facility (WRF) (Oak Creek, WI). Results from the development of the foaming potential method using linear alkylbenzene sulfonate (LAS) showed that the method was reproducible (relative standard deviation Zoogloea, Flavobacterium, Variovorax, and Bdellovibrio. This is the first report that Variovorx and Bdellovibrio relative abundance was correlated with foaming events in activated sludge. Furthermore, the foaming potential positively correlated (ρ = 0.24) with soluble total nitrogen. Characterizing foaming events through frequent sampling and monitoring of specific genera and functional parameters may allow for predictions and preemptive mitigation efforts to avoid negative consequences in the future. Practitioner Points A reproducible method to measure foaming potential in activated sludge is available. Genera Zoogloea, Flavobacterium, Variovorax, and Bdellovibrio correlated with foaming events. A year-long sampling campaign of activated sludge measuring foaming potential and microbial community composition was conducted at South Shore Water Reclamation Facility in Oak Creek, WI. More research at other facilities with this method is needed to understand links between microbes and foamin

Cell Surface-Expression of the Phosphate-Binding Protein PstS: System Development, Characterization, and Evaluation for Phosphorus Removal and Recovery

Simultaneous overabundance and scarcity of inorganic phosphate (Pi) is a critical issue driving the development of innovative water/wastewater treatment technologies that not only facilitate Pi removal to prevent eutrophication, but also recover Pi for agricultural reuse. Here, a cell-surface expressed high-affinity phosphate binding protein (PstS) system was developed, and its Pi capture and release potential was evaluated. E. coli was genetically modified to express PstS on its outer membrane using the ice nucleation protein (INP) as an anchoring motif. Verification of protein expression and localization were performed utilizing SDS-polyacrylamide gel electrophoresis (SDS-PAGE), western blot, and outer membrane separation analyses. Cell surface characterization was investigated through acid-base titration, X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FTIR). These tests provided information on the macromolecular structure and composition of the bacteria surface as well as the proton-exchange properties of the surface functional groups (i.e., pKa values). Phosphate desorption and adsorption batch experiments were conducted to evaluate the effects of temperature, pH, and ionic strength on phosphate capture and release. The PstS surface-displayed cells demonstrated greater potential to release and capture phosphate compared to non-modified cells. Higher temperatures up to 40°C, basic pH conditions (pH = 10.5), and higher ionic strength up to 1.0 mol/L KCl promoted 20%–50% higher phosphate release