Anaerobic digester design and operation influences the biomass degradation efficiency performed by complex and diverse microbial communities. Optimum anaerobic digester design and operational parameters in residential on-site wastewater treatment sites (OWTS) establishes physiochemical environments suitable for the growth and stability of the microbial communities responsible for organic waste degradation. A comparative study of the microbial communities and their functional profiles between different OWTS designs and operational parameters have not been done despite their functional importance in residential organic waste removal. Using whole-metagenome shotgun sequencing, microbial community compositions and functions were compared between two digester designs: conventional box septic tanks and septic tanks equipped with a novel closed-conduit tube called the InnerTubeTM. Wastewater was sampled along the length of each digester to explore the microbial community stratification during the anaerobic digestion treatment process. Additionally, the effect of effluent, aerobic recirculating-lines on the digester microbiome was also explored. Physiochemical characteristics in the form of oxygen demand, nitrogen and solids content was used as endpoints and correlated with microbial community and functional gene abundances to explore the microbes driving anaerobic digestion. Conventional digesters were characterized by syntrophic proprionate-oxidizing microbes and acetoclastic methanogens, while InnerTube™ digesters were characterized by syntrophic sulfate-reducing microbes and hydrogenotrophic methanogens. Recirculating digesters were enriched with denitrifying microbial consortia in syntrophy with hydrogenotrophic methanogens. Microbial communities were organized according to hydrolytic, acidogenic, acetogenic, and methanogenic groups along the digester treatment process. Insight into the core microbiome of OWTS can inform bioaugmentation and digester design and operation optimization strategies to improve the treatment of decentralized residential sewage sources. / Thesis / Master of Science (MSc) / Anaerobic digesters are used throughout North America to treat residential sewage. Despite their prevalence, the composition and function of the microbial communities driving sewage degradation in residential digesters has not been studied. We used DNA sequencing to compare the microbial communities and functional genes in different anaerobic digester designs across Southern Ontario. Our findings suggest there are successive microbial groups along the length of septic tanks and that different septic tank designs harbor characteristic sulfidogenic and methanogenic microbes. Characterization of these microbes could inform septic tank bioaugmentation, design and operational optimization strategies to improve sewage treatment performance.

Yes / A novel Euler-Lagrangian (EL) computational uid dynamics (CFD) nite volume-based model to simulate the gas mixing of sludge for anaerobic digestion is developed and described. Fluid motion is driven by momentum transfer from bubbles to liquid. Model validation is undertaken by assessing the ow eld in a labscale model with particle image velocimetry (PIV). Conclusions are drawn about the upscaling and applicability of the model to full-scale problems, and recommendations are given for optimum application.

Anaerobic digestion of wastewater sludge has widely been in application for stabilization of sludge. With the increase in hauling cost and many environmental and health concerns regarding land application of biosolids, digestion processes generating minimized sludge with better effluent characteristics is becoming important for many public and wastewater utilities. This study was designed to investigate the performance of anaerobic-aerobic-anaerobic digestion of sludge and compare it to anaerobic-aerobic digestion and single stage mesophilic digestion of sludge. Experiments were carried out in three stages: Single-stage mesophilic anaerobic digestion (MAD) 20d SRT; Sequential Anaerobic/Aerobic digestion (Ana/Aer); and Anaerobic/Aerobic/Anaerobic digestion (An/Aer/An). The Anaerobic/Aerobic/Anaerobic digestion of sludge was studied with two options to determine the best option in terms of effluent characteristics. The two sludge withdrawal options were to withdraw effluent from the anaerobic digester (An/Aer/An – A) or withdraw effluent from the aerobic digester (An/Aer/An – B). Different operational parameters, such as COD removal, VS destruction, biogas production, Nitrogen removal, odor removal and dewatering properties of the resulting biosolids were studied and the results were compared among different processes. From the study, it was found that An/Aer/An – B (wastage from aerobic reactor) provided better effluent characteristics than An/Aer/An – A (wastage from anaerobic reactor), Ana/Aer or conventional MAD. The study also shows that the Anaerobic/Aerobic/Anaerobic (An/Aer/An, with wastage from the aerobic or anaerobic digester) digestion of the sludge can improve the biosolids quality by improving the dewatering capabilities, with lower optimum polymer dose, reduced CST and increased cake solid concentration, and reduce the odor generation from the biosolids. Both An/Aer/Ana – A and An/Aer/An – B gave 70% VS removal, compared to 50% with single MAD and 62% with only Ana/Aer. COD removal of both An/Aer/An – A and An/Aer/An – B was 70%, while it was 50% and 66% for single MAD and Ana/Aer respectively. In the aerobic reactors of Ana/Aer and An/Aer/An - B, nitrification and denitrification with removal of nitrogen was observed. The An/Aer/An – B system had more ammonia and TKN removal (70%) than Ana/Aer (64%). The effluent from each stage was analyzed for dewatering ability, cake solid concentration and odor production potential. Compared with a single Ana/Aer system, the extra anaerobic step in An/Aer/An – A and – B reduced polysaccharides in the effluent. The Ana/Aer system released less protein than the conventional MAD system and the addition of the second anaerobic step - especially with system An/Aer/An – B (discharge from aerobic reactor) - greatly reduced protein, resulting in improved dewaterability and less polymer demand. An/Aer/An (both of the options: A and B) had lower CST than single MAD (both 15d and 20d SRT) and Ana/Aer. Compared to Ana/Aer, a reduction of 52% for An/Aer/An – A and 20% for An/Aer/An – B in polymer dose requirement was observed, indicating improved dewatering characteristics. The An/Aer/An – B has higher biosolid cake concentration than MAD or Ana/Aer. The results showed that An/Aer/An (both options: A and B) biosolid had lower odor generation potential than single MAD (15d and 20d SRT) or Ana/Aer. Of all the stages,the An/Aer/An – A and – B system, generated odor which peaked at shorter time and lasted for shorter duration of time. / Master of Science

The increased demand for advanced techniques in anaerobic digestion over the last few years has led to the employment of various pre-treatment methods prior to anaerobic digestion to increase gas production. These pre-treatment methods alter the physical and chemical properties of sludge in order to make it more readily degradable by anaerobic digestion. The thermal hydrolysis process has been used in several treatment plants around the world, but none currently operate in the US. Thermal hydrolysis causes cell walls to rupture under the effect of high temperature and high pressure and results in highly solubilized product which is readily biodegradable. The performance of the process was evaluated for a treatment plant located in Dallas, TX. The performance assessment was based on various characteristics including pH, solids removal, COD removal and gas production. The study was conducted in two phases to investigate the effect of change in mesophilic temperature (37°C and 42°C) and the effect of solids retention time (SRT) (15 days and 20 days). Thermally hydrolyzed combined (1:1) primary and waste activated sludge was fed to a Thermal Hydrolysis (TH) anaerobic digester and its performance was compared to a conventional mesophilic anaerobic digester receiving non pre-treated sludge. The thermal hydrolysis pre-treatment was found to be more effective as compared to the conventional anaerobic digester. The efficiency of the process varied slightly with increase in temperature but the change in SRT was seen to have a greater impact on the digester's performance. The pre-treatment technique was observed to deliver the best results at a 20 day SRT. / Master of Science

Anaerobic digestion and struvite precipitation are two effective ways of treating dairy manure for recovering biogas and phosphorus. Anaerobic digestion of dairy manure is commonly limited by slow fiber degradation, while one of the limitations to struvite precipitation is the availability of orthophosphate. The aim of this work was to study the use of microwave-based thermochemical pretreatment to simultaneously enhance manure anaerobic digestibility (through fiber degradation) and struvite precipitation (through phosphorus solubilization). Microwave heating combined with different chemicals (NaOH, CaO, H₂SO₄, or HCl) enhanced solubilization of manure and degradation of glucan/xylan in dairy manure. However, sulfuric acid-based pretreatment resulted in a low anaerobic digestibility, probably due to the sulfur inhibition and side reactions. The pretreatments released 20-40% soluble phosphorus and 9-14% ammonium. However, CaO-based pretreatment resulted in lower orthophosphate releases and struvite precipitation efficiency as calcium reacts with phosphate to form calcium phosphate. Collectively, microwave heating combined with NaOH or HCl led to a high anaerobic digestibility and phosphorus recovery. Using these two chemicals, the performance of microwave- and conventional-heating in thermochemical pretreatment was further compared. The microwave heating resulted in a better performance in terms of COD solubilization, glucan/xylan reduction, phosphorus solubilization and anaerobic digestibility. Lastly, temperature and heating time used in microwave treatment were optimized. The optimal values of temperature and heating time were 147°C and 25.3 min for methane production, and 135°C and 26 min for orthophosphate release, respectively. Applying manure or slurry directly to the land can contribute to pathogen contamination of land, freshwater and groundwater. Thus it is important to study the fate of pathogens in diary manure anaerobic digestion systems. The goal of the project was to establish a molecular based quantitative method for pathogen identification and quantification, compare the molecular based method with culture based method and study pathogen fate in dairy manure and different anaerobic digesters. Result showed that molecular based method detected more E.coli than the culture based method with less variability. Thermophilic anaerobic digestion can achieve more than 95% pathogen removal rate while mesophilic anaerobic digester had increased E.coli number than fresh manure, indicating temperature is a key factor for pathogen removal. In general, the overall goal of the study is to develop an integrated dairy manure treatment system. The microwave based pretreatment enhanced the subsequent biogas production and struvite precipitation, and the molecular tool based method provided a more precise and faster way to study the pathogen fate in various anaerobic digestions. / Ph. D.

Sludge dewatering and chemical conditioning requirements were examined from the perspective of biopolymer and cation release from activated sludge flocs. Both aerobic and anaerobic digestion processes were considered from two different activated sludge sources at a temperature of 20° C. Polymer demand and specific resistance to filtration increased with an increase in total soluble biopolymer concentration for all temperature ranges. In anaerobic digestion, the protein release was three times greater than the polysaccharide release. Conversely, aerobic digestion of the same sludge resulted in a greater release of polysaccharides than proteins. Polymer conditioning requirements in the anaerobic digestors were an order of magnitude higher than in the aerobic digestors; proteins were considered to be the biopolymer fraction responsible for the high polymer conditioning requirements and poor dewatering properties. Biopolymer is released to the supernatant as colloids bound by divalent cations. Peptidase and glucosidase activity were used to monitor enzymatic activity relative to biopolymer release and degradation. The reasons for the increases and decreases in hydrolase activity are unknown. / Master of Science

Microalgae are photosynthetic organisms considered to be one of the most promising high-value chemicals and biofuel-producing organisms. However, there are several challenges for the widespread implementation of industrial processes using microalgae. The work presented in this dissertation proposes solutions to the different challenges involving the use of microalgae as microbial cell factories. To investigate the application of anaerobic digestion as a way to generate nutrients for microbial growth, salmon offal was used as substrate for anaerobic digestion, and soil from a flooded run-off pond on the Virginia Tech campus in Blacksburg, VA. A fast reduction in volatile solids and the short-chain fatty acid production profile is favorable for the growth of microalgae. A novel algae strain Scenedesmus sp. A6 was isolated from a decorative waterfountain in a hotel in Madison, IN. Mixotrophic growth trials were conducted using wastewater from the salmon offal digestion, that demostrated the A6 isolate grows six times faster in the wastewater then autotrophically. Bioassays of ethanolic cell extracts of A6 cultures demonstrated antimicrobial activity against E. coli cells at concentrations above 50 µg/ml. Genome sequencing and assembly revealed multiple copies of genes involved with acetate and ammonia metabolism, and several genes involved with secondary metabolite synthesis. An alternative to the high capital investment of photobioreactors for the cultivation of microalgae is the use of open-source and open-hardware bioreactor controller. Here, the concept of an open-hardwate bioreactor control called ``BioBrain'' is introduced. The BioBrain device is based on the Arduino Mega micro-controller board, and is capable of monitoring and controlling culture conditions during simple strain characterization studies, with an estimated construction cost of less than $800 USD. Finally, a new primer design tool for the ligation-independant cloning technique 𝜆-PCR was developed called lambdaPrimeR. The contributions of this work are the discovery and development of different tools that can overcome the challenges of the use of microalgae as microbial cell factories in industrial processes. / Ph. D. / Microalgae are single-celled organisms capable of photosynthesis and have the potential to revolutionize fuel and high-value chemical production. However, the high process costs involving the cultivation and biomass harvesting of these organisms limits the number of industrial applications of microalgae. Therefore, reduction of the overall costs of any process involving microalgae is vital for the widespread use of these organisms in industry. On this dissertation, I explore different approaches to tackle the challenges of using microalgae as a high-value chemicals cell factories. First, the use of anaerobic digestion of salmon offal to generate low-cost nutrients for algae growth is successfully demonstrated, with the discovery of a novel algae isolate Scenedesmus sp. A6, capable of very robust growth on the anaerobic digestion wastewater. Further characterization of this novel isolate showed that it has antimicrobial activity against E. coli cells. Therefore, the Scenedesmus sp. A6 isolate has the potential to be used as a high-value chemical cell factory. Reduction in equipment and instrumentation costs was also achieved by the design and construction of an open-hardware and open-source bioreactor controller device called the “BioBrain”, and a low-cost modular bubble column photobioreactor called “The Big Large Tube”. Together, these two devices represent a significant reduction in equipment costs for the cultivation of microalgae. Finally, an open-source Bioinformatics tool called “lambdaPrimeR” was developed to facilitate the use of a novel Genetic Engineering technique called λ-PCR, that has the potential to make genetic engineering of microalgae much easier.

In 2023, approximately 2.36 million metric tons of olive oil were produced globally. Olive pomace, a byproduct of the flesh and pits left after olive oil extraction, presents environmental challenges when used as landfill due to its high polyphenol and organic contents, or when combusted due to greenhouse gas emissions. Its potential as animal feed is limited, yet it holds promise for methane production via anaerobic digestion (AD), providing a source of renewable energy. However, the highly crystallized polysaccharides in olive pomace, such as cellulose, hemicellulose, and pectin, impede its conversion to methane, and the high polyphenol content inhibits methanogen growth. To address this, phenolics were extracted from olive pomace, producing a phenolics-extracted olive pomace (PEOP) and a phenolics-rich olive liquid. After further resin-based extraction of phenolics-rich olive liquid, approximately two-thirds of the phenolics were removed, yielding phenolics-extracted olive liquid (PEOL). Enzymatic hydrolysis was conducted on several olive byproduct streams: olive pomace with water, PEOP with PEOL, and PEOP with water, to convert insoluble polysaccharides into reducing sugars that are more readily utilized by methane-producing microorganisms. Various enzymes, including cellulase, hemicellulase, xylanase, and pectinase, were individually treated with olive pomace to determine the optimal hydrolysis time and enzyme concentrations. Response surface methodology (RSM) identified the optimal enzyme cocktail ratio (1.1% cellulase, db, and 0.9% pectinase, db) for achieving the highest reducing sugar contents (22.3 mg/mL), which had 79.1% increase when compared to the control sample (12.5 mg/mL). After 19 days of anaerobic digestion at 37 °C, olive samples before phenolics extraction (olive pomace with water) and olive samples after phenolics extraction (phenolics-extracted olive pomace with phenolics-extracted olive liquid), produced similar amounts of methane (~175 mL CH4/g VS). This indicated that in our experimental settings, the phenolics reduction did not significantly impact methane yields. Carbohydrate profiles may also influence biofuel yields, as hexoses (C6 sugars) are preferred over pentoses (C5 sugars) for end-product production during biotechnical conversion. To explore the effect of carbohydrate profiles on methane production from olive byproducts, two response surface methodology (RSM) coded enzyme cocktail-treated samples with different carbohydrate profiles underwent anaerobic digestion for 19 days at 37 °C, yielding similar amounts of methane (~156 mL CH4/g VS), comparable to the control sample. This suggested that anaerobic digestion can utilize different hexoses and pentoses at similar rates. These findings demonstrated that olive pomace can be used for biomethane production instead of being landfilled or combusted. While enzymatic hydrolysis increased reducing sugar contents, it did not enhance methane yields. Reducing phenolic contents of 2/3 did not improve biomethane yield, and the impact of greater reduction requires further assessment.

Bacterial resistance to antibiotics has become a worldwide health problem, resulting in untreatable infections and escalating healthcare costs. Wastewater treatment plants are a critical point of control between anthropogenic sources of pathogens, antibiotic resistant bacteria (ARBs), antibiotic resistance genes (ARGs), and the environment through discharge of treated effluent and land application of biosolids. Recent studies observing an apparent resuscitation of pathogens and pathogen indicators and the widening realization of the importance of addressing environmental reservoirs of ARGs all lead toward the need for improved understanding of ARG fate and pathogen inactivation kinetics and mechanisms in sludge stabilization technologies. This research has investigated the fate of two pathogens, methicillin-resistant Staphylococcus aureus (MRSA) and Escherichia coli, and various ARGs under pasteurization, anaerobic digestion, biosolids storage, and land application conditions. Pathogen die-off occurs at a rate specific to each pathogen and matrix in ambient and mesophilic temperature environments. Viable but nonculturable (VBNC) states are initiated by thermal treatments, such as thermophilic digestion and possibly pasteurization, and allow the persistence of pathogen cells and any ARGs contained therein through treatment and into the receiving environment where resuscitation or transformation could occur. Raw sludge ARG content does affect digester effluent quality, although the predominant mechanisms of ARG persistence may be different in mesophilic versus thermophilic digestion. In both thermophilic and mesophilic digestion, a correlation was observed between raw sludge and digester ARGs associated with Class 1 integrons, possibly as a result of horizontal gene transfer. ARB survival was shown to contribute to ARG content in mesophilic digestion, but not thermophilic digestion. Thermophilic digestion may achieve a higher ARG reduction because of reduced microbial diversity compared to mesophilic digestion. However, it is evident that horizontal gene transfer still does occur, particularly with highly mobile integrons, so that complete reduction of all ARGs would not be possible with thermophilic digestion alone. Surprisingly, the experiments that introduced various concentrations of antibiotic sulfamethoxazole and antimicrobial nanosilver did not induce enhanced rates of horizontal gene transfer. Finally, ARG concentrations in biosolids increased during cold temperature storage suggesting that there is a stress induction of horizontal gene transfer of integron-associated ARGs. / Ph. D.

Le processus de dégradation anaérobie de la matière organique est un phénomène naturel largement répandu sur terre (ex. marais, lacs, rizières, systèmes digestifs d'animaux et humains). Une très grande diversité microbienne est entretenue durant ce processus, traduisant une diversité de voies métaboliques impliquées. Lorsqu'elle est complète, la digestion anaérobie aboutie à la formation de biogaz (mélange de méthane et de dioxyde de carbone). En termes de biotechnologie, le traitement par voie anaérobie de pollutions organiques permet de réduire le volume de déchets en générant du méthane valorisable sous plusieurs formes (électricité, chaleur, gaz naturel, biocarburant). Cependant, les digesteurs industriels sont optimisés pour un fonctionnement à 35°C ou à 55°C, ce qui nécessite un apport exogène d'énergie de maintenance. Ainsi, les travaux de thèse se sont intéressés à l'étude de la capacité d'adaptation de divers écosystèmes anaérobies industriels couvrant une variété de procédés et de conditions opératoires à convertir l'éthanol en biogaz à différentes températures. La première phase de l'étude avait pour but le conditionnement, en réacteurs de laboratoire d'écosystèmes à leur température d'origine avec un substrat facilement biodégradable (éthanol). Ensuite, les performances des communautés microbiennes (le potentiel méthanogène maximal et la cinétique de dégradation) ont été estimées sur un gradient de température de 5°C à 55°C en fioles. La phase de conditionnement des écosystèmes en réacteur batch a montré que la production de biogaz avoisinait la production théorique et que cette production s'accompagnait d'une diminution de la durée de réaction avec ajout successif du substrat. De plus, les cinétiques de production de biogaz obtenues les variaient fortement d'un écosystème à l'autre. Des profils d'empreintes moléculaires (CE-SSCP) des communautés bactériennes et archées ont été réalisés au début et à la fin du conditionnement. Ces profils de communauté ont été comparés entre eux par analyse en composante principale (ACP). Les populations bactériennes qui assuraient une performance efficiente étaient différentes de celles qui garantissaient une bonne capacité d'adaptation. Par ailleurs, le potentiel d'adaptation dépendait de la présence de populations d'Archaea méthanogènes bien spécifiques. En plaçant ensuite les écosystèmes conditionnés dans des conditions de température éloignées de la température d'origine, seuls les écosystèmes mésophiles se sont acclimatés aux températures psychrophiles. Comme attendu, l'activé spécifique maximale des méthanogènes était toujours obtenue à la température d'origine de l'écosystème. L'analyse des communautés bactériennes et archées à la fin de la période d'acclimatation a révélé que l'acclimatation des écosystèmes thermophiles et mésophiles à des températures plus faibles ne modifiait que légèrement la structure des communautés microbiennes. En revanche, des changements plus importants étaient obtenus lorsque la température d'incubation était augmentée par rapport à la température d'origine de l'écosystème. En résumé, l'étude de l'effet de la température d'incubation (de 5°C à 55°C) sur l'activité fermentaire et sur la structure des populations microbiennes est un bon modèle d'étude au laboratoire pour appréhender l'impact d'un facteur abiotique sur la dynamique structurelle et fonctionnelle d'une communauté microbienne complexe. / The process of anaerobic degradation of organic matter is a natural phenomenon widespread on Earth (eg, marshes, lakes, rice fields, digestive systems of animals and humans). A high microbial diversity is maintained during this process, reflecting a diversity of metabolic pathways involved. When complete, the anaerobic digestion accomplished in the formation of biogas (methane mixture and carbon dioxide). In terms of biotechnology, anaerobic treatment of organic pollution reduces the volume of waste and generates methane recoverable in several forms (electricity, heat, natural gas, biofuels). However, industrial digesters are optimized for operation at 35 ° C or 55 ° C, which requires exogenous energy maintenance. Thus, the thesis is interested in the study of the adaptability of various anaerobic ecosystems covering a variety of industrial processes and operating conditions to convert ethanol into biogas at different temperatures. The first phase of the study was to the conditioning, in laboratory reactors ecosystems to their original temperature with a readily biodegradable substrate (ethanol). Then, the performances of microbial communities (the maximum methanogenic potential and degradation kinetics) were estimated on a temperature gradient of 5 ° C to 55 ° C in glass bottles. The conditioning phase of the ecosystems in batch reactor showed that the biogas averaged theoretical production and this production was followed by a decrease in reaction time with successive addition of the substrate. In addition, the kinetics of the biogas obtained varied greatly from one ecosystem to another. Molecular fingerprinting profiles (CE-SSCP) of bacterial and archaeal communities were performed at the beginning and at the end of conditioning. These community profiles were compared with each other by principal component analysis (PCA). Bacterial populations that ensured efficient performance were different from those that ensured a good adaptability. In addition, the potential for adaptation depended on the presence of very specific methanogenic Archaea populations. When placing ecosystems conditioned in temperature away from the original temperature, only mesophilic ecosystems adapted to psychrophilic temperatures. As expected, specific methanogenic activity was always obtained at the original temperature of the ecosystem. Analysis of bacterial and archaeal communities at the end of the acclimation period revealed that acclimation thermophilic and mesophilic ecosystems to lower temperatures only modified slightly the structure of microbial communities. On the other hand, more significant changes were obtained when the incubation temperature was increased in comparison to the original temperature of the ecosystem. In summary, the study of the effect of incubation temperature (5 ° C to 55 ° C) on the fermentation activity and microbial population structure is a good model for laboratory study to understand the impact of abiotic factor on the structural and functional dynamics of a complex microbial community.