Anaerobic digestion (AD) is widely used in wastewater treatment plants for stabilisation of primary and waste activated sludges. Increasingly energy prices as well as stringent environmental and public health regulations ensure the ongoing popularity of anaerobic digestion. Reduction of volatile solids, methane production and pathogen reduction are the major objectives of anaerobic digestion. Phased anaerobic digestion is a promising technology that may allow improved volatile solids destruction and methane gas production. In AD models, microbially-mediated processes are described by functionally-grouped microorganisms. Ignoring the presence of functionally-different species in the separate phases may influence the output of AD modeling. The objective of this research was to thoroughly investigate the kinetics of hydrolysis, acetogenesis (i.e., propionate oxidation) and methanogenesis (i.e., acetoclastic) in phased anaerobic digestion systems. Using a denaturing gradient gel electrophoresis (DGGE) technique, bacterial and archaeal communities were compared to complement kinetics studies. Four phased digesters including Mesophilic-Mesophilic, Thermophilic-Mesophilic, Thermophilic-Thermophilic and Mesophilic-Thermophilic were employed to investigate the influence of phase separation and temperature on the microbial activity of the digestion systems. Two more digesters were used as control, one at mesophilic 35 0C (C1) and one at thermophilic 55 0C (C2) temperatures. The HRTs in the first-phase, second-phase and single-phase digesters were approximately 3.5, 14, and 17 days, respectively. All the digesters were fed a mixture of primary and secondary sludges. Following achievement of steady-state in the digesters, a series of batch experiments were conducted off-line to study the impact of the digester conditions on the kinetics of above-mentioned processes. A Monod-type equation was used to study the kinetics of acetoclastic methanogens and POB in the digesters, while a first-order model was used for the investigation of hydrolysis kinetics. Application of an elevated temperature (55 0C) in the first-phase was found to be effective in enhancing solubilisation of particulate organics. This improvement was more significant for nitrogen-containing material (28%) as compared to the PCOD removal (5%) when the M1 and T1 digesters were compared. Among all the configurations, the highest PCOD removal was achieved in the T1T2 system (pvalue<0.05). In contrast to the solubilisation efficiencies, the mesophilic digesters (C1, M1M2 and T1M3) outperformed the thermophilic digesters (C2, T1T2 and M1T3) in COD removal. The highest COD removal was obtained in the T1M3 digestion system, indicating a COD removal efficiency of 50.7±2.1%. The DGGE fingerprints from digesters demonstrated that digester parameters (i.e., phase separation and temperature) influenced the structure of the bacterial and archaeal communities. This resulted in distinct clustering of DGGE profiles from the 1st-phase digesters as compared to the 2nd-phase digesters and from the mesophilic digesters as compared to the thermophilic ones. Based on the bio-kinetic parameters estimated for the various digesters and analysis of the confidence regions of the kinetic sets (kmax and Ks), the batch experiment studies revealed that the kinetic characteristics of the acetoclastic methanogens and POB developed in the heavily loaded digesters (M1 and T1) were different from those species developed in the remaining mesophilic digesters (M2, M3 and C1). As with the results from the mesophilic digesters, a similar observation was made for the thermophilic digesters. The species of acetoclastic methanogens and POB within the T1 digester had greater kmax and Ks values in comparison to the values of the T3 and C2 digesters. However, the bio-kinetic parameters of the T2 digester showed a confidence region that overlapped with both the T1 and T3 digesters. The acetate and propionate concentrations in the digesters supported these results. The acetate and propionate concentrations in the M1 digesters were, respectively, 338±48 and 219±17 mgCOD/L, while those of the M2, M3 and C1 digesters were less than 60 mg/L as COD. The acetate and propionate concentrations were, respectively, 872±38 and 1220±66 in T1 digester, whereas their concentrations ranged 140-184 and 209-309 mg/L as COD in the T2, T3 and C2 digesters. In addition, the DGGE results displayed further evidence on the differing microbial community in the 1st- and 2nd-phase digesters. Two first-order hydrolysis models (single- and dual-pathway) were employed to study the hydrolysis process in the phased and single-stage digesters. The results demonstrated that the dual-pathway hydrolysis model better fit the particulate COD solubilisation as compared to the single-pathway model. The slowly (F0,s) and rapidly (F0,r) hydrolysable fractions of the raw sludge were 36% and 25%, respectively. A comparison of the estimated coefficients for the mesophilic digesters revealed that the hydrolysis coefficients (both Khyd,s and Khyd,r) of the M1 digester were greater than those of the M2 and M3 digesters. In the thermophilic digesters it was observed that the Khyd,r value of the T1 digester differed from those of the T2, T3 and C2 digesters; whereas, the hydrolysis rate of slowly hydrolysable matter (i.e., Khyd,s) did not differ significantly among these digesters. The influence of the facultative bacteria, that originated from the WAS fraction of the raw sludge, and/or the presence of hydrolytic biomass with different enzymatic systems may have contributed to the different hydrolysis rates in the M1 and T1 digesters from the corresponding mesophilic (i.e, M2 and M3) and thermophilic (i.e., T2 and T3) 2nd-phase digesters.

Thesis (M.P.H.)--University of Michigan, 1944. / Cover title: The effect of certain industrial wastes on anaerobic decomposition.

Thesis (M.P.H.)--University of Michigan, 1944. / Cover title: The effect of certain industrial wastes on anaerobic decomposition.

Thesis (Ph.D.) - Carleton University, 2007. / Includes bibliographical references (p. 248-258). Also available in electronic format on the Internet.

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1993. / Vita. Abstract. Includes bibliographical references (leaves 94-98). Also available via the Internet.

Submitted in fulfillment of the requirements of the degree of Masters of Engineering: Chemical Engineering, Durban University of Technology, Durban, South Africa, 2018. / The anaerobic process is increasingly becoming a subject for many as it reduces greenhouse gas emissions and recovers carbon dioxide energy as methane. Even though these benefits are attainable, proper control and design of the process variables has to be done in order to optimize the system productivity and improve stability. The aim of this research was to optimize methane and biogas yields on the anaerobic co-digestion of sewage sludge using bio-chemical substrates as co-substrates. The first objective was to find the bio-chemical substrate that will generate the highest biogas and methane yields. The anaerobic digestion of these substrates was operated using 6 L digesters at 37.5℃. The substrate which generated the highest biogas and methane yield in the first batch experiment was then used for the second batch test. The objective was to optimize the anaerobic conditions (substrate to inoculum ratio, co-substrate concentration and temperature) in-order to optimize the biogas and methane yields. The second batch test was achieved using the conventional One-Factor-At-A-Time (OFAT) and the Design of Experiment (DOE) methods. Final analysis showed that the bio-chemical substrates could be substrates of interest to biogas generators. Amongst the substrates tested in the first batch experiment glycerol (Oleo-Chemical Product waste) generated the highest methane and biogas yields of 0.71 and 0.93 L. (g volatile solids added)-1, respectively. It was believed that glycerol contains significant amount of other organic substances such as lipids that have higher energy content than the other bio-chemical substrates, thus generating larger biogas and methane yields. Moreover, digestion of sewage sludge alone produced biogas yields of 0.19 L /g VS and 0.33 L/g COD, and methane yields of 0.16 L/g VS and 0.28 L/g COD. Generally, co-digestion yields were higher than digestion yields of sewage alone. Using the OFAT method the results of the second batch test on glycerol demonstrated highest amounts of volatile solids (VS) reduction, chemical oxygen demand (COD) reduction, biogas yield and methane yield of 99.7%, 100%, 0.94 L (g VS added)-1 and 0.75 L (g VS added)-1 at a temperature, substrate to inoculum ratio and glycerol volume of 50℃, 1 (on VS basis) and 10 mL, respectively. Above 22 mL and substrate to inoculum ratio of 1, the process was inhibited. The DOE results suggested that the highest methane and biogas yields were 0.75 and 0.94 L (g VS added)-1, respectively. These results were similar to the OFAT results, thus the DOE software may be used to define the biogas and methane yields equations for glycerol. In conclusion, anaerobic co-digestion of bio-chemical substrates as co-substrates on sewage sludge was successfully applied to optimize methane and biogas yields. / M

During anaerobic digestion bacteria inside the digester require a carbon source for their growth and metabolism, sewage sludge was used as a carbon source in this study. The COD content was used to measure the disappearance of the substrate. COD content was reduced by 48.3% and 49% in the methanogenic and sulphidogenic bioreactors, respectively, while sulphate concentration was reduced by 40%, producing 70mg/L of hydrogen sulphide as the end product over the first 5-7 days. Sulphate (which is used as a terminal electron acceptor of sulphur reducing bacteria) has little or no effect on the sulphidogenic and methanogenic proteases. Sulphite and sulphide (the intermediate and end product of sulphate reduction) increased protease activity by 20% and 40%-80%, respectively. Maximum protease activity occurred on day 21 in the methanogenic reactor and on day 9 in the sulphidogenic reactor. The absorbance, which indicates the level of amino acid increased to 2 and 9 for methanogenic and sulphidogenic bioreactors, respectively. Proteases that were active during anaerobic digestion were associated with the pellet (organic particulate matter) of the sewage. These enzymes have an optimum activity at pH 10 and at temperature of 50°C. The proteases that were active at pH 5 and 7, had optimum temperatures at 30°C and 60°C, respectively. Due to their association with organic particulate matter, these enzymes were stable at their optimum temperatures for at least five hours at their respective pH. Inhibition by PMSF, TPCK and 1.10-phenanthroline suggested that proteases inside the anaerobic digester are a mixture of cysteine, serine and metalloproteases. At pH 5, however, EDTA appeared to enhance protease activity by 368% (three-fold). Acetic acid decreased protease activity by 21%, while both propionic and butyric acid at 200 mg/L cause total inhibition of protease activity while these acids at higher pH (where they exist as their corresponding salts) exerted little effect. Copper, iron and zinc inhibited protease activity by 85% at pH 5 with concentrations ranging between 200 and 600 mg/L. On the other hand, nickel, showed an increase in protease activity of nearly 250%. At pH 7 and 10, copper had no effect on protease activity while iron, nickel and zinc inhibited these enzymes by 20-40%. Proteases at pH 7 were extracted from the pellet by sonication, releasing 50% of the total enzymes into the solution. The enzymes were precipitated by ammonium sulphate precipitation, and further purified by ion exchange chromatography and gel filtration. Ion exchange chromatography revealed that most of the enzymes that hydrolyse proteins are negatively charged while gel filtration showed that their molecular weight is approximately 500 kDa.

The start-up and step-up operation of two 16-liter, continuously operated, upflow anaerobic reactors receiving baker's yeast wastewater is presented in this thesis. The two reactors (A and B) were almost identical in construction. Reactor A was a conventional upflow anaerobic sludge blanket (UASB) reactor, and reactor B was a hybrid reactor. In addition to all the features of a UASB reactor, a fixed-film structure was installed in the mid section of the reactor B. Both reactors were operated at 35 °C and at a constant hydraulic retention time of 7 days. The waste strength, expressed in chemical oxygen demand (COD), was varied from 8 g COD liter⁻¹ (during the start-up) to 58 g COD liter⁻¹. The organic loading rate ranged from 1.1 to 9.4 g COD liter⁻¹ day⁻¹. The start-up lasted for the first 46 days. Towards the end of the start-up, methane production rates of 0.23 and 0.28 liter CH₄ liter⁻¹ day⁻¹ and COD reductions of 62.2% and 67.2% were achieved at organic loading rates of 1.1 and 1.3 g COD liter⁻¹ day⁻¹ for reactors A and B respectively. During the step-up operation, maximum methane production rates were, for reactors A and B respectively, 0.91 and 0.95 liter CH₄ liter⁻¹ day⁻¹ at organic loading rates of 5.8 and 6.4 g COD liter⁻¹ day⁻¹. In addition, reactor profiles for sludge concentration, pH, volatile fatty acids, and COD are also presented. / Applied Science, Faculty of / Chemical and Biological Engineering, Department of / Graduate

Extracellular polymeric substances (EPS) in activated sludge are known to account for the flocculent nature of activated sludge. Extensive studies over the last few decades have attempted to extract and characterize activated sludge EPS, but a lack of agreement between studies has also been quite common. The molecular makeup of EPS has, however, remained nearly unexplored, leaving their identity, function, and fate over various stages in the activated sludge system mainly unknown. In spite of their critical involvement in bioflocculation and long history of related research, our understanding of EPS is still greatly limited and better elucidation of their composition and structure is needed. The hypothesis of this research was that activated sludge floc contains different fractions of EPS that are distinguishable by their association with certain cations and that each fraction behaves differently when subjected to shear, aerobic digestion, anaerobic digestion and other processes. In order to examine this floc hypothesis, the research mainly consisted of three sections: 1) development of EPS extraction methods that target cations of interest (divalent cations, especially calcium and magnesium, iron, and aluminum) from activated sludge; 2) molecular investigations on activated sludge EPS using metaproteomic analyses, comprising sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and protein identification by liquid chromatography tandem mass spectrometry (LC/MS/MS), and hemaagglutination (HA)/HA inhibition assays; and 3) investigating the fate of EPS in sludge digestion using SDS-PAGE. Evaluation of prior research and data from preliminary studies led to the development of the three extraction methods that were used to target specific cations from activated sludge and to release their associated EPS into solution. These methods are the cation exchange resin (CER) procedure for extracting Ca²⁺+Mg²⁺, sulfide extraction for removing Fe, and base treatment (pH 10.5) for dissolving Al. The cation selectivity in the three extraction methods, the composition of EPS (protein/polysaccharide), amino acid composition, and a series of sequential extraction data established initial research evidence that activated sludge EPS that are associated with different cations are not the same. SDS-PAGE was successfully applied to study extracellular proteins from several sources of both full- and bench-scale activated sludges. The three extraction methods led to different SDS-PAGE profiles, providing direct evidence that proteins released by the three methods were indeed different sludge proteins. Another important outcome from this stage of research was finding the similarity and differences of extracellular proteins between different sources of activated sludge. SDS-PAGE data showed that many of CER-extracted proteins were well conserved in all the sludges investigated, indicating that a significant fraction of Ca²⁺ and Mg²⁺-bound proteins are universal in activated sludge. On the other hand, protein profiles resulting from sulfide and base extraction were more diverse for different sludges, indicating that Al and Fe and their associated proteins are quite dynamic in activated sludge systems. Protein bands at high densities were analyzed for identifications by LC/MS/MS and several bacterial proteins and polypeptides originating from influent sewage were identified in this study. This was also thought to be the first account of protein identification work for full-scale activated sludge. The analysis of SDS-PAGE post sludge digestion revealed that CER-extracted proteins remained intact in anaerobic digestion while they were degraded in aerobic digestion. While the fate of sulfide-and base-extracted proteins in aerobic digestion was not as clearly resolved, their changes in anaerobic digestion were well determined in this research. Sulfide-extracted protein bands were reduced by anaerobic digestion, indicating that Fe-bound EPS were degraded under anaerobic conditions. While parts of base-extracted proteins disappeared after anaerobic digestion, others became more extractable along with the extraction of new proteins, indicating that the fate of base-extractable proteins, including Al-bound proteins, is more complex in anaerobic digestion than CER-extracted and sulfide-extracted proteins. These results show that Ca²⁺+Mg²⁺, Fe³⁺, and Al³⁺ play unique roles in floc formation and that each cation-associated EPS fraction imparts unique digestion characteristics to activated sludge. Finally, since a considerably different cation content is quite common for different wastewaters, it is postulated that this variability is one important factor that leads to different characteristics of activated sludge and sludge digestibility across facilities. The incorporation of the impact of cations and EPS on floc properties into an activated sludge model might be challenging but will assure a better engineering application of the activated sludge process. / Ph. D.

"Anaerobic Stabilization" (AnS) is a phenomenon previously observed in biological nutrient removal (BNR) systems that use anaerobic-aerobic sequencing for phosphorus and/or nitrogen removal. AnS manifests itself in the form of less-than-theoretical oxygen requirements for the extent of organics stabilization observed. The objectives of this study were to develop an improved methodology for the quantitative determination of AnS, verify the occurrence and validate the statistical significance of AnS, identify components of the AnS-related redox balance, and investigate possible explanations for AnS. A lab-scale continuous-flow A/O¹ system was operated with chemical inhibition of nitrification at a 12-hour nominal HRT, 10-day BSRT (Biological Solids Retention Time), 1Q RAS flow, and varying synthetic feed compositions. Data from this system were used to demonstrate that, by eliminating the need to quantify the clarifier OUR, the Boundary Exchange AnS determination method developed in this study afforded a major advantage over earlier methods. Non-zero AnS was shown to be a statistically significant, reproducible phenomenon. Carbon, oxygen, and sulfur were identified as the three main elements affecting AnS in the A/O system studied. A second lab-scale A2/O system operating at a 6-hour nominal HRT, 5-day BSRT, 1Q RAS flow, and 2Q RNX flow, and receiving raw municipal wastewater feed spiked with acetate, was uSed in conjunction with the A/O system to study possible AnS explanations. A combination of processes accounted for varying percentages of observed AnS. Hydrogen production explained 0.1 percent or less, while methane production explained almost 19 percent with formate in the feed but no more than 0.8 percent without it. Aeration-induced Stripping of reduced volatiles explained up to 6 percent. Attempts to identify the reduced volatiles revealed traces of ethanol but no n-butanol in the A2/O system. Limitations of the COD test were identified as a possible explanation for AnS that warrants further investigation. A unified speculative biochemical model consistent with all results of this study and with established theory, and capable of partially explaining observed AnS, is proposed in this study. ¹A/O and A2/O are trademarks of Air Products and Chemicals, Inc., Allentown, PA, U.S.A. / Ph. D.