The Blue Plains Wastewater Treatment Plant in Washington, D.C. is in the process of updating its anaerobic digesters, with a completion date set for 2008. This research was undertaken to aid designers at Blue Plains by evaluating alternative digestion approaches. The technologies applicable to the plant included thermophilic anaerobic digestion, acid/gas phased digestion, and temperature phased anaerobic digestion. To obtain design data, a year long study was conducted using bench scale digestion systems operating at different solids retention times (SRT) and organic loading rates (OLR). The digesters were fed with mixed primary and secondary waste (50/50 by weight) from the Blue Plains wastewater treatment facility. The digesters were operated until they reached steady state as observed by volatile solids reduction (VSR), methane production, and volatile fatty acid (VFA) levels. At this point, samples of digested waste sludge were analyzed for residual biological activity, dewatering properties and headspace organo-sulfur production. Results from the study demonstrated that the TPAD digestion process had the lowest residual biological activity (RBA) after digestion, and that the single stage thermophilic digesters had the highest RBA. Sludge from single stage mesophilic digestion had the highest odor potential, with headspace gas tests generating over 1400 mg organo-sulfur per m3 of headspace gas, while both single stage thermophilic and TPAD systems generated less than 400 mg/m3 at all SRTs studied. / Master of Science

The performance and stability of a thermophilic anaerobic digestion system are inherently dependent on the engineered environment within each reactor. While the selection of operational parameters such as mixing, solids retention time, and digestion temperature are often selected on the basis of certain desirable outcomes such as the deactivation of human pathogens, these parameters have been shown to have a broad impact on the overall sludge digestion process. Since the current time-temperature requirements for biosolids pathogen reduction are most easily met at elevated digestion temperatures within the thermophilic range, it is certainly worth examining the effect of specific digestion temperatures on ancillary factors such as operational stability and the aesthetic quality of biosolids. A series of experiments were carried out in which wastewater sludge was digested at a range of temperatures (35°C, 49°C, 51°C, 53°C, 55°C, 57.5°C). Each reactor was operated for a period at steady state in order to make observations of microbial activity, digestion performance, and biosolids aesthetics as affected solely by digestion temperature. Results of this study show that poor operational stability arises in reactors operated at 57.5°C. Elevated concentrations of hydrogen and short-chain fatty acids in the 57.5°C digesters are evidence that the observed temperature-induced digester failures are related to the temperature sensitivity of hydrogenotrophic (CO₂-reducing) methanogens. Reactors operated at other temperatures performed equally well with respect to solids removal and operational stability. In addition, peak volatile organic sulfur compound (VOSC) production from biosolids treated at 51°C and above was greatly reduced in comparison with mesophilic anaerobic digestion and a lower temperature (49°C) thermophilic system. Since the biosolids methanogenic community appeared to be equally capable of degrading VOSC over the range of thermophilic temperatures, the conclusion is that the activity of VOSC producing organisms in digested and dewatered biosolids is greatly reduced when operating temperature in excess of 51°C are used. This study shows that small changes in an operationally defined parameter such as digestion temperature can have a large impact on the performance and stability of a digestion process. Single minded selection of digestion temperature in order to achieve effective pathogen reduction can result in poor digester performance and the production of an aesthetically unacceptable product. Careful selection, however, of an appropriate digestion temperature can not only ensure successful pathogen reduction in compliance with current regulations, but can also improve the performance, stability, and aesthetic quality of digestion systems employing thermophilic anaerobic digestion. / Master of Science

An anaerobic, cellulose- decomposing bacteria was isolated in pure culture from the inghetata of a bovine rumen. The organism was a small, curved, Gram negative rod that occurred singly or in chains. The isolated bacterium was identified as a member of the genus Butyrivibrio because of its morphology and because of its production of certain volatile fatty acids in a rumen fluid-glucose medium fermentation. The characteristic fermentation waa the production of a large amount of butyric acid and 1ome lactic acid or succinic acid, and the lack of production of propionic acid. It was found that when rumen fluid was omitted from the fermentation medium the production of butyric acid decreased markedly. The organism was found to have a relatively narrow pH tolerance for the initiation of growth, pH 6.3 to 7.2, with an optimum of pH 6.9 to 7.0. Growth stopped at about pH 6.4, and continued fermentation decreased the pH to 6.0. The organism was cultured continuously on a medium which chemically defined except that vitamin-free casein hydrolysate was included, when the hydrolysate was replaced by a mixture of pure L-isomers of amino acids similar in composition to the hydrolysate, good growth occurred. However, when each amino acid was supplied at a uniform weight concentration, much weaker growth was obtained. Evidence was obtained indicating that amino acids were inhibitory, although the reason for the mild inhibition was not disclosed. The following amino acids were found to be critical nutrients, but in some cases their status, whether essential or stimulatory, was found to be dependent on the composition of the medium: - cysteine, histidine, isoleucine. methionine, lysine, tyrosine, asparagine and leucine. A medium containing only these amino acids supported only very weak growth. Aspartic acid was found to be mildly inhibitory to growth, and glutamic acid was found to reverse the inhibition. A mixture of the common purine and pyrimidine bases, and a mixture of volatile fatty acids, were each found to be inhibitory to growth. Biotin, folic acid, and pyridoxal were essential vitamins, but the other eight B-vitamins examined were found to be neither stimulatory nor essential. Carbon dioxide was found to be required in relatively high concentration in order for growth to start in a medium devoid of rumen fluid. Purines, pyrimidines, cobalamin, and Tween-80 in a mixture were found not to replace the carbon dioxide. Rumen fluid was found to contain material very stimulatory for growth of the Butyrivibrio. The stimulatory material was found to be possibly two compounds or types of compounds, one a peptide or peptide like substance, and the other an anionic substance which probably was polynucleotide-like. / Ph. D.

Strict anaerobic culture techniques were used to enumerate the anaerobic bacteria present in three soil sites located along a drainage catena near Blacksburg, Virginia. An anaerobic cooked meat plus 0.5% glucose medium cultured the largest number of anaerobes from the poorly drained soil. The population of obligate anaerobes ranged from 10⁶/ g dry weight soil on the poorly drained soil (% moisture = 112.06) to 10⁵/g dry weight soil on the intermediate soil (% moisture = 34.51) to 10⁴/g dry weight soil on the well drained soil (% moisture 20.81). The population of organisms able to grow anaerobically (facultative plus obligate) ranged from 10⁶/g dry weight soil on the poorly drained site to 10⁵/g dry weight soil on the well drained site. This same population on the poorly drained site was relatively constant over a nine month period with the exception of a sharp rise in early spring. The clostridia constituted at least one third of the obligately anaerobic bacteria present on the poorly drained soil. A sizeable percentage of the obligate anaerobic isolates on this site were either clostridia which formed spores unable to germinate in the medium employed, clostridia which were very pleomorphic in cell shape and gram reaction, or nonsporeforming obligate anaerobes. These results indicate that strict anaerobes and possibly nonsporeforming strict anaerobes exist in soils of different drainage character even though facultative organisms appear to be more successful competitors on the more well drained sites. / Master of Science

Temperature-phased anaerobic digestion (TPAD) has become increasingly appealing in recent years due to the pathogen destruction capabilities of the system. However, there has also been concern about the dewatering properties of the sludges created by these systems. A laboratory study was conducted at Virginia Tech to determine the effect of thermophilic solids retention time (SRT) on sludge dewatering properties, to characterize system parameters associated with dewatering, and to understand the mechanisms causing changes in dewatering properties between the thermophilic and mesophilic phases. The study showed that while anaerobic digestion caused dewatering properties to deteriorate, sludges varied little with thermophilic SRT. Acidogenesis was essentially complete after 1.5 days. Subsequent mesophilic digestion resulted in little change to dewatering properties and modest reductions in conditioning doses, but substantial reductions in biopolymer (protein + polysaccharides) occurred. It appears that thermophilic anaerobic digestion creates or releases colloidal materials that cause dewatering to be poor and subsequent mesophilic digestion for 15 days does little to improve sludge properties of TPAD systems. / Master of Science

This study aims to provide the latest understanding of cutting-edge technologies that enable wastewater organic carbon diversion and recovery through the enhancement of sludge production and blending, digestibility, dewaterability, and dewatered cake odor emission control. A comprehensive literature review showed that iron-based coagulants tend to show less negative impact than aluminum-based coagulants. This can be attributed to the reduction of ferric to ferrous ions in the course of anaerobic digestion (AD), which leads to a suite of changes in protein bioavailability, alkalinity, and hydrogen sulfide levels, and in turn the sludge dewaterability and odor potential. In terms of the roles of thermal hydrolysis pretreatment (THP), the mechanism review indicated that the improvement of sludge dewaterability and anaerobic digestibility as a result of THP was because of the destruction of extracellular polymeric substances and increase of hydrolysis rate. However, THP also brings side effects such as high free residual ammonia and recalcitrant dissolved organic nitrogen (rDON) in the effluent. Besides, a comprehensive understanding of the formation of the odorous compounds in the sludge treatment processes indicated that sulfurous and nitrogenous compounds are usually regarded as the major odor-causing substances. A Pilot THP-AD study indicated that adding aluminum to produce primary sludge can improve overall plant sludge digestibility, dewaterability, and well as the rDON reduction. Moreover, results from a pilot THP-AD and biochemical methane potential (BMP) test study indicated that adding a secondary thermal hydrolysis after a primary thermal hydrolysis-AD system can still create new BMP. Finally, a pilot study was conducted to evaluate the effect of aeration in the sludge holding tank on biosolids odor emission. The two rounds of bench-scale aeration studies indicated that aerating the sludge in holding tanks reduced peak emission concentrations of sulfurous odorous compounds. Further full-scale validation confirmed that aeration can be used by utilities as a simple means for biosolids odor control. / Doctor of Philosophy / Public wastewater treatment annually consumed 3-4% energy production and contributed 1% greenhouse gas emission in the U.S. Meanwhile, the chemical energy contained in wastewater was estimated to be 9.3 times the energy it takes to treat it. Therefore, harvesting wastewater energy is proposed as a viable means for achieving energy and carbon neutral wastewater treatment. The approach to sending wastewater energy as much as possible to anaerobic digesters in which microorganisms help harvest useful energy in the form of flammable methane was evaluated in this study. From literature, we learned that chemicals used for upstream wastewater energy capture and nutrient removal may make the downstream energy recovery difficult. While, thermal hydrolysis pretreatment, an industrial-scale pressure cooker, can be used to improve the ease of microbial bioenergy harvesting by making organics more biodegradable. However, thermal hydrolysis may also bring side effect in terms of recalcitrant organic formation. Also, in the course of energy recovery, the production and emission of nuisance odor may occur but can be controlled. Building on this existing knowledge, this study evaluated the pros and cons of the approach to using chemicals to capture and recover energy from wastewater. The results showed that the extents of energy recovery and savings was greater than the compromised solids reduction from the process. Moreover, results from a biochemical methane potential test study indicated that adding a secondary thermal hydrolysis can recover even more chemical energy from wastewater. In the end, a pilot study was conducted to develop a simple and economical approach to mitigating the odor emission issue during sludge handling. Results showed that pumping air into the sludge holding tank can substantially reduce peak odor emission. This approach was later verified in a full-scale test and recommended to utilities as a simple means for biosolids odor control.

Waste sludge is frequently treated by anaerobic digestion to kill pathogens, generate methane gas and reduce odors so the sludge can be safely land applied. In an attempt to reduce sludge volumes and improve sludge dewatering properties, the use of thermal hydrolysis (TH), a sludge pretreatment method, has been adopted by numerous wastewater treatment plants, among them being the District of Columbia Water and Sewage Authority (DC WASA). The use of anaerobic digestion in collaboration with thermal hydrolysis has been shown to increase VS removal, COD removal and biogas production. The sludge generated also dewaters to a higher cake solids than from conventional anaerobic digestion. Unfortunately, DC WASA has found that the use of thermal hydrolysis had brought about two major issues. These are: (a) does thermal hydrolysis increase destruction of fats, oils and greases compared to conventional digestion? and (b) is the mixing method used at Virginia Tech (recirculating gas mixing) capable of stripping ammonia from the digester? Therefore the main purpose of this study is to evaluate these issues which occur with the use of the thermal hydrolysis process. Experiments were conducted in two phases. The first phase was to assess the performance of anaerobic digesters via their biogas production with and without long chain fatty acid addition and with or without thermal hydrolysis. This research was further carried out in two stages. First a mixture of unsaturated long chain fatty acids (hydrolyzed and unhydrolyzed) was used. The fatty acid mixture included oleic, linoleic and linolenic acids, which contain one, two and three double bonds, respectively. In the second stage, the effect of a single unsaturated fatty acid (hydrolyzed and unhydrolyzed) was analyzed. If extra gas is generated, grease addition to the digesters will be implemented. If thermal hydrolysis produces more gas, the greases will be added through the thermal hydrolysis unit rather than being added directly to the digester. The results showed that addition of long chain fatty acids greatly increased gas production and the long chain fatty acids that were thermally hydrolyzed generated more gas than the untreated long chain fatty acids, although the gain was not large. The second phase of the study was carried out by alternating the type of recirculating gas mixing (partial and continuous) in the anaerobic bioreactor. To achieve this goal, short-term anaerobic bioreactor studies were conducted by varying the frequency of the gas. The result showed that continuous gas recirculation at the bottom of the digester was responsible for stripping ammonia from the system. It appeared that up to 500 mg/L of ammonia was being stripped from the digester operating at 20 day solids retention time. This suggests that ammonia can be stripped if a reduction of ammonia in the digester was desired. / Master of Science

Washington D.C. Water and Sewage Authority is planning to construct a new anaerobic digestion facility at its Blue Plains WWTP by 2008. The research conducted in this study is to aid the designers of this facility by evaluating alternative digestion technologies. Alternative anaerobic digestion technologies include thermophilic, acid/gas phased, and temperature phased digestion. In order to evaluate the relative merits of each, a year long study evaluated the performance of bench scale digestion systems at varying solids retention times (SRT) and organic loading rates (OLR). The digesters were fed a blend of primary and secondary residuals from the Blue Plains wastewater treatment facility. In each study phase, temperature phased anaerobic digestion was compared to single stage mesophilic digestion (the industry standard) at the same SRT. Single stage thermophilic digestion was evaluated by sampling the first thermophilic stage of the temperature phased digestion systems throughout the study. Additionally, the first phase study compared acid/gas phased digestion to temperature phased and single stage mesophilic digestion. Results of the study demonstrated that the temperature phased digestion system consistently performed better than the other systems during each study phase by having higher volatile solids reduction (VSR), higher methane production, and lower residual biological activity. The highest observed VSR during the study (67%) occurred in a temperature phased digestion system operated at 7.5 days in each stage. Based on these results, it seems a suitable candidate for the Blue Plains digestion facility. Additionally, odor studies performed in conjunction with the research presented in this paper have shown distinct advantages for the temperature phased process. / Master of Science

Thesis (MSc)--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Fermentation in Zymomonas mobilis has been described as a catabolic highway, with 50 % of soluble protein comprising glycolytic and fermentative enzymes. In conjunction with one of the fastest observed fermentations, the conversion of glucose to ethanol forms one of the least efficient energy extractions found in nature. The low energy yield of fermentation in Z. mobilis is a result of the usage of the Entner-Doudoroff glycolytic pathway, which has half the energy yield per mol substrate compared to the well known Embden-Meyerhof-Parnas glycolytic pathway. The work presented in this thesis forms part of a larger project to compare glycolytic regulation in different micro-organisms (i.e., Z. mobilis, Escherichia coli, Saccharomyces cerevisiae and Lactococcus lactis). These organisms were chosen based on their usage of different glycolytic mechanisms. By using supply-demand analysis for quantifying glycolytic regulation as well as similar experimental conditions (e.g. using non-growing cell cultures), we can compare the regulatory behaviour of mechanistically distinct freeenergy supplies. The aim of this thesis was to quantify the importance of anaerobic free-energy generation for the regulation of the Entner-Doudoroff glycolytic pathway in Z. mobilis. We used metabolic control analysis (MCA) and supply-demand analysis to realize this goal. The central message of MCA is that when a metabolic parameter (e.g., a conserved metabolic moiety) is deemed important for affecting a particular steady-state variable (i.e., fermentation flux), its effect on the steady state variable should be tested. An extension to MCA, supply-demand analysis, provides a quantitative framework for analyzing the regulatory importance of cellular commodities such as anaerobic free-energy. This is done through comparing the elasticities of anaerobic free-energy supply and demand, which yields the degree to which the respective reaction blocks control the flux through anaerobic free-energy metabolism, as well as determine the cellular free-energy state (ATP/ADP ratio). The regulation of anaerobic free-energy metabolism in Z. mobilis was investigated with an experimental approach. The key features of our experimental setup were the use of NMR spectroscopy for detecting metabolites, as well as employing non-growing conditions for supply-demand experiments. With NMR spectroscopy metabolites could be detected in real time without using invasive sampling techniques; the use of nongrowing conditions further simplified the analysis by enabling us to correlate fermentative behaviour exclusively with the anaerobic free-energy state. Fermentation of glucose was investigated in the wild type Z. mobilis, a recombinant containing a non-expressing plasmid, or expressing plasmids for over-expressing the glucose facilitator (TCDB 2.A.1.1.4) or glucose-6-phosphate dehydrogenase (EC 1.1.1.49). In addition, ATP demand in the non-expressing recombinant and wild type was perturbed by titrating with the uncoupler acetic acid. Our results show that the anaerobic free-energy demand, the glucose facilitator and glucose-6-phospate dehydrogenase all control the flux of ethanol production in Z. mobilis. The Entner-Doudoroff glycolytic supply activity was found to be sensitive to changes in the ratios of ATP/ADP (elasticity varied between –0.31 and –0.49) and NTP/NDP (elasticity varied between –0.31 and – 0.50). / AFRIKAANSE OPSOMMING: Fermentasie in Zymomonas mobilis word beskryf as ‘n kataboliese snelweg, waar glikolitiese en fermentatiewe ensieme 50% van totale oplosbare proteïene in die sel uitmaak. Hoewel dié fermentasie een van die vinnigstes is wat tot op hede waargeneem is, is die omskakeling van glukose na etanol een van die mees ondoeltreffende energieekstraksies in die natuur. Dié lae energie-opbrengs, soos waarneembaar in fermentasie in Zymomonas mobilis, kan toegeskryf word aan die Entner-Doudoroff metaboliese pad. Hierdie metaboliese pad lewer slegs die helfte van die energie-opbrengs per mol substraat vergeleke met die meer bekende Embden-Meyerhof-Parnas glikolitiese pad. Die navorsing in hierdie tesis is deel van ‘n omvattende projek wat poog om die regulering van glikolise in verskillende mikro-organismes (Z. mobilis, Escherichia coli, Saccharomyces cerevisiae en Lactococcus lactis) te vergelyk. Dié organismes is gekies op grond van die uiteenlopende glikolitiese meganismes waarvan hulle gebruik maak. Ten einde die reguleringsgedrag van meganisties verskillende vry-energie produksieweë m.b.v. vraag-aanbod analise te vergelyk, moet glikolitiese regulering eers onder eenderse eksperimentele kondisies (b.v. nie-groeiende selkulture) gekwantifiseer kan word. Die hoofdoel van hierdie tesis was om die belang van anaerobiese vry-energie produksie vir die regulering van die Entner-Doudoroff glikolitiese pad in Z. mobilis te kwantifiseer. Hiervoor is van Metaboliese kontrole-analise (MKA) en vraag-aanbodanalise (‘n uitbreiding van MKA) gebruik gemaak. MKA is ‘n tegniek waarmee die effek wat ‘n metaboliese parameter (soos metaboliese deel-konservering) op ‘n spesifieke bestendige toestand-veranderlike (soos fermentasiefluksie) het, gekwantifiseer kan word. Vraagaanbodanalise daarenteen, bied ‘n kwantitatiewe raamwerk waardeur die regulatoriese belang van sellulêre kommoditeite (byvoorbeeld anaerobiese vry-energie) geanaliseer kan word. Tydens laasgenoemde proses word die elastisiteit van die anaerobiese vry-energie aanbod en die elastisiteit van die vraag vergelyk. Op hierdie manier kan die mate van beheer wat die onderskeie reaksieblokkie oor die fluksie deur anaerobiese vry-energie metaboliese paaie, sowel as oor die sellulêre vry-energie toestand (ATP/ADP verhouding), bepaal word. In hierdie werk is die regulering van anaerobiese vry-energie metabolisme in Z. mobilis ondersoek deur van ‘n eksperimentele benadering gebruik te maak. Die sleuteleienskappe van dié benadering was om kernmagnetiese-resonansiespektroskopie (KMR spektroskopie) te gebruik om metabolietkonsentrasies te meet, en om van niegroeiende kondisies gebruik te maak vir die vraag-aanbod eksperimente. Metabolietkonsenstrasies kon aaneenlopend bepaal word sonder die gebruik van monsternemingstegnieke wat die reaksie sou kon beïnvloed. Eksterne invloede op die fermentasiegedrag kon ook uitgesluit word deur van nie-groeiende kondisies gebruik te maak, sodat die waargenome fermentasiegedrag uitsluitelik aan die anaerobiese vryenergie toestand toegeskryf kan word. Glukose fermentasie was ondersoek in wilde tipe Z. mobilis, en in drie rekombinante wat onderskeidelik ‘n glukose fasiliteerder ooruitdrukkingsplasmied (TCDB 2.A.1.1.4), ‘n glukose-6-fosfaat dehidrogenase ooruitdrukkingsplasmied (EC 1.1.1.49), en ‘n nieuitdrukkingsplasmied bevat het. Die ATP vraag in die wilde tipe en die nieuitdrukkingsrekombinant is geperturbeer deur titrasies met asynsuur as ontkoppelaar. Die resultate toon dan die anaerobiese vry-energievraag, sowel as die glukose fasiliteerder en glukose-6-fosfaat dehidrogenase, die fluksie van etanolproduksie in Z. mobilis beheer. Die Entner-Doudoroff glikolitiese produksie-aktiwiteit was sensitief vir veranderinge in die ATP/ADP verhouding (elastisiteite was tussen -0.31 en -0.49) en die NTP/NDP verhouding (elastisiteite was tussen -0.31 en -0.50).

O presente trabalho propõe-se a verificar a viabilidade de utilização do reator anaeróbio horizontal do leito fixo (RAHLF) para o tratamento da vinhaça sob condições termofílicas, com ênfase na etapa de partida do reator. O RAHLF tem sido utilizado com êxito no tratamento de diversos efluentes sob condições mesofílicas, em experimentos de bancada. Referências bibliográficas indicam que reatores do tipo UASB tratando vinhaça sob condições termofílicas, atingem remoção de DQD da ordem de 70% e composição do gás com até 60% de metano. Neste experimento, os dados obtidos de remoção de DQD foram de aproximadamente 70% para valores de DQD afluente da ordem de 10.000 miligramas por litro e porcentagem de metano no gás de 63%, dados esses que indicam a viabilidade do tratamento da vinhaça neste reator. Resulta-se a natureza de pesquisa exploratória desta etapa dos trabalhos, sendo necessária a realização de etapa complementar visando otimizar o tempo de detenção hidráulica e a carga orgânica máxima a ser utlizada. Como um estudo preliminar os resultados foram satisfatórios. / The present work proposes to verify the viability to use the Horizontal Anaerobic Immobilized Sludge (HAIS) reactor for the vinasse treatment in termophilic conditions, with emphasis in the reactor departure. The HAIS reactor has being to utilize with success on the treatment of many effluents under mesophilic conditions in workbench experiments. Bibliographic references indicate that reactors the UASB kind treat vinasse under termophilic conditions, arrive removal the COD (chernical oxygen demand) in the order the 70% and gas composition with 60% of methane. In this experiment, the data obtained the removal of COD were about 70% for values de affluent COD the order the 10.000 mg/L and percentage the methane in the gas the 63%, data these that indicate the viability the termophilic treatment of vinasse in this reactor. Stick out it the nature the exploratory research in this stage the work being necessary the realization the complementary stage aiming at optirnize the hydraulic detention time and the maxim organic load to be utilized. How a preliminary study the results were satisfactory.