Дисертації з теми "Anaerobic hydrolysis"

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

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 (37oC and 42oC) 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 digesterâ s performance. The pre-treatment technique was observed to deliver the best results at a 20 day SRT.
Master of Science

Endoglucanases play an important function in cellulose hydrolysis and catalyse the initial attack on the polymer by randomly hydrolysing the β-1,4 glucosidic bonds within the amorphous regions of cellulose chains. Cellulolytic bacteria have been isolated and characterised from the sewage sludge and the activation of several hydrolytic enzymes under biosulphidogenic conditions of sewage hydrolysis has been reported. The aims of this study were to: identify, induce production, locate and isolate, characterise (physicochemical and kinetic) and purify endoglucanases from anaerobic biosulphidogenic sludge. The endoglucanase activities were shown to be associated with the pellet particulate matter and exhibited a pH optimum of 6 and temperature optimum of 50 °C. The enzymes were thermally more stable when immobilised to the floc matrix of the sludge than when they were released into the aqueous solution via sonication. For both immobilised and released enzymes, sulphate was slightly inhibitory; activity was reduced to 84 % and 77.5 % of the initial activity at sulphate concentrations between 200 and 1000 mg/l, respectively. Sulphite was stimulatory to the immobilised enzymes between 200 and 1000 mg/l. Sulphide stimulated the activities of the immobilised endoglucanases, but inhibited activities of the soluble enzymes above 200 mg/l. The enzyme fraction did not hydrolyse avicel (a crystalline substrate), indicating the absence of any exocellulase activity. For CMC (carboxymethylcellulose) and HEC (hydroxylethylcellulose) the enzyme had K_m,app_ values of 4 and 5.1 mg/ml respectively and V_max,app_ values of 0.297 and 0.185 μmol/min/ml respectively. Divalent ions (Cu²⁺, Ni²⁺ and Zn²⁺) proved to be inhibitory while Fe²⁺, Mg²⁺ and Ca²⁺ stimulated the enzyme at concentrations between 200 and 1000 mg/l. All the volatile fatty acids studied (acetic acid, butyric acid, propionic acid and valeric acid) inhibited the enzymes, with acetic acid eliciting the highest degree of inhibition. Sonication released ~74.9 % of the total enzyme activities into solution and this was partially purified by PEG 20 000 concentration followed by DEAE-Cellulose ion exchange chromatography, which resulted in an appreciable purity as measured by the purification factor, 25.4 fold.

Lipids represent an important fraction of the particulate organic charge in slaughterhouse wastewater. Anaerobic treatment of slaughterhouse wastewater has been reported to be slowed down or impaired because of high concentrations of suspended solids, particularly fats. However, the fate of lipids during anaerobic digestion has been poorly defined, especially for wastewaters from the meat processing industry. The objectives of this thesis were thus (1) to evaluate the effect of hydrolysis pretreatment on the anaerobic digestion of fat particles in slaughterhouse wastewater; (2) to characterise and quantify neutral fat hydrolysis and long-chain fatty acid (LCFA) oxidation during anaerobic degradation of slaughterhouse wastewater with and without hydrolysis pretreatment; and (3) to determine the effect of particle size on fat hydrolysis. The efficiency of four pretreatments to hydrolyse and reduce the size of pork and beef fat particles during mixing at room temperature was tested: NaOH and three commercial lipases of plant, bacterial and animal origins. The most promising pretreatment was the pancreatic lipase PL-250 that could significantly reduce the initial average particle size (Din) of pork fat by a maximum of 40% after 4 h of mixing at room temperature. Approximately 35% of the neutral fat was hydrolysed after 5.5-h of pretreatment with 250 mg/l of PL-250 in a substrate containing approximately 2000 mg/l of pork fat particles. Most of the free LCFAs released during the hydrolytic pretreatment remained adsorbed on the fat particle surface. The effect of pretreatment with PL-250 on subsequent anaerobic digestion of the substrate was evaluated by feeding control and enzyme pretreated slaughterhouse wastewater containing pork fat particles to anaerobic sequencing batch reactors (ASBRs) operated at 25°C. The main conclusions from the experiment were: (1) Pretreatment with PL-250 only had a small effect on pork fat particle digestion at 25°C, marked by a decrease of about 5% in digestion time to achieve 80% reduction in initial neutral fat and free LCFA concentrations. (2) Anaerobic degradation of pork fat particles is mainly controlled by free LCFA oxidation and, in ASBRs operated at 25°C, near maximum oxidation rate is reached at low free LCFA concentration. Consequently, increasing the initial free LCFA concentration by prehydrolysing the substrate will have limited effect on fat degradation rate. (3) At Din ranging from 60 to 450 mum, pork fat hydrolysis rate in anaerobic reactors is not a function of particle size. The fat particles became more filamentous and plate-like as their size was increased. Bacteria could probably colonise the inside as well as the outside of the particles. Consequently, specific surface area (m2/m3) available for hydrolysis was not significantly increased by decreasing the pork fat particle size. (4) Neutral fat hydrolysis and free LCFA oxidation rates can be adequately modelled using first-order and Monod-type kinetics, respectively. The first-order hydrolysis rate constant averaged 0.63 +/- 0.07 d-1, while the maximum oxidation rate (kmax) and half-saturation concentration (Ks) were estimated at 164 +/- 37 mg free LCFA /l/d and 35 +/- 31 mg free LCFA/1, respectively. (5) Fat hydrolysis rate will be underestimated if based on the increase in soluble compounds with respect to particulate organics. An analytical method that removes bound LCFAs from solids surface must be used to measure lipid hydrolysis.

The increased demand for advanced sludge stabilization in wastewater treatment facilities over the past decade has led to the implementation of various pretreatment techniques prior to anaerobic digestion. In an attempt to reduce sludge volumes and improve sludge conditioning properties, the use of thermal hydrolysis process before anaerobic digestion has been adopted with an increase in solids destruction, COD removal, and methane gas. In this study, the evaluation of thermal hydrolysis process as a viable pretreatment strategy to anaerobic digestion has been conducted in order to assess its capacity for solids solubilization. Solubilization experiments were conducted at temperatures ranging from 130 to 170℃ and reaction times between 10 and 60 min. Anaerobic biogas production by thermally pre-treated sludge was carried out through a mesophilic anaerobic digester. The results showed that solids solubilization increased with increases in temperature and time, while temperatures above 160℃ for 30 min strongly affected the sludge characteristics. Ammonia production via deamination by thermal hydrolysis was less significant than protein solubilization at a temperature of 170℃. Both protein and carbohydrate solubilization were more dependent on temperature than reaction time. The enhancement of the biogas production was achieved with increases in temperature as pretreatment of 170℃ yielded 20% more biogas than at 130℃. However, it seems the enhancement was linked to the initial biodegradability of the sludge.
Master of Science

A series of lab-scaled digestion studies including conventional mesophilic anaerobic digestion(MAD), thermophilic anaerobic digestion (TAD) at a range of treatment temperatures, and mesophilic high solids digestion of thermally pretreated wastewater sludge (THD) were carried out. Enhanced digestion performance in terms of solids destruction and methane generation by THD relative to MAD was achieved, and was largely attributable to the solubilization and subsequent biodegradation of energy-rich substrates within blended primary and secondary sludge. TAD was observed to underperform MAD, especially at elevated temperatures as methanogenic inhibition resulted in the accumulation of headspace hydrogen, thus resulting in poor removal of volatile fatty acids. The thermodynamics of fatty acid metabolism was favorable at each digestion temperature, thus it was concluded that microbial inhibition was the controlling factor in poor thermophilic performance. Inhibition by free unionized ammonia (NH₃) was characterized for THD and MAD biomass. Acetic acid degradation was equally affected over a range of NH₃ concentrations; however, methane generation by THD was less sensitive to ammonia inhibition, thus suggesting that methanogenesis by THD was less dependent on the NH₃-sensitive process of aceticlastic methanogenesis. Total ammonia nitrogen (TAN) and bicarbonate alkalinity were stoichiometrically produced from proteinaceous material during thermal hydrolytic pretreatment and subsequent high solids anaerobic digestion. Combined effects of TAN and high pH resulted in NH₃-inhibition during THD. Kinetic evaluations suggested that a growth rate reduction of approximately 65% was associated with in-situ NH₃ concentrations of the THD reactor. NH₃-inhibition was apparently responsible for a shift in dominant methanogenic community of the aceticlastic Methanosarcina barkeri in MAD to the hydrogenotrophic Methanoculleus bourgensis in THD. A similar shift in methanogenic community was observed between low temperature thermophilic digestion at 47°C, where the dominant order was Methanosarcinales, to high temperature thermophilic digestion at 59°C where the dominant order was Methanobacteriales. These findings support a process-driven pathway shift from aceticlastic to non-aceticlastic methanogenesis between 180 and 290 mg/L NH₃-N. Such a threshold is supported by previous literature related to ammonia tolerance of pure cultures of methanogens and has significant implications for the kinetic design of advanced anaerobic digestion processes.
Ph. D.

Tese de Doutoramento em Ciência e Tecnologia Animal
Cellulosomes are one of nature’s most elaborate and highly efficient nanomachines. These cell bound multi-enzyme complexes orchestrate the deconstruction of cellulose and hemicellulose, two of the most abundant polymers on earth, thus playing a major role in carbon turnover. Integration of cellulosomal components occurs via highly ordered protein:protein interactions between cohesins and dockerins, whose specificities allow the precise incorporation of cellulases and hemicellulases onto a molecular scaffold. Clostridium thermocellum and C. cellulolyticum cellulosomes have been extensively characterized and constitute the paradigm for the organization of cellulases and hemicellulases in multi-enzyme complexes by thermophilic and mesophilic anaerobic bacteria, respectively. The recent sequencing of C. thermocellum and C. cellulolyticum genomes allowed the identification of the complete set of cohesins, dockerins and cellulosomal domains encoded by these bacteria. Here, several unresolved issues concerning cohesin-dockerin specificity, cellulosome assembly and the role of cellulosomal catalytic components in plant cell wall hydrolysis will be explored. The ligand specificities of some newly identified C. thermocellum cohesin and dockerin domains were described (Chapter 2). A novel cell-bound protein, termed OlpC, which contains a type I cohesin domain was discovered in C. thermocellum. A restricted set of dockerins were shown to interact, primarily, with OlpC. All the remaining dockerin containing polypeptides expressed by C. thermocellum are directed to cellulosomes. Significantly, the structure of two C. cellulolyticum cohesin-dockerin complexes revealed that, as it was previously reported for C. thermocellum, mesophilic dockerins also express a dual binding mode for cohesins (Chapter 3). Initial crystallization studies with the two N-terminal domains of C. thermocellum cellulosomal xylanase Xyn10B anticipate the elucidation of its 3D structure, which may provide insightful data concerning the function of this enzyme in plant cell wall hydrolysis (Chapter 4). Finally, a cellulosomal family 2 CE (CtCE2), which grafts a second discrete non-catalytic binding functionality into its active site, was characterized (Chapter 5). CtCE2 provides a rare example of “gene sharing” where the introduction of a second functionality into the active site of an enzyme does not compromise the original activity of the biocatalyst.
RESUMO - Nova perspectiva no mecanismo de integração do celulossoma e na degradação da parede celular vegetal por bactérias anaeróbias - Os celulossomas são um dos mais intricados e eficientes complexos multi-enzimáticos existentes na Natureza. Estes complexos, que se encontram ligados à parede celular bacteriana, desempenham um papel importante na degradação da celulose e da hemicelulose, dois dos mais abundantes polímeros na terra. A integração dos componentes celulossomais ocorre através de interacções proteína-proteína, muito ordenadas, estabelecidas entre coesinas e doquerinas, cuja especificidade permite a incorporação precisa de celulases e hemicelulases numa proteína de integração celulossomal. Os celulossomas dos organismos Clostridium thermocellum e C. cellulolyticum têm sido extensivamente caracterizados e constituem o paradigma para a organização de celulases e hemicelulases em complexos multienzimáticos de bactérias anaeróbias, tanto termófilas como mesófilas, respectivamente. A recente sequenciação dos genomas do C. thermocellum e do C. cellulolyticum permitiu a identificação de um conjunto completo de coesinas, doquerinas e domínios celulossomais codificados por estas bactérias. Neste trabalho, várias questões relativas à especificidade coesina-doquerina, à formação do celulossoma e ao papel dos componentes celulossomais catalíticos serão investigadas. A especificidade de doquerinas e coesinas do C. thermocellum recentemente identificados foi descrita (Capítulo 2). Uma nova proteína da parede celular, designada como OlpC, que contém um domínio doquerina, foi descoberta no C. thermocellum. Demonstrou-se que um conjunto restrito de doquerinas reage preferencialmente com a OlpC. Os restantes polipéptidos expressos pela bactéria C. thermocellum, contendo também doquerinas, ligam-se ao celulossoma. A estrutura de dois complexos coesina-doquerina do C. cellulolyticum revelou, como previamente comunicado para a bactéria C.thermocellum, que as doquerinas de organismos mesófilos também apresentam uma dupla ligação para com as coesinas (Capítulo 3). Estudos preliminares de cristalização dos dois domínios Nterminais da xilanase celulossomal Xyn10B antecipam a futura elucidação da sua estrutura 3D, o que poderá esclarecer a função deste enzima na hidrólise da parede celular vegetal (Capítulo 4). Finalmente, foi descrita uma esterase de hidratos de carbono da família 2 (CtCE2), que apresenta uma funcionalidade discreta, não-catalítica de ligação a glúcidos no seu centro catalítico. A CtCE2 fornece um raro exemplo de “gene sharing”, onde a introdução de uma segunda funcionalidade no centro catalítico de uma enzima não compromete a actividade original do biocatalisador.
This work was funded by Fundação para a Ciência e a Tecnologia, grant SFRH/BD/25439/2005 Co-funded by POCTI/BIA-PRO/59118/2004 and PPCDT/BIA-PRO/59118/2004 from Ministério da Ciência, Tecnologia e Ensino Superior

The hydrolysis of complex organic heteropolymers contained in municipal wastewater to simpler monomers by extracellular hydrolytic enzymes is generally considered the rate-limiting step of the biodegradation process. Previous studies of the Recycling Sludge Bed Reactor (RSBR) revealed that the hydrolysis of complex particulate organics, such as those contained in primary sludge (PS), was enhanced under anaerobic biosulphidogenic conditions. Although the mechanism was not fully understood, it appeared to involve the interaction of sulfide and sludge flocs. The current study was conducted using a 3500 ml laboratory-scale RSBR fed sieved PS at a loading rate of 0.5 kg COD/m³.day and an initial chemical oxygen demand (COD) to sulfate ratio (COD:SO₄) of 1:1. There was no significant accumulation of undigested sludge in the reactor over the 60-day experimental period and the quantity of SO₄ reduced indicated that the yield of soluble products from PS was at least as high as those reported previously for this system (> 50%). In the current study, the specific activities of a range of extracellular hydrolytic enzymes (L-alanine aminopeptidase, L-leucine aminopeptidase, arylsulphatase, α-glucosidase, β- glucosidase, protease and lipase) were monitored in a sulfide gradient within a biosulphidogenic RSBR. Data obtained indicated that the specific enzymatic activities increased with the depth of the RSBR and also correlated with a number of the physicochemical parameters including sulfide, alkalinity and sulfate. The activities of α- glucosidase and β-glucosidase were higher than that of the other enzymes studied. Lipase activity was relatively low and studies conducted on the enzyme-enzyme interaction using specific enzyme inhibitors indicated that lipases were probably being digested by the proteases. Further studies to determine the impact of sulfide on the enzymes, showed an increase in the enzyme activity with increasing sulfide concentration. Possible direct affects were investigated by looking for changes in the Michaelis constant (Km) and the maximal velocity (Vmax) of the crude enzymes with varying sulfide concentrations (250, 400 and 500 mg/l) using natural and synthetic substrates. The results showed no significant difference in both the Km and the Vmax for any of the hydrolytic enzymes except for the protease. The latter showed a statistically significant increase in the Km with increasing sulfide concentration. Although this indicated a direct interaction, this difference was not large enough to be of biochemical significance and was consequently not solely responsible for the enhanced hydrolysis observed in the RSBR. Investigation into the floc characteristics indicated that the biosulphidogenic RSBR flocs were generally small in size and became more dendritic with the depth of the RSBR. Based on the above data, the previously proposed descriptive models of enhanced hydrolysis of particulate organic matter in a biosulphidogenic RSBR has been revised. It is thought that the effect of sulfide on the hydrolysis step is primarily indirect and that the reduction in floc size and alteration of the floc shape to a more dendritic form is central to the success of the process.

Two different sludge pretreatments were investigated in an attempt to improve the management and performance of processes for the co-digestion of biosolids with fats, oils, and grease (FOG). The mechanisms of long chain fatty acids (LCFA) degradation in thermal hydrolysis pretreatment and the influence of pH on LCFA degradation in two-phase co-digestion systems were studied. LCFA thermal hydrolysis was investigated at different temperatures (90-250 °C) and reaction times (30 minutes and 8 hours). Approximately 1% of saturated fatty acids were degraded to shorter chain fatty acids at 140 and 160 °C (8-hr thermal hydrolysis). Only 1% or less of unsaturated fatty acids were degraded from 90 to 160 °C (8-hr thermal hydrolysis). Little degradation (< 1%) of both saturated and unsaturated LCFAs was observed at a 30-min reaction time. Both groups of LCFAs were stable up to 250 °C (30-min hydrolysis). The use of chemical-thermal treatments was also investigated. Only unsaturated LCFAs, C18:1 and C18:2, were degraded when thermally hydrolyzed with hydrogen peroxide coupled with activated carbon or copper sulfate. Semi-continuous, acid-phase digesters (APDs) under different pH conditions were studied in order to understand the effects of pH on FOG degradation. Increases in soluble chemical oxygen demand (SCOD) were observed in all APDs. However, the APDs with pH adjustment appeared to perform better than the controls in terms of solubilizing organic compounds. Approximately 38% and 29% of total COD (TCOD) was solubilized, and maximum volatile fatty acid (VFA) concentrations of 10,700 and 7,500 mg/L TCOD were achieved at pH 6 and 7, respectively; It is useful to note that the feed sludge had a VFA concentration of 2,700 mg/L COD. Higher pH (6.0-7.0) showed less accumulation of LCFA materials and more soluble LCFAs in the APDs. This indicates that the lower pH in the APDs was most likely the cause of precipitation and accumulation of LCFAs due to saturation of unsaturated LCFAs.
Master of Science

The slow degradation of complex organics such as waste activated sludge (WAS) is a well-known limitation that impacts the process rate of conventional anaerobic digestion (AD). Thermal pretreatment can accelerate the digestion process by disrupting the structure of WAS before AD. The present research was initiated by comparing the two commonly used thermal pretreatment methods, conductive (conventional) heating (CH) and microwave (MW) hydrolysis, for enhanced sludge disintegration and AD performance. A bench-scale programmable MW oven operated at a frequency of 2.45 GHz was used for MW pretreatment. The CH was performed using a custom-built pressure sealed vessel which could simulate the MW pretreatment under any arbitrary heating profiles. After comparing the CH and MW pretreatments, a novel and highly efficient radio frequency (RF) pretreatment system at a frequency of 13.56 MHz was designed, manufactured, and tested for the first time. The RF system was custom-designed based on the dielectric characteristics of thickened WAS (TWAS) to achieve very efficient as well as uniform heating. The effects of the novel RF pretreatment system on sludge solubilization and AD performance were compared with those of the commercially available MW ovens. Considering the obtained results and analyses, under identical thermal profiles, the thermal pretreatment methods (CH, MW at 2.45 GHz, and RF at 13.56 MHz) achieved similar sludge disintegration as well as AD performance (p-value>0.05). However, the pretreatment temperature, heating rate, and holding time were significant factors in determining the sludge solubilization ratio and AD performance. Ohmic heating was found as the primary heating mechanism at a frequency of 13.56 MHz. It causes the ionic conduction flow to dominate the heating mechanism in the custom-designed RF pretreatment system by contributing to more than 99% of the total dissipated power. Considering the impedance measurement results, the power transfer efficiency of the RF heating system was above 88% throughout the operation. The overall energy efficiency of the RF pretreatment system was measured between 67.3 to 95.5% for the temperature range of 25 to 120°C which was significantly higher than the MW system efficiency which varied from 37 to 43%.
Applied Science, Faculty of
Engineering, School of (Okanagan)
Graduate

Landfill leachates and thermal hydrolysis pretreated anaerobic digestion centrate can quench UV light at publically owned treatment works (POTWs). Increased eutrophication, has led to tightening of nutrient discharge limits in some regions of the country. Biologically recalcitrant organic nitrogen, adds to effluent nitrogen making it difficult to meet these requirements.

The study aimed at characterizing landfill leachate and centrate fractions to develop an understanding that might help design on-site treatment methods. Leachates varying in on-site treatment and ages were fractionated on basis of hydrophobic nature. Humic substances were the major UV light quenching fractions. Majority of the humic substances were > 1 kDa molecular weight cut off (MWCO) indicating that membrane treatment might be effective for UV quenching substances removal. UV absorbing substances were found to be more bio-refractory than organic carbon. Significant decrease in humic substances with long term landfilling indicated that age was important in determining the potential for leachates to impact the UV disinfection. Organic nitrogen was observed to be hydrophilic in nature (mostly < 1 kDa). Proteins which are easily biodegradable contributed around one-third of the organic nitrogen.

For thermal hydrolysis centrate, the optimum treatment depended on particle size and hydrophobic nature. Biological treatment was observed to be more effective for the removal of  
organic matter and UV254 quenching substances for fractions < 300 kDa. Biological treatment had little impact on organic nitrogen. Coagulation-flocculation is an effective treatment for higher molecular weight (MW) fractions whereas a membrane bioreactor would be more suitable for smaller MW fractions.
Master of Science

This study is aimed to provide comprehensive evaluation and mechanistic understanding of the impact of process intensification techniques applied in main and side stream wastewater treatment on biosolids management in terms of anaerobic digestion enhancement, dewaterability improvement, odor mitigation, as well as phosphorus and nitrogen removal. The first part of this study was conducted to understand the effect of anaerobic digester solids retention time (SRT) on odor emission from biosolids. A kinetic model and inhibitory studies showed the emission of methanethiol (MT), a representative odor compound, was primarily determined by the dynamic concurrence of MT production from amino acid and utilization by methanogens in the course of anaerobic digestion. MT emission pattern follows a bell-shape curve with SRT in anaerobic digesters. However, for digested and dewatered biosolids, SRT ranging from 15 to 50 days in anaerobic digesters demonstrated insignificant effect on the odor emission from biosolids. In contrast, the peak odor emission was found to exponentially increase with both shear intensity and polymer dose applied during dewatering. The second part of this study investigated the impact of process intensification practices on sludge dewatering performance. The integration of high-rate activated sludge process and anaerobic digestion elevated the sludge orthophosphate level, leading to struvite scaling and dewaterability deterioration. Superior orthophosphate removal, significant improvement of sludge dewaterability, and favorable economics were achieved through sludge conditioning by cerium chloride. Continuous flow aerobic granulation technology offered significant process intensification of mainstream treatment trains. However, its impact on biosolids management was not studied. This study showed that there was little dewaterability difference between aerobic granular sludge and activated sludge when polymer was not added. However, about 75% polymer saving and improved dewatering performance were observed with polymer addition. When subjected to high shear, a greater dewaterability deterioration was observed for granular sludge than activated sludge. The last part of this study is focused on the impact of anaerobic digestion process intensification through thermal treatment including pre-pasteurization, thermophilic anaerobic digestion, temperature phased anaerobic digestion, and thermal hydrolysis pretreatment. Improved methane production, pathogen reduction, dewatering performance, and odor mitigation were observed with the involvement of these high-temperature processes. However, special cautions and measure should be taken during the start-up of these high rate processes as they are more liable to digester souring. In addition, the in-depth understanding of the mechanism of recalcitrant dissolved organic nitrogen formation during sludge thermal pretreatment was provided.
Doctor of Philosophy
This study is aimed to provide comprehensive evaluation and mechanistic understanding of the impact of process intensification techniques applied in main and side stream wastewater treatment on biosolids management in terms of anaerobic digestion enhancement, dewaterability improvement, odor mitigation, as well as phosphorus and nitrogen removal. The first part of this study was conducted to understand the effect of anaerobic digester solids retention time (SRT) on odor emission from biosolids. A kinetic model and inhibitory studies showed the emission of methanethiol (MT), a representative odor compound, was primarily determined by the dynamic concurrence of MT production from amino acid and utilization by methanogens in the course of anaerobic digestion. MT emission pattern follows a bell-shape curve with SRT in anaerobic digesters. However, for digested and dewatered biosolids, SRT ranging from 15 to 50 days in anaerobic digesters demonstrated insignificant effect on the odor emission from biosolids. In contrast, the peak odor emission was found to exponentially increase with both shear intensity and polymer dose applied during dewatering. The second part of this study investigated the impact of process intensification practices on sludge dewatering performance. The integration of high-rate activated sludge process and anaerobic digestion elevated the sludge orthophosphate level, leading to struvite scaling and dewaterability deterioration. Superior orthophosphate removal, significant improvement of sludge dewaterability, and favorable economics were achieved through sludge conditioning by cerium chloride. Continuous flow aerobic granulation technology offered significant process intensification of mainstream treatment trains. However, its impact on biosolids management was not studied. This study showed that there was little dewaterability difference between aerobic granular sludge and activated sludge when polymer was not added. However, about 75% polymer saving and improved dewatering performance were observed with polymer addition. When subjected to high shear, a greater dewaterability deterioration was observed for granular sludge than activated sludge. The last part of this study is focused on the impact of anaerobic digestion process intensification through thermal treatment including pre-pasteurization, thermophilic anaerobic digestion, temperature phased anaerobic digestion, and thermal hydrolysis pretreatment. Improved methane production, pathogen reduction, dewatering performance, and odor mitigation were observed with the involvement of these high-temperature processes. However, special cautions and measure should be taken during the start-up of these high rate processes as they are more liable to digester souring. In addition, the in-depth understanding of the mechanism of recalcitrant dissolved organic nitrogen formation during sludge thermal pretreatment was provided.

The UK buries about 100 million tonnes of waste a year, of which 25% is municipal solid waste (refuse). The environmental impacts from gas and leachate releases are known and direct risks to health from landfill are reported. Europe has agreed to a Landfill Directive which has set targets for the stepwise reduction in biodegradable municipal waste going to landfill. The anaerobic digestion of municipal solid waste in controlled bioreactors is an area that could play an important role in overall evolution towards sustainability by recovering biogas and organic matter. Separated hydrolysis and subsequent anaerobic codigestion was demonstrated from the literature review to have the best potential for biodegradable municipal waste diverted from landfill. The rate of hydrolysis of solids wastes remains an outstanding problem. In this research, firstly the codigestion of industrial effluent (coffee wastewater), food wastes and garden wastes were investigated for their impact on hydrolysis and digestion. The results show that there were no treatability problems for coffee wastes up to 37.5% of volume feed per day at the HRT of 9 days. The results supported the view that dilute biodegradable streams such as coffee waste may improve digestion by promoting mixing. Fruit and vegetable wastes were highly biodegradable and can have a major improvement in biogas production of the whole codigestion process, whereas garden waste was not as successful as a cosubstrate, probably because of the predominant celluloses and lignocelluloses with a low biodegradability. The literature review also revealed that washing or elutriation can remove organic matter from municipal waste. This is an important hydrolytic process in which a solubilised acidic organic matter is obtained. The codigestion of refuse hydrolysate with sewage sludge was therefore studied. A control digester treating sewage sludge only was compared with an experimental reactor fed mixed refuse hydrolysate with sewage sludge. It was possible to add the solubilised hydrolysate to existing anaerobic digesters designed at a standard sludge solids loading rate without causing overloading. (Continues...).

Anaerobic digestion has been the preferred method for reducing and stabilizing waste sludge from biological wastewater treatment for over a century; however, as sludge volumes and disposal costs increase, there has been a desire to develop various methods for reducing the volume of sludge to be treated, improving the performance of the digesters, and increasing the energy value of the sludge. To this end, there have been numerous pretreatment and side-stream systems studied and developed over the past several decades with the overall goal of reducing the volume of biosolids to be disposed of in landfills or by land application. These technologies can be broken into four large groups: mechanical, thermal, chemical and biological, although there is often overlap between groups.

This research approached the evaluations of these technologies through several methods in the hopes of developing effective tools for predicting the performance of each technology. Batch digestion studies mimicking several of these treatment methods and extensive analytical work on samples from full-scale installations were conducted to determine the effectiveness of each technology. From these studies a simple batch digestion methodology was developed to analyze the effectiveness of the Cannibal solids reduction process on wastewater streams that have never been exposed to it. Batch digestion of sludges pretreated with ozone, mechanical shear and sonication provided insight into the effectiveness of each technology. Extensive analytical work on samples collected from full-scale installations of thermal hydrolysis, mechanical shear and the Cannibal process provided some insight into the workings of each process and potential leads as to how to further characterize and evaluate each process.
Master of Science

Class A biosolids are solid by-product of wastewater treatment which meet Environmental Protection Agency requirements to be used as fertilizer in farms, vegetable gardens, and can be sold directly to consumers. In 2014, this study’s target nutrient recovery facility adopted thermal hydrolysis pretreatment and anaerobic digestion to upgrade biosolids quality from Class B (previously lime-stabilized) to Class A. In order to certify if this newly produced material met all regulatory requirements, we performed laboratory analysis to characterize fecal coliforms, volatile solids, and metals content. In addition, we showed a baseline for nutrient management of total nitrogen, phosphorus, and the change in levels of polybrominated diphenyl ethers (PBDEs). Samples were collected for over a year since the start of THP-AD operation. Results were compared with the Class B biosolids produced at the same facility. Based on EPA standards, Class A biosolids were produced with stable quality after March, 2015, 16 weeks after process initiation. This work suggests that THP-AD is effective in producing Class A biosolids. In general, PBDEs in biosolids decreased from 1790 ± 528 (Class B) to 720 ± 110 µg/kg d.w. Our results suggest that the total levels of PBDEs decrease, however, the impact of the THP-AD on specific congeners are complex.

The doctoral thesis is focused on the microbial production of lactic acid and ethanol using food processing waste as substrate. Coffee processing waste (spent coffee grounds), wine production waste (grape pomace) and orange processing waste (orange peel) were chosen as substrates for experiments. The theoretical part is dedicated to summarizing current knowledge about waste from food production and possibilities of its processing. It also deals with selected metabolites (lactic acid, ethanol) to which these wastes can be used. Part of the experiments was focused on the characterization and optimization of hydrolysis to maximize the amount of fermentable saccharides. Different combinations of chemical, physical and enzymatic hydrolysis of selected substrates have been tested. Subsequently, a suitable strain for lactic acid and ethanol production was searched for. In the case of lactic acid production, 7 bacterial strains were selected (Lactobacillus casei CCM 4798, Bacillus coagulans CCM 2013, Bacillus coagulans CCM 2658, Lactobacillus rhamnosus CCM 1825T, Lactobacillus delbruckii subsp. bulgaricus CCM 7190, Lactobacillus plantarum CCM 7039T, Streptococcus thermophilus CCM 4757). These strains were first cultivated on the synthetic media containing different kind of saccharides. Afterward, the cultivation on the waste biomass hydrolysates were tested. In the case of ethanol production, 2 yeast strains kmeny (S. cerevisiae CNCTC 6646 a S. cerevisiae CNCTC 6651) were cultivated on hydrolysates of individual waste substrates. Subsequently, the experiments focused on the production of lactic acid and ethanol on hydrolysates of waste biomass in bioreactor were done. The last part of this doctoral thesis deals with the microaerobic pretreatment of lignocellulosic biomass to increase the production of organic acids during the acetogenic phase of anaerobic digestion.

O presente trabalho avaliou a geração de biogás em um reator de duas fases, operado em bateladas sequencias, com resíduos coletados no CEAGESP de São Paulo-SP. O reator com volume efetivo de 10,7 L, era composto por um biofiltro anaeróbio em sua parte inferior, seguido por um separador de gases e posteriormente por um depósito de resíduos a digerir na parte superior. O inóculo era proveniente do fundo de uma lagoa de lixiviados do aterro de São Carlos-SP. O experimento foi conduzido em temperatura controlada em 30 ± 2 ºC nas Etapas 1, 2 e 3. A Etapa 1 consistiu na ativação e adaptação da biomassa, utilizando-se etanol e posteriormente resíduo orgânico do CEAGESP. A Etapa 2 consistiu na operação anaeróbia do sistema com resíduo do CEAGESP com lodo já adaptado. Na Etapa 3, verificou-se o efeito da aeração no compartimento de resíduos. Na Etapa 4, avaliou-se a influência da temperatura na digestão anaeróbia e com isto foi possível a obtenção do coeficiente de Arrhenius. Na Etapa 5, comparou-se o sistema de duas fases com um segundo reator anaeróbio, com configuração parecida com a convencional. Os resultados obtidos de todas as etapas na geração de biogás no reator de duas fases foram de 0,44; 0,44; 0,47 m3 /Kg SV e as eficiências de remoção de Sólidos Voláteis foram de 82,1%; 84,5% e 84,8% nas Etapa 1, 2 e 3, respetivamente, com um tempo de ração de 14 d nas três etapas. As concentrações de metano foram de 68,4; 67,1 e 66,6%, respectivamente. Na Etapa 4, os resultados da geração de biogás foram de 0,36; 0,38; 0,41; 0,41 m3 biogás/Kg SV nas temperaturas 25,6 ºC; 28,9 ºC; 34,0 ºC e 38,1 ºC, respetivamente. Denotando que a temperatura é um fator importante na geração de biogás em digestão anaeróbia. No reator convencional, a geração de biogás foi de 0,32 m3 biogás/Kg SV.
The present work evaluated the biogas generation in a two-phase reactor, operated in batch sequences, with residues collected at CEAGESP in São Paulo-SP. The reactor with an effective volume of 10.7 L was composed of an anaerobic biofilter in its lower part, followed by a gas separator and later by a deposit of waste to digest in the upper part. The inoculum was from the bottom of a leachate pond in the São Carlos-SP landfill. The experiment was conducted at a temperature of 30 ± 2 º C in Steps 1, 2 and 3. Step 1, consisted of the activation and adaptation of the biomass using ethanol and then organic waste from CEAGESP. Step 2, consisted of the anaerobic operation of the system with residue of the CEAGESP with already adapted sludge. In Step 3, the effect of aeration on the waste compartment was verified. In Step 4, the influence of temperature on the anaerobic digestion was evaluated and with this it was possible to obtain the Arrhenius coefficient. In Step 5, the two-phase system was compared with a second anaerobic reactor, with the same configuration as conventional. The results of all stages in the biogas generation in the two-phase reactor were 0.44; 0.44; 0.47 m3 / Kg SV and the removal efficiencies of Volatile Solids were 82.1%; 84.5% and 84.8% in Step 1, 2 and 3 respectively; and a feed time were of 14 d in the three steps. Consequently, the percentage of methane was 68.4; 67.1 and 66.6%. In stage 4 the results in the biogas generation were 0.36; 0.38; 0.41; 0.41 m3 biogas / Kg SV at temperatures 25.6 ° C; 28.9 ° C; 34.0 ° C and 38.1 ° C respectively. In the conventional reactor the biogas generation was 0.32 m3 biogas / Kg SV.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq
The biological sludge approprieate treatment must be developed in order to take advantages of the products generated during the process. However, the anaerobic sludge treatment is arduous due to the complexity of the substances present in sludge flakes, granules and biofilms, becoming indispensable a pre-treatment step in order to raise the solubilization of the materials. At this study, anaerobic sludges produced by anaerobic reactos with low cell retention time (CRT) (2, 4, 6, 8, 10 and 20 days) were characterized and the 2 and 8 day sludge were submitted to solubilization from pre- treatment commonly called alkaline hydrolysis, consisting of the use of NaOH to reach pH 12, and subjected to stirring for 48 hours at room temperature. The objective of this study was to characterize the anaerobic sludges and evaluate the increase of organic materials solubilization, and make comparisons. The results of the soluble organic materials at 2 and 8 days, showed an increase in the concentrations of 14 and 28 times COD, 20 and 40 times to DQOS, 21 and 63 times to carbohydrates and 31 to 60 times to proteins respectively The solubilization of the 8-day solubilized sludge showed higher solubilization than the 2-day sludge, showing that the use of anaerobic low-CRT reactor to generate sludge is interesting in order to produce a sludge that releases higher amounts of soluble materials after a hidrolisis process, be rapidly degradeted by the anaerobic digestion.
O tratamento adequado do lodo biológico deve ocorrer visando tirar proveito dos produtos gerados durante o processo. No entanto o tratamento anaeróbio do lodo biológico é dificultado pela complexidade das substâncias formadoras dos flocos, grânulos e biofilmes, fazendo-se indispensável uma etapa de pré-tratamento empregada de modo a elevar a solubilização dos materiais. No presente trabalho foram caracterizados 6 lodos anaeróbios provenientes de reatores com baixo tempo de retenção celular (2, 4, 6, 8, 10 e 20 dias) e destes, os lodos de 2 e 8 dias (fase acidogênica) foram submetidos à solubilização a partir do pré-tratamento químico comumente chamado de hidrólise alcalina, consistindo na utilização de NaOH para alcançar pH 12, sob agitação por 48 horas em 28°C. Objetivou-se caracterizar os lodos anaeróbios de baixo tempo de retenção celular (TRC) e avaliar o aumento da solubilização dos materiais orgânicos nos lodos de 2 e 8 dias. Os resultados dos materiais orgânicos solúveis nos lodos de 2 e 8 dias, apresentaram incremento nas concentrações de 14 e 28 vezes para COD, 20 e 40 vezes para DQOS, 21 e 63 vezes para carboidratos e 31 e 60 vezes para proteínas respectivamente. Evidencia-se em todos os parâmetros analisados uma maior solubilização do lodo solubilizado de 8 dias em relação ao lodo de 2 dias, mostrando assim ser interessante a utilização de reatores anaeróbios de baixo TRC para gerar um lodo que após hidrolisado, se solubilize seus materiais e seja degradado mais rapidamente.

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Este trabalho teve como objetivos avaliar a biodegradabilidade anaerÃbia e o potencial de produÃÃo de metano (PPM) de trÃs resÃduos lignocelulÃsicos proveniente da cadeia produtiva do biocombustÃvel (fibra do mesocarpo do dendà - FMD), (bagaÃo de cana-de-aÃÃcar - BCA) e lÃnter de algodÃo tipo 4 - (LA4). Para tanto, empregou-se trÃs diferentes tipos de prÃ-tratamentos fÃsico-quÃmicos (hidrÃlise hidrotÃrmica, hidrÃlise Ãcida e hidrÃlise alcalina), onde foram usados diversos tempos de reaÃÃo, temperaturas, razÃes massa/volume e concentraÃÃes de Ãcido ou Ãlcali, de forma a buscar o melhor mÃtodo para facilitar a digestÃo anaerÃbia do material. Os prÃ-tratamentos foram avaliados usando-se planejamento fatorial multivariado 22 ou 23, com ponto central em triplicata. O PPM e a biodegradabilidade anaerÃbia obtidos com FMD, BCA e LA4 sem prÃ-tratamento foram, respectivamente, 77,8, 35,6 e 165,3 L CH4/kg substrato e 8,7, 4,4 e 24,1%. Os resultados obtidos com a FMD mostraram que o melhor PPM (199 L CH4/kg substrato) foi obtido utilizando o prÃ-tratamento Ãcido com [HCl] de 1,97 M , durante 34 min, a 103 ÂC, o qual promoveu 19% de biodegradabilidade. A digestÃo anaerÃbia do BCA à mais beneficiada quando se utiliza hidrÃlise hidrotÃrmica (10 min, 200 ÂC), resultando em PPM de 199 LCH4/kg Subst. e biodegradabilidade anaerÃbia de 27,4%. Os melhores resultados de PPM e biodegradabilidade do LA4 foram de 397,1 L CH4/kg Subst e 49,1%, obtidos com o prÃ-tratamento Ãcido ([HCl] 1M, 136 ÂC, 20 min). Apesar dos diversos prÃ-tratamento causarem aumento significativo da hidrÃlise anaerÃbia destes resÃduos lignocelulÃsicos, a energia gerada a partir do metano (FMD = 6,9 MJ/kg Subst.; BCA = 6,8 MJ/kg Subst. e LA4 = 13,2 MJ/kg Subst.) foi menor do que a obtida por uma eventual queima direta da fibra (FMD = 9,6 MJ/kg Subst.; BCA = 7,2 MJ/kg Subst. e LA4 = 17,3 MJ/kg Subst. â na forma de briquete). Uma alternativa à utilizar o prÃ-tratamento alcalino para reaproveitamento da lignina extraÃda, alÃm da geraÃÃo de energia. Desta forma, pode-se extrair atà 91% da lignina presente em FMD e 80% em BCA, que pode ser utilizada na indÃstria quÃmica em geral, e gerar 180 e 313,4 L CH4/kg de FMD e BCA hidrolisados, respectivamente. Estes valores sÃo suficientes para gerar 6,2 e 11,2 MJ/kg Subst, respectivamente.
This study aimed the evaluation of the anaerobic biodegradability and methane production potential (MPP) of three lignocellulosic wastes derived from the biofuels production chain: palm oil mesocarp fiber (PMF), sugarcane bagasse (SCB) and cotton linter type 4 (CL4). Three different types of physico-chemical pretreatments were used (hydrothermal hydrolysis, acid hydrolysis and alkaline hydrolysis), which were evaluated based on the solubilisation of sugars or extraction of lignin. Different reaction times, temperatures, mass/volume ratios, and concentrations of acid or alkali were used for seeking the best pretreatment that improves the anaerobic digestion of the material. The data of the pretreatments were analysed using multivariate factorial design 22 or 23, with the central point in triplicate (level 0) and six star-points (when necessary). The MPP and anaerobic biodegradability obtained with PMF, SCB and CL4 without pretreatment were, respectively, 77.8, 35.6 and 165.3 L CH4/kg substrate and 8.7, 4.4 and 24.1%. The results obtained with the PMF showed that the best MPP (199 L CH4/kg substrate) was obtained using acid hydrolysis with [HCl] of 1.97 M, during 34 min, at 103 Â C, which promoted 19% of biodegradability. Anaerobic digestion SCB is improved when using hydrothermal hydrolysis (10 min, 200 ÂC) resulting in a MPP of 199 L CH4/kg substrate and an anaerobic biodegradability of 27.4%. The best results of CL4 were MPP of 397.1 L CH4/kg substrate and biodegradability of 49.1% obtained with acid hydrolysis ([HCl] of 1 M, 136 ÂC, 20 min). Despite the several pretreatment cause significant increase in the anaerobic hydrolysis of these lignocellulosic wastes, the power generated from methane (PMF = 6.9 MJ/kg Subst, SCB = 6.8 MJ/kg Subst, CL4 = 13.2 MJ/kg Subst.) were lower than that obtained by the eventual direct combustion of the fibre (PMF = 9.6 MJ/kg Subst, SCB = 7.2 MJ/kg Subst, CL4 = 17.3 MJ/kg Subst. CL4 in the form of briquette). An alternative is to use the alkaline hydrolysis for extracting lignin and further use in the chemical industry, as well as for power generation. The results show that it is possible to extract up to 91% of the lignin present in the PMF and 80% in the SCB, which can generate up to 180 and 313.4 L CH4/kg of the hydrolysed PMF and SCB, respectively. These values are sufficient to produce 6.2 and 11.2 MJ/kg Subst, respectively.

La bioconversion en méthane de biomasses lignocellulosiques est l’une des alternatives les plus prometteuses pour la production de méthane issu de la digestion anaérobie. Toutefois, les biomasses lignocellulosiques présentent des caractéristiques bio-physico-chimiques très variables en raison leur composition biochimique et de l’organisation structurale très diverses. Par ailleurs, leur faible biodégradabilité en conditions anaérobie nécessite de les prétraiter avant méthanisation pour optimiser la production de méthane. Ce travail vise à évaluer l’influence des caractéristiques d’une large gamme de substrats lignocellulosiques sur leur biodégradabilité anaérobie et les corrélations entre leurs caractéristiques bio-physico-chimiques et le potentiel biométhanogène, et d’étudier les effets du prétraitement fongique en présence de Ceriporiopsis subvermispora sur le potentiel biométhanogène de biomasses lignocellulosiques sélectionnées dans la présente étude et de caractériser les changements de leurs caractéristiques après le prétraitement fongique. La caractérisation de 36 biomasses lignocellulosiques représentatives d’une large gamme de gisements potentiellement mobilisables a permis de mettre en évidence les corrélations linéaires entre le potentiel biométhanogène des biomasses et certaines de leur caractéristiques bio-physico-chimiques, dont la teneur en lignine et la demande biochimique en oxygène. Les biomasses sylvicoles et agricoles ont montré des caractéristiques distinctes de la biodégradabilité aérobie et anaérobie. Les résultats de prétraitement fongique sur les 5 biomasses ont permis de mettre en évidence que le champignon de pourriture blanche Ceriporiopsis subvermispora réagit distinctement selon la biomasse prétraitée. Pour certaines biomasses, le prétraitement fongique conduit à augmenter significativement la production de méthane et la vitesse de bioconversion en méthane. Cette espèce présente la capacité de dégrader sélectivement la lignine sur certaines biomasses et, sur d’autres, celle de dégrader de manière non-sélective des polysaccharides et des lignines. De plus, pour les deux souches de Ceriporiopsis subvermispora testées, des métabolismes différents ont été mis en évidence sur une même biomasse. Les résultats de compositions et ceux de l’analyse structurale des biomasses (initiales, autoclavées, contrôles, et prétraitées par Ceriporiopsis subvermispora) ont montré que leur structure peut être modifiée sans toutefois observer une transformation significative de leur composition biochimique
Bioconversion to methane lignocellulosic biomass is one of the most promising alternatives for the production of methane from anaerobic digestion. However, lignocellulosic biomass has various bio-physicochemical characteristics due to their biochemical composition and diverse structural organization. Moreover, their low biodegradability in anaerobic condition requires pretreatment before methanation to optimize methane production. This work aims to evaluate the influence of the characteristics of a wide range of lignocellulosic substrates on their anaerobic biodegradability and correlations between their bio-physical-chemical characteristics and biomethane potential, and study the effects of fungal pretreatment in the presence of Ceriporiopsis subvermispora on the biogas potential of lignocellulosic biomass selected in this study and characterize their changes of their characteristics before and after the fungal pretreatment. The characterization of 36 representative lignocellulosic biomass of a wide range of potentially mobilized deposits allowed to highlight the linear correlations between biomethane potential of biomass and some of their bio-physical-chemical characteristics, of which the lignin content and biochemical oxygen demand. The forest and agricultural biomass exhibited distinct characteristics of the aerobic and anaerobic biodegradability. The results of fungal pretreatment of the 5 biomass indicated that the white rot fungus Ceriporiopsis subvermispora reacts distinctly depending on the pretreated biomass. For some biomass, fungal pretreatment leads to significant increase of methane production and the bioconversion rate of methane. This species presents the ability to selectively degrade lignin on some biomasses, in others, the ability to non-selectively degrade polysaccharides and lignins. In addition, for both strains of Ceriporiopsis subvermispora tested, different metabolisms were highlighted on the same biomass. The results of compositions and those of the structural analysis of biomass (initials, autoclaved, controls, and pretreated with Ceriporiopsis subvermispora) showed that their structure can be modified without observing a significant transformation of their biochemical composition

The cheese whey represents the most important reject of the industry of dairy products, mainly due to its expressive generated volume. The cheese whey is a problematic substrate under the environmental point of view, presenting high amounts of carbohydrates, proteins and fats, giving it a chemical demand of oxygen of approximately a hundred times larger than the one of the domestic waste. An alternative for its treatment would be the anaerobic fermentation, which reduces its pollutant impact, making possible even the recovery of the energy from the formed biogas. However, the low biodegradation rate of the fats in the cheese whey difficulties the anaerobic treatment, reducing the mass transfer, leading to biomass loss and to the collapse of the reactor. In this context, this work had as objective to evaluate the effect of the preliminary stage from the alkaline hydrolyses of the fats in the anaerobic biodegradation of the cheese whey. A complet experimental design, being considered as the independent variables factors as time and the concentration of NaOH indicated the best hydrolyses conditions as 0,1% of NaOH, reaction time at 15h in 35°C and 200 rpm. In the study of the biodegradability of the cheese whey was used as inoculum a sludge colleted in a anaerobic reactor from the effluents treatment of a food industry. The COD removal and the biogas production were monitored by time, in different concentrations of the cheese whey solution in the basal medium. The removal of COD and the biogas production were higher in the experiments with the previously hydrolyzed solutions, especially for larger concentrations of cheese whey. These results shows that the alkaline hydrolyzes may be an alternative in the biological treatment of effluents with high fat concentration.
O soro de queijo representa o mais importante rejeito da indústria de laticínios, devido principalmente ao expressivo volume gerado. O soro é um substrato problemático sob o ponto de vista ambiental, pois apresenta elevados teores de carboidratos, proteínas e gorduras, que lhe conferem uma Demanda Química de Oxigênio cerca de cem vezes maior que a do esgoto doméstico. Uma alternativa de tratamento do mesmo seria a fermentação anaeróbia, através da qual se reduziria seu impacto poluidor, além de possibilitar a recuperação de energia do biogás formado. No entanto, a baixa taxa de biodegradação das gorduras presentes no soro dificulta o tratamento anaeróbio, reduzindo a transferência de massa, levando à perda de biomassa e até o colapso do reator. Neste contexto, o trabalho teve como objetivo avaliar o efeito de uma etapa preliminar de hidrólise alcalina das gorduras sobre a biodegradação anaeróbia do soro. Um planejamento experimental fatorial 32, considerando-se como variáveis independentes os fatores tempo e a concentração de NaOH indicou as melhores condições de hidrólise como sendo 0,1% de NaOH, tempo de reação de 15 h a 35°C e 200 rpm. No estudo da biodegradabilidade do soro foi empregado como inóculo um lodo coletado em um reator anaeróbio de tratamento de efluentes de uma indústria alimentícia. A remoção de DQO e a produção de biogás foram monitoradas ao longo do tempo, mediante diferentes concentrações da solução de soro no meio basal. A remoção de DQO e a produção de biogás foram mais elevadas nos experimentos com a solução previamente hidrolisada, especialmente para maiores teores de solução de soro. Estes resultados mostram que a hidrólise alcalina pode ser uma alternativa no tratamento biológico de efluentes com altos teores de gordura.
Mestre em Engenharia Química

A new technology for biotechnological treatment of mine waters with both high concentrations of heavy metals and sulphate was developed. The technology is based on the technical coupling of microbially mediated hydrolysis, fermentation and microbial sulphate reduction in a self-stabilising process. Electron donor for sulphate reduction is supplied by degradation of a solid substrate (silage). Elimination of metals is primarily achieved by sulphide precipitation within the sulphate reduction zone. The organic compounds are either supplied by elution or by hydrolysis of polymeric compounds which was named active elution. The concept was realised as a two-phase process with (active) elution in the first phase (R1) and sulphate reduction and metal elimination in the second phase (R2). With this process setup the supply of sufficient amounts of electron donor in R1, a stable and effective sulphate reduction yield as the basis of metal elimination in R2 and a stable separation of microbial processes in R1 and R2 was achieved at hydraulic retention times of 69 h in R1 and 40 h in R2. Almost complete elimination of heavy metals was achieved from wastewaters with 0.2 mM Ni2+, Cu2+, Zn2+, Fe2+ and Mn2. A structurised mathematical model describing the two-phase process was developed on the basis of literature values and tested with data from continuous experiments. Microbial processes were significantly influenced in the presence of precipitated heavy metal sulfides. The effect was dependent on both the bound metal (Ni2+ or Fe2+) and the relative distance between sediment and biomass
Es wurde eine neuartige Technologie zur biotechnologischen Reinigung von schwermetallbelasteten, sulfathaltigen Bergbauwässern entwickelt. Die Technologie basiert auf der technischen Kopplung von mikrobiell vermittelter Hydrolyse, Fermentation und mikrobieller Sulfatreduktion in einem selbststabilisierenden Prozess, wobei aus Abbau eines festen Substanzgemisches (Silage) Elektronendonor zur Sulfatreduktion bereitgestellt wird. Die Schwermetallelimination erfolgt vorrangig durch sulfidische Fällung, die technisch einstufig mit der mikrobiellen Sulfatreduktion realisiert wurde. Die organischen Verbindungen wurden durch Elution bereitgestellt bzw. durch hydrolytischen Abbau von polymeren Verbindungen. Hierfür wurde der Begriff der ?Aktiven Elution? geprägt. Die Konzeption wurde technisch zweistufig umgesetzt. In der ersten Stufe (R1) erfolgt die (Aktive) Elution, in der zweiten Stufe (R2) erfolgen Sulfatreduktion und Schwermetallelimination. Mit der verfahrenstechnischen Umsetzung wurde die Bereitstellung einer ausreichenden Menge an Elektronendonor in R1, eine effektive und stabile Sulfatreduktionsausbeute als Bedingung der Schwermetallelimination in R2 und eine weitgehende Trennung der mikrobiellen Prozesse in R1 und R2 bei Verweilzeiten von 69 h in R1 und 40 h in R2 erreicht. Bei Behandlung von wässrigen Lösungen mit 0,2 mM Ni2+, Cu2+, Zn2+, Fe2+ und Mn2+ konnte eine nahezu vollständige Elimination der Schwermetalle aus der Lösung erreicht werden. Es wurde ein strukturiertes mathematisches Modell für den zweistufigen Prozess auf der Basis von Literaturangaben entwickelt und anhand der kontinuierlichen Laborversuche überprüft. Es wurde ein erheblicher Einfluss schwermetallsulfidischer Präzipitate auf die mikrobiellen Prozesse festgestellt. Dabei wurde dieser Einfluss in Abhängigkeit von der Art der gebundenen Metallionen (Ni2+ oder/und Fe2+) und in Abhängigkeit der relativen räumlichen Anordnung von Sediment und Biomasse festgestellt

A new technology for biotechnological treatment of mine waters with both high concentrations of heavy metals and sulphate was developed. The technology is based on the technical coupling of microbially mediated hydrolysis, fermentation and microbial sulphate reduction in a self-stabilising process. Electron donor for sulphate reduction is supplied by degradation of a solid substrate (silage). Elimination of metals is primarily achieved by sulphide precipitation within the sulphate reduction zone. The organic compounds are either supplied by elution or by hydrolysis of polymeric compounds which was named active elution. The concept was realised as a two-phase process with (active) elution in the first phase (R1) and sulphate reduction and metal elimination in the second phase (R2). With this process setup the supply of sufficient amounts of electron donor in R1, a stable and effective sulphate reduction yield as the basis of metal elimination in R2 and a stable separation of microbial processes in R1 and R2 was achieved at hydraulic retention times of 69 h in R1 and 40 h in R2. Almost complete elimination of heavy metals was achieved from wastewaters with 0.2 mM Ni2+, Cu2+, Zn2+, Fe2+ and Mn2. A structurised mathematical model describing the two-phase process was developed on the basis of literature values and tested with data from continuous experiments. Microbial processes were significantly influenced in the presence of precipitated heavy metal sulfides. The effect was dependent on both the bound metal (Ni2+ or Fe2+) and the relative distance between sediment and biomass.
Es wurde eine neuartige Technologie zur biotechnologischen Reinigung von schwermetallbelasteten, sulfathaltigen Bergbauwässern entwickelt. Die Technologie basiert auf der technischen Kopplung von mikrobiell vermittelter Hydrolyse, Fermentation und mikrobieller Sulfatreduktion in einem selbststabilisierenden Prozess, wobei aus Abbau eines festen Substanzgemisches (Silage) Elektronendonor zur Sulfatreduktion bereitgestellt wird. Die Schwermetallelimination erfolgt vorrangig durch sulfidische Fällung, die technisch einstufig mit der mikrobiellen Sulfatreduktion realisiert wurde. Die organischen Verbindungen wurden durch Elution bereitgestellt bzw. durch hydrolytischen Abbau von polymeren Verbindungen. Hierfür wurde der Begriff der ?Aktiven Elution? geprägt. Die Konzeption wurde technisch zweistufig umgesetzt. In der ersten Stufe (R1) erfolgt die (Aktive) Elution, in der zweiten Stufe (R2) erfolgen Sulfatreduktion und Schwermetallelimination. Mit der verfahrenstechnischen Umsetzung wurde die Bereitstellung einer ausreichenden Menge an Elektronendonor in R1, eine effektive und stabile Sulfatreduktionsausbeute als Bedingung der Schwermetallelimination in R2 und eine weitgehende Trennung der mikrobiellen Prozesse in R1 und R2 bei Verweilzeiten von 69 h in R1 und 40 h in R2 erreicht. Bei Behandlung von wässrigen Lösungen mit 0,2 mM Ni2+, Cu2+, Zn2+, Fe2+ und Mn2+ konnte eine nahezu vollständige Elimination der Schwermetalle aus der Lösung erreicht werden. Es wurde ein strukturiertes mathematisches Modell für den zweistufigen Prozess auf der Basis von Literaturangaben entwickelt und anhand der kontinuierlichen Laborversuche überprüft. Es wurde ein erheblicher Einfluss schwermetallsulfidischer Präzipitate auf die mikrobiellen Prozesse festgestellt. Dabei wurde dieser Einfluss in Abhängigkeit von der Art der gebundenen Metallionen (Ni2+ oder/und Fe2+) und in Abhängigkeit der relativen räumlichen Anordnung von Sediment und Biomasse festgestellt.

Im Laborversuch konnte der positive Einfluss einer thermobarischen Vorbehandlung auf die Hydrolysier- und Vergärbarkeit von Rinderfestmist und Rindergülle nachgewiesen werden. Die Laborergebnisse wurden innerhalb eines theoretischen Modells in den Praxismaßstab übertragen, um den Einfluss auf Treibhausgasemissionen, Energiebilanz und Ökonomie zu bewerten. Die Vorbehandlungstemperaturen im Labor lagen zwischen 140 und 220°C in Schritten von 20 K und einer Vorbehandlungszeit von jeweils 5 Minuten. Die höchste Methanmehr¬ausbeute von 58 % konnte bei einer Temperatur von 180°C ermittelt werden. Das Auftreten von Inhibitoren und nicht vergärbaren Bestandteilen führte bei einer Aufbereitungstemperatur von 220°C zu Methanausbeuten, die geringer waren als die des unaufbereiteten Einsatzstoffes. In einer erweiterten Analyse konnte ein funktioneller Zusammenhang zwischen der Methanausbeute nach 30 Tagen und der Methanbildungsrate und -ausbeute während der Beschleunigungsphase gezeigt werden. Mittels einer Regressionsanalyse der so ermittelten Werte wurde nachgewiesen, dass die optimale Aufbereitungstemperatur 164°C ist und die minimale größer als 115°C zu sein hat. Treibhausgasemissionen und Energiebilanz wurden im Rahmen einer Ökobilanz nach ISO 14044 (2006) ermittelt, sowie eine Kosten-Nutzen-Analyse durchgeführt. Dazu wurde eine Anlage zur thermobarischen Vorbehandlung entwickelt und innerhalb eines Modells in eine Biogasanlage integriert. Weiterhin wurde in diesem Modell Maissilage durch Rinderfestmist und / oder Rindergülle als Einsatzstoff ersetzt. Rinderfestmist, ein Einsatzstoff mit hohem organischen Trockenmassegehalt, der ohne Vorbehandlung nicht einsetzbar wäre, erreichte eine energetische Amortisationszeit von 9 Monaten, eine Vermeidung in Höhe der während der Herstellung emittierten Treibhausgase innerhalb von 3 Monaten und eine ökonomische Amortisationszeit von 3 Jahren 3 Monaten, wohingegen Rindergülle keine positiven Effekte zeigte.
Hydrolysis and digestibility of cattle waste as feedstock for anaerobic digestion were improved by thermobarical treatment in lab-scale experiments. The effects of this improvement on greenhouse gas emissions, energy balance and economic benefit was assessed in a full-scale model application. Thermobarical treatment temperatures in lab-scale experiments were 140 to 220°C in 20 K steps for a 5-minute duration. Methane yields could be increased by up to 58 % at a treatment temperature of 180°C. At 220°C, the abundance of inhibitors and other non-digestible substances led to lower methane yields than those obtained from untreated material. In an extended analysis, it could be demonstrated that there is a functional correlation between the methane yields after 30 days and the formation rate and methane yield in the acceleration phase. It could be proved in a regression of these correlation values that the optimum treatment temperature is 164°C and that the minimum treatment temperature should be above 115°C. The theoretical application of a full-scale model was used for assessing energy balance and greenhouse gas emissions following an LCA approach according to ISO 14044 (2006) as well as economy. A model device for thermobarical treatment has been suggested for and theoretically integrated in a biogas plant. The assessment considered the replacement of maize silage as feedstock with liquid and / or solid cattle waste. The integration of thermobarical pretreatment is beneficial for raw material with high organic dry matter content that needs pretreatment to be suitable for anaerobic digestion: Solid cattle waste revealed very short payback times, e.g. 9 months for energy, 3 months for greenhouse gases, and 3 years 3 months for economic amortization, whereas, in contrast, liquid cattle waste did not perform positive replacement effects in this analysis.

To develop anaerobic digestion (AD), innovative solutions to increase methane yields in existing AD processes are needed. In particular, the adoption of low energy pre-treatments to enhance biomass biodegradability is needed to provide efficient digestion processes increasing profitability. To obtain these features, hydrodynamic cavitation has been evaluated as an innovative solutions for AD of waste activated sludge (WAS), food waste (FW), macro algae and grass, in comparison with steam explosion (high energy pre-treatment). The effect of these two pre-treatments on the substrates, e.g. particle size distribution, soluble chemical oxygen demand (sCOD), biochemical methane potential (BMP) and biodegradability rate, have been evaluated. After two minutes of hydrodynamic cavitation (8 bar), the mean fine particle size decreased from 489- 1344 nm to 277- 381 nm (≤77% reduction) depending of the biomasses. Similar impacts were observed after ten minutes of steam explosion (210 °C, 30 bar) with a reduction in particle size between 40% and 70% for all the substrates treated.  In terms of BMP value, hydrodynamic cavitation caused significant increment only within the A. nodosum showing a post treatment increment of 44% compared to the untreated value, while similar values were obtained before and after treatment within the other tested substrates. In contrast, steam explosion allowed an increment for all treated samples, A. nodosum (+86%), grass (14%) and S. latissima (4%). However, greater impacts where observed with hydrodynamic cavitation than steam explosion when comparing the kinetic constant K. Overall, hydrodynamic cavitation appeared an efficient pre-treatment for AD capable to compete with the traditional steam explosion in terms om kinetics and providing a more efficient energy balance (+14%) as well as methane yield for A. nodosum.
Det behövs innovativa lösningar för att utveckla anaerob rötning i syfte att öka metangasutbytet från biogassubstrat. Beroende på substratets egenskaper, kan förbehandling möjliggöra sönderdelning av bakterieflockar, uppbrytning av cellväggar, elimination av inhiberande ämnen och frigörelse av intracellulära organiska ämnen, som alla kan leda till en förbättring av den biologiska nedbrytningen i rötningen. För att uppnå detta har den lågenergikrävande förebehandlingsmetoden hydrodynamisk kavitation prövats på biologiskt slam, matavfall, makroalger respektive gräs, i jämförelse med ångexplosion. Effekten på substraten av dessa två förbehandlingar har uppmäts genom att undersöka distribution av partikelstorlek, löst organiskt kol (sCOD), biometan potential (BMP) och nedbrytningshastigheten. Efter 2 minuters hydrodynamisk kavitation (8 bar) minskade partikelstorleken från 489- 1344 nm till 277- 281 nm (≤77 % reduktion) för de olika biomassorna. Liknande påverkan observerades efter tio minuters ångexplosion (210 °C, 30 bar) med en partikelstorlekreducering mellan 40 och 70 % för alla behandlade substrat. Efter behandling med hydrodynamisk kavitation, i jämförelse med obehandlad biomassa, ökade metanproduktionens hastighetskonstant (K) för matavfall (+65%), makroalgen S. latissima (+3%), gräs (+16 %) samtidigt som den minskade för A. nodosum (-17 %). Förbehandlingen med ångexplosion ökade hastighetskonstanten för S. latissima (+50 %) och A. nodosum (+65 %) medan den minskade för gräs (-37 %), i jämförelse med obehandlad biomassa. Vad gäller BMP värden, orsakade hydrodynamisk kavitation små variationer där endast A. nodosum visade en ökning efter behandling (+44 %) i jämförelse med obehandlad biomassa. Biomassa förbehandlade med ångexplosion visade en ökning för A .nodosum (+86 %), gräs (14 %) och S. latissima (4 %). Sammantaget visar hydrodynamisk kavitation potential som en effektiv behandling före rötning och kapabel att konkurrera med den traditionella ångexplosionen gällande kinetik och energibalans (+14%) samt metanutbytet för A. nodosum.

Stockholm Water is a water and sewage company with Henriksdal as one of two wastewater treatment plants (WWTPs). At Henriksdal wastewater sludge generated in the wastewater treatment process is digested which generate biogas; a mixture of mainly methane and carbon dioxide. If purified to methane content of 96 - 98 % this gas is called biomethane.

Biogasmax is a project aiming to reduce the use of fossile fuels in Europe by providing that biogas is a good technical, economical and environmental alternative as vehicle fuel. The specific aim for Stockholm Water is to increase the biogas production at the existing plant in Henriksdal. Enzymatic treatment of wastewater sludge is an innovative technique earlier proofed to increase the biogas production from wastewater sludge with up to 60 %. The enzyme activity is in turn proven to significantly increase in the presence of a cation binding agent.

One aim with this thesis was to investigate if the sludge from Henriksdal wastewater treatment process at all is affected of enzymatic treatment in presence of a cation binding agent since this has shown to have some significance. The chemical oxygen demand (COD) was measured in the liquid phase of sludge after treatment and used as a measurement of treatment effect. Another aim of this thesis was to look into the possibility to increase the methane production from sludge at Henriksdal WWTP. This was investigated through batch laboratory digestion tests.

The sludge from Henriksdal WWTP was shown to be a good substrate for the enzymes added. COD in the liquid phase was increased with 17 – 32 % depending on the dose of enzymes and sodium citrate added. Digestion of sludge with a total addition of 18.6 mg enzymes per 1 g total solids (TS) and a concentration of 5 mM sodium citrate increased the methane production with almost 18 % compared to untreated sludge. This equals an increase of 18.3 % when converted to represent a totally blended and continuous digestion chamber at Henriksdal WWTP. The increased methane production also results in a sludge reduction out from the digestion chambers. The increased methane production and sludge reduction though does not fulfil the increased costs for the enzymes and sodium citrate applied. These doses must be decreased and the costs for both enzymes and sodium citrate must be reduced for this technique to be economically feasible in a full scale operation.

Dans le cadre des législations européennes relatives au traitement des déchets et aux énergies renouvelables, la méthanisation apparaît comme une alternative prometteuse pour la stabilisation et la valorisation des Ordures Ménagères Résiduelles (OMR). D'un point de vue opérationnel l'hétérogénéité et les difficultés de mise en mouvement d'une matrice aussi complexe que les OMR sont à l'origine de pertes de rendement voire de l'arrêt d'installations de méthanisation. Les performances de méthanisation sont en particulier limitées par l'étape d'hydrolyse des fractions lignocellulosiques qui représentent la majorité du potentiel méthanogène des OMR. Dans ce contexte, l'objectif principal du travail de thèse, était l'étude d'un procédé de percolation dans lequel le déchet n'est pas mis en mouvement. Au travers de ce travail nous avions également pour ambition de produire des connaissances à caractère plus générique sur l'hydrolyse afin d'en améliorer les performances. Des expériences préliminaires ont d'abord permis la définition d'un système expérimental adéquat pour l'étude à l'échelle laboratoire de l'hydrolyse des OMR. La représentativité d'un déchet reconstitué, reproductible et d'utilisation aisée, a notamment été vérifiée en termes de potentiel méthanogène, de profil hydrolytique et de flore microbienne. Suite à la définition de ce système expérimental, son comportement hydrolytique a été comparé à celui d'un test de lixiviation de référence (NF EN 12457-4) afin de valider l'intérêt opérationnel de la percolation pour l'hydrolyse des OMR. De façon inattendue, l'extraction de 38,90% de la matière carbonée initiale du déchet a ainsi été mise en évidence lors de l'hydrolyse par percolation contre 17,84% lors de l'hydrolyse par lixiviation, renforçant l'intérêt suscité par la percolation pour l'hydrolyse des OMR. L'optimisation des performances d'hydrolyse par percolation a ensuite été réalisée par le criblage de huit paramètres opérationnels afin de déterminer leur influence sur les performances d'hydrolyse des OMR, au travers de deux plans d'expérience. L'ajout d'alcalinité (12 gHCO3-.L-1) et la recirculation du percolat pendant 6 h par jour ont ainsi permis d'augmenter significativement les performances d'hydrolyse, passant de 17 à 43% d'extraction de la matière organique (DCO) initiale du déchet (autrement dit de 26 à 69% de la matière biodégradable initiale). L'étude des communautés microbiennes et de leur activité a également été réalisée. Le séquençage des pyrotags d'ADNr 16S a ainsi permis de mettre en évidence le caractère dominant des Classes Clostridia et Bacteroidia au sein des communautés hydrolytiques. Le couplage de cette démarche qualitative à une approche quantitative par qPCR sur une série de biomarqueurs taxonomiques et fonctionnels a permis de montrer qu'il existe une corrélation positive entre l'ajout de carbonates, la neutralisation du pH, la quantité de matière hydrolysée à 14 jours et soit l'abondance de la Classe Bacteroidia soit celle des gènes de la famille hydA, impliqués dans la fermentation. Finalement, l'analyse microbiologique a été approfondie au jour 4, c'est-à-dire durant la phase d'hydrolyse intense, grâce à une approche de métatranscriptomique. L'analyse des transcrits fonctionnels indique que l'alcalinité influence l'activité des microorganismes de la Classe Clostridia dès le jour 4 des essais d'hydrolyse. Plus spécifiquement, l'ajout de carbonates semble corrélé à une modification du métabolisme des sucres chez des microorganismes non cultivables apparentés à Clostridium cellulolyticum et à l'augmentation de l'expression de l'opéron nif, impliqué dans la fixation de l'azote, chez différents groupes de microorganismes
In the framework of the European green policy, anaerobic digestion appears as a promising technology for stabilization and valorization of Municipal Solid Waste (MSW). In practice, mechanical mixing of a complex and heterogeneous matrix such as MSW induces major operational constraints. Anaerobic digestion performances are especially limited by hydrolysis of lignocellulosic fractions which represent the main part of MSW methanogenic potential. In this context, this PhD project was aiming to characterize and optimize of a percolation process in which MSW stands still. Preliminary experiments were conducted in order to define an experimental system suitable for lab-scale study of MSW hydrolysis. Therefore, the representativeness of an easy-to-use and reproducible reconstituted waste was verified in terms of methanogenic potential, hydrolytic profiles and associated microbial communities. Following system definition, hydrolysis behavior by percolation was compared to a reference lixiviation test (NF EN 12457-4). Surprisingly, hydrolysis by percolation permitted the extraction of 39% of carbonated matter initially contained in waste whereas 18% were extracted during hydrolysis by lixiviation, thus validating operational benefit of percolation for MSW hydrolysis. Optimization of hydrolysis performance was then conducted through the screening of eight operational parameters for their influence on MSW hydrolysis performances thanks to two Designs Of Experiment (DOE). Cumulative effect of alkalinity addition (12 gHCO3-.L-1) and percolate recirculation (6 hour.day-1) significantly improved hydrolysis yield, from 17 to 43% of extracted organic matter compared to the initial content of waste (corresponding to an extraction of 26 and 69% of biodegradable matter). Structure and activity of hydrolytic microbial communities were also studied. 16S rDNA-pyrotags sequencing brought out the dominance of classes Clostridia and Bacteroidia. Additionally, a quantitative approach led by qPCR revealed a correlation between carbonates addition, pH neutralization, amounts of hydrolyzed matter at day 14 and either class Bacteroidia or genes from hydA family, involved in fermentation. Finally, metatranscriptomic approach was conducted at day 4 in order to further study microbial activity during the intense hydrolysis phase. According to functional analysis, alkalinity seems have positive influence on class Clostridia activity. More specifically, carbonates addition seems correlated to a modification of carbohydrates metabolism of organisms affiliated to Clostridium cellulolyticum and to transcriptional up-regulation of nif operon, involved in nitrogen fixation, among various types of microorganisms

Lignocellulose is an ubiquitous source of fixed carbon that is presently underexploited for renewable energy technologies. Currently, producing enzyme cocktails that robustly degrade these feedstocks is a significant economic bottleneck. Anaerobic gut fungi native to the digestive tracts of ruminants and hindgut fermenters are widely understudied despite their inherent ability to degrade a significant portion (~50%) of the lignocellulose in herbivorous animals. Challenges in cultivation due to their strict oxygen sensitivity, and the lack of a central repository to maintain axenic stocks substantially impede the progress with anaerobic fungi. Yet, these microbes have evolved elegant strategies and may harbor novel biomass degrading enzymes that could be used to more efficiently hydrolyze lignocellulose. Developing these organisms through characterization and genome engineering will yield significant contributions to the bioenergy community by improving hydrolysis technologies.

In this work, we report the isolation of four novel species of anaerobic gut fungi. A more complete characterization of one of our four fungal isolates is investigated, whereby the effects of substrate composition and the corresponding fungal growth rates are compared. I also explore the growth of one of our fungal isolates on transgenic poplar to understand how fungal growth and enzyme secretion adapt to variable lignin composition. Notably, no significant reductions in growth were observed highlighting the ability of anaerobic fungi to degrade diverse feedstocks regardless of lignin composition. I have additionally included preliminary work intended to identify what epigenetic regulational strategies exist for anaerobic fungi, and how they relate to carbohydrate active enzyme expression. We hope to leverage this knowledge to engineer base enzyme cocktails that release significant portions of the fermentable sugars in untreated or mildly treated plant biomass as a means to make bioenergy technologies more efficient.

Microalgae can be used to polish secondary treated wastewater by removing nutrients and carbon without the addition of oxygen making it a reduced energy treatment compared to traditional extended aeration systems. The recovered microalgae in turn can be used for biofuels applications such as biogas production via anaerobic digestion and biodiesel production via lipid transesterification. Anaerobic digestion is a more feasible option due to its low energy requirement and on-site power generation ability for water utilities. Nevertheless, anaerobic digestion of microalgae has several challenges with the most difficult being the recalcitrant nature of the cell wall of most microalgae resisting microbial attack during digestion. This has resulted in low methane yields after long retention times during anaerobic digestion. Also, the rigidity of the cell walls has led to low lipids release from microalgae cells due to difficulty in extracting the intracellular cell components, affecting other biofuels production processes. Due to this, several authors have suggested a pretreatment process as a means to disrupt the cell wall structure and improve degradation of microalgae. To determine the efficiency of microalgae pretreatment, a proper quantitative technique is useful to analyse cell disruption rate. This research began by comparing different pretreatment technologies using a light microscope. The light microscope was fitted with a Neubauer haemocytometer cell counter, in addition to the use of image-J cell counting software for visual analysis to quantify cell wall disruption using Chlorella vulgaris (C.vulgaris) as the model alga. C.vulgaris was selected as the microalgae species in this project as it has been widely established as a suitable species for biofuel production and anaerobic digestion due its dominance, being a local species in Australia, higher growth rates and higher lipids content when compared to other species. Pretreatment techniques compared included thermal processes using a water bath and autoclave, mechanical processing using a high-speed homogeniser, combinations of water bath and high-speed homogeniser as well as enzymatic pretreatment using lysozyme. The results of the experiments conducted showed over 80% cell disruption using high speed homogeniser and lysozyme enzyme. Thermal pretreatment using Autoclave produced the lowest cell disruption results at 42%. The results of the combination of water bath at roiling boil for 5 minutes and 5 minutes high speed homogeniser treatment at 4,000rpm showed a 50% cell disruption rate. For the water bath thermal pretreatment alone, 20 minutes was found to be most effective producing a 65% disruption rate. However, using microscopic analysis, although effective, is time-consuming for larger cell counts, making industrial pretreatment efficiency determination a challenge. Besides, the degree of pretreatment necessary to disrupt the cell is affected by the mechanical strength of the cell wall. Currently, there is little or no test for cell wall strength measurement that is shown to impact cell wall disruption and using anaerobic digestion to quantify cell strength can be slow due to long retention times. Understanding microalgae mechanical strength would enable better selection of microalgae pretreatment methods and improve energy production from microalgae, making it a more efficient process resulting in improvements in subsequent anaerobic digestion rates. From the study, a reproducible technique using high-speed homogeniser (at speeds between 4,000rpm to 33,000rpm) to evaluate the relative cell wall strength of C.vulgaris was developed and cell disruption was determined from lipid concentration following extraction. During the technique development, several solvents including diethyl ether, hexane and dichloromethane were investigated and compared for their use in extracting broken-only algae cells from solution. Dichloromethane proved to be the most suitable solvent for wet algae lipids extraction. From the results, it was determined that significant lipids extraction was from 8,500 rpm, which was identified -1 as the critical speed with shear rate of 18,227s . The maximum shear rate at 33,000rpm was found to -1 be 70,765s . Total lipids available in the cell was calculated using a modified Bligh and dyer method of dichloromethane to methanol of 2:1. It was found that the percentage of lipids from broken only cells compared to the total lipids in the cells was about a quarter at maximum cell disruption speed of 33,000rpm. Experimental verification was conducted using chlorophyll analysis and lysozyme addition which displayed a similar trend as the lipid extraction results show that the critical speed was also observed at 8,500rpm. Lysozyme enzymatic pretreatment was investigated for cell wall disruption and its impact in anaerobic hydrolysis as previous research had shown its ability to degrade C.vulgaris cells for biofuel processes. Lysozyme was later deduced in this project to initiate cell disruption, making further cell degradation by other hydrolytic enzymes easier, leading to improved lipids extraction and better anaerobic hydrolysis. The novel technique developed will assist biofuel technologies to determine the efficiency of microalgae pretreatment and has also provided knowledge on the critical shear rate when disruption occurs. Furthermore, microalgae cells showed resistance to microbial hydrolysis during previous anaerobic digestion studies using recovered microalgae from wastewater systems. Commercial anaerobic digestion using microalgae from wastewater utilises bacteria inoculum already present in the wastewater system. The effectiveness of this, however, has been low generating low yields of methane. Researching and identifying key micro-organisms in microalgae anaerobic digestion will promote the technology and improve bio-methane yields. To achieve this, bacteria such as Escherichia coli (E.coli), Streptococcus thermophilus (S.thermophilus), Lactobacillus plantarum (L.plantarum), Acetobacter aceti (A.aceti), as well as hydrolytic enzymes such as lysozyme, amylase, cellulase, pectinase, and Aspergillus oryzae (A.oryzae) fungus were utilised in separate and combined experiments’ to show the effectiveness of microbial selection and enzymes as inoculum for degrading C.vulgaris cell wall during anaerobic hydrolysis to produce volatile fatty acids (VFA) as intermediates. The amount of VFAs produced was used as a means of experimental process efficiency and to predict potential bio-methane production. Two separate experiment batches were conducted with batch 1 having retention times of 30, 45 and 60 days. Batch 2 had a retention time of 15 days as the results from batch 1 showed optimum VFA production at 30 days retention time. From the results, optimum total VFA concentration was obtained after 15 days retention using inoculum containing mixed enzymes (lysozyme, cellulase, pectinase and amylase) at 195 mg/l. This is followed by mixed bacteria containing E.coli, S.thermophilus and L.plantarum at total VFA concentration of 161 mg/l. Literature review on the selected bacteria shows the capability of these bacteria being able to produce the selected hydrolytic enzymes. Hence, the efficiency of the bacteria in producing total VFA results close to the values obtained from the mixed enzymes. Lowest VFA production was observed in test containing A.oryzae alone at 23mg/l. The low digestion efficiency observed from the fungus has been suggested to be as a result of the possibility of no cellulose wall detected in C.vulgaris cell. Another possibility is the aerobic property of the fungus limiting its growth efficiency during digestion. To investigate this further, C.vulgaris was flocculated with A.oryzae for 24 hours as well as 72 hours and used to harvest the microalgae. The harvested C.vulgaris cells were then subjected to high-speed homogeniser treatment using the technique developed earlier before undergoing anaerobic digestion using a retention time of 13 days with sampling every two days in a separate experiment. The initial tests involving harvesting of the microalgae by flocculation shows 72 hours to produce greater flocculation efficiency with almost 100% of the cells observed to flocculate and clump together under visual observation using a motic light microscope at 400X magnification. For the cell strength tests, 72-hours flocculated algae also displayed better performance with lipids extraction efficiency of 27% more than the control containing C.vulgaris alone. The 24-hour flocculated microalgae also showed good results with 20% more lipids production compared to the control containing C.vulgaris alone. However, when the flocculated microalgae at 72-hours was investigated for anaerobic hydrolysis, the results were again low providing only 14.7mg/l of total VFA at peak observed at day 5. The results confirm the earlier findings of the possibility of the absence of cellulose in the cell wall of C.vulgaris. Hence, the use of fungus A.oryzae maybe useful only in microalgae harvesting technology and not anaerobic digestion. In addition, the project provides a detailed energy calculation of the different pretreatment strategies employed and discussed the amount of energy consumed. Thermal pretreatment was found to have a lower energy consumption at 86kJ/L feed with energy recovery for both autoclave and waterbath compared. Also, without energy recovery, thermal pretreatment was still quite low at 497 kJ/feed for autoclave and 393 kJ/L feed for waterbath. Contrarily, high speed homogeniser was found to be energy intensive at maximum speed of 33000rpm with energy consumption of 1,080 kJ/L. However, at the critical speed of 8,500 rpm, energy consumption of the high speed homogeniser was low and close to thermal pretreatment with energy recovery utilising only 88.7 kJ/L feed. Moreover, potential biomethane to be produced from the optimum anaerobic hydrolysis experiment conducted at 15-days was evaluated. An energy balance and cost analysis were documented from the various biological and enzymatic pretreatments employed. A positive energy balance was observed across the various inoculum employed. Optimum net energy production was recorded by inoculum containing mixed enzymes (lysozyme, pectinase, cellulase and amylase) at 3362 J/L feed. This is followed by mixed bacteria (E.coli, S.thermophilus and L.plantarum) inoculum with net energy production at 2769.5 J/L feed. Investigating and proposing an effective method of microalgae digestion will enable microalgae disposal from wastewater and promote energy recovery making microalgal treatment of wastewater more likely in water and waste treatment facilities.

碩士
國立成功大學
環境工程學系碩博士班
100
The objective of this study is to construct a system that hydrolyzes cellulose and produces hydrogen simultaneously using anaerobic fluidized bed (AnFB) reactor. A thermophilic cellulolytic microbe, Clostridium sp. TCW1 was chosen as an inoculation for AnFB. There were three Runs in AnFB process operation. 1st Run was to start up this system and enrich biomass of cellulose hydrolyzing microbes, 2nd Run was to increase cellulose volumetric loading rate (VLR) to 1.16 g-COD/L/d, and 3rd Run was adding starch kitchen waste (SKW) to increase hydrogen production. In 1st Run, the hydrogen production rate was 0.02±0.01 L-H2/L/d, hydrogen yield was 0.77±0.57 mmole-H2/g-COD, and the total solid amount at Day 111 was 2,046 g SS. In 2nd Run, the hydrogen production rate was 0.004 L-H2/L/d, hydrogen yield was 0.10 mmole-H2/g-COD, and the total solid amount at Day 232 and Day 248 were 538 g SS and 206 g SS respectively, there were biomass washed out and degraded during 2nd Run. In 3rd Run, the hydrogen production rate is up to 1.0 L-H2/L/d. The enzyme activity of Endo-β-1,4-glucanase (CMCase) of AnFB operation process at Day 232 in 2nd Run was 0.15 U/mL, it near the maximum activity 0.17 U/mL of batch culture though biomass content in AnFB was not enough (538 g); at Day 280 in 3rd Run, CMCase activity down to 0.02 U/mL when vegetable kitchen waste (VKW) was fed to AnFB, it seems VKW was not favored for cellulase production in this process operation.

碩士
逢甲大學
化學工程學系
102
Because of excessive human development and extensively use of fossil fuels created environmental pollution, also appeared short supply situation and inflation, one way to solve this energy problem is that create a new energy. The purpose of this study is hydrolyzed agricultural waste, and then the hydrolysis solution is used to the nutrition source of hydrogen fermentation. Physical chemistry and enzymatic method places mainly in the hydrolysis process, however, in order to improve hydrogen production capacity, the source of bacteria is screening and domesticating from mix-culture, the result is that Clostridium butyricum VP13266. The capability of hydrogen fermentation was influenced on the concentration of inhibitors, it can be removed by the method of organic solvent detoxification. The removal rate of acetic acid is 0.791 (g/l/ml)by using methyl acetate, the removal rate of hydroxyl-methyl furfural and furfural is 0.144 and 0.381(g/l/ml)by using ethyl acetate. The total accumulation and yield of hydrogen is 925.79±97.86 ml/L and 297.68 ml H2/g Tsused by using the enzyme hydrolysis solution, and then the butyric acid and acetic acid ratio achieve 2.29±0.03. The total accumulation and yield of hydrogen is 1394.7±39.4 ml/L and 472.0 ml H2/g Tsused by using the physical chemistry hydrolysis solution, and then the butyric acid and acetic acid ratio achieve 2.73±0.35.

Biogas production is one of the most promising methods of producing renewable energy with the use and management of by-products of agriculture, industry and municipal waste in the anaerobic digestion process. Over the past decades, the biogas production technology has been focused on the development and optimization of systems characterized by a high rate of anaerobic digestion of energy crops, as well as solid agro-industrial wastes. The anaerobic digestion process consists of four stages: (i) hydrolysis (degradation) of high-molecular mass organic compounds to smaller monomers (proteins are degraded to amino acids, carbohydrates – to simple sugars, lipids to glycerin and fatty acids); (ii) acidogenesis, i.e. decomposition of the hydrolyzed substances to, among others, organic acids, alcohols, etc.; (iii) acetogenesis – oxidation of the produced organic acids to acetates and acetic acid, and (iv) methanogenesis, i.e. the degradation of acetates and acetic acid to methane and carbon dioxide. Therefore, the end products of anaerobic digestion of biomass are: biogas, consisting mainly of methane (30-70%) and carbon dioxide (30-70%), as well as sludge with a reduced amount of organic compounds. The key process, often limiting the speed of these biochemical changes, is hydrolysis. In order to facilitate effective biomass degradation, its pretreatment is often required. Biological pretreatment, which can be an alternative to the physical and chemical methods, is based on the activity of microorganisms, mainly bacteria and fungi, which produce hydrolytic enzymes (mainly cellulolytic, proteolytic and lipolytic). The biological pretreatment of the substrate may include: (i) the addition of specific microorganisms or consortia of hydrolytic microorganisms to the bioreactor (bioaugmentation) or (ii) supplementation of the system only with hydrolytic enzymes. Although the latter solution can improve the efficiency of the process, the activity of exogenous enzymes is usually affected by many different factors, including the type and variability of the substrate, incubation time, process configuration and physico-chemical conditions (e.g., temperature and pH). Bioaugmentation of the system also increases the enzymatic activity, as the enzymes are produced in situ by the microorganisms introduced to the system. The simultaneous production of complexes of various types of hydrolytic enzymes by live microorganisms allows for the application of this method in a potentially wide range of processes of utilization of different types of substrates (depending on the availability of various organic compounds in the bioreactor tank). 9 Among the main problems and limitations of the use of bioaugmentation are: low enzymatic activity of the microorganisms and sensitivity to changing stress conditions (including pH, temperature and the presence of heavy metals and antibiotics/pharmaceuticals). For this reason, the use of microbial consortia rather than singlestrain inocula is preferred, as different strains often vary in enzymatic properties or the range of tolerance to stress factors. This helps to maintain the activity of at least some members of the consortium over a wide range of conditions, on different types of substrates. Therefore, the most important step in the development of microbial vaccines is the selection and use of specialized microorganisms with a high biotechnological potential. In addition, the developed vaccines must meet the (bio)safety requirements for specific applications. The results obtained in this work increase the knowledge on the selection of microorganisms with cellulolytic, proteolytic and lipolytic activities, and constitute guidelines for the development of microbiological vaccines to be used for the degradation of organic compounds contained in plant biomass and sewage sludge, and increased biogas production. AIM The aims of the research were the following: (i) the selection of natural consortia of cellulolytic microorganisms and analysis of the impact of the source of microorganisms on the enzymatic activity of these consortia (ii) isolation and physiological characterization of pure cultures of cellulolytic bacteria and the development of a new (artificial) microbial consortium with a high cellulolytic activity, (iii) determination of the impact of the use of the developed consortia (both the natural and artificial) on the efficiency of lignocellulosic biomass hydrolysis and biogas production from it, (iv) determination of the total biotechnological (metabolic) potential and biosafety of the selected proteolytic and lipolytic strains in the context of their use for degradation of organic compounds contained in the sewage sludge, (v) demonstration of the impact of application of the proteolytic, lipolitic and cellulolytic strains on the efficiency of hydrolysis of organic compounds (proteins, lipids and carbohydrates) and biogas production from sewage sludge, (vi) analysis of the impact of the source of isolation of bacterial strains (sewage sludge, agricultural biogas plant) on their metabolic potential.

碩士
國立中興大學
食品暨應用生物科技學系所
99
Each year, worldwide orange production is more than 60 million tons. Thirty percent of the orange production is processed to making orange products sach as orange juice, but the utilization rate of oranges fruit is only 50%. There are rich in carbohydrate in the by-product of orange (orange peel waste), it is a good ingredient to ferment for alcohol with non-graining crop. It can not only increase the orange by-product`s extra value, but can reduce the pollution of environment. In this experiment, we used three kinds of commercial enzymes (Pectinase, Cellulase and Viscozyme) to hydrolysis of insoluble carbohydrate in orange peel, and found optimized enzyme loading activity units. Orange peel waste was pretreated under different heating to reduce D-limoene content (98.2%) of orange peel. We investigated orange peel wastes as fermentation substrate adjust initial pH value and remove solids with Saccharomyces cerevisiae BCRC 20271 in flask to observe viable count of yeast, sugar consumption, rate of alcohol and yield during the fermentation. In addition, we utilized autofermenter to compare repeated flask fermentation conditions with simultaneously sacchrarification and fermentation, studying changes in alcohol during fermentation. The result showed that orange pulp treats Viscozyme(30 U/g dry OPW) and Pectinase (25 U/g dry OPW) able to increase soluble carbohydrate concentration. Pretreatments at 121℃, 15 psi for 15 min effectively reduce D-limoene content of orange peel, will not inhibit the growth of S.cerevisiae. In flask fermentation, Maillard reaction occurs after enzymatic hydrolysis of orange pulp with high temperature, may produce substances interfere with the growth of S.cerevisiae. If we remove fermentation substrate of solids, will reduce concentration of interfering substances and increase oxygen permeability result in increasing specific growth rate (μ) of S.cerevisiae and shortening lag phase of the growth. Fermentation substrate was adjusted initial pH = 5 (maintain at pH4~5 during fermentation), S.cerevisiae will not take place death phenomenon and alcohol yield could be 74. 5%. During fermentation maintain pH = 5, the growth of S.cerevisiae is better, result from significantly competing small molecular sugar with alcohol fermentation, and alcohol yield has dropped (64.50%). Orange peel treated at 121℃, 15 psi for 15 min, and simultaneously added S.cerevisiae and enzymes carried out sacchrarification and fermentation in fermenter. To avoid not producing substances to interfere with the growth of S.cerevisiae from Maillard reaction, the growth of yeast will not lag phase occur. The pH is not adjusted during fermentation ( maintain at pH3.8~3.6), result in the growth of S.cerevisiae is worse than adjusted initial pH = 5. Low pH value may increase sugar concentration to convert alcohol, so alcohol yield achieves 89.2% more than before all experiments.

abstract: Eighty-two percent of the United States population reside in urban areas. The centralized treatment of the municipal wastewater produced by this population is a huge energy expenditure, up to three percent of the entire energy budget of the country. A portion of this energy is able to be recovered through the process of anaerobic sludge digestion. Typically, this technology converts the solids separated and generated during the wastewater treatment process into methane, a combustible gas that may be burned to generate electricity. Designing and optimizing anaerobic digestion systems requires the measurement of degradation rates for waste-specific kinetic parameters. In this work, I discuss the ways these kinetic parameters are typically measured. I recommend and demonstrate improvements to these commonly used measuring techniques. I provide experimental results of batch kinetic experiments exploring the effect of sludge pretreatment, a process designed to facilitate rapid breakdown of recalcitrant solids, on energy recovery rates. I explore the use of microbial electrochemical cells, an alternative energy recovery technology able to produce electricity directly from sludge digestion, as precise reporters of degradation kinetics. Finally, I examine a fundamental kinetic limitation of microbial electrochemical cells, acidification of the anode respiring biofilm, to improve their performance as kinetic sensors or energy recovery technologies.
Dissertation/Thesis
Doctoral Dissertation Civil, Environmental and Sustainable Engineering 2020

碩士
朝陽科技大學
環境工程與管理系
103
This study mainly focused on public sewage treatment plants to conduct the effectiveness evaluation of anaerobically digested sludge and respectively conducted the sludge ultrasonic analysis and mass balance analysis. After the sludge ultrasonic analysis, the organic matter release, sludge reduction, and gas effects of hydrolysis products were discussed. Furthermore, the study simultaneously carried out domestic sewage plant data collection and conducted a mass balance analysis to establish the assessment framework for the sludge treatment units. The experimental results show that in the ultrasonic sludge hydrolysis in terms of dissolution of CODs, concentrated and digested sludge had notable effects, but in the sludge reduction portion, digested sludge had no significant change. In the concentrated sludge after the lengthening of ultrasonic processing time, the reduced rate of TS or SS significantly decreased; thus, the sludge after hydrolysis, biochemical methane production potential test, and concentrated sludge biogas production were as follows: untreated > 1 min > 5 min > 10 min; digested sludge 10 min ≥ 5 min > untreated > 1 min > 30 min. For concentrated and digested sludge after ultrasound hydrolysis, although dissolution of CODs increased and sludge (concentrated sludge only) decreased, gas production situation did not increase in the BMP test. From the data provided by each sewage treatment plant, the reported information showed no unity, and most of the sewage treatment plants did not have statistics on sludge volume. As a balance analysis could not be effectively conducted on each unit, the evaluation results of onsite field interviews and sampling showed that each unit reached the mass balance, the effectiveness of anaerobic digestion was also above standard, and the operating conditions were good. For the sludge unit to carry out an effectiveness evaluation, it is recommended that the test items and frequency be unified.

Technologies for pretreatment of waste activated sludges (WAS) prior to digestion are of increasing interest to wastewater treatment utilities because of their promise for improving sludge digestibility and reducing the mass of biosolids remaining after digestion. While there has been considerable study of pretreatment processes, a common approach to describing the impact of pretreatments on sludge biodegradability has not been developed. The overall objective of this study was to develop protocols that can be employed to characterize the impact of pretreatment processes on WAS digestion. Sonication and ozonation were employed as models of physical and chemical pretreatment technologies respectively. A range of physical, chemical and biological responses were evaluated to assess the impact of pretreatment on WAS properties as well as digestibility. WAS samples that were generated over a range of solids residence times (SRTs) under controlled operating conditions were employed to facilitate an assessment of the interaction between pretreatment and WAS properties on digestibility. The VS, COD and soluble TKN responses indicated that a significant fraction of the WAS solids were solublized by sonication and ozonation, however, it appeared that the types of materials which were solublized was affected by the SRT at which the WAS was generated and the level of pretreatment. The results indicated that the impact of pretreatment on biodegradability of WAS was not described by solublization values exclusively without considering the SRT of the sludge and the level and type of pretreatment. A higher level of proteinaceous materials was preferentially solublized as the result of pretreatment. Respirometry revealed that both sonication and ozonation substantially reduced the viable heterotrophs in the sludge and modestly increased the readily biodegradable fraction of COD. The ultimate yields of CH4 and NH4 in BMP tests and VFAs in BAP tests revealed that pretreatment marginally increased the ultimate digestibility of the sludges. Only a high dose of ozonation substantially increased the digestibility of the 15 day SRT sludge. However, both sonication and ozonation substantially increased the rate of hydrolysis which is typically the rate limiting process in WAS digestion. The BMP test was not a useful test to evaluate the rate of methane generation due to inhibition of methanogens in the early days of BMP test for pretreated sludges. The comparison between VFA and ammonia responses in day 10 of BAP test and ultimate values of these responses after 60 days in BMP test revealed linear relationships between these responses. According to these relationships, a set of models were introduced in this study. The models can be employed to predict the ultimate methane and ammonia generation using soluble COD, VFA or ammonia responses in day 10 of BAP tests. The BAP test was determined to be a shorter test (10 days) than the BMP (55 to 60 days) test and could provide information on the rates of hydrolysis and acidification/ammonification processes. Characterization of biodegradable and non-biodegradable material in WAS samples was conducted using a simplified ADM1 model. The characterization also revealed that proteins are a substantial fraction of biodegradable materials. The estimated ammonia, VFA and methane values from the stoichiometric model were similar to the corresponding values from the experiments. This supported the validity of the simplified model for all sludges employed in this study.

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.

Polymer hydrolysis is the first (and rate limiting) step for biomethanation of heterogeneous biomass feedstock’s. Satisfactory hydrolysis has been difficult to achieve, understand and predict adequately, to run anaerobic bioreactors with such feedstock’s efficiently. The fraction of hot water soluble extracts (crude proteins and extractables, Fcpe), the nature and material of intercellular binding and the extent and complexity of lignin present have been considered as key parameters for hydrolysis and has been analyzed for a variety of biomass degradation data available at the Centre for Sustainable Technologies, Indian Institute of Science. Feedstocks were grouped into those bound with high levels of pectic/protein materials or lignin-bound types. The data on the initial (10-15d) as well as the overall rates of hydrolysis (0-50d) has been analyzed. The extent of hydrolysis achieved for pectin bound substrates were high (≥65%) and that of lignin bound substrate was low (≤30% VS, Acacia). The initial hydrolysis rates were strongly correlated to the content of extractables (=0.117Fcpe). Subsequently, the hydrolysis rates rise to reach maxima and then begin to fall. Most fresh feedstock had somewhat similar rates of the increase in hydrolysis rates but the time to reach maximum and its value varied among feed stocks. Many lignin bound feed stocks did not have such a pattern. With regards to the overall hydrolysis rate constant, it was found that these clustered into two groups that represented pectin bound (0.154/d) and lignin bound (0.045/d) types. Therefore from this study it was concluded that anaerobic decomposition of heterogeneous biomass could be predicted using two rate parameters and one intrinsic property of the biomass feedstock, namely, a. the initial rate of hydrolysis (based on the extent of extractables =0.117 Fcpe) b.the maximum rate achieved and the time when it is reached (an intrinsic property based on feed stock and but not determined in this study) c. the overall hydrolysis rate (choosing between 0.154 /d or 0.045 /d depending upon the nature of inter-cellular binding material, pectin or lignin, respectively). This research provides new insights into the prediction of hydrolysis rate a key limiting step for heterogeneous biomass biomethanation (hydrolysis) based on the level of extractables, the type of cellular cementing material and the maxima that can be achieved.

Τα απόβλητα των ελαιοτριβείων αποτελούν ένα από τα σημαντικότερα περιβαλλοντικά προβλήματα της Μεσογείου, λόγω της άκριτης διάθεσης τους. Είναι χαρακτηριστικό ότι, περίπου το 95% της παγκόσμιας παραγωγής ελαιόλαδου παράγεται από μικρές, οικογενειακές επιχειρήσεις Μεσογειακών χωρών. Στόχος της παρούσας διατριβής ήταν η βιοτεχνολογική αξιοποίηση των αποβλήτων των ελαιοτριβείων για την αναερόβια παραγωγή υδρογόνου. Ειδικότερα, μελετήθηκε η δυνατότητα παραγωγής υδρογόνου σε μεσόφιλες συνθήκες από το ημι-στερεό υπόλειμμα διφασικών ελαιοτριβείων (ελαιοπολτός ή olive pulp) και από τα υγρά απόβλητα τριφασικών ελαιοτριβείων (OMW) με χρήση μικτής αναερόβιας καλλιέργειας μικροοργανισμών. Τα απόβλητα αραιώθηκαν με νερό βρύσης σε αναλογία όγκων 1:4 αντίστοιχα, ώστε να καταστεί δυνατή η βιολογική επεξεργασία τους. Πειράματα σε αντιδραστήρες τύπου CSTR κατέδειξαν ότι, η συνεχής μεσόφιλη αναερόβια παραγωγή υδρογόνου είναι εφικτή τόσο από αραιωμένο ελαιοπολτό (1:4) όσο και από αραιωμένο απόβλητο OMW (1:4). Η απόδοση της συνεχούς διεργασίας σε υδρογόνο από αραιωμένο ελαιοπολτό (1:4) προσδιορίστηκε μικρότερη από τη μέγιστη θεωρητική απόδοση (4 mol H2/mol γλυκόζης που καταναλώθηκε) πιθανότατα λόγω της αρνητικής επίδρασης της μερικής πίεσης του υδρογόνου. Στα πλαίσια αξιοποίησης των πειραματικών αποτελεσμάτων της παρούσας διατριβής το μαθηματικό μοντέλο αναερόβιας χώνευσης ADM1 τροποποιήθηκε κατάλληλα, ώστε να καταστεί δυνατή η περιγραφή της αναερόβιας παραγωγής υδρογόνου. Αρχικά, όλες οι κρίσιμες παράμετροι του μοντέλου προσδιορίστηκαν από τα πειραματικά δεδομένα της συνεχούς αναερόβιας παραγωγής υδρογόνου από αραιωμένο ελαιοπολτό (1:4), ενώ πειράματα διαλείποντος έργου πραγματοποιήθηκαν για την επαλήθευσή τους. Προκειμένου να εξεταστεί η εγκυρότητα του τροποποιημένου μοντέλου και η δυνατότητα αξιόπιστης περιγραφής της αναερόβιας παραγωγής υδρογόνου από απόβλητα ελαιοτριβείων, το μοντέλο χρησιμοποιήθηκε για την περιγραφή της αναερόβιας επεξεργασίας του αραιωμένου αποβλήτου OMW (1:4) με στόχο την παραγωγή υδρογόνου. Στη συνέχεια, αναπτύχθηκαν και εφαρμόστηκαν μέθοδοι προεπεξεργασίας του αραιωμένου ελαιοπολτού (1:4) (φυσικοχημικές μέθοδοι και ενζυμική υδρόλυση) με κύριο στόχο την αύξηση της συγκέντρωσης των διαλυτών υδατανθράκων του, ενώ στις περιπτώσεις που αυτό επιτεύχθηκε, διερευνήθηκε η επίδραση τους στην απόδοση της διεργασίας σε υδρογόνο. Η προσπάθεια αυτή βασίστηκε στο συμπέρασμα που προέκυψε από πειράματα διαλείποντος έργου, σύμφωνα με τα οποία, οι αδιάλυτοι υδατάνθρακες συνεισέφεραν ελάχιστα στην αναερόβια παραγωγή υδρογόνου με την εκατοστιαία κατά βάρος περιεκτικότητα τους να αντιστοιχεί περίπου στο 50% της περιεκτικότητας του αποβλήτου σε ολικούς υδατάνθρακες. Μεταξύ των φυσικοχημικών μεθόδων που εφαρμόστηκαν (προσθήκη αλκαλικού μέσου, οζονισμός, επεξεργασία με ατμό) ως βέλτιστη μέθοδος επιλέχθηκε η επεξεργασία με ατμό (1 bar, 121oC) για 60 min, καθώς οδήγησε στο μεγαλύτερο ποσοστό αύξησης των διαλυτών υδατανθράκων (περίπου 26% επί της αρχικής τους συγκέντρωσης), με το μικρότερο δυνατό οικονομικό κόστος, αυξάνοντας την απόδοση της διεργασίας σε υδρογόνο περίπου κατά 45% (εκφρασμένη ως mL Η2/g διαλυτών υδατανθράκων που καταναλώθηκαν). Τα εμπορικά διαλύματα ενζύμων Celluclast 1.5L (διάλυμα ενδο-β-γλυκανάσης) και Novozyme 188 (διάλυμα β-γλυκοσιδάσης) χρησιμοποιήθηκαν για την ενζυμική υδρόλυση του αραιωμένου ελαιοπολτού (1:4). Συμπερασματικά, πειράματα διαλείποντος έργου κατέδειξαν ότι, η απόδοση της αναερόβιας διεργασίας παραγωγής υδρογόνου από αραιωμένο ελαιοπολτό (1:4) καθίσταται βέλτιστη με την προσθήκη μόνο Celluclast 1.5L σε συγκέντρωση 50 FPU/g αδιάλυτων υδατανθράκων υποστρώματος και σε αναλογία όγκων υποστρώματος/μαγιάς μικροοργανισμών (S/X) ίση με 1 σε διεργασία ενός σταδίου. Τέλος, μελετήθηκε η επίδραση της προσθήκης του ενζύμου Celluclast 1.5L στην απόδοση της συνεχούς διεργασίας παραγωγής υδρογόνου από αραιωμένο ελαιοπολτό (1:4) στον αντιδραστήρα τύπου CSTR.
Olive mill wastes constitute one of the most important environmental problems of Mediterranean region, because of their thoughtless disposal. It is characteristic that, approximately 95% world’s olive oil production is derived from small, familiar enterprises which are mainly located in Mediterranean countries. The biotechnological exploitation of olive mill wastes for anaerobic hydrogen production was the aim of this thesis. In details, the possibility of hydrogen production from semi-solid residue derived from two-phase centrifugation process (olive pulp) and olive mill wastewater derived from three-phase centrifugation process (OMW) was examined with mixed anaerobic cultures under mesophilic conditions. The wastes were previously diluted with tap water (1:4), in order to be susceptible for biological treatment. Various experiments in CSTR type reactors showed that, the continuous mesophilic anaerobic hydrogen production is feasible from diluted olive pulp (1:4) and diluted OMW (1:4) as well. The potential of hydrogen production from diluted olive pulp (1:4) was lower than the maximum theoretical potential (4 mol H2/mol consumed glucose) probably due to the negative effect of partial pressure of hydrogen. The anaerobic digestion model No 1 (ADM1) was properly modified in order to describe the anaerobic hydrogen production. All the model’s critical parameters were determined by fitting the experimental data of continuous anaerobic hydrogen production from diluted olive pulp (1:4), while batch experiments were conducted for their verification. In order to examine the validity and the reliability of the modified model for the description of anaerobic hydrogen production from various types of olive mill wastes, it was also tested in the case of diluted ΟMW (1:4) anaerobic treatment. Pretreatment methods of diluted olive pulp (1:4) were developed and evaluated (physicochemical methods and enzyme hydrolysis) targeting to the increase of soluble carbohydrates available concentration, while in the cases where this was achieved the effect on hydrogen potential was investigated. This attempt was based on the conclusion derived from batch experiments, indicated that, the non-soluble carbohydrates contribute to anaerobic hydrogen production only to a very small extent, with their concentration correspond approximately to 50% of waste content in total carbohydrates. Among the physicochemical methods that were applied (addition of alkaline solution, ozonation, treatment with steam), the treatment with steam (1 bar, 121oC) for 60 min was selected as the optimum method, because the achieved increase in soluble carbohydrates concentration was the highest (about 26%) with the least economic cost. The potential of anaerobic hydrogen production was increased approximately 45% (expressed as mL H2/g soluble carbohydrates consumed). Two commercial enzyme solutions, Celluclast 1.5L (endo-β-glucanase) and Novozyme 188 (β-glucosidase), were used for the enzymatic hydrolysis of diluted olive pulp (1:4). Conclusively, the potential of anaerobic hydrogen production from diluted olive pulp (1:4) was optimum with the addition of Celluclast 1.5L (50 FPU/g non soluble carbohydrates from substrate) and substrate/mixed culture volume ratio (S/X) equal to 1 in one stage process (Simultaneous Saccharification and Fermentation, SSF) Finally, enzyme (Celluclast 1.5L) was added into the CSTR-type reactor in order to determine the effect in the potential of anaerobic hydrogen production from diluted olive pulp (1:4).

碩士
國立成功大學
環境工程學系碩博士班
99
In the recent years, clear and renewable energy has become an emerging topic to ensure the environment sustainability in the future. That effectively converts lignocellulosic biomass to the energy and reusable solvents already drew much attention, while the biological processes such as the simultaneous saccharification and co-fermentation (SSCF) or the recent consolidated bioprocessing (CBP) were developed to demonstrate the feasibility. Whichever the process was actually required the cellulase production, hydrolysis and fermentation mediated by microbial populations to increase the efficiency of energy recovering. In this research, it was focused on the selected members in the Genus Clostridium with a goal obtain the cellulolytic and fermentative anaerobes at the thermophilic anaerobic conditions, and then preliminary characterization on biochemical and physiological properties of these microorganisms in cellulose hydrolysis were studied accordingly. To achieve so, the cattle manure was selected as the microbial source, and succeeded in enriching the key microbial populations capable of hydrolyzing filter paper. Based on microbial community analysis, it was found that the various Clostridium groups were present in the enrichment, including Clostridial cluster I, III, XII, and XVIII. Of those, the strain H1 close to Clostridium clariflavum within the cluster III was successfully isolated in the pure culture and then further studied for the cellulolytic capability. The scanning electron microscope observation showed that the strain H1 could attach on the fibers of the filter paper during hydrolyzing and form the terminal endospores in the aged medium environment. In particular, the strain H1 seemed to produce yellow affinity substance (YAS) to enhance the cellulose hydrolysis. Besides, the strain H1 had the optimal growth temperature between 55~60℃ and the optimal initial pH between 7.5~8. Using cellobiose as the growth substrate, the strain H1 needed a lag time of 16 hrs to adapt the environment and then reached the plateau phase in 44 hrs in the growth curve. In addition to cellobiose, the strain H1 could favorably utilize cellulose and xylan to produce hydrogen and ethanol with accumulation of reducing sugar. For example, when the initial concentration of α-cellulose at 5 g / L, it was found the highest concentration of reducing sugar accumulated up to 2.23 ± 0.3 g / L. This might be because the strain H1 had a slower ability on fermenting oligosaccharides like cellobiose, sucrose and monosaccharides like glucose, xylose. However, as compared with the strain Thermoanaerobacterium thermosaccharolyticum RCB isolated from bagasse compost previously in our laboratory, it had a good ability utilizing xylan, oligosaccharides and monosaccharides to produce hydrogen, ethanol and butanol but not good in utilizing cellulose substrates. Further, the result of the cellulase activity analysis showed that the strain H1 reached the stationary level with the increasing rate of reducing sugar concentration 1.70 g/L/day. It was discovered that the highest activity of cell-free and cell-bound xylanase was 7.45 ± 2.98 U/mg and 2.03 ± 0.30 U/mg, respectively, whereas the expression of exoglucanase and endoglucanase enzyme activities was not prominent. Finally, the strain H1 was applied to hydrolyze the natural cellulose materials like bagasse, rice straw, and napier grass after alkali treatment. The results showed that when the initial concentration of carbon at 5 g / L was hydrolyzed by the strain H1, the highest concentration of reducing sugar could accumulate up to 1.32 ± 0.3 g /L in the culture with xylanase activity of 1.91 ~ 3.01U/mg. The overall result from this study suggested that the strain H1 has a great potential in converting the cellulose feedstock to sugars, which can be further converted to bioenergy using the hydrogen-producing or alcohol-producing specialists such as T. thermosaccharolyticum RCB. From this research, we succeeded in getting the cellulolytic bacteria which produced the cellulase and the fermentative bacteria, and understand the characterization. It could provide this datum for the actual reactor operation or the reference of other biomass energy.

碩士
東海大學
環境科學與工程學系
97
As technology is developed fast, there is an urgent need for development of renewable energy source. Among different kinds of renewable energy, biofuels produced by conversion of cellulosic materials have attacted attention worldwide in recent years. Hydrogen is considered a clean and efficient energy among renewable energy and will not cause the secondary pollution. Hydrogen can be produced from organic wastes by fermentative microorganisms. Therefore, hydrogen has the potential for replacing conventional fossil fuels. The purpose of this study was to find the optimal conditions for a thermophilic anaerobic isolate Clostridium thermocellum strain TCW1 for its conversion of cellulosic materials to hydrogen gas. The experimental results showed the optimal temperature of strain TCW1 for hydrogen production was 60℃, optimal pH was 6.99, optimal substrate concentration was 5 g/L and Fe2+ concentration and Ni2+ concentration were 5 mg/L and 0.01 mg/L, respectively. The highest quantity of hydrogen production was 462.3 mL/L • liquid under optimal conditions with 160 rpm agitation. Hydrogen concentration produced by strain TCW1 could reach 34.12％, and H2 yield could reach 106.7 mL/g • cellulose. Strain TCW1 could also produce H2 by fermenting different natural cellulosic materials such as rice straw , napier grass, orange peel and vegetable leaf. Cumulative hydrogen produced from natural substrates could reach 416.0(napier grass) and 358.1(rice straw) mL H2/L, respectively. Hydrogen concentration were 27.73(napier grass) and 23.88(rice straw)％, and H2 yield were 83.20 and 71.63 mL/g • substrate.