The activated sludge process efficiency depends on separation of microbial cells from treated wastewater. Separation can fail due to a number of problems. One of these problems is sludge bulking which is non-settling situation of biomass. Former studies showed that phosphorus deficiency caused filamentous sludge bulking with increasing magnesium ion concentrations. The main objectives of this study are to find out the effect of magnesium ions on sludge properties in phosphorus deficient medium and to determine if there is any bulking. Three different concentrations of magnesium (0.5, 5, 15 meq/L) were added to three bioreactors which contained phosphorus deficient medium. In first set C: N: P ratio was 100:5:0.05. In second set, C:N:P ratio was elevated to 100:5:1. At steady state, physical characteristics including sludge volume index (SVI), viscosity, turbidity and dewaterability were determined. Besides concentration of extracellular polymeric substances (EPS) as well as conductivity was measured. By using API kits, bacterial identification was achieved. In first set phosphorus deficiency and increasing magnesium ion concentration caused filamentous bulking. Carbohydrate content of extracellular polymeric substance significantly increased by magnesium addition. Dewaterability of the system got worse and viscosity decreased. Sludge Volume Index (SVI) indicated severe bulking at all magnesium concentrations. By using biochemical tests microorganisms dominant in the system were determined In second set, all of the parameters indicated healthy flocculation. By magnesium addition, EPSp and EPSc increased. Dewaterability and settleability, improved by the presence of phosphorus with close values measured at different magnesiuim concentrations. Nocardia related genera of Corynebacterium and Enteric microorganisms were identified.

Biological filtration (biofiltration) of drinking-water is gaining popularity due the potential for biodegradation of an array of contaminants not removed by traditional drinking-water processes. However, previous research has suggested that biomass growth on biofilter media may lead to increased headloss, and thus, greater energy and water requirements for backwashing. Research has suggested that the main cause of headloss might be due to extracellular polymeric substances (EPS) rather than the bacterial cells themselves. As EPS production has been shown to increase under nitrogen- and phosphorus-limited or -depleted conditions, the goal of this research was to add to the body of knowledge regarding biofiltration by studying the relationship between EPS production and nutrient limitations in drinking-water. Batch experiments with a synthetic groundwater were run with a mixed community of drinking-water bacteria under nutrient-balanced (a molar carbon to nitrogen to phosphorus ratio [C:N:P] of 100:10:1), nutrient-limited (e.g., C:N:P of 100:10:0.1), and nutrient-depleted conditions (C:N:P of 100:0:1 or 100:10:0). After 5 days, growth was measured as the optical density at 600 nanometers (OD600), and the concentrations of free and bound carbohydrates and proteins, the main components of EPS, were measured. In batch experiments with 2.0 and 0.2 g/L as carbon (mixture of acetic acid, mannitol and sucrose) increases in EPS production per OD600 and decreases in growth were noted under nutrient-depleted conditions. When the same experiments were conducted with a pure culture of Bacillus cereus, bound polysaccharides normalized to OD600 increased under nitrogen- and phosphorus-depleted conditions. Since previous research suggested that Bradyrhizobium would be an important player in EPS production in drinking-water biofilters, similar batch experiments were conducted with Bradyrhizobium. However, due to experimental challenges with Bradyrhizobium japonicum USDA 110, differences in EPS production under nutrient limitations could not be reliably assessed. Additional work is required with Bradyrhizobium. Recommendations for future work include the replication of these batch conditions in steady-state chemostats containing biofilm attachment media and in bench-scale columns. Additionally, future work should include experiments at carbon concentrations as low as 2 mg/L to match typical carbon concentrations in drinking-water biofilters. / text

Activated sludge systems are the most widely used biological wastewater treatment processes all over the world. The main working principles of an activated sludge system are the oxidation of biologically degradable wastes by microorganisms and the subsequent separation of the newly formed biomass from the treated effluent. Separation by settling is the most troublesome stage of an activated sludge process. A decrease in the efficiency of the separation of microbial biomass from the treated effluent causes a decrease in the overall efficiency of the treatment plant. The efficiency of the separation process is related to the bioflocculation, which can be briefly defined as the aggregation of the bacteria into flocs through flocculation. Bioflocculation depends on the extracellular polymers (EPS) that are produced by microorganisms. The operating conditions of the activated sludge system is a key determinant of the synthesis of EPS and bioflocculation. The main objective of this study is to find out the effect of magnesium ions on the bioflocculation process under phosphorus deficient and sufficient conditions. In order to achieve this aim, the effects of magnesium ion in 4 different concentrations (0.9, 5, 10 and 20 meq/L) are investigated in semi continuous reactors. The reactors are operated at a mean cell residence time of 8 days and 20&ordm / C temperature. When reactors are confirmed to be at steady state, several sets of analysis are conducted. In particular, the surface chemical parameters including EPS and its components, electrical charge, and hydrophobicity as well as physical properties such as settlability, filterability, viscosity, floc strength, and turbidity are examined. It has been understood that phosphorus deficiency causes severe filamentous bulking under magnesium rich conditions. Increasing the phosphorus concentration in the influent can cure this problem. After the sludges are cured some granular structures were observed in the microscopic investigations and they are thought to be polyphosphate granules in which microorganisms tend to accumulate phosphorus when they find the adequate source after a starvation period. To consider the reactors operated at phosphate present conditions, it has been found that EPS increases with increasing influent magnesium concentration. However, protein type EPS (EPSP) exhibits a sharper increase when compared to the carbohydrate type EPS (EPSC) indicating the selective attitude of magnesium ions to protein type of polymers. It has been understood that the increase in the influent magnesium concentration results in an increase in dewaterability and zone settling velocity, and a decrease in the viscosity. Hydrophobicity was found to exhibit a maximum value at 10 meq/L magnesium fed sludge and then it dropped back. Surface charge values also made a minimum at 10 meq/L reactor and then no change occurred at the increase of the magnesium concentration to 20 meq/L. Finally, COD values were found to increase with the increasing magnesium concentration due to the increasing EPS.

No description available.

Although high rate algae ponds (HRAPs) are a proven wastewater treatment technology with numerous environmental, social, and economic benefits, their widespread use has been hindered by inconsistent and unreliable settling performance. Hence, the goal of this thesis is to investigate how specific operational parameters affect the settling performance of HRAPs. Nine HRAPs (30 m2 surface area, 0.3 m depth) were operated as three triplicate sets, with each set run on either a 2, 3, or 4 day HRT continuously from January 25, 2012 through April 11, 2013. Settling performance was determined (i) by measuring the TSS of Imhoff cone supernatant after 2 and 24 hours of settling and (ii) by measuring the TSS of tube settler effluent. Ponds operating on 2 - 3 day HRTs (loading rate was 24 - 36 g/m3-day csBOD5 and food to microorganism (F/M) ratio was 0.13 - 0.21 day-1) were able to settle consistently with residual TSS averaging less than 40 mg/L after 2 hours of settling time. Tube settlers showed potential as effective harvesting devices; ponds operating on a 2-day HRT averaged 27.9 ± 9.2 mg/L TSS in tube settler effluent at an overflow rate (OFR) of 9.4 L/min-m2. Microscopy analysis was performed and relationships were made between settling performance and algae dominance and floc structure.

Although the A-stage high-rate activated sludge (HRAS) process destroys some of the chemical energy present in municipal wastewater, this process has been gaining attention as a viable technology for achieving energy neutrality at water resource recovery facilities. In addition to carbon capture for energy recovery, A-stages are also being utilized upstream of shortcut biological nitrogen removal (BNR) processes as these BNR processes often require a controlled influent carbon to nitrogen ratio that is lower than required for conventional BNR processes. While there is extensive knowledge on conventional activated sludge processes, including process controllers and activated sludge models, there has been little detailed research on the carbon removal mechanisms of A-stage processes operated at solids retention times (SRT) less than about one day. The overall objective of this study was to elucidate the chemical oxygen demand (COD) removal mechanisms of short SRT activated sludge processes with a specific focus on the removal of the different COD fractions under varying operating conditions including dissolved oxygen, hydraulic retention time, temperature, and SRT. Once understood, automatic process control logic was developed with the purpose of producing the influent characteristics required for emerging shortcut BNR processes and capturing the remaining COD with the intent of redirecting it to an energy recovery process. To investigate the removal and assimilation of readily biodegradable substrate (SS), this study evaluated a respirometric method to estimate the SS and active heterotrophic biomass (XH) fractions of the raw wastewater influent and effluent of an A-stage pilot process. The influent SS values were comparable to the SS values determined using a physical-chemical method, but the effluent values did not correlate well. This led to the measurement of the heterotrophic aerobic yield coefficient and decay rate of the pilot process. The yield coefficient was estimated to be 0.79±0.02 gCOD/gCOD, which was higher than the accepted value of 0.67 g/g. It was speculated that the batch respirometry tests resulted in the aerobic storage of SS and this likely contributed to the error associated with the determination of SS and XH. Therefore, physical-chemical fractionation methods were used to determine the removal of the individual COD fractions. It was concluded that the SRT was the primary control parameter and below a 0.5 day SRT the dominate COD removal mechanisms were assimilation and oxidation of readily degradable substrate and sedimentation of particulate matter. At SRTs between 0.5-1 days, COD removal became a function of hydrolysis, as adsorption of particulate and colloidal matter was maximized but not complete because of limited adsorption sites. Once adequate adsorption sites were available, effluent quality became dependent on the efficiency of bioflocculation and solids separation. While the SRT of the pilot process could not be directly controlled because of severe biofouling issues when using in situ sensors, a MLSS-based SRT controller was successfully implemented instead. The controller was able to reduce total COD removal variation in the A-stage by 90%. This controller aslo provided the capability to provide a consistent carbon to nitrogen ratio to the downstream B-stage pilot process. To ascertain the settling, dewaterability, and digestibility of the sludge produced by the pilot A-stage process, several standardized and recently developed methods were conducted. The results from these tests indicated that the A-stage had similar dewaterability and digestibility characteristics to primary sludge with average achievable cake solids of 34.3±0.4% and average volatile solids reduction (VSR) of 82±4%. The A-stage sludge also had an average specific methane yield of 0.45±0.06 m3CH4/kgVS. These results were attributed to low extracellular polymeric substance (EPS) content. However, further research is needed to better quantify EPS and determine the effect of HRAS operating parameters on EPS production. Overall the A/B pilot study was able to capture 47% of the influent COD as waste sludge while only oxidizing 45% of the influent COD. Of the COD captured, the A-stage contributed over 70% as dry solids. Coupled with high sludge production, VSR, and methane yield the A/B process was able to generate 10-20% more biogas and 10-20% less dry solids after anaerobic digestion than a comparable single-sludge BNR process. / Ph. D.

Activated sludge treatment systems rely on a microbial consortium structurally organized in bioflocs to treat pollutants present in wastewater. The treatment process efficiency in these systems can be severely affected by toxic chemicals present in the influent wastewater. The effects of chemical toxins at the treatment process level are determined by the mechanisms that occur at the biofloc and cellular levels, which can be physical, chemical and physiological in nature. We believe that the overall process effects of chemical toxins on activated sludge systems likely result from a combination of all three types of mechanisms and that they are interdependent, in the sense that specific bacterial stress response mechanisms (physiological mechanisms that protect the cell from toxic conditions) may lead to physical/chemical alterations at the floc level, and vice-versa. Ultimately, understanding the mechanisms that occur at the floc and metabolic scales will help to design more robust and efficient treatment systems, and to develop tools to prevent and mitigate the effects of toxic chemicals on activated sludge systems. In this research, we set out to establish the link between the effects of chemical toxins on activated sludge cultures at the process, floc and metabolic scales. First, the effects of shock loads of different toxic sources (1-chloro-2,4-dinitrobenzene (CDNB), cadmium, 1-octanol, 2,4-dinitrophenol (DNP), weakly complexed cyanide, pH 5, 9 and 11, and high ammonia levels) on activated sludge process parameters (biomass growth, respiration rate, flocculation, chemical oxygen demand (COD) removal, dewaterability and settleability) were studied. For all chemical shocks except ammonia and pH, concentrations that caused 15, 25 and 50% respiration inhibition were used to provide a single pulse chemical shock to sequencing batch reactor (SBR) systems containing a nitrifying (10 day solids retention time – SRT) and a non-nitrifying (2 day SRT) biomass. We found that cadmium and pH 11 shocks were the conditions that most detrimentally affected all the processes, followed by CDNB. DNP and cyanide primarily led to effects on respiration, while pH 5, 9, octanol and various ammonia concentrations did not impact the treatment process to a significant extent. Additionally, there was a clear correlation between biomass deflocculation and increases in the effluent soluble COD of the shocked reactors for different chemical sources. With this study, we were able to establish a source-effect matrix linking classes of chemical toxins to their potential inhibitory effects on activated sludge processes, thereby contributing to a better understanding of the potential effects of toxic industrial discharges into biological treatment systems. The findings of the first phase of the research, specifically the correlation between chemical-induced deflocculation and increases in soluble COD, served as a motivation to explore the role of floc structure in the response of activated sludge cultures to toxic compounds, and to conduct a more in-depth analysis of the supernatant (soluble phase) of toxin-exposed activated sludge. In one study, we evaluated the respiration inhibition induced by octanol, cadmium, N-ethylmaleimide (NEM), cyanide and DNP on activated sludge biomasses with different floc structures but similar physiological characteristics, with the objective of assessing the role of the extracellular polymeric substances (EPS) in flocs as a protection barrier against chemical toxins. Mechanical shearing was applied to fresh mixed liquor to produce biomasses with different floc structure properties and specific oxygen uptake rate assays were conducted on the sheared and unsheared mixed liquors. The results showed that the respiration inhibition by octanol and cadmium was more intense in sheared mixed liquor (which had less EPS material available in the flocs and smaller floc sizes) than in the unsheared biomass. Conversely, the respiration inhibition induced by NEM and cyanide was similar for the different mixed liquors tested. These results allowed us to conclude that the EPS matrix functions as a protective barrier for the bacteria inside activated sludge flocs to chemicals that it has the potential to interact with, such as hydrophobic (octanol) and positively-charged (cadmium) compounds, but that the toxicity response for soluble, hydrophilic toxins (NEM and cyanide) is not significantly influenced by the presence of the polymer matrix. In the final study that was conducted, we used the metabolomics-based technique metabolic footprinting to assess if the soluble phase of mixed liquor exposed to different chemical toxins exhibited a toxin-specific biochemical composition. We hypothesized that toxin-specific effects could be distinguished through footprint patterns of those soluble samples. The impact of cadmium, DNP and NEM shock loads on the composition of the soluble fraction of activated sludge mixed liquor was analyzed by liquid chromatography-mass spectrometry (LC-MS). The results from this study indicated that there was a significant release of biomolecules (proteins, carbohydrates and humic acids) from the floc structure into the bulk liquid due to chemical stress. More importantly, using a multivariate statistical method called discriminant function analysis with genetic algorithm variable selection (GA-DFA), we were able to show that the soluble phase samples from the different reactors could be differentiated, thereby indicating that the footprints generated by LC-MS were different for the four conditions tested and, therefore, toxin-specific. These footprints, thus, contain information about specific biomolecular differences between the samples, and we found that only a limited number of m/z (mass to charge) ratios from the mass spectra data was needed to differentiate between the control and each chemical toxin-derived samples. In addition, since the experiments were conducted with mixed liquor from four distinct wastewater treatment plants, the discriminating m/z ratios may potentially be used as universal stress biomarkers. These results are promising and indicate that LC-MS may be used for the discovery of activated sludge stress biomarkers, to allow the development of new toxin detection technologies for prevention of upset events in activated sludge systems. / Ph. D.

The aim of this master thesis study was improvement of the required operational conditions for aerobic granulation in sequencing batch reactors (SBRs). In the first part of the study, membrane bioreactor sludge (MBS) and conventional activated sludge (CAS), were used to investigate the effect of suspended seed sludge type on granulation in SBRs. The MBS granules were found to be advantageous in terms of size, resistance to toxic effects, stability and recovery compared to CAS granules. During non-inhibitory conditions, sCOD removal efficiencies were 70&plusmn / 13% and 67&plusmn / 11% for MBS and CAS, and total nitrogen (TN) removal efficiencies were 38&plusmn / 8% and 26&plusmn / 8%, respectively. In the second part of the study, the effects of period sequence (anoxic-aerobic and aerobic-anoxic) on aerobic granulation from MBS, and sCOD, N removal efficiencies were investigated. Granules developed in anoxic-aerobic period sequence were more stable and larger (1.8-3.5 mm) than granules developed in aerobic-anoxic sequence. Under steady conditions, almost 95% sCOD, 90% Total Ammonia Nitrogen (TAN) and around 39-47 % of TN removal was achieved. Almost 100% denitrification in anoxic period was achieved in anoxic-aerobic period sequence and it was observed around 40% in aerobic-anoxic period sequence. The effects of influent sulfate (from 35.1 mg/L to 70.2 mg/L) on treatment efficiencies of aerobic granules were also investigated. The influent SO42- concentrations of 52.6 mg/L to 70.2 mg/L promoted sulfate reduction. The produced sulfide (0.24 mg/L to 0.62 mg/L) inhibited the ammonia-oxidizing bacteria (AOB) performance by 10 to 50%.

Diplomová práce se zabývá vlivem uhlíkatého plniva na životaschopnost a produkci extracelulárních látek vybrané bakterie Bacillus subtilis (CCM 1999) a kvasinky Yarrowia lipolytica (CCY 29-26-52). Antimikrobiální aktivita těchto částic, přítomných v kultivačním mediu, byla sledována pomocí následujících parametrů: růst daného mikroorganismu, produkce extracelulárních proteinů a v poslední řadě byla monitorována produkce extracelulárních polymerních substancí, které mají úzkou souvislost s tvorbou biofilmu. Suspenze materiálů (0,135 mg/mL) byly připraveny ve dvou rozdílných kultivačních mediích; tzn. živné medium s obsahem glukózy pro Bacillus subtilis a bazální medium s přídavkem Tweenu 80 pro Yarrowia lipolytica, a media byla inokulována příslušným typem mikroorganismu. Experimenty probíhaly po dobu 6 dnů při rychlosti třepání 160 rpm a teplotě 30 °C pro Bacillus subtilis a 28 °C pro Yarrowia lipolytica. Testovány byly celkem tři typy uhlíkatého nanomateriálu, získané z Katedry anorganické chemie, Vysoké školy chemicko-technologické v Praze. Tyto materiály specifikované jako materiál “A”, “B” a “C” se navzájem lišily velikostí částic a stupněm oxidace. Na základě skríningových studií byla vybrána koncentrace testovaného materiálu 0,135 mg/mL a rychlost třepání 160 rpm. Metodou měření optické hustoty vzorku při 600 nm byly sestaveny a porovnány růstové křivky obou mikroorganismů v přítomnosti testovaných nanočástic po dobu 5 dní. Tímto způsobem bylo zjištěno, že přítomnost nanočástic v mediu nemá velký vliv na růst zkoumaného mikroorganismu. Tato metoda, je však pouze orientační, protože se nevyhneme chybě díky přítomnosti mrtvých buněk. Dále byla testována produkce celkových a extracelulárních proteinů daným mikroorganismem v přítomnosti testovaných nanočástic. Nebyla však pozorována výrazná odchylka hodnot od hodnot kontrolního vzorku, který neobsahoval testovaný materiál. Na základě metod počítání kolonií (Bacillus subtilis) a buněk (Yarrowia lipolytica) byly určeny ztráty životaschopnosti mikroorganismu ve 3 časech (6, 48 a 144 hodin); v kratším časovém intervalu byl růst spíše podporován. Dále byla monitorována produkce extracelulárních polymerních substancí (EPS), tedy proteinů, redukujících substancí a polysacharidů. Tyto látky byly vylučovány daným mikroorganismem do prostředí v průběhu 24 hodin. Bacillus subtilis produkoval EPS ve větší míře než Yarrowia lipolytica. Předpokládáme, že produkce EPS by mohla souviset s tvorbou biofilmu, který chrání buňky před toxicitou nanočástic.

This study is the first to consider how the combination of root physical effects, microbial production of EPS, and root effects on the hydrodynamic boundary layer could influence streambank soil erodibility. Specifically, the goal of this research was to quantify the physical and biological effects of roots on streambank fluvial erosion. A series of laboratory-scale erosion tests were conducted using a mini jet erosion testing device and a recirculating flume channel to address this goal. Several soil and vegetation factors that influence fluvial entrainment, like extracellular polymeric substances (EPS), soil aggregate stability and root length density, were measured following erosion testing. For flume experiments, three streambank boundary conditions were constructed to simulate unvegetated streambanks, as well as streambanks with herbaceous and woody roots. Soil treatments were also created to represent unamended and organic matter (OM) amended soil either without roots (bare soil), with synthetic roots, or with living roots (Panicum virgatum). Median soil erosion rates along the simulated rooted boundaries were two to ten times higher compared to the unvegetated boundary due to protruding root impacts on the boundary layer. In flume experiments, median erosion rates were 30% to 72% lower for unamended soils containing compacted synthetic root fibers as compared to bare soil samples. Adding both OM and fibers to the soil had a greater effect; the median erosion rate reductions of live rooted treatments (95% to 100%) and synthetic rooted + OM treatments (86% to 100%) were similar and statistically lower than bare soil controls. Stimulated microbial production of EPS proteins were significantly correlated with increased erosion resistance in OM-amended treatments while OM treatments had significantly lower EPS carbohydrates compared to unamended treatments. In summary, while sparsely spaced roots exposed on streambanks may increase soil erosion rates due to impacts on the hydrodynamic boundary layer, overall results highlight how the synergistic relationship between root fibers and soil microbes can significantly reduce streambank soil erodibility due to fiber reinforcement and EPS production. / Doctor of Philosophy / Plant roots are known to protect streambank soils from erosion by water; however, exactly how roots provide this protection has remained unclear. Among other things, roots can influence streambank soil erosion by holding soil together through a thick root network, interacting with soil microorganisms to stimulate the release of "sticky" organic compounds called extracellular polymeric substances (EPS), and altering the force of the water against the streambank. This research aimed to quantify and compare the relative importance of these three mechanisms on streambank soil erosion using a mini jet erosion testing device and an indoor recirculating flume channel. To do this in the flume, three walls were constructed to simulate unvegetated streambanks, as well as streambanks with herbaceous and woody roots. In greenhouse settings, soil treatments were also created to represent unamended and organic matter (OM) amended soil either without roots (bare soil), with artificial roots, or with living roots (Panicum virgatum). While roots protruding out of streambanks appeared to increase median soil erosion rates due to the impact of roots on near-bank flow, artificial roots in the soil and OM amended soils reduced soil erosion rates. Specifically, OM amendments stimulated the production of EPS proteins, leading to improved soil stability and increased soil resistance to erosion by water. Overall results highlight how the synergistic relationship between root fibers (living roots and artificial roots) and soil microbes can significantly reduce streambank soil erodibility due to root binding and microbial production of EPS. While plant roots naturally provide both fibers and EPS to soils, these materials could be incorporated into fill soils during construction to rapidly increase soil erosion resistance following levee construction and stream restoration projects.