The extracellular polymers produced by bacteria play an important role in bacterial aggregation or bacterial flocculation in secondary waste treatment. The mechanisms responsible for this floc formation are thought to be polymer induced adsorption and interparticle bridging among bacterial cells or between bacterial cells and inorganic colloids. The efficiency of the processes following flocculation in the treatment line such as sedimentation, sludge thickening, and sludge dewatering depends on the extent of this bacterial flocculation. In this research, sludge samples from under various substrate conditions were examined for type, molecular weight, physical characteristics„ and quantity of extracellular polymers so that the general characteristics of the various polymers could be established. An attempt was made to determine if a relationship exists between the state of bacterial aggregation and the polymer characteristics. This research also investigated the sludge physical properties. The effect of various parameters such as pH, divalent cation (mixture and concentration), and mixing (period and intensity) on dewatering properties were studied. A major goal of this study was to develop a flocculation model for activated sludge. This model could be used to determine if plants can increase the efficiency of waste treatment and sludge thickening and sludge dewatering processes. / Ph. D.

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

This research first examined nitrification inhibition caused by different classes of industrially relevant chemicals on activated sludge and found that conventional aerobic nitrification was inhibited by single pulse inputs of every chemical tested, with 1-chloro-2,4-dinitrobenzene (oxidant) having the most severe impact, followed by alkaline pH 11, cadmium (heavy metal), cyanide, octanol (hydrophobic) and 2,4-dinitrophenol (respiratory uncoupler). Of the different chemicals tested, the oxidative and hydrophobic chemicals showed severe nitrification inhibition relative to other treatment processes and therefore deserved further investigation. For oxidative chemicals, we hypothesized that the more severe inhibition was because nitrifying bacteria lack one or more of the microbial stress response mechanisms used to mediate the toxic effect of oxidative chemicals. During these experiments, we showed that a rapid (minutes) antioxidant potassium efflux mechanism does not exist in two nitrifying bacteria, Nitrosomonas europaea and Nitrospira moscoviensis. Furthermore, we showed that another important antioxidant molecule, glutathione, was not oxidized as readily as in a non-nitrifying bacterium. Furthermore, we hypothesized that hydrophobic chemical-induced nitrification inhibition recovered more quickly because of the presence of membrane modification stress response mechanisms. While testing this hypothesis, we showed that N. europaea modified its cell membrane in response to hydrophobic chemicals using a long-term (hours) membrane modification mechanism that required the synthesis of new fatty acids, but it did not contain a short-term (minutes) response mechanism involving a cis/trans isomerase. Therefore, investigating these nitrifier stress responses showed that nitrifiers lack short-term stress responses that may be used to rapidly detect inhibition, indicating that conventional methods of detecting nitrification inhibition, like differential respirometry and nitrate generation rate (NGR), are still the fastest and easiest methods to use. Because several conventional methods exist, we also investigated differences between differential respirometry and a UV method we developed to measure NGR. During these tests, we showed that the UV NGR method provided a more reliable measure of nitrification inhibition than differential respirometry, and that the time to maximum nitrification inhibition depended on the properties of the chemical toxin, which implies that longer exposure times may be needed to accurately predict nitrification inhibition. / Ph. D.

M.S.

An activated sludge system that incorporates a sidestream anaerobic bioreactor, called the Cannibal process, was the focus of this study. A prior study of this process (Novak et al., 2007) found that this system generated about 60% less solids than conventional activated sludge without any negative effects on the effluent quality. Although that study showed substantial solids reduction, questions remain concerning the specific mechanism(s) that account for the solids loss. In this study, the mechanisms that account for the loss of biological solids was the focus of the investigation. The first part of this study was conducted to evaluate those effects in terms of the role of iron in the influent wastewater and feeding patterns on the performance of the Cannibal system. It was found that the Cannibal system with high iron in the influent produced less biological solids than the system receiving low iron. The data also showed that the Cannibal system operated under fast feed (high substrate pressure) produced much less solids than the system with slow feed (low substrate pressure). The high substrate pressure was achieved by feeding the influent wastewater to the Cannibal system over a short time period so that the substrate concentration would initially peak and then decline as degradation occurred. This is called "fast feed." For low substrate pressure, the influent was added slowly so the substrate concentration remained low at all times. This is called "slow feed." Later, an attempt to increase substrate pressure in the slow feed Cannibal system was conducted by either manipulating the aeration patterns or adding a small reactor in front of the main reactor (selector). It was found that either interrupting aeration in the aerobic reactor or providing a small aerobic reactor in front of the main reactor resulted in an increase in solids reduction. The second part of this study was to investigate the mechanisms of floc destruction in the fast and the slow feed Cannnibal systems. It was found that higher accumulation of biopolymers (proteins and polysaccharides) occurred in the fast feed system and this was associated with a greater solids reduction in the fast than the slow feed system. In addition, more protein hydrolysis and more Fe(III)-reducing microorganism activity in the fast feed environment were found to be factors in higher solids reduction. The last part of this study was to investigate the structure of the Cannibal sludge flocs generated under the fast and the slow feed conditions. It was found that the readily biodegradable (1 kDa.) protein is larger in the flocs from the fast feed than the slow feed Cannibal system. This resulted in higher floc destruction in the fast feed condition. / Ph. D.

The purpose of this investigation was to conduct batch and continuous flow laboratory experiments with bench scale activated sludge units to determine the effects of nickel on the completely mixed activated sludge process. The model units were located in a constant temperature room maintained at 20±2°C. The batch reactors were operated under acclimated and shock loaded condition with various nickel concentration to determine its effects on the rate of COD removal. The continuous flow units were operated until steady state conditions were obtained at each mean cell residence time studied and then data were recorded for an approximate 7 days period and averaged to obtain one steady state data point. Nickel was added to the waste water at various concentration to determine its effects on COD removal efficiency, degree of nitrification and on the biokinetic constants Ymax and kd. / Ph. D.

The possible effects of changes in the feeding pattern on activated sludge properties related to bioflocculation have been analyzed in lab scale sequencing batch reactors (SBR) in order to determine if these changes in effluent water quality and settling and dewatering properties are significant, so they can be considered in future studies or if they can be recommended as crucial when operating and designing wastewater treatment plants. The activated sludge process is widely used to treat wastewater from both industrial and municipal sources. Biomass from industrial facilities containing high monovalent to divalent ion content usually settles poorly, which leads to low quality effluents that fail to meet environmental requirements. Therefore, the combined effect of feeding pattern plus the addition of sodium to activated sludge reactors was studied in this experiment. A series of SBRs were operated at different sodium concentrations that ranged from 1.5 - 15 meq/L and different feeding times that ranged from 1 minute to 4 hours. Biomass samples were taken from each reactor to study the settling and dewatering properties and effluent samples were used to analyze the amount of organic matter and exocellular polymeric substances present due to deflocculation. As expected, the changes in feeding strategies affected all of the properties measured. When the feeding time was maintained low (pulse feed) the effluent quality and settling properties were the best. As the feeding time was increased the effluent quality, settling, and dewatering properties increased suggesting that the way in which the reactors were fed affected the overall bioflocculation process. The causes of the high deflocculation observed are not well understood, but data suggest that a microbial community change could have affected exocellular biopolymers which are believed to play an important role on bioflocculation. This research demonstrates the importance of the interaction between cation content and feeding pattern when operating a wastewater treatment plants and when reporting lab-scaled results related to settling and bioflocculation. / Master of Science

The Cannibal system, comprised of an activated sludge process integrated with a side stream anaerobic bioreactor, is capable of reducing excess sludge up to 60% compared to the conventional activated sludge process. The hydraulic retention time (HRT) in the Cannibal bioreactor and the interchange rate (the percent of sludge by mass interchanged between the activated sludge system and the bioreactor on daily basis) are the two important operational parameters in the optimization of the Cannibal process. This research was designed to investigate the effect of the Cannibal bioreactor hydraulic retention time and the interchange rate on the solids destruction in the system. The first phase of this study has looked at the effect of three different HRTs, 5 day, 7 day and 10 day. The interchange rate during phase I was 10%. The results showed that the 7 day HRT can be recommended as the minimum retention period for the Cannibal process. The 5 day HRT Cannibal system had some settling problems and high volatile fatty acid content compared to the 7 day HRT Cannibal system. The protein and polysaccharide tests showed that the Cannibal bioreactor is primarily involved in the release of biopolymers which are degraded in the aerobic environment. The second part of this study focused on the effect of the interchange rate (IR) on the solids destruction in the system. The interchange rates that were applied in the system were 15%, 10%, 7%, 5% and 4%.The HRT in the Cannibal bioreactor was 7 day. The results showed that the 10% interchange rate gave maximum solids destruction than the other interchange rates. This implies that 10% is an optimum IR for the Cannibal system. Apart from higher solids wastage, the 4% and 5% IR Cannibal systems had higher volatile fatty acid production. / Master of Science

This study consists of three research phases. First, we developed corrective action strategies to mitigate the impact of calcium hypochlorite and cadmium pulse shocks for the Plum Island Wastewater Treatment Plant (WWTP) in Charleston, SC. The corrective action strategies were developed in consultation with industrial consultants and operational personnel from the utility. These strategies were tested using a laboratory scale system, which was constructed and operated similar to the parent facility. Two corrective actions were tested for calcium hypochlorite, while only one strategy was tested for the cadmium at the laboratory scale. This study shows that no corrective action strategies are required for an acute hypochlorite stress. This is due to the fact that hypochlorite is highly reactive and dissipates rapidly on contact with the wastewater matrix, thus causing only low level process deterioration. In fact, implementation of corrective action strategies results in greater process deterioration as compared to the non-intervention approach. The corrective action tested for cadmium stress showed potential for reducing the peak impact of the toxin and allowed for faster process recovery as compared to the unstressed control. For the second phase, the corrective actions were tested at a pilot scale facility operated at the Plum Island wastewater treatment plant. We tested two different corrective action strategies for cadmium, while only one strategy was tested for hypochlorite during the pilot scale study. Similar to the laboratory scale experiments, we conclude that no mitigative approaches are necessary for an acute hypochlorite stress. Additionally, the implementation of mitigative approaches for the pilot scale cadmium stress events resulted in greater process deterioration as compared to the non-intervention approach. In contrast to the laboratory scale experiments, theoretical effluent blending calculations showed that corrective actions may not reduce the impact of the cadmium stress. This was attributed to the lower intensity of process deterioration caused by the simulated cadmium stress. The pilot scale study shows that prior to implementing a corrective action strategy, the operator should determine the probable extent of process deterioration due to the detected chemical contaminant before deciding if a corrective action is needed. The pilot scale study also evaluated the effectiveness of current sensor technologies towards the upstream detection of influent anomalies and reliable monitoring of process performance during an upset event. Multivariate analysis on the rate of change of influent sensor signals was reliably able to detect the presence of both toxins tested during this study. For the third phase of this research, we investigated the impact of cadmium stress on the structure and function of bioreactor microbial communities. We observed significant increases in post-stress heterotrophic and autotrophic bacterial respiration rates for the bioreactors subjected to cadmium stress. The higher respiration rates were due to an increase in bacterial abundance in the cadmium stressed reactors. We were also able to show that the increase in bacterial abundance was not due to changes in community structure or due to cadmium induced deflocculation. In fact, this study demonstrates that transient cadmium stress reduces predator abundance within the activated sludge community and this reduction in predator grazing was responsible for the increase in bacterial abundance. This research highlights the importance of higher life forms, specifically eukaryotic microorganisms, in regulating bacterial community dynamics in systems undergoing chemical perturbations. / Ph. D.

Novel settling characteristic metrics were developed based on the fundamental mechanisms of coagulation, flocculation, and settling. The settling metrics determined parameters that are essential in monitoring and optimizing the activated sludge process without the need for expensive or specialized equipment. Current settling characteristic measurements that don't require specialized instruments such as sludge volume index (SVI) or initial settling velocity (ISV), have no fundamental basis in solid-liquid separation and only indicate whether settling is good or bad without providing information as to limitations present in a sludge matrix. Furthermore, the emergence of aerobic granulation as a potential pathway to mitigate solid-separation issues further stresses the need for new settling characteristic metrics to enable integration of the new technology with the current infrastructure. The granule or intrinsic aggregate fraction in different types was of sludge was quantified by simulating different surface overflow rates (SOR). The technique named Intrinsic Settling Classes (ISC) was able to separate granules and floc by simulating high SOR values due to the lack of a flocculation time needed for granules. The method had to be performed in a discrete settling environment to characterize a range of flocculation behavior and was able to classify the granular portion of five different types of sludge. ISC was proven to accurately (±2%) determine the granule fraction and discrete particle distribution. The major significance of the test is its ability to show if a system is producing particles that will eventually grow to become granules. This methodology proved to be very valuable in obtaining information as to the granular fraction of sludge and the granular production of a system. Flocculent settling (stokesian) was found to be predominant within ideally operating clarifiers, and the shift to 'slower' hindered settling (non-stokesian) causes both failure and poor effluent quality. Therefore, a new metric for settling characteristics was developed and classified as Limit of Stokesian Settling (LOSS). The technique consisted of determining the total suspended solids (TSS) concentration at which mixed liquor settling characteristics transition from stokesian to non–stokesian settling. An image analytical technique was developed with the aid of MATLAB to identify this transition. The MATLAB tool analyzed RGB images from video, and identified the presence of an interface by a dramatic shift in the Red indices. LOSS data for Secondary activated-sludge systems were analyzed for a period of 60 days at the Blue Plains Advanced Wastewater Treatment Plant. LOSS numbers collected experimentally were validated with the Takacs et al. (1991) settling model. When compared to flux curves with small changes in the sludge concentration matrix, LOSS was found to be faster at characterizing the hindered settling velocity and was less erratic. Simple batch experiments based on the critical settling velocity (CSV) selection were used as the basis for the development of two novel parameters: threshold of flocculation/flocculation limitation (TOF/a), and floc strength. TOF quantified the minimum solids concentration needed to form large flocs and was directly linked to collision efficiency. In hybrid systems, an exponential fitting on a CSV matrix was proposed to quantify the collision efficiency of flocs (a). Shear studies were conducted to quantify floc strength. The methods were applied to a wide spectrum of sludge types to show the broad applicability and sensitivity of the novel methods. Three different activated sludge systems from the Blue Plains AWWTP were monitored for a 1 year period to explore the relationship between effluent suspended solids (ESS) and activated sludge settling and flocculation behavior. Novel metrics based on the transitional solids concentration (TOF, and LOSS) were also collected weekly. A pilot clarifier and settling column were run and filmed to determine floc morphological properties. SVI was found to lose sensitivity (r < 0.20) when characterizing ISV above a hindered settling rate of 3 m h-1. ISV and LOSS had a strong correlation (r = 0.71), but ISV was subject to change, depending on the solids concentration. Two sludge matrix limitations influencing ESS were characterized by transition concentrations; pinpoint floc formation, and loose floc formation. Pinpoint flocs had TOF values above 400 mg TSS L-1; loose floc formation sludge had TOF and LOSS values below 400 mg TSS L-1 and 900 mg TSS L-1, respectively. TOF was found to correlate with the particle size distribution while LOSS correlated to the settling velocity distribution. The use of both TOF and LOSS is a quick and effective way to characterize limitations affecting ESS. / Ph. D.