Elucidating the Response of Activated Sludge Cultures to Toxic Chemicals at the Process, Floc and Metabolic Scales

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Virginia Tech


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.



discriminant function analysis, extracellular polymeric substances, floc structure, toxic chemicals, activated sludge, metabolic footprinting, liquid chromatography-mass spectrometry