Gregor Knopp

Ozone treatment of conditioned wastewater selects antibiotic resistance genes, opportunistic bacteria, and induce strong population shifts

An ozone treatment system was investigated to analyze its impact on clinically relevant antibiotic resistant bacteria (ARB) and antibiotic resistant genes (ARGs). A concentration of 0.9±0.1g ozone per 1g DOC was used to treat conventional clarified wastewater. PCR, qPCR analyses, Illumina 16S Amplicon Sequencing, and PCR-DGGE revealed diverse patterns of resistances and susceptibilities of opportunistic bacteria and accumulations of some ARGs after ozone treatment. Molecular marker genes for enterococci indicated a high susceptibility to ozone. Although they were reduced by almost 99%, they were still present in the bacterial population after ozone treatment. In contrast to this, Pseudomonas aeruginosa displayed only minor changes in abundance after ozone treatment. This indicated different mechanisms of microorganisms to cope with the bactericidal effects of ozone. The investigated ARGs demonstrated an even more diverse pattern. After ozone treatment, the erythromycin resistance gene (ermB) was reduced by 2 orders of magnitude, but simultaneously, the abundance of two other clinically relevant ARGs increased within the surviving wastewater population (vanA, blaVIM). PCR-DGGE analysis and 16S-Amplicon-Sequencing confirmed a selection-like process in combination with a substantial diversity loss within the vital wastewater population after ozone treatment. Especially the PCR-DGGE results demonstrated the survival of GC-rich bacteria after ozone treatment.

Integrated Evaluation Concept to Assess the Efficacy of Advanced Wastewater Treatment Processes for the Elimination of Micropollutants and Pathogens

A multidisciplinary concept has been developed to compare advanced wastewater treatment processes for their efficacy of eliminating micropollutants and pathogens. The concept is based on (i) the removal/formation of selected indicator substances and their transformation products (TPs), (ii) the assessment of ecotoxicity via in vitro tests, and (iii) the removal of pathogens and antibiotic resistant bacteria. It includes substances passing biological wastewater treatment plants regulated or proposed to be regulated in the European Water Framework Directive, TPs formed in biological processes or during ozonation, agonistic/antagonistic endocrine activities, mutagenic/genotoxic activities, cytotoxic activities, further activities like neurotoxicity as well as antibiotics resistance genes, and taxonomic gene markers for pathogens. At a pilot plant, ozonation of conventionally treated wastewater resulted in the removal of micropollutants and pathogens and the reduction of estrogenic effects, whereas the in vitro mutagenicity increased. Subsequent post-treatment of the ozonated water by granular activated carbon (GAC) significantly reduced the mutagenic effects as well as the concentrations of remaining micropollutants, whereas this was not the case for biofiltration. The results demonstrate the suitability of the evaluation concept to assess processes of advanced wastewater treatment including ozonation and GAC by considering chemical, ecotoxicological, and microbiological parameters.

Survival, reproduction, growth, and parasite resistance of aquatic organisms exposed on-site to wastewater treated by advanced treatment processes

Advanced wastewater treatment technologies are generally known to be an effective tool for reducing micropollutant discharge into the aquatic environment. Nevertheless, some processes such as ozonation result in stable transformation products with often unknown toxicity. In the present study, whole effluents originating from nine different steps of advanced treatment combinations were compared for their aquatic toxicity. Assessed endpoints were survival, growth and reproduction of Lumbriculus variegatus, Daphnia magna and Lemna minor chronically exposed in on-site flow-through tests based on standard guidelines. The treatment combinations were activated sludge treatment followed by ozonation with subsequent filtration by granular activated carbon or biofilters and membrane bioreactor treatment of raw wastewater followed by ozonation. Additionally, the impact of treated wastewater on the immune response of invertebrates was investigated by challenging D. magna with a bacterial endoparasite. Conventionally treated wastewater reduced reproduction of L. variegatus by up to 46%, but did not affect D. magna and L. minor with regard to survival, growth, reproduction and parasite resistance. Instead, parasite susceptibility was significantly reduced in D. magna exposed to conventionally treated as well as ozonated wastewater in comparison to D. magna exposed to the medium control. None of the three test organisms provided clear evidence that wastewater ozonation leads to increased aquatic toxicity. Rather than to the presence of toxic transformation products, the affected performance of L. variegatus could be linked to elevated concentrations of ammonium and nitrite that likely resulted from treatment failures.

Ecotoxicological impacts of surface water and wastewater from conventional and advanced treatment technologies on brood size, larval length, and cytochrome P450 (35A3) expression in Caenorhabditis elegans

Anthropogenic micropollutants and transformation products (TPs) negatively affect aquatic ecosystems and water resources. Wastewater treatment plants (WWTP) represent major point sources for (micro)pollutants and TPs in urban water cycles. The aim of the current study was to assess the removal of micropollutants and toxicity during conventional and advanced wastewater treatment. Using wild-type and transgenic Caenorhabditis elegans, the endpoint reproduction, growth, and cytochrome P450 (CYP) 35A3 induction (via cyp-35A3::GFP) were assessed. Samples were collected at four WWTPs and a receiving surface water. One WWTP included the advanced treatments: ozonation followed by granular activated carbon (GAC) or biological filtration (BF), respectively. Relevant micropollutants and WWTP parameters (n = 111) were included. Significant reproductive toxicity was detected for one WWTP effluent (31–83% reduced brood size). Three of four effluents significantly promoted the growth of C. elegans larvae (49–55% increased lengths). This effect was also observed for the GAC (34–41%) and BF (30%) post-treatments. Markedly, significant cyp-35A3::GFP induction was detected for one effluent before and after ozonation, being more pronounced for the ozonated samples (5- and 7.4-fold above controls). While the advanced treatments decreased the concentrations of most micropollutants, the observed effects may be attributed to effects of residual target compounds and/or compounds not included in the target chemical analysis. This highlights the need for an integrated assessment of (advanced) wastewater treatment covering both biological and chemical parameters.