Application of low-cost and sustainable solar oxidation treatment to prevent microbial resistance in effluents in Brazil

This project used a sustainable solar oxidation system (photo-Fenton) as post-treatment of secondary wastewater from a sewage treatment plant to remove bacteria and antibiotic-resistant genes (ARB and ARG). The project´s working hypothesis was that this technology would allow inactivation of resistant bacteria and the elimination of antibiotic-resistance genes from these secondary effluents in Brazil. Oxidation of these contaminants would prevent the spread of antimicrobial resistance in the environment via release of the effluent into receiving bodies of water.

The team collected samples from a secondary effluent (biological treatment with activated iodine) at a sewage treatment plant in Belo Horizonte. The experiments were performed on the bench scale, in a solar radiation simulation chamber (SUNTEST), and on a semi-pilot scale, using a compound parabolic concentrator (CPC) type reactor with a capacity of 45L of effluent. The solar photo-Fenton reaction uses ferrous ion (Fe2+) as a catalyst in the presence of an oxidant to generate hydroxyl radicals (OH•), while solar radiation potentiates the generation of these radicals, responsible for degradation of contaminants in samples. Due to the high reactivity of the hydroxyl radical, leading to a shorter permanence time in the system, alternative oxidants have been tested for these processes to increase their efficiency.

Three different oxidants were tested in this project, peroxide, persulfate, and peroxide + persulfate. The occurrence of ARBs and ARGs in samples was analyzed using classical culture techniques (bacterial plating) and molecular biology (qPCR) and advanced metagenomic techniques through high-performance genetic sequencing such as 16S, which allowed analysis of microbiological diversity in the sample before and after the proposed treatment, as well as whole genome sequencing (WGS), permitting the analysis of all genes present in the samples before and after treatment.

Treatment with hydrogen peroxide as the oxidant showed a significant decrease in the microbial diversity of effluent samples after applying solar photo-Fenton. Six of the 12 pathogens on the WHO priority list were identified in the secondary effluent samples, and some were eliminated after treatment, such as Staphylococcus sp. and Enterococcus sp. As for ARGs, the treatment removed more than 90% of the genes accounting for resistance in the β-lactam class and more than 60% of resistance genes tet(X)_1, tet(X)_2, mph (E)_3, and msr(E)_4.

Switching the oxidant to persulfate allowed 88% and 99% reductions, respectively, compared to the presence of Proteobacteria and Bacteriodetes in the secondary effluent before treatment. Regarding removal of ARGs, the treatment efficiently reduced the genes responsible for broad-spectrum antibiotic resistance, such as to aminoglycosides, beta-lactams, macrolides, and sulfonamides. Removal of > 50% was obtained for the primary resistance genes present in the effluent before treatment, namely macB, sul1, aadA, arnA, tetA, and sul2. The combined use of the two oxidants reduced Bacteriodetes by 92%, with increasing profusion of Proteobacteria and Firmicutes. Meanwhile, this treatment completely fragmented the samples´ DNA, thereby preventing total genome analysis.

Sewage treatment plants receive effluents from widely diverse sources, including hospitals, factories, and especially residences. The effluents contain a multiplicity of resistant bacteria and antibiotic resistance genes and thus act as disseminators of antimicrobial resistance in the environment. This proposal involved a multidisciplinary team of engineers, biologists, and chemists who collaborated in the use of solar oxidative processes for inactivation of these antibiotic-resistant microorganisms and degradation of resistance genes in sewage station effluents. The proposed technique uses solar energy, a key potential energy source, particularly in tropical regions. When converted to chemical energy in the solar photo-Fenton process, sunlight potentiates the generation of oxidant radicals capable of damaging essential organic components of microorganism cells, leading to disinfection and DNA degradation. The results showed that treatment with sunlight and easily accessible reagents (iron salts, hydrogen peroxide, and sodium persulfate) effectively combats priority organisms on the WHO list and their resistance genes.

Improvement in environmental quality, hindering the spread of ARBs and ARGs through the environment-human, environment-animal, and thus animal-human routes.

The next steps involve scaling up the proposed treatment process for use in sewage treatment plants and expanding the technology to compact treatment modules for hospital effluents to avoid the release of ARBs and ARGs into local sewage and thus into sewage treatment plants.