MICROBIOLOGICAL ANALYSIS OF WASTEWATER AND SETUP OF THE MICROBIAL CONSORTIUM
Among biological treatments for wastewater treatment, the use of microbial consortia is one of the most efficient and sustainable technologies: the consortia consist of the association of photosynthetic microorganisms (eukaryotic microalgae and/or prokaryotic cyanobacteria) with aerobic heterotrophic bacteria. The combination of microorganisms with different metabolic capacities allows the development of a robust biological system capable of operating in various environmental conditions and on different pollutant and nutritional loads. This project proposes to apply a biofilter development process already successfully tested for the creation of another biofilter, capable of reusing dishwasher wastewater, the ZeroMile system. The core of the ZeroMile system, patented by the Tor Vergata University and the Polytechnic of Milan on the national (2019) and international scale (2020), is a new generation biofilter based upon an ad hoc engineered microbial consortium whose "beta version" consists of two types of microorganisms: a nitrogen-fixing cyanobacterium (Trichormus variabilis) and three strains of aerobic heterotrophic bacteria, isolated and chosen among the colonizers of dishwasher wastewater.
The washing machine's biofilter will also be based on an ad hoc engineered microbial consortium; to produce the engineered microbial consortium, two types of preliminary procedures will be carried out on the wastewater: isolation and identification of the cultivable aerobic heterotrophic bacterial strains present in the washing machine wastewater (using standard microbiology techniques) and identification of the structure and composition of the entire microbial community present in the wastewater, via DNA Metabarcoding (in Next Generation Sequencing).
The first preliminary procedure involves the sowing of wastewater samples on suitable culture media, to isolate and then identify the components of the cultivable aerobic heterotrophic microbial community. The strains will be taxonomically identified through Sanger sequencing and then used in co-culture tests with Trichormus variabilis. The second preliminary procedure, the DNA Metabarcoding, will help to understand, in relation to their dominance or rarity in the wastewater and any possible pathogenicity, which of the isolated bacteria will be the most suitable to build the consortium.
The realization of the microbial consortium involves the cultivation of the photosynthetic nitrogen-fixing cyanobacterium (Trichormus variabilis), the photosynthetic partner of the biofilter, and co-culture tests of Trichormus variabilis with three strains of wastewater bacteria. The final co-culture will be tested in the wastewater to evaluate its survival and growth: survival is evaluated with different methods (optical density in spectrophotometry; amount of biomass in dry weight and chlorophyll content), while the vitality of co-cultures will be evaluated in transmitted light optical microscopy, with interferential contrast.