Exploring microbial diversity in Atlantic Forest ponds for biotechnological applications

Furnas and Grande are the names of two ponds located in a mountain region near the city of Iporanga (state of São Paulo, Brazil), in the vast area of Atlantic Forest. A team of researchers from University of São Paulo (USP), and members of the HELVA project, conducted a fieldwork sampling campaign in those two ponds in December 2023.

The fieldwork team, led by Prof. Cassius Stevani, comprised the researchers Pedro Miragaia, Dielle Procópio, Renato Freire and Elen Perpétuo. The primary object of the fieldwork campaign, which spanned a week, was to collect water and sediment samples from the two ponds to found strains of bacteria able to produce polyhydroxyalkanoate (PHA), a type of polyesters produced in nature by microorganisms through bacterial fermentation of sugars and lipids.

“Furnas and Grande pounds offers unique environmental context”, explained Prof. Cassius Stevani. Furnas is a human-made pond that served in the past as a water reservoir for miner houses. Grande pond, located at the top of a mountain near Bairro da Serra, is a natural pond dating more than 1,000 years. “We previously conducted a metagenomics analysis of the bacteria present in both pounds and we knew that are several species of bacteria from the genus Cupriavidus known to produce PHA”, said Stevani.

The sampling effort involved collecting 5L of water that would be used for metagenomics analysis and six 50-mL vials of each for bacteria isolation, containing both water and sediments.The sampling process included metagenomic analysis to identify species found in each pond followed by isolating the desired bacteria through cultivation in a medium containing acetate as the sole carbon source.

In the laboratory stage, the researchers extracted DNA from bacteria to properly identify the species and to sequence their genome. This is crucial to verify the genes involved in the PHA biosynthesis. “The next step is to maximize PHA production by cultivation under different conditions, such as pH, illumination, nutrients or temperature, and genetic modification”, explained Cassius Stevani.

The research team is specifically targeting PHA producing bacteria from acetate, therefore the bacteria are isolated by using an acetate-rich medium. “Bacteria from genus Cupriavidus are known to produce PHA in high yields, but we are interested in other bacteria too. During the isolation process we can discover new bacteria that can produce PHA in even higher yields than Cupriavidus.”, observed Stevani.

The obtained samples will be stored in the lab ultrafreezer. The USP researchers will use culture of micro-organisms for PHA producing optimization and genetic transformation overexepressing the specific genes involved in the PHA biosynthesis.“We are always looking for new cultures of bacteria that can be used for biotechnological purposes”, explained Stevani.

“We’re pretty confident we’ll isolate interesting bacteria from the samples collected in this fieldwork”, he added.So far, Stevani’s team successfully isolated cultures capable of consuming acetate and has confirmed that such cultures can produce PHA.