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.

Metagenomic Analysis of Bacteria for PHA Production from Furnas and Grande Ponds
Professor Cassius Stevani explained that Furnas and Grande ponds offer unique environmental contexts. Humans made Furnas, and it previously served as a water reservoir for miner houses. Grande pond sits at the top of a mountain near Bairro da Serra. It is a natural pond more than 1,000 years old. Stevani said they previously conducted a metagenomics analysis of the bacteria present in both ponds and knew there are several species of bacteria from the genus Cupriavidus known to produce PHA.
The sampling effort involved the collection of 5L of water for metagenomics analysis and six 50-mL vials of each for bacteria isolation, containing both water and sediments. We performed 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.

Isolation of PHA-Producing Bacteria
The research team specifically targets PHA-producing bacteria from acetate. Therefore, they isolate the bacteria by using an acetate-rich medium. Stevani observed, “Bacteria from the genus Cupriavidus are known to produce PHA in high yields, but we are also interested in other bacteria. During the isolation process, we may discover new bacteria that can produce PHA in even higher yields than Cupriavidus.
We will store the obtained samples in the lab ultrafreezer. The USP researchers will use cultures of micro-organisms to optimize PHA production and genetically transform them to overexpress specific genes involved in PHA biosynthesis. Stevani explained, “We are always looking for new cultures of bacteria that we can use for biotechnological purposes.”
“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.