A BIOTECHNOLOGICAL PLATFORM FOR METABOLIC CHARACTERIZATION OF ANAEROBIC BACTERIA UNDER CO₂-ENRICHED CULTIVATION: PROOF OF CONCEPT USING Clostridium saccharobutylicum DSM 13864

The continuous increase in atmospheric CO₂ emissions has intensified the search for sustainable technologies capable of supporting the transition toward a low-carbon bioeconomy. Among these, microbial processes have emerged as promising alternatives for future carbon valorization. However, advancing this field requires robust and standardized biotechnological platforms capable of systematically characterizing the physiological and metabolic behavior of anaerobic microorganisms cultivated under CO₂-enriched conditions. Such platforms are essential for building microbial strain portfolios, accelerating the discovery of microorganisms with biotechnological potential, and supporting the development of innovative bioprocesses for the production of value-added biochemicals. Here, we report the development and validation of a biotechnological platform for the integrated metabolic characterization of anaerobic bacteria cultivated under CO₂-enriched conditions, using Clostridium saccharobutylicum DSM 13864 as a proof of concept. Batch cultivations were performed for 96 h in 50 mL glass serum bottles containing mineral medium under anaerobic, CO₂-enriched conditions. The platform integrates temporal monitoring of optical density (OD₆₀₀), biomass production, pH, and organic acid production, quantified by high-performance liquid chromatography (HPLC) using a Dionex Ultimate 3000 system (Thermo Scientific, USA) with external calibration using certified organic acid standards. An abiotic control was included to validate the experimental workflow and distinguish biological activity from possible chemical transformations. The abiotic control remained stable throughout the experiment, with no detectable production of organic acids and no evidence of spontaneous substrate conversion. In contrast, inoculated cultures exhibited rapid glucose depletion within the first 24 h, progressive bicarbonate consumption, biomass formation, and medium acidification. HPLC analyses identified acetate as the predominant metabolite, increasing from 0.019 to 0.421 g L⁻¹ during cultivation, while lactate reached 0.235 g L⁻¹. Propionate was detected only at trace concentrations (<0.01 g L⁻¹), whereas formate was not detected. The integrated physiological and metabolic analyses demonstrated that the proposed platform provides a standardized, robust, and reproducible workflow for characterizing anaerobic microorganisms cultivated under CO₂-enriched conditions while reliably distinguishing biological activity from abiotic processes. Beyond validating the methodology, this proof of concept establishes a versatile technological platform for screening and evaluating diverse anaerobic microorganisms, expanding microbial strain portfolios, and accelerating the development of sustainable bioprocesses for carbon valorization and the production of industrially relevant biochemicals, including acetate and lactate.

SENAI DR/MS, ISI Biomassa, and BiotransformaCO₂ Internal Project n. 815593.