The growing need for sustainable biotechnological solutions to address environmental challenges, such as climate change and resource depletion, has intensified interest in microbial-based production systems. Synthetic biofilms, which mimic natural microbial consortia, offer a promising platform for optimizing complex metabolic processes that can convert renewable feedstocks into valuable chemicals. In this context, understanding and harnessing the interactions between co-immobilized microorganisms are critical for advancing bioprocesses that contribute to circular bioeconomy goals. In this study, we investigated the viability and metabolic activity of Clostridium carboxidivorans and Clostridium kluyveri within a synthetic, dual-layered biofilm composed of agar hydrogel. This setup compartmentalized each bacterial species. Embedding the bacteria in a structured biofilm offers numerous opportunities for bioproduction, but the inability to monitor cell growth or movement within the immobilization matrix limits process insights. To address this, we adapted a fluorescence in situ hybridization (FISH) protocol, enabling precise, species-specific visualization of bacterial distribution and growth within the gel matrix. Batch processes with the dual-layered biofilm in anaerobic flasks, designed with a metabolic advantage for C. kluyveri, revealed distinct growth dynamics. C. kluyveri exhibited significant metabolic activity, forming clusters at low initial cell concentrations and converting ethanol and acetate into 1-butyrate and 1-hexanoate, indicating viability and cell growth. C. carboxidivorans remained evenly distributed without significant growth or product formation, suggesting that while the cells were viable, they were not metabolically active under the experimental conditions. Both bacterial species were confined to their respective compartments throughout the process, with C. kluyveri showing enhanced substrate conversion at higher initial cell densities in the hydrogel. The pH drop throughout the batch experiment likely contributed to incomplete substrate consumption, particularly for C. kluyveri, which thrives within a narrow pH range. These findings highlight synthetic biofilms as a promising platform for optimizing microbial interactions and improving bioprocess efficiency, especially in applications involving complex metabolic exchanges between co-immobilized microorganisms. Further research will focus on applying conditions to support the growth and metabolic activity of C. carboxidivorans to explore spatial dynamics of bacterial migration and cooperative relationships in the synthetic biofilm.

A powerful strategy to accelerate bioprocess development is to complement parallel bioreactor systems with an automated approach, often achieved using liquid handling stations. The benefit of such high-throughput experiments is determined by the employed monitoring procedures. To gain a molecular understanding of the microbial production strains in miniaturized parallel single-use bioreactors, we extended the at-line monitoring procedures to transcriptome analysis in a parallel approach using RNA-Seq. To perform automated RNA-Seq experiments, we developed a sample preparation workflow consisting of at-line cell disruption by enzymatic cell lysis, total RNA extraction, nucleic acid concentration normalization, and Nanopore cDNA Library preparation. The pH-controlled aerobic batch growth of Saccharomyces cerevisiae was studied with six different carbon sources (glucose, pyruvate, fructose, galactose, sucrose, and mannose) on a 11 mL scale using 24 parallel stirred tank bioreactors integrated into a liquid handling station while performing at-line sample preparation for RNA-Seq on the same deck. With four biological replicates per condition, 24 cDNA libraries were prepared over 11.5 h. Off-line Nanopore sequencing yielded 20.97 M classified reads with a Q-score > 9. Differential gene expression analysis revealed significant differences in transcriptomic profiles when comparing growth with glucose (exponential growth) to growth with pyruvate (stress conditions), allowing identification of 674 downregulated and 709 upregulated genes. Insignificant changes in gene expression patterns were measured when comparing growth with glucose and fructose, yielding only 64 differentially expressed genes. The expected differences in cellular responses identified in this study show a promising approach for transcriptomic profiling of bioreactor cultures, providing valuable insights on a molecular level at-line in a high-throughput fashion.

In this study, we explore the use of C. kluyveri in synthetic biofilms for the production of 1-butyrate and 1-hexanoate, investigating the impact of inoculation temperature during biofilm formation and the presence of yeast extract. Therefore, a novel synthetic biofilm reactor has been designed and constructed. Prior to investigating synthetic biofilms in this reactor, we carried out preliminary batch experiments in anaerobic flasks containing an inoculated agar hydrogel fixed at the bottom and overlaid medium. For the operation of the novel synthetic biofilm reactor, specific volumes of inoculated agar hydrogel were dispensed into a cylindrical mold with a diameter of 102 mm, forming the synthetic biofilm with a height of 4 mm, which was then transferred into the biofilm reaction chamber onto the support grid. The biofilm support grid separates the gas phase (CO₂, N₂) above the synthetic biofilm from the aqueous phase (medium) below. Our results show that C. kluyveri remains metabolically active at biofilm preparation temperatures of up to 45 °C, with extended lag phases observed at 70 °C. The synthetic biofilm demonstrated efficient chain elongation in batch processes, converting ethanol and acetate into 1-butyrate and 1-hexanoate, with final concentrations of 2.7 g L⁻¹ and 10.1 g L⁻¹, respectively, with yeast extract in the circulating liquid medium of the synthetic biofilm reactor setup. The maximum estimated space-time yields for 1-butyrate and 1-hexanoate, referenced to the biofilm volume, were 1.331 g L⁻¹ h⁻¹ and 4.947 g L⁻¹ h⁻¹, respectively. Experiments without yeast extract lead to final concentrations of 2.0 g L⁻¹ 1-butyrate, and 7.3 g L⁻¹ 1-hexanoate and maximum estimated space-time yields, referenced to the biofilm volume, were 0.332 g L⁻¹ h⁻¹ and 1.123 g L⁻¹ h⁻¹, respectively. The use of synthetic biofilms, even without yeast extract, eliminates the need for significant cell growth during chain elongation. However, product concentrations were lower without yeast extract.

Syngas fermentation in combination with chain elongation offers great promise for sustainable medium-chain fatty acid production. While immobilization has proven effective for stabilizing monocultures of C. kluyveri for chain elongation, its applicability to co-cultures involving C. carboxidivorans for simultaneous syngas fermentation remains unexplored. This study investigates the physiological compatibility of C. carboxidivorans with agar-based hydrogel immobilization and its co-cultivation potential with C. kluyveri in a synthetic dual-layer biofilm reactor. First, we conducted autotrophic batch fermentations using suspended and immobilized cells, proving metabolic activity similar for both. Applying different sulfur feeding rates, experiments showed best ethanol formation with C. carboxidivorans at increased sulfur feeding, enabling better conditions for chain elongation with C. kluyveri. In the synthetic dual-layer biofilm reactor, with the C. carboxidivorans biofilm in contact with the CO-containing gas phase above the C. kluyveri biofilm, the formation of 1-butyrate and 1-hexanoate was observed with product formation rates of 0.46 g L⁻¹ d⁻¹ 1-butyrate, and 0.91 g L⁻¹ d⁻¹ 1-hexanoate, respectively. The formation rate of 1-hexanoate in the dual-layer biofilm reactor was approximately 7.6 times higher than that reported with suspended cells in a stirred tank bioreactor. Spatial analysis revealed species-specific migration behavior and confirmed that C. carboxidivorans reduced local CO concentrations, improving the environment for C. kluyveri.