Design and validation of a multiplex PCR method for the simultaneous quantification of Clostridium acetobutylicum, Clostridium carboxidivorans and Clostridium cellulovorans

Abstract

Co-cultures of clostridia with distinct physiological properties have emerged as an alternative to increase the production of butanol and other added-value compounds from biomass. The optimal performance of mixed tandem cultures may depend on the stability and fitness of each species in the consortium, making the development of specific quantification methods to separate their members crucial. In this study, we developed and tested a multiplex qPCR method targeting the 16S rRNA gene for the simultaneous quantification of Clostridium acetobutylicum, Clostridium carboxidivorans and Clostridium cellulovorans in co-cultures. Designed primer pairs and probes could specifically quantify the three Clostridium species with no cross-reactions thus allowing significant changes in their growth kinetics in the consortia to be detected and correlated with productivity. The method was used to test a suitable medium composition for simultaneous growth of the three species. We show that higher alcohol productions were obtained when combining C. carboxidivorans and C. acetobutylicum compared to individual cultures, and further improved (> 90%) in the triplet consortium. Altogether, the methodology could be applied to fermentation processes targeting butanol productions from lignocellulosic feedstocks with a higher substrate conversion efficiency

This study has received funding from the Spanish Ministry of Science and Innovation (ref. PCI2019-111932-2, project SynConsor4Butanol). L. F.-P. is grateful for the Research Training grant from the Catalan Government (2021 FISDU 00132). S.P. is a Serra Hunter Fellow (UdG-AG-575) and acknowledges the funding from the ICREA Academia award. LEQUIA and EcoAQUA have been recognized as a consolidated research groups (2021 SGR01352 and 2021 SGR01142, respectively) by the Catalan Government