@article{sandoval-espinola_chinn_thon_bruno-bárcena_2017, title={Evidence of mixotrophic carbon-capture by n-butanol-producer Clostridium beijerinckii}, volume={7}, ISSN={2045-2322}, url={http://dx.doi.org/10.1038/s41598-017-12962-8}, DOI={10.1038/s41598-017-12962-8}, abstractNote={Recent efforts to combat increasing greenhouse gas emissions include their capture into advanced biofuels, such as butanol. Traditionally, biobutanol research has been centered solely on its generation from sugars. Our results show partial re-assimilation of CO2 and H2 by n-butanol-producer C. beijerinckii. This was detected as synchronous CO2/H2 oscillations by direct (real-time) monitoring of their fermentation gasses. Additional functional analysis demonstrated increased total carbon recovery above heterotrophic values associated to mixotrophic assimilation of synthesis gas (H2, CO2 and CO). This was further confirmed using 13C-Tracer experiments feeding 13CO2 and measuring the resulting labeled products. Genome- and transcriptome-wide analysis revealed transcription of key C-1 capture and additional energy conservation genes, including partial Wood-Ljungdahl and complete reversed pyruvate ferredoxin oxidoreductase / pyruvate-formate-lyase-dependent (rPFOR/Pfl) pathways. Therefore, this report provides direct genetic and physiological evidences of mixotrophic inorganic carbon-capture by C. beijerinckii.}, number={1}, journal={Scientific Reports}, publisher={Springer Science and Business Media LLC}, author={Sandoval-Espinola, W. J. and Chinn, M. S. and Thon, M. R. and Bruno-Bárcena, J. M.}, year={2017}, month={Oct} }
 @article{sandoval-espinola_chinn_bruno-barcena_2015, title={Inoculum optimization of Clostridium beijerinckii for reproducible growth}, volume={362}, ISSN={1574-6968}, url={http://dx.doi.org/10.1093/femsle/fnv164}, DOI={10.1093/femsle/fnv164}, abstractNote={Spore-forming solventogenic Clostridium spp. are receiving renewed attention due to their butanol production abilities. However, there is an absence of literature describing the preparation of dense, vigorous and homogeneous seed cultures of Clostridium spp., guaranteeing reproducibility during fermentation. Therefore, we performed a series of growth experiments of Clostridium beijerinckii NCIMB 8052 and its offspring SA-1 to evaluate the influence of inoculum age (harvest time) on the subsequent population's maximum specific growth rate, as a signal of population homogeneity. The organisms were cultivated in Reinforced Clostridial Medium and supplemented sweet sorghum juice. The best inoculum ages coincided with the late-exponential growth phase: between 9 and 11 h in the conditions tested. Additionally, the harvest time was delayed up to 4 h by pre-adapting the seed culture with 0.75 g L−1 butyric acid. These findings were validated by performing a series of bench-top batch fermentations showcasing reproducibility in growth kinetics with 95% confidence limits. Overall, these experiments allowed us to understand the transient nature of seed cultures of C. beijerinckii NCIMB 8052 and SA-1, while enabling reproducibility and consistent culture performance.}, number={19}, journal={FEMS Microbiology Letters}, publisher={Oxford University Press (OUP)}, author={Sandoval-Espinola, Walter J. and Chinn, Mari and Bruno-Barcena, Jose M.}, editor={Sauer, MichaelEditor}, year={2015}, month={Sep}, pages={fnv164} }
 @article{sandoval-espinola_makwana_chinn_thon_andrea azcarate-peril_bruno-barcena_2013, title={Comparative phenotypic analysis and genome sequence of Clostridium beijerinckii SA-1, an offspring of NCIMB 8052}, volume={159}, ISSN={["1465-2080"]}, url={http://europepmc.org/abstract/med/24068240}, DOI={10.1099/mic.0.069534-0}, abstractNote={Production of butanol by solventogenic clostridia is controlled through metabolic regulation of the carbon flow and limited by its toxic effects. To overcome cell sensitivity to solvents, stress-directed evolution methodology was used three decades ago on Clostridium beijerinckii NCIMB 8052 that spawned the SA-1 strain. Here, we evaluated SA-1 solventogenic capabilities when growing on a previously validated medium containing, as carbon- and energy-limiting substrates, sucrose and the products of its hydrolysis d-glucose and d-fructose and only d-fructose. Comparative small-scale batch fermentations with controlled pH (pH 6.5) showed that SA-1 is a solvent hyper-producing strain capable of generating up to 16.1 g l−1 of butanol and 26.3 g l−1 of total solvents, 62.3 % and 63 % more than NCIMB 8052, respectively. This corresponds to butanol and solvent yields of 0.3 and 0.49 g g−1, respectively (63 % and 65 % increase compared with NCIMB 8052). SA-1 showed a deficiency in d-fructose transport as suggested by its 7 h generation time compared with 1 h for NCIMB 8052. To potentially correlate physiological behaviour with genetic mutations, the whole genome of SA-1 was sequenced using the Illumina GA IIx platform. PCR and Sanger sequencing were performed to analyse the putative variations. As a result, four errors were confirmed and validated in the reference genome of NCIMB 8052 and a total of 10 genetic polymorphisms in SA-1. The genetic polymorphisms included eight single nucleotide variants, one small deletion and one large insertion that it is an additional copy of the insertion sequence ISCb1. Two of the genetic polymorphisms, the serine threonine phosphatase cbs_4400 and the solute binding protein cbs_0769, may possibly explain some of the observed physiological behaviour, such as rerouting of the metabolic carbon flow, deregulation of the d-fructose phosphotransferase transport system and delayed sporulation.}, number={PART 12}, journal={MICROBIOLOGY-SGM}, author={Sandoval-Espinola, Walter J. and Makwana, Satya T. and Chinn, Mari S. and Thon, Michael R. and Andrea Azcarate-Peril, M. and Bruno-Barcena, Jose M.}, year={2013}, month={Dec}, pages={2558–2570} }