Effects of orphan histidine kinases on clostridial sporulation progression and metabolism.

Solventogenesis and sporulation of clostridia are the main responsive adaptations to the acidic environment during acetone-butanol-ethanol (ABE) fermentation. It was hypothesized that five orphan histidine kinases (HKs) including Cac3319, Cac0323, Cac0903, Cac2730, and Cac0437 determined the cell fates between sporulation and solventogenesis. In this study, the comparative genomic analysis revealed that a mutation in cac0437 appeared to contribute to the nonsporulating feature of ATCC 55025. Hence, the individual and interactive roles of five HKs in regulating cell growth, metabolism, and sporulation were investigated. The fermentation results of mutants with different HK expression levels suggested that cac3319 and cac0437 played critical roles in regulating sporulation and acids and butanol biosynthesis. Morphological analysis revealed that cac3319 knockout abolished sporulation (Stage 0) whereas cac3319 overexpression promoted spore development (Stage VII), and cac0437 knockout initiated but blocked sporulation before Stage II, indicating the progression of sporulation was altered through engineering HKs. By combinatorial HKs knockout, the interactive effects between two different HKs were investigated. This study elucidated the regulatory roles of HKs in clostridial differentiation and demonstrated that HK engineering can be effectively used to control sporulation and enhance butanol biosynthesis.

Enforcing ATP hydrolysis enhanced anaerobic glycolysis and promoted solvent production in Clostridium acetobutylicum.

BACKGROUND: The intracellular ATP level is an indicator of cellular energy state and plays a critical role in regulating cellular metabolism. Depletion of intracellular ATP in (facultative) aerobes can enhance glycolysis, thereby promoting end product formation. In the present study, we examined this s trategy in anaerobic ABE (acetone-butanol-ethanol) fermentation using Clostridium acetobutylicum DSM 1731. RESULTS: Following overexpression of atpAGD encoding the subunits of water-soluble, ATP-hydrolyzing F1-ATPase, the intracellular ATP level of 1731(pITF1) was significantly reduced compared to control 1731(pIMP1) over the entire batch fermentation. The glucose uptake was markedly enhanced, achieving a 78.8% increase of volumetric glucose utilization rate during the first 18 h. In addition, an early onset of acid re-assimilation and solventogenesis in concomitant with the decreased intracellular ATP level was evident. Consequently, the total solvent production was significantly improved with remarkable increases in yield (14.5%), titer (9.9%) and productivity (5.3%). Further genome-scale metabolic modeling revealed that many metabolic fluxes in 1731(pITF1) were significantly elevated compared to 1731(pIMP1) in acidogenic phase, including those from glycolysis, tricarboxylic cycle, and pyruvate metabolism; this indicates significant metabolic changes in response to intracellular ATP depletion. CONCLUSIONS: In C. acetobutylicum DSM 1731, depletion of intracellular ATP significantly increased glycolytic rate, enhanced solvent production, and resulted in a wide range of metabolic changes. Our findings provide a novel strategy for engineering solvent-producing C. acetobutylicum, and many other anaerobic microbial cell factories.

RRNPP-type quorum sensing affects solvent formation and sporulation in Clostridium acetobutylicum.

The strictly anaerobic bacterium Clostridium acetobutylicum is well known for its ability to convert sugars into organic acids and solvents, most notably the potential biofuel butanol. However, the regulation of its fermentation metabolism, in particular the shift from acid to solvent production, remains poorly understood. The aim of this study was to investigate whether cell-cell communication plays a role in controlling the timing of this shift or the extent of solvent formation. Analysis of the available C. acetobutylicum genome sequences revealed the presence of eight putative RRNPP-type quorum-sensing systems, here designated qssA to qssH, each consisting of an RRNPP-type regulator gene followed by a small open reading frame encoding a putative signalling peptide precursor. The identified regulator and signal peptide precursor genes were designated qsrA to qsrH and qspA to qspH, respectively. Triplicate regulator mutants were generated in strain ATCC 824 for each of the eight systems and screened for phenotypic changes. The qsrB mutants showed increased solvent formation during early solventogenesis and hence the QssB system was selected for further characterization. Overexpression of qsrB severely reduced solvent and endospore formation and this effect could be overcome by adding short synthetic peptides to the culture medium representing a specific region of the QspB signalling peptide precursor. In addition, overexpression of qspB increased the production of acetone and butanol and the initial (48 h) titre of heat-resistant endospores. Together, these findings establish a role for QssB quorum sensing in the regulation of early solventogenesis and sporulation in C. acetobutylicum.

Two stirred-tank bioreactors in series enable continuous production of alcohols from carbon monoxide with Clostridium carboxidivorans.

Microbial batch production of alcohols by fermentation of CO-rich gases with Clostridia is limited by low volumetric productivities due to the need for formation of organic acids first (acidogenic phase) followed by re-consumption of the acids to form alcohols (solventogenic phase). Continuous autotrophic production of alcohols was made possible with C. carboxidivorans by use of two continuously operated stirred-tank bioreactors in series without cell retention. The pH in the first reactor was controlled to pH 6.0 for continuous growth of the cells. Steady-state concentrations of 3.0 g L-1 acetate and 0.1 g L-1 butyrate were measured at a mean hydraulic residence time of 8.3 h. The pH in the second reactor was controlled to pH 5.0 for enhancing continuous formation of alcohols resulting in steady-state concentrations of 6.1 g L-1 ethanol, 0.7 g L-1 butanol, and 0.1 g L-1 hexanol at a mean hydraulic residence time of 12.5 h. Continuous formation of alcohols from CO was already observed in the first stirred-tank reactor parallel to the formation of acids, whereas re-consumption of acids as well as de-novo syntheses of alcohols from CO was shown in the second stirred-tank reactor. Thus, high final alcohol-to-acid ratios of 3.9 gethanol gacetate-1 and 4.4 gbutanol gbutyrate-1 were achieved in the continuous syngas-fermentation process with C. carboxidivorans.

Bioconversion of waste office paper to hydrogen using pretreated rumen fluid inoculum.

In this study, a microbial consortium from an acid-treated rumen fluid was used to improve the yields of H2 production from paper residues in batch reactors. The anaerobic batch reactors, which contained paper and cellulose, were operated under three conditions: (1) 0.5 g paper/L, (2) 2 g paper/L, and (3) 4 g paper/L. Cellulase was added to promote the hydrolysis of paper to soluble sugars. The H2 yields were 5.51, 4.65, and 3.96 mmol H2/g COD, respectively, with substrate degradation ranging from 56 to 65.4 %. Butyric acid was the primary soluble metabolite in the three reactors, but pronounced solventogenesis was detected in the reactors incubated with increased paper concentrations (2.0 and 4.0 g/L). A substantial prevalence of Clostridium acetobutylicum (99 % similarity) was observed in the acid-treated rumen fluid, which has been recognized as an efficient H2-producing strain in addition to ethanol and n-butanol which were also detected in the reactors.

CO2 supply is a powerful tool to control homoacetogenesis, chain elongation and solventogenesis in ethanol and carboxylate fed reactor microbiomes.

Anaerobic fermentation technology enables the production of medium chain carboxylates and alcohols through microbial chain elongation. This involves steering reactor microbiomes to yield desired products, with CO2 supply playing a crucial role in controlling ethanol-based chain elongation and facilitating various bioprocesses simultaneously. In the absence of CO2 supply (Phase I), chain elongation predominantly led to n-caproate with a high selectivity of 96 Cmol%, albeit leaving approximately 80% of ethanol unconverted. During this phase, C. kluyveri and Proteiniphilum-related species dominated the reactors. In Phase II, with low CO2 input (2.0 NmL L-1 min-1), formation of n-butyrate, butanol, and hexanol was stimulated. Increasing CO2 doses in Phase III (6 NmL L-1 min-1) led to CO2 utilization via homoacetogenesis, coinciding with the enrichment of Clostridium luticellarii, a bacterium that can use CO2 as an electron acceptor. Lowering CO2 dose to 0.5 NmL L-1 min-1 led to a shift in microbiome composition, diminishing the dominance of C. luticellarii while increasing C. kluyveri abundance. Additionally, other Clostridia, Proteiniphilum, and Lactobacillus sakei-related species became prevalent. This decrease in CO2 load from 6 to 0.5 NmL L-1 min-1 minimized excessive ethanol oxidation from 30%-50% to 0%-3%, restoring a microbiome favoring net n-butyrate consumption and n-caproate production. The decreased ethanol oxidation coincided with the resurgence of hydrogen formation at partial pressures above 1%. High concentrations of butyrate, caproate, and ethanol in the reactor, along with low acetate concentration, promoted the formation of butanol and hexanol. It is evident that CO2 supply is indispensable for controlling chain elongation in an open culture and it can be harnessed to stimulate higher alcohol formation or induce CO2 utilization as an electron acceptor.

Enrichment of homoacetogens converting H2/CO2 into acids and ethanol and simultaneous methane production.

An anaerobic granular sludge was enriched to utilize H2/CO2 in a continuous gas-fed up-flow anaerobic sludge reactor by applying operating conditions expected to produce acetic acid, butyric acid, and ethanol. Three stages of fermentation were found: Stage I with acetic acid accumulation with the highest concentration of 35 mM along with a pH decrease from initial 6 to 4.5. In Stage II, H2/CO2 was replaced by 100% H2 to induce solventogenesis, whereas butyric acid was produced with the highest concentration of 2.5 mM. At stage III with 10 µM tungsten (W) addition, iso-valeric acid, valeric acid, and caproic acid were produced at pH 4.5-5.0. In the batch tests inoculated with the enriched sludge taken from the bioreactor (day 70), however, methane production occurred at pH 6. Exogenous 15 mM acetate addition enhanced both the H2 and CO2 consumption rate compared to exogenous 10, 30, and 45 mM acetate by the enriched sludge. Exogenous acetate was failed to be converted to ethanol using H2 as electron donor by the enriched acetogens.

Exploring the Influence of pH on the Dynamics of Acetone-Butanol-Ethanol Fermentation.

Clostridium acetobutylicum is an anaerobic bacterium that is extensively studied for its ability to produce butanol. Over the past two decades, various genetic and metabolic engineering approaches have been used to investigate the physiology and regulation system of the biphasic metabolic pathway in this organism. However, there has been a relatively limited amount of research focused on the fermentation dynamics of C. acetobutylicum. In this study, we developed a pH-based phenomenological model to predict the fermentative production of butanol from glucose using C. acetobutylicum in a batch system. The model describes the relationship between the dynamics of growth and the production of desired metabolites and the extracellular pH of the media. Our model was found to be successful in predicting the fermentation dynamics of C. acetobutylicum, and the simulations were validated using experimental fermentation data. Furthermore, the proposed model has the potential to be extended to represent the dynamics of butanol production in other fermentation systems, such as fed-batch or continuous fermentation using single and multi-sugars.

Selective butanol production from carbon monoxide by an enriched anaerobic culture.

An anaerobic mixed culture able to grow on pure carbon monoxide (CO) as well as syngas (CO, CO2 and H2), that produced unusual high concentrations of butanol, was enriched in a bioreactor with intermittent CO gas feeding. At pH 6.2, it mainly produced acids, generally acetic and butyric acid. After adaptation, under stress conditions of CO exposure at a partial pressure of 1.8 bar and low pH (e.g., 5.7), the enrichment accumulated ethanol, but also high amounts of butanol, up to 6.8 g/L, never reported before, with a high butanol/butyric acid molar ratio of 12.6, highlighting the high level of acid to alcohol conversion. At the end of the assay, both the acetic acid and ethanol concentrations decreased, with concomitant butyric acid production, suggesting C2 to C4 acid bioconversion, though this was not a dominant bioconversion process. The reverse reaction of ethanol oxidation to acetic acid was observed in the presence of CO2 produced during CO fermentation. Interestingly, butanol oxidation with simultaneous butyric acid production occurred upon production of CO2 from CO, which has to the best of our knowledge never been reported. Although the sludge inoculum contained a few known solventogenic Clostridia, the relative taxonomic abundance of the enriched sludge was diverse in Clostridia and Bacilli classes, containing known solventogens, e.g., Clostridium ljungdhalii, Clostridium ragsdalei and Clostridium coskatii, confirming their efficient enrichment. The relative abundance of unassigned Clostridium species amounted to 27% with presumably novel ethanol/butanol producers.

Reduction of acetone to isopropanol using producer gas fermenting microbes.

Gasification-fermentation is an emerging technology for the conversion of lignocellulosic materials into biofuels and specialty chemicals. For effective utilization of producer gas by fermenting bacteria, tar compounds produced in the gasification process are often removed by wet scrubbing techniques using acetone. In a preliminary study using biomass generated producer gas scrubbed with acetone, an accumulation of acetone and subsequent isopropanol production was observed. The effect of 2 g/L acetone concentrations in the fermentation media on growth and product distributions was studied with "Clostridium ragsdalei," also known as Clostridium strain P11 or P11, and Clostridium carboxidivorans P7 or P7. The reduction of acetone to isopropanol was possible with "C. ragsdalei," but not with P7. In P11 this reaction occurred rapidly when acetone was added in the acidogenic phase, but was 2.5 times slower when added in the solventogenic phase. Acetone at concentrations of 2 g/L did not affect the growth of P7, but ethanol increased by 41% and acetic acid concentrations decreased by 79%. In the fermentations using P11, growth was unaffected and ethanol concentrations increased by 55% when acetone was added in the acidogenic phase. Acetic acid concentrations increased by 19% in both the treatments where acetone was added. Our observations indicate that P11 has a secondary alcohol dehydrogenase that enables it to reduce acetone to isopropanol, while P7 lacks this enzyme. P11 offers an opportunity for biological production of isopropanol from acetone reduction in the presence of gaseous substrates (CO, CO2, and H2).

Autotrophic (C1-gas) versus heterotrophic (fructose) accumulation of acetic acid and ethanol in Clostridium aceticum.

The influence of the carbon source on the metabolism and growth of Clostridium aceticum was investigated, supplying either CO or fructose as sole carbon source. The acid and solvent production patterns were determined under either autotrophic or heterotrophic conditions, elucidating the effect of pH on the substrate's bioconversion pattern. The highest maximum specific growth rate was observed with CO, under the organism's optimal growth conditions, reaching 0.052 h-1 and an acetic acid concentration of 18 g·L-1. The production of 4.4 g·L-1 ethanol was also possible, after medium acidification, during CO bioconversion. Conversely, formic acid inhibition was observed during fructose fermentation under optimal growth conditions. In the latter experiments, it was not possible to stimulate solvent production when growing C. aceticum on fructose, despite applying the same medium acidification strategy as with CO, showing the selective effect of the carbon source (autotrophic vs heterotrophic) on the metabolic pattern and solventogenesis.

Ribozyme-Mediated Downregulation Uncovers DNA Integrity Scanning Protein A (DisA) as a Solventogenesis Determinant in Clostridium beijerinckii.

Carbon catabolite repression (CCR) limits microbial utilization of lignocellulose-derived pentoses. To relieve CCR in Clostridium beijerinckii NCIMB 8052, we sought to downregulate catabolite control protein A (CcpA) using the M1GS ribozyme technology. A CcpA-specific ribozyme was constructed by tethering the catalytic subunit of Escherichia coli RNase P (M1 RNA) to a guide sequence (GS) targeting CcpA mRNA (M1GSCcpA). As negative controls, the ribozyme M1GSCcpA-Sc (constructed with a scrambled GSCcpA) or the empty plasmid pMTL500E were used. With a ∼3-fold knockdown of CcpA mRNA in C. beijerinckii expressing M1GSCcpA (C. beijerinckii_M1GSCcpA) relative to both controls, a modest enhancement in mixed-sugar utilization and solvent production was achieved. Unexpectedly, C. beijerinckii_M1GSCcpA-Sc produced 50% more solvent than C. beijerinckii_pMTL500E grown on glucose + arabinose. Sequence complementarity (albeit suboptimal) suggested that M1GSCcpA-Sc could target the mRNA encoding DNA integrity scanning protein A (DisA), an expectation that was confirmed by a 53-fold knockdown in DisA mRNA levels. Therefore, M1GSCcpA-Sc was renamed M1GSDisA. Compared to C. beijerinckii_M1GSCcpA and _pMTL500E, C. beijerinckii_M1GSDisA exhibited a 7-fold decrease in the intracellular c-di-AMP level after 24 h of growth and a near-complete loss of viability upon exposure to DNA-damaging antibiotics. Alterations in c-di-AMP-mediated signaling and cell cycling likely culminate in a sporulation delay and the solvent production gains observed in C. beijerinckii_M1GSDisA. Successful knockdown of the CcpA and DisA mRNAs demonstrate the feasibility of using M1GS technology as a metabolic engineering tool for increasing butanol production in C. beijerinckii.

Combination of Trace Metal to Improve Solventogenesis of Clostridium carboxidivorans P7 in Syngas Fermentation.

Higher alcohols such as butanol (C4 alcohol) and hexanol (C6 alcohol) are superior biofuels compared to ethanol. Clostridium carboxidivorans P7 is a typical acetogen capable of producing C4 and C6 alcohols natively. In this study, the composition of trace metals in culture medium was adjusted, and the effects of these adjustments on artificial syngas fermentation by C. carboxidivorans P7 were investigated. Nickel and ferrous ions were essential for growth and metabolite synthesis during syngas fermentation by P7. However, a decreased dose of molybdate improved alcohol fermentation performance by stimulating carbon fixation and solventogenesis. In response to the modified trace metal composition, cells grew to a maximum OD600 nm of 1.6 and accumulated ethanol and butanol to maximum concentrations of 2.0 and 1.0 g/L, respectively, in serum bottles. These yields were ten-fold higher than the yields generated using the original composition of trace metals. Furthermore, 0.5 g/L of hexanol was detected at the end of fermentation. The results from gene expression experiments examining genes related to carbon fixation and organic acid and solvent synthesis pathways revealed a dramatic up-regulation of the Wood-Ljungdahl pathway (WLP) gene cluster, the bcs gene cluster, and a putative CoA transferase and butanol dehydrogenase, thereby indicating that both de novo synthesis and acid re-assimilation contributed to the significantly elevated accumulation of higher alcohols. The bdh35 gene was speculated to be the key target for butanol synthesis during solventogenesis.

Enrichment of a solventogenic anaerobic sludge converting carbon monoxide and syngas into acids and alcohols.

An anaerobic granular sludge was acclimatized to utilise CO in a continuously gas-fed stirred tank bioreactor by applying operating conditions expected to stimulate solventogenesis, i.e. the production of alcohols, and allowing to enrich for solventogenic populations. A cycle of high (6.2) and low (4.9) pH was applied in order to produce volatile fatty acids first at high pH, followed by their bioconversion into alcohols at low pH. The addition of yeast extract stimulated biomass growth, but not necessarily solventogenesis. The highest concentrations of metabolites achieved were 6.18 g/L acetic acid (30th day), 1.18 g/L butyric acid (28th day), and 0.423 g/L hexanoic acid (32nd day). Subsequently, acids were metabolized at lower pH, producing alcohols at concentrations of 11.1 g/L ethanol (43rd day), 1.8 g/L butanol (41st day) and 1.46 g/L hexanol (42nd day), confirming the successful enrichment strategy. Similarly, the enriched sludge could also convert syngas into acids and alcohols.

N-Acyl-Homoserine Lactone Quorum Sensing Switch from Acidogenesis to Solventogenesis during the Fermentation Process in Serratia marcescens MG1.

N-acyl-homoserine lactone quorum sensing (AHL-QS) has been shown to regulate many physiological behaviors in Serratia marcescens MG1. In the current study, the effects of AHL-QS on the biosynthesis of acid and neutral products by S. marcescens MG1 and its isogenic ∆swrI with or without supplementing exogenous N-hexanoyl-L-homoserine lactone (C6-HSL) were systematically investigated. The results showed that swrI disruption resulted in rapid pH drops from 7.0 to 4.8, which could be restored to wild type by supplementing C6-HSL. Furthermore, fermentation product analysis indicated that ∆swrI could lead to obvious accumulation for acidogenesis products such as lactic acid and succinic acid, especially excess acetic acid (2.27 g/l) produced at the early stage of fermentation, whereas solventogenesis products by ∆swrI appeared to noticeably decrease by an approximate 30% for acetoin during 32-48 h and by an approximate 20% for 2,3-butanediol during 24-40 h, when compared to those by wild type. Interestingly, the excess acetic acid produced could be removed in an AHL-QS-independent manner. Subsequently, quantitative real-time PCR was used to determine the mRNA expression levels of genes responsible for acidogenesis and solventogenesis and showed consistent results with those of product synthesis. Finally, by close examination of promoter regions of the analyzed genes, four putative luxI box-like motifs were found upstream of genes encoding acetyl-CoA synthase, lactate dehydrogenase, α-acetolactate decarboxylase, and Lys-like regulator. The information from this study provides a novel insight into the roles played by AHL-QS in switching from acidogenesis to solventogenesis in S. marcescens MG1.

Production of acids and alcohols from syngas in a two-stage continuous fermentation process.

A two-stage continuous system with two stirred tank reactors in series was utilized to perform syngas fermentation using Clostridium carboxidivorans. The first bioreactor (bioreactor 1) was maintained at pH 6 to promote acidogenesis and the second one (bioreactor 2) at pH 5 to stimulate solventogenesis. Both reactors were operated in continuous mode by feeding syngas (CO:CO2:H2:N2; 30:10:20:40; vol%) at a constant flow rate while supplying a nutrient medium at different flow rates of 8.1, 15, 22 and 30 ml/h. A cell recycling unit was added to bioreactor 2 in order to recycle the cells back to the reactor, maintaining the OD600 around 1 in bioreactor 2 throughout the experimental run. When comparing the flow rates, the best results in terms of solvent production were obtained with a flow rate of 22 ml/h, reaching the highest average outlet concentration for alcohols (1.51 g/L) and the most favorable alcohol/acid ratio of 0.32.

Part by Part: Synthetic Biology Parts Used in Solventogenic Clostridia.

The solventogenic Clostridia are of interest to the chemical industry because of their natural ability to produce chemicals such as butanol, acetone and ethanol from diverse feedstocks. Their use as whole cell factories presents multiple metabolic engineering targets that could lead to improved sustainability and profitability of Clostridium industrial processes. However, engineering efforts have been held back by the scarcity of genetic and synthetic biology tools. Over the past decade, genetic tools to enable transformation and chromosomal modifications have been developed, but the lack of a broad palette of synthetic biology parts remains one of the last obstacles to the rapid engineered improvement of these species for bioproduction. We have systematically reviewed existing parts that have been used in the modification of solventogenic Clostridia, revealing a narrow range of empirically chosen and nonengineered parts that are in current use. The analysis uncovers elements, such as promoters, transcriptional terminators and ribosome binding sites where increased fundamental knowledge is needed for their reliable use in different applications. Together, the review provides the most comprehensive list of parts used and also presents areas where an improved toolbox is needed for full exploitation of these industrially important bacteria.

Continuous lactose fermentation by Clostridium acetobutylicum--assessment of solventogenic kinetics.

This work reports the results of a series of tests on the specific butanol production rate by Clostridium acetobutylicum continuous cultures. The tests were carried out using lactose as carbon source to mimic cheese-whey. A continuous stirred tank reactor equipped with a microfiltration unit was used. The dilution rate (D) ranged between 0.02 and 0.15h(-1) and the ratio R of the permeate stream rate to the stream fed to the reactor ranged between 14% and 95%. For each set of D and R values, the continuous cultures were characterized in terms of concentration of cells, acids and solvents. Results were processed to assess the concentration of acidogenic cells, solventogenic cells, spores and the specific butanol production rate. The max butanol productivity was 0.5gL(-1)h(-1) at D=0.1h(-1) and R=95%. The butanol productivity referred to solventogenic cells was expressed as a function of concentration of lactose, acids and butanol.

Continuous xylose fermentation by Clostridium acetobutylicum--Assessment of solventogenic kinetics.

This work deals with the specific butanol production rate of Clostridium acetobutylicum using xylose--a relevant fraction of lignocellulosic feedstock for biofuel production--as carbon source. The tests were carried out in a CSTR equipped with a microfiltration unit. The dilution rate (D) ranged between 0.02 and 0.22 h(-1) and the ratio R between the permeate stream rate and the stream fed to the reactor ranged between 14% and 88%. The biomass present in the broth was identified as a heterogeneous cell population consisting of: acidogenic cells, solventogenic cells and spores. The results were processed to assess the concentration of acidogenic cells, solventogenic cells and spores. The specific butanol production rate was also assessed. The max butanol productivity was 1.3 g L(-1) h(-1) at D = 0.17 h(-1) and R = 30%. A comparison between the results reported in a previous work carried out with lactose was made.

Formación de endosporas en Clostridium y su interacción con el proceso de solventogénesis / Endospore formation in Clostridium and its interaction with solventogenesis

La solventogénesis y la esporulación son mecanismos de las células de Clostridium para resistir ambientes hostiles. Este segundo proceso ha sido estudiado utilizando como modelo lo que sucede con Bacillus, aunque se reconocen diferencias marcadas entre los dos géneros especialmente en el inicio, específicamente en los eventos por medio de los que se da la fosforilación del regulador maestro Spo0A. En la actualidad se ha avalado la teoría que afirma que tres histidin quinasas huérfanas, diferentes a las proteínas Spo0B (histidin quinasa) y Spo0F(fosfotransferasa) en Bacillus, son las encargadas de fosforilar en Clostridium de forma directa a Spo0A, que posteriormente activa la transcripción de diferentes factores sigma relacionados, de forma similar en los dos géneros bacterianos. La proteína Spo0A, perteneciente a la familia de reguladores de respuesta, se comporta como una entidad regulatoria global que tiene injerencia sobre los procesos de formación de esporas y solventes, modulando genes necesarios para que se produzcan acetona y butanol, además de genes de esporulación. El entendimiento de este proceso ha llevado a los investigadores a emplear diferentes técnicas moleculares que permitan incrementar la producción de solventes, así como eliminar la propiedad de las células de producir endosporas. Por tal razón, este escrito presenta un resumen de estas dos redes de expresión de genes, conectadas por el regulador maestro Spo0A.

Solventogenesis and sporulation are mechanisms used by Clostridium cells to resist hostile environments. Sporulation has been studied using as a model what happens with Bacillus, but marked differences were recognized, particularly in the events that led the phosphorylation of the master controller Spo0A. Currently, a theory that claims that three orphan histidine kinases, different from Spo0B (histidin kinase) and spo0F (phosphotransferase) proteins in Bacillus, phosphorilate directly Spo0A in Clostridium activating transcription of different sigma factors which are similar in the two bacterial genera, has been supported. Spo0A protein, which belongs to the family of response regulators, behaves as an entity that has global regulatory interference on the processes of spore formation and solvents, modulating genes necessary to produce acetone and butanol. The understanding of this process has led researchers to employ different molecular techniques that increase the production of solvents, and remove the property of the cells to produce endospores. Reason why, this paper presents an updated summary of these two gene expression networks, connected by the master regulator spo0A.