Effects of Spo0A on Clostridium acetobutylicum with an emphasis on biofilm formation.

Clostridium acetobutylicum is a well-known strain for biofuel production. In previous work, it was found that this strain formed biofilm readily during fermentation processes. Biofilm formation could protect cells and enhance productivities under environmental stresses in our previous work. To explore the molecular mechanism of biofilm formation, Spo0A of C. acetobutylicum was selected to investigate its influences on biofilm formation and other physiological performances. When spo0A gene was disrupted, the spo0A mutant could hardly form biofilm. The aggregation and adhesion abilities of the spo0A mutant as well as its swarming motility were dramatically reduced compared to those of wild type strain. Sporulation was also negatively influenced by spo0A disruption, and solvent production was almost undetectable in the spo0A mutant fermentation. Furthermore, proteomic differences between wild type strain and the spo0A mutant were consistent with physiological performances. This is the first study confirming a genetic clue to C. acetobutylicum biofilm and will be valuable for biofilm optimization through genetic engineering in the future.

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.

The Small RNA sr8384 Is a Crucial Regulator of Cell Growth in Solventogenic Clostridia.

Small RNAs (sRNAs) are crucial regulatory molecules in organisms and are well-known not only for their roles in the control of diverse crucial biological processes but also for their value in regulation rewiring. However, to date, in Gram-positive anaerobic solventogenic clostridia (a group of important industrial bacteria with exceptional substrate and product diversity), sRNAs remain minimally explored, and thus there is a lack of detailed understanding regarding these important molecules and their use as targets for genetic improvement. Here, we performed large-scale phenotypic screens of a transposon-mediated mutant library of Clostridium acetobutylicum, a typical solventogenic clostridial species, and discovered a novel sRNA (sr8384) that functions as a crucial regulator of cell growth. Comparative transcriptomic data combined with genetic and biochemical analyses revealed that sr8384 acts as a pleiotropic regulator and controls multiple targets that are associated with crucial biological processes through direct or indirect interactions. Notably, the in vivo expression level of sr8384 determined the cell growth rate, thereby affecting the solvent titer and productivity. These findings indicate the importance of the sr8384-mediated regulatory network in C. acetobutylicum Furthermore, a homolog of sr8384 was discovered and proven to be functional in another important Clostridium species, C. beijerinckii, suggesting the potential broad role of this sRNA in clostridia. Our work showcases a previously unknown potent and complex role of sRNAs in clostridia, providing new opportunities for understanding and engineering these anaerobes.IMPORTANCE The uses of sRNAs as new resources for functional studies and strain modifications are promising strategies in microorganisms. However, these crucial regulatory molecules have hardly been explored in industrially important solventogenic clostridia. Here, we identified sr8384 as a novel determinant sRNA controlling the cell growth of solventogenic Clostridium acetobutylicum Based on a detailed functional analysis, we further reveal the pleiotropic function of sr8384 and its multiple direct and indirect crucial targets, which represents a valuable source for understanding and optimizing this anaerobe. Of note, manipulation of this sRNA achieves improved cell growth and solvent synthesis. Our findings provide a new perspective for future studies on regulatory sRNAs in clostridia.

A novel regulatory pathway consisting of a two-component system and an ABC-type transporter contributes to butanol tolerance in Clostridium acetobutylicum.

Despite the long-term interest in solventogenic clostridia-based ABE (acetone-butanol-ethanol) fermentation, clostridial butanol tolerance and its underlying mechanism remain poorly understood, which is a major obstacle hindering further improvements of this important fermentative process. In this study, a two-component system (TCS), BtrK/BtrR, was identified and demonstrated to positively regulate butanol tolerance and ABE solvent formation in Clostridium acetobutylicum, a representative species of solventogenic clostridia. The transcriptomic analysis results showed that BtrK/BtrR has a pleiotropic regulatory function, affecting a large number of crucial genes and metabolic pathways. Of the differentially expressed genes, btrTM, encoding a putative ABC-type transporter (named BtrTM), was shown to be under the direct control of BtrR, the response regulator of the BtrK/BtrR TCS. Furthermore, BtrTM was shown to contribute to more butanol tolerance (46.5% increase) by overexpression, revealing a novel regulatory mechanism consisting of the BtrK/BtrR TCS and the BtrTM transporter in C. acetobutylicum. Based on these findings, we achieved faster growth and solvent production of C. acetobutylicum by overexpressing BtrK/BtrR or its direct target BtrTM, although no significant improvement in the final butanol titer and yield. These results further confirm the importance of BtrK/BtrR and BtrTM in this organism. Also, of significance, a specific number of btrR-btrT-btrM-btrK-like gene clusters were identified in other Clostridium species, including the pathogens Clostridium perfringens and Clostridium botulinum, indicating a broad role for this regulatory module in the class Clostridia.

Development of an in vivo fluorescence based gene expression reporter system for Clostridium tyrobutyricum.

C. tyrobutyricum, an acidogenic Clostridium, has aroused increasing interest due to its potential to produce biofuel efficiently. However, construction of recombinant C. tyrobutyricum for enhanced biofuel production has been impeded by the limited genetic engineering tools. In this study, a flavin mononucleotide (FMN)-dependent fluorescent protein Bs2-based gene expression reporter system was developed to monitor transformation and explore in vivo strength and regulation of various promoters in C. tyrobutyricum and C. acetobutylicum. Unlike green fluorescent protein (GFP) and its variants, Bs2 can emit green light without oxygen, which makes it extremely suitable for promoter screening and transformation confirmation in organisms grown anaerobically. The expression levels of bs2 under thiolase promoters from C. tyrobutyricum and C. acetobutylicum were measured and compared based on fluorescence intensities. The capacities of the two promoters in driving secondary alcohol dehydrogenase (adh) gene for isopropanol production in C. tyrobutyricum were distinguished, confirming that this reporter system is a convenient, effective and reliable tool for promoter strength assay and real time monitoring in C. tyrobutyricum, while demonstrating the feasibility of producing isopropanol in C. tyrobutyricum for the first time.

Butanol and butyric acid production from Saccharina japonica by Clostridium acetobutylicum and Clostridium tyrobutyricum with adaptive evolution.

Optimal conditions of hyper thermal (HT) acid hydrolysis of the Saccharina japonica was determined to a seaweed slurry content of 12% (w/v) and 144 mM H2SO4 at 160 °C for 10 min. Enzymatic saccharification was carried out at 50 °C and 150 rpm for 48 h using the three enzymes at concentrations of 16 U/mL. Celluclast 1.5 L showed the lowest half-velocity constant (Km) of 0.168 g/L, indicating a higher affinity for S. japonica hydrolysate. Pretreatment yielded a maximum monosaccharide concentration of 36.2 g/L and 45.7% conversion from total fermentable monosaccharides of 79.2 g/L with 120 g dry weight/L S. japonica slurry. High cell densities of Clostridium acetobutylicum and Clostridium tyrobutyricum were obtained using the retarding agents KH2PO4 (50 mM) and NaHCO3 (200 mM). Adaptive evolution facilitated the efficient use of mixed monosaccharides. Therefore, adaptive evolution and retarding agents can enhance the overall butanol and butyric acid yields from S. japonica.

Acetone, butanol, and ethanol production from the green seaweed Enteromorpha intestinalis via the separate hydrolysis and fermentation.

Acetone, butanol, and ethanol (ABE) were produced following the separate hydrolysis and fermentation (SHF) method using polysaccharides from the green macroalgae Enteromorpha intestinalis as biomass. We focused on the optimization of enzymatic saccharification as pretreatments for the fermentation of E. intestinalis. Pretreatment was carried out with 10% (w/v) seaweed slurry and 270-mM H2SO4 at 121 °C for 60 min. Monosaccharides (mainly glucose) were obtained from enzymatic hydrolysis with a 16-U/mL mixture of Celluclast 1.5 L and Viscozyme L at 45 °C for 36 h. ABE fermentation with 10% (w/v) E. intestinalis hydrolysate was performed using the anaerobic bacteria Clostridium acetobutylicum with either uncontrolled pH, pH controlled at 6.0, or pH controlled initially at 6.0 and then 4.5 after 4 days, which produced ABE contents of 5.6 g/L with an ABE yield (YABE) of 0.24 g/g, 4.8 g/L with an YABE of 0.2 g/g, and 8.5 g/L with an YABE of 0.36 g/g, respectively.

Small and Low but Potent: the Complex Regulatory Role of the Small RNA SolB in Solventogenesis in Clostridium acetobutylicum.

The recently revived Clostridium acetobutylicum-based acetone-butanol-ethanol (ABE) fermentation is widely celebrated and studied for its impact on industrial biotechnology. C. acetobutylicum has been studied and engineered extensively, yet critical areas of the molecular basis for how solvent formation is regulated remain unresolved. The core solventogenic genes (adhE1/aad, ctfA, ctfB, and adc) are carried on the sol locus of the pSOL1 megaplasmid, whose loss leads to asporogenous, "degenerate" cells. The sol locus includes a noncoding small RNA (sRNA), SolB, whose role is presumed to be critical for solventogenesis but has eluded resolution. In the present study, SolB overexpression downregulated the sol-locus genes at the transcript level, resulting in attenuated protein expression and a solvent-deficient phenotype, thus suggesting that SolB affects expression of all sol-locus transcripts and seemingly validating its hypothesized role as a repressor. However, deletion of solB resulted in a total loss of acetone production and severe attenuation of butanol formation, with complex effects on sol-locus genes and proteins: it had a small impact on adc mRNA or its corresponding protein (acetoacetate decarboxylase) expression level, somewhat reduced adhE1 and ctfA-ctfB mRNA levels, and abolished the ctfA-ctfB-encoded coenzyme A transferase (CoAT) activity. Computational predictions support a model whereby SolB expressed at low levels enables the stabilization and translation of sol-locus transcripts to facilitate tuning of the production of various solvents depending on the prevailing culture conditions. A key predicted SolB target is the ribosome binding site (RBS) of the ctfA transcript, and this was verified by expressing variants of the ctfA-ctfB genes to demonstrate the importance of SolB for acetone formation.IMPORTANCE Small noncoding RNAs regulate many important metabolic and developmental programs in prokaryotes, but their role in anaerobes has been explored minimally. Regulation of solvent formation in the important industrial organism C. acetobutylicum remains incompletely understood. While the genes for solvent formation and their promoters are known, the means by which this organism tunes the ratios of key solvents, notably the butanol/acetone ratio to balance its electron resources, remains unknown. Significantly, the roles of several coding and noncoding genes in the sol locus in tuning the solvent formation ratios have not been explored. Here we show that the small RNA SolB fine-tunes the expression of solvents, with acetone formation being a key target, by regulating the translation of the acetone formation rate-limiting enzyme, the coenzyme A transferase (CoAT). It is notable that SolB expressed at very low levels enables CoAT translation, while at high, nonphysiological expression levels, it leads to degradation of the corresponding transcript.

Acetone-Butanol-Ethanol Production from Waste Seaweed Collected from Gwangalli Beach, Busan, Korea, Based on pH-Controlled and Sequential Fermentation Using Two Strains.

The optimal conditions for acetone-butanol-ethanol (ABE) production were evaluated using waste seaweed from Gwangalli Beach, Busan, Korea. The waste seaweed had a fiber and carbohydrate, content of 48.34%; these are the main resources for ABE production. The optimal conditions for obtaining monosaccharides based on hyper thermal (HT) acid hydrolysis of waste seaweed were slurry contents of 8%, sulfuric acid concentration of 138 mM, and treatment time of 10 min. Enzymatic saccharification was performed using 16 unit/mL Viscozyme L, which showed the highest affinity (Km = 1.81 g/L). After pretreatment, 34.0 g/L monosaccharides were obtained. ABE fermentation was performed with single and sequential fermentation of Clostridium acetobutylicum and Clostridium tyrobutyricum; this was controlled for pH. A maximum ABE concentration of 12.5 g/L with YABE 0.37 was achieved using sequential fermentation with C. tyrobutyricum and C. acetobutylicum. Efficient ABE production from waste seaweed performed using pH-controlled culture broth and sequential cell culture.

A dynamic metabolic flux analysis of ABE (acetone-butanol-ethanol) fermentation by Clostridium acetobutylicum ATCC 824, with riboflavin as a by-product.

The present study reveals that supplementing sodium acetate (NaAc) strongly stimulates riboflavin production in acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum ATCC 824 with xylose as carbon source. Riboflavin production increased from undetectable concentrations to ∼0.2 g L-1 (0.53 mM) when supplementing 60 mM NaAc. Of interest, solvents production and biomass yield were also promoted with fivefold acetone, 2.6-fold butanol, and 2.4-fold biomass adding NaAc. A kinetic metabolic model, developed to simulate ABE biosystem, with riboflavin production, revealed from a dynamic metabolic flux analysis (dMFA) simultaneous increase of riboflavin (ribA) and GTP (precursor of riboflavin) (PurM) synthesis flux rates under NaAc supplementation. The model includes 23 fluxes, 24 metabolites, and 72 kinetic parameters. It also suggested that NaAc condition has first stimulated the accumulation of intracellular metabolite intermediates during the acidogenic phase, which have then fed the solventogenic phase leading to increased ABE production. In addition, NaAc resulted in higher intracellular levels of NADH during the whole culture. Moreover, lower GTP-to-adenosine phosphates (ATP, ADP, AMP) ratio under NaAc supplemented condition suggests that GTP may have a minor role in the cell energetic metabolism compared to its contribution to riboflavin synthesis.

Cauliflower waste utilization for sustainable biobutanol production: revelation of drying kinetics and bioprocess development.

Efficient yet economic production of biofuel(s) using varied second-generation feedstock needs to be explored in the current scenario to cope up with global fuel demand. Hence, the present study was performed to reveal the use of cauliflower waste for acetone-butanol-ethanol (ABE) production using Clostridium acetobutylicum NRRL B 527. The proximate analysis of cauliflower waste demonstrated to comprise 17.32% cellulose, 9.12% hemicellulose, and 5.94% lignin. Drying of cauliflower waste was carried out in the temperature range of 60-120 °C to investigate its effect on ABE production. The experimental drying data were simulated using moisture diffusion control model. The cauliflower waste dried at 80 °C showed maximum total sugar yield of 26.05 g L-1. Furthermore, the removal of phenolics, acetic acid, and total furans was found to be 90-97, 10-40, and 95-97%, respectively. Incidentally, maximum ABE titer obtained was 5.35 g L-1 with 50% sugar utilization.

Improving cellular robustness and butanol titers of Clostridium acetobutylicum ATCC824 by introducing heat shock proteins from an extremophilic bacterium.

In recent years, increasing concerns over environment, energy and climate have renewed interest in biotechnological production of butanol. However, growth inhibition by fermentation products and inhibitory components from raw biomass has hindered the development of acetone-butanol-ethanol (ABE) fermentation. Improving the cellular robustness of Clostridium acetobutylicum is of great importance for efficient ABE production. In this study, we attempted to improve the robustness and butanol titers of C. acetobutylicum ATCC824 by overexpressing GroESL and DnaK from the extremely radioresistant bacterium Deinococcus wulumuqiensis R12 and from C. acetobutylicum ATCC824 itself. Three recombinant strains were obtained and designated 824(dnaK R12), 824(groESL R12) and 824(groESL824). These three recombinants were found to have significantly improved tolerances to stresses including butanol, furfural, oxidation and acid. Meanwhile, the butanol titers increased to 13.0g/L, 11.2g/L and 10.7g/L, which were 49.4%, 28.7% and 23.0% higher than that from the wild-type strain (8.7g/L), respectively. For 824(dnaK R12), the production of acetic and butyric acids decreased by 97.1% (1.4g/L vs. 0.04g/L) and 100% (0.3g/L vs. 0g/L), respectively, compared with the wild-type strain. Overexpressing GroESL and DnaK from D. wulumuqiensis R12 also resulted in better growth and ABE production than the wild-type strain on fermentation in the presence of 2.5g/L furfural. Strain 824(groESL R12) was superior to 824(groESL 824) in diverse types of stress-tolerance and butanol titer, indicating that GroESL from the extremophilic bacterium could perform its function more efficiently in the heterologous host than native GroESL. Our study provides evidence that extremophilic bacteria can be excellent resources for engineering C. acetobutylicum to improve its robustness and butanol titer.

High-efficient n-butanol production by co-culturing Clostridium acetobutylicum and Saccharomyces cerevisiae integrated with butyrate fermentative supernatant addition.

Butanol is not only an important chemical intermediate and solvent in pharmaceutical and cosmetics industries, but also considered as an advanced biofuel. Although species of the natural host Clostridium have been engineered, butanol titers in the anaerobe seem to be limited by its intolerance to butanol less than 13 g/L. Here we aimed to develop a technology for enhancing butanol production by a co-culture system with butyrate fermentative supernatant addition. First, when adding 4.0 g/L butyrate into the acetone-butanol-ethanol (ABE) fermentation broth with single-shot at 24 h, the "acid crash" phenomenon occurred and the ABE fermentation performance deteriorated. Subsequently, we found that adding certain amino acids could effectively enhance butyrate re-assimilation, butanol tolerance and titer (from 11.1 to 14.8 g/L). Additionally, in order to decrease the raw material cost, butyrate fermentative supernatant produced by Clostridium tyrobutyricum was applied to butanol production in the Clostridium acetobutylicum/Saccharomyces cerevisiae co-culture system, instead of adding synthetic butyrate. Final butanol and total ABE concentrations reached higher levels of 16.3 and 24.8 g/L with increments of 46.8 and 37.8%, respectively. These results show that the proposed fermentation strategy has great potential for efficiently butanol production with an economic approach.

Model-based quantification of metabolic interactions from dynamic microbial-community data.

An important challenge in microbial ecology is to infer metabolic-exchange fluxes between growing microbial species from community-level data, concerning species abundances and metabolite concentrations. Here we apply a model-based approach to integrate such experimental data and thereby infer metabolic-exchange fluxes. We designed a synthetic anaerobic co-culture of Clostridium acetobutylicum and Wolinella succinogenes that interact via interspecies hydrogen transfer and applied different environmental conditions for which we expected the metabolic-exchange rates to change. We used stoichiometric models of the metabolism of the two microorganisms that represents our current physiological understanding and found that this understanding - the model - is sufficient to infer the identity and magnitude of the metabolic-exchange fluxes and it suggested unexpected interactions. Where the model could not fit all experimental data, it indicates specific requirement for further physiological studies. We show that the nitrogen source influences the rate of interspecies hydrogen transfer in the co-culture. Additionally, the model can predict the intracellular fluxes and optimal metabolic exchange rates, which can point to engineering strategies. This study therefore offers a realistic illustration of the strengths and weaknesses of model-based integration of heterogenous data that makes inference of metabolic-exchange fluxes possible from community-level experimental data.

Simplifying multidimensional fermentation dataset analysis and visualization: One step closer to capturing high-quality mutant strains.

In this study, we analyzed mutants of Clostridium acetobutylicum, an organism used in a broad range of industrial processes related to biofuel production, to facilitate future studies of bioreactor and bioprocess design and scale-up, which are very important research projects for industrial microbiology applications. To accomplish this, we generated 329 mutant strains and applied principal component analysis (PCA) to fermentation data gathered from these strains to identify a core set of independent features for comparison. By doing so, we were able to explain the differences in the mutant strains' fermentation expression states and simplify the analysis and visualization of the multidimensional datasets related to the strains. Our study has produced a high-efficiency PCA application based on a data analytics tool that is designed to visualize screening results and to support several hundred sets of data on fermentation interactions to assist researchers in more precisely screening and capturing high-quality mutant strains. More importantly, although this study focused on the use of PCA in microbial fermentation engineering, its results are broadly applicable.

Performance comparison of ethanol and butanol production in a continuous and closed-circulating fermentation system with membrane bioreactor.

Since both ethanol and butanol fermentations are urgently developed processes with the biofuel-demand increasing, performance comparison of aerobic ethanol fermentation and anerobic butanol fermentation in a continuous and closed-circulating fermentation (CCCF) system was necessary to achieve their fermentation characteristics and further optimize the fermentation process. Fermentation and pervaporation parameters including the average cell concentration, glucose consumption rate, cumulated production concentration, product flux, and separation factor of ethanol fermentation were 11.45 g/L, 3.70 g/L/h, 655.83 g/L, 378.5 g/m2/h, and 4.83, respectively, the corresponding parameters of butanol fermentation were 2.19 g/L, 0.61 g/L/h, 28.03 g/L, 58.56 g/m2/h, and 10.62, respectively. Profiles of fermentation and pervaporation parameters indicated that the intensity and efficiency of ethanol fermentation was higher than butanol fermentation, but the stability of butanol fermentation was superior to ethanol fermentation. Although the two fermentation processes had different features, the performance indicated the application prospect of both ethanol and butanol production by the CCCF system.

Kinetic Study of Acetone-Butanol-Ethanol Fermentation in Continuous Culture.

Acetone-butanol-ethanol (ABE) fermentation by clostridia has shown promise for industrial-scale production of biobutanol. However, the continuous ABE fermentation suffers from low product yield, titer, and productivity. Systems analysis of the continuous ABE fermentation will offer insights into its metabolic pathway as well as into optimal fermentation design and operation. For the ABE fermentation in continuous Clostridium acetobutylicum culture, this paper presents a kinetic model that includes the effects of key metabolic intermediates and enzymes as well as culture pH, product inhibition, and glucose inhibition. The kinetic model is used for elucidating the behavior of the ABE fermentation under the conditions that are most relevant to continuous cultures. To this end, dynamic sensitivity analysis is performed to systematically investigate the effects of culture conditions, reaction kinetics, and enzymes on the dynamics of the ABE production pathway. The analysis provides guidance for future metabolic engineering and fermentation optimization studies.

Thermochemical hydrolysis of macroalgae Ulva for biorefinery: Taguchi robust design method.

Understanding the impact of all process parameters on the efficiency of biomass hydrolysis and on the final yield of products is critical to biorefinery design. Using Taguchi orthogonal arrays experimental design and Partial Least Square Regression, we investigated the impact of change and the comparative significance of thermochemical process temperature, treatment time, %Acid and %Solid load on carbohydrates release from green macroalgae from Ulva genus, a promising biorefinery feedstock. The average density of hydrolysate was determined using a new microelectromechanical optical resonator mass sensor. In addition, using Flux Balance Analysis techniques, we compared the potential fermentation yields of these hydrolysate products using metabolic models of Escherichia coli, Saccharomyces cerevisiae wild type, Saccharomyces cerevisiae RN1016 with xylose isomerase and Clostridium acetobutylicum. We found that %Acid plays the most significant role and treatment time the least significant role in affecting the monosaccharaides released from Ulva biomass. We also found that within the tested range of parameters, hydrolysis with 121 °C, 30 min 2% Acid, 15% Solids could lead to the highest yields of conversion: 54.134-57.500 gr ethanol kg(-1) Ulva dry weight by S. cerevisiae RN1016 with xylose isomerase. Our results support optimized marine algae utilization process design and will enable smart energy harvesting by thermochemical hydrolysis.

Enhanced butanol production from cassava with Clostridium acetobutylicum by genome shuffling.

To obtain strains exhibiting high levels of solvent tolerance and butanol production, wild type strains of Clostridium acetobutylicum butanol-producing strain GX01 and Lactobacillus mucosae butanol-tolerant strain M26 were subjected to mutagenesis combining N-methyl-N-nitro-N-nitrosoguanidine induction with genome shuffling. After four successive rounds of genome shuffling, the C. acetobutylicum shuffled strain GS4-3 showing greater levels of fermentation performances (such as secreting a higher level of amylase, improving the thermal stability, and possessing greater environmental robustness) compared to the wild type strains was isolated. As a result, after optimization of culture conditions, mutant GS4-3 produced 32.6 g/L of total solvent, 20.1 g/L of butanol production, and 0.35 g/L/h of butanol productivity, which were, respectively, increased by 23.5, 23.3, and 40.0 % than the wild-type strain GX01, in a 10 L bioreactor. The enhanced production of butanol and tolerance of solvent of mutant associated with GS4-3 make it promising for acetone/butanol/ethanol fermentation from cassava (Manihot esculenta).

Calorimetric studies of the growth of anaerobic microbes.

This article aims to validate the use of calorimetry to measure the growth of anaerobic microbes. It has been difficult to monitor the growth of strict anaerobes while maintaining optimal growth conditions. Traditionally, optical density and ATP concentration are usually used as measures of the growth of anaerobic microbes. However, to take these measurements it is necessary to extract an aliquot of the culture, which can be difficult while maintaining anaerobic conditions. In this study, calorimetry was used to continuously and nondestructively measure the heat generated by the growth of anaerobic microbes as a function of time. Clostridium acetobutylicum, Clostridium beijerinckii, and Clostridium cellulovorans were used as representative anaerobic microbes. Using a multiplex isothermal calorimeter, we observed that peak time (tp) of C. acetobutylicum heat evolution increased as the inoculation rate decreased. This strong correlation between the inoculation rate and tp showed that it was possible to measure the growth rate of anaerobic microbes by calorimetry. Overall, our results showed that there is a very good correlation between heat evolution and optical density/ATP concentration, validating the use of the method.