Screening Antibacterial Photodynamic Effect of Monascus Red Yeast Rice (Hong-Qu) and Mycelium Extracts.

The fungus Monascus is a well-known source of secondary metabolites with interesting pharmaceutical and nutraceutical applications. In particular, Monascus pigments possess a wide range of biological activities (e.g. antimicrobial, antioxidant, anti-inflammatory or antitumoral). To broaden the scope of their possible application, this study focused on testing Monascus pigment extracts as potential photosensitizing agents efficient in antimicrobial photodynamic therapy (aPDT) against bacteria. For this purpose, eight different extracts of secondary metabolites from the liquid- and solid-state fermentation of Monascus purpureus DBM 4360 and Monascus sp. DBM 4361 were tested against Gram-positive and Gram-negative model bacteria, Bacillus subtilis and Escherichia coli and further screened for ESKAPE pathogens, Staphylococcus aureus and Pseudomonas aeruginosa. To the bacterial culture, increasing concentration of extracts was added and it was found that all extracts showed varying antimicrobial activity against Gram-positive bacteria in dark, which was further increased after irradiation. Gram-negative bacteria were tolerant to the extracts' exposure in the dark but sensitivity to almost all extracts that occurred after irradiation. The Monascus sp. DBM 4361 extracts seemed to be the best potential candidate for aPDT against Gram-positive bacteria, being efficient at low doses, i.e. the lowest total concentration of Monascus pigments exhibiting aPDT effect was 3.92 ± 1.36 mg/L for E. coli. Our results indicate that Monascus spp., forming monascuspiloin as the major yellow pigment and not-forming mycotoxin citrinin, is a promising source of antimicrobials and photoantimicrobials.

Whole genome sequencing and characterization of Pantoea agglomerans DBM 3797, endophyte, isolated from fresh hop (Humulus lupulus L.).

Background: This paper brings new information about the genome and phenotypic characteristics of Pantoea agglomerans strain DBM 3797, isolated from fresh Czech hop (Humulus lupulus) in the Saaz hop-growing region. Although P. agglomerans strains are frequently isolated from different materials, there are not usually thoroughly characterized even if they have versatile metabolism and those isolated from plants may have a considerable potential for application in agriculture as a support culture for plant growth. Methods: P. agglomerans DBM 3797 was cultured under aerobic and anaerobic conditions, its metabolites were analyzed by HPLC and it was tested for plant growth promotion abilities, such as phosphate solubilization, siderophore and indol-3-acetic acid productions. In addition, genomic DNA was extracted, sequenced and de novo assembly was performed. Further, genome annotation, pan-genome analysis and selected genome analyses, such as CRISPR arrays detection, antibiotic resistance and secondary metabolite genes identification were carried out. Results and discussion: The typical appearance characteristics of the strain include the formation of symplasmata in submerged liquid culture and the formation of pale yellow colonies on agar. The genetic information of the strain (in total 4.8 Mb) is divided between a chromosome and two plasmids. The strain lacks any CRISPR-Cas system but is equipped with four restriction-modification systems. The phenotypic analysis focused on growth under both aerobic and anaerobic conditions, as well as traits associated with plant growth promotion. At both levels (genomic and phenotypic), the production of siderophores, indoleacetic acid-derived growth promoters, gluconic acid, and enzyme activities related to the degradation of complex organic compounds were found. Extracellular gluconic acid production under aerobic conditions (up to 8 g/l) is probably the result of glucose oxidation by the membrane-bound pyrroloquinoline quinone-dependent enzyme glucose dehydrogenase. The strain has a number of properties potentially beneficial to the hop plant and its closest relatives include the strains also isolated from the aerial parts of plants, yet its safety profile needs to be addressed in follow-up research.

Transcriptomic studies of solventogenic clostridia, Clostridium acetobutylicum and Clostridium beijerinckii.

Solventogenic clostridia are not a strictly defined group within the genus Clostridium but its representatives share some common features, i.e. they are anaerobic, non-pathogenic, non-toxinogenic and endospore forming bacteria. Their main metabolite is typically 1-butanol but depending on species and culture conditions, they can form other metabolites such as acetone, isopropanol, ethanol, butyric, lactic and acetic acids, and hydrogen. Although these organisms were previously used for the industrial production of solvents, they later fell into disuse, being replaced by more efficient chemical production. A return to a more biological production of solvents therefore requires a thorough understanding of clostridial metabolism. Transcriptome analysis, which reflects the involvement of individual genes in all cellular processes within a population, at any given (sampling) moment, is a valuable tool for gaining a deeper insight into clostridial life. In this review, we describe techniques to study transcription, summarize the evolution of these techniques and compare methods for data processing and visualization of solventogenic clostridia, particularly the species Clostridium acetobutylicum and Clostridium beijerinckii. Individual approaches for evaluating transcriptomic data are compared and their contributions to advancements in the field are assessed. Moreover, utilization of transcriptomic data for reconstruction of computational clostridial metabolic models is considered and particular models are described. Transcriptional changes in glucose transport, central carbon metabolism, the sporulation cycle, butanol and butyrate stress responses, the influence of lignocellulose-derived inhibitors on growth and solvent production, and other respective topics, are addressed and common trends are highlighted.

Effect of a Monascus sp. Red Yeast Rice Extract on Germination of Bacterial Spores.

The pink-red color of traditional sausages (cured meat) is the result of nitrite addition and the formation of nitrosomyoglobin. However, the pleasant color of processed meat products is a side effect of nitrite addition while the main anticipated goal is to suppress the germination of clostridial spores. The fungus Monascus is known as a producer of oligoketide pigments, which are used in Asian countries, especially in China, for coloring foods, including meat products. Although, different biological activities of Monascus pigments have been tested and confirmed in many studies, their effect on germination of bacterial spores has never been investigated. This study is focused on testing the activity of red yeast rice (RYR) extract, containing monascin, rubropunctatin, rubropunctamine complexes and monascuspiloin as the main pigments, on germination of Clostridium and Bacillus spores. It was found that addition of nitrite alone, at the permitted concentration, had no effect on spore germination. However, the combined effects of nitrite with NaCl, tested after addition of pickling salt, was efficient in inhibiting the germination of C. beijerinckii spores but had no effect on B. subtilis spores. In contrast, total suppression of C. beijerinckii spore germination was reached after addition of RYR extract to the medium at a concentration of 2% v/v. For B. subtilis, total inhibition of spore germination was observed only after addition of 4% v/v RYR extract to the medium containing 1.3% w/w NaCl.

Identification and Validation of Reference Genes in Clostridium beijerinckii NRRL B-598 for RT-qPCR Using RNA-Seq Data.

Gene expression analysis through reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) depends on correct data normalization by reference genes with stable expression. Although Clostridium beijerinckii NRRL B-598 is a promising Gram-positive bacterium for the industrial production of biobutanol, validated reference genes have not yet been reported. In this study, we selected 160 genes with stable expression based on an RNA sequencing (RNA-Seq) data analysis, and among them, seven genes (zmp, rpoB1, rsmB, greA, rpoB2, topB2, and rimO) were selected for experimental validation by RT-qPCR and gene ontology (GO) enrichment analysis. According to statistical analyses, zmp and greA were the most stable and suitable reference genes for RT-qPCR normalization. Furthermore, our methodology can be useful for selection of the reference genes in other strains of C. beijerinckii and it also suggests that the RNA-Seq data can be used for the initial selection of novel reference genes, however, their validation is required.

Changes in efflux pump activity of Clostridium beijerinckii throughout ABE fermentation.

Pumping toxic substances through a cytoplasmic membrane by protein transporters known as efflux pumps represents one bacterial mechanism involved in the stress response to the presence of toxic compounds. The active efflux might also take part in exporting low-molecular-weight alcohols produced by intrinsic cell metabolism; in the case of solventogenic clostridia, predominantly acetone, butanol and ethanol (ABE). However, little is known about this active efflux, even though some evidence exists that membrane pumps might be involved in solvent tolerance. In this study, we investigated changes in overall active efflux during ABE fermentation, employing a flow cytometric protocol adjusted for Clostridia and using ethidium bromide (EB) as a fluorescence marker for quantification of direct efflux. A fluctuation in efflux during the course of standard ABE fermentation was observed, with a maximum reached during late acidogenesis, a high efflux rate during early and mid-solventogenesis and an apparent decrease in EB efflux rate in late solventogenesis. The fluctuation in efflux activity was in accordance with transcriptomic data obtained for various membrane exporters in a former study. Surprisingly, under altered cultivation conditions, when solvent production was attenuated, and extended acidogenesis was promoted, stable low efflux activity was reached after an initial peak that appeared in the stage comparable to standard ABE fermentation. This study confirmed that efflux pump activity is not constant during ABE fermentation and suggests that undisturbed solvent production might be a trigger for activation of pumps involved in solvent efflux. KEY POINTS: • Flow cytometric assay for efflux quantification in Clostridia was established. • Efflux rate peaked in late acidogenesis and in early solventogenesis. • Impaired solventogenesis led to an overall decrease in efflux.

Phenotypic and genomic analysis of isopropanol and 1,3-propanediol producer Clostridium diolis DSM 15410.

Clostridium diolis DSM 15410 is a type strain of solventogenic clostridium capable of conducting isopropanol-butanol-ethanol fermentation. By studying its growth on different carbohydrates, we verified its ability to utilize glycerol and produce 1,3-propanediol and discovered its ability to produced isopropanol. Complete genome sequencing showed that its genome is a single circular chromosome and belongs to the cluster I (sensu scricto) of the genus Clostridium. By cultivation analysis we highlighted its specific behavior in comparison to two selected closely related strains. Despite the fact that several CRISPR loci were found, 16 putative prophages showed the ability to receive foreign DNA. Thus, the strain has the necessary features for future engineering of its 1,3-propanediol biosynthetic pathway and for the possible industrial utilization in the production of biofuels.

Deeper below the surface-transcriptional changes in selected genes of Clostridium beijerinckii in response to butanol shock.

The main bottleneck in the return of industrial butanol production from renewable feedstock through acetone-butanol-ethanol (ABE) fermentation by clostridia, such as Clostridium beijerinckii, is the low final butanol concentration. The problem is caused by the high toxicity of butanol to the production cells, and therefore, understanding the mechanisms by which clostridia react to butanol shock is of key importance. Detailed analyses of transcriptome data that were obtained after butanol shock and their comparison with data from standard ABE fermentation have resulted in new findings, while confirmed expected population responses. Although butanol shock resulted in upregulation of heat shock protein genes, their regulation is different than was assumed based on standard ABE fermentation transcriptome data. While glucose uptake, glycolysis, and acidogenesis genes were downregulated after butanol shock, solventogenesis genes were upregulated. Cyclopropanation of fatty acids and formation of plasmalogens seem to be significant processes involved in cell membrane stabilization in the presence of butanol. Surprisingly, one of the three identified Agr quorum-sensing system genes was upregulated. Upregulation of several putative butanol efflux pumps was described after butanol addition and a large putative polyketide gene cluster was found, the transcription of which seemed to depend on the concentration of butanol.

Phenotypic and Genomic Analysis of Clostridium beijerinckii NRRL B-598 Mutants With Increased Butanol Tolerance.

N-Butanol, a valuable solvent and potential fuel extender, can be produced via acetone-butanol-ethanol (ABE) fermentation. One of the main drawbacks of ABE fermentation is the high toxicity of butanol to producing cells, leading to cell membrane disruption, low culture viability and, consequently, low produced concentrations of butanol. The goal of this study was to obtain mutant strains of Clostridium beijerinckii NRRL B-598 with improved butanol tolerance using random chemical mutagenesis, describe changes in their phenotypes compared to the wild-type strain and reveal changes in the genome that explain improved tolerance or other phenotypic changes. Nine mutant strains with stable improved features were obtained by three different approaches and, for two of them, ethidium bromide (EB), a known substrate of efflux pumps, was used for either selection or as a mutagenic agent. It is the first utilization of this approach for the development of butanol-tolerant mutants of solventogenic clostridia, for which generally there is a lack of knowledge about butanol efflux or efflux mechanisms and their regulation. Mutant strains exhibited increase in butanol tolerance from 36% up to 127% and the greatest improvement was achieved for the strains for which EB was used as a mutagenic agent. Additionally, increased tolerance to other substrates of efflux pumps, EB and ethanol, was observed in all mutants and higher antibiotic tolerance in some of the strains. The complete genomes of mutant strains were sequenced and revealed that improved butanol tolerance can be attributed to mutations in genes encoding typical stress responses (chemotaxis, autolysis or changes in cell membrane structure), but, also, to mutations in genes X276_07980 and X276_24400, encoding efflux pump regulators. The latter observation confirms the importance of efflux in butanol stress response of the strain and offers new targets for rational strain engineering.

Role of efflux in enhancing butanol tolerance of bacteria.

N-butanol, a valued solvent and potential fuel extender, could possibly be produced by fermentation using either native producers, i.e. solventogenic Clostridia, or engineered platform organisms such as Escherichia coli or Pseudomonas species, if the main process obstacle, a low final butanol concentration, could be overcome. A low final concentration of butanol is the result of its high toxicity to production cells. Nevertheless, bacteria have developed several mechanisms to cope with this toxicity and one of them is active butanol efflux. This review presents information about a few well characterized butanol efflux pumps from Gram-negative bacteria (P. putida and E. coli) and summarizes knowledge about putative butanol efflux systems in Gram-positive bacteria.

Comparison of Lactic Acid Production by L. casei in Batch, Fed-batch and Continuous Cultivation, Testing the use of Feather Hydrolysate as a Complex Nitrogen Source

Abstract A comprehensive comparison of the main fermentation parameters, productivity, yield and final L-lactic acid concentration, obtained through batch, fed-batch and continuous cultivations using Lactobacillus casei CCDM 198 and a model cultivation medium was carried out. Using this data, a pulse-feed fed-batch process was established for testing chicken feather hydrolysate as a replacement for all complex nitrogen sources (yeast and beef extracts and peptone) in the medium. As comparably high values of productivity (about 4.0 g/L/h) and yield (about 98 %) were reached under all cultivation conditions, the maximum final L-lactic acid concentration (116.5 g/L), as achieved through pulse-feed fed-batch fermentation, was chosen as the most important criterion for process selection. Fed-batch cultivation with chicken feather hydrolysate as both a complex nitrogen source and a neutralizing agent for maintaining constant culture pH yielded half the concentration of L-lactic acid compared to the model medium. We demonstrate here that chicken feather hydrolysate has potential for use in the production of L-lactic acid but its utilization requires further optimization

Transient and Steady Pervaporation of 1-Butanol-Water Mixtures through a Poly[1-(Trimethylsilyl)-1-Propyne] (PTMSP) Membrane.

The transient and steady pervaporation of 1-butanol-water mixtures through a poly[1-(trimethylsilyl)-1-propyne] (PTMSP) membrane was studied to observe and elucidate the diffusion phenomena in this high-performing organophilic glassy polymer. Pervaporation was studied in a continuous sequence of experiments under conditions appropriate for the separation of bio-butanol from fermentation broths: feed concentrations of 1.5, 3.0 and 4.5 w/w % of 1-butanol in nutrient-containing (yeast extract) water, temperatures of 37, 50 and 63 °C, and a time period of 80 days. In addition, concentration polarization was assessed. As expected, the total flux and individual component permeabilities declined discernibly over the study period, while the separation factor (average ß = 82) and selectivity towards 1-butanol (average α = 2.6) remained practically independent of the process conditions tested. Based on measurements of pervaporation transients, for which a new apparatus and model were developed, we found that the diffusivity of 1-butanol in PTMSP decreased over time due to aging and was comparable to that observed using microgravimetry in pure vapor in 1-butanol. Hence, despite the gradual loss of free volume of the aging polymer, the PTMSP membrane showed high and practically independent selectivity towards 1-butanol. Additionally, a new technique for the measurement and evaluation of pervaporation transients using Fourier transform infrared spectroscopy (FTIR) analysis of permeate was proposed and validated.

Transcriptional analysis of amino acid, metal ion, vitamin and carbohydrate uptake in butanol-producing Clostridium beijerinckii NRRL B-598.

In-depth knowledge of cell metabolism and nutrient uptake mechanisms can lead to the development of a tool for improving acetone-butanol-ethanol (ABE) fermentation performance and help to overcome bottlenecks in the process, such as the high cost of substrates and low production rates. Over 300 genes potentially encoding transport of amino acids, metal ions, vitamins and carbohydrates were identified in the genome of the butanol-producing strain Clostridium beijerinckii NRRL B-598, based on similarity searches in protein function databases. Transcriptomic data of the genes were obtained during ABE fermentation by RNA-Seq experiments and covered acidogenesis, solventogenesis and sporulation. The physiological roles of the selected 81 actively expressed transport genes were established on the basis of their expression profiles at particular stages of ABE fermentation. This article describes how genes encoding the uptake of glucose, iron, riboflavin, glutamine, methionine and other nutrients take part in growth, production and stress responses of C. beijerinckii NRRL B-598. These data increase our knowledge of transport mechanisms in solventogenic Clostridium and may be used in the selection of individual genes for further research.

Effect of initial pH, different nitrogen sources, and cultivation time on the production of yellow or orange Monascus purpureus pigments and the mycotoxin citrinin.

Monascus purpureus was grown in submerged liquid culture using ammonium sulfate, sodium nitrate, and peptone as nitrogen sources while initial medium pH was adjusted to 2.5, 5.5, 6.5, or 8.0. The combined effect of culture pH and nitrogen source on the biosynthesis of yellow (ankaflavin and monascin) and orange (rubropunctatin and monascorubrin) pigments, plus the mycotoxin citrinin, was evaluated chromatographically. Optimum cultivation conditions, that is, initial pH 2.5 and 8.8 g/L peptone as a nitrogen source, resulted in high levels of production of yellow and orange pigments (sum of pigment concentration 1,138 mg/L) and negligible citrinin concentration (2 mg/L).

A transcriptional response of Clostridium beijerinckii NRRL B-598 to a butanol shock.

BACKGROUND: One of the main obstacles preventing solventogenic clostridia from achieving higher yields in biofuel production is the toxicity of produced solvents. Unfortunately, regulatory mechanisms responsible for the shock response are poorly described on the transcriptomic level. Although the strain Clostridium beijerinckii NRRL B-598, a promising butanol producer, has been studied under different conditions in the past, its transcriptional response to a shock caused by butanol in the cultivation medium remains unknown. RESULTS: In this paper, we present a transcriptional response of the strain during a butanol challenge, caused by the addition of butanol to the cultivation medium at the very end of the acidogenic phase, using RNA-Seq. We resequenced and reassembled the genome sequence of the strain and prepared novel genome and gene ontology annotation to provide the most accurate results. When compared to samples under standard cultivation conditions, samples gathered during butanol shock represented a well-distinguished group. Using reference samples gathered directly before the addition of butanol, we identified genes that were differentially expressed in butanol challenge samples. We determined clusters of 293 down-regulated and 301 up-regulated genes whose expression was affected by the cultivation conditions. Enriched term "RNA binding" among down-regulated genes corresponded to the downturn of translation and the cluster contained a group of small acid-soluble spore proteins. This explained phenotype of the culture that had not sporulated. On the other hand, up-regulated genes were characterized by the term "protein binding" which corresponded to activation of heat-shock proteins that were identified within this cluster. CONCLUSIONS: We provided an overall transcriptional response of the strain C. beijerinckii NRRL B-598 to butanol shock, supplemented by auxiliary technologies, including high-pressure liquid chromatography and flow cytometry, to capture the corresponding phenotypic response. We identified genes whose regulation was affected by the addition of butanol to the cultivation medium and inferred related molecular functions that were significantly influenced. Additionally, using high-quality genome assembly and custom-made gene ontology annotation, we demonstrated that this settled terminology, widely used for the analysis of model organisms, could also be applied to non-model organisms and for research in the field of biofuels.

Effective continuous acetone-butanol-ethanol production with full utilization of cassava by immobilized symbiotic TSH06.

BACKGROUND: Butanol production by fermentation has recently attracted increasingly more attention because of its mild reaction conditions and environmentally friendly properties. However, traditional feedstocks, such as corn, are food supplies for human beings and are expensive and not suitable for butanol production at a large scale. In this study, acetone, butanol, and ethanol (ABE) fermentation with non-pretreated cassava using a symbiotic TSH06 was investigated. RESULTS: In batch fermentation, the butanol concentration of 11.6 g/L was obtained with a productivity of 0.16 g/L/h, which was similar to that obtained from glucose system. A full utilization system of cassava was constructed to improve the fermentation performance, cassava flour was used as the substrate and cassava peel residue was used as the immobilization carrier. ABE fermentation with immobilized cells resulted in total ABE and butanol concentrations of 20 g/L and 13.3 g/L, which were 13.6% and 14.7% higher, respectively, than those of free cells. To further improve the solvent productivity, continuous fermentation was conducted with immobilized cells. In single-stage continuous fermentation, the concentrations of total ABE and butanol reached 9.3 g/L and 6.3 g/L with ABE and butanol productivities of 1.86 g/L/h and 1.26 g/L/h, respectively. In addition, both of the high product concentration and high solvent productivity were achieved in a three-stage continuous fermentation. The ABE productivity and concentration was 1.12 g/L/h and 16.8 g/L, respectively. CONCLUSIONS: The results indicate that TSH06 could produce solvents from cassava effectively. This study shows that ABE fermentation with cassava as a substrate could be an efficient and economical method of butanol production.

Acidogenesis, solventogenesis, metabolic stress response and life cycle changes in Clostridium beijerinckii NRRL B-598 at the transcriptomic level.

Clostridium beijerinckii NRRL B-598 is a sporulating, butanol and hydrogen producing strain that utilizes carbohydrates by the acetone-butanol-ethanol (ABE) fermentative pathway. The pathway consists of two metabolic phases, acidogenesis and solventogenesis, from which the latter one can be coupled with sporulation. Thorough transcriptomic profiling during a complete life cycle and both metabolic phases completed with flow cytometry, microscopy and a metabolites analysis helped to find out key genes involved in particular cellular events. The description of genes/operons that are closely involved in metabolism or the cell cycle is a necessary condition for metabolic engineering of the strain and will be valuable for all C. beijerinckii strains and other Clostridial species. The study focused on glucose transport and catabolism, hydrogen formation, metabolic stress response, binary fission, motility/chemotaxis and sporulation, which resulted in the composition of the unique image reflecting clostridial population changes. Surprisingly, the main change in expression of individual genes was coupled with the sporulation start and not with the transition from acidogenic to solventogenic metabolism. As expected, solvents formation started at pH decrease and the accumulation of butyric and acetic acids in the cultivation medium.

Transcription profiling of butanol producer Clostridium beijerinckii NRRL B-598 using RNA-Seq.

BACKGROUND: Thinning supplies of natural resources increase attention to sustainable microbial production of bio-based fuels. The strain Clostridium beijerinckii NRRL B-598 is a relatively well-described butanol producer regarding its genotype and phenotype under various conditions. However, a link between these two levels, lying in the description of the gene regulation mechanisms, is missing for this strain, due to the lack of transcriptomic data. RESULTS: In this paper, we present a transcription profile of the strain over the whole fermentation using an RNA-Seq dataset covering six time-points with the current highest dynamic range among solventogenic clostridia. We investigated the accuracy of the genome sequence and particular genome elements, including pseudogenes and prophages. While some pseudogenes were highly expressed, all three identified prophages remained silent. Furthermore, we identified major changes in the transcriptional activity of genes using differential expression analysis between adjacent time-points. We identified functional groups of these significantly regulated genes and together with fermentation and cultivation kinetics captured using liquid chromatography and flow cytometry, we identified basic changes in the metabolism of the strain during fermentation. Interestingly, C. beijerinckii NRRL B-598 demonstrated different behavior in comparison with the closely related strain C. beijerinckii NCIMB 8052 in the latter phases of cultivation. CONCLUSIONS: We provided a complex analysis of the C. beijerinckii NRRL B-598 fermentation profile using several technologies, including RNA-Seq. We described the changes in the global metabolism of the strain and confirmed the uniqueness of its behavior. The whole experiment demonstrated a good reproducibility. Therefore, we will be able to repeat the experiment under selected conditions in order to investigate particular metabolic changes and signaling pathways suitable for following targeted engineering.

Flow cytometry analysis of Clostridium beijerinckii NRRL B-598 populations exhibiting different phenotypes induced by changes in cultivation conditions.

BACKGROUND: Biobutanol production by clostridia via the acetone-butanol-ethanol (ABE) pathway is a promising future technology in bioenergetics , but identifying key regulatory mechanisms for this pathway is essential in order to construct industrially relevant strains with high tolerance and productivity. We have applied flow cytometric analysis to C. beijerinckii NRRL B-598 and carried out comparative screening of physiological changes in terms of viability under different cultivation conditions to determine its dependence on particular stages of the life cycle and the concentration of butanol. RESULTS: Dual staining by propidium iodide (PI) and carboxyfluorescein diacetate (CFDA) provided separation of cells into four subpopulations with different abilities to take up PI and cleave CFDA, reflecting different physiological states. The development of a staining pattern during ABE fermentation showed an apparent decline in viability, starting at the pH shift and onset of solventogenesis, although an appreciable proportion of cells continued to proliferate. This was observed for sporulating as well as non-sporulating phenotypes at low solvent concentrations, suggesting that the increase in percentage of inactive cells was not a result of solvent toxicity or a transition from vegetative to sporulating stages. Additionally, the sporulating phenotype was challenged with butanol and cultivation with a lower starting pH was performed; in both these experiments similar trends were obtained-viability declined after the pH breakpoint, independent of the actual butanol concentration in the medium. Production characteristics of both sporulating and non-sporulating phenotypes were comparable, showing that in C. beijerinckii NRRL B-598, solventogenesis was not conditional on sporulation. CONCLUSION: We have shown that the decline in C. beijerinckii NRRL B-598 culture viability during ABE fermentation was not only the result of accumulated toxic metabolites, but might also be associated with a special survival strategy triggered by pH change.