The kinetics of growth, acid and solvent production in batch culture of Clostridium pasteurianum DSMZ 525 were examined in mixed or mono-substrate fermentations. In pH-uncontrolled batch cultures, the addition of butyric acid or glucose significantly enhanced n-butanol production and the ratio of butanol/1,3-propanediol. In pH-controlled batch culture at pH = 6, butyric acid addition had a negative effect on growth and did not lead to a higher n-butanol productivity. On the other hand, mixed substrate fermentation using glucose and glycerol enhanced the growth and acid production significantly. Glucose limitation in the mixed substrate fermentation led to the reduction or inhibition of the glycerol consumption by the growing bacteria. Therefore, for the optimal growth and n-butanol production by C. pasteurianum, a limitation of either substrate should be avoided. Under optimized batch conditions, n-butanol concentration and maximum productivity achieved were 21 g/L, and 0.96 g/L × h, respectively. In comparison, mixed substrate fermentation using biomass hydrolysate and glycerol gave a n-butanol concentration of 17 g/L with a maximum productivity of 1.1 g/L × h. In terms of productivity and final n-butanol concentration, the results demonstrated that C. pasteurianum DSMZ 525 is well suitable for n-butanol production from mixed substrates of biomass hydrolysate and glycerol and represents an alternative promising production strain.

Diols are compounds with two hydroxyl groups and have a wide range of appealing applications as chemicals and fuels. In particular, five low molecular diol compounds, namely 1,3-propanediol (1,3-PDO), 1,2-propanediol (1,2-PDO), 2,3-butanediol (2,3-BDO), 1,3-butanediol (1,3-BDO), and 1,4-butanediol (1,4-BDO), can be biotechnologically produced by direct microbial bioconversion of renewable materials. In this review, we summarize recent developments in the microbial production of diols, especially regarding the engineering of typical microbial strains as cell factory and the development of corresponding bioconversion processes.

Background Clostridium pasteurianum as an emerging new microbial cell factory can produce both n-butanol (BuOH) and 1,3-propanediol (1,3-PDO), and the pattern of product formation changes significantly with the composition of the culture medium. Among others iron content in the medium was shown to strongly affect the products selectivity. However, the mechanism behind this metabolic regulation is still unclear. For a better understanding of such metabolic regulation and for process optimization, we carried out fermentation experiments under either iron excess or iron limitation conditions, and performed metabolic, stoichiometric and proteomic analyses.Results1,3-PDO is most effectively produced under iron limited condition (Fe−), whereas 1,3-PDO and BuOH were both produced under iron rich condition (Fe+). With increased iron availability the BuOH/1,3-PDO ratio increased significantly from 0.27 mol/mol (at Fe−) to 1.4 mol/mol (at Fe+). Additionally, hydrogen production was enhanced significantly under Fe+ condition. Proteomic analysis revealed differentiated expression of many proteins including several ones of the central carbon metabolic pathway. Among others, pyruvate: ferredoxin oxidoreductase, hydrogenases, and several electron transfer flavoproteins was found to be strongly up-regulated under Fe+ condition, pointing to their strong involvement in the regeneration of the oxidized form of ferredoxin, and consequently their influences on the product selectivity in C. pasteurianum. Of particular significance is the finding that H2 formation in C. pasteurianum is coupled to the ferredoxin-dependent butyryl-CoA dehydrogenase catalyzed reaction, which significantly affects the redox balance and thus the product selectivity.ConclusionsThe metabolic, stoichiometric and proteomic results clearly show the key roles of hydrogenases and ferredoxins dependent reactions in determining the internal redox balance and hence product selectivity. Not only the NADH pool but also the regulation of the ferredoxin pool could explain such product variation under different iron conditions.

BackgroundClostridium pasteurianum is becoming increasingly attractive for the production of chemicals and fuels such as n-butanol and 1,3-propanediol. Previously we have shown that dual substrate fermentation using glucose and glycerol enhanced the cell growth and butanol production significantly. Although C. pasteurianum can grow efficiently with either glucose or glycerol alone, under certain conditions, glucose limitation in the mixed substrate fermentation leads to growth cessation. To understand this phenomenon and for process optimization, fermentation experiments were performed in the presence of excess glycerol but with varied initial concentrations of glucose which were followed by physiological, metabolic and proteomic analyses.ResultsPhysiological characterization showed that the observed cease of growth is not due to the toxicity of n-butanol. Furthermore, the growth can be resumed by addition of glucose or the intermediate oxaloacetate. Proteomic analysis shed more light on the system-level regulation of many proteins directly or indirectly associated with this phenomenon. Surprisingly, it is found that the specific growth rate of C. pasteurianum in the different growth phases (e.g. before and after glucose limitation) correlated well with the expression level of the ATP dependent pyruvate carboxylase and with the expression level of biotin synthase which provides the cofactor biotin for the formation of oxaloacetate from pyruvate. Bioenergetic analysis based on the formation rates of metabolites further show that ATP supply is not a limiting factor for the pyruvate carboxylation to oxaloacetate.ConclusionsThe results of physiological and proteomic analyses clearly show that the anaplerotic synthesis of oxaloacetate plays a key role in determining the growth behaviour of C. pasteurianum in fermentations with mixed substrates of glucose and glycerol. This study provides interesting targets for metabolic engineering of this emerging industrial microorganism.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0497-4) contains supplementary material, which is available to authorized users.

Clostridium pasteurianum is a promising producer of the bulk chemicals 1,3‐propanediol and n‐butanol (BuOH), with significantly different product patterns and physiology from other typical Clostridia. Nevertheless, its growth and product formation are ultimately limited by the accumulation of inhibiting products, especially by BuOH. In this study, we implemented in situ gas stripping for BuOH removal and compared the stripping performance of external nitrogen (N2) and fermentation effluent gas (FG) from the process itself. Gas stripping was studied in fermentations of glycerol and a mixture of glycerol and glucose. In general, N2 exhibits favourable physical properties to strip out BuOH from an aqueous phase. However, in situ removal of butanol in C. pasteurianum culture with N2 stripping strongly perturbs the culture conditions such as the redox potential and thus the physiology of the microorganism, leading to enhanced formation of organic acids, especially in cosubstrate fermentation, whereas the use of FG does not show such perturbations. In an effort to explore the use of FG for gas stripping the effects of FG circulation rate and stirring speed of the bioreactor on BuOH stripping efficiency and the fermentation performance were studied in more detail. Mass transfer coefficient (kSa) of BuOH in the bioreactor was also characterized at different gas circulation rates and stirring speeds. In a fermentation of glycerol with FG stripping at a relatively high gas flow rate (7 vvm) as high as 39.2 g/L BuOH (total) and 53.7 g/L 1,3‐PDO can be simultaneously produced. The results are discussed in view of further process optimization and scale up.