Impact of anaerobiosis strategy and bioreactor geometry on the biochemical response of Clostridium butyricum VPI 1718 during 1,3-propanediol fermentation

The impact of anaerobiosis strategy on 1,3-propanediol production during cultivation of Clostridium butyricum VPI 1718 in different size bioreactors was studied. In batch trials with N2 gas infusion, the fermentation was successfully accomplished, regardless of initial glycerol concentration imposed and bioreactor geometry. However, in the absence of N2 continual sparging, significant variations concerning the biochemical response of the strain were observed. Specifically, at 1-L bioreactor, the absence of N2 infusion at high initial glycerol concentration induced lactate dehydrogenase activity and thus lactic acid synthesis, probably due to partial blockage of phosphoroclastic reaction caused by insufficient self-generated anaerobiosis environment. During fed-batch cultivation with continual N2 sparging, the strain produced ～71 g L(-1) of 1,3-propanediol, whereas under self-generated anaerobiosis, 1,3-propanediol pathway was evidently restricted, as only 30.5 g L(-1) of 1,3-propanediol were finally produced. Apparently, N2 infusion strategy paired with bioreactor geometry can alter the biochemical behavior of the particular strain.     

Construction of glycerol synthesis pathway in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> for bioconversion of glucose into 1,3-propanediol

1,3-Propanediol (1,3-PDO) is an important three-carbon compound widely used in new polyester polymer materials. Natural organisms that can produce 1,3-PDO from glycerol were well studied. However, no natural microorganisms found could directly convert glucose to 1,3-PDO due to its insufficient glycerol synthesis pathway. In this study, two essential glycerol synthesis genes, CgGPD gene (encoding glycerol-3-phosphate dehydrogenase from Candida glycerinogenes) and ScGPP2 gene (encoding glycerol-3-phosphatase from Saccharomyces cerevisiae), were expressed in wild-type Klebsiella pneumoniae, a natural 1,3-PDO producers with reduction pathway for 1,3-PDO synthesis from glycerol. The results of fermentation, key enzyme activities, and metabolites analysis confirmed that recombinant K. pneumoniae now possessed a metabolic pathway capable of converting glucose to 1,3-PDO. The strain could produce 1,3-PDO from glucose with a final titer of 17.27 g/L with 40 g/L glucose in the medium, showing a 1.26-fold increase compared with 30 g/L glucose. Also, adding certain concentrations of glycerol could quickly initiate the 1,3-PDO synthetic pathway and promote the accumulation of 1,3-PDO, which could shorten the fermentation cycle. These results have important implications for further studies involving the use of one strain for bioconversion of glucose to 1,3-PDO.     

Efficient production of 1,3-propanediol from fermentation of crude glycerol with mixed cultures in a simple medium

The objective of this study was to examine the applicability of mixed cultures for 1,3-propanediol (1,3-PDO) production from crude glycerol. Three different sources of mixed cultures were tested, where the mixed culture from a municipal wastewater treatment plant showed the best results. 1,3-PDO can be produced as the main product in this mixed culture with typical organic acids like acetic and butyric acids as by-products. The yield was in the range of 0.56–0.76 mol 1,3-PDO per mol glycerol consumed depending on the glycerol concentration. A final product concentration as high as 70 g/L was obtained in fed-batch cultivation with a productivity of 2.6 g/L h. 1,3-PDO can be kept in the culture several days after termination of the fermentation without being degraded. Degradation tests showed that 1,3-PDO is degraded much slower than other compounds in the fermentation broth. In comparison to 1,3-PDO production in typical pure cultures, the process developed in this work with a mixed culture achieved the same levels of product titer, yield and productivity, but has the decisive advantage of operation under complete non-sterile conditions. Moreover, a defined fermentation medium without yeast extract can be used and nitrogen gassing can be omitted during cultivation, leading to a strong reduction of investment and production costs.     

Continuous production of 1,3-propanediol using raw glycerol with immobilized Clostridium beijerinckii NRRL B-593 in comparison to suspended culture

The continuous production of 1,3-propanediol (1,3-PDO) was investigated with Clostridium beijerinckii NRRL B-593 using raw glycerol without purification obtained from a biodiesel production process. Ceramic rings and pumice stones were used for cell immobilization in a packed-bed bioreactor. For comparison purpose, a control bioreactor with suspended culture was also run. The effect of hydraulic retention time (HRT) on the production of 1,3-PDO in both immobilized and suspended bioreactors were also investigated. The study revealed that HRT is an important factor for both immobilized and suspended systems and a HRT of 2 h is the best one in terms of volumetric production rate (g 1,3-PDO/L/h). Furthermore, cell immobilization had also obvious benefits especially for the robustness and the reliability of the production. The results indicated that cell immobilization achieved a 2.5-fold higher productivity in comparison to suspended cell system. Based on our results, continuous production of 1,3-PDO with immobilized cells is an efficient method, and raw glycerol can be utilized without any pretreatment.     

Systems metabolic engineering of Corynebacterium glutamicum for high-level production of 1,3-propanediol from glucose and xylose

Corynebacterium glutamicum is a versatile chassis which has been widely used to produce various amino acids and organic acids. In this study, we report the development of an efficient C. glutamicum strain to produce 1,3-propanediol (1,3-PDO) from glucose and xylose by systems metabolic engineering approaches, including (1) construction and optimization of two different glycerol synthesis modules; (2) combining glycerol and 1,3-PDO synthesis modules; (3) reducing 3-hydroxypropionate accumulation by clarifying a mechanism involving 1,3-PDO re-consumption; (4) reducing the accumulation of toxic 3-hydroxypropionaldehyde by pathway engineering; (5) engineering NADPH generation pathway and anaplerotic pathway. The final engineered strain can efficiently produce 1,3-PDO from glucose with a titer of 110.4 g/L, a yield of 0.42 g/g glucose, and a productivity of 2.30 g/L/h in fed-batch fermentation. By further introducing an optimized xylose metabolism module, the engineered strain can simultaneously utilize glucose and xylose to produce 1,3-PDO with a titer of 98.2 g/L and a yield of 0.38 g/g sugars. This result demonstrates that C. glutamicum is a potential chassis for the industrial production of 1,3-PDO from abundant lignocellulosic feedstocks.     

Bioconversion of glycerol to 1,3-propanediol in thin stillage-based media by engineered Lactobacillus panis PM1

Thin stillage (TS) is a waste residue that remains after bioethanol production, and its disposal reflects the high costs of bioethanol production. Thus, the development of cost-effective ways to process TS is a pending issue in bioethanol plants. The aim of this study was to evaluate the utilization of TS for the production of the valuable chemical, 1,3-propanediol (1,3-PDO), by Lactobacillus panis PM1. Different fermentation parameters, including temperature, pH and strains [wild-type and a recombinant strain expressing a NADPH-dependent aldehyde reductase (YqhD) gene] were tested in batch and fed-batch cultivations. The highest 1,3-PDO concentration (12.85 g/L) and yield (0.84 g/g) were achieved by batch fermentation at pH-4.5/30 °C by the YqhD recombinant strain. Furthermore, pH-controlled batch fermentation reduced the total fermentation period, resulting in the maximal 1,3-PDO concentration of 16.23 g/L and yield of 0.72 g/g in TS without an expensive nutrient or nitrogen (e.g., yeast extract, beef extract, and peptone) supplementation. The addition of two trace elements, Mg²⁺ and Mn²⁺, in TS increased 1,3-PDO yield (0.74 g/g) without 3-hydroxypropionaldehyde production, the only intermediate of 1,3-PDO biosynthetic pathway in L. panis PM1. Our results suggest that L. panis PM1 can offer a cost-effective process that utilizes the TS to produce a value-added chemical, 1,3-PDO.     

Development of recombinant <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> ∆<Emphasis Type="Italic">dhaT</Emphasis> strain for the co-production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol

Klebsiella pneumoniae converts glycerol to the specialty chemical 1,3-propanediol (1,3-PDO), which is used for the production of polytrimethylene terepthalate (PTT). In this study, an NAD⁺-dependent gamma-glutamyl-gamma-aminobutyraldehyde dehydrogenase (PuuC) of K. pneumoniae DSM 2026, which oxidizes 3-hydroxypropionaldehyde to a platform chemical 3-hydroxypropionic acid (3-HP), was cloned and overexpressed in K. pneumoniae DSM 2026 for the co-production of 3-HP and 1,3-PDO from glycerol. In addition, the gene dhaT, encoding NADH-dependent 1,3-propanediol oxidoreductase (1,3-PDOR), was deleted from the chromosome for the balanced production of 3-HP and 1,3-PDO. The recombinant K. pneumoniae ∆dhaT, expressing puuC, produced 3.6 g 3-HP and 3.0 g 1,3-PDO per liter with an average yield of 81% on glycerol carbon in shake flask culture under microaerobic conditions. When a fed-batch culture was carried out under microaerobic conditions at pH 7.0 in a 5-l bioreactor, the recombinant K. pneumoniae ∆dhaT (puuC) strain produced 16.0 g 3-HP and 16.8 g 1,3-PDO per liter with a cumulative yield of 51% on glycerol carbon in 24 h. The production of 1,3-PDO in the dhaT-deletion mutant was attributed to the expression of NAD(P)H-dependent hypothetical oxidoreductase. This study demonstrates the feasibility of obtaining two commercially valuable chemicals, 3-HP and 1,3-PDO, at a significant scale.     

Production of 1,3-Propanediol from Glucose by Recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> BL21(DE3)

A range of recombinant strains of Escherichia coli were developed to produce 1,3-propanediol (1,3-PDO), an important C3 diol, from glucose. Two modules, the glycerol-producing pathway converting dihydroxyacetone phosphate to glycerol and the 1,3-PDO-producing pathway converting glycerol to 1,3-PDO, were introduced into E. coli. In addition, to avoid oxidative assimilation of the produced glycerol, glycerol oxidative pathway was deleted. Furthermore, to enhance the carbon flow to the Embden- Meyerhof-Parnas pathway, the Entner-Doudoroff pathway was disrupted by deleting 6-phosphogluconate dehydratase and 2-keto-3-deoxy-6-phosphogluconate aldolase. Finally, the acetate production pathway was removed to minimize the production of acetate, a major and toxic by-product. Flask experiments were carried out to examine the performance of the developed recombinant E. coli. The best strain could produce 1,3-PDO with a yield of 0.47 mol/mol glucose. Along with 1,3-PDO, glycerol was produced with a yield of 0.33 mol/mol glucose.     

Isolation and characterization of the new <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> J2B strain showing improved growth characteristics with reduced lipopolysaccharide formation

Klebsiella pneumoniae is a suitable biocatalyst for the production of 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid (3-HP) from glycerol. However, its commercial applications have been impeded due to its poor growth characteristics and the excessive production of lipopolysaccharide (LPS). To overcome these limitations, a new K. pneumoniae J2B (KpJ2B) strain was isolated from municipal waste anaerobic digester samples. The shake flask cultivation of this new strain under aerobic conditions showed a specific growth rate of 0.92/h, which is 1.13 times higher than that achieved using the well studied K. pneumoniae DSMZ2026 (KpDSMZ). When the new strain was grown in a bioreactor under aerobic conditions using a fed-batch mode for 36 h, the biomass concentration (4.03 g/L CDW) and productivity (0.15 g/L/h) were almost 2.2 times higher than the corresponding values with KpDSMZ. Growth was accompanied by the production of 1,3-PDO (186 mM), lactic acid (235 mM), ethanol (170 mM), and acetic acid (92.2 mM) at significant levels, indicating the resistance of the strain to the inhibitory effects of these metabolites. A comparison of the SEM images and 2-keto-3-deoxyoctonate content (KpJ2B, 1.4 μg/g CDW; KpDSMZ, 1.9 μg/g CDW) confirmed the lower LPS content in the KpJ2B strain. Furthermore, this new isolate exhibited higher sensitivity towards a range of antibiotics and better sedimentation properties than the KpDSMZ strain. This suggests that KpJ2B is an attractive strain for industrial applications.     

Human faecal microbiota display variable patterns of glycerol metabolism

Significant amounts of glycerol reach the colon microbiota daily through the diet and/or by in situ microbial production or release from desquamated epithelial cells. Some gut microorganisms may anaerobically reduce glycerol to 1,3-propanediol (1,3-PDO), with 3-hydroxypropanal as an intermediate. Accumulation of the latter intermediate may result in the formation of reuterin, which is known for its biological activity (e.g. antimicrobial properties). To date, glycerol metabolism in mixed cultures from the human colon has received little attention. Using in vitro batch incubations of faeces from 10 human individuals, we demonstrated that glycerol addition (140 mM) significantly affects the metabolism and composition of the microbial community. About a third of the samples exhibited rapid glycerol conversion, yielding proportionally higher levels of acetate and 1,3-PDO. In contrast, a slower glycerol metabolism resulted in higher levels of propionate. Furthermore, rapid glycerol metabolism correlated with significant shifts in the Lactobacillus-Enterococcus community, which were not observed in slower glycerol-metabolizing samples. As the conversion of glycerol to 1,3-PDO is a highly reducing process, we infer that the glycerol metabolism may act as an effective hydrogen sink. Given the importance of hydrogen-consuming processes in the gut, this work suggests that glycerol may have potential as a tool for modulating fermentation kinetics and profiles in the gastrointestinal tract.     

Development of Klebsiella pneumoniae J2B as microbial cell factory for the production of 1,3-propanediol from glucose

1,3-Propanediol (1,3-PDO) is an important platform chemical which has a wide application in food, cosmetics, pharmaceutical and textile industries. Its biological production using recombinant Escherichia coli with glucose as carbon source has been commercialized by DuPont, but E. coli cannot synthesize coenzyme B₁₂ which is an essential and expensive cofactor of glycerol dehydratase, a core enzyme in 1,3-PDO biosynthesis. This study aims to develop a more economical microbial cell factory using Klebsiella pneumoniae J2B which can naturally synthesize coenzyme B₁₂. To this end, the heterologous pathway for the production of glycerol from dihydroxyacetone-3-phosphate (DHAP), a glycolytic intermediate, was introduced to J2B and, afterwards, the strain was extensively modified for carbon and energy metabolisms including: (i) removal of carbon catabolite repression, (ii) blockage of glycerol export across the cell membrane, (iii) improvement of NADH regeneration/availability, (iv) modification of TCA cycle and electron transport chain, (v) overexpression of 1,3-PDO module enzyme, and (vi) overexpression of glucose transporter. A total of 33 genes were modified and/or overexpressed, and one resulting strain could produce 814 mM (62 g/L) of 1,3-PDO with the yield of 1.27 mol/mol glucose in fed-batch bioreactor culture with a limited supplementation of coenzyme B₁₂ at 4 μM, which is ~10 fold less than that employed by DuPont. This study highlights the importance of balanced use of glucose in the production of carbon backbone of the target chemical (1,3-PDO) and regeneration of reducing power (NADH). This study also suggests that K. pneumoniae J2B is a promising host for the production of 1,3-PDO from glucose.     

以甘油为底物利用重组大肠杆菌生产聚3-羟基丙酸

【目的】解决前期研究中所构建的以甘油为底物合成聚3-羟基丙酸(P3HP)的代谢途径中存在两个主要的问题——细胞内还原力不平衡和质粒丢失,以提高P3HP的产量。【方法】克隆来源于肺炎克雷伯氏菌的1,3-丙二醇(1,3-PDO)氧化还原酶基因,构建P3HP和1,3-PDO联产的菌株,解决细胞内还原力不平衡的问题。利用自杀性载体系统介导的同源重组技术,将甘油脱水酶及其激活因子的基因整合到大肠杆菌基因组中,提高质粒的稳定性。同时,对发酵条件进行优化。【结果】菌种改造和发酵条件优化显著提高了P3HP产量,在摇瓶条件下到达2.7 g/L,比以前的报道提高2倍,并可同时得到2.4 g/L 1,3-PDO。【结论】该重组大肠杆菌合成P3HP的产量得到提高,具有较好的工业化生产前景。     

High production of 1,3-propanediol from industrial glycerol by a newly isolated Clostridium butyricum strain

Batch and continuous cultures of a newly isolated Clostridium butyricum strain were carried out on industrial glycerol, the major by-product of the bio-diesel production process. For both types of cultures, the conversion yield obtained was around 0.55 g of 1,3-propanediol formed per 1 g of glycerol consumed whereas the highest 1,3-propanediol concentration, achieved during the single-stage continuous cultures was 35-48 g l-1. Moreover, the strain presented a strong tolerance at the inhibitory effect of the 1,3-propanediol, even at high concentrations of this substance at the chemostat (e.g. 80 g l-1). 1,3-Propanediol was associated with cell growth whereas acetate and butyrate seemed non growth-associated products. At low and medium dilution rates (until 0.1 h-1), butyrate production was favoured, whereas at higher rates acetate production increased. The maximum 1,3-propanediol volumetric productivity obtained was 5.5 g l-1 h-1. A two-stage continuous fermentation was also carried out. The first stage presented high 1,3-propanediol volumetric productivity, whereas the second stage (with a lower dilution rate) served to further increase the final product concentration. High 1,3-propanediol concentrations were achieved (41-46 g l-1), with a maximum volumetric productivity of 3.4 g l-1 h-1. A cell concentration decrease was reported between the second and the first fermentor.     

Converting crude glycerol to 1,3-propandiol using resting and immobilized Klebsiella sp. HE-2 cells

In this study, 1,3-propanediol (1,3-PDO) was produced from crude glycerol through the fermentation of resting and immobilized cells of a Klebsieblla sp. HE-2 strain isolated from a hydrogen producing anaerobic sludge collected in Southern Taiwan. The Klebsieblla sp. HE-2 cells were first grown on a fermentation medium (FM medium). The medium was then switched to resting-cell medium (RC medium) tailored to improve the production of 1,3-PDO. Using a glycerol-amended FM medium, the soluble metabolites consisted of 1,3-PDO, 2,3-butanediol, and ethanol and byproducts (such as acetic acid and lactic acid) at a content of 18, 28, 49, and 5% (of total soluble metabolites), respectively. When the culture was transferred from the FM medium to the RC medium, the concentration of 1,3-PDO was doubled from 5 g/L to 10 g/L. Using immobilized cells of Klebsieblla sp. HE-2 greatly improved the operational stability and reusability of the cells, as the immobilized cells could be used for 6 cycles without significant activity loss. The immobilized cells were able to directly utilize non-pretreated crude glycerol obtained from a local biodiesel manufacturing plant for 1,3-PDO production with an efficiency comparable to that obtained from using pure glycerol.     

Simultaneous production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol by a recombinant strain of Klebsiella pneumoniae

In this study, an aldehyde dehydrogenase (ALDH) was over-expressed in Klebsiella pneumoniae for simultaneous production of 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO). Various genes encoding ALDH were cloned and expressed in K. pneumoniae, and expression of Escherichia colialdH resulted in the highest 3-HP titer in anaerobic cultures in shake flasks. Anaerobic fed-batch culture of this recombinant strain was further performed in a 5-L reactor. The 3-HP concentration and yield reached 24.4 g/L and 0.18 mol/mol glycerol, respectively, and at the same time 1,3-PDO achieved 49.3 g/L with a yield of 0.43 mol/mol in 24 h. The overall yield of 3-HP plus 1,3-PDO was 0.61 mol/mol. Over-expression of the E. coli AldH also reduced the yields of by-products except for lactate. This study demonstrated the possibility of simultaneous production of 3-HP and 1,3-PDO by K. pneumoniae under anaerobic conditions without supply of vitamin B₁₂.     

Fermentation strategies for 1,3-propanediol production from glycerol using a genetically engineered Klebsiella pneumoniae strain to eliminate by-product formation

We generated a genetically engineered Klebsiella pneumoniae strain (AK-VOT) to eliminate by-product formation during production of 1,3-propanediol (1,3-PD) from glycerol. In the present study, the glycerol-metabolizing properties of the recombinant strain were examined during fermentation in a 5 L bioreactor. As expected, by-product formation was completely absent (except for acetate) when the AK-VOT strain fermented glycerol. However, 1,3-PD productivity was severely reduced owing to a delay in cell growth attributable to a low rate of glycerol consumption. This problem was solved by establishing a two-stage process separating cell growth from 1,3-PD production. In addition, nutrient co-supplementation, especially with starch, significantly increased 1,3-PD production from glycerol during fed-batch fermentation by AK-VOT in the absence of by-product formation.     

Optimization of medium compositions favoring butanol and 1,3-propanediol production from glycerol by Clostridium pasteurianum

Medium compositions favoring butanol and 1,3-propanediol (1,3-PDO) production from glycerol by Clostridium pasteurianum DSM525 were investigated using statistical experimental designs. Medium components affecting butanol and 1,3-PDO production were screened using a fractional factorial experimental design. Among the six tested variables (phosphate buffer, MnSO4·H2O, MgSO4·7H2O, FeSO4·7H2O, (NH4)2SO4, and yeast extract), FeSO4·7H2O, (NH4)2SO4, and yeast extract were found to be significant variables for further optimization of medium using a Box-Behnken design. Optimal butanol (0.98 g/L/h) and 1,3-PDO (1.19 g/L/h) productivities were predicted by the corresponding quadratic model for each product and the models were validated experimentally under optimized conditions. The optimal medium composition for butanol production was significantly different from that for 1,3-PDO production (0.06 vs. 0 g/L for FeSO4·7H2O, 7.35 vs. 0 g/L for (NH4)2SO4, and 5.08 vs. 8.0 g/L for yeast extract), suggesting that the product formation from glycerol by C. pasteurianum DSM525 can be controlled by changing medium compositions.     

Optimization of growth and production of toxins by three dinoflagellates in photobioreactor cultures

A bioreactor system for biotoxin production was appraised against traditional methods of growing dinoflagellate cultures. In an optimised bioreactor culture (5.4?L) operated in batch mode, growth of Karenia selliformis was more efficient than in 15-L bulk carboy culture in terms of growth rate (μ?=?0.07?day^?1 versus 0.05?day^?1) and growth maximum (G _max, 169.10⁶ versus 41.10⁶ cells L^?1). Maximal gymnodimine concentration (1200?μg L^?1) in bioreactor culture was 8-fold higher than in bulk carboy culture, and the yield per cell (pg cell^?1) was 2-fold higher. Similarly the bioreactor batch culture of Alexandrium ostenfeldii performed more efficiently than carboy cultures in terms of growth rate (1.6-fold higher), growth maximum (15-fold higher) and desmethyl C spirolide (SPX-desMe-C) yield (5-fold higher [μg L^?1], though the yield [pg cell^?1basis] was lower). When bioreactor cultures of K. selliformis were operated in continuous mode, the yield of gymnodimine was substantially higher than a carboy or the bioreactor run in batch mode to growth max (793?μg day^?1 over 58?days in continuous culture was achieved versus an average of 60?μg day^?1 [carboy over 40?days] or 249?μg day^?1 [batch mode] over 26?days). Likewise in continuous bioreactor cultures of A. ostenfeldii run over 25?days, the yield of SPX-desMe-C (29?μg day^?1) was substantially higher than in same cultures run in batch mode or carboys (10.2 day^?1 and 7.7?μg day^?1 respectively). Similarly 5.4?L bioreactor batch cultures of K. brevisulcata reached 3.8-fold higher cell densities than carboy cultures, and when operated in continuous mode, the brevisulcatic acids were more efficiently produced than in batch culture (12?μg day^?1 versus 7?μg day^?1). When the bioreactor system was upscaled to 52?L, the maximum cell densities and toxin yields of K. brevisulcata cultures were somewhat less than those achieved in the smaller reactor, which was attributed to reduced light penetration.     

Improved n-butanol production by a non-acetone producing Clostridium pasteurianum DSMZ 525 in mixed substrate fermentation

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.