Metabolism, morphology and transcriptome analysis of oscillatory behavior of Clostridium butyricum during long-term continuous fermentation for 1,3-propanediol production.

BACKGROUND: Oscillation is a special cell behavior in microorganisms during continuous fermentation, which poses threats to the output stability for industrial productions of biofuels and biochemicals. In previous study, a spontaneous oscillatory behavior was observed in Clostridium butyricum-intensive microbial consortium in continuous fermentation for 1,3-propanediol (1,3-PDO) production from glycerol, which led to the discovery of oscillation in species C. butyricum. RESULTS: Spontaneous oscillations by C. butyricum tended to occur under glycerol-limited conditions at low dilution rates. At a glycerol feed concentration of 88 g/L and a dilution rate of 0.048 h-1, the oscillatory behavior of C. butyricum was observed after continuous operation for 146 h and was sustained for over 450 h with an average oscillation period of 51 h. During oscillations, microbial glycerol metabolism exhibited dramatic periodic changes, in which productions of lactate, formate and hydrogen significantly lagged behind that of other products including biomass, 1,3-PDO and butyrate. Analysis of extracellular oxidation-reduction potential and intracellular ratio of NAD+/NADH indicated that microbial cells experienced distinct redox changes during oscillations, from oxidized to reduced state with decreasing of growth rate. Meanwhile, C. butyricum S3 exhibited periodic morphological changes during oscillations, with aggregates, elongated shape, spores or cell debris at the trough of biomass production. Transcriptome analysis indicated that expression levels of multiple genes were up-regulated when microbial cells were undergoing stress, including that for pyruvate metabolism, conversion of acetyl-CoA to acetaldehyde as well as stress response. CONCLUSION: This study for the first time systematically investigated the oscillatory behavior of C. butyricum in aspect of occurrence condition, metabolism, morphology and transcriptome. Based on the experimental results, two hypotheses were put forward to explain the oscillatory behavior: disorder of pyruvate metabolism, and excessive accumulation of acetaldehyde.

Sequential fed-batch fermentation of 1,3-propanediol from glycerol by Clostridium butyricum DL07.

The demand for 1,3-propanediol (1,3-PDO) has increased sharply due to its role as a monomer for the synthesis of polytrimethylene terephthalate (PTT). Although Clostridium butyricum is considered to be one of the most promising bioproducers for 1,3-PDO, its low productivity hinders its application on industrial scale because of the longer time needed for anaerobic cultivation. In this study, an excellent C. butyricum (DL07) strain was obtained with high-level titer and productivity of 1,3-PDO, i.e., 104.8 g/L and 3.38 g/(L•h) vs. 94.2 g/L and 3.04 g/(L•h) using pure or crude glycerol as substrate in fed-batch fermentation, respectively. Furthermore, a novel sequential fed-batch fermentation was investigated, in which the next bioreactor was inoculated by C. butyricum DL07 cells growing at exponential phase in the prior bioreactor. It could run steadily for at least eight cycles. The average concentration of 1,3-PDO in eight cycles was 85 g/L with the average productivity of 3.1 g/(L•h). The sequential fed-batch fermentation could achieve semi-continuous production of 1,3-PDO with higher productivity than repeated fed-batch fermentation and would greatly contribute to the industrial production of 1,3-PDO by C. butyricum. KEY POINTS: • A novel C. butyricum strain was screened to produce 104.8 g/L 1,3-PDO from glycerol. • Corn steep liquor powder was used as a cheap nitrogen source for 1,3-PDO production. • A sequential fed-batch fermentation process was established for 1,3-PDO production. • An automatic glycerol feeding strategy was applied in the production of 1,3-PDO.

Stability and oscillatory behavior of microbial consortium in continuous conversion of crude glycerol to 1,3-propanediol.

Microbial consortium is an alternative for bioconversion of crude glycerol to value-added products whereas concerns about the process stability in long-term operation existed. The aim of this study is to evaluate the feasibility of using an anaerobic microbial consortium as inoculum for continuous conversion of crude glycerol to 1,3-propanediol (1,3-PDO). Performances of continuous fermentations with the consortium inoculum were evaluated under different dilution rates and glycerol feed concentrations. The highest 1,3-PDO production of 57.86 g/L was achieved with a productivity of 5.55 g/(L·h). Analyses of kinetic data showed that the consortium maintained a consistent pattern for 1,3-PDO production under different operating conditions despite changes in community composition. The continuous fermentation by the consortium was able to operate for a longer period of time (31 volume changes) than that using pure culture (24 volume changes) with the average 1,3-PDO concentration of 53.52 g/L and productivity of 6.69 g/(L·h) under glycerol-excess condition, which could be contributed to the intraspecies diversity among Clostridium butyricum in the consortium. Under glycerol-limited conditions, however, a spontaneous oscillation of the consortium was observed after continuous operation for about 120 h, along with severe fluctuations of the microbial community. The oscillatory behavior could be reduced by increasing the dilution rates and was likely the metabolic feature of C. butyricum.

Selection and characterization of an anaerobic microbial consortium with high adaptation to crude glycerol for 1,3-propanediol production.

Crude glycerol is an ideal feedstock for bioproduction of 1,3-propanediol (1,3-PDO) while pure culture always shows low substrate tolerance and limited productivity. In this study, an anaerobic microbial consortium for conversion of crude glycerol was selected and its 1,3-PDO production capacity was evaluated. The consortium was obtained from anaerobic activated sludge by 19 serial transfers and mainly consisted of 94.64% Clostridiaceae and 4.47% Peptostreptococcaceae. The consortium adapted well with high glycerol concentration of 120 g/L as well as wide substrate concentration fluctuation from 15 to 80 g/L, producing 60.61 and 82.66 g/L 1,3-PDO in the batch and fed-batch fermentation, with the productivity of 3.79 and 3.06 g/(L∙h), respectively, which are among the best results published so far. Furthermore, mini consortia isolated by serial dilution exhibited similar microbial composition but gradually decreasing tolerance to crude glycerol. Four randomly selected Clostridium butyricum displayed different substrate tolerance and insufficient 1,3-PDO production capacity. This work demonstrated that the high adaptation to crude glycerol of the consortium was the collaborative effort of different individuals.

Analytical performance of and real sample analysis with an HBV gene visual detection chip.

A novel hepatitis B virus (HBV) gene detection chip has been developed. The HBV-specific probes immobilized on glass slides were hybridized with polymerase chain reaction (PCR) products of different serum samples. The hybridization signal can be easily visualized upon a sandwich assay with nanoparticle amplification. The analytical performance (e.g., specificity, sensitivity, and accuracy) of this method has been evaluated. The chip-based detection method possesses a greater sensitivity and a better reproducibility than some of the conventional immunological or molecular biological methods (e.g., enzyme-linked immunosorbent assay, ELISA) and is simple, cost-effective, and highly selective.

Visual gene diagnosis of HBV and HCV based on nanoparticle probe amplification and silver staining enhancement.

A visual gene-detecting technique using nanoparticle-supported gene probes is described. With the aid of gold nanoparticle-supported 3'-end-mercapto-derivatized oligonucleotide serving as detection probe, and 5'-end -amino-derivatized oligonucleotide immobilized on glass surface acting as capturing probe, target DNA was detected visually by sandwich hybridization based on highly sensitive "nano-amplification" and silver staining. Different genotypes of Hepatitis B and C viruses in the serum samples from infected patients were detected using home-made HBV, HCV, and HBV/HCV gene chips by the gold/silver nanoparticle staining amplification method. The present visual gene-detecting technique may avoid limitations with the reported methods, for its high sensitivity, good specificity, simplicity, speed, and cheapness. This technique has potential applications in many fields, especially in multi-gene detection gene chips coupled with the detection will find applications in clinic. Additionally, resonance Rayleigh light scattering (RLS) spectroscopy is used, for the first time, to judge and monitor the immobilization of gene probes on gold nanoparticle surfaces.