Yaqin Sun

High tolerance to glycerol and high production of 1,3‐propanediol in batch fermentations by microbial consortium from marine sludge

1,3‐Propanediol (1,3‐PD) is a versatile bulk chemical and widely used as a monomer to synthesis polymers, such as polyesters, polyethers and polyurethanes. 1,3‐PD can be produced by microbial fermentation with the advantages of the environmental protection and sustainable development. Low substrate tolerance and wide by‐product profile limit microbial production of 1,3‐PD by Klebsiella pneumonia on industrial scale. In this study, microbial consortia were investigated to overcome some disadvantages of pure fermentation by single strain. Microbial consortium named DL38 from marine sludge gave the best performance. Its bacterial community composition was analyzed by 16S rRNA gene amplicon high‐throughput sequencing and showed that Enterobacteriaceae was the most abundant family. Compared with three K. pneumonia strains isolated from DL38, the microbial consortium could grow well at an initial glycerol concentration of 200 g/L to produce 81.40 g/L of 1,3‐PD with a yield of 0.63 mol/mol. This initial glycerol concentration is twice the highest concentration by single isolated strain and more than the critical value (188 g/L) extrapolated from the fermentation kinetics for K. pneumonia. On the other hand, a small amount of by‐products were produced in batch fermentation of microbial consortium DL38, especially no 2,3‐butanediol detected. The mixed culture of strain W3, Y5 and Y1 improved the tolerance to glycerol and changed the metabolite profile of single strain W3. The batch fermentation with the natural proportion (W3: Y5: Y1 = 208: 82: 17) was superior to that with other proportions and single strain. This study showed that microbial consortium DL38 possessed excellent substrate tolerance, narrow by‐product profile and attractive potential for industrial production of 1,3‐PD.

Nonlinear Mathematical Simulation and Analysis of Dha Regulon for Glycerol Metabolism in Klebsiella pneumoniae

Abstract Glycerol may be converted to 1,3-propanediol (1,3-PD) by Klebsiella pneumoniae under anaerobic conditions and glycerol dismutation involves two parallel pathways controlled by the dha regulon. In this study, a fourteen-dimensional nonlinear dynamic system is presented to describe the continuous culture and multiplicity analysis, in which two regulated negative-feedback mechanisms of repression and enzyme inhibition are investigated. The model describing the expression of gene-mRNA-enzyme-product was established according to the repression of the dha regulon by 3-hydroxypropionaldehy (3-HPA). Comparisons between simulated and experimental results indicate that the model can be used to describe the production of 1,3-PD under continuous fermentation. The new model is translated into the corresponding S-system version. The robustness of this model is discussed by using the S-system model and the sensitivity analysis shows that the model is sufficiently robust. The influences of initial glycerol concentration and dilution rate on the biosynthesis of 1,3-PD and the stability of the dha regulon model are investigated. The intracellular concentrations of glycerol, 1,3-PD, 3-HPA, repressor mRNA, repressor, mRNA and protein levels of glycerol dehydratase (GDHt) and 1,3-PD oxydoreductase (PDOR) can be predicted for continuous cultivation. The results of simulation and analysis indicate that 3-HPA accumulation will repress the expression of the dha regulon at the transcriptional level. This model gives new insights into the regulation of glycerol metabolism in K. pneumoniae and explain some of the experimental observations.

Partition behavior of hydrophilic diols in an ethanol/ammonium sulfate salting-out extraction system

It is important to investigate the partition behavior of hydrophilic alcohols at different process parameters in order to have a deep understanding of the salting‐out extraction technique and to design suitable downstream processes. First, phase diagram data of ethanol/ammonium sulfate at 293.15 K were obtained, and the reliability of binodal curve and tie line data were proved by Merchuk, Othmer–Tobias, and Bancroft equations, respectively. Then, the partition behavior of five short‐chain diols was studied. For different tie lines, the partition coefficient changed linearly with an increase in tie line length. The concentration of diols also had a significant effect on the partition behavior due to the similarity between diols and ethanol. Further, the effect of temperature was increased as the hydrophobicity of the diols increased, and the partition behavior of diols was correlated with their hydrophobicity, implying that the solutes with higher hydrophobicity could be extracted more effectively. These findings are useful for designing an economic and efficient salting‐out extraction process for diverse products.

Metabolic Flux Analysis of Bioconversion of Glycerol into 1,3-Propandiol by Klebsiella Pneumoniae

This study presents an analysis of metabolic fluxes of the anaerobic glycerol metabolism by Klebsiella pneumoniae for the production of 1,3-propanediol (1,3-PD). A metabolic network was reconstructed from the partly annotated genome sequence as well as from biochemical and physiological literatures. Extracellular fluxes measured in continuous culture under steady condition were used to estimate intracellular fluxes. A comparison of changes in the branch point of 3- hydroxypropionaldehyde (3-HPA) flux distribution reveals that glycerol dehydratase (GDHt) is the rate-limiting enzyme at high glycerol concentration and 1,3-propanediol oxidoreductase (PDOR) is the rate-limiting enzyme at low initial glycerol concentration for the production of 1,3-PD due to an insufficient supply of reducing equivalents.

基于傅里叶变换近红外光谱实时分析 1,3- 丙二醇发酵过程生物量的在线监测方法

Biomass is an important parameter reflecting the fermentation dynamics. Real-time monitoring of biomass can be used to control and optimize a fermentation process. To overcome the deficiencies of measurement delay and manual errors from offline measurement, we designed an experimental platform for online monitoring the biomass during a 1,3-propanediol fermentation process, based on using the fourier-transformed near-infrared (FT-NIR) spectra analysis. By pre-processing the real-time sampled spectra and analyzing the sensitive spectra bands, a partial least-squares algorithm was proposed to establish a dynamic prediction model for the biomass change during a 1,3-propanediol fermentation process. The fermentation processes with substrate glycerol concentrations of 60 g/L and 40 g/L were used as the external validation experiments. The root mean square error of prediction (RMSEP) obtained by analyzing experimental data was 0.341 6 and 0.274 3, respectively. These results showed that the established model gave good prediction and could be effectively used for on-line monitoring the biomass during a 1,3-propanediol fermentation process.

Robustness and Nonlinear Dynamic Analysis for Trp Operon and Optimization of Tryptophan Production: An Integrated Model Considering Gene Regulation, Genes Interaction and Product Excretion

The tryptophan operon (trp operon), paradigm for repressible operons, is regulated by three different negative-feedback mechanisms of repression, transcription attenuation and enzyme inhibition. Initiation of transcription is controlled by the interaction of the tryptophan repressor with its target site on the operator. Moreover, the interaction among the regulator genes, the operator genes and the structure genes and excretion of tryptophan play an important factor according to previous works. In this study, an expended mathematical model for the tryptophan operon regulation on the effects of repression, feedback enzyme inhibition, attenuation, interaction among genes and excretion of tryptophan is presented. The new model is first translated into the corresponding S-system version. The robustness of this model is then discussed by using the S-system model and the sensitivity analysis shows that the model is robust enough. The influences of cell growth rate on the biosynthesis of tryptophan, stability and dynamic behavior of the trp operon are also well investigated. The transportation of tryptophan through cytoplasmic membranes, especially the inhibition of tryptophan transport can influence the level of intracellular tryptophan significantly. The theoretical analysis indicates that an increase of inhibition constant of tryptophan transport is favorable for the biosynthesis of tryptophan. Furthermore, a steady-state optimization model is established based on trp operon models. The optimization results indicate that it is possible to attain a stable and robust steady state with a rate of tryptophan production increased more than 4.8 times in which the growth rate is kept as 0.00624h-1 and some key parameters is modulated.