Zhongping Shi

Yeast extract promotes phase shift of bio-butanol fermentation by Clostridium acetobutylicum ATCC824 using cassava as substrate.

When fermenting on cassava (15-25%, w/v) with Clostridium acetobutylicum ATCC824, a severe delay (18-40 h) was observed in the phase shift from acidogenesis to solventogenesis, compared to the cases of fermenting on corn. By adding yeast extract (2.5 g/L-broth) into cassava meal medium when the delay appeared, the phase shift was triggered and fermentation performances were consequently improved. Total butanol concentrations/butanol productivities, compared to those with cassava substrate alone, increased 15%/80% in traditional fermentation while 86%/79% in extractive fermentation using oleyl alcohol as the extractant, and reached the equivalent levels of those using corn substrate. Analysis of genetic transcriptional levels and measurements of free amino acids in the broth demonstrated that timely and adequate addition of yeast extract could promote phase shift by increasing transcriptional level of ctfAB to 16-fold, and indirectly enhance butanol synthesis through accelerating the accumulation of histidine and aspartic acid families.

Enhancing acetone biosynthesis and acetone-butanol-ethanol fermentation performance by co-culturing Clostridium acetobutylicum/Saccharomyces cerevisiae integrated with exogenous acetate addition.

Acetone is the major by-product in ABE fermentations, most researches focused on increasing butanol/acetone ratio by decreasing acetone biosynthesis. However, economics of ABE fermentation industry strongly relies on evaluating acetone as a valuable platform chemical. Therefore, a novel ABE fermentation strategy focusing on bio-acetone production by co-culturing Clostridium acetobutylicum/Saccharomyces cerevisiae with exogenous acetate addition was proposed. Experimental and theoretical analysis revealed the strategy could, enhance C. acetobutylicum survival oriented amino acids assimilation in the cells; control NADH regeneration rate at moderately lower level to enhance acetone synthesis but without sacrificing butanol production; enhance the utilization ability of C. acetobutylicum on glucose and direct most of extra consumed glucose into acetone/butanol synthesis routes. By implementing the strategy using synthetic or acetate fermentative supernatant, acetone concentrations increased to 8.27-8.55g/L from 5.86g/L of the control, while butanol concentrations also elevated to the higher levels of 13.91-14.23g/L from 11.63g/L simultaneously.

Evaluation of high butanol/acetone ratios in ABE fermentations with cassava by graph theory and NADH regeneration analysis

Higher butanol/acetone ratio is always desirable in ABE fermentation, and this ratio is closely associated with the complicated patterns of metabolic reactions and NADH generation rate. The patterns of acetate/butyrate formation and re-assimilation in multiple closed reaction loops, as well as NADH regeneration in ABE fermentation using different substrates varies. In this study, we evaluated butanol/acetone ratio in ABE fermentations utilizing cassava and corn based media by graph theory and NADH regeneration analysis. The theoretical calculations and experimental data revealed that a lower metabolic strength in butyrate loop and enhanced NADH generation rate were responsible for the achievement of higher butanol/acetone ratio when fermenting cassava based substrate. In traditional fermentations and extractive fermentations with oleyl alcohol/bio-diesel as the extractants when using cassava based substrate, butanol/acetone ratios reached 2.24, 2.84, and 2.19 with the increasing increments of 14.9, 61.4, and 6.8% respectively, while butanol productivities stayed at comparably high levels as compared with those of the fermentations when cultivating on corn based substrate.

Enhancing Butanol Production under the Stress Environments of Co-Culturing Clostridium acetobutylicum/Saccharomyces cerevisiae Integrated with Exogenous Butyrate Addition

In this study, an efficient acetone-butanol-ethanol (ABE) fermentation strategy integrating Clostridium acetobutylicum/Saccharomyces cerevisiae co-culturing system with exogenous butyrate addition, was proposed and experimentally conducted. In solventogenic phase, by adding 0.2 g-DCW/L-broth viable S. cerevisiae cells and 4.0 g/L-broth concentrated butyrate solution into C. acetobutylicum culture broth, final butanol concentration and butanol/acetone ratio in a 7 L anaerobic fermentor reached the highest levels of 15.74 g/L and 2.83 respectively, with the increments of 35% and 43% as compared with those of control. Theoretical and experimental analysis revealed that, the proposed strategy could, 1) extensively induce secretion of amino acids particularly lysine, which are favorable for both C. acetobutylicum survival and butanol synthesis under high butanol concentration environment; 2) enhance the utilization ability of C. acetobutylicum on glucose and over-produce intracellular NADH for butanol synthesis in C. acetobutylicum metabolism simultaneously; 3) direct most of extra consumed glucose into butanol synthesis route. The synergetic actions of effective amino acids assimilation, high rates of substrate consumption and NADH regeneration yielded highest butanol concentration and butanol ratio in C. acetobutylicum under this stress environment. The proposed method supplies an alternative way to improve ABE fermentation performance by traditional fermentation technology.

Enhanced porcine circovirus Cap protein production by Pichia pastoris with a fuzzy logic DO control based methanol/sorbitol co-feeding induction strategy

Porcine circovirus Cap protein production by P. pastoris with strong AOX promoter suffered with the problems with traditional pure methanol induction: (1) inefficient methanol metabolism; (2) extensive oxygen supply load; (3) difficulty in stable DO control; (4) low protein titer. In this study, based on the difference of DO change patterns in response to methanol and sorbitol additions, a novel fuzzy control system was proposed to automatically regulate the co-feeding rates of methanol and sorbitol for efficient Cap protein induction. With aid of the proposed control system when setting DO control level at 10%, overall fermentation performance was significantly improved: (1) DO could be stably controlled under mild aeration condition; (2) methanol consumption rate could be restricted at moderate level and the major enzymes involved with methanol metabolism were largely activated; (3) Cap protein concentration reached a highest level of 198mg/L, which was about 64% increase over the best one using the pure methanol induction strategies.

Simulation of computational fluid dynamics and comparison of cephalosporin C fermentation performance with different impeller combinations

Cephalosporin C (CPC) fermentation by Acremonium chrysogenum is an extremely high oxygen-consuming process and oxygen transfer rate in a bioreactor directly affects fermentation performance. In this study, fluid dynamics and oxygen transfer in a 7 L bioreactor with different impellers combinations were simulated by computational fluid dynamics (CFD) model. Based on the simulation results, two impeller combinations with higher oxygen transfer rate (KLa) were selected to conduct CPC fermentations, aiming at achieving high CPC concentration and low accumulation of major by-product, deacetoxycephalosporin (DAOC). It was found that an impeller combination with a higher KLa and moderate shear force is the prerequisite for efficient CPC production in a stirred bioreactor. The best impeller combination, which installed a six-bladed turbine and a four-pitched-blade turbine at bottom and upper layers but with a shortened impellers inter-distance, produced the highest CPC concentration of 35.77 g/L and lowest DAOC/CPC ratio of 0.5%.

Inhibitory effects of recombinant porcine interferon-α on high- and low-virulence porcine reproductive and respiratory syndrome viruses.

The inhibitory effects of recombinant porcine interferon alpha (rPoIFN-α) on the propagation of low-virulence PRRSV (lvPRRSV) in MARC-145 cells, and on the progress and severity of high virulence PRRSV (hvPRRSV)-induced infections in pigs, were determined. Pre-treatment of MARC-145 cells with increasing concentrations of rPoIFN-α prior to infection with lvPRRSV decreased the observed cytopathic effects (CPEs) in a concentration-dependent manner. Viral propagation and antibody response were temporarily delayed in swine treated with rPoIFN-α either at the same time as the hvPRRSV challenge was administered or post-challenge. Exposure of challenged animals to rPoIFN-α after the onset of disease symptoms alleviated associated hyperthermia. Variations in lymphocyte subsets indicated that rPoIFN-α treatment might alleviate damage to the immune system or enhance propagation of host cytotoxic T-lymphocytes when the treatment was applied simultaneously with the virus or 1dpc, respectively.

Improved production of 2,5-furandicarboxylic acid by overexpression of 5-hydroxymethylfurfural oxidase and 5-hydroxymethylfurfural/furfural oxidoreductase in Raoultella ornithinolytica BF60

2,5-Furandicarboxylic acid (FDCA) is a promising bio-based building block and can be produced by biotransformation of 5-hydroxymethylfurfural (HMF). To improve the FDCA production, two genes-one encoding HMF oxidase (HMFO; from Methylovorus sp. strain MP688) and another encoding for HMF/Furfural oxidoreductase (HmfH; from Cupriavidus basilensis HMF14)-were introduced into Raoultella ornithinolytica BF60. The FDCA production in the engineered whole-cell biocatalyst increased from 51.0 to 93.6mM, and the molar conversion ratio of HMF to FDCA increased from 51.0 to 93.6%.

Electron receptor addition enhances butanol synthesis in ABE fermentation by Clostridium acetobutylicum

The techniques for enhancing butanol production in ABE fermentation by Clostridium acetobutylicum generally focus on adding electron carrier to strengthen NADH synthesis, repressing hydrogenase by aerating CO, supplementing butyrate, etc. However, those methods suffer from the problems of total solvent decrease, high purification cost, using expensive supplemental substances, etc. In this study, we added small amount of electron receptors (Na2SO4/CaSO4, 2g/L) into ABE fermentation broth: to alter electron/proton distributions in the intracellular electron transport shuttle system, directing more electron/proton pairs into NADH synthesis route; to stimulate intracellular accumulation of those amino acids favorable for cells survival/butanol synthesis. In ABE fermentation in a 7L fermentor, adding 2g/L Na2SO4 could raise butanol concentration to a higher level of 12.96g/L, which was 34.8% higher than that of the control. Addition of tiny amount cheap electron receptor would provide a new way to enhance bio-butanol production.

Models construction for acetone–butanol–ethanol fermentations with acetate/butyrate consecutively feeding by graph theory

Several fermentations with consecutively feeding of acetate/butyrate were conducted in a 7 L fermentor and the results indicated that exogenous acetate/butyrate enhanced solvents productivities by 47.1% and 39.2% respectively, and changed butyrate/acetate ratios greatly. Then extracellular butyrate/acetate ratios were utilized for calculation of acids rates and the results revealed that acetate and butyrate formation pathways were almost blocked by corresponding acids feeding. In addition, models for acetate/butyrate feeding fermentations were constructed by graph theory based on calculation results and relevant reports. Solvents concentrations and butanol/acetone ratios of these fermentations were also calculated and the results of models calculation matched fermentation data accurately which demonstrated that models were constructed in a reasonable way.