Currently, biomanufacturing technology and industry are receiving worldwide attention. However, there are still great challenges on bioprocess optimization and scale-up, including: lacing the process detection methods, which makes it difficult to meet the requirement of monitoring of key indicators and parameters; poor understanding of cell metabolism, which arouses problems to rationally achieve process optimization and regulation; the reactor environment is very different across the scales, resulting in low efficiency of stepwise scale-up. Considering the above key issues that need to be resolved, here we summarize the key technological innovations of the whole chain of fermentation process, i.e., real-time detection-dynamic regulation-rational scale-up, through case analysis. In the future, bioprocess design will be guided by a full lifecycle in-silico model integrating cellular physiology (spatiotemporal multiscale metabolic models) and fluid dynamics (CFD models). This will promote computer-aided design and development, accelerate the realization of large-scale intelligent production and serve to open a new era of green biomanufacturing.
Bioreactors ; Computer Simulation ; Fermentation ; Hydrodynamics

A method for measuring 13C isotopic abundance of intracellular metabolites of Saccharopolysporaerythraea by ultra-high performance liquid chromatography (UPLC)-triple quadrupole mass spectrometry was established.First, the chromatographic conditions of UPLC were optimized, and then the MS conditions such as unique tube lens voltage, collision energy, and ion pair were optimized.On the bases of length of the parent and daughter ions carbon chains and whether the daughter ions contain 13C atoms, the one-to-one method, one-to-many method and SIM method were established for measuring 13C isotopic abundance.Then these methods were used to measure naturally labeled intracellular metabolite standards and 13C labeled samples, and according to the gap between the experimental value and the theoretical value, the best method was established for each metabolite of different characteristics.The results showed that one-to-one method was most effective for measuring the metabolites of daughter ions not containing 13C atoms represented by sugar phosphates, one-to-many method was the best for measuring the metabolites of both parent and daughter ions containing 13C short carbon chains represented by carboxylic acids, SIM method could play a role in measuring the metabolites of both parent and daughter ions containing 13C long carbon chains represented by coenzyme A.This method had a good measurement precision and could be applied to the measurement of Saccharopolysporaerythraea intracellular metabolites, which contributed to the consequent study of metabolic mechanism and the efficient expression of erythromycin.

Objective To explore the value of apolipoprotein B/A-1 ratio combined with carotid plaque nature examination in predicting the risk of patients with coronary heart disease.Methods A total of 250 patients with CHD were selected and divided into acute coronary syndrome group (ACS group,n=190) and stable angina pectoris group (SAP group,n=60).A total of 98 normal individuals by CAG were selected as control group.The carotid color ultrasound was performed for all the patients.The nature of carotid atherosclerostic plaque was estimated according to the acoustic characteristics of plaques.The ApoB and ApoA-1 contents as well as ApoB/A-1 ratio were detected and calculated,and correlations between indexes and risk factors were analyzed.Results There were no significant differences in levels of TC,TG,and LDL-C between the three groups (P>0.05).HDL-C level in the ACS group was significantly higher than that in the control group (P<0.05).ApoB and ApoB/A1 in the ACS group were significantly higher than those in the control group and ACS group (P<0.05).There was no significant difference in ApoA1 between the three groups (P>0.05).The total occurrence rates of plaques in ACS group and SAP group were significantly higher than the control group (P<0.05).The occurrence rate of carotid soft plaque in ACS group was significantly higher than SAP group and the control group (P<0.05).Conclusion Unstable plaques are able to detect by carotid ultrasound in CHD patients,and the ApoB level and ApoB/A-1 ratio increased abnormally,which indicates the severity of coronary artery lesions increases in CHD patients.

Objective To explore the value of apolipoprotein B/A-1 ratio combined with carotid plaque nature examination in predicting the risk of patients with coronary heart disease.Methods A total of 250 patients with CHD were selected and divided into acute coronary syndrome group (ACS group,n=190) and stable angina pectoris group (SAP group,n=60).A total of 98 normal individuals by CAG were selected as control group.The carotid color ultrasound was performed for all the patients.The nature of carotid atherosclerostic plaque was estimated according to the acoustic characteristics of plaques.The ApoB and ApoA-1 contents as well as ApoB/A-1 ratio were detected and calculated,and correlations between indexes and risk factors were analyzed.Results There were no significant differences in levels of TC,TG,and LDL-C between the three groups (P>0.05).HDL-C level in the ACS group was significantly higher than that in the control group (P<0.05).ApoB and ApoB/A1 in the ACS group were significantly higher than those in the control group and ACS group (P<0.05).There was no significant difference in ApoA1 between the three groups (P>0.05).The total occurrence rates of plaques in ACS group and SAP group were significantly higher than the control group (P<0.05).The occurrence rate of carotid soft plaque in ACS group was significantly higher than SAP group and the control group (P<0.05).Conclusion Unstable plaques are able to detect by carotid ultrasound in CHD patients,and the ApoB level and ApoB/A-1 ratio increased abnormally,which indicates the severity of coronary artery lesions increases in CHD patients.

A rapid and accurate determination method of lipids in microalgae plays a significant role in an efficient breeding process for high-lipid production of microalgae. Using low field nuclear magnetic resonance (LF-NMR), we developed a direct quantitative method for cellular lipids in Chlorella protothecoides cells. The LF-NMR signal had a linear relationship with the lipid content in the microalgae cells for both dry cell samples and algal broth samples (R2 > 0.99). These results indicated that we could use this method for accurate determination of microalgal lipids. Although LF-NMR is a rapid and easy lipid determination method in comparison to conventional methods, low efficiency would limit its application in high throughput screening. Therefore, we developed a novel combined high throughput screening method for high-lipid content mutants of C. protothecoides. Namely, we initially applied Nile red staining method for semi-quantification of lipid in the pre-screening process, and following with LF-NMR method for accurate lipid determination in re-screening process. Finally, we adopted this novel screening method in the breeding process of high-lipid content heterotrophic cells of C. protothecoides. From 3 098 mutated strains 108 high-lipid content strains were selected through pre-screening process, and then 9 mutants with high-lipid production were obtained in the re-screening process. In a consequence, with heterotrophical cultivation of 168 h, the lipid concentration could reach 5 g/L, and the highest lipid content exceeded 20% (W/W), which was almost two-fold to that of the wild strain. All these results demonstrated that the novel breeding process was reliable and feasible for improving the screening efficiency.
Chlorophyta ; chemistry ; Heterotrophic Processes ; High-Throughput Screening Assays ; Lipids ; analysis ; Magnetic Resonance Spectroscopy ; Microalgae ; chemistry ; Staining and Labeling

Carbon-limited continuous culture was used to study the relationship between the growth of Aspergillus niger and the production of glucoamylase. The result showed that when the specific growth rate was lower than 0.068 h(-1), the production of glucoamylase was growth-associated, when the specific growth rate was higher than 0.068 h(-1), the production of glucoamylase was not growth-associated. Based on the result of continuous culture, the Monod dynamics model of glucose consumption of A. niger was constructed, Combining Herbert-Pirt equation of glucose and oxygen consumption with Luedeking-Piret equation of enzyme production, the black-box model of Aspergillus niger for enzyme production was established. The exponential fed-batch culture was designed to control the specific growth rate at 0.05 h(-1) by using this model and the highest yield for glucoamylase production by A. niger reached 0.127 g glucoamylase/g glucose. The black-box model constructed in this study successfully described the glucoamylase production by A. niger and the result of the model fitted the measured value well. The black-box model could guide the design and optimization of glucoamylase production by A. niger.
Aspergillus niger ; metabolism ; Batch Cell Culture Techniques ; Carbon ; Culture Media ; Glucan 1,4-alpha-Glucosidase ; biosynthesis ; Glucose ; Industrial Microbiology ; methods ; Oxygen

Filamentous fungi are widely used in industrial fermentation. Particular fungal morphology acts as a critical index for a successful fermentation. To break the bottleneck of morphological analysis, we have developed a reliable method for fungal morphological analysis. By this method, we can prepare hundreds of pellet samples simultaneously and obtain quantitative morphological information at large scale quickly. This method can largely increase the accuracy and reliability of morphological analysis result. Based on that, the studies of Aspergillus niger morphology under different oxygen supply conditions and shear rate conditions were carried out. As a result, the morphological responding patterns of A. niger morphology to these conditions were quantitatively demonstrated, which laid a solid foundation for the further scale-up.
Aspergillus niger ; cytology ; Fermentation ; Industrial Microbiology ; Reproducibility of Results

13 C isotopic abundance of intracellular free amino acid with a characteristic of fast- turnover can quickly reflect changes in intracellular metabolic state. But the concentration of intracellular free amino acid is low, the existed 13 C isotope detection method based on GC-MS can not satisfy the requirement with full scan mode. In this study, the selected ion monitoring method was used to detect accuracy higher likelihood of analysis of 13 C isotopic abundance of free intracellular amino acid. First, in the full scan mode we analyzed of the fracture law of different amino acids, found the feature corresponding to each amino acid fragments, and established 16 kinds of free intracellular amino acids characteristic fragment library. Then using this characteristic fragment library, only specific m/z signal was detected in sample analysis, which realized the selected ion monitoring and improved the quality of signal. The results of amino acid standards showed that the signal-to-noise ratio, measurement precision and accuracy were improved by 17, 2. 0 and 3. 8 times compared with the full scan mode. In the analysis of coenzyme Q10 producing strains of samples, this method was successfully used to detect isotopic abundance of 8 kinds of free intracellular amino acids. This method plays an important role in the detection of 13 C isotopic abundance of the intracellular free amino acid in cell metabolism research.

We observed the influence of methanol concentration on purification recovery of recombinant human consensus interferon-a (cIFN) produced by Pichia pastoris. Fermentations controlled at 0.75% (W/V) methanol showed a 24% increase in clFN expression compared to using 0.25% methanol. However, the purification recovery rate of cIFN at 0.25% methanol was 3.75-fold higher than that at 0.75% methanol. To seek the reason, we analyzed the stability of clFN by SDS-PAGE and Native-PAGE as well as Western blotting. The electrophoresis results revealed that cIFN formed a lot of aggregates in media when the induction was controlled at 0.75% methanol, and two different aggregate forms were found: disulfide bond covalent aggregates and non-covalent aggregates. However, these aggregates almost disappeared when the methanol concentration was controlled at 0.25%, at the same time, cIFN bioactivity of supernatant increased almost 4.48-fold. Finally, 0.73g monomer cIFN was obtained after purification from 1 liter supernatant at 0.25% methanol induction, showed a 2.84-fold increase compare to the induction at 0.75% methanol.
Antiviral Agents ; isolation & purification ; Fermentation ; Humans ; Interferon-alpha ; biosynthesis ; genetics ; isolation & purification ; Methanol ; analysis ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification

Forty eight patients with knee joint lipohemarthrosis underwent X-ray, CT, MRI and ultrasonography examinations, the diagnosis was confirmed by knee operation or puncture.The findings of 4 imaging methods were compared.Plain radiographs displayed fracture lines in 39 cases and fat-liquid layer of suprapatellar bursa in 40 cases, it displayed suprapatellar fat-liquid layer in 5 out of 9 occult fracture cases.CT displayed fracture lines in 48 cases and joint lipohemarthrosis in 48 cases; 3D imaging showed fracture lines better.MRI displayed fracture lines in 45 cases and joints lipohemarthrosis in 48 cases, and also showed the intracapsular structure injury.Ultrasonography revealed fracture lines in 15 cases and joint lipohemarthrosis in 48 cases, lipohemarthrosis was displayed in 7 out of 9 occult fracture cases.All 4 diagnostic imaging methods have advantages and disadvantages for diagnosis of lipohemarthrosis, and should be chosen on the basis of clinical needs.