An effective and simplified pH-stat control strategy for the industrial fermentation of vitamin B12 by Pseudomonas denitrificans

In order to improve the productivity of vitamin B(12) by Pseudomonas denitrificans carried out in a 120-m(3) fermenter, the effect of pH on vitamin B(12) biosynthesis was investigated. Results obtained from shake flask experiments showed that the feeding of carbon source (beet molasses or glucose) and methyl-group donor (betaine or choline chloride) significantly influenced the pH and the biosynthesis of vitamin B(12). In contrast to beet molasses or choline chloride, using glucose as a feed medium and betaine as a methyl-group donor, pH could be maintained at a stable range. As a result, higher vitamin B(12) production was achieved. Accordingly, an effective and simplified pH-stat control strategy was established for the fermentation of vitamin B(12) in a 120-m(3) industrial fermenter. When the new pH control strategy was applied, pH was stably kept in the range of 7.15-7.30 during fermentation. Thus, 214.3 mug/mL of vitamin B(12) was achieved.     

An effective automated glucose sensor for fermentation monitoring and control

An industrial glucose analyser was partnered to an automated injection system to evaluate glucose in the culture medium of a bioreactor. This sensor has been validated on continuous cultures ofSchizosaccharomyces pombe and continuous and fed-batch cultures ofSaccharomyces cerevisiae. In addition to the advantage of a more accurate process monitoring, the main interest of this sensor deals with the control of the substrate concentration to a prespecified reference signal. Several experiments have been carried out first to validate the sensor, then to control the process evolution.     

An energy cost minimizing strategy for fermentation dissolved oxygen control

A cost-minimizing mathematical model for on-line control of dissolved oxygen using agitation speed and aeration rate was developed. In pilot scale monensin fermentation using Streptomyces cinnamonensis, this algortihm provided stable control of dissolved oxygen at 40%, reducing energy usage 27.8%. The agitation and aeration profiles provided by the algorithm respresent the pathway of least energy cost for control at the desired dissolved oxygen level. Other observed advantages of bivariable control were reduction of foaming, evaporation, and gas holdup. Reduced maintenance of compressors and agitator motors could also be expected due to decreased load. Monensin productivity equivalent to fermentation with constant agitation and aeration was not obtained, however, with potency reduced 14.8% with the dissolved oxygen control strategy.List of Symbols A m² cross sectional area of fermentor - A ₁, A ₂, A ₃, A ₄ constants of polynomial fit to Calderbank's equations - BP N/m² gauge back pressure - C _ag $/W/s cost of electrical power - C _Q $/m³ cost of compressed air - CE mol/m³/s carbon dioxide evolution rate - D m impeller diameter - DO, DO _meas, DO _sp % dissolved oxyen saturation at any time, measured, and setpoint respectively - h m height of liquid in fermentor - H N/m²/mmol Henry's constant for oxygen in water - H _av average gas holdup in fermentor - k _L a, k _L a _meas, k _L k _sp s^–1 oxygen mass transfer coefficient at any time, measured, and setpoint respectively - N, N _sp s^–1 agitation speed at any time and setpoint respectively - N _a, N _{a, sp} aeration number at any time and setpoint respectively - N _i total number of impellers - N _p impeller power number - N _s number of impellers into which air is directly sparged - OU, OU _meas mol/m³/s Oxygen uptake rate at any time and measured respectively - P W ungassed agitation power - P _g, P _g,meas, P _g,sp W gassed agitation power at any time, measured, and set point respectively - Q, Q _meas, Q _sp m³/s aeration rate at any time, measured, and setpoint respectively - T K fermentation temperature - u _g m/s linear gas velocity - V m³ fermentation liquid volume - mole fraction of oxygen in fermentation off-gas - calculation constant - motor efficiency - $/s sum of agitation and aeration costs - kg/m³ liquid density     

An effective docking strategy for virtual screening based on multi-objective optimization algorithm

Background  

Development of a fast and accurate scoring function in virtual screening remains a hot issue in current computer-aided drug research. Different scoring functions focus on diverse aspects of ligand binding, and no single scoring can satisfy the peculiarities of each target system. Therefore, the idea of a consensus score strategy was put forward. Integrating several scoring functions, consensus score re-assesses the docked conformations using a primary scoring function. However, it is not really robust and efficient from the perspective of optimization. Furthermore, to date, the majority of available methods are still based on single objective optimization design.     

An alkaline pH control strategy for methionine adenosyltransferase production in Pichia pastoris fermentation

Pichia pastoris is a successful system for expressing heterologous proteins and its fermentation pH is always maintained below 7.0. However, particular proteins are unstable under acidic conditions, such as methionine adenosyltransferase (MAT), and thus fermentation under acidic pH conditions is unsuitable because protein activity is lost owing to denaturation. Here, a strategy employing alkaline pH in the late fermentation period was developed to improve MAT production. Initially, P. pastoris KM71 was transformed with the mat gene to overexpress MAT. After 72 h of in vitro incubation at different pH values, the expressed MAT displayed highest stability at pH 8.0; however, pH 8.0 inhibited cell growth and induced cell rupture, thus affecting protein production. To balance MAT stability and Pichia cell viability, different pH control strategies were compared. In strategy A (reference), the induction pH was maintained at 6.0, whereas in strategy B, it was gradually elevated to 8.0 through a 25 h transition period (80 ～ 105 h). MAT activity was 0.86 U/mg (twofold higher than the control). However, MAT content was reduced by 50% when compared with strategy A, because of proteases released upon cell lysis. To improve cell viability under alkaline conditions, glycerol was added in addition to methanol (strategy C). When compared with strategy B, the MAT-specific activity remained nearly constant, whereas the expression level increased to 1.27 g/L. The alkaline pH control strategy presented herein for MAT production represents an excellent alternative for expressing proteins that are stable only under alkaline conditions.     

An effective surfactant-assisted hydrothermal pretreatment strategy for bioethanol production from chili post-harvest residue by separate hydrolysis and fermentation

Bioprocess and Biosystems Engineering - Surfactants play major role in the delignification of lignocellulosic biomass. Surfactant-assisted hydrothermal pretreatment was evaluated for chili...     

Mixed carbon source control strategy for enhancing alginate lyase production by marine Vibrio sp. QY102

Medium and culture conditions for alginate lyase production by marine Vibrio sp. QY102 were first optimized using statistical methods including Plackett–Burman design and central composite design. Then, fermentation in 5-L bioreactor showed that alginate acted as easily used carbohydrate for Vibrio sp. QY102, while starch extended its growth phase and stabilized pH variations. Thus, a novel strategy using mixed carbon sources was proposed that starch supported growth while enzyme synthesis was induced by pulse feedings of solid alginate. The optimized process followed that Vibrio sp. QY102 grew on starch until the end of the logarithmic growth phase, and then solid alginate was added as 1 g/L every 3 h. Meanwhile, initial pH 5.0 and natural pH during fermentation was favorable for alginate lyase production. After optimization, the highest alginate lyase production reached 52.8 U/mL, which was 329 % higher than the control. Finally, fermentation scale-up was performed in 30-L bioreactor and the maximum alginate lyase production was obtained as 46.8 U/mL.     

人工智能时代发酵优化与放大技术的机遇与挑战

人工智能(artificial intelligence, AI)技术正引发一场新的产业革命,其成功应用正从信息产业迅速渗透到各行各业。传统的发酵工程技术受到巨大挑战的同时更多地迎来了发展变革的机遇。首先,合成生物技术飞速发展使高性能菌株的可获得性及获取效率显著提升,对传统低效的发酵优化放大技术提出很大挑战,亟需对发酵优化放大技术进行升级,以满足高通量菌种性能验证及工艺开发能力的需求;其次,发酵装备技术的持续发展为高效发酵优化技术的进步奠定了良好基础,加之人工智能技术特别是数字孪生与知识图谱等技术的应用,将为传统发酵技术的颠覆性发展带来巨大推动力。本文分别从合成生物时代对发酵优化技术的挑战、发酵优化与放大的核心技术、高通量发酵装备技术、数据可视化技术、数字孪生及知识图谱等智能技术在发酵优化放大中的应用等几个方面进行综述,并对未来工业发酵优化技术的场景以及未来发酵技术对人才培养等提出的新要求进行了展望。     

An oxidoreduction potential shift control strategy for high purity propionic acid production by Propionibacterium freudenreichii CCTCC M207015 with glycerol as sole carbon source

The effects of oxidoreduction potential (ORP) regulation on the process of propionic acid production by Propionibacterium freudenreichii CCTCC M207015 have been investigated. Potassium ferricyanide and sodium borohydride were determined as ORP control agents through serum bottle experiment. In batch fermentation, cell growth, propionic acid and by-products distribution were changed with ORP levels in the range of 0–160 mV. Based on these analysis results, an ORP-shift control strategy was proposed: at first 156 h, ORP was controlled at 120 mV to obtain higher cell growth rate and propionic acid formation rate, and then it was shifted to 80 mV after 156 h to maintain the higher propionic acid formation rate. By applying this strategy, the optimal parameters were obtained as follows: the propionic acid concentration 45.99 g L^?1, productivity 0.192 g L^?1 h^?1, the proportion of propionic acid to total organic acids 92.26 % (w/w) and glycerol conversion efficiency 76.65 %. The mechanism of ORP regulation was discussed by the ratio of NADH/NAD⁺, ATP levels, and metabolic flux analysis. The results suggest that it is possible to redistribute energy and metabolic fluxes by the ORP-shift control strategy, and the strategy could provide a simple and efficient tool to realize high purity propionic acid production with glycerol as carbon source.     

A novel osmolality-shift fermentation strategy for improving acarbose production and concurrently reducing byproduct component C formation by Actinoplanes sp. A56

Component C (Acarviosy-1,4-Glc-1,1-Glc) was a highly structural acarbose analog, which could be largely formed during acarbose fermentation process, resulting in acarbose purification being highly difficult. By choosing osmolality level as the key fermentation parameter of acarbose-producing Actinoplanes sp. A56, this paper successfully established an effective and simplified osmolality-shift strategy to improve acarbose production and concurrently reduce component C formation. Firstly, the effects of various osmolality levels on acarbose fermentation were firstly investigated in a 50-l fermenter. It was found that 400–500 mOsm/kg of osmolality was favorable for acarbose biosynthesis, but would exert a negative influence on the metabolic activity of Actinoplanes sp. A56, resulting in an obviously negative increase of acarbose and a sharp formation of component C during the later stages of fermentation (144–168 h). Based on this fact, an osmolality-shift fermentation strategy (0–48 h: 250–300 mOsm/kg; 49–120 h: 450–500 mOsm/kg; 121–168 h: 250–300 mOsm/kg) was further carried out. Compared with the osmolality-stat (450–500 mOsm/kg) fermentation process, the final accumulation amount of component C was decreased from 498.2 ± 27.1 to 307.2 ± 9.5 mg/l, and the maximum acarbose yield was increased from 3,431.9 ± 107.7 to 4,132.8 ± 111.4 mg/l.     

Sorbitol as a non-repressing carbon source for fed-batch fermentation of recombinant Pichia pastoris

Glycerol/methanol and sorbitol/methanol mixed-feed fermentation strategies for the production of recombinant proteins by Pichia pastoris were compared in order to examine sorbitol's potential as a carbon source. Although P. pastoris does have a lower cell yield on sorbitol than on glycerol, the specific rate of product formation is higher (60 g protein g^–1 dry wth for sorbitol/methanol, vs 45 g protein g^–1 dry wth for glycerol/methanol), resulting in comparable final recombinant expression levels. Importantly, the presence of residual sorbitol in the growth medium appears to be less repressive to the alcohol oxidase promoter in this organism, providing a more forgiving means of operating mixed-feed fed-batch recombinant P. pastoris fermentations.     

An effective alternate cloning strategy for unstable mouse genomic sequences

Unstable mammalian genomic sequences frequently underwent spontaneous rearrangement during the bacterial cloning process. When the flanking sequences of an INSM1 gene comprised of 3.0 and 4.5 kb were subcloned into a targeting vector for a gene deletion study, both the genomic sequences underwent spontaneous rearrangement. Neither the usage of recombinase-free Escherichia coli competent cells nor lowering the culture incubation temperature averted the recombination events. Co-transformation of a methyltransferase vector, pAIT2, with the targeting vector had little effect in preventing recombination through methylation of the plasmid DNA. Here, we show that a single-copy cloning technique is effective to clone the unstable mouse genomic DNA into the targeting vector.     

Design of a tubular loop bioreactor for scale-up and scale-down of fermentation processes

Microorganisms traveling through circulation loops in large-scale bioreactors experience variations in their environment such as dissolved oxygen concentration and pH gradients. The same changes are not experienced in small bioreactors, and it is suggested that herein lies one of the major reasons for the problems encountered when translating fermentation data from one scale to another. One approach to study this problem is to look at the circulation loop itself. The present work concerns an attempt to simulate the circulation loops inside stirred tank reactors, using a tubular loop reactor specially constructed for the purpose. The reactor carries a number of ports and probes along its length for the determination of concentration gradients within. The broth is circulated around the loop by the use of peristaltic pumps, and the circulation time (t(c), s) is used as a measure of simulated reactor size. The reactor system has been evaluated using the citric acid fermentation by Aspergillus niger as a test process. Acid production and fungal morphology, in terms of the mean convex perimeter of mycelial clumps quantified by image analysis, were used as the parameters of evaluation for the two systems in comparison. From comparative experiments carried out in 10 and 200 L stirred tank bioreactors, it appears that the loop reactor simulates the corresponding stirred tank representing a valuable tool in scaling up and scaling down of fermentation process.     

灭虫灵乳油中试应用的研究

灭虫灵乳油是由敌敌畏和氯氰菊酯复配而成的二元复配杀虫剂。它具有触杀、胃毒和熏蒸作用。对菜青虫等多种害虫具有击倒快、效果好、无药害,增效显著,共毒系数为875．32。使用浓度为1:2000～4000,防治效果达92.3％～100％残效期15天左右。对高等动物毒性中等,雌、雄性大白鼠的LD50分别为794和126.0mg／kg。     

An autoclavable glucose biosensor for microbial fermentation monitoring and control

The design, construction, and characterization of a prototype-regenerable glucose biosensor based on the reversible immobilization of glucose oxidase (GOx) using cellulose binding domain (CBD) technology is described. GOx, chemically linked to CBD, is immobilized by binding to a cellulose matrix on the sensor-indicating electode. Enzyme immobilization can be reversed by perfusing the cellulose matrix with a suitable eluting solution. An autocavable sensor membrane system is employed which is shown to be practical for use in real microbial fermentations. The prototype glucose biosensor was used without failure or deterioration during fed-batch fermentations of Escherichia coli reaching a maximum cell density of 85 g (dry weight)/L. Medium glucose concentration based on sensor output correlated closely with off-line glucose analysis and was controlled manually at 0.44 +/- 0.2 g/L for 2 h based on glucose sensor output. The sensor enzyme component could be eluted and replaced without interrupting the fermentation. To our knowledge, no other in situ biosensor has been used for such an extended period of time in such a high-cell-density fermentation. (c) 1995 John Wiley & Sons, Inc.     

A robust fed-batch feeding strategy independent of the carbon source for optimal polyhydroxybutyrate production

A three-stage control strategy independent of the organic substrate was developed for automated substrate feeding in a two-phase fed-batch culture of Cupriavidus necator DSM 545 for the production of the biopolymer polyhydroxybutyrate (PHB). The optimal feeding strategy was determined using glucose as the substrate. A combined substrate feeding strategy consisting of exponential feeding and a novel method based on alkali-addition monitoring resulted in a maximal cell concentration in the biomass growth phase. In the PHB accumulation phase, a constant substrate feeding strategy based on the estimated amount of biomass produced in the first phase and a specific PHB accumulation rate was implemented to induce PHB under limiting nitrogen at different biomass concentrations. Maximal cell and PHB concentrations of 164 and 125 g/L were obtained when nitrogen feeding was stopped at 56 g/L of residual biomass; the glucose concentration was maintained within its optimal range. The developed feeding strategy was validated using waste glycerol as the sole carbon source for PHB production, and the three-stage control strategy resulted in a PHB concentration of 65.6 g/L and PHB content of 62.7% while keeping the glycerol concentration constant. It can thus be concluded that the developed feeding strategy is sensitive, robust, inexpensive, and applicable to fed-batch culture for PHB production independent of the carbon source.