Event-driven time-optimal control for a class of discontinuous bioreactors

Discontinuous bioreactors may be further optimized for processing inhibitory substrates using a convenient fed-batch mode. To do so the filling rate must be controlled in such a way as to push the reaction rate to its maximum value, by increasing the substrate concentration just up to the point where inhibition begins. However, an exact optimal controller requires measuring several variables (e.g., substrate concentrations in the feed and in the tank) and also good model knowledge (e.g., yield and kinetic parameters), requirements rarely satisfied in real applications. An environmentally important case, that exemplifies all these handicaps, is toxicant wastewater treatment. There the lack of online practical pollutant sensors may allow unforeseen high shock loads to be fed to the bioreactor, causing biomass inhibition that slows down the treatment process and, in extreme cases, even renders the biological process useless. In this work an event-driven time-optimal control (ED-TOC) is proposed to circumvent these limitations. We show how to detect a "there is inhibition" event by using some computable function of the available measurements. This event drives the ED-TOC to stop the filling. Later, by detecting the symmetric event, "there is no inhibition," the ED-TOC may restart the filling. A fill-react cycling then maintains the process safely hovering near its maximum reaction rate, allowing a robust and practically time-optimal operation of the bioreactor. An experimental study case of a wastewater treatment process application is presented. There the dissolved oxygen concentration was used to detect the events needed to drive the controller.     

Probing of C-terminal lysine variation in a recombinant monoclonal antibody production using Chinese hamster ovary cells with chemically defined media

C-terminal lysine (C-K) variants are commonly observed in therapeutic monoclonal antibodies and recombinant proteins. Heterogeneity of C-K residues is believed to result from varying degree of proteolysis by endogenous carboxypeptidase(s) during cell culture production. The achievement of batch-to-batch culture performance and product quality reproducibility is a key cell culture development criterion. Understanding the operational parameters affecting C-K levels provides valuable insight into the cell culture process. A CHO cell line X expressing a recombinant antibody was selected as the model cell line due to the exhibited sensitivity of its C-K level to the process conditions. A weak cation exchange chromatography (WCX) method with or without carboxypeptidase B (CpB) treatment was developed to monitor the C-K level for in-process samples. The effects of operating conditions (i.e., temperature and culture duration) and media trace elements (copper and zinc) on C-K variants were studied. The dominant effect on C-K level was identified as the trace elements concentration. Specifically, increased C-K levels were observed with increase of copper concentration and decrease of zinc concentration in chemically defined medium. Further, a hypothesis for C-K processing with intracellular and extracellular carboxypeptidase activity was proposed, based on preliminary intracellular carboxypeptidase Western blot results and the extracellular HCCF holding study.     

Comparative metabolite analysis to understand lactate metabolism shift in Chinese hamster ovary cell culture process

A metabolic shift from lactate production (LP) to net lactate consumption (LC) phenotype was observed in certain Chinese hamster ovary (CHO) cell lines during the implementation of a new chemically defined medium (CDM) formulation for antibody production. In addition, this metabolic shift typically leads to process performance improvements in cell growth, productivity, process robustness, and scalability. In our previous studies, a correlation between a key media component, copper, and this lactate metabolism shift was observed. To further investigate this phenomenon, two complementary studies were conducted. In the first study, a single cell line was cultivated in two media that only differed in their copper concentrations, yet were known to generate an LP or LC phenotype with that cell line. In the second study, two different cell lines, which were known to possess inherently different lactate metabolic characteristics, were cultivated in the same medium with a high level of copper; one cell line produced lactate throughout the duration of the culture, and the other consumed lactate after an initial period of LP. Cell pellet and supernatant samples from both studies were collected at regular time intervals, and their metabolite profiles were investigated. The primary finding from the metabolic analysis was that the cells in LP conditions exhibited a less efficient energy metabolism, with glucose primarily being converted into pyruvate, sorbitol, lactate, and other glycolytic intermediates. This decrease in energy efficiency may be due to an inability of pyruvate and acetyl-CoA to progress into the TCA cycle. The lack of progression into the TCA cycle or overflow metabolism in the LP phenotype resulted in the inadequate supply of ATP for the cells. As a consequence, the glycolysis pathway remained the major source of ATP, which in turn, resulted in continuous LP throughout the culture. In addition, the accumulation of free fatty acids was observed; this was thought to be a result of phospholipid catabolism that was being used to supplement the energy produced through glycolysis in order to meet the needs of LP cells. A thorough review of the metabolic profiles indicated that the lactate metabolic shift could be related to the oxidative metabolic capacity of cells.     

DO-stat fed-batch production of 2-keto-d-gluconic acid from cassava using immobilized Pseudomonas aeruginosa

Bioconversion of cassava-derived glucose to 2-keto-d-gluconic acid (2-KDG) using resting cells of immobilized Pseudomonas aeruginosa IFO 3448 was investigated. The tuberous roots of cassava were selected as the feedstock as they are inexpensive and widely available, and possess high amounts of starch (approximately 70% (w/w) of dry mass). Immobilized bacteria was used in a fed-batch fermenter and recycled over a period of 2 weeks. Given that the formation of 2-KDG from glucose requires oxygen as a reagent, and that high glucose concentrations are detrimental to the production yield of 2-KDG by resting cells, a DO-stat control strategy was used, whereby the feed rate of cassava hydrolysate was regulated by coupling it with the control variable, dissolved oxygen. For 319 h of operation including three cycles of repeated fed batch, 72 g of 2-KDG was produced from hydrolysate derived from 110 g of dried cassava at a maximum production rate of 0.55 g/L/h and an average concentration of 35 g/L.     

Fed-batch cultivation of the marine bacterium Sulfitobacter pontiacus using immobilized substrate and purification of sulfite oxidase by application of membrane adsorber technology

Sulfitobacter pontiacus, a gram-negative heterotrophic bacterium isolated from the Black Sea is well known to produce a soluble AMP-independent sulfite oxidase (sulfite: acceptor oxidoreductase) of high activity. Such an enzyme can be of great help in establishing biosensor systems for detection of sulfite in food and beverages considering the high sensitivity of biosensors and the increasing demand for such biosensor devices. For obtaining efficient amounts of the enzyme, an induction of its biosynthesis by supplementing sufficient concentrations of sodium sulfite to the fermentation broth is required. Owing to the fact that a high initial concentration of sodium sulfite decreases dramatically the enzyme expression, different fed-batch strategies can be applied to circumvent such inhibition or repression of the enzyme respectively. By the use of sulfite species immobilized in polyvinyl alcohol gels, an approach to the controlled and continuous feeding of sulfite to the cultivation media could be established to diminish inhibitory concentrations. Furthermore, the purification of the enzyme is described by using membrane adsorber technology.     

Sex‐Dependent Dietary Obesity,Induction of UCPs,and Leptin Expression in Rat Adipose Tissues

Objective: The aim of this study was to determine the sex‐dependent differences in the response of key parameters involved in thermogenesis and control of body weight in brown adipose tissue (BAT) and white adipose tissue (WAT) in postcafeteria‐fed rats, a model of dietary obesity. Research Methods and Procedures: BAT and WAT were obtained from male and female control and postcafeteria‐fed Wistar rats. Postcafeteria‐fed rats were initially fed with cafeteria diet from day 10 of life until day 110 (cafeteria period) and with standard chow diet from then until day 180 of life (postcafeteria period). Body mass and energy intake were evaluated. Biometric parameters were analyzed in interscapular BAT (IBAT). Levels of uncoupling protein 1 (UCP1), α₂‐adrenergic receptor (AR), and β₃‐AR proteins and UCP1, UCP2, UCP3, β₃‐AR, and leptin mRNAs, in IBAT or WAT, were studied by Western blot and Northern blot analyses, respectively. Results: Rats attained 59% (females) and 39% (males) increase in body weight at the end of the cafeteria period. During the postcafeteria period, the rats showed a loss of body weight, which was higher in females. Postcafeteria‐fed female rats also presented higher activation of thermogenic parameters in IBAT, including UCP1, UCP2, and UCP3 mRNAs. Female control rats showed lower levels of both α2 and β₃‐ARs in BAT compared with male rats, but these levels in postcafeteria‐fed female and male rats were the same, because males tended to down‐regulate them. Levels of leptin mRNA in response to the postcafeteria state depended on gender and the specific WAT depot studied. Discussion: It is suggested that in postcafeteria‐fed female rats, BAT thermogenic capacity becomes more efficiently activated than in males. Female rats also showed a bigger weight loss. The parallel regulation of the levels of UCP2 and UCP3 mRNAs, with respect to UCP1 mRNA, with higher activation in female postcafeteria‐fed rats, suggests a possible role of both UCP2 and UCP3 in the regulation of energy expenditure and in the control of body weight. The distinct responses to overweight of α2 and β₃‐ARs—which were sex dependent—and leptin mRNA—which depended on both sex and WAT depot—also support the different response of thermogenesis‐related parameters between overweight males and females.     

面包酵母海藻糖积累条件的研究

对面包酵母的海藻糖积累条件及补料分批发酵进行了研究。结果表明 pH为 5 .0 ,温度 37℃有利于海藻糖积累。少量多次的补加葡萄糖可持续增加海藻糖的含量 ,但随着发酵时间的延长 ,葡萄糖对海藻糖转化率有逐渐减少的趋势。     

Altitudinal distribution limits of aquatic macroinvertebrates: an experimental test in a tropical alpine stream

1. Temperature and oxygen are recognised as the main drivers of altitudinal limits of species distributions. However, the two factors are linked, and both decrease with altitude, why their effects are difficult to disentangle. 2. This was experimentally addressed using aquatic macroinvertebrates; larvae of Andesiops (Ephemeroptera), Claudioperla, (Plecoptera), Scirtes (Coleoptera) and Anomalocosmoecus (Trichoptera), and the amphipod Hyalella in an Ecuadorian glacier‐fed stream (4100–4500 m a.s.l.). The following were performed: (i) quantitative benthic sampling at three sites to determine altitudinal patterns in population densities, (ii) transplants of the five taxa upstream of their natural altitudinal limit to test the short‐term (14 days) effect on survival, and (iii) in situ experiments of locomotory activity as a proxy for animal response to relatively small differences in temperature (5 °C vs. 10 °C) and oxygen saturation (55% vs. 62%). 3. The transplant experiment reduced survival to a varying degree among taxa, but Claudioperla survived well at a site where it did not naturally occur. In the in situ experiment, Scirtes and Hyalella decreased their activity at lower oxygen saturation, whereas Andesiops and Anomalocosmoecus did so at a low temperature. The decrease in activity from a high to a low temperature and oxygen for the five taxa was significantly correlated with their mortality in the transplant experiment. 4. Together the present experiments indicate that even relatively small differences in temperature and oxygen may produce effects explaining ecological patterns, and depending on the taxon, either water temperature or oxygen saturation, without clear interacting effects, are important drivers of altitudinal limits.