THE FUZZY NEURAL NETWORK CONTROLLER IN YEAST FED-BATCH FERMENTATION

On the basis of the optimized substrate feeding rate of glucose acquired from an artificial neural network model of yeast fed-batch fermentation, an adaptive fuzzy neural network controller (FNNC) was developed, and on-line experiments were performed to examine the feasibility of the controller for actual use. The objective is to obtain a high concentration of cell mass in the shortest fed-batch culture time. Experimental results reveal that FNNC-controlled on-line feeding of substrate performs well.     

Efficient fuzzy control strategies for the application of pH-stat to fed-batch cultivation of genetically engineered Escherichia coli

In the cultivation of genetically engineered Escherichia coli it is very important to control the substrate concentration at an appropriate level in order to avoid the accumulation of acetate, thereby elevating the expression level of plasmid-encoded protein. In this paper, a pH-stat mode of fuzzy control was considered for the overexpression of β-galactosidase in the fed-batch cultivation of recombinant E. coli. In the simple pH-stat fuzzy control, the response of pH change in the culture broth to the feeding rate of glucose was used to estimate the glucose consumption rate. In the modified pH-stat fuzzy control, the glucose consumption rate was accurately estimated by using pH change and the change in the carbon dioxide content of the exhaust gas. With this control strategy, the cell density could be increased to 72 g DCW dm^?3, which was twofold higher than that attained in the cultivation with the simple pH-stat fuzzy control. The bulk β-galactosidase concentration was increased to 4150 U cm^?3, which was threefold higher than when the simple pH-stat control was used.     

Enhancement of secreted production of glucoamylase through fed-batch bioreactor culture of recombinant yeast harboring glucose-controllable <Emphasis Type="Italic">SUC2</Emphasis> promoter

Glucoamylase that hydrolyses starch to glucose is one of the important industrial enzymes for ethanol production industry. Therefore, genetic production of recombinant glucoamylase has been widely studied. Previously, we reported secreted production of Saccharomyces diastaticus-originated glucoamylase in Saccharomyces cerevisiase expression system using its own signal sequence and the SUC2 promoter that is regulated by glucose level in culture medium. In the present work, we performed a comparative study between batch and fed-batch bioreactor cultures for secreted production of recombinant glucoamylase. Through maintaining low glucose levels in the culture broth, we obtained about 7-fold higher secreted production levels of glucoamlyase in fed-batch culture. Fed-batch culture strategy also enhanced (∼3.1-fold) secretion efficiency of recombinant glucoamylase in S. cerevisiae.     

A novel strategy on the high-cell-density cultivation of Candida utilis for the enhanced production of glutathione

Efficient glutathione production by high-cell-density cultivation of Candida utilis was investigated. A series of batch glutathione fermentations were carried out and the optimal initial glucose concentration was found to be about 26 g/L. Then, fed-batch fermentation under diverse feeding strategies was used to enhance glutathione production with a total glucose concentration of 150 g/L. Constant glucose feeding strategy cannot meet the requirement of cells at the late period of feeding, while exponential glucose feeding strategy cannot satisfy the needs of cells at the beginning of feeding. Based on the results above, a polynomial glucose feeding strategy was developed to provide enough glucose for cells along with the cultivation, under which both the cell and glutathione productivity were satisfactorily improved. Furthermore, fed-batch fermentation under this strategy with a total glucose concentration of 200 g/L was successfully performed, the dry cell weight and glutathione concentration reached 91.2 g/L and 825 mg/L, respectively.     

Investigations on the growth ofRhodococcus rubra in relation to the formation of stable biological foams

The growth characteristics of a foam-forming species,Rhodococcus rubra were studied on different substrates. The basic medium contained Czapek (3.34%), yeast extract (0.2%), potassium dihydrogen phosphate (0.12%), dipotassium hydrogen phosphate (0.25%) and ammonium chloride (0.1%). This was supplemented with varying concentrations of glucose (0–2%). The same basic medium was also used to examine the growth ofR. rubra in combination with varying concentration of n-hexadecane (0.0–0.5%) as a source of energy while varying the concentration of ammonium chloride in the range 1–3 gl ⁻¹. Studies based on determining the biomass concentration, the surface activity related to the cell suspensions and measuring the variations in broth pH revealed that glucose encouraged the growth ofR. rubra, compared to the control. However, increasing the glucose concentration from 0.1 to 2.0% had no further effect on growth. The surface activity of the cell suspensions increased with increasing glucose concentration. Results similar to glucose were exhibited by the addition of n-hexadecane, suggesting same degree of growth among different concentrations with higher surface activity increasing with increase in substrate concentration. Results have also shown that the pH of all the culture broths decreased as the ammonium chloride concentration increased, suggesting that there was a production of hydrogen ions during the course of its metabolism.     

Fate of iridoid glycosides in different life stages of the Buckeye,Junonia coenia (Lepidoptera: Nymphalidae)

The buckeye butterfly,Junonia coenia (Lepidoptera: Nymphalidae), specializes on plants that contain iridoid glycosides. To determine the fate of these compounds in larvae, pupae, and adults of this species, we reared larvae on artificial diets with and without iridoid glycosides, and on leaves of a host plant,Plantago lanceolata (Plantaginaceae). Quantification by gas chromatography showed that newly molted third-, fourth-, and fifth-instar larvae reared on leaves ofP. lanceolata contained means of 5.13, 2.88, and 6.83% dry weight iridoid glycoside. In contrast, the mean iridoid glycoside concentration of actively feeding fifth-instar larvae was 0.28% dry weight, that of pupae was 0.19% dry weight iridoids, and adults contained no detectable iridoids. Feeding experiments suggested that this reduction in actively feeding larvae was due to the metabolism of iridoid glycosides.P. lanceolata leaves in these experiments contained a mean of 1.00% dry weight iridoid glycoside, with a 2:1 ratio of aucubin to catalpol. Calculation of iridoid consumption and utilization indices showed that larvae fed artificial diets consumed, digested, and sequestered aucubin and catalpol in similar ways. When these indices were calculated for larvae fed leaves ofP. lanceolata, catalpol was sequestered twice as efficiently as aucubin.     

Three-stage fermentation and kinetic modeling of bioflocculant by Corynebacterium glutamicum

Fermentation of bioflocculant with Corynebacterium glutamicum was studied by way of kinetic modeling. Lorentzian modified Logistic model, time-corrected Luedeking–Piret and Luedeking–Piret type models ...     

三段式谷氨酸棒杆菌发酵生产生物絮凝剂的过程及动力学模拟(英文)

Fermentation of bioflocculant with Corynebacterium glutamicum was studied by way of kinetic modeling. Lorentzian modified Logistic model, time-corrected Luedeking–Piret and Luedeking–Piret type models were pro-posed and applied to describe the cell growth, bioflocculant synthesis and consumption of substrates, with the correlation of initial biomass concentration and initial glucose concentration, respectively. The results showed that these models could well characterize the batch culture process of C. glutamicum at various initial glucose con-centrations from 10.0 to 17.5 g·L?1. The initial biomass concentration could shorten the lag time of cel growth, while the maximum biomass concentration was achieved only at the optimal initial glucose concentration of 16.22 g·L?1. A novel three-stage fed-batch strategy for bioflocculant production was developed based on the model prediction, in which the lag phase, quick biomass growth and bioflocculant production stages were sequentially proceeded with the adjustment of glucose concentration and dissolved oxygen. Biomass of 2.23 g·L?1 was obtained and bioflocculant concentration was enhanced to 176.32 mg·L?1, 18.62% and 403.63%higher than those in the batch process, respectively, indicating an efficient fed-batch culture strategy for bioflocculant production.     

Optimization of Propionic Acid Feeding for Production of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in Fed-Batch Culture of Ralstonia eutropha

The feeding method of propionic acid for production of poly(3-hydroxybutyrate-co-3-hydro xyvalerate) [P(3HB-co-3HV)] by fed-batch culture of Ralstonia eutropha was optimized to achieve high cell density and high 3HV yield. Effects of different feeding strategies of propionic acid on the production of P(3HB-co-3HV) were investigated. A decline of specific synthesis rate of copolymer and the yield of 3HV unit from propionic acid were observed due to the propionic acid accumulation in culture broth when the feeding solution with high P/G(propionic acid to glucose) ratio was employed. It was further confirmed by controlling propionic acid concentration at a low level in the separate feeding of propionic acid. An optimal feeding strategy was demonstrated to reduce the propionic acid accumulation. The cell concentration, P(3HB-co-3HV) productivity and 3HV unit fraction reached to 163.9kg.m-3, 1.8kg.m-3.h-1, and 10.6%(by mass), respectively, resulting in a yield of 0.33g HV per g propionic acid.     

基于生长代谢耦联的1,3-丙二醇发酵过程底物流加控制策略

研究了克雷伯氏杆菌转化甘油生产1,3-丙二醇的底物流加控制策略,其特征是批式流加发酵过程与细胞生长和代谢相耦联. 通过细胞生长动力学分析,建立了对数生长期和稳定期底物消耗与生物量和碱液消耗量之间的函数关系. 2次在线反馈补料发酵实验结果表明,甘油浓度控制在20±2 g/L时,1,3-丙二醇的终浓度分别达80.3和78.8 g/L以上,与手动反馈补料相比,1,3-丙二醇浓度分别提高了25.0和23.5 g/L.     

ON-LINE OPTIMAL FEED OF SUBSTRATE DURING FED-BATCH CULTURE OF CELL MASS

In the cultivation of cell mass, a mathematical model for the specific growth rate of the cells can be used to design a strategy for the optimal feed of substrate to the culture. In this paper, a model of specific growth rate valid at both low and high cell concentration is developed for Candida utilis and Candida brassicae. An on-line control algorithm is then proposed to determine the optimal substrate concentration for the production of cell mass in fed-batch culture. An on-line estimation scheme using the sensitivity method is also included for cases in which the cell mass can not be measured. Simulation results demonstrate that the proposed method can minimize the production time of fed-batch culture.     

(Z)-oxacyclotridec-10-en-2-one does not appear to be responsible for resistance to adult alfalfa weevil feeding exhibited byMedicago rugosa

Steam distillate prepared from leaves ofMedicago rugosa Desr. could inhibit feeding by adult alfalfa weevils (Hypera postica (Gyll.) on membrane filters, whereas an equivalent amount of steam distillate prepared usingM. sativa L. had no effect on weevil feeding. Earlier work established that a 12-carbon lactone, (Z)-oxacyclotridec-10-en-2-one, was responsible for the feeding deterrent properties of the steam distillate fromM. rugosa. The concentration of this volatile varied with plant age and leaf position. The maximum concentration in leaves ofM. rugosa (area basis) was estimated, on the basis of tests using an inert substrate, to be too low to cause a detectable inhibition of feeding. No correlation between lactone content and weevil feeding was detected when leaves from similar positions on plants of different ages were used in a multiple choice bioassay. Although small amounts (< 12.5g) of (Z)-oxacyclotridec-10-en-2-one can inhibit adult alfalfa weevil feeding on 13-mm-diameter membrane filters, because leaf concentrations (area basis) of this compound are lower than required to cause a detectable inhibition of feeding on the inert substrate, and because concentrations do not correlate with weevil feeding, it is unlikely that this lactone is responsible for resistance to adult alfalfa weevil feeding exhibited byM. rugosa.Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the USDA and does not imply approval to the exclusion of other products or vendors that may also be suitable.Technical paper No. 9416 of the Agricultural Experiment Station, Oregon State University, Corvallis, Oregon 97331.     

Optimal culture condition for the production of phenyalanine ammonia lyase from <Emphasis Type="Italic">E. coli</Emphasis>

The effects of culturing conditions on phenylalanine ammonia lyase production by a recombinant E. coli strain were investigated by using a controlled fed-batch fermentation system. In a 5 L fermentor, the optimal composition of the batch medium was 2% glucose, 1% yeast extract, 0.7% K₂HPO₄, 0.8% KH₂PO₄, 0.5% (NH₄)₂SO₄, 0.1% MgSO₄·7H₂O. The optimal feed glucose solution was 50%. Glucose concentration and pH of the culture broth were maintained at about 2.0 g/L and 7.0 during the fed-batch phase, respectively. Following 24-h cultivation, 0.2 mmol/L isopropyl-β-D-thiogalactopyranoside (IPTG) was added and temperature was shifted from 37°C to 42°C to induce pal gene expression. Under optimal conditions, a high productivity of 300 U/g could be achieved after 48 h culture, and a cell density of OD₆₀₀ about 82 was obtained at 52 h culture at 500 r/m stirrer speed and 1 vvm, respectively.     

Effect of gross cardiac glycoside content of seeds of common milkweed,Asclepias syriaca,on cardiac glycoside uptake by the milkweed bugOncopeltus fasciatus

Milkweed bugs,Oncopeltus fasciatus, were fed seeds of common milkweed,Asclepias syriaca, that contained differing concentrations of cardiac glycoside. Whole seeds had a mean cardiac glycoside concentration of 4.01 mg equivalents to digitoxin per g dry weight, and seed embryos had a mean concentration of 5.56 mg/g dry weight. Bugs fed these seeds concentrated cardiac glycoside: their mean concentration was 6.85 mg/g dry weight. Milkweed bugs fed seeds of lower cardiac glycoside content sequestered a greater percent of the available glycoside than bugs fed seeds of high glycoside content. The quantitative variation of cardiac glycoside content of the seeds of this single species did not significantly affect the growth of bugs. In a separate feeding preference experiment, bugs were offered seeds of both high (5.18 mg/g dry weight) and low (2.30 mg/g dry weight) cardiac glycoside content. The bugs showed no indication of selecting seeds of either high or low glycoside content.     

Fuzzy control of ethanol concentration for emulsan production in a fed-batch cultivation ofAcinetobacter Calcoaceticus RAG-1

A fuzzy control system was organized and applied to the control of ethanol concentration in a fed-batch cultivation process for emulsan production byAcinetobacter calcoaceticus RAG-1. The membership functions and fuzzy rules were determined by sets of data and experiences obtained from the preliminary culture experiments. The input variables, error (the difference between the set point value and the process variable) and the change of the error, were fuzzified by using the membership functions and the output variable, change of the ethanol feed rate, was inferred based on the membership functions and the given fuzzy rules. To obtain the numerical value for the output variable, the center-of-gravity method was used in the defuzzification procedure. The results showed that the ethanol concentration was well regulated around optimal level and the emulsan yield was increased compared with that of the cultivation controlled by the conventional feedback control loop.     

谷氨酰胺限制对杂交瘤细胞生长、代谢和单抗生产的影响

利用批培养和流加培养方式以及不同的流加培养成分,考察了谷氨酰胺限制对杂交瘤细胞生长、代谢和单抗生成的影响.谷氨酰胺限制降低了最大细胞密度和单抗比生产速率,说明谷氨酰胺限制阻碍了细胞生长和单抗生产.谷氨酰胺限制流加培养的YLac/Gluc和YLac/Cells高于批培养和谷氨酰胺非限制流加培养的,说明谷氨酰胺限制促进了葡萄糖向乳酸的生成,增强了葡萄糖的溢流代谢.与批培养和谷氨酰胺非限制流加培养相比,谷氨酰胺限制流加培养的氨和丙氨酸浓度及其比生成速率较低,而且YAmm/Gln高,YAla/Gln和YAla/Cells低,说明谷氨酰胺限制使得较多的谷氨酰胺参与脱氢反应,提高了谷氨酰胺的利用率,降低了细胞对氨和丙氨酸的生成,从而削弱了氨对细胞生长和单抗生产的毒害.在流加培养中应严格控制谷氨酰胺的流加浓度及与葡萄糖的配比,要避免谷氨酰胺过分限制导致的葡萄糖溢流代谢和对细胞生长和单抗产率的不利效应.     

超声波提取黄芩药渣中的黄芩苷

为了探索黄芩植物药废渣的再利用价值,首次采用超声波萃取技术对黄芩植物药废渣中的药用有效成分-黄芩苷进行再次提取,考察溶剂浓度、超声提取时间以及溶剂量等因素对黄芩苷提取率的影响。设计正交实验对超声波提取工艺参数进行优化。结果表明超声波最佳提取工艺条件为：15倍量70％浓度的乙醇,在20℃条件下超声提取50min,黄芩苷提取率达6．0％以上。通过试验得出的结论为：黄芩植物药废渣中尚含有一定量的药用有效成分-黄芩苷,具有再资源化利用的价值。     

Glucosinolate levels in cotyledons of mustard,Brassica juncea L. and rape,B. napus L. do not determine feeding rates of flea beetle,Phyllotreta cruciferae (Goeze)

Sinigrin (allyl glucosinolate), the major glucosinolate in the cotyledons ofBrassica juncea cv. Cutlass, occurred in the highest concentration and amount at seedling emergence and declined during growth. Glucobrassicin (3-indolylmethyl glucosinolate), the major glucosinolate in the cotyledons ofB. napus cv. Westar, occurred in the lowest concentration and amount at seedling emergence. The amount of glucobrassicin per cotyledon pair increased about fourfold during 14 days of growth, but its concentration remained relatively unchanged because of dilution by increasing cotyledon biomass. These different glucosinolate profiles indicate a different metabolic control and different biological function for sinigrin and glucobrassicin. The flea beetle,Phyllotreta cruciferae Goeze, does not discriminate between cotyledons having sinigrin or glucobrassicin since the two crucifers were fed upon equally in choice tests. Restricting the concentration of sulfur in the nutrient medium accelerated the decline of sinigrin inB. juncea cv. Cutlass but did not alter the feeding rate ofP. cruciferae compared to controls. Sulfur restriction reduced glucobrassicin inB. napus cv. Westar to undetectable levels and somewhat reduced the feeding rate of P.Cruciferae. Nevertheless,P. cruciferae still fed actively on cotyledons ofB. napus cv. Westar depleted of glucosinolates and severely damaged many of them. Since glucosinolate type and concentration had little effect on feeding response, reduction or elimination of foliar glucosinolates alone would not seem a useful strategy for protecting seedlings of these two crucifers from flea beetle damage.     

Observer-based non-linear control of a fed-batch autoinductive fermentation process

Optimal regulatory control of an autoinductive recombinant culture in a fed-batch reactor is considered. End point optimization results in a three-stage process: biomass growth, inducer synthesis and product synthesis. It is shown that in the last stage the substrate concentration should be maintained constant. This is achieved using an input—output linearizing controller accompanied by a novel non-linear state observer for the estimation of unmeasured state variables on the basis of on-line off-gas carbon dioxide concentration measurements. Experimental runs of luminous recombinant E. coli strain in a laboratory fermenter demonstrate the rapid convergence of the observer estimates as well as the effectiveness and robustness of the overall control system.     

FED-BATCH FERMENTATION OF BACILLUS CLAUSII FOR EFFICIENT PRODUCTION OF ALKALINE PROTEASE USING DIFFERENT FEEDING STRATEGIES

The effect of different feeding strategies, involving constant rate and linear feeding with negative and positive slopes, on protease production of an indigenous Bacillus clausii was investigated. The results indicated that alkaline protease was produced at high levels soon after glucose was completely consumed. Alkaline protease activity was at a maximum during the constant feeding rate and in the absence of glucose and presence of mineral salts and yeast extract in the feed medium. Maximum protease production in the fed-batch culture using an optimized level of feeding composition was 2430 ± 67 U/mL, which increased by up to 35% when compared to the 1800 ± 14 U/mL produced during batch culture. During batch fermentation, the protease yield and productivity obtained were 90000 U/g and 64285 U/L · h; however, under fed-batch conditions, these were 121500 U/g and 71470 /L · h, respectively. Hence, the suggested strategy has the potential to be applied to industrial production of protease used in detergent products.