ABSTRACT
The purpose of this study was to assess the processes of plant community formation on recultivated dumps of spent uranium-containing industrial waste from uranium deposit mines, as well as to identify the degree of impact of agro-climatic factors, agrochemical indicators of soils of recultivated dumps, and the level of residual ionizing radiation on the productivity of the emerging vegetation cover. Studies of plant colonization of recultivated Grachevsky and Shantobinsky uranium mine dumps located in Northern Kazakhstan were carried out. The mining and technical stage of reclamation consisted of planning a dump with slopes of 15° and covering it with a 1 m layer of chestnut soil. In total, 30-35 plant species are present in the dumps, the projective coverage is approximately the same (56.6-70.0%), and the herbage density is 15-16.6 plants/100 m2. As a result of the measures taken to recultivate the dumps, the intensity of the background ionizing radiation at the Grachevsky mine dump was in the range of 25-35 µR/hr and at the Shantobinsky mine dump 10-25 µR/hr, which indicates compliance with safety standards. The plant species which can be used for artificial plant colonization of uranium-containing waste dumps, were identified.
Subject(s)
Uranium , Kazakhstan , Mining , Industrial Waste , PlantsABSTRACT
Heterologous protein production often causes a significant metabolic burden in Escherichia coli cells which manifests itself in a substantial decrease in their physiological characteristics such as the maximal specific growth rate on a given substrate, the maximal substrate uptake rate as well as the maximal specific oxygen uptake rate. In high-cell-density cultures, the substrate feed rate must be adapted to this changing capabilities of the cells in order to avoid overfeeding and thus the formation of by-products that inhibit the cell performance further. This requires the precise knowledge about the changes in these specific rates, particularly during the product formation phase. In order to precisely investigate the time profile of the critical specific substrate uptake rate σcrit of microorganisms, i.e. the maximal rate at which the cells can fully oxidize their substrate, a new online tracking technique is presented. The feed rate F is modulated in such a way that the specific substrate uptake rate σ is linearly raised towards its critical value σcrit. When this is reached the feed rate is automatically reduced and the procedure is repeated. In this way the method automatically follows the changing time profile of σcrit during the entire cultivation and avoids significant acetate formation rates. This procedure considerably increases the identifiability of σcrit. The high precision of the technique also results from replacing the pO2 measurements that seem to suggest themselves for monitoring maximal oxygen uptake rate, by measuring the total oxygen consumption rate tOUR, which is available at a much higher signal-to-noise ratio and is not as prone to distortions. An important advantage of measuring tOUR is that it allows keeping pO2 controlled at its optimal value. The applicability of the new tracking technology is demonstrated at E. coli cultures. The resulting σcrit(t) profile allows determining the substrate feed rate profile and other key variables that can be used for advanced feedback control in protein production processes. The technique can be considered a PAT tool and is well suited to industrial fermentations.
Subject(s)
Batch Cell Culture Techniques , Escherichia coli/metabolism , Green Fluorescent Proteins/biosynthesis , Acetates/metabolism , Biomass , Escherichia coli/growth & development , Glucose/metabolism , Recombinant Proteins/biosynthesisABSTRACT
By means of improved feedback control kLa measurements become possible at a precision and reproducibility that now allow a closer look at the influences of power input and aeration rate on the oxygen mass transfer. These measurements are performed online during running fermentations without a notable impact on the biochemical conversion processes. A closer inspection of the mass transfer during cultivations showed that at least the number of impellers influences mass transfer and mixing: On the laboratory scale, two hollow blade impellers clearly showed a larger kLa than the usually employed three impeller versions when operated at the same agitation power and aeration rate. Hollow blade impellers are preferable under most operational conditions because of their perfect gas handling capacity. Mixing time studies showed that these two impeller systems are also preferable with respect to mixing. Furthermore the widths of the baffle bars depict a significant influence on the kLa. All this clearly supports the fact that it is not only the integral power density that finally determines kLa.
Subject(s)
Escherichia coli/growth & development , Fermentation , Oxygen/chemistry , Aerobiosis , Bioreactors , Escherichia coli/chemistry , Green Fluorescent Proteins/chemistry , Models, TheoreticalABSTRACT
An new cascade control system is presented that reproducibly keeps the cultivation part of recombinant protein production processes on its predetermined track. While the system directly controls carbon dioxide production mass and carbon dioxide production rates along their setpoint profiles in fed-batch cultivation, it simultaneously keeps the specific biomass growth rates and the biomass profiles on their desired paths. The control scheme was designed and tuned using a virtual plant environment based on the industrial process control system SIMATIC PCS 7 (Siemens AG). It is shown by means of validation experiments that the simulations in this straightforward approach directly reflect the experimentally observed controller behaviour. Within the virtual plant environment, it was shown that the cascade control is considerably better than previously used control approaches. The controller significantly improved the batch-to-batch reproducibility of the fermentations. Experimental tests confirmed that it is particularly suited for cultivation processes suffering from long response times and delays. The performance of the new controller is demonstrated during its application in Escherichia coli fed-batch cultivations as well as in animal cell cultures with CHO cells. The technique is a simple and reliable alternative to more sophisticate model-supported controllers.
Subject(s)
Bioreactors , Models, Theoretical , Recombinant Proteins/biosynthesis , Animals , CHO Cells , Cricetinae , Cricetulus , Escherichia coli/cytology , Escherichia coli/growth & development , HumansABSTRACT
Cell cultures must tightly be kept under control in order to guarantee a sufficiently small variability in the protein product quality. A simple and efficient technique for CHO-cell cultures is presented that allows keeping the viable cell count X(v) and the specific growth rate µ of the cells on predefined trajectories. As X(v) and µ cannot directly be measured online, they are controlled indirectly via the total mass of oxygen consumed. Online values of the latter can precisely be estimated from off gas analysis, i.e. from the O2 volume ratio measured in the vent line and air flow rate measurements. In glutamine-limited fed-batch cultivations, the glutamine feed rate can be manipulated in such a way that the viable cell density and the specific growth rate are kept on predefined profiles for nearly the entire cultivation time. The viability of the cells is not affected by the closed loop control actions. The technique was validated with CHO-cells cultured in a 2.5-L fully instrumented stirred tank bioreactor. It is shown that the controller is able to run the process exactly on predefined tracks with a high batch-to-batch reproducibility. By means of six fed-batch cultivations of CHO cells it was shown that a remarkable reproducibility of viable cell concentration could be achieved throughout 140 h cultivation time.
Subject(s)
Cell Culture Techniques/methods , Animals , Bioreactors , CHO Cells , Cell Count , Cell Culture Techniques/instrumentation , Cell Survival/drug effects , Cricetinae , Cricetulus , Feedback, Physiological/drug effects , Glutamine/pharmacology , Oxygen/metabolism , Reference StandardsABSTRACT
In order to guarantee the quality of recombinant therapeutic proteins produced in mammalian cell systems, the straightforward approach in industry is to run the processes as reproducible as possible. It is first shown that considerable distortions in the currently operated processes appear when the initial cell density deviates from its nominal value. Small deviations in the initial cell mass may lead to severe deviations from the desired biomass trajectory. Next, it is shown how to design a fed-batch production process in such a way that it is robust with respect to variations in the viable cell density. A simple open loop strategy is proposed for that purpose. Here we show for the first time at animal cell cultures (CHO cells) that by means of an appropriate glutamine feed rate profile F(t), which keeps the specific growth rate of the cells on a predefined value below its maximal value while maintaining the viabilities on a high level, the diverging viable cell count profiles change over into a robust converging set of profiles. The CHO cells used to validate the procedure could be focused to any specific growth rates below µ(max).
ABSTRACT
A simple well-performing adaptive control technique for pH control in fermentations of recombinant protein production processes is described and its design procedure is explained. First, the entire control algorithm was simulated and parameterized. Afterwards it was tested in real cultivation processes. The results show that this simple technique leads to significant reductions in the fluctuations of the pH values in microbial cultures at a minimum of expenditures. The signal-to-noise ratio and thus the information captured by the pH signal were increased by about an order of magnitude. This leads to a substantial improvement in the noise of many other process signals that are used to monitor and control the process. For instance, respiratory off-gas data of CO(2) and its derived carbon dioxide production rate signals from the cultures carry much less noise as compared to those values obtained with conventional pH control. Detailed process analysis revealed that even very small pH jumps of 0.03 values during the fermentation were shown to result in pronounced deflections in CO(2)-volume fraction of 8% (peak to peak). The proposed controller, maintaining the pH within the interval of 0.01 around the setpoint, reduces the noise considerably.
Subject(s)
Bioreactors , Hydrogen-Ion Concentration , Algorithms , Carbon Dioxide/metabolism , Computer Simulation , Equipment Design , Escherichia coli/metabolism , Fermentation , Oxygen/metabolism , Recombinant Proteins/metabolismABSTRACT
Design and experimental validation of advanced pO(2) controllers for fermentation processes operated in the fed-batch mode are described. In most situations, the presented controllers are able to keep the pO(2) in fermentations for recombinant protein productions exactly on the desired value. The controllers are based on the gain-scheduling approach to parameter-adaptive proportional-integral controllers. In order to cope with the most often appearing distortions, the basic gain-scheduling feedback controller was complemented with a feedforward control component. This feedforward/feedback controller significantly improved pO(2) control. By means of numerical simulations, the controller behavior was tested and its parameters were determined. Validation runs were performed with three Escherichia coli strains producing different recombinant proteins. It is finally shown that the new controller leads to significant improvements in the signal-to-noise ratio of other key process variables and, thus, to a higher process quality.