Development of a strategy to control the dissolved concentrations of oxygen and carbon dioxide at constant shear in a plant cell bioreactor

To examine the effects of volatile components on plant cell growth, a bioreactor control system was developed to simultaneously control the dissolved concentrations of both oxygen and carbon dioxide. The first step in this work was to develop a mathematical model to account for gas-liquid mass transfer; biological utilization and production of O(2) and CO(2); and the series of chemical reactions of CO(2) in water. Using this model and dynamic measurements for dissolved O(2) and CO(2), it was observed that (1) both absorption and desorption of a volatile component could be described by a single mass transfer coefficient, K(l)a, and (2) K(l)a values for oxygen and carbon dioxide transfer were directly proportional. The second step of this work was to employ the mathematical model in an adaptive feed-forward strategy to control the dissolved O(2) and CO(2) concentrations by manipulating the inlet gas composition to the bioreactor. This strategy allowed dissolved concentrations to be controlled without the need for changing either the total gas flow rate or agitator speed. Adaptive control was required because the volumetric rates of O(2) and CO(2) consumption and production vary with time during long term operation and therefore these rates must be continually updated. As the final step, we demonstrated that this control strategy was capable of controlling the dissolved gas concentrations in both short- and long-term studies involving the cultivation of Catharanthus roseus plant cells.     

Optimized aeration by carbon dioxide gas for microalgal production and mass transfer characterization in a vertical flat-plate photobioreactor

To optimize the aeration conditions for microalgal biomass production in a vertical flat-plate photobioreactor (VFPP), the effect of the aeration rate on biomass productivity was investigated under given conditions. Air enriched with 5% or 10% (v/v) CO(2) was supplied for the investigation at rates of 0.025-1 vvm. The CO(2) utilization efficiency, change of pH in the medium, and the optimum aeration rate were determined by evaluating biomass productivity. To investigate the VFPP mass transfer characteristics, the overall volumetric mass transfer coefficient, k(L)a, was evaluated for several different flat-plate sizes. Increasing the height of the VFPP could improve both the mass transfer of CO(2) and the illumination conditions, so this appeared to be a good method for scaling up. Based on a comparison of the k(L)a value at the optimum aeration rate with previously reported results, it was confirmed that the range of CO(2) concentration used in the experiments was cost-effective for mass culture.     

Oxygen and carbon dioxide mass transfer and the aerobic, autotrophic cultivation of moderate and extreme thermophiles: a case study related to the microbial desulfurization of coal

Mass transfers of O(2), CO(2), and water vapor are among the key processes in the aerobic, autotrophic cultivation of moderate and extreme thermophiles. The dynamics and kinetics of these processes are, in addition to the obvious microbial kinetics, of crucial importance for the industrial desulfurization of high-pyritic coal by such thermophiles. To evaluate the role of the temperature on the gas mass transfer, k(L)a measurements have been used to supplement the existing published data. Oxygen mass transfer from gas (air) to liquid (5 mM H(2)SO(4) in water) phase as a function of the temperature has been studied in a laboratory-scale fermentor. At 15, 30, 45, and 70 degrees C, (k(L)a)(o) values (for oxygen) were determined under three different energy input conditions by the dynamic gassing in/out method. The (k(L)a)(o) was shown to increase under these conditions with increasing temperature, and straight lines were obtained when the logarithm of (k(L)a)(o) was plotted against the temperature. By multiplying the equilibrium concentration of O(2) in water with (k(L)a)(o) maximal, O(2) transfer capacities were calculated. It appeared that in finite of a decreased solubility of O(2) at elevated temperature in mechanically mixed fermentors the calculated transfer capacities showed only minor changes for the range between 15 and 70 degrees C. However, in an air-mixed fermentor the transfer capacity of O(2) decreased slowly but steadily.Carbon dioxide mass transfer was predicted by calculations on the basis of the data for oxygen transfer. The maximal CO(2) transfer capacity, calculated as the product of the equilibrium CO(2) concentration times (k(L)a)(c), decreased slowly as the temperature increased over the range 15-70 degrees C under all three energy input conditions. Subsequent process design calculations showed that for aerobic, autotrophic cultures, CO(2) limitation is more likely to occur than O(2) limitation.     

Study of gaseous substrate fermentations: carbon monoxide conversion to acetate. 2. Continuous culture

The fermentation of gaseous substrates such as CO, H(2), and CO(2) may be performed in a continuous stirred tank reactor, as well as the traditional batch reactor. In this article, the conversion of carbon monoxide by Peptostreptococcus productus is demonstrated in a stirred tank reactor under both mass transfer-controlled and nonmass transfer-controlled conditions. Utilizing a non-steady-state procedure, intrinsic rates are evaluated under non-mass transfer-controlled conditions in a time period of only 5-6 hours. A steady-state procedure was used to evaluate CSTR performance under mass transfer-controlled conditions. The mass transfer coefficient was calculated, followed by the development of a model to predict CSTR behavior for this gas phase substrate.     

Determination of dissolved CO(2) concentration and CO(2) production rate of mammalian cell suspension culture based on off-gas measurement

The determination of dissolved CO(2) and HCO(3)(-) concentrations as well as the carbon dioxide production rate in mammalian cell suspension culture is attracting more and more attention since the effects on major cell properties, such as cell growth rate, product quality/production rate, intracellular pH and apoptosis, have been revealed. But the determination of these parameters by gas analysis is complicated by the solution/dissolution of carbon dioxide in the culture medium. This means that the carbon dioxide transfer rate (CTR; which can easily be calculated from off-gas measurement) is not necessarily equal to carbon dioxide production rate (CPR). In this paper, a mathematical method to utilize off-gas measurement and culture pH for cell suspension culture is presented. The method takes pH changes, buffer and medium characteristics that effect CO(2) mass transfer into account. These calculations, based on a profound set of equations, allow the determination of the respiratory activity of the cells, as well as the determination of dissolved CO(2), HCO(3)(-) and total dissolved carbonate. The method is illustrated by application to experimental data. The calculated dissolved CO(2) concentrations are compared with measurements from an electrochemical CO(2) probe.     

Transfer of carbon dioxide within cultures of microalgae: plain bubbling versus hollow-fiber modules

In attempts to improve the metabolic efficiency in closed photosynthetic reactors, availability of light and CO(2) are often considered as limiting factors, as they are difficult to control in a culture. The carbon source is usually provided via bubbling of CO(2)-enriched air into the culture medium; however, this procedure is not particularly effective in terms of mass transfer. Besides, it leads to considerable waste of that gas to the open atmosphere, which adds to operation costs. Increase in the interfacial area of contact available for gas exchange via use of membranes might be a useful alternative; microporous membranes, in hollow-fiber form, were tested accordingly. Two hollow-fiber modules, different in both hydrophilicity and outer surface area, were tested and duly compared, in terms of mass transfer, versus traditional plain bubbling. Overall volumetric coefficients (K(L)a) for CO(2) transfer were 1.48 x 10(-2) min(-1) for the hydrophobic membrane, 1.33 x 10(-2) min(-1) for the hydrophilic membrane, and 7.0 x 10(-3) min(-1) for plain bubbling. A model microalga, viz. Nannochloropsis sp., was cultivated using the two aforementioned membrane systems and plain bubbling. The produced data showed slight (but hardly significant) increases in biomass productivity when the hollow-fiber devices were used. However, hollow-fiber modules allow recirculation of unused CO(2), thus reducing feedstock costs. Furthermore, such indirect way of supplying CO(2) offers the additional possibility for use of lower gas pressures, as no need to counterbalance hydrostatic heads exists.     

Mass transfer resistance of sterile plugs in shaking bioreactors

One of the mass transfer resistances for the gas exchange of shaking flasks is the sterile plug. The gas exchange through the sterile plug is described by an extended model of Henzler and Schedel [Bioprocess Eng. 7 (1991) 123]. Based on this model, a new method was developed to obtain the mass transfer resistance of various sterile closures. It consists of measuring the water evaporation rate of the shaking flask and is therefore very easily applied. Sterile plugs made of cotton, wrapped paper, urethane foam and fibreglass and caps made out of aluminium and silicone have been examined. Instead of the oxygen transfer coefficient (k(O(2))), which is commonly found in the literature, the carbon dioxide diffusion coefficient (D(CO(2))) is used to describe the mass transfer resistance of the sterile plug. The investigation revealed that this resistance is mainly dependent on the neck geometry and to a lesser extent on the plug material and density. The gas exchange of aluminium-caps was not reproducible.     

Use of silicone membranes to enhance gas transfer during microbial fuel cell operation on carbon monoxide

Electricity generation in a microbial fuel cell (MFC) using carbon monoxide (CO) or synthesis gas (syngas) as a carbon source has been demonstrated recently. A major challenge associated with CO or syngas utilization is the mass transfer limitation of these sparingly soluble gases in the aqueous phase. This study evaluated the applicability of a dense polymer silicone membrane and thin wall silicone tubing for CO mass transfer in MFCs. Replacing the sparger used in our previous study with the membrane systems for CO delivery resulted in improved MFC performance and CO transformation efficiency. A power output and CO transformation efficiency of up to 18 mW (normalized to anode compartment volume) and 98%, respectively, was obtained. The use of membrane systems offers the advantage of improved mass transfer and reduced reactor volume, thus increasing the volumetric power output of MFCs operating on a gaseous substrate such as CO.     

Sensing and transfer of respiratory gases at the fish gill

The gill is both a site of gas transfer and an important location of chemoreception or gas sensing in fish. While often considered separately, these two processes are clearly intricately related because the gases that are transferred between the ventilatory water and blood at the gill are simultaneously sensed by chemoreceptors on, and within, the gill. Modulation of chemoreceptor discharge in response to changes in O(2) and CO(2) levels, in turn, is believed to initiate a series of coordinated cardiorespiratory reflexes aimed at optimising branchial gas transfer. The past decade has yielded numerous advances in terms of our understanding of gas transfer and gas sensing at the fish gill, particularly concerning the transfer and sensing of carbon dioxide. In addition, recent research has moved from striving to construct a single model that covers all fish species, to recognition of the considerable inter-specific variation that exists with respect to the mechanics of gas transfer and the cardiorespiratory responses of fish to changes in O(2) and CO(2) levels. The following review attempts to integrate gas transfer and gas sensing at the fish gill by exploring recent advances in these areas.     

An efficient system for carbonation of high-rate algae pond water to enhance CO2 mass transfer

High-rate algal ponds have the potential to produce 59 T of dry biomass ha(-1)year(-1) based on the specific productivity of 20 g m(-2) day(-1). Atmospheric air provides only 5% of the CO(2) to the pond surface required for photosynthesis. Hence, CO(2) is usually provided via bubbling of concentrated CO(2)-air mixture into the algae ponds. This process is, however, not significantly effective in terms of mass transfer. Use of bubble column to increase the interfacial area of contact available for gas exchange is proposed as an efficient alternative. A carbonation column (CC) was modeled and designed to measure CO(2) absorptivity in-pond water at various pH regimes. The CC performed at 83% CO(2) transfer efficiency. An air-to-pond mass transport coefficient of 0.0037 m min(-1) was derived. The proposed device can be used with any exhaust gas stream with higher concentrations of CO(2) in conjunction with raceways for optimizing algae production.     

Study of gaseous substrate fermentations: carbon monoxide conversion to acetate. 1. Batch culture

Biological processes may be used to convert gas phase substrates, such as H(2)S, CH(4), CO, H(2), and CO(2), to useful products. Utilization of these substrates is often a mass transfer limited process, first requiring absorption across the gas-liquid interface and diffusion through the culture medium to the cell surface, prior to reaction. This article presents a method for determining fermentation parameters of a gaseous substrate in convenient batch vessels using a modified Monod model. The procedure is illustrated with experimental data for the conversion of carbon monoxide to acetate by the strict anaerobe Peptostreptococcus productus.     

Study of Plasma Induced Chemistry by DC Discharges in CO<Subscript>2</Subscript>/N<Subscript>2</Subscript>/H<Subscript>2</Subscript>O Mixtures Above a Water Surface

The chemistry induced by atmospheric pressure DC discharges above a water surface in CO(2)/N(2)/H(2)O mixtures was investigated. The gaseous mixtures studied represent a model prebiotic atmosphere of the Earth. The most remarkable changes in the chemical composition of the treated gas were the decomposition of CO(2) and the production of CO. The concentration of CO increased logarithmically with the increasing input energy density and an increasing initial concentration of CO(2) in the gas. The highest achieved concentration of CO was 4.0 +/- 0.6 vol. %. The production of CO was crucial for the synthesis of organic species, since reactions of CO with some reactive species generated in the plasma, e. g. H* or N* radicals, were probably the starting point in this synthesis. The presence of organic species (including the tentative identification of some amino acids) was demonstrated by the analysis of solid and liquid samples by high-performance liquid chromatography, infrared absorption spectroscopy and proton-transfer-reaction mass spectrometry. Formation of organic species in a completely inorganic CO(2)/N(2)/H(2)O atmosphere is a significant finding for the theory of the origins of life.     

Changes in mass and energy transfer between the canopy and the atmosphere: model development and testing with a free-air CO2 enrichment (FACE) experiment

The rationale for this study is found in the probable higher temperatures and changes in rainfall patterns that are expected in the future as a result of increasing levels of CO2 in the atmosphere. In particular, higher air temperatures may cause an increase in evapotranspiration demand while a reduction in rainfall could increase the severity and duration of drought in arid and semi-arid regions. Representation of the water transfer scheme includes water uptake by roots and the interaction between evapotranspiration and CO2 enrichment. The predicted response of a spring wheat (Triticum aestivum L. cv. Yecora rojo) canopy in terms of energy exchange processes to elevated atmospheric CO2 level was tested against measurements collected at the FACE (Free Air Enrichment Experiment) site in 1994. Simulated and measured canopy conductances were reduced by about 30% under elevated [CO2] under optimum conditions of water supply. Reductions in latent heat fluxes under elevated instead of ambient [CO2] caused reductions in both simulated and measured seasonal water use of 6% under optimum and 2% under suboptimum irrigation. The soil-plant-atmosphere water transfer scheme proposed here offers several advances in the simulation of land surface interactions. First, the stomatal resistance model minimizes assumptions in existing land surface schemes about the effects of interactions among environmental conditions (radiation, temperature, CO2) upon stomatal behavior. These interactions are resolved in the calculation of CO2 in which processes are already well understood.     

Identification of mass-transfer parameters and process simulation of SCP production process in airlift tower reactors with an external loop

A distributed parameter model for simulation of SCP-production processes in tower reactors with an outer loop was developed by considering substrate, cell, and CO(2) balances in the liquid phase, and O(2) and CO(3) balances in the ges phase and taking into account variations of dissolved oxygen concentration, pressure, and k(L)a along the column, as well as double substrate Monod kinetics. This model was used to describe the cultivation of Hansenula polymorpha in a tower-loop reactor (height 275 cm, diameter 15 cm). Parameter identification and process simulation were carried out by a hybrid computer. The variation of identified mass transfer parameters with fermentation time and operation mode is considered employing ethanol and glucose substrate, respectively. Relationships among k(L)a, substrate concentration, and superficial gas velocity were developed to facilitate the layout and simulation of pilot-plant reactors.     

Proton transfer within the active-site cavity of carbonic anhydrase III

The maximal turnover rate of CO2 hydration catalyzed by the carbonic anhydrases is limited by proton transfer steps from the zinc-bound water to solution, steps that regenerate the catalytically active zinc-bound hydroxide. Catalysis of CO2 hydration by wild-type human carbonic anhydrase III (HCA III) (k(cat) = 2 ms (-1)) is the least efficient among the carbonic anhydrases in its class, in part because it lacks an efficient proton shuttle residue. We have used site-directed mutagenesis to test positions within the active-site cavity of HCA III for their ability to carry out proton transfer by replacing various residues with histidine. Catalysis by wild-type HCA III and these six variants was determined from the initial velocity of hydration of CO2 measured by stopped-flow spectrophotometry and from the exchange of 18O between CO2 and H2O at chemical equilibrium by mass spectrometry. The results show that histidine at three positions (Lys64His, Arg67His and Phe131His) have the capacity to transfer protons during catalysis, enhancing maximal velocity of CO2 hydration and 18O exchange from 4- to 15-fold compared with wild-type HCA III. Histidine residues at the other three positions (Trp5His, Tyr7His, Phe20His) showed no firm evidence for proton transfer. These results are discussed in terms of the stereochemistry of the active-site cavity and possible proton transfer pathways.     

Cultivation of microplantlets derived from the marine red alga Agardhiella subulata in a stirred tank photobioreactor

Macrophytic marine red algae are a diverse source of bioactive natural compounds. "Microplantlet" suspension cultures established from red algae are potential platforms for biosynthesis of these compounds, provided suitable bioreactor configurations for mass culture can be identified. The stirred tank bioreactor offers high rates of gas-liquid mass transfer, which may facilitate the delivery of the CO(2) in the aeration gas to the phototrophic microplantlet suspension culture. Therefore, the effects of impeller speed and CO(2) delivery on the long-term production of microplantlet biomass of the model red alga Agardhiella subulata was studied within a stirred tank photobioreactor equipped with a paddle blade impeller (D(i)/D(T) = 0.5). Nutrient medium replacement was required for sustained biomass production, and the biomass yield coefficient based on nitrate consumption was 1.08 +/- 0.09 g dry biomass per mmol N consumed. Biomass production went through two exponential phases of growth, followed by a CO(2) delivery limited growth phase. The CO(2)-limited growth phase was observed only if the specific growth rate in the second exponential phase of growth was at least 0.03 day(-)(1), the CO(2) delivery rate was less than 0.258 mmol CO(2) L(-)(1) culture h(-)(1), and the plantlet density was at least 10 g fresh mass L(-)(1). Increasing the aeration gas CO(2) partial pressure from 0.00035 to 0.0072 atm decreased the cultivation pH from 8.8 to 7.8, prolonged the second exponential phase of growth by increasing the CO(2) delivery rate, and also increased the photosynthetic oxygen evolution rate. Impeller speeds ranging from 60 to 250 rpm, which generated average shear rates of 2-10 s(-)(1), did not have a significant effect on biomass production rate. However, microplantlets cultivated in a stirred tank bioreactor ultimately assumed compact spherical shape, presumably to minimize exposure to hydrodynamic stress.     

Effects of elevated CO2 concentration on seed production in C3 annual plants

The response of seed production to CO(2) concentration ([CO(2)]) is known to vary considerably among C(3) annual species. Here we analyse the interspecific variation in CO(2) responses of seed production per plant with particular attention to nitrogen use. Provided that seed production is limited by nitrogen availability, an increase in seed mass per plant results from increase in seed nitrogen per plant and/or from decrease in seed nitrogen concentration ([N]). Meta-analysis reveals that the increase in seed mass per plant under elevated [CO(2)] is mainly due to increase in seed nitrogen per plant rather than seed [N] dilution. Nitrogen-fixing legumes enhanced nitrogen acquisition more than non-nitrogen-fixers, resulting in a large increase in seed mass per plant. In Poaceae, an increase in seed mass per plant was also caused by a decrease in seed [N]. Greater carbon allocation to albumen (endosperm and/or perisperm) than the embryo may account for [N] reduction in grass seeds. These differences in CO(2) response of seed production among functional groups may affect their fitness, leading to changes in species composition in the future high-[CO(2)] ecosystem.     

Interrelationship of the mass transfer coefficient with regard to O2 and CO2 in the process of antibiotic biosynthesis]

A complex of studies on the effect of technological parameters on the mass transfer coeficients with respect to O2 and CO2 was carried out. It was shown that the ratio between the mass transfer coefficients with respect to O2 and CO2 was constant and equal to 20 for the fermentation broths of the antibiotic-producing organisms studied.     

Increase in leaf mass per area benefits plant growth at elevated CO2 concentration

An increase in leaf mass per area (MLA) of plants grown at elevated [CO2] is often accompanied by accumulation of non-structural carbohydrates, and has been considered to be a response resulting from source-sink imbalance. We hypothesized that the increase in MLA benefits plants by increasing the net assimilation rate through maintaining a high leaf nitrogen content per area (NLA). To test this hypothesis, Polygonum cuspidatum was grown at ambient (370 micro mol mol-1) and elevated (700 micro mol mol-1) [CO2] with three levels of N supply. Elevated [CO2] significantly increased MLA with smaller effects on NLA and leaf mass ratio (fLM). The effect of change in MLA on plant growth was investigated by the sensitivity analysis: MLA values observed at ambient and elevated [CO2] were substituted into a steady-state growth model to calculate the relative growth rate (R). At ambient [CO2], substitution of a high MLA (observed at elevated [CO2]) did not increase R, compared with R for a low MLA (observed at ambient [CO2]), whereas at elevated [CO2] the high MLA always increased R compared with R at the low MLA. These results suggest that the increase in MLA contributes to growth enhancement under elevated [CO2]. The optimal combination of fLM and MLA to maximize R was determined for different [CO2] and N availabilities. The optimal fLM was nearly constant, while the optimal MLA increased at elevated [CO2], and decreased at higher N availabilities. The changes in fLM of actual plants may compensate for the limited plasticity of MLA.     

Proton transport by phosphate diffusion--a mechanism of facilitated CO2 transfer

We have measured CO2 fluxes across phosphate solutions at different carbonic anhydrase concentrations, bicarbonate concentration gradients, phosphate concentrations, and mobilities. Temperature was 22-25 degrees C, the pH of the phosphate solutions was 7.0-7.3. We found that under physiological conditions of pH and pCO2 a facilitated diffusion of CO2 occurs in addition to free diffusion when (a) sufficient carbonic anhydrase is present, and (b) a concentration gradient of HCO3- is established along with a pCO2 gradient, and (c) the phosphate buffer has a mobility comparable to that of bicarbonate. When the phosphate was immobilized by attaching 0.25-mm-long cellulose particles, no facilitation of CO2 diffusion was detectable. A mechanism of facilitated CO2 diffusion in phosphate solutions analogous to that in albumin solutions was proposed on the basis of these findings: bicarbonate diffusion together with a facilitated proton transport by phosphate diffusion. A mathematical model of this mechanism was formulated. The CO2 fluxed predicted by the model agree quantitatively with the experimentally determined fluxes. It is concluded that a highly effective proton transport mechanism acts in solutions of mobile phosphate buffers. By this mechanism; CO2 transfer may be increased up to fivefold and proton transfer may be increased to 10,000-fold.