Dynamics and control of continuous microbial propagators to subject substrate inhibition

Inhibitory substrate levels are common in industrial fermentations and in biological waste-water treatment of many industrial wastes. Continuous microbial cultures are unstable to certain disturbances, such as shock loading by inhibitory substrates. Two feedback proportional control strategies are analyzed and compared for a simple model culture assumed represent able by the culture concentrations of biomass and a single rate-limiting and growth-limiting nutrient (substrate). One control strategy, the well known turbidostat, consists of adjusting culture holding time (e.g., by flow rate adjustment) in response to deviations in turbidity or some other measure of culture biomass concentration. The other control strategy is to adjust holding time in response to deviations in limiting nutrient concentrations in the culture. This second control strategy, termed the nutristat, can be superior to the turbidostat in many applications. The sign and magnitude of the dimensionless group {(X /YD )[dμ/dS]s }, is shown to be an important determinant, in the behavior of the open loop and the two closed loop processes. This characteristic group is positive when the specific growth rate is increased by increases in the nutrient concentration, zero when the growth rate is unaffected by the nutrient concentration, and negative in the presence of nutrient or substrate inhibition. The effects of process modifications and of modeling assumptions on the control of the process are discussed and more sophisticated control schemes are also proposed.     

Characteristics of transformedPanax ginseng C.A. Meyer hairy roots: Growth and nutrient profile

Ginseng (Panax ginseng C.A. Meyer) hairy root cultures, which are established via the infection of ginseng root discs withRhizobium rhizogenes, have been used to construct profiles of both biomass growth and nutrient consumption in flask cultures. In a 250 mL shake flask culture, the maximum biomass was observed on the 59th day of the culture period, at 216.8 g (fresh wt) per liter or 11.4 g (dry wt) per liter. The hairy roots were determined to have a growth rate of 0.355 g-DW/g cells/day during the exponential growth phase and a maximum specific growth rate on day 7. Total ginseng saponin and phenolic compound contents were noted to have increased within the latter portion of the culture period. Linear correlations between increases in biomass weight and nutrient uptake were used to imply the conductivity yield 2.60 g-DW/(L·mS) and carbon yield 0.45 g-DW/(g sugar) in the 250 mL flask cultures. The biomass yield when two different nitrogen sources were used (ammonia and nitrate) was shown to remain approximately constant, at 0.47 g-DW/(l·mM NH₄) and 0.33 g-DW/(L·mM NO₃); it remained at these levels for 16 days with the ammonia, and for 24 days with the nitrate. The biomass yield when a phosphate source was used was also shown to remain approximately constant for 9 days, at 3.17 g-DW/(L·mM PO₄), with an R² of 0.99.     

Development of a kinetic metabolic model: application to Catharanthus roseus hairy root

A kinetic metabolic model describing Catharanthus roseus hairy root growth and nutrition was developed. The metabolic network includes glycolysis, pentose-phosphate pathway, TCA cycle and the catabolic reactions leading to cell building blocks such as amino acids, organic acids, organic phosphates, lipids and structural hexoses. The central primary metabolic network was taken at pseudo-steady state and metabolic flux analysis technique allowed reducing from 31 metabolic fluxes to 20 independent pathways. Hairy root specific growth rate was described as a function of intracellular concentration in cell building blocks. Intracellular transport and accumulation kinetics for major nutrients were included. The model uses intracellular nutrients as well as energy shuttles to describe metabolic regulation. Model calibration was performed using experimental data obtained from batch and medium exchange liquid cultures of C. roseus hairy root using a minimal medium in Petri dish. The model is efficient in estimating the growth rate.     

Comparative evaluation of modified bioreactors for enhancement of growth and secondary metabolite biosynthesis usingPanax ginseng hairy roots

Hairy root cultures have demonstrated great promise in terms of their biosynthetic capability toward the production of secondary metabolites, but continue to constitute a major challenge with regard to large-scale cultures. In order to assess the possibility of conducting mass production of biomass, and the extraction of useful metabolites fromPanax ginseng. P. ginseng hairy roots, transformed byRhizobium rhizogenes KCTC 2744, were used in bioreactors of different types and sizes. The most effective mass production of hairy roots was achieved in several differently sized air bubble bioreactors compared to all other bioreactor types. Hairy root growth was enhanced by aeration, and the production increased with increasing aeration rate in a 1 L bioreactor culture. It was determined that the hairy root growth rate could be substantially enhanced by increases in the aeration rate upto 0.5 wm, but at aeration rates above 0.5 wm, only slight promotions in growth rates were observed. In 20 L air bubble bioreactors, with a variety of inoculum sizes, the hairy roots exhibited the most robust growth rates with an inoculum size of 0.1% (w/v), within the range 0.1 to 0.7% (w/v). The specific growth rates of the hairy roots decreased with increases in the inoculum size.     

In vitro propagation of plant virus using different forms of plant tissue culture and modes of culture operation

Plant virus accumulation was investigated in vitro using three different forms of plant tissue culture. Suspended cells, hairy roots and shooty teratomas of Nicotiana benthamiana were infected with tobacco mosaic virus (TMV) using the same initial virus:biomass ratio. Viral infection did not affect tissue growth or morphology in any of the three culture systems. Average maximum virus concentrations in hairy roots and shooty teratomas were similar and about an order of magnitude higher than in suspended cells. Hairy roots were considered the preferred host because of their morphological stability in liquid medium and relative ease of culture. The average maximum virus concentration in the hairy roots was 0.82 ± 0.14 mg g⁻¹ dry weight; viral coat protein represented a maximum of approximately 6% of total soluble protein in the biomass. Virus accumulation in hairy roots was investigated further using different modes of semi-continuous culture operation aimed at prolonging the root growth phase and providing nutrient supplementation; however, virus concentrations in the roots were not enhanced compared with simple batch culture. The relative infectivity of virus in the biomass declined by 80–90% during all the cultures tested, irrespective of the form of plant tissue used or mode of culture operation. Hairy root cultures inoculated with a transgenic TMV-based vector in batch culture accumulated green fluorescent protein (GFP); however, maximum GFP concentrations in the biomass were relatively low at 39 μg g⁻¹ dry weight, probably due to genetic instability of the vector. This work highlights the advantages of using hairy roots for in vitro propagation of TMV compared with shooty teratomas and suspended plant cells, and demonstrates that batch root culture is more effective than semi-continuous operations for accumulation of high virus concentrations in the biomass.     

Fed-batch hairy root cultures within situ separation

Fed-batch cultures ofL. erythrorhizon hairy root were carried out by controlling sucrose concentration and media conductivity in a shake flask and a modified stirred tank reactor. For the efficient product recovery from the culture,in situ adsorption by XAD-2 was also conducted. When sucrose was used as a carbon source, the highest shikonin production and hairy root growth were obtained. When glucose or fructose was used instead, the growth was severely inhibited. In addition, it was found that alternating feeding of sucrose could be used as an effective strategy for enhancing the productivity of shikonin derivatives., As the XAD-2 amount was increased up to 1.5 g/L, shikonin production was enhanced by removing shikonin produced and other products which might be inhibitory to cell growth. Most amount of shikonin produced was successfully recovered in XAD-2 (Over 99%). Using hairy root culture in a modified stirred tank reactor, the shikonin productivity and hairy root growth rate on the average were 9.34 mg/L day and 0.49 g DCW/L · day, respectively.     

Mixotrophy in gyrodinium galatheanum (DINOPHYCEAE): grazing responses to light intensity and inorganic nutrients*

This paper presents results of field and laboratory studies on mixotrophy in the estuarine dinoflagellate Gyrodinium galatheanum (Braarud) Taylor. We tested the hypotheses that this primarily photosynthetic organism becomes phagotrophic when faced with suboptimal light and/or nutrient environments. In Chesapeake Bay, incidence of feeding of this species on cryptophytes is positively correlated with prey density and concentrations of nitrate and nitrite, but negatively correlated with depth, salinity, and phosphate concentration. Feeding in natural assemblages and cultures increased hyperbolically with light intensity. The stoichiometric proportions of dissolved inorganic P and N (DIP:DIN) at the stations where G. galatheanum was present were far below the optimal growth P:N (1:10). Incidence of feeding was negatively related to the ratio of DIP to DIN, suggesting that P limitation may have induced feeding. Addition of nitrate, or addition of both nitrate and phosphate, inhibited feeding in a natural population, indicating that N limitation may also induce feeding. Ingestion of the cryptophyte, Storeatula major, by cultured G. galatheanum was higher in media low in nitrate or phosphate or both, but moderate rates of feeding occurred in nutrient‐replete cultures. When cells were grown in media with varying concentrations of nitrate and phosphate, N deficiency resulted in greater cellular N and Chl a losses than did P deficiency, but P deficiency stimulated feeding more than N deficiency. Both N and P deficiency, or P:N ratios that deviated greatly from 1:10, result in an increase of cellular carbon content and an increase in propensity to feed. Our results suggest that feeding in G. galatheanum is partly a strategy for supplementing major nutrients (N and P) that are needed for photosynthetic carbon assimilation. Feeding in G. galatheanum may also be a strategy for supplementing C metabolism or acquiring trace organic growth factors, since feeding occurs, although at a reduced rate, in nutrient‐replete cultures.     

The extent to which external oxygen transfer limits growth in shake flask culture of hairy roots

This study demonstrates the effects of oxygen limitations on growth of Atropa belladonna hairy roots in 250-mL shake flasks. Exponential growth was observed only for the first 6-8 d culture; after this time, growth continued with declining specific rate. Increasing the shaking speed and decreasing the medium volume improved biomass production and, to some extent, the specific growth rate of the roots. Direct enhancement of oxygen delivery, either by periodic exposure of the roots to air or oxygen enrichment of the atmosphere, also increased growth rates and/or final biomass levels. These results show that oxygen limitations are likely to affect biomass production and kinetic measurements in shake flask cultures of hairy roots. (c) 1997 Wiley & Sons, Inc. Biotechnol Bioeng 55: 520-526, 1997.     

Inhibitory role of root hairs on transport within root culture bioreactors

An experimental system was developed to produce root cultures of Hyoscyamus muticus with and without the profuse root hairs. Growth in the presence of 7.6 microM pyrene butyric acid (PBA) and 2.2 mM phosphate virtually eliminated root hairs, whereas growth rate, general morphology and nutrient yields remained unchanged in well-mixed flask culture. These root cultures were used to demonstrate decreased flow resistance in a tubular reactor as a result of root hair removal. To assess the impact on bioreactor performance, hairy and hairless root cultures were grown in a highly characterized 15-L bubble column bioreactor. In the absence of root hairs, the mixing was greatly enhanced; mixing times became shorter for the hairless culture at roughly 100 g (fresh weight)/L. By the end of the 3-week culture period, the mixing time of the hairy culture was 29 times longer than that of the hairless culture. The growth rate of the hairless culture in the bioreactor was as much as 2.4 times greater than growth of the hairy culture under the same conditions. The improved reactor performance was reflected in greater biomass accumulation and respiratory activity. These results show that the root hairs-which facilitate nutrient uptake in a static soil environment-are detrimental to growth in a liquid environment as an effect of their stagnating fluid flow and limiting oxygen availability.     

Tanshinone biosynthesis in Salvia miltiorrhiza and production in plant tissue cultures

Salvia miltiorrhiza Bunge (Lamiaceae) root, generally called Danshen, is an important herb in Chinese medicine widely used for treatment of cardiovascular diseases. Diterpenoid tanshinons are major bioactive constituents of Danshen with notable pharmacological activities and the potential as new drug candidates against some important human diseases. The importance of Danshen for traditional and modern medicines has motivated the research interest over two decades in the biosynthesis and biotechnological production of tanshinones. Although diterpenes in plants are presumably derived from the non-mevalonate (MVA) pathway, tanshinone biosynthesis in S. miltiorrhiza may also depend on the MVA pathway based on some key enzymes and genes detected in the early steps of these pathways. Plant tissue cultures are the major biotechnological processes for rapid production of tanshinones and other bioactive compounds in the herb. Various in vitro cultures of S. miltiorrhiza have been established, including cell suspension, adventitious root, and hairy root cultures, which can accumulate the major tanshinones as in the plant roots. Tanshinone production in cell and hairy root cultures has been dramatically enhanced with various strategies, including medium optimization, elicitor stimulation, and nutrient feeding operations. This review will summarize the above developments and also provide our views on future trends.     

Bioreactor for cultivation of red beet hairy roots and in situ recovery of primary and secondary metabolites

To arrive at an appropriate bioreactor design and in situ recovery of the products, red beet hairy roots were used as a model system where the levels of betalain pigments (betacyanins and betaxanthins) were followed as secondary metabolite and the peroxidase enzyme as primary metabolite. Medium volume and other kinetic parameters were found to play significant roles by way of directly affecting the biomass yield rather than a specific metabolite. The hydrodynamic stress created on the roots by large culture volume could be minimized by pulse‐feeding of medium in shake‐flasks; and by separating the biomass chamber from the aerated medium reservoir in circulatory fed‐batch bioreactor. Accordingly the bioreactor was modified to provide anchorage and air‐enrichment chamber which resulted in higher formation of both the metabolites than in shake‐flasks. Various down‐stream processing aspects such as in situ release of pigments by non‐destructive methods, followed by adsorption through a column and recovery by desorption were optimized for betalains. A strategy for simultaneous recovery of pigment and peroxidase was worked out using aqueous two phase extraction (ATPE).     

Performance of hairy root cultures of Cichorium intybus L. In bioreactors of different configurations

Summary A transformed root culture of Cichorium intybus L. cv. Lucknow Local grown in different configurations of bioreactors was examined. The roots grown in an acoustic mist bioreactor showed the best performance in terms of increased specific growth rate (0.072d⁻¹) and esculin content (18.5gl⁻¹), the latter of which was comparable to that of shake flask data. C. intybus hairy root cultures grown in an acoustic mist bioreactor produced nearly twice as much esculin as compared to roots grown in bubble column and nutrient sprinkle bioreactors. Studies relating to on-line estimation of conductivity and osmolarity to predict the growth of hairy root cultures are also discussed. The results demonstrate the efficacy and the advantages of an acoustic mist bioreactor for the cultivation of hairy root cultures, especially with reference to C. intybus hairy roots.     

Batch and fed-batch production of betalains by red beet (Beta vulgaris) hairy roots in a bubble column reactor

Hairy root cultures from red beet (Beta vulgaris L.), which could be used for the commercial production of biologically active betalain pigments, were cultivated in a 3 L bubble column bioreactor in batch mode with various rates of air supply. Both the growth of the roots and betalain volumetric yields were highest (12.7 g accumulated dry biomass/L and 330.5 mg/ L, respectively) with a 10 L/h (0.083 vvm) air supply. The air flow rate also influenced the betacyanins/betaxanthins ratios in the cultures. Growth and betalains production were then examined in two fed-batch regimes (with a 10 L/h air supply), in which nutrient medium was fed just once or on five occasions, designated FBI and FBII, respectively. The root mass accumulation was increased in the FBI feeding regime (to 13.3 g accumulated dry biomass/ L), while in FBII the betalains content was ca. 11% higher (15.1 mg betacyanins/g dry weight and 14.0 mg betaxanthins/g dry weight) than in the most productive batch regime. Data on the time course of the utilization of major components in the medium during both operational modes were also collected. The implications of the information acquired are discussed, and the performance of the hairy roots (in terms of both growth and betalains production) in the bubble column reactor and previously investigated cultivation systems is compared.     

Enhanced truncated-t-PA (CT-b) expression in high-cell-density fed-batch cultures of <Emphasis Type="Italic">Pichia pastoris</Emphasis> through optimization of a mixed feeding strategy by response surface methodology

Recently, Pichia pastoris has been the focal point of interest as an expression system for production of many recombinant proteins. The study and optimization of feeding strategy are of major importance to achieve maximum volumetric productivity in fed-batch cultivations. Among different feeding strategies used in P. pastoris fed-batch cultures, those trying to maintain a constant specific growth rate have usually resulted in superior productivities. The objective of the present study was to investigate and optimize the co-feeding of glycerol and methanol to attain maximum expression of t-PA in P. pastoris fed-batch cultures with constant specific growth rate. The experiments were designed by response surface methodology, considering the specific feeding rates of methanol and glycerol as independent variables. In each experiment, glycerol and methanol were fed according to a predetermined equation to maintain a constant specific growth rate. It was found that with glycerol feeding for higher specific growth rates, the inhibitory properties of glycerol are more pronounced, while the best expression level was achieved when the ratio of µ _set glycerol to that of methanol was around 1.67. In all specific growth rates tested, almost a similar ratio of the specific glycerol feeding rate to that of methanol led to the maximum protein production and activity. The statistical model predicted the optimal operating conditions for µ _set glycerol and that of methanol to be 0.05 and 0.03 h^?1, respectively. Applying the optimum strategy, maximum of 52 g/L biomass, 300 mg/L t-PA and 340,000 IU/mL enzyme activity were obtained.     

Population balance approach to modeling hairy root growth

Though numerous models have been developed to describe the growth of microbial cell cultures, far fewer models are available to describe the growth of hairy root cultures. Here a population balance model is proposed to simulate the growth of hairy roots. The model accounts for the increase in biomass due to elongation of a branch by cell division as well as the formation of new branches. The model incorporates the fact that although the likelihood of the formation of a new lateral branch is a maximum at a specific age of the parent branch, lateral branches can form over a distribution of ages of the parent branch. Model parameters are estimated using the genetic algorithm based on experimental data for batch and continuous bioreactors. The model proposed here may provide a better understanding of the increase in biomass of hairy root cultures.     

Biomass production of hairy roots of Artemisia annua and Arachis hypogaea in a scaled‐up mist bioreactor

Hairy roots have the potential to produce a variety of valuable small and large molecules. The mist reactor is a gas phase bioreactor that has shown promise for low‐cost culture of hairy roots. Using a newer, disposable culture bag, mist reactor performance was studied with two species, Artemisia annua L. and Arachis hypogaea (peanut), at scales from 1 to 20 L. Both species of hairy roots when grown at 1 L in the mist reactor showed growth rates that surpassed that in shake flasks. From the information gleaned at 1 L, Arachis was scaled further to 4 and then 20 L. Misting duty cycle, culture medium flow rate, and timing of when flow rate was increased were varied. In a mist reactor increasing the misting cycle or increasing the medium flow rate are the two alternatives for increased delivery of liquid nutrients to the root bed. Longer misting cycles beyond 2–3 min were generally deemed detrimental to growth. On the other hand, increasing the medium flow rate to the sonic nozzle especially during the exponential phase of root growth (weeks 2–3) was the most important factor for increasing growth rates and biomass yields in the 20 L reactors. A. hypogaea growth in 1 L reactors was µ = 0.173 day^?1 with biomass yield of 12.75 g DW L^?1. This exceeded that in shake flasks at µ = 0.166 day^?1 and 11.10 g DW L^?1. Best growth rate and biomass yield at 20 L was µ = 0.147 and 7.77 g DW L^?1, which was mainly achieved when medium flow rate delivery was increased. The mist deposition model was further evaluated using this newer reactor design and when the apparent thickness of roots (+hairs) was taken into account, the empirical data correlated with model predictions. Together these results establish the most important conditions to explore for future optimization of the mist bioreactor for culture of hairy roots. Biotechnol. Bioeng. 2010;107: 802–813. © 2010 Wiley Periodicals, Inc.     

Optimal feeding under stoichiometric constraints: a model of compensatory feeding with functional response

When the nutrient content of food is limited, herbivores often increase their feeding rates. Such an increase in the feeding rate is called ‘compensatory feeding’. Although it has a number of implications for herbivore population and plant–forager dynamics, the compensatory feeding is not yet functionally formulated especially in relation with ecological stoichiometry. Therefore, we constructed a simple mathematical model by incorporating the optimal feeding rate into the type II functional response to maximize a forager's growth rate under constraints of carbon or nutritionally important element like phosphorus (P). We used the planktonic herbivore Daphnia as a model herbivore. The model revealed that the optimal feeding rate increased by using excess carbon when relative P content of food was less than a certain level, which is known as the threshold elemental ratio. This level changed with the change of food abundance. It also showed that whether or not foragers should exhibit compensatory feeding depends on their stoichiometric characteristics and digestive traits, and also on the assimilability of a given food. These findings are helpful to test the feeding conditions under which compensatory feeding is advantageous for a given animal. Our model can be easily incorporated into forager population dynamics and prey‐consumer interaction models because the optimal feeding rate can be analytically given.     

Control of the specific growth rate of Bacillus subtilis for the production of biosurfactant lipopeptides in bioreactors with foam overflow

This paper formulates a feeding law for a bioprocess dedicated to the production of an antibiotic surfactant using Bacillus subtilis. The specificity of the process relies on the use of the surface active property of the product to extract it by foaming. The control law is designed to maintain a constant specific biomass growth rate while taking into account the particularity of the process. This law can be regarded as a generalization of the conventional exponential feeding strategy and is generic enough to encompass the case of continuous processes with partial recycling. Conventional exponential feeding strategies indeed fail to account for the loss of biomass induced by the foaming. Previous experiments have provided a model of the process and values for its parameters. From this information, a feeding rate law was computed using the feeding strategy proposed in this paper and applied to an experimental culture. This experiment allows discussion of the modeling of the biomass extraction method used in this study. The results on the estimated specific growth rate highlight the complete agreement between the expected and experimental features. Further process optimization studies can now be performed on the basis of the constant specific biomass growth rate.     

Effects of sodium sulfate on the freshwater microalga Chlamydomonas moewusii: implications for the optimization of algal culture media

The study of the microalgal growth kinetics is an indispensable tool in all fields of phycology. Knowing the optimal nutrient concentration is an important issue that will help to develop efficient growth systems for these microorganisms. Although nitrogen and phosphorus are well studied for this purpose, sulfur seems to be less investigated. Sulfate is a primary sulfur source used by microalgae; moreover, the concentration of this compound is increasing in freshwater systems due to pollution. The aim of this study was to investigate the effects of different sodium sulfate concentrations in the culture medium on growth and growth kinetics of the freshwater microalga Chlamydomonas moewusii. Production of biomass, chl content, kinetic equations, and a mathematical model that describe the microalgal growth in relation with the concentration of sodium sulfate were obtained. The lowest concentration of sodium sulfate allowing optimal growth was 0.1 mM. Concentrations higher than 3 mM generated a toxic effect. This work demonstrates that this toxic effect was not directly due to the excess of sulfate ion but by the elevation of the ionic strength. An inhibition model was successfully used to simulate the relationship between specific growth rate and sodium sulfate in this microalga.