State and parameter estimation of microalgal photobioreactor cultures based on local irradiance measurement

Local photosynthetic photon flux fluence rate (PPFFR) determined by a submersible 4pi quantum micro-sensor was used in developing a versatile on-line state estimator for stirred-tank microalgal photobioreactor cultures. A marine micro-alga Dunaliella salina was used as a model organism in this study. On-line state estimation was realized using the extended Kalman filter (EKF), based on a state model of the photobioreactor and on-line local PPFFR measurement. The dynamic state model for the photobioreactor was derived based on mass-balance equations of the relevant states. The measurement equation was established based on an empirical correlation between the microalgal biomass concentration and the local PPFFR measured at a fixed point inside the photobioreactor. An internal model approach was used to estimate the specific growth rate without the need of state-based kinetic expression. The estimator was proven to be capable of estimating biomass concentration and specific growth rate, as well as phosphate and dissolved oxygen concentrations in a photobioreactor illuminated with either fixed or time-varying incident radiation. The quantum sensor was shown to be robust and able to quickly respond to dynamic changes in local PPFFR. In addition, the quantum sensor outputs were not affected by bubble aeration or agitation within the typical operating range. The strong filtering capacity of EKF gives the state estimator superior performance compared to direct calculation from the empirical biomass/local PPFFR correlation. This state estimation system makes use of inexpensive and reliable sensor hardware to report key process dynamics of microalgal photobioreactor cultures on-line, enabling improved operation of such a process.     

Comparative study of the fermentation of D-glucose/D-xylose mixtures with Pachysolen tannophilus and Candida shehatae

We have performed a comparative analysis of the fermentation of the solutions of the mixtures of D-glucose and D-xylose with the yeasts Pachysolen tannophilus (ATCC 32691) and Candida shehatae (ATCC 34887), with the aim of producing bioethanol. All the experiments were performed in a batch bioreactor, with a constant aeration level, temperature of 30v°C, and a culture medium with an initial pH of 4.5. For both yeasts, the comparison was established on the basis of the following parameters: maximum specific growth rate, biomass productivity, specific rate of substrate consumption (q_s) and of ethanol production (q_E), and overall ethanol and xylitol yields. For the calculation of the specific rates of substrate consumption and ethanol production, differential and integral methods were applied to the kinetic data. From the experimental results, it is deduced that both Candida and Pachysolen sequentially consume the two substrates, first D-glucose and then D-xylose. In both yeasts, the specific substrate-consumption rate diminished over each culture. The values q_s and q_E proved higher in Candida, although the higher ethanol yield was of the same order for both yeasts, close to 0.4 kg kg^у.     

The tuning of a model-based estimator for the specific growth rate of Candida utilis

Control of microbial conversion processes is frequently inhibited by the infeasibility of measuring important process variables. In order to circumvent this lack of measurements, an accurate or valuable and conveniently measurable on-line hardware measurement can be combined with the balance equations describing the process to obtain estimates of less easily measurable variables. In this article the on-line estimation of the specific growth rate of Candida utilis is evaluated. The observer-based estimator requires a hardware measurement of the biomass during fermentations in conjuction with a model of the process; therefore the Biomass Monitor, giving an on-line measurement of viable biomass, is used in the bioreactor experiments described. The optimal tuning of the estimation for the experimental conditions is described and several alternative adaptations of the design of the estimator are presented. The influence of implemented time intervals for discretization of the estimator on the reliability of the estimated growth rate values is discussed. Additionally, the necessary choice of an initial value of the estimated specific growth rate has proven to be of great importance in practice.     

Modeling of growth and energy metabolism of Pichia pastoris producing a fusion protein

A fusion protein composed of a cellulose binding domain from Neocallimastix patriciarum cellulase A and Candida antarctica lipase B (CBD-lipase) was produced by Pichia pastoris methanol utilization plus phenotype in high cell-density cultures. The genes expressing CBD-lipase were fused to the alpha-factor secretion signal sequence of Saccharomyces cerevisiae and placed under the control of the alcohol oxidase gene (AOX1) promoter. To control the repression and induction of AOX1 and oxygen demand at high cell density, a four-stage process was used. Batch growth on glycerol was used in the first step to provide biomass (28 g L^-1) while product formation was prevented due to repression of the AOX1. The second stage was exponential fed-batch growth on glycerol, which caused a slight increase of the enzyme alcohol oxidase activity due to derepression of the AOX1. This procedure resulted in smooth transition to exponential fed-batch growth on methanol, the third stage, in which the AOX1 was strongly induced. The fourth stage was constant fed-batch growth on methanol used to control the oxygen demand at the high cell density. A kinetic model was developed that could predict biomass growth and oxygen consumption in processes with and without oxygen-enriched air. With oxygen enrichment to 34% O₂ in the inlet air the methanol feed rate could be increased by 50% and this resulted in 14% higher final cell density (from 140 to 160 g L^-1 cell dry weight). The increased methanol feed rate resulted in a proportionally increased specific rate of product secretion to the medium. After an initial decrease, the synthesis capacity of the cell was kept constant throughout the cultivation, which made the product concentration increase almost constantly during the process. The kinetic model also describes how the low maintenance demand of P. pastoris compared with E. coli enables this organism to grow to such high cell densities.     

Combining yield coefficients and exit-gas analysis for monitoring of the baker's yeast fed-batch fermentation

Baker's yeast is one of the micro-organisms that is studied most in literature. Therefore, a lot of knowledge on the biochemical pathways and corresponding yield coefficients is available. This knowledge is combined with measurements of oxygen and carbon dioxide in the exit-gas to determine the coefficients appearing in the stoichiometric equations. In this manner, two measurements are sufficient to yield on-line estimates for biomass, glucose, ethanol and the specific growth rate, and information about the (ill-defined) nitrogen source NH_q. This is not possible if the yield coefficients are not included in the estimation procedure. A sensitivity analysis illustrates that this estimation scheme is rather insensitive to uncertainties on the yield coefficients.     

Light gradient partitioning by tropical tree seedlings in the absence of canopy gaps

To explore the importance of light availability for seedling growth in low light environments, we examined light-dependent growth, biomass allocation and mortality of tree seedlings growing in sites with 0.2-6.5% full sun, the range of light commonly encountered in the understory of closed canopy, lowland tropical forests. We transplanted seedlings of the canopy tree species, Dipteryx panamensis, Virola koschnyii, and Brosimum alicastrum into second-growth forest and native tree plantations at La Selva Biological Station, Costa Rica. We assessed seedling survival, growth, and seedling light environments bimonthly for 14 months. Plants were harvested at the end of the study to assess leaf area, total biomass, biomass partitioning and root growth. Survivorship of all species exceeded 60% across all microsites, although both D. panamensis and B. alicastrum had lower probabilities of survival in the darkest microsites. All species showed a strong positive relationship between light availability and growth, increasing in total biomass as light increased. However, the strength of the growth response differed among species causing a change in the rank order of species growth rates as light availability increased. Although D. panamensis showed the lowest growth rates in the darkest microsites, a strong response to increasing light led to a cross-over in performance, such that D. panamensis had the highest growth rate at the highest light levels studied. These data suggest that resource gradient partitioning could occur even in low light environments (0.2-6.5%). Given the limited range of light regimes sampled (i.e., non-gap microsites), our data demonstrate that growth of tropical tree seedlings beneath closed canopies is highly sensitive to light availability and that shade-tolerant species vary in these responses. Our results show that understory light heterogeneity, in the absence of canopy gaps, can significantly affect recruitment processes for shade-tolerant tree species.     

Multirate state and parameter estimation in an antibiotic fermentation with delayed measurements

This article discusses issues related to estimation and monitoring of fermentation processes that exhibit endogenous metabolism and time-varying maintenance activity. Such culture-related activities hamper the use of traditional, software sensor-based algorithms, such as the extended kalman filter (EKF). In the approach presented here, the individual effects of the endogenous decay and the true maintenance processes have been lumped to represent a modified maintenance coefficient, m(c). Model equations that relate measurable process outputs, such as the carbon dioxide evolution rate (CER) and biomass, to the observable process parameters (such as net specific growth rate and the modified maintenance coefficient) are proposed. These model equations are used in an estimator that can formally accommodate delayed, infrequent measurements of the culture states (such as the biomass) as well as frequent, culture-related secondary measurements (such as the CER). The resulting multirate software sensor-based estimation strategy is used to monitor biomass profiles as well as profiles of critical fermentation parameters, such as the specific growth for a fed-batch fermentation of Streptomyces clavuligerus. (c) 1994 John Wiley & Sons, Inc.     

Modelling of continuous microbial cultivation taking into account the memory effects

The problem of chemostat dynamics modelling for the purpose of control is considered. The "memory" of the culture is explicitly taken into account. Two possibilities for improving the quality of the proposed modelling approaches are discussed. A general model that accounts for the culture `memory' by means of different `memory' functions in the expressions of the specific growth rate and of the specific consumption rate and a polynomial function of the substrate concentration for the yield factor is proposed. The case where the maintenance energy is taken into account is also discussed. Two modifications of the general model (w-type and S-type) are presented. A zero-order `memory' function and a i-function with delay are applied in order to describe the `memory' effects. Continuous growth of the strain Saccharomyces cerevisiae on a glucose limited medium is considered as a case study. Detailed investigations of the variety of models, derived from the general model by applying different `memory' functions and different assumptions are carried out. The results are compared with those previously reported for the same process. It is shown that a significant improvement in predicting the substrate dynamics (not accompanied by any decrease in the quality of the model with respect to the biomass concentration) could be achieved, involving a first- or second-order polynomial function for the yield factor. It is also shown that the quality of the model mainly depends on the way that `memory' function is incorporated. The detailed investigations give priority to the w-type models. In this case past values of both biomass and substrate variables are considered. The time delay models with pure (constant) delay and those which account for the culture `memory' by zero-order `memory' function (adaptability parameter) are compared with respect to their utilization for the purpose of model-based control.     

Evaluation of on-line viable biomass measurements during fermentations of Candida utilis

In this article the suitability of the Biomass Monitor for on-line measurement of viable biomass is thoroughly evaluated during aerobic fermentations of Candida utilis. Successively a number of specifications of the measuring device are discussed for the studied biological system. The optimal measurement frequency for the given experimental conditions is determined. Furthermore, reliable calibrations of the capacitance readings versus well-known off-line analysis of dry weight and plate counts of the yeast have been established. In addition, the impact of varying fermentation conditions such as stirrer speed and air flow rate together with the influence of the oxygen concentration and conductance of the medium on the capacitance signal have been studied and quantified when a significant influence was observed. It is illustrated that knowledge of the viable biomass during fermentations is very useful in the estimation of the specific growth rate of the organism.     

Joint aerobic biodegradation of wastewater from table olive manufacturing industries and urban wastewater

Wastewater generated in the elaboration of table olives has been treated using activated sludge from a municipal wastewater plant after adequate acclimation. To avoid bactericide properties of some chemical structures present in this type of effluents, synthetic urban wastewater has been used to dilute the original wastewater. The main parameters affecting efficiency of biological processes have been studied. Thus, initial biomass concentration, temperature up to 303 K (upper working temperature limit = 313 K) and initial substrate concentration exerted a positive influence on COD degradation rate. The optimum pH was found to be around 7, experiencing a slight inhibition on cell activity at pH 4. Under the experimental conditions investigated other parameters like polyphenol content, absorbance at 254 nm and total organic carbon were also reduced to some extent. Only nitrates amount was increased after the biological process took place. A kinetic model based on Monod equation was proposed and applied to experimental results. The maximum specific growth rate was calculated by means of the aforementioned kinetic model. The value of this parameter as a function of temperature was fitted to an Arrhenius expression, w_max = 9.43 2 10¹⁰ exp(72021/RT) h^у (R in J mol^у K^у283 K < T < 303 K, pH , 7-10).     

Stable adaptive algorithm for simultaneous estimation of time-varying parameters and state variables in aerobic bioprocesses

The application of modern model based control algorithms in the bioprocesses is hampered by the lack of accurate and cheap on-line sensors, capable of providing on-line measurements of the main process variables and parameters. In this paper, a new approach for estimation of immeasurable time-varying parameters and state variable is presented for a class of aerobic bioprocesses using only on-line measurements of the oxygen uptake rate. The approach consists in the design of a new parameter estimator of biomass growth rate and yield coefficient for oxygen consumption on the basis of the theory of adaptive estimation. The dynamical equation of the measurable reaction rate, oxygen uptake rate, is presented as a linear one with respect to the biomass growth rate and the yield coefficient for oxygen consumption. In this way, the structure of the proposed estimator becomes linear time-varying one. After some mathematical transformations, that structure is presented in a form, allowing to be derived the stability conditions using some theoretical results concerning the stability of adaptive observers. The estimates of the yield coefficient for oxygen consumption, the biomass concentration and specific growth rate are obtained then on the basis of the generated estimates using well known kinetic models of bioprocesses. With respect to previous similar approaches, the new estimation algorithm gives stable estimates not only of immeasurable state variable and reaction rates but likewise of an yield coefficient. The behavior of the proposed estimator is studied under inexact initial conditions, step changes of dilution rate and in the presence of measurement noise by simulations using a process model, which belongs to the investigated class of bioprocesses.     

Fluctuations of macrobenthic populations: a link between climate-driven river run-off and sole fishery yields in the Gulf of Lions

The Rhone river is the most important input to the Mediterranean Sea, responsible for 50% of the primary productivity of the Gulf of Lions. A highly variable amount of 1-23᎒⁶ t year^-1 of terrestrial material is exported to the sea by the Rhone and stocked on the continental shelf for the most part. Soft-bottom communities off the Rhone delta were dominated by polychaetes both in species richness and abundance, and exhibited strong temporal fluctuations mainly related to flooding events. Floods caused pulses of organic matter followed, with different time lags, by peaks of polychaetes. Opportunistic, short-lived species, such as Mediomastus sp. and Aricidea claudiae, exhibited high short-term peaks in density and biomass a few months after flooding events. Conversely, long-lived species, such as Laonice cirrata and Sternaspis scutata, peaked in density and biomass with a time lag of 1-3 years, and their population increase lasted for a few years. The common sole, Solea solea, is a voracious predator of polychaetes which represent >80% of its prey. A positive correlation was found between the mean annual discharge of the Rhone river and the annual commercial landings of S. solea with a time lag of 5 years in the two fishing harbours (Sete and Martigues) located close to the Rhone delta. The long-term increase in food (i.e. polychaete density and biomass) after flooding events might favour the different stages of the sole life cycle, enhancing its population size for several years. Fluctuations of sole fishery yields in the Gulf of Lions could be influenced by climate, as the Rhone river flow is related to the North Atlantic Oscillation that drives precipitation over Western Europe.     

Zooplankton biomass variation in relation to climatic conditions in the Barents Sea

Over the last two decades, there have been large changes in the zooplankton biomass in the Barents Sea. These biomass variations are mainly attributed to predation pressure and environmental factors (e.g. advective transport). When stock size of capelin (Mallotus villosus), a major planktivorous fish in the Barents Sea ecosystem, was quite low as in 1986 and 1994, the zooplankton biomass showed marked increase. However, the increase in the zooplankton biomass occurred in different water masses during 1986 and 1994. In 1986, a climatically cold year, the plankton biomass was highest in the Arctic waters of the northeastern Barents Sea. This is probably due to the increase in larger Arctic amphipod species, such as Themisto libellula. In 1994, a climatically warm year, the zooplankton biomass was high in the Atlantic waters of the southwestern Barents Sea. The large increase in zooplankton biomass in the Atlantic waters in 1994 was presumably due to the higher inflow of advected organisms, e.g. Calanus spp., as well as high temperatures, which may lead to high growth rates of zooplankton. Throughout the studied region, the plankton biomass in the "cold year" of 1986 was generally much lower than in the "warm year" of 1994.     

Oxygen availability and the growth of Escherichia coli W3110: A problem exacerbated by scale-up

By examining the effects of oxygen availability on the batch growth of Escherichia coli on glucose the present study seeks to determine the responses of growing cells to varying degrees of oxygen excess, limitation or starvation as might be experienced in an industrial-scale bioreactor. It was found that as the degree of oxygen limitation increases so too does the byproduct acetate production in addition to a concomitant decrease in the substrate based biomass yield coefficient and maximum specific growth rate. Similar, although not as severe, responses to excess oxygen growth conditions were also observed. This result supports the concept of oxygen being potentially toxic to growing organisms, resulting in, at the very least, a degree of inhibition.     

Competition between macroalgae and corals: effects of herbivore exclusion and increased algal biomass on coral survivorship and growth

Recent declines in coral abundance accompanied by increases in macroalgal cover on Florida reefs highlight the importance of competition for space between these groups. This paper documents the frequency of coral-algal interactions on the Northern Florida Reef Tract and evaluates the effects of grazer exclusions and experimental algal addition on growth and tissue mortality of three coral species, Siderastrea siderea, Porites astreoides, and Montastraea faveolata. The frequency of interactions between corals and macroalgae was high as more than 50% of the basal perimeter of colonies was in contact with macroalgae; turf forms, Halimeda spp., and Dictyota spp. were the most common groups in contact with corals. Decreased grazing pressure resulted in significant increases in algal biomass within cages, and caged corals showed species-specific susceptibility to increased algal biomass. While no effects were detected for S. siderea, significant decreases in growth rates were documented for caged P. astreoides which had growth rates three to four times lower than uncaged colonies. When an algal addition treatment was included to duplicate maximum algal biomass levels documented for reefs in the area, colonies of P. astreoides in the algal addition treatment had growth rates up to ten times lower than uncaged colonies. High susceptibility to algal overgrowth was also found for the reef-building coral M. faveolata, which experienced significant tissue mortality under both uncaged (5.2% decrease in live tissue area per month) and caged (10.2% per month) conditions. The documented effects of increased algal biomass on coral growth and tissue mortality suggest a potential threat for the long-term survivorship and growth of corals in the Florida Reef Tract if present rates of algal growth and space utilization are maintained.     

Effect of time and temperature of fermentation on the microflora of grape pomace

The effects of fermentation time and temperature of grape pomace on the number of bacteria and yeasts were assessed using ANOVA of experimentally generated data, as well as analysis of models derived from first principles and fitted by non-linear regression to such data. Specific rates of death of yeasts and bacteria were experimentally obtained at different fermentation times (pilot scale), and at different fermentation times and different temperatures (laboratory scale) for pomace of Alvarinho and Loureiro grape varieties obtained after vinification at two different locations. Viable numbers of yeasts and bacteria in grape pomace were high, especially in the first 3 week of fermentation; for bacteria, there was an increase of their levels during the first 3 week, followed by a significant decrease towards 9 week; for yeasts there was a monotonic decrease throughout such whole period. The numbers of viable microorganisms were mathematically correlated with time and temperature for both wineries using mechanistic models and following a methodology of increasing model complexity. After having checked the validity of underlying assumptions of normal distribution and constant variance of residuals of the experimental data, determination of the best nested model was based on an F-test; such model considered that the behavior of the microbial population is well described by a constant specific growth rate and an increasing specific death rate, both of which vary with temperature following Arrhenius relationships. Activation energies for the specific death rates of yeasts and bacteria were (1.469-1.560)᎒⁴ and (2.584-4.152)᎒⁴ cal/mol, respectively, for the temperature range 20-35 ^:C. Prediction of the time profile of viable numbers of the major families in the adventitious microflora of grape pomace, as a function of fermentation parameters that are easily manipulated is important in attempts to eventually standardize this solid-state, ill-defined fermentation and so eventually optimize the manufacture of marcs obtained by steam distillation of such fermented pomaces.     

Growth dynamics of oak seedlings (Quercus macrocarpa Michx. and Quercus muhlenbergii Engelm.) from gallery forests: implications for forest expansion into grasslands

Seedling growth dynamics of Quercus macrocarpa Michx. and Quercus muhlenbergii Engelm. were compared over a 3-month period under optimal growth conditions. These two species are the dominant trees at the western limit of the eastern deciduous forest, and are typically confined to gallery forests along stream beds in tallgrass prairie. Since tallgrass prairie is characterized by a highly variable climate and is prone to periodic drought, we hypothesized that these oaks would have rapid root growth and produce deep taproots as seedlings, enabling them to avoid drought stress and persist in this region. These traits may also facilitate forest expansion into the more xeric tallgrass prairie if fires are suppressed. Taproots of Q. macrocarpa and Q. muhlenbergii grew to approximately 140 cm and 100 cm in length, respectively, after 104 days. In both species, 65% or more of seedling biomass was allocated below ground, and root/total biomass was significantly greater in Q. muhlenbergii at 0-20 and 21-40 days after germination. The seedling taproot elongation rates reported here are much greater than rates reported in other eastern deciduous forest trees. Long-term precipitation data and soil moisture patterns from tallgrass prairie, when combined with rapid taproot elongation rates, suggest that soil moisture may not limit oak establishment or growth in tallgrass prairie in most years, although water uptake by roots was not measured in this study. Other factors, such as fire, herbivory, and seed predation and dispersal may be equally important in constraining the distribution of these species to gallery forests.     

Dynamic modelling of aerobic bioprocesses in gel particles with immobilised cells

A dynamic model for aerobic growing cells immobilised into gel beads is developed and its operation is illustrated for the case of gluconic acid production by a strictly aerophilic strain of Gluconobacter oxydans. The model consists of both kinetic and mass transfer equations predicting the time course of bulk and intraparticle concentrations of substrates, products, and biomass. The model includes a product inhibition term. The parameter values are taken from own studies and from the literature. A sensitivity analysis of the model shows that the most significant parameters for the process are the biotransformation rate constant, the specific cell growth rate in the bulk, and the Thiele modulus for glucose. The computer simulation reveals that depending on the parameter values the gel particles might perform as a source or a sink of the product, thus enhancing or retarding the net process. For a specific parameter selection, the biotransformation in the pellets can prevail compared with the bulk in the beginning of the process as long as the direction of the product diffusion flux is from the beads toward the bulk. Since the process in the free culture dominates, the system is more sensitive to parameters associated with the bulk phase (aeration rate, specific microbial growth rate, oxygen uptake rate). The model can be applied for prediction and fast evaluation of the performance of aerobic processes accomplished by immobilised growing cells.     

On-line estimation of the specific growth rate based on viable biomass measurements: experimental validation

The application of model based control techniques to biotechnological processes is often hampered due to the lack of reliable on-line sensors. This problem can be tackled by the application of software sensors, in which the available hardware measurements are combined with the model equations. The resulting estimates serve as additional measurements useful for process monitoring and control. In this paper, an observer based estimator for the specific growth rate based on on-line viable biomass measurements is studied. Several fed-batch experiments with baker's yeast in a stirred tank bioreactor illustrate the design, tuning, and implementation from a practical point of view. The main contributions of this paper are to illustrate (i) the implementation and validation of the presented algorithm in real-time, (ii) the use of an advanced on-line biomass measurement, and (iii) the design and tuning of the algorithm from a practical point of view. Real-time knowledge of the specific growth rate is important because it yields information on the viability of the cells and it can be used in real-time feedback control algorithms.     

Optimized fed-batch fermentation of L-β-hydroxy isobutyric acid by Yarrowia lipolytica

Yarrowia lipolytica KCCM50506, which transforms isobutyric acid to L-#-hydroxy isobutyric acid (L-#-HIBA), was screened. Chemostat cultures were carried out in jar fermentors at dilution rates of 0.02 h^у to 0.12 h^у. L-#-HIBA fermentation-regulating factors were determined to be specific growth rate, and concentrations of glucose and isobutyric acid in fermentor from analysis of steady-state data. The specific productivity of L-#-HIBA increased as the specific growth rate increased, apparently as a growth-associated type of product formation. A fed-batch culture was carried out under optimum conditions where the concentrations of glucose and isobutyric acid in the fermentor were maintained at 23 g l^у and 9 g l^у, respectively. The concentrations of cells and L-#-HIBA obtained at the end of fermentation were 20 g l^у and 49 g l^у, respectively, corresponding to 2.0 and 2.7 times more than concentrations in batch culture.