On-line biomass monitoring by capacitance measurement.

An in-situ, steam-sterilizable capacitance probe was used to follow the biomass concentration on-line, in bioreactors from 20 to 2000 l total volume. Microbial cultures of Saccharomyces cerevisiae, Pichia pastoris and Streptomyces virginiae were grown in batch and fed-batch culture in both defined and complex media in order to demonstrate the wide dynamic operating range of the instrument. A linear correlation was found between the on-line capacitance measurement and the off-line measurements (optical density, OD620; packed mycelial volume, PMV; biomass concentration X, and colony forming units, CFU ml-1) for biomass concentrations (dry cell weight) up to 30 g l-1 (St. virginiae), 106 g l-1 (S. cerevisiae) and 89 g l-1 (P. pastoris). The on-line capacitance measurement was slightly influenced by variations in agitation speed and strong extraneous radio frequencies. A specific capacitance constant (Cs) was defined for all microbial cells which was dependent on cell viability and cell size. The Cs was easy to calculate using the on-line capacitance measurement and an off-line estimation of biomass concentration. The Biomass Monitor proved suitable for precise on-line monitoring of both homogeneous (uni-cellular) and heterogeneous (mycelial) cultures in bioreactors.     

A novel fiber optic probe for on-line monitoring of biomass concentrations

A fiber optic biomass probe for on-line measurement of biomass concentration was designed. Results of biomass concentration monitoring experiments with suspended cells of baker's yeast as well as an experimental cultivation of S. cerevisiae are presented. The device was able to observe biomass concentrations of 14 g l^у S. cerevisiae. By means of correlations the capability of estimating the biomass concentration from the probe signal is demonstrated.     

On-line biomass measurements in bioreactor cultivations: comparison study of two on-line probes

Two on-line probes for biomass measurement in bioreactor cultivations were evaluated. One probe is based on near infrared (NIR) light absorption and the other on dielectric spectroscopy. The probes were used to monitor biomass production in cultivations of several different microorganisms. Differences in NIR probe response compared to off-line measurement methods revealed that the most significant factor affecting the response was cell shape. The NIR light absorption method is more developed and reliable for on-line in situ biomass estimation than dielectric spectroscopy. The NIR light absorption method is, however, of no significant use, when the cultivation medium is not clear, and especially in processes using adsorbents or solid matrix for the microorganism to grow on. The possibilities offered by dielectric spectroscopy are impressive, but the on-line probe technology needs to be improved.     

Computer-aided baker's yeast fermentations.

The economics of yeast production depend heavily upon the cellular yield coefficient on the carbon source and the volumetric productivity of the process. The application of an on-line computer to maximize these two terms during the fermentation requires a continuous method of measuring cell density and growth rate. Unfortunately, a direct sensor for biomass concentration suitable for use in industrial fermentations is not available. Material balancing, with the aid of on-line computer monitoring, offers an indirect method of measurement. Laboratory results from baker's yeast production in a 14-liter fermentor (with a PDP-11/10 computer for on-line analyses) show this indirect measurement technique to be a viable alternative. From the oxygen uptake and carbon dioxide production data, gas flow rate, and ammonia addition rate, the cell density during the fermentation has been estimated and found to compare well with actual fermentation data.     

Estimation of multiple biomass growth rates and biomass concentration in a class of bioprocesses

In this paper, an approach to the estimation of multiple biomass growth rates and biomass concentration is proposed for a class of aerobic bioprocesses characterized by on-line measurements of dissolved oxygen and carbon dioxide concentrations, as well as off-line measurements of biomass concentration. The approach is based on adaptive observer theory and includes two steps. In the first step, an adaptive estimator of two out of three biomass growth rates is designed. In the second step, the third biomass growth rate and the biomass concentration are estimated, using two different adaptive estimators. One of them is based on on-line measurements of dissolved oxygen concentration and off-line measurement of biomass concentrations, while the other needs only on-line measurements of the carbon dioxide concentration. Simulations demonstrated good performance of the proposed estimators under continuous and batch-fed conditions.     

Real time monitoring biomass concentration in Streptomyces clavuligerus cultivations with industrial media using a capacitance probe

On-line monitoring biomass concentration in mycelial fed-batch cultivations of Streptomyces clavuligerus grown with soluble and partially insoluble complex media, was investigated with an in-situ capacitance probe fitted to an industrial pilot-plant tank. Standard off-line and on-line biomass determinations, including cell dry weight, packed mycelial volume, viscosity, DNA concentration and total CO(2) evolution in the exhaust gases, were performed throughout the experiments and compared to on-line capacitance measurements. Linear relations between capacitance and all other measurements were developed for both media that hold only in defined process phases, depending on the biomass state and the amount of insoluble matter present. For the industrial complex culture media good linear relations were obtained in the fast growth phase between capacitance and DNA concentration and total CO(2) evolution, while in the subsequent transition and stationary phases only with apparent viscosity was a reasonable correlation found. The capacitance probe was shown to be a valuable tool for real-time monitoring biomass concentration in industrial-like cultivation of mycelial streptomycetes.     

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.     

The dielectric permittivity at radio frequencies and the bruggeman probe: novel techniques for the on-line determination of biomass concentrations in plant cell cultures

A novel technique is described for the measurement of the volume fraction of biomass in a suspension by the simultaneous measurement of the conductivity of a suspension containing cells and of the medium in which the cells are suspended. The presence of non-conducting particulate matter in a suspension will cause the conductivity of a suspension to be decreased relative to that of the medium in which the particles are suspended. A simple equation (the Bruggeman equation) describes the relationship between the volume fraction of non-conducting particulate matter and the decrease in conductivity. The accuracy of this method for the determination of the biomass concentration of plant cells (Festuca arundinacea) in culture was shown. The method was successfully applied to the on-line determination of biomass concentrations during the growth of F. arundinacea cultures, and gave good agreement with biomass levels as determined from measurements of the radio-frequency dielectric permittivity of such cultures.     

Isolation and biochemical characterization of cell wall tight protein complex involved in self-flocculation of Kluyveromyces bulgaricus

Flocculation of yeasts is a cell–cell aggregation phenomenon which is driven by interactions between cell wall lectins and cell wall heteropolysaccharides. In Sabouraud medium, Kluyveromyces bulgaricus was highly flocculent. Incubation of flocculent K. bulgaricus cells with EDTA or Hecameg® led to extracts showing hemagglutinating and flocculating properties. Purification of the extracts by native PAGE gave two bands which allowed flocculation of deflocculated K. bulgaricus. Both bands with specific reflocculating activity were composed of five subunits, of which only three possessed weak reflocculating activity upon deflocculated yeast. The mixture of these three proteins allow the recovery of initial specific reflocculating activity of the complex. These three proteins, denoted p28, p36 and p48, presented, in their first 15 amino acids, homologies with glycolysis enzymes, i.e., 3-phosphoglycerate mutase, glyceraldehyde-3-phosphate dehydrogenase and enolase, respectively. However, no such enzymatic activity could be detected in the crude extract issued from treatment with EDTA and Hecameg® of flocculent yeast cells. When yeasts had grown in glucose poor medium, flocculation was drastically affected. The EDTA and Hecameg® crude extracts showed weak reflocculating activity. After PAGE, the protein complexes did not appear in the EDTA extract, but they did appear in the Hecameg® crude extract. These results suggest that: (i) self-flocculation of K. bulgaricus depends on the expression of different floc-forming protein complex, (ii) these proteins are galactose specific lectins showing homologies in their primary structure with glycolysis enzymes.     

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.     

Potential relationship between glutathione metabolism and flocculation in the yeast Kluyveromyces lactis

Reduced glutathione (GSH) is involved in biochemical and physiological processes in cells. Flocculation is an important mechanism in microorganisms. The present study concerned the potential relationship between GSH metabolism and flocculation. Two yeast strains, a flocculent (Kluyveromyces lactis 5c) and a nonflocculent (Kluyveromyces lactis 5a) strain, were used. The level of intracellular GSH measured during the growth period was significantly higher in the nonflocculent than in the flocculent strain; in contrast, the flocculent strain exhibited brighter staining of vacuoles than the nonflocculent strain when observed using epifluorescence microscopy. Compounds acting either on flocculation (EDTA, galactose) or on GSH metabolism (buthionine sulfoximine, and N-acetylcysteine) were tested on the flocculent strain during the growth period. Both EDTA and galactose fully inhibited flocculation and induced GSH overproduction of 58% and 153%, respectively. Buthionine sulfoximine decreased GSH level by 76% but had no effect on flocculation; N-acetylcysteine increased the GSH level and flocculation by 106% and 41%, respectively. Combination of EDTA and N-acetylcysteine produced similar effects than with each of them. Combination of galactose and N-acetylcysteine increased the GSH level but decreased flocculation. These results demonstrated that GSH homeostasis is linked to the flocculation mechanism. A hypothesis related to stress is given.     

基于傅里叶变换近红外光谱实时分析1,3-丙二醇发酵过程生物量的在线监测方法

生物量是反映生物发酵过程进展的重要参数,对生物量进行实时监测可用于对发酵过程的调控优化。为克服目前主要采用的离线方法检测生物量时间滞后和人工测量误差较大等缺点,本研究针对1,3-丙二醇发酵过程设计了一个基于傅里叶变换近红外光谱实时分析技术的生物量在线监测实验平台,通过对实时采集光谱预处理以及敏感光谱段分析,应用偏最小二乘算法,建立了1,3-丙二醇发酵过程生物量变化的动态预测模型。以底物甘油浓度为60 g/L和40 g/L的发酵过程作为外部验证实验,分析得到模型的预测均方根误差分别为0.341 6和0.274 3,结果表明所建立的模型具有较好的实时预测能力,能够实现对1,3-丙二醇发酵过程中生物量的有效在线监测。     

Studies of on-line viable yeast biomass with a capacitance biomass monitor

A commercially available biomass monitor has been employed in a number of applications. For capacitance monitors, a relationship between capacitance measurement and cell counts or colony forming units has been reported in the literature. However, for use as an online instrument, a more practical correlation with the biomass concentration is needed. In this study, we followed the batch growth of brewer's yeast and a correlation with viable biomass concentration (g DW/L) was demonstrated. This correlation was utilized with the capacitance biomass monitor in a control loop to maintain setpoint biomass levels in a cyclic reactor under perturbations. Not only did the system demonstrate the capability of the biomass monitor to control biomass in such a system, but it also confirmed the correlation reported in our earlier work. (c) 1994 John Wiley & Sons, Inc.     

The effects of aeration and agitation on the measurement of yeast biomass using a laser turbidity probe

The paper reports the measurement of biomass concentration using a laser turbidity probe. A suspension of Bakers’ yeast (0.5?50?gl^-1) was subjected to various conditions of agitation and aeration in a stirred tank reactor and the turbidity measured using the probe. Both agitation and aeration were found to influence the turbidity. At any constant biomass concentration, the effect on the turbidity measurement of changing agitation or aeration rate independently was linear, while at any constant conditions of agitation and aeration rate, the relationship between turbidity and biomass concentration was non-linear. The results indicate that, in a bioprocess with non-particulate medium, it is possible to correct for the effects of aeration and agitation on turbidity measurement using a multivariate calibration model. A procedure for calibration and correction of measurements for the effects of agitation and aeration is presented and is verified using experimental data. This procedure may be generalised for other applications.     

Effect of nitrogen limitation on the ergosterol production by fed-batch culture of Saccharomyces cerevisiae

The diversity and content of available nitrogen sources in the growth medium both are very important in the accumulation of ergosterol in the yeast cell membrane. Growth on the good nitrogen sources such as ammonia can harvest more yeast cells than on poor ones, but ergosterol content in those yeast cells is relatively lower. Ergosterol content, one of the most variable parameters in ergosterol production by yeast cultivation, is greatly influenced by nitrogen limitation. The aim of our work was to study how the nitrogen sources affected the membrane ergosterol content and increase the total ergosterol yield. On the premise of keeping high ergosterol content in yeast cell, the ergosterol yield was enhanced by increasing the yeast biomass. Direct feed back control of glucose using an on-line ethanol concentration monitor was introduced to achieve high cell density. Ammonia, which acted as nitrogen source, was added to adjust pH during fermentation process, but its addition needed careful control. Cultivation in 5 L bioreactor was carried out under following conditions: culture temperature 30+/-1 degrees C, pH 5.5+/-0.1, agitation speed 600 rpm, controlling ethanol concentration below 1% and controlling ammonium ion concentration below 0.1 mol/L. Under these conditions the yeast dry weight reached 95.0+/-2.6 g/L and the ergosterol yield reached 1981+/-34 mg/L.     

Application of hydrocyclone for separation of Pichia pastoris produced r-HBsAg from fermentation culture: impact of concentration and pressure on hydrocyclone performance

Abstract

Separation of biomass from culture media by centrifugation and then washing the biomass are mandatory steps in the fermentation process of recombinant Pichia pastoris expressed HBsAg intracellularly. Biomass has to be washed many times to eliminate the culture media residues thoroughly. In this study, we tried to develop the hydrocyclone as an alternative method for separation of biomass from fermentation culture, an attractive replacement for centrifugation processes. The advantages of using hydrocyclone in biomass separation could be summarized in its suitability for continuous separation and its low risk of contamination. To evaluate the performance of hydrocyclone, concentration ratio in underflow to feed stream, capacity, and centrifugal force by considering three parameters of pressure drop, concentration, and the type of hydrocyclone were investigated.

Using three level factorial design a concentration ratio equation was developed, with the correlation coefficient R² = 0.977 ensured the good fitness of the predicted data with the experimental results. In optimal conditions, maximum concentration ratio was 1.246, for flow rate 13.5 LPM and C-force equal to 1276.11 at maximum pressure drop (3?bar) and minimum concentration (0.5% w/w) in hydrocyclone 1. Herein, two different hydrocyclones with the cylindrical diameters of 19?mm and 21?mm were used for separating the yeast cells.     

Use of focussed beam reflectance measurement (FBRM) for monitoring changes in biomass concentration

The potential of focussed beam reflectance measurement (FBRM) as a tool to monitor changes in biomass concentration was investigated in a number of biological systems. The measurement technique was applied to two morphologically dissimilar plant cell suspension cultures, Morinda citrifolia and Centaurea calcitrapa, to a filamentous bacteria, Streptomyces natalensis, to high density cultures of Escherichia coli and to a murine Sp2/0 hybridoma suspension cell line, 3-2.19. In all cases, the biomass concentration proved to be correlated with total FBRM counts. The nature of the correlation varied between systems and was influenced by the concentration, nature, size and morphology of the particle under investigation.     

On-line estimation of the biomass activity during animal-cell cultivations

A software sensor was developed to determine the volumetric biomass activity of animal cell cultivations on-line. It was based on the on-line estimation of the ATP-production rate from the oxygen uptake and the lactic-acid production rate. The sensor was verified for a batch culture of Vero cells, and a batch and a continuous culture of hybridoma cells. For the hybridoma cells, the sensor showed a good correlation with the biomass concentration. However, this was not the case for the Vero cells. As soon as glutamine was exhausted, the biomass activity stabilized, whereas the amount of biomass almost doubled. Because the sensor developed responds to nutrient limitations much faster than becomes visible through cell density measurements, and because the volumetric biomass activity can be related to the volumetric consumption rates and production rates of important metabolites, it shows excellent possibilities for control purposes. (c) 1996 John Wiley & Sons, Inc.     

Elemental balancing of biomass and medium composition enhances growth capacity in high-density Chlorella vulgaris cultures

The basic requirements for high-density photoautotrophic microalgal cultures in enclosed photobioreactors are a powerful light source and proper distribution of light, efficient gas exchange, and suitable medium composition. This article introduces the concept of balancing the elemental composition of growth medium with biomass composition to obtain high-density cultures. N-8 medium, commonly used for culturing Chlorella vulgaris was evaluated for its capacity to support high-density cultures on the basis of elemental stoichiometric composition of C. vulgaris. This analysis showed that the N-8 medium is deficient in iron, magnesium, sulfur, and nitrogen at high cell densities. N-8 medium was redesigned to contain stoichiometrically balanced quantities of the four deficient elements to support a biomass concentration of 2% (v/v). The redesigned medium, called M-8 medium, resulted in up to three- to fivefold increase in total chlorophyll content per volume of culture as compared to N-8 medium. Further experiments showed that addition of each of the four elements separately to N-8 medium did not improve culture performance and that balanced supplementation of all four deficient elements was required to yield the improved performance. Long-term (24 d) C. vulgaris culture in M-8 medium showed continuous increase in chlorophyll content and biomass throughout the period of cultivation. In contrast, the increase in chlorophyll content and biomass ceased after 7 and 12 d, respectively in N-8 medium, demonstrating the higher capacity of M-8 medium to produce biomass. Thus, the performance of high cell density photobioreactors can be significantly enhanced by proper medium design. The elemental composition of the biomass generated is an appropriate basis for medium design.     

Enhanced production of L-(+)-lactic acid in chemostat by Lactobacillus casei DSM 20011 using ion-exchange resins and cross-flow filtration in a fully automated pilot plant controlled via NIR

Due to the lack of suitable in-process sensors, on-line monitoring of fermentation processes is restricted almost exclusively to the measurement of physical parameters only indirectly related to key process variables, i.e., substrate, product, and biomass concentration. This obstacle can be overcome by near infrared (NIR) spectroscopy, which allows not only real-time process monitoring, but also automated process control, provided that NIR-generated information is fed to a suitable computerized bioreactor control system. Once the relevant calibrations have been obtained, substrate, biomass and product concentration can be evaluated on-line and used by the bioreactor control system to manage the fermentation. In this work, an NIR-based control system allowed the full automation of a small-scale pilot plant for lactic acid production and provided an excellent tool for process optimization. The growth-inhibiting effect of lactic acid present in the culture broth is enhanced when the growth-limiting substrate, glucose, is also present at relatively high concentrations. Both combined factors can result in a severe reduction of the performance of the lactate production process. A dedicated software enabling on-line NIR data acquisition and reduction, and automated process management through feed addition, culture removal and/or product recovery by microfiltration was developed in order to allow the implementation of continuous fermentation processes with recycling of culture medium and cell recycling. Both operation modes were tested at different dilution rates and the respective cultivation parameters observed were compared with those obtained in a conventional continuous fermentation. Steady states were obtained in both modes with high performance on lactate production. The highest lactate volumetric productivity, 138 g L(-1) h(-1), was obtained in continuous fermentation with cell recycling.