Real-time monitoring and automatic density control of large-scale microalgal cultures using near infrared (NIR) optical density sensors

Signals from near infrared (NIR) light transmittance sensors were used for both real-time monitoring of algal biomass density in growing mass cultures (200l tubular biofences), and also as feedback in a system that controlled the density of the culture by automatic injection of fresh growth medium. When operated in a semi-continuous production mode between predefined density values, diurnal growth patterns were recorded on-line that provided information on the dynamics of the microalgal cultures with respect to environmental conditions. The bioreactor system was also programmed to operate in constant biomass density mode, thereby maintaining the culture at the optimal population density (OPD), and sustaining high biomass production levels. The system has potential for operating a dynamic density set point for microalgal cultures where the optimal population density varies as a function of ambient growing conditions.     

Metabolic active-high density VERO cell cultures on microcarriers following apoptosis prevention by galactose/glutamine feeding

The control of cell death occurring in high density cultures performed in bioreactors is an important factor in production processes. In this work, medium nutrient removal or feeding was used to determine at which extension apoptosis could be, respectively, involved or prevented in VERO cell cultures on microcarriers. Glutamine and galactose present in the VERO cell culture medium was consumed after, respectively, 6 and 12 days of culture. Kinetics studies showed that fresh medium replacement and, to some extent, galactose or glutamine depleted-fresh medium replacement provided a nutritional environment, allowing the VERO cell cultures to attain high densities. Galactose was shown to be a more critical nutrient when cultures reached a high density. In agreement with that, VERO cell cultures supplemented with galactose and/or glutamine were shown to confirm previous findings and, again at high densities, galactose was shown to be a critical nutrient for VERO cell growth. These observations also indicated that in VERO cell cultures, for feeding purposes, the glutamine could be replaced by galactose. The inverse was not true and led, at high densities, to a decrease of cell viability. In the absence of glutamine and galactose, apoptosis was observed in VERO cell cultures by cytofluorometry, Acridine orange staining or light and electron microscopy, reaching high levels when compared to cultures performed with complete medium. VERO cells apoptosis process could be prevented by the galactose and/or glutamine feeding and, at high densities, galactose was more efficient in protecting the cultures. These cultures, prevented from apoptosis, were shown to synthesize high levels of measles virus following infection. Our data show that apoptosis prevention by glutamine/galactose feeding, led to high productive and metabolic active VERO cell cultures, as indicated by the high cell density obtained and the virus multiplication leading to higher virus titers.     

山玉兰花粉离体萌发和花粉管生长特性的研究

山玉兰(Magnolia delavayi)是木兰科木兰属的常绿乔木或大型灌木,是重要的园林造景、庭院绿化素材,也是重要的育种资源。山玉兰花粉的研究对其杂交育种的成败具有重要影响,但目前尚未见其花粉活力的相关报道。该研究以新鲜的山玉兰花粉为对象,采用悬滴培养法分析了温度、光照以及培养液的蔗糖和硼酸浓度对山玉兰花粉萌发的影响。结果表明:(1)山玉兰花粉萌发时,最适宜的温度为27℃。(2)光暗条件下,山玉兰花粉以浓度为5%的蔗糖培养效果最佳,其萌发率在16%以上;而硼酸浓度则以0.001%的培养效果最佳。(3)蔗糖与硼酸共同作用可有效促进花粉萌发和花粉管生长。其中,在光照条件下,以5.0%蔗糖+0.001%硼酸为最适宜的培养液,花粉萌发率达41.27%,花粉管长达281.49μm;而在黑暗条件下,则以5.0%蔗糖+0.01%硼酸为最适宜的培养液,花粉萌发率达45.71%,花粉管长达254.00μm。该研究结果为进一步开展人工辅助授粉、发掘山玉兰的种质资源工作奠定了基础。     

Problems in developing the biotechnology of algal biomass production

Summary The effects of environmental conditions (solar irradiance and temperature) and population density on the production of Spirulina biomass with brackish water are reported for cultures grown in outdoor ponds. Higher specific growth rates were observed at lower population densities. Lower growth rates were associated with limitation by light in dense cultures under optimum conditions in the summer. Seasonal variation in productivity was observed. In summer, light was the limiting factor, whereas in winter the low daytime temperature appeared to constitute the major limitation. The oxygen concentration in the culture can serve as a useful indicator of limiting factors and can also be used to estimate the extent of such limitations.     

红光下培养方式对雨生红球藻细胞增殖及其活力的影响

研究重点针对雨生红球藻绿色游动细胞的增殖培养阶段,分析了在利于细胞增殖的红光条件下,几种培养方式的调整对增殖过程和细胞活力的影响。结果显示:（1）在红光下,增殖平台期维持时间长,细胞活力稳定,细胞中性脂无累积,但进入平台期前,细胞中性脂有规律波动,进入平台期后相对稳定;通过更新率为20%的半连续培养,细胞数产出较批次培养提高57%;半连续培养中细胞呈现胁迫调节的时间较批次培养晚。随着培养时间增加,半连续培养下细胞营养盐吸收能力降低。（2）初始接种密度与细胞增殖速率及细胞光合活力呈负相关:初始密度低的细胞增殖速率较高,细胞光合作用活力高。（3）在培养过程中添加CO2时,最大密度均有提高,达6.0105 cells/mL,较无添加组提高54%;细胞分裂速率均有提高,但红光下较白光下增殖速率高（分别为0.223/d和0.198/d）;添加CO2降低培养液pH,利于维持适宜增殖的pH环境。叶绿素荧光参数以及细胞粒径在红光和白光下有显著差异:红光下,Fv/Fm显著高于白光下;红光下补充CO2显著减小细胞粒径,而白光下粒径无显著变化。研究结果显示,在红光下,采用间断式半连续培养补充CO2培养绿色游动细胞,有利于提升细胞活力与产出。     

Effects of cell density,light intensity and mixing on Undaria pinnatifida gametophyte activity in a photobioreactor

An on-line controlled 7 l sterilizable photobioreactor was used for the optimisation of a culture of gametophytes of Undaria pinnatifida. The gametophytes, which had been stored for three years in a culture cabinet at 16 degrees C, could rapidly grow in the photobioreactor under controlled conditions. The rate of increase of dissolved oxygen and pH were used to monitor the photosynthetic activity. Optimal gametophytes density changed varying the light intensity. The optimal cell densities were 3.24 and 3.45 g FW l(-1) when the cultures were exposed to 61.7 and 82.3 microE m(-2) s(-1), respectively. The optimal cell density was higher under a high photon flux density (PFD) than under low PFD. On the other hand, the optimal light intensities were different for different cell density cultures. The light saturation point was higher at high cell density cultures than at low cell density cultures. The optimal rotational speed was 150 rpm for high cell density culture in the photobioreactor.     

Isolation, culture and characteristics of epididymal epithelial cells from adult cats

A tissue-culture system in which cells retain defined ultrastructural and functional characteristics was established to provide a basis for functional investigations of the epididymal duct in the cat. A widely used culture protocol for rat epididymal epithelium was used as a starting point and subsequently modified. The cellular population of the cat's epididymal epithelium was isolated by successive collagenase and trypsin digestion. A high yield of isolated cells obtained with good viability, were cultured in DMEM/F12 medium supplemented with foetal bovine serum, in absence or in presence of additional dihydrotestosterone (1 nM). The plated primary cultures reached confluence within 5-8 days, producing a monolayer of cohesive cells. Samples taken after 6 days in culture were processed for transmission and scanning electron microscopies. Immunocytochemical staining was used to estimate the purity of the epithelial cell population in the monolayers. The cell cultures displayed several functional traits of in vivo epithelia, including [35S] hypotaurine and [35S] taurine production. These results demonstrate that primary cultures of epididymal epithelial cells isolated from sexually mature cats maintain several differentiated characteristics of the intact organ and therefore provide a valuable system for the study of epididymal epithelial cell functions, metabolic activities and their regulation in cats.     

The growth-limiting factor of a population of cyanobacteria in the hypertrophic Dutch Lake Brielle

Summary Some methods to identify nutrient deficiencies in natural populations of algae are described. A bloom of the cyanobacteriumMicrocystis aeruginosa in Lake Brielle, The Netherlands, is taken as example of a natural situation. Some results obtained from continuous cultures ofM. aeruginosa contribute to the understanding of the characteristics of the natural population. Attention is paid to nutrient levels of the lake, pigment contents and N:P ratio of the algal population, the primary productivity and the uptake kinetics of nitrate and ammonia. The natural algal population of the lake showed a preferential use of ammonia. It is assumed that the growth-limiting factor forM. aeruginosa under the prevailing temperature is light.     

Problems of optimisation of plant cell culture processes.

The adoption of plant cell cultures as an industrial process depends greatly on the economics of such a process. The multicycle or draw-fill culture technique is one method for improving the productivity and, hence, cost of a process. Mathematical models have been devised for the functional relationships between the nominal costs of biomass and secondary metabolites and the plant cell growth characteristics in a multicycle growth system. The models were used to evaluate the data obtained with cultures of Dioscorea deltoidea (which produces diosgenin) and Panax ginseng, grown in various types of bioreactors. The multicycle system gave an increase of 1.5-2 in biomass productivity compared with batch culture, but was probably only commercially viable if the cost of the process in the bioreactor was at least 30 times that of the medium and if an inoculum of about 30% of the culture of the previous cycle was left in the bioreactor. In the multicycle system incompletely utilised nutrient or metabolite accumulation can only reach 1.43 times or less that of the initial values. With the P. ginseng culture, about 75% of the calculated maximum cell packing density per fresh weight (approximately 530 g 1-1) in this regime was achieved. The possibility of growth in the standard bioreactor of a shear sensitive type culture was shown with a marine impeller speed up to 330 cm s-1.     

On-line monitoring of marine cyanobacterial cultivation based on phycocyanin fluorescence.

A novel on-line fluorescence monitoring system for marine cyanobacterial cultivation was developed. This method is based on the measurement of intracellular phycocyanin content, which is the major light harvesting protein. A fluorescence spectrophotometer, equipped with a flow cell connected with a culture liquid recycling tube was used. Experiments were carried out using a marine unicellular cyanobacteria Synechococcus sp. NKBG 042902 isolated from Japanese coastal sea water. We have optimized excitation wavelength to avoid the light scattering, using non-pigmented old cells which no longer contained phycocyanin. At an excitation wavelength of 590 nm, light scattering was minimized. Viable cell concentration could be measured in the range of 2 x 10(6) to 2 x 10(8) cells per ml, without pronounced light scattering. Continuous monitoring of marine cyanobacteria cultivation was performed. Cell concentrations were determined by both culture fluorescence and by using a hemacytometer. A good linear correlation was obtained. We conclude that on-line monitoring of cyanobacterial culture fluorescence based on phycocyanin is a rapid, efficient and also versatile method for determining viable cell concentration.     

Cage Culture Turbidostat: a Device for Rapid Determination of Algal Growth Rate

The present cage culture turbidostat consists of a growth chamber and a control unit. The microorganisms (photoautotrophic algae) are kept in the growth chamber by porous membranes (pore size 1 to 3 μm) which retain the algae but allow efficient exchange of the growth medium. Flow rate and composition of the medium can therefore be varied independently of algal population density. A reciprocating pumping mode of the medium is introduced to obtain more gentle clearance of membranes than that provided by rotation or stirring in other membrane fermentors. Pulsed light and a light-emitting diode/light-sensitive transistor couple are used to monitor the turbidity of the culture, independent of external light needed for growth. The control unit keeps the turbidity constant by frequent activation of the dilution pump. Theoretical analysis of growth in the turbidostat shows that integrated activation time of the dilution pump is proportional to the growth rate of the organism. Theoretical analysis was also used to determine minimum flow-rate and nutrient concentration of medium to cover the requirement of the algae. Experiments with three different marine diatoms were carried out, and they demonstrated that the growth rate could be determined every hour and that the cultures could be kept at constant turbidity over 10 to 14 days at least.     

Light as an Energy Source in Continuous Cultures of Bacteriorhodopsin-Containing Halobacteria

The role of light as an energy source for slightly aereated cultures of halobacteria was studied, using continuous cultures with low nutrient concentrations and a low oxygen supply. A series of experiments were carried out with non-illuminated and differently illuminated cultures and with different oxygen transfer rates. Under low oxygen availability, light proved to be a decisively important energy source that allowed the populations to reach higher growth rates and much higher population densities. Oxygen influenced the growth over only a minimal level, below which neither the illuminated nor the dark cultures were affected by the oxygen transfer rate. From these results, it appears that the bacteriorhodopsin-mediated energy supply could have a very important role for the ecology of halobacteria in their microaerophilic habitats. In the illuminated cultures, cells that originated purple colonies on plates appeared. These cells, which could be bacteriorhodopsin-constitutive mutants, are now being studied.     

Outdoor production of Phaeodactylum tricornutum biomass in a helical reactor

The production of microalga Phaeodactylum tricornutum in an outdoor helical reactor was analysed. The influence of temperature, solar irradiance and air flow rate on the yield of the culture was evaluated. Biomass productivities up to 1.5 g l(-1) per day and photosynthetic efficiency up to 14% were obtained by maintaining the cultures below 30 degrees C, dissolved oxygen levels less than 400% Sat. (with respect to air saturated culture) and controlling the cell density in order to achieve an average irradiance within the culture below 250 microE m(-2) s(-1). Under these conditions, the fluorescence parameter, Fv/Fm, which reflects the maximal efficiency of PSII photochemistry, remained roughly 0.6-0.7 and growth rates up to 0.050 h(-1) were achieved. The average irradiance and the light/dark cycle frequency, were the variables determining the behaviour of the cultures. A hyperbolic relationship between growth rate and biomass productivity with the average irradiance was observed, whereas both biomass productivity and photosynthetic efficiency linearly increased with the light/dark cycle frequencies. Optimum design and operational conditions which maximise the production of P. tricornutum biomass in outdoor helical reactors were determined.     

Effect of nutrients and light intensity on growth of Mallomonascaudata (Synurophyceae)

We used batch cultures of three strains of the unicellular synurophyte Mallomonascaudata to investigate the effects of nitrate, phosphate, silicate and light intensity on population growth and growth rate. The three strains were isolated from three different reservoirs in Kyungpook Province, Korea. For all three strains, we observed high population growth under all nutrient concentrations studied, except at nitrate concentration below 0.8 μM. The maximum growth rate (μ_max) occurred at 8.2 μM or 16.5 μM nitrate, depending on the strain, and at 11.5 μM phosphate. Silicate concentration had no effect on growth rate. With respect to light intensity, the maximum population growth and maximum growth rates (μ_max) occured between 42 and 104 μmol m^?2 s^?1 depending on strain and culture temperature. Population growth of these three strains under batch culture occurred over a wide range of nutrient and light intensities, but there seemed to be strain‐specific differences that may represent adaptations to local environments.     

Biomass recycling and species competition in continuous cultures

A chemostat with cell feedback is analyzed for three kinds of limiting nutrient: a substrate dissolved in the inflow, a gas bubbled directly into the reactor, and light. The effects of recycle are distinct in each case, because the relationships between hydraulic detention time and nutrient inflow are different for each type of nutrient, Effluent recycle, in which the recycle stream is more dilute than the reactor, is discussed in terms of cell detention time and nutrient limitation. Results from chemostat cultures of the blue-green alga, Spirulina geitleri, demonsrtat cell feedback under light limitation. Maximum Productivity is fixed by the incident light intensity. At a particular dilution rate recycling increases or decreases productivity by taking cell density closer or further from the optimum density. Cell recycle with heterogeneous populations can change the outcome of species competition. Selective recycling of one species can reverse this outcome or stabilize coexistence by its selective effect on cell detention time. Experimental results from light-limited mixed cultures of S. geitleri and a Chlorella sp. verify this.     

Development of a Sensitive Microbioassay Using Amplified Cultivation1

A novel ultramicro microbioassay was developed. The present method, referred to as amplified cultivation, is composed of two successive kinds of culture. The first culture is performed until a maximum difference is obtained in the ratio of numbers of viable cells appearing in a basal medium and that contained in a nutrient. The second culture is continued after adding a complete medium to the cultures and stopped just after the growth of microorganisms in the blank reaches a certain detectable limit, e.g., by optical density or acid production. Lactose was analyzed by this amplified cultivation method with an extremely low concentration of viable cells of Bifidobacterium bifidum N4 as the inoculum. The detectable limits of lactose were 0.05 μg and 1 μg by the test tube method and by the pulp disc method respectively, with an increase in sensitivity by a factor of more than 10³ compared with the corresponding conventional microbioassays. Moreover, a relationship between the diameters of the growth zones and the logarithm of the amount of lactose was achieved in a range of 50–1000 μg of lactose in the pulp disc method.     

Enrichment for heterokaryocytes by the use of iso-osmotic density gradients after plant protoplast fusion

Summary Buoyant density differences between different types of protoplasts were used in an iso-osmotic density gradient system to enrich protoplast fusion mixtures for heterokaryocytes. Protoplasts of maize stem and wheat mesophyll, as well as epidermis, stem parenchyma and mesophyll protoplasts of two amphihaploid, light sensitive tobacco mutants were fused with polyethylene glycol using conventional methods and a new rolling tube technique. The protoplast combinations used for fusion involved protoplast types with considerably different buoyant densities. Enriched fractions of maize-wheat heterokaryocytes of intermediate density were recovered which contained up to 31% mostly binucleate heterokaryocytes (a 2–7 fold relative enrichment). Tobacco heterokaryocytes recovered analogously from enriched intermediate fractions readily divided and gave rise to an increased number of light resistant calluses when compared with cultures from non-fractionated fusion mixtures. Maize-wheat fusion products, however, failed to divide.     

Reaggregation of fetal rat brain cells in a stationary culture system I: Methodology and cell identification

Summary A stationary tissue culture system for reaggregation cultures of rat brain cells is described. Aggregates were formed by placing cells at high concentrations in liquid overlay cultures on a nonadherent nutrient agar surface. No physical stress in the form of rotation or shaking was applied to the aggregating cell population. Transmission electron microscopy and immunohistochemistry showed that the cells developed from homogeneously dispersed, immature cells in Day 4 aggregates, to mature astrocytes, oligodendrocytes, and neurons in Day 20 aggregates. Twenty days and older aggregates had a tightly packed neuropil which was most prominent in a cell-sparse outer layer of the aggregates. When the aggregates were allowed to adhere to a substrate, both glial fibrillary acidic protein (GFAP) positive and negative cells were observed migrating out from the aggregates. Cells giving a positive reaction for neuron specific enolase (NSE) were also present. This reaggregation procedure, with transfer of selected brain cell aggregates into agar-coated multiwells is an alternative three-dimensional culture system which can be potentially useful in the study of morphogenesis and cell interactions in the nervous system. This project was supported by the Norwegian Cancer Society.     

A PHOTOELECTRIC DENSITOMETER FOR USE WITH SUSPENSIONS

A device for quickly and accurately measuring the population density of a suspension of microorganisms, permitting the preparation of yeast suspensions of known density to within 1 per cent error, was constructed with two Visitron photoelectric cells, a single light of high intensity and a good Wheatstone bridge for balancing the currents from the two photoelectric cells. A large Pyrex milk culture tube holding the suspension is placed in the path of one beam of light coming through a small longitudinal slit and thence to one photocell; a second similar slit directs another beam of light upon the second photocell, thus causing dissimilar currents to flow, the ratio of whose magnitudes may be measured by the bridge resistances. A relation between these currents and the relative light intensities is shown, and the one significant unmeasurable variable (the characteristic constant of a photocell) is practically eliminated by the use of a method of ratios. After careful standardization of technique the apparatus proved more accurate than other methods available for the purpose indicated. In rapid use its accuracy may be put safely at 1 per cent for measuring the densities of cultures of approximately the same age and composed of cells having comparable optical characteristics.     

Myocytes and fibroblasts exhibit functional synergism in mixed cultures of neonatal rat heart cells

Cardiac cells obtained from neonatal rat heart contain a mixed population of cell types that can be enriched in culture in either myocytes or fibroblast-like cells. A metabolic comparison of mixed heart cell cultures with enriched cultures of the same age-in-culture and initial cell density showed that mixed cultures used glucose more rapidly than either enriched myocytes or fibroblasts. Mixed cultures were shown to respond to deprivation of insulin and of serum with decreases in the rate of glucose usage and decreases in the protein content of cells, whereas enriched cultures did not respond in the expected manner to insulin deprivation. Mixed, 11-day-old cells also exhibited greater increases in cellular protein and greater resistance to the stress of starvation than enriched cultures. Palmitate usage, however, was similar in all cultures examined. We conclude that mixed cultures may serve as a better model system to study cardiac metabolism and to monitor the effects of drugs and hormones on the neonatal myocardium. In addition, it is clear from our results that myocytes and fibroblastic-like cells coexist in a metabolically functional synergism.