A structured model for simulation of cultures of the cyanobacterium Spirulina platensis in photobioreactors: II. Identification of kinetic parameters under light and mineral limitations

A structured model for the culture of cyanobacteria in photobioreactors is developed on the basis of Schuster's approximations for radiative light transfer. This model is therefore limited to monodimensional geometries and kinetic aspects.Light-harvesting pigments play a crucial role in defining the profile of radiative transfer inside the culture medium and in controlling the metabolism, particularly the metabolic deviations induced by mineral limitations. Modeling therefore requires the biomass to be divided into several compartments, among which the light-harvesting compartment allows a working illuminated volume to be defined within the photobioreactor. This volume may change during batch cultures, largely decreasing as pigment concentration increases during growth but increasing as pigments are consumed during mineral limitation. This approach enables, in photobioreactors of simple parallelepipedic, geometries, kinetic parameters to be determined with high accuracy; this may then be extended to vessels of more complex geometries, such as cylindrical photobioreactors.The model is applied to controlled batch cultures of the cyanobacterium Spirulina platensis in parallelepipedic photobioreactors to assess its ability to predict the behavior of these microorganisms in conditions of light and mineral limitations. Results allowed the study of optimal operating condition for continuous cultures to be approached (c) 1992 John Wiley & Sons, Inc.     

Cultivation of Marine Sponges

There is increasing interest in biotechnological production of marine sponge biomass owing to the discovery of many commercially important secondary metabolites in this group of animals. In this article, different approaches to producing sponge biomass are reviewed, and several factors that possibly influence culture success are evaluated. In situ sponge aquacultures, based on old methods for producing commercial bath sponges, are still the easiest and least expensive way to obtain sponge biomass in bulk. However, success of cultivation with this method strongly depends on the unpredictable and often suboptimal natural environment. Hence, a better-defined production system would be desirable. Some progress has been made with culturing sponges in semicontrolled systems, but these still use unfiltered natural seawater. Cultivation of sponges under completely controlled conditions has remained a problem. When designing an in vitro cultivation method, it is important to determine both qualitatively and quantitatively the nutritional demands of the species that is to be cultured. An adequate supply of food seems to be the key to successful sponge culture. Recently, some progress has been made with sponge cell cultures. The advantage of cell cultures is that they are completely controlled and can easily be manipulated for optimal production of the target metabolites. However, this technique is still in its infancy: a continuous cell line has yet to be established. Axenic cultures of sponge aggregates (primmorphs) may provide an alternative to cell culture. Some sponge metabolites are, in fact, produced by endosymbiotic bacteria or algae that live in the sponge tissue. Only a few of these endosymbionts have been cultivated so far. The biotechnology for the production of sponge metabolites needs further development. Research efforts should be continued to enable commercial exploitation of this valuable natural resource in the near future. Received November 5, 1998; accepted June 20, 1999.     

Bioprocess considerations for production of secondary metabolites by plant cell suspension cultures

Plant cell culture provides a viable alternative over whole plant cultivation for the production of secondary metabolites. In order to successfully cultivate the plant cells at large scale, several engineering parameters such as, cell aggregation, mixing, aeration, and shear sensitivity are taken into account for selection of a suitable bioreactor. The media ingredients, their concentrations and the environmental factors are optimized for maximal synthesis of a desired metabolite. Increased productivity in a bioreactor can be achieved by selection of a proper cultivation strategy (batch, fed-batch, two-stageetc.), feeding of metabolic precursors and extraction of intracellular metabolites. Proper understanding and rigorous analysis of these parameters would pave the way towards the successful commercialization of plant cell bioprocesses.     

Comparative analysis of the outdoor culture of Haematococcus pluvialis in tubular and bubble column photobioreactors

The present paper makes a comparative analysis of the outdoor culture of H. pluvialis in a tubular photobioreactor and a bubble column. Both reactors had the same volume and were operated in the same way, thus allowing the influence of the reactor design to be analyzed. Due to the large changes in cell morphology and biochemical composition of H. pluvialis during outdoor culture, a new, faster methodology has been developed for their evaluation. Characterisation of the cultures is carried out on a macroscopic scale using a colorimetric method that allows the simultaneous determination of biomass concentration, and the chlorophyll, carotenoid and astaxanthin content of the biomass. On the microscopic scale, a method was developed based on the computer analysis of digital microscopic images. This method allows the quantification of cell population, average cell size and population homogeneity. The accuracy of the methods was verified during the operation of outdoor photobioreactors on a pilot plant scale. Data from the reactors showed tubular reactors to be more suitable for the production of H. pluvialis biomass and/or astaxanthin, due to their higher light availability. In the tubular photobioreactor biomass concentrations of 7.0 g/L (d.wt.) were reached after 16 days, with an overall biomass productivity of 0.41 g/L day. In the bubble column photobioreactor, on the other hand, the maximum biomass concentration reached was 1.4 g/L, with an overall biomass productivity of 0.06 g/L day. The maximum daily biomass productivity, 0.55 g/L day, was reached in the tubular photobioreactor for an average irradiance inside the culture of 130 microE/m2s. In addition, the carotenoid content of biomass from tubular photobioreactor increased up to 2.0%d.wt., whereas that of the biomass from the bubble column remained roughly constant at values of 0.5%d.wt. It should be noted that in the tubular photobioreactor under conditions of nitrate saturation, there was an accumulation of carotenoids due to the high irradiance in this reactor, their content in the biomass increasing from 0.5 to 1.0%d.wt. However, carotenoid accumulation mainly took place when nitrate concentration in the medium was below 5.0mM, conditions which were only observed in the tubular photobioreactor. A similar behaviour was observed for astaxanthin, with maximum values of 1.1 and 0.2%d.wt. measured in the tubular and bubble column photobioreactors, respectively. From these data astaxanthin productivities of 4.4 and 0.12 mg/L day were calculated for the tubular and the bubble column photobioreactors. Accumulation of carotenoids was also accompanied by an increase in cell size from 20 to 35 microm, which was only observed in the tubular photobioreactors. Thus it may be concluded that the methodology developed in the present study allows the monitoring of H. pluvialis cultures characterized by fast variations of cell morphology and biochemical composition, especially in outdoor conditions, and that tubular photobioreactors are preferable to bubble columns for the production of biomass and/or astaxanthin.     

Secondary metabolism of hairy root cultures in bioreactors

Summary In vitro cultures are being considered as an alternative to agricultural processes for producing valuable secondary metabolites. Most efforts that use differentiated cultures instead of cell suspension cultures have focused on transformed (hairy) roots. Bioreactors used to culture hairy roots can be roughly divided into three types: liquid-phase, gas-phase, or hybrid reactors that are a combination of both. The growth and productivity of hairy root cultures are reviewed with an emphasis on successful bioreactors and important culture considerations. The latter include strain selection, production of product in relation to growth phase, media composition, the gas regime, use of elicitors, the role of light, and apparent product loss. Together with genetic engineering and process optimization, proper reactor design plays a key role in the development of successful large scale production of secondary metabolites from plant cultures.     

Modeling of biomass productivity in tubular photobioreactors for microalgal cultures: effects of dilution rate, tube diameter, and solar irradiance

A macromodel is developed for estimating the year-long biomass productivity of outdoor cultures of microalga in tubular photobioreactors. The model evaluates the solar irradiance on the culture surface as a function of day of the year and the geographic location. In a second step, the geometry of the system is taken into account in estimating the average irradiance to which the cells are exposed. Finally, the growth rate is estimated as a function of irradiance, taking into account photoinhibition and photolimitation. The model interconnects solar irradiance (an environmental variable), tube diameter (a design variable), and dilution rate (an operating variable). Continuous cultures in two different tubular photobioreactors were analyzed using the macromodel. The biomass productivity ranged from 0.50 to 2.04 g L-1 d-1, and from 1.08 to 2. 76 g L-1 d-1, for the larger and the smaller tube diameter photobioreactors, respectively. The quantum yield ranged from 1.1 to 2.2 g E-1; the higher the incident solar radiation, the lower the quantum yield. Simultaneous photolimitation and photoinhibition of outdoor cultures was observed. The model reproduced the experimental results with less than 20% error. If photoinhibition was neglected, and a growth model that considered only photolimitation was used to fit the data, the error increased to 45%, thus reflecting the inadequacy of previous outdoor growth models that disregard photoinhibition. Copyright 1998 John Wiley & Sons, Inc.     

Hairy root culture for mass-production of high-value secondary metabolites

Plant cell cultivations are being considered as an alternative to agricultural processes for producing valuable phytochemicals. Since many of these products (secondary metabolites) are obtained by direct extraction from plants grown in natural habitat, several factors can alter their yield. The use of plant cell cultures has overcome several inconveniences for the production of these secondary metabolites. Organized cultures, and especially root cultures, can make a significant contribution in the production of secondary metabolites. Most of the research efforts that use differentiated cultures instead of cell suspension cultures have focused on transformed (hairy) roots. Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic (cancerous) roots produced by A. rhizogenes infection are characterized by high growth rate, genetic stability and growth in hormone free media. These genetically transformed root cultures can produce levels of secondary metabolites comparable to that of intact plants. Hairy root cultures offer promise for high production and productivity of valuable secondary metabolites (used as pharmaceuticals, pigments and flavors) in many plants. The main constraint for commercial exploitation of hairy root cultivations is the development and scaling up of appropriate reactor vessels (bioreactors) that permit the growth of interconnected tissues normally unevenly distributed throughout the vessel. Emphasis has focused on designing appropriate bioreactors suitable to culture the delicate and sensitive plant hairy roots. Recent reactors used for mass production of hairy roots can roughly be divided as liquid-phase, gas-phase, or hybrid reactors. The present review highlights the nature, applications, perspectives and scale up of hairy root cultures for the production of valuable secondary metabolites.     

Scaling up microalgal cultures to commercial scale

ABSTRACT

Scaling up algal cultures to the very large volumes required for commercial production is a complex task and requires skilled and experienced personnel. First it is necessary to consider how to optimize the process of producing enough inoculum for the large ponds or photobioreactors in order to minimize the time and cost required. In order to minimize the need for re-inoculation from stock cultures it is also essential to manage the large-scale cultures to avoid significant contamination or collapse. The maintenance of long-term, stable, high-productivity, large-scale cultures, usually under prevailing outdoor conditions of variable irradiance, temperature and rainfall, presents additional challenges most of which are not seen in the constant environment experienced by small-scale laboratory cultures. Methods and protocols to deal with these can only be developed at the large-scale and they will mostly be specific for the alga being cultured, the culture system being used and the location of the production plant. A common feature of all large-scale operations known to us is that, over time (years), both productivity and reliability of the cultures improve as the operators gather experience in managing their cultures.     

Transformed root cultures for biotechnology

This review is concerned with the application of hairy roots, i.e. plant roots formed from plant cells after transformation by Agrobacterium rhizogenes for the production of bioactive compounds. Transformed root cultures have been established from numerous species of dicotyledonous plants. The plants, as well as the main products accumulated in hairy root cultures derived from these plants, are listed in this paper. Data are presented on novel compounds, hitherto detected only in transformed roots but not occurring in the corresponding intact plants. The possible use of hairy root cultures for the over-production of secondary metabolites and biotransformation of chemicals is discussed. In order to enhance the productivity of hairy root cultures, various methods have been derived, and optimized procedures are proposed. They include selection of high-producing clones, elicitation, composition of growth media, culture conditions and genetic approach. Hairy roots usually store secondary metabolites in vacuoles inside the cells. Therefore, several methods have been used to increase the amount of products released into the medium. Unfortunately, no general procedure is known that works in all cases, and the excretion behaviour of hairy root cultures varies from one species to another and even within one species from one clone to another. Special attention is given to the cultivation methods and bioreactor systems for hairy root cultures. Hairy roots are cultivated usually in shake flasks; however, shake flask culture is not suitable for the complex optimization and continuous control of the culture conditions. In this paper, we are going to present bioreactors proposed for the cultivation of hairy roots under more or less controlled conditions. Modifications of typical bacterial bioreactors, i.e. stirred tanks, airlift loop reactors and other constructions, are presented. A very special type of bioreactor providing good conditions for loose root mass multiplication without oxygen or substrate limitations, is the mist bioreactor. Nowadays, it is practically impossible to select the one best bioreactor type for hairy root culture.     

Ammonium photo-production by heterocytous cyanobacteria: potentials and constraints

Over the last decades, production of microalgae and cyanobacteria has been developed for several applications, including novel foods, cosmetic ingredients and more recently biofuel. The sustainability of these promising developments can be hindered by some constraints, such as water and nutrient footprints. This review surveys data on N₂-fixing cyanobacteria for biomass production and ways to induce and improve the excretion of ammonium within cultures under aerobic conditions. The nitrogenase complex is oxygen sensitive. Nevertheless, nitrogen fixation occurs under oxic conditions due to cyanobacteria-specific characteristics. For instance, in some cyanobacteria, the vegetative cell differentiation in heterocyts provides a well-adapted anaerobic microenvironment for nitrogenase protection. Therefore, cell cultures of oxygenic cyanobacteria have been grown in laboratory and pilot photobioreactors (Dasgupta et al., 2010; Fontes et al., 1987; Moreno et al., 2003; Nayak & Das, 2013). Biomass production under diazotrophic conditions has been shown to be controlled by environmental factors such as light intensity, temperature, aeration rate, and inorganic carbon concentration, also, more specifically, by the concentration of dissolved oxygen in the culture medium. Currently, there is little information regarding the production of extracellular ammonium by heterocytous cyanobacteria. This review compares the available data on maximum ammonium concentrations and analyses the specific rate production in cultures grown as free or immobilized filamentous cyanobacteria. Extracellular production of ammonium could be coupled, as suggested by recent research on non-diazotrophic cyanobacteria, to that of other high value metabolites. There is little information available regarding the possibility for using diazotrophic cyanobacteria as cellular factories may be in regard of the constraints due to nitrogen fixation.     

Diatom cultivation and biotechnologically relevant products. Part II: Current and putative products

While diatoms are widely present in terms of diversity and abundance in nature, few species are currently used for biotechnologically applications. Most studies have focussed on intracellularly synthesised eicosapentaenoic acid (EPA), a polyunsaturated fatty acid (PUFA) used for pharmaceutical applications. Applications for other intracellular molecules, such as total lipids for biodiesel, amino acids for cosmetic, antibiotics and antiproliferative agents, are at the early stage of development. In addition, the active principle component must be identified amongst the many compounds of biotechnological interest. Biomass from diatom culture may be applied to: (1). aquaculture diets, due to the lipid- and amino-acid-rich cell contents of these microorganisms, and (2). the treatment of water contaminated by phosphorus and nitrogen in aquaculture effluent, or heavy metal (bioremediation). The most original application of microalgal biomass, and specifically diatoms, is the use of silicon derived from frustules in nanotechnology. The competitiveness of biotechnologically relevant products from diatoms will depend on their cost of production. Apart from EPA, which is less expensive when obtained from Phaeodactylum tricornutum than from cod liver, comparative economic studies of other diatom-derived products as well as optimisation of culture conditions are needed. Extraction of intracellular metabolites should be also optimised to reduce production costs, as has already been shown for EPA. Using cell immobilisation techniques, benthic diatoms can be cultivated more efficiently allowing new, biotechnologically relevant products to be investigated.     

Hairy Root Culture for Mass-Production of High-Value Secondary Metabolites

ABSTRACT

Plant cell cultivations are being considered as an alternative to agricultural processes for producing valuable phytochemicals. Since many of these products (secondary metabolites) are obtained by direct extraction from plants grown in natural habitat, several factors can alter their yield. The use of plant cell cultures has overcome several inconveniences for the production of these secondary metabolites. Organized cultures, and especially root cultures, can make a significant contribution in the production of secondary metabolites. Most of the research efforts that use differentiated cultures instead of cell suspension cultures have focused on transformed (hairy) roots. Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic (cancerous) roots produced by A. rhizogenes infection are characterized by high growth rate, genetic stability and growth in hormone free media. These genetically transformed root cultures can produce levels of secondary metabolites comparable to that of intact plants. Hairy root cultures offer promise for high production and productivity of valuable secondary metabolites (used as pharmaceuticals, pigments and flavors) in many plants. The main constraint for commercial exploitation of hairy root cultivations is the development and scaling up of appropriate reactor vessels (bioreactors) that permit the growth of interconnected tissues normally unevenly distributed throughout the vessel. Emphasis has focused on designing appropriate bioreactors suitable to culture the delicate and sensitive plant hairy roots. Recent reactors used for mass production of hairy roots can roughly be divided as liquid-phase, gas-phase, or hybrid reactors. The present review highlights the nature, applications, perspectives and scale up of hairy root cultures for the production of valuable secondary metabolites.     

Microalgal mass culture systems and methods: Their limitation and potential

Cultivation of microalgae using natural and man-made open-ponds istechnologically simple, but not necessary cheap due to the high downstream processing cost. Products of microalgae cultured in open-pondscould only be marketed as value-added health food supplements, specialityfeed and reagents for research. The need to achieve higher productivityand to maintain monoculture of algae led to the development of enclosedtubular and flat plate photobioreactors. Despite higher biomassconcentration and better control of culture parameters, data accumulatedin the past 25 years have shown that the illuminated areal, volumetricproductivity and cost of production in these enclosed photobioreactors arenot better than those achievable in open-pond cultures. The technicaldifficulty in sterilizing these photobioreactors has hindered their applicationfor the production of high value pharmaceutical products. The alternativeof cultivating microalgae in heterotrophic mode in sterilizable fermentorshas achieved some commercial success. The maximum specific growth ratesof heterotrophic algal cultures are in general slower than those measured inphotosynthetic cultures. The biomass productivity of heterotrophic algalcultures has yet to achieve a level that is comparable to industrialproduction of yeast and other heterotrophic microrganisms. Mixotrophiccultivation of microalage takes advantage of their ability to utilise organicenergy and carbon substrates and perform photosynthesis concurrently. Moreover, production of some algal metabolites is light regulated. Futuredesign of sterilizable bioreactors for mixotrophic cultivation of microalgaemay have to consider the organic substrate the main source of energy andlight the supplemental source of energy, a change in mindset.     

In Vitro Haustorium Development in Roots and Root Cultures of the Hemiparasitic Plant Triphysaria Versicolor

Parasitic plants in the Orobanchaceae invade host plant roots through root organs called haustoria. Parasite roots initiate haustorium development when exposed to specific secondary metabolites that are released into the rhizosphere by host plant roots. While molecular approaches are increasingly being taken to understand the genetic mechanism underlying these events, a limitation has been the lack of a transformation system for parasitic plants. Since the haustorium development occurs in roots of Orobanchaceae, root cultures may be suitable material for transient or stable transformation experiments. To this end, root cultures were obtained from explants, and subsequently calluses, from the hemiparasitic plant Triphysaria versicolor. The cultured roots retained their competence to form haustoria when exposed to host roots, host root exudates, or purified haustorium-inducing factors. The root culture haustoria invaded host roots and initiated a vascular continuity between the parasite and host roots. The ontogeny of haustoria development on root cultures was indistinguishable from that on seedlings roots. Root cultures should provide useful material for molecular studies of haustorium development.     

Method for the passaging of microcarrier cultures to a production scale for producing high titre Disabled Infectious Single Cycle-Herpes Simplex Virus Type-2

A complementary cell line CR2 is currently used to propagate the Disabled Infectious Single Cycle Herpes Simplex Virus Type 2 (DISC HSV-2) on a small laboratory scale upto 15 L. These cultures are initiated by passaging the cells from roller bottle cultures. Whilst this is suitable for the laboratory scale it is totally impractical for use in seeding an industrial manufacturing scaled version of the culture. It is paramount to have a robust system for passaging cells from a small microcarrier culture system to a larger one by a serial subculturing regime. Here we report on the successes we have had in our laboratory in scaling up our production system for the DISC HSV-2 from small 1-L cultures to a 50-L vessel with the maintenance of the viral productivity. Ease of use, reproducibility and the need to minimise overall production times were factors which were taken into consideration whilst developing our procedures. We were aware of the need to keep a production train simple and as short as possible as this was the small scale study for an envisaged manfacturing process.     

光生物反应器培养微藻研究进展

微藻具有固定CO2和净化有机废水的能力,在环保、食品饲（饵）料、医药和生物能源开发等领域备受关注,但规模化培养及其产业化仍是研究的难点,亟待解决。就常用于大规模培养微藻的光生物反应器的特点和结构进行了综述。其中,封闭式微藻光生物反应器能够较好地调控藻种的培养条件、不易遭受污染,藻种的纯度容易控制,但培养规模小,生产成本较高;而开放式微藻光生物反应器无法精确控制藻种生长环境,但生产规模大、产量高、生产成本低,因此应用广泛。最佳的方法是综合两者优点,即首先利用封闭式微藻光生物反应器进行中试放大,大量繁殖藻种,然后投入开放式微藻光生物反应器内进行大规模商业生产,此方法有望成为微藻光生物反应器的发展方向,以期为微藻大规模培养提供参考借鉴。     

Biotechnology of algal biomass production: a review of systems for outdoor mass culture

Microalgae are very efficient solar energy converters and they can produce a great variety of metabolites. Man has always tried to take advantage of these proporties through algal mass culture. Despite the fact that many applications for microalgae have been described in the literature, these micro-organisms are still of minor economic importance. Industrial reactors for algal culture are at present, all designed as open race-ways (shallow open ponds where culture is circulated by a paddle-wheel). Technical and biological limitations of these open systems have given rise to the development of enclosed photoreactors (made of transparent tubes, sleeves or containers and where light source may be natural or artificial). The present review surveys advances in these two technologies for cultivation of microalgae. Starting from published results, the advantages and disadvantages of open systems and closed photobioreactors are discussed. A few open systems are presented for which particularly reliable results are available. Emphasis is then put on closed systems, which have been considered as capital intensive and are justified only when a fine chemical is to be produced.     

Kinetic analysis of red pigment and citrinin production by Monascus ruber as a function of organic acid accumulation

In submerged cultures performed in synthetic medium containing glucose and glutamate, the filamentous fungus Monascus ruber produced a red pigment and a mycotoxin, citrinin. In oxygen-limiting conditions, the production of these two metabolites was growth-associated, as was the production of primary metabolites. In oxygen-excess conditions, the profile of citrinin production was typical of a secondary metabolite, since it was produced mostly during the stationary phase. In contrast, the production of the pigment decreased rapidly throughout the culture, showing a profile characteristic of an inhibitory mechanism. The organic acids produced during the culture, L-malate and succinate, were shown to be slightly inhibitory against pigment production, while citrinin production was unaffected. However, this inhibition could not account for the observed profile of pigment production in batch cultures. Other dicarboxylic acids such as fumarate or tartrate showed a similar effect to that provoked by malate and succinate as regards pigment production. It was concluded that the decrease in red pigment production during the culture was due to the inhibitory effect of an unknown product whose accumulation was favored in aerobic conditions.     

Photobioreactors for mass cultivation of algae

Algae have attracted much interest for production of foods, bioactive compounds and also for their usefulness in cleaning the environment. In order to grow and tap the potentials of algae, efficient photobioreactors are required. Although a good number of photobioreactors have been proposed, only a few of them can be practically used for mass production of algae. One of the major factors that limits their practical application in algal mass cultures is mass transfer. Thus, a thorough understanding of mass transfer rates in photobioreactors is necessary for efficient operation of mass algal cultures. In this review article, various photobioreactors that are very promising for mass production of algae are discussed.     

Medicinal plant cell suspension cultures: pharmaceutical applications and high-yielding strategies for the desired secondary metabolites

The development of plant tissue (including organ and cell) cultures for the production of secondary metabolites has been underway for more than three decades. Plant cell cultures with the production of high-value secondary metabolites are promising potential alternative sources for the production of pharmaceutical agents of industrial importance. Medicinal plant cell suspension cultures (MPCSC), which are characterized with the feature of fermentation with plant cell totipotency, could be a promising alternative “chemical factory”. However, low productivity becomes an inevitable obstacle limiting further commercialization of MPCSC and the application to large-scale production is still limited to a few processes. This review generalizes and analyzes the recent progress of this bioproduction platform for the provision of medicinal chemicals and outlines a range of trials taken or underway to increase product yields from MPCSC. The scale-up of MPCSC, which could lead to an unlimited supply of pharmaceuticals, including strategies to overcome and solution of the associated challenges, is discussed.