Animal signals: Ethological and games-theory approaches are not incompatible

Recent authors have contrasted the ‘traditional ethological approach’ to the study of animal signals with that derived from games theory. It is argued here that the ‘traditonal ethological approach’ they portray is not in keeping with main stream of ethological research on animal signals. In particular, it has not been generally assumed that the evolution of animal signals was based on selection for mutual benefit of actor and reactor, nor that signals carry precise information of what the actor will do next. A synthesis of the ethological and games-theory approaches is possible. It is suggested that many threat displays may signal ‘Will stay, but attack if provoked’ or ‘Will stay, but will flee if provoked’, and that the subsequent behaviour of the displaying bird depends in part on that of the reactor.     

Scale-up of cell culture bioreactors using biomechatronic design

Scale-up of cell culture bioreactors is a challenging engineering work that requires wide competence in cell biology, mechanical engineering and bioprocess design. In this article, a new approach for cell culture bioreactor scale-up is suggested that is based on biomechatronic design methodology. The approach differs from traditional biochemical engineering methodology by applying a sequential design procedure where the needs of the users and alternative design solutions are systematically analysed. The procedure is based on the biological and technical functions of the scaled-up bioreactor that are derived in functional maps, concept generation charts and scoring and interaction matrices. Basic reactor engineering properties, such as mass and heat transfer and kinetics are integrated in the procedure. The methodology results in the generation of alternative design solutions that are thoroughly ranked with help of the user needs. Examples from monoclonal antibodies and recombinant protein production illuminate the steps of the procedure. The methodology provides engineering teams with additional tools that can significantly facilitate the design of new production methods for cell culture processes.     

Scale-up from shake flasks to fermenters in batch and continuous mode with Corynebacterium glutamicum on lactic acid based on oxygen transfer and pH

Scale-up from shake flasks to fermenters has been hampered by the lack of knowledge concerning the influence of operating conditions on mass transfer, hydromechanics, and power input. However, in recent years the properties of shake flasks have been described with empirical models. A practical scale-up strategy for everyday use is introduced for the scale-up of aerobic cultures from shake flasks to fermenters in batch and continuous mode. The strategy is based on empirical correlations of the volumetric mass transfer coefficient (k(L) a) and the pH. The accuracy of the empirical k(L) a correlations and the assumptions required to use these correlations for an arbitrary biological medium are discussed. To determine the optimal pH of the culture medium a simple laboratory method based on titration curves of the medium and a mechanistic pH model, which is solely based on the medium composition, is applied. The effectiveness of the scale-up strategy is demonstrated by comparing the behavior of Corynebacterium glutamicum on lactic acid in shake flasks and fermenters in batch and continuous mode. The maximum growth rate (micro(max) = 0.32 h(-1)) and the oxygen substrate coefficient (Y O2 /S= 0.0174 mol/l) of C. glutamicum on lactic acid were equal for shake flask, fermenter, batch, and continuous cultures. The biomass substrate yield was independent of the scale, but was lower in batch cultures (Y(X/S) = 0.36 g/g) than in continuous cultures (Y(X/S) = 0.45 g/g). The experimental data (biomass, respiration, pH) could be described with a simple biological model combined with a mechanistic pH model.     

Factors influencing the production of a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid, by Pseudomonas aeruginosa PR3 (NRRL B-18602) in batch cultures

Pseudomonas aeruginosa PR3 (NRRL B-18602) converts oleic acid to a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD). Parameters that included medium volume, cell growth time, gyration speed, pH, substrate concentration, and dissolved oxygen concentration were evaluated for a scale-up production of DOD in batch cultures using Fernbach flasks and a bench-top bioreactor. Maximum production of about 2 g DOD (38% yield) was attained in Fernbach flasks containing 500 ml medium when cells were grown at 28 degrees C and 300 rpm for 16-20 h and the culture was adjusted to pH 7 prior to substrate addition. Increases of medium volume and substrate concentration failed to enhance yield. When batch cultures were initially conducted in a reactor, excessive foaming occurred that made the bioconversion process inoperable. This was overcome by a new aeration mechanism that provided adequate dissolved oxygen to the fermentation culture. Under the optimal conditions of 650 rpm, 28 degrees C, and 40-60% dissolved oxygen concentration, DOD production reached about 40 g (40% yield) in 4.5 L culture medium using a 7-L reactor vessel. This is the first report on a successful scale-up production of DOD.     

Scale-up and design of a pilot-plant photobioreactor for the continuous culture of Spirulina platensis

Scale-up of bioreactors has the intrinsic difficulty of establishing a reliable relationship among physical parameters involved in the design of the new bioreactor and the physiology of the cultured cells. This is more critical in those cases where a more complex operation of the bioreactor is needed, such as in photobioreactors. A key issue in the operation of photobioreactors is establishing a quantification for the interaction between external illumination, internal light distribution and cell growth. In this paper an approach to the scale-up of a photobioreactor for the culture of Spirulina platensis, based on a mathematical model describing this interaction, and the operation of a previous reactor 10 times smaller is presented. The paper describes the approach followed in the scale-up, the analysis of different design constraints, the physical realization of the new bioreactor design, innovative use of plastic material walls to improve reactor safety, and finally the corroboration of its satisfactory operation.     

Development and optimization of an adenovirus production process

Adenoviral vectors have a number of advantages such as their ability to infect post-mitotic tissues. They are produced at high titers and are currently used in 28% of clinical protocols targeting mainly cancer diseases through different strategies. The major disadvantages of the first generation of recombinant adenoviruses are addressed by developing new recombinant adenovirus vectors with improved capacity and safety and reduced inflammatory response. To meet increasing needs of adenovirus vectors for gene therapy programs, parallel development of efficient, scalable and reproducible production processes is required. HEK-293 complementing cell line physiology, metabolism and viral infection kinetics were studied at small scale to identify optimal culture conditions. Batch, fed-batch and perfusion culture modes were evaluated. Development of new monitoring tools (in situ GFP probe) and quantification techniques (HPLC determination of total viral particles) contributed to acceleration of process development. On-line monitoring of physiological parameters such as respiration and biovolume of the culture allowed real-time supervision and control of critical phases of the process. Use of column chromatographic steps instead of CsCl gradient purification greatly eased process scale-up. The implementation of the findings at large scale led to the development of an optimized and robust integrated process for adenovirus production using HEK-293 cells cultured in suspension and serum-free medium. The two-step column-chromatography purification was optimized targeting compliance with clinical material specifications. The complete process is routinely operated at a 20-L scale and has been scaled-up to 100 L. Scale-up of adenoviral vector production in suspension and serum-free medium, and purification according to regulatory requirements, are achievable. To overcome metabolic limitations at high cell densities, use of perfusion mode with low-shear cell retention devices is now a common trend in adenovirus manufacturing. Further process improvements will rely on better understanding of the mechanisms of virus replication and maturation in complementing host cells.     

Modeling Complex Biological Macromolecules: Reduction of Multibead Models

The shape of simple and complex biological macromolecules can be approximated by bead modeling procedures. Such approaches are required, for example, for the analysis of the scattering and hydrodynamic behavior of the models under analysis and the prediction of their molecular properties. Using the atomic coordinates of proteins for modeling inevitably leads to models composed of a multitude of beads. In particular, for hydrodynamic modeling, a drastic reduction of the bead number may become unavoidable to enable computation. A systematic investigation of different approaches and computation modes shows that the ‘running mean’, ‘cubic grid,’ and ‘hexagonal grid’ approaches are successful, provided that the extent of reduction does not exceed a factor of 100 and the grid approaches use beads of unequal size and the beads are located at the centers of gravity. Further precautions to be taken include usage of appropriate interaction tensors for overlapping beads of unequal size and appropriate volume corrections when calculating intrinsic viscosities. The applied procedures were tested with the small protein lysozyme in a case study and were then applied to the huge capsid of the phage fr and its trimeric building block. The appearance of the models and the agreement of molecular properties and distance distribution functions of unreduced and reduced models can be used as evaluation criteria.     

Coupled metabolic-hydrodynamic modeling enabling rational scale-up of industrial bioprocesses

Metabolomics aims to address what and how regulatory mechanisms are coordinated to achieve flux optimality, different metabolic objectives as well as appropriate adaptations to dynamic nutrient availability. Recent decades have witnessed that the integration of metabolomics and fluxomics within the goal of synthetic biology has arrived at generating the desired bioproducts with improved bioconversion efficiency. Absolute metabolite quantification by isotope dilution mass spectrometry represents a functional readout of cellular biochemistry and contributes to the establishment of metabolic (structured) models required in systems metabolic engineering. In industrial practices, population heterogeneity arising from fluctuating nutrient availability frequently leads to performance losses, that is reduced commercial metrics (titer, rate, and yield). Hence, the development of more stable producers and more predictable bioprocesses can benefit from a quantitative understanding of spatial and temporal cell-to-cell heterogeneity within industrial bioprocesses. Quantitative metabolomics analysis and metabolic modeling applied in computational fluid dynamics (CFD)-assisted scale-down simulators that mimic industrial heterogeneity such as fluctuations in nutrients, dissolved gases, and other stresses can procure informative clues for coping with issues during bioprocessing scale-up. In previous studies, only limited insights into the hydrodynamic conditions inside the industrial-scale bioreactor have been obtained, which makes case-by-case scale-up far from straightforward. Tracking the flow paths of cells circulating in large-scale bioreactors is a highly valuable tool for evaluating cellular performance in production tanks. The “lifelines” or “trajectories” of cells in industrial-scale bioreactors can be captured using Euler-Lagrange CFD simulation. This novel methodology can be further coupled with metabolic (structured) models to provide not only a statistical analysis of cell lifelines triggered by the environmental fluctuations but also a global assessment of the metabolic response to heterogeneity inside an industrial bioreactor. For the future, the industrial design should be dependent on the computational framework, and this integration work will allow bioprocess scale-up to the industrial scale with an end in mind.     

Trickle-bed root culture bioreactor design and scale-up: growth, fluid-dynamics, and oxygen mass transfer

Trickle-bed root culture reactors are shown to achieve tissue concentrations as high as 36 g DW/L (752 g FW/L) at a scale of 14 L. Root growth rate in a 1.6-L reactor configuration with improved operational conditions is shown to be indistinguishable from the laboratory-scale benchmark, the shaker flask (mu=0.33 day(-1)). These results demonstrate that trickle-bed reactor systems can sustain tissue concentrations, growth rates and volumetric biomass productivities substantially higher than other reported bioreactor configurations. Mass transfer and fluid dynamics are characterized in trickle-bed root reactors to identify appropriate operating conditions and scale-up criteria. Root tissue respiration goes through a minimum with increasing liquid flow, which is qualitatively consistent with traditional trickle-bed performance. However, liquid hold-up is much higher than traditional trickle-beds and alternative correlations based on liquid hold-up per unit tissue mass are required to account for large changes in biomass volume fraction. Bioreactor characterization is sufficient to carry out preliminary design calculations that indicate scale-up feasibility to at least 10,000 liters.     

Comparison of air-lift and stirred tank reactors for itaconic acid production by Aspergillus terreus

In a 2-l stirred tank reactor (STR), maximum production rate ofitaconic acid was 0.48g/l.h , for an agitation rate of 400 rpm andan aeration rate of 0.5 vvm. In an air-lift reactor (ALR) themaximum production rate was 0.64 g/l.h at an O supply rate of0.41 l O /l. min. Power input per unit volume which gave themaximum production rates for STR and ALR were 1180 and 542 W/m 3,respectively. If O -enriched air was used in place of air for ALR,the corre-sponding power input per unit volume was decreased to 34W/m 3 . ALR requires less power input per unit volume in comparisonwith that of STR whether therefore air or O -enriched air is used.ALR would be a suitable bioreactor for a large production of itaconicacid.     

Factors Influencing the Production of a Novel Compound, 7,10-Dihydroxy-8(<Emphasis Type="Italic">E</Emphasis>)-octadecenoic Acid,by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PR3 (NRRL B-18602) in Batch Cultures

Pseudomonas aeruginosa PR3 (NRRL B-18602) converts oleic acid to a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD). Parameters that included medium volume, cell growth time, gyration speed, pH, substrate concentration, and dissolved oxygen concentration were evaluated for a scale-up production of DOD in batch cultures using Fernbach flasks and a bench-top bioreactor. Maximum production of about 2 g DOD (38% yield) was attained in Fernbach flasks containing 500 ml medium when cells were grown at 28°C and 300 rpm for 16–20 h and the culture was adjusted to pH 7 prior to substrate addition. Increases of medium volume and substrate concentration failed to enhance yield. When batch cultures were initially conducted in a reactor, excessive foaming occurred that made the bioconversion process inoperable. This was overcome by a new aeration mechanism that provided adequate dissolved oxygen to the fermentation culture. Under the optimal conditions of 650 rpm, 28°C, and 40–60% dissolved oxygen concentration, DOD production reached about 40 g (40% yield) in 4.5 L culture medium using a 7-L reactor vessel. This is the first report on a successful scale-up production of DOD. Received: 26 September 2002 / Accepted: 24 October 2002     

Ecomarkets for conservation and sustainable development in the coastal zone

Because conventional markets value only certain goods or services in the ocean (e.g. fish), other services provided by coastal and marine ecosystems that are not priced, paid for, or stewarded tend to become degraded. In fact, the very capacity of an ecosystem to produce a valued good or service is often reduced because conventional markets value only certain goods and services, rather than the productive capacity. Coastal socio‐ecosystems are particularly susceptible to these market failures due to the lack of clear property rights, strong dependence on resource extraction, and other factors. Conservation strategies aimed at protecting unvalued coastal ecosystem services through regulation or spatial management (e.g. Marine Protected Areas) can be effective but often result in lost revenue and adverse social impacts, which, in turn, create conflict and opposition. Here, we describe ‘ecomarkets’ – markets and financial tools – that could, under the right conditions, generate value for broad portfolios of coastal ecosystem services while maintaining ecosystem structure and function by addressing the unique problems of the coastal zone, including the lack of clear management and exclusion rights. Just as coastal tenure and catch‐share systems generate meaningful conservation and economic outcomes, it is possible to imagine other market mechanisms that do the same with respect to a variety of other coastal ecosystem goods and services. Rather than solely relying on extracting goods, these approaches could allow communities to diversify ecosystem uses and focus on long‐term stewardship and conservation, while meeting development, food security, and human welfare goals. The creation of ecomarkets will be difficult in many cases, because rights and responsibilities must be devolved, new social contracts will be required, accountability systems must be created and enforced, and long‐term patterns of behaviour must change. We argue that efforts to overcome these obstacles are justified, because these deep changes will strongly complement policies and tools such as Marine Protected Areas, coastal spatial management, and regulation, thereby helping to bring coastal conservation to scale.     

Scale-up and application of a cyclone reactor for fermentation processes

A cyclone reactor for microbial fermentation processes was developed with high oxygen transfer capabilities. Three geometrically similar cyclone reactors with 0.5?l, 2.5?l and 15?l liquid volume, respectively, were characterized with respect to oxygen mass transfer, mixing time and residence time distribution. Semi-empirically correlations for prediction of oxygen mass transfer and mixing times were identified for scale-up of cyclone reactors. A volumetric oxygen mass transfer coefficient k _L a of 1.0?s^?1 (available oxygen transfer rate with air: 29?kg?m^?3?h^?1) was achieved with the cyclone reactor at a volumetric power input of 40?kW?m^?3 and an aeration gas flow rate of 0.2?s^?1. Continuous methanol controlled production of formate dehydrogenase (FDH) with Candida boidinii in a 15?l cyclone reactor resulted in more than 100% improvement in dry cell mass concentration (64.5?g?l^?1) and in about 100% improvement in FDH space-time yield (300?U?l^?1?h^?1) compared to steady state results of a continuous stirred tank reactor.     

大数据-模型混合驱动下生物过程优化与放大的新机遇与挑战

当前,生物制造技术和产业是世界关注的热点.然而,生物过程优化与放大过程中普遍面临以下几个难题,包括:过程检测手段缺乏,难以满足关键指标参数的监控;细胞代谢认知匮乏,无法理性实现过程最优化调控;反应器环境差异大,导致逐级放大效率低下.文中针对以上亟待解决的关键问题,通过案例分析介绍发酵过程实时检测-动态调控-理性放大全链...     

Continuous ethanol fermentation in a tower reactor with flocculating yeast recycle: scale-up effects on process performance, kinetic parameters and model predictions

An unsegregated and unstructured model developed for a small-scale process of ethanol production in a tower reactor with cell recycle was applied to describe the experimental data obtained in a large-scale process. The model was developed considering the following points: reactor hydrodynamic behavior analogous to that of ideal CSTR, substrate limitation, inhibition phenomena linked both to ethanol and to biomass, absence of fermentation in the settler, and no loss of cell viability. The scale-up criterion consisted in maintaining an identical relation settler volume/fermentor volume on the two scales. All large-scale experiments were carried out using a flocculating yeast strain IR-2, isolated from fermented food, and identified as Saccharomyces cerevisiae. Sugarcane juice was used as the substrate source with sugar concentrations of 150?g/l. Different values of dilution rate and recycle ratio were employed (D?=?0.11–0.33?h^?1, α?=?5.4–18.0) and the temperature was of 32?°C. The kinetic parameters were similar on both scales and the model predictions agreed well with the large-scale experimental data.     

Biotechnological aspects of the production of the anticancer drug podophyllotoxin

The natural lignan podophyllotoxin, a dimerized product of two phenylpropanoid moieties which occurs in a few plant species, is a pharmacologically important compound for its anticancer activities. It is used as a precursor for the chemical synthesis of the anticancer drugs etoposide, teniposide and etopophose. The availability of this lignan is becoming increasingly limited because of the scarce occurrence of its natural sources and also because synthetic approaches for its production are still commercially unacceptable. Biotechnological production using cell culture may be considered as an alternative source. Selection of the best performing cell line, its maintenance and stabilization are necessary prerequisites for its production in bioreactors and subsequent scale-up of the cultivation process to the industrial level. Scale-up of growth and product yield depends on a multitude of factors, such as growth medium, physicochemical conditions, seed inoculum, type of reactor and processing conditions. The composition of the growth medium, elicitors and precursors, etc. can markedly influence the production. Optimum levels of parameters that facilitate high growth and product response in cell suspensions of Podophyllum hexandrum have already been determined by statistical design. P. hexandrum cells have successfully been cultivated in a 3-l stirred-tank bioreactor under low shear conditions in batch and fed-batch modes of operation. The batch kinetic data were used to identify the mathematical model which was then used to develop nutrient-feeding strategies for fed-batch cultivation to prolong the productive log phase of cultivation. An improvement in the production of podophyllotoxin to 48.8 mg l^–1 in a cell culture of P. hexandrum was achieved, with a corresponding volumetric productivity of 0.80 mg l^–1 day^–1, when the reactor was operated in continuous cell-retention mode. Efforts are being made to further enhance its production levels by the development of hairy root culture or by varying the channeling of precursors towards the desired biosynthetic pathway by molecular approaches.     

Design,Construction, and Starting-up of an Anaerobic Reactor for the Stabilisation,Handling, and Disposal of Excess Biological Sludge Generated in a Wastewater Treatment Plant

Design, construction, and starting-up of an upflow anaerobic sludge blanket reactor was carried out. This system was proposed for excess sludge stabilisation, particularly that generated at an activated sludge wastewater treatment facility installed in a sugarcane mill. The upflow anaerobic sludge blanket (UASB) reactor built, had a working volume of 22.3 m³and a hydraulic residence time of 22 days. Methane production was at a maximum of 79% volume with an average of 60% for this treatment. For starting up the anaerobic reactor, a suitable inoculum from a neighboring plant was used. As the waste characteristics in both plants were different, an acclimation procedure was followed to achieve granulation. Control and stability of anaerobic reactions were monitored with alkalinity data, using the so-called ‘alfa alkalinity’ to try to keep its value at around 0.4. Once pseudosteady-state conditions were reached (chemical oxygen demand reduction and methane-rich biogas production within ±10 percent), the organic load was steadily increased up to feeding 100% excess sludge. The UASB reactor used to stabilise the excess biomass generated a sludge with a much lower volume than that originally fed. Its design ensured adequate hydraulic flow and biogas production with a high methane content. The bacteria were attached constituting spheres and very minor maintenance operations were required.     

Proteomic tools against the neglected pathology of snake bite envenoming

This article covers the application of proteomic tools (‘venomics’, ‘antivenomics’ and ‘venom phenotyping’) to study the composition and natural history of snake venoms, and the cross-reactivity of antivenoms with homologous and heterologous venoms, to help address the neglected pathology of snake bite envenoming. The identification of evolutionary and immunological trends may help to replace the traditional geographic- and phylogenetic-driven hypotheses for antivenom production strategies with a more rational approach based on proteome phenotype and immunological profile similarities. Antivenomics and venom phenotyping may also contribute to expand the clinical range of currently existing antidotes.     

Applicability of near-infrared spectroscopy for process monitoring in bioethanol production

The applicability of near-infrared (NIR) spectroscopy to bioethanol production is investigated. The NIR technique can provide assistance for rapid process monitoring, because organic compounds absorb radiation in the wavelength range 1100–2300 nm. For quantification of a sample's chemical composition, a calibration model is required that relates the measured spectral NIR absorbances to concentrations. For calibration, the concentrations in g/l are determined by the analytical reference method high performance liquid chromatography (HPLC). The calibration models are built and validated for moisture, protein, and starch in the feedstock material, and for glucose, ethanol, glycerol, lactic acid, acetic acid, maltose, fructose, and arabinose in the processed broths. These broths are prepared in laboratory experiments: The ground cereal samples are fermented to alcoholic broths (‘mash’), which are divided into an ethanol fraction and the residual fraction ‘stillage’ by distillation. The NIR technology together with chemometrics proved itself beneficial for fast monitoring of the current state of the bioethanol process, primarily for higher concentrated substances (>1 g/l).     

Pattern, process and prediction in aquatic ecology. A limnological view of some general ecological problems

1. New scientific concepts such as models of chaos, complex dynamics and non-linear interactions have the potential to contribute to an improved understanding of ecological patterns and processes. This paper discusses some of the known dynamics of phytoplankton, pelagic food chains and nutrient cycles in the light of some of these new concepts. The paper brings these new conceptual models together with data from a wide range of sources in an attempt to produce a synthesis of system behaviour which allows us to understand why some things are inherently more predictable than others. In particular it looks at the limnological management tools of empirical biomass models and biomanipulation and at the need for prediction of species composition. 2. The structures observed in ecosystems (nutrient pools, sizes, species, temporal/spatial patterns) show properties at a spectrum of scales, as do the processes (fluxes, grazing, competition). Both respond to a spectrum of external perturbations that may be climatologically or anthropogenically induced. Empirical biomass models work because of the annual averaging of pattern and process and because of some inherent properties of the functioning of pelagic ecosystems. Many aspects of ecosystem pattern and process vary in a regular way with trophic state. Examination of empirical data sets can lead to an improved understanding of system behaviour if questions are asked about why things happen the way they do. 3. Feedbacks between pattern, process and periodicity are seen to be an inherent property of the system. Understanding the fundamental dynamics of non-linear interactions in ecosystems may make it possible to exploit the external spectrum of environmental perturbations and to control system function. For example, by imposing external physical perturbations on pelagic systems it may be possible to manipulate the species composition of the phytoplankton community. Because of the complexity of possible interactions both ‘horizontally’ between species and ‘vertically’ within the food chain, any prediction of species composition will necessarily be probabilistic.