Deceleration-stats save much time during phototrophic culture optimization.

In case of phototrophic cultures, photobioreactor costs contribute significantly to the total operating costs. Therefore one of the most important parameters to be determined is the maximum biomass production rate, if biomass or a biomass associated product is the desired product. This is traditionally determined in time consuming series of chemostat cultivations. The goal of this work is to assess the experimental time that can be saved by applying the deceleration stat (D-stat) technique to assess the maximum biomass production rate of a phototrophic cultivation system, instead of a series of chemostat cultures. A mathematical model developed by Geider and co-workers was adapted in order to describe the rate of photosynthesis as a function of the local light intensity. This is essential for the accurate description of biomass productivity in phototrophic cultures. The presented simulations demonstrate that D-stat experiments executed in the absence of pseudo steady-state (i.e., the arbitrary situation that the observed specific growth rate deviates <5% from the dilution rate) can still be used to accurately determine the maximum biomass productivity of the system. Moreover, this approach saves up to 94% of the time required to perform a series of chemostat experiments that has the same accuracy. In case more information on the properties of the system is required, the reduction in experimental time is reduced but still significant.

Modeling growth, lipid accumulation and lipid turnover in submerged batch cultures of Umbelopsis isabellina.

The production of lipids by oleaginous yeast and fungi becomes more important because these lipids can be used for biodiesel production. To understand the process of lipid production better, we developed a model for growth, lipid production and lipid turnover in submerged batch fermentation. This model describes three subsequent phases: exponential growth when both a C-source and an N-source are available, carbohydrate and lipid production when the N-source is exhausted and turnover of accumulated lipids when the C-source is exhausted. The model was validated with submerged batch cultures of the fungus Umbelopsis isabellina (formerly known as Mortierella isabellina) with two different initial C/N-ratios. Comparison with chemostat cultures with the same strain showed a significant difference in lipid production: in batch cultures, the initial specific lipid production rate was almost four times higher than in chemostat cultures but it decreased exponentially in time, while the maximum specific lipid production rate in chemostat cultures was independent of residence time. This indicates that different mechanisms for lipid production are active in batch and chemostat cultures. The model could also describe data for submerged batch cultures from literature well.

Modeling lipid accumulation in oleaginous fungi in chemostat cultures. II: Validation of the chemostat model using yeast culture data from literature.

A model that predicts cell growth, lipid accumulation and substrate consumption of oleaginous fungi in chemostat cultures (Meeuwse et al. in Bioproc Biosyst Eng. doi: 10.1007/s00449-011-0545-8 , 2011) was validated using 12 published data sets for chemostat cultures of oleaginous yeasts and one published data set for a poly-hydroxyalkanoate accumulating bacterial species. The model could describe all data sets well with only minor modifications that do not affect the key assumptions, i.e. (1) oleaginous yeasts and fungi give the highest priority to C-source utilization for maintenance, second priority to growth and third priority to lipid accumulation, and (2) oleaginous yeasts and fungi have a growth rate independent maximum specific lipid production rate. The analysis of all data showed that the maximum specific lipid production rate is in most cases very close to the specific production rate of membrane and other functional lipids for cells growing at their maximum specific growth rate. The limiting factor suggested by Ykema et al. (in Biotechnol Bioeng 34:1268-1276, 1989), i.e. the maximum glucose uptake rate, did not give good predictions of the maximum lipid production rate.

Modeling lipid accumulation in oleaginous fungi in chemostat cultures: I. Development and validation of a chemostat model for Umbelopsis isabellina.

Lipid-accumulating fungi may be able to produce biodiesel precursors from agricultural wastes. As a first step in understanding and evaluating their potential, a mathematical model was developed to describe growth, lipid accumulation and substrate consumption of the oleaginous fungus Umbelopsis isabellina (also known as Mortierella isabellina) in submerged chemostat cultures. Key points of the model are: (1) if the C-source supply rate is limited, maintenance has a higher priority than growth, which has a higher priority than lipid production; (2) the maximum specific lipid production rate of the fungus is independent of the actual specific growth rate. Model parameters were obtained from chemostat cultures of U. isabellina grown on mineral media with glucose and NH(4) (+). The model describes the results of chemostat cultures well for D > 0.04 h(-1), but it has not been validated for lower dilution rates because of practical problems with the filamentous fungus. Further validation using literature data for oleaginous yeasts is described in part II of this paper. Our model shows that not only the C/N-ratio of the feed, but also the dilution rate highly influences the lipid yield in chemostat cultures.

Expression of phosphofructokinase in Neisseria meningitidis.

Neisseria meningitidis serogroup B is a pathogen that can infect diverse sites within the human host. According to the N. meningitidis genomic information and experimental observations, glucose can be completely catabolized through the Entner-Doudoroff pathway and the pentose phosphate pathway. The Embden-Meyerhof-Parnas pathway is not functional, because the gene for phosphofructokinase (PFK) is not present. The phylogenetic distribution of PFK indicates that in most obligate aerobic organisms, PFK is lacking. We conclude that this is because of the limited contribution of PFK to the energy supply in aerobically grown organisms in comparison with the energy generated through oxidative phosphorylation. Under anaerobic or microaerobic conditions, the available energy is limiting, and PFK provides an advantage, which explains the presence of PFK in many (facultatively) anaerobic organisms. In accordance with this, in silico flux balance analysis predicted an increase of biomass yield as a result of PFK expression. However, analysis of a genetically engineered N. meningitidis strain that expressed a heterologous PFK showed that the yield of biomass on substrate decreased in comparison with a pfkA-deficient control strain, which was associated mainly with an increase in CO(2) production, whereas production of by-products was similar in the two strains. This might explain why the pfkA gene has not been obtained by horizontal gene transfer, since it is initially unfavourable for biomass yield. No large effects related to heterologous expression of pfkA were observed in the transcriptome. Although our results suggest that introduction of PFK does not contribute to a more efficient strain in terms of biomass yield, achievement of a robust, optimal metabolic network that enables a higher growth rate or a higher biomass yield might be possible after adaptive evolution of the strain, which remains to be investigated.

Quantification and characterization of enzymatically produced hyaluronan with fluorophore-assisted carbohydrate electrophoresis.

Hyaluronan (HA) is a polysaccharide with high-potential medical applications, depending on the chain length and the chain length distribution. Special interest goes to homogeneous HA oligosaccharides, which can be enzymatically produced using Pasteurella multocida hyaluronan synthase (PmHAS). We have developed a sensitive, simple, and fast method, based on fluorophore-assisted carbohydrate electrophoresis (FACE), for characterization and quantification of polymerization products. A chromatographic pure fluorescent template was synthesized from HA tetrasaccharide (HA4) and 2-aminobenzoic acid. HA4-fluor and HA4 were used as template for PmHAS-mediated polymerization of nucleotide sugars. All products, fluorescent and nonfluorescent, were analyzed with gel electrophoresis and quantified using lane densitometry. Comparison of HA4- and HA4-fluor-derived polymers showed that the fluorophore did not negatively influence the PmHAS-mediated polymerization. Only even-numbered oligosaccharide products were observed using HA4-fluor or HA4 as template. The fluorophore intensity was linearly related to its concentration, and the limit of detection was determined to be 7.4pmol per product band. With this assay, we can now differentiate oligosaccharides of size range DP2 (degree of polymerization 2) to approximately DP400, monitor the progress of polymerization reactions, and measure subtle differences in polymerization rate. Quantifying polymerization products enables us to study the influence of experimental conditions on HA synthesis.

Gene-expression-based quality scores indicate optimal harvest point in Bordetella pertussis cultivation for vaccine production.

The evolution of vaccine product quality during batch cultivation of Bordetella pertussis, the causative agent of whooping cough, was investigated with the goal to determine the optimal harvest point. The process was explored by measuring mRNA expression at frequent intervals during cultivation. The genes that are involved in virulence are already known for this product and changes in their expression levels are proposed to be indicative for product quality. A quantitative product quality score is calculated based on the expression levels of these virulence genes, which allows comparison of expected product quality between culture samples. Product quality scores were maximal throughout the logarithmic growth phase, but dropped significantly at the start of the stationary phase. This showed that the decreasing lactate and glutamate concentrations towards the end of the batch are critical for product quality. On-line measurement of these nutrients allows the cultivation process to be harvested at the optimal harvest point, increasing process robustness and consistency.

Evaluation of a critical process parameter: oxygen limitation during cultivation has a fully reversible effect on gene expression of Bordetella pertussis.

Modern (bio)pharmaceutical process development requires thorough investigation of all process parameters that are critical to product quality. The impact of a disturbance of such a parameter during processing needs to be known so that a rational decision can be made about the release of the product. In cultivation processes the dissolved oxygen (DO) concentration is generally accepted as being a critical parameter. In this article the impact of a 90 min period of oxygen limitation during the cultivation of the strictly aerobic Bordetella pertussis bacterium is investigated. The cultivation is the most important process step for the manufacturing of a vaccine against whooping cough disease. Samples were taken immediately before and after oxygen limitation and at the end of cultivation of four oxygen limited and three control cultivations. DNA microarray analysis of the full transcriptome of the B. pertussis bacterium revealed that a 90 min period of oxygen limitation has a substantial effect on overall gene expression patterns. In total 104 genes were identified as a significant hit at any of the sample points, of which 58 were directly related to oxygen limitation. The other genes were mainly affected towards the end of cultivation. Of all genes involved in oxygen limitation none were identified to show a significant difference between the oxygen limited and control cultivations at the end of the batch. This indicates a fully reversible effect of oxygen limitation on gene expression. This finding has implications for the risk assessment of dissolved oxygen concentration as a critical process parameter.

Novel applications for microbial transglutaminase beyond food processing.

Transglutaminase (EC 2.3.2.13) initially attracted interest because of its ability to reconstitute small pieces of meat into a 'steak'. The extremely high cost of transglutaminase of animal origin has hampered its wider application and has initiated efforts to find an enzyme of microbial origin. Since the early 1990s, many microbial transglutaminase-producing strains have been found, and production processes have been optimized. This has resulted in a rapidly increasing number of applications of transglutaminase in the food sector. However, applications of microbial transglutaminase in other sectors have been explored to a much lesser extent. Here, we will present the wider potential of transglutaminases and discuss recent efforts that could contribute to the realization of their potential.

Modeling Neisseria meningitidis B metabolism at different specific growth rates.

Neisseria meningitidis is a human pathogen that can infect diverse sites within the human host. The major diseases caused by N. meningitidis are responsible for death and disability, especially in young infants. At the Netherlands Vaccine Institute (NVI) a vaccine against serogroup B organisms is currently being developed. This study describes the influence of the growth rate of N. meningitidis on its macro-molecular composition and its metabolic activity and was determined in chemostat cultures. In the applied range of growth rates, no significant changes in RNA content and protein content with growth rate were observed in N. meningitidis. The DNA content in N. meningitidis was somewhat higher at the highest applied growth rate. The phospholipid and lipopolysaccharide content in N. meningitidis changed with growth rate but no specific trends were observed. The cellular fatty acid composition and the amino acid composition did not change significantly with growth rate. Additionally, it was found that the PorA content in outer membrane vesicles was significantly lower at the highest growth rate. The metabolic fluxes at various growth rates were calculated using flux balance analysis. Errors in fluxes were calculated using Monte Carlo Simulation and the reliability of the calculated flux distribution could be indicated, which has not been reported for this type of analysis. The yield of biomass on substrate (Y(x/s)) and the maintenance coefficient (m(s)) were determined as 0.44 (+/-0.04) g g(-1) and 0.04 (+/-0.02) g g(-1) h(-1), respectively. The growth associated energy requirement (Y(x/ATP)) and the non-growth associated ATP requirement for maintenance (m(ATP)) were estimated as 0.13 (+/-0.04) mol mol(-1) and 0.43 (+/-0.14) mol mol(-1) h(-1), respectively. It was found that the split ratio between the Entner-Doudoroff and the pentose phosphate pathway, the sole glucose utilizing pathways in N. meningitidis, had a minor effect on ATP formation rate but a major effect on the fluxes going through for instance the citric-acid cycle. For this reason, we presented flux ranges for underdetermined parts of metabolic network rather than presenting single flux values, which is more commonly done in literature.

Design process of an area-efficient photobioreactor.

This article describes the design process of the Green Solar Collector (GSC), an area-efficient photobioreactor for the outdoor cultivation of microalgae. The overall goal has been to design a system in which all incident sunlight on the area covered by the reactor is delivered to the algae at such intensities that the light energy can be efficiently used for biomass formation. A statement of goals is formulated and constraints are specified to which the GSC needs to comply. Specifications are generated for a prototype which form and function achieve the stated goals and satisfy the specified constraints. This results in a design in which sunlight is captured into vertical plastic light guides. Sunlight reflects internally in the guide and eventually scatters out of the light guide into flat-panel photobioreactor compartments. Sunlight is focused on top of the light guides by dual-axis positioning of linear Fresnel lenses. The shape and material of the light guide is such that light is maintained in the guides when surrounded by air. The bottom part of a light guide is sandblasted to obtain a more uniform distribution of light inside the bioreactor compartment and is triangular shaped to ensure the efflux of all light out of the guide. Dimensions of the guide are such that light enters the flat-panel photobioreactor compartment at intensities that can be efficiently used by the biomass present. The integration of light capturing, transportation, distribution and usage is such that high biomass productivities per area can be achieved.

Effect of feed and bleed rate on hybridoma cells in an acoustic perfusion bioreactor: metabolic analysis.

For the development of optimal perfusion processes, insight into the effect of feed and bleed rate on cell growth, productivity, and metabolism is essential. In the here presented study the effect of the feed and bleed rate on cell metabolism was investigated using metabolic flux analysis. Under all tested feed and bleed rates the biomass concentration as calculated from the nitrogen balance (biomass-nitrogen) increased linearly with an increase in feed rate, as would be expected. However, depending on the size of the feed and bleed rate, this increase was attained in two different ways. At low feed and bleed rates (Region I) the increase was obtained through an increase in viable-cell concentration, while the cellular-nitrogen content remained constant. At high feed and bleed rates (Region II) the increase was attained through an increase in cellular-nitrogen content, while the cell concentration remained constant. Per gram biomass-nitrogen, the specific consumption and production rates of the majority of the nutrients and products were identical in both regions, as were most of the fluxes. The major difference between the two regions was an increased flux from pyruvate to lactate and a decreased flux of pyruvate toward citrate in region II. The decreased in-flux at the level of citrate can either be balanced by a decreased out-flux toward lipid biosynthesis leading to a lower fraction of lipids in the cell, by a decreased out-flux toward the citric acid cycle resulting in a decreased energy generation, or by a combination of these. Finally, the specific productivity increases less than the nitrogen content per cell in region II, which implies that for obtaining maximum production rates it is important to increase the cell density and not only the biomass density.

Modeling conversion and transport phenomena in solid-state fermentation: a review and perspectives.

Solid-state fermentation (SSF) is accompanied inevitably by development of concentration and temperature gradients within the substrate particles and microbial biofilms. These gradients are needed for driving the transport of substrates and products. In addition, concentration gradients have been suggested to be crucial for obtaining the characteristics that define the products of SSF; nevertheless, gradients are also known to result in reduced productivity and unwanted side reactions. Solid-state fermentations are generally batch processes and this further complicates their understanding as conditions change with time. Mathematical models are therefore needed for improving the understanding of SSF processes and allowing their manipulation to achieve the desired outcomes. Existing models of SSF processes describe coupled substrate conversion and diffusion and the consequent microbial growth. Existing models disregard many of the significant phenomena that are known to influence SSF. As a result, available models cannot explain the generation of the numerous products that form during any SSF process and the outcome of the process in terms of the characteristics of the final product. This review critically evaluates the proposed models and their experimental validation. In addition, important issues that need to be resolved for improved modeling of SSF are discussed.

Hypothesized kinetic models for describing the growth of globular and encrusting demosponges.

The marine sponges Dysidea avara and Chondrosia reniformis (globular forms) were cultured in the laboratory on a diet of viable Phaeodactylum tricornutum cells and dissolved nutrients (algae and fish powders). Our growth data were combined with literature data for Pseudosuberites andrewsi (a globular sponge) and for the encrusting sponges Oscarella lobularis, Hemimycale columella, and Crambe crambe. The suitability of three growth models-linear, exponential, and radial accretive-for describing the growth of globular and encrusting sponges was assessed. Radial accretive growth was determined to be the best model to describe growth of both encrusting and globular sponges. Average growth rates of 0.051+/-0.016 and 0.019+/-0.003 mm/day (calculated as the increase of the radius of the sponge per day) were obtained experimentally for D. avara and C. reniformis, respectively.

Effect of low oxygen concentrations on growth and alpha-amylase production of Aspergillus oryzae in model solid-state fermentation systems.

Oxygen transfer in the fungal mat is a major concern in solid-state fermentation (SSF). Oxygen supply into the mycelial layers is hampered by diffusion limitation. For aerobic fungi, like Aspergillus oryzae, this oxygen depletion can be a severely limiting factor for growth and metabolite production. This paper describes the effects of a low oxygen concentration on growth at the levels of individual hyphae, colonies and overcultures, and on alpha-amylase production in overcultures. PDA medium was used to study the effect of a low oxygen concentration on hyphal elongation rate and branching frequency of hyphae, and radial extension rate of colonies of A. oryzae. We found similar saturation constants (K(O2)) of 0.1% (v/v in the gas phase) for oxygen concentration described with Monod kinetics, for branching frequency of hyphae and colony extension rate. When A. oryzae was grown as an over-culture on wheat-flour model substrate at 0.25% (v/v) oxygen concentration, the reduction in growth was more pronounced than as individual hyphae and a colony on PDA medium. Experimental results also showed that the specific alpha-amylase production rate under the condition of 0.25% (v/v) oxygen was reduced. Because the value of K(O2) is relatively low, it is reasonable to simplify the kinetics of growth of A. oryzae to zero-order kinetics in coupled diffusion/reaction models.

Significance of bed porosity, bran and specific surface area in solid-state cultivation of Aspergillus oryzae.

In this paper, the effects of bed porosity, bran and specific surface area on the oxygen uptake rate and alpha-amylase production during growth of Aspergillus oryzae on wheat grain and wheat-flour substrate are reported. The high oxygen uptake rate found during cultivation of A. oryzae on wheat-flour substrate was not reached on wheat grain. This is mainly due to the bran of the wheat grain. Using wheat-flour substrates, it was shown that extra bed porosity increased the alpha-amylase production and oxygen uptake rates. Furthermore, the peak oxygen uptake rate decreased with increasing surface area-volume ratio of the substrate particles, while the alpha-amylase production and the cumulative oxygen uptake per gram of initial substrate dry matter increased. The present work does not support a direct correlation between aerial mycelia and enzyme production. There is, however, a correlation between the alpha-amylase yield and the cumulative oxygen uptake (not the uptake rate). This implies that aerial mycelia could accelerate alpha-amylase production even if they do not increase the yield.

Marine sponges as pharmacy.

Marine sponges have been considered as a gold mine during the past 50 years, with respect to the diversity of their secondary metabolites. The biological effects of new metabolites from sponges have been reported in hundreds of scientific papers, and they are reviewed here. Sponges have the potential to provide future drugs against important diseases, such as cancer, a range of viral diseases, malaria, and inflammations. Although the molecular mode of action of most metabolites is still unclear, for a substantial number of compounds the mechanisms by which they interfere with the pathogenesis of a wide range of diseases have been reported. This knowledge is one of the key factors necessary to transform bioactive compounds into medicines. Sponges produce a plethora of chemical compounds with widely varying carbon skeletons, which have been found to interfere with pathogenesis at many different points. The fact that a particular disease can be fought at different points increases the chance of developing selective drugs for specific targets.

What can spores do for us?

Many organisms have the ability to form spores, a remarkable phase in their life cycles. Compared with vegetative cells, spores have several advantages (e.g. resistance to toxic compounds, temperature, desiccation and radiation) making them well suited to various applications. The applications of spores that first spring to mind are bio-warfare and the related, but more positive, field of biological control. Although they are often considered metabolically inert, spores can also be used as biocatalysts. Other uses for spores are found in the fields of probiotics, tumour detection and treatment, biosensing and in the "war against drugs".

What to do in marine biotechnology?

During the symposium "Marine Biotechnology: Basics and Applications", held 25 February-1 March, 2003 in Matalascañas, Spain, a special brainstorm session was organized. Two questions were addressed: 1, "What is the most desirable development in marine biotechnology"?; 2, "What is the most spectacular development in this field in your 'wildest' dreams"? The outcome of this session is reported in this paper. From the more than 250 ideas generated, concern for the environment and human health emerged as the most significant issues.

Progress towards a controlled culture of the marine sponge Pseudosuberites andrewsi in a bioreactor.

Explants of the tropical sponge Pseudosuberites andrewsi were fed with the marine diatom Phaeodactylum tricornotum. The food was supplied either as intact algae or as a filtered crude extract. Growth (measured as an increase in underwater weight) was found in both experiments. The explants fed with intact algae increased to an average underwater weight of 255% of the initial weight in 45-60 days. The explants fed with crude extract increased to an average of 200% of the initial weight in 30 days. These results show that it is possible to grow a sponge using a single microorganism species as a food source. In addition, it was demonstrated that sponges are also capable of growing on non-particulate food. Therefore, this study is an important step forward towards the development of controlled, in vivo sponge cultures.