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Supplemental Digital Content is available in the text., IMPORTANCE: A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 is hypothesized to play a role in the pathogenesis of invasive infection, but studies in sepsis are lacking. OBJECTIVES: To study A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 protein level in pediatric sepsis and to study the association with outcome. DESIGN: Data from two prospective cohort studies. SETTING AND PARTICIPANTS: Cohort 1 is from a single-center study involving children admitted to PICU with meningococcal sepsis (samples obtained at three time points). Cohort 2 includes patients from a multicenter study involving children admitted to the hospital with invasive bacterial infections of differing etiologies (samples obtained within 48 hr after hospital admission). MAIN OUTCOMES AND MEASURES: Primary outcome measure was mortality. Secondary outcome measures were PICU-free days at day 28 and hospital length of stay. RESULTS: In cohort 1 (n = 59), nonsurvivors more frequently had A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 levels above the detection limit than survivors at admission to PICU (8/11 [73%] and 6/23 [26%], respectively; p = 0.02) and at t = 24 hours (2/3 [67%] and 3/37 [8%], respectively; p = 0.04). In cohort 2 (n = 240), A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 levels in patients within 48 hours after hospital admission were more frequently above the detection limit than in healthy controls (110/240 [46%] and 14/64 [22%], respectively; p = 0.001). Nonsurvivors more often had detectable A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 levels than survivors (16/21 [76%] and 94/219 [43%], respectively; p = 0.003), which was mostly attributable to patients with Neisseria meningitidis. CONCLUSIONS AND RELEVANCE: In children with bacterial infection, detection of A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 within 48 hours after hospital admission is associated with death, particularly in meningococcal sepsis. Future studies should confirm the prognostic value of A Disintegrin and Metalloproteinase with Thrombospondin Motifs-1 and should study pathophysiologic mechanisms.

Patients with Cystic Fibrosis (CF) suffer from pancreatic insufficiency, lipid malabsorption and gastrointestinal complaints, next to progressive pulmonary disease. Altered mucosal homoeostasis due to malfunctioning chloride channels results in an adapted microbial composition of the gastrointestinal and the respiratory tract. Additionally, antibiotic treatment has the potential to distort resident microbial communities dramatically. This study aims to investigate early life development of the gut microbial community composition of children with CF compared to healthy infants and to study the independent effects of antibiotics taking into account other clinical and lifestyle factors.Faecal samples from 20 infants with CF and 45 healthy infants were collected regularly during the first 18 months of life and microbial composition was determined using 16S rRNA based sequencing.We observed significant differences in the overall microbiota composition between infants with CF and healthy infants (p0.001). Akkermansia and Anaerostipes were significantly more abundant in control infants, whereas Streptococci and E. coli were significantly more abundant in infants with CF, also after correction for several clinical factors (p0.05). Antibiotic use in infants with CF was associated with a lower alpha diversity, a reduced abundance of Bifidobacterium and Bacteroides, and a higher abundance of Enterococcus.Microbial development of the gut is different in infants with CF compared to healthy infants from the first months of life on, and further deviates over time, in part as a result of antibiotic treatment. The resulting dysbiosis may have significant functional consequences for the microbial ecosystem in CF patients.

Lower respiratory tract infections (LRTIs) in children are common and, although often mild, a major cause of mortality and hospitalization. Recently, the respiratory microbiome has been associated with both susceptibility and severity of LRTI. In this current study, we combined respiratory microbiome, viral, and clinical data to find associations with the severity of LRTI. Nasopharyngeal aspirates of children aged one month to five years included in the STRAP study (Study to Reduce Antibiotic prescription in childhood Pneumonia), who presented at the emergency department (ED) with fever and cough or dyspnea, were sequenced with nanopore 16S-rRNA gene sequencing and subsequently analyzed with hierarchical clustering to identify respiratory microbiome profiles. Samples were also tested using a panel of 15 respiratory viruses and Mycoplasma pneumoniae, which were analyzed in two groups, according to their reported virulence. The primary outcome was hospitalization, as measure of disease severity. Nasopharyngeal samples were isolated from a total of 167 children. After quality filtering, microbiome results were available for 54 children and virology panels for 158 children. Six distinct genus-dominant microbiome profiles were identified, with Haemophilus-, Moraxella-, and Streptococcus-dominant profiles being the most prevalent. However, these profiles were not found to be significantly associated with hospitalization. At least one virus was detected in 139 (88%) children, of whom 32.4% had co-infections with multiple viruses. Viral co-infections were common for adenovirus, bocavirus, and enterovirus, and uncommon for human metapneumovirus (hMPV) and influenza A virus. The detection of enteroviruses was negatively associated with hospitalization. Virulence groups were not significantly associated with hospitalization. Our data underlines high detection rates and co-infection of viruses in children with respiratory symptoms and confirms the predominant presence of Haemophilus-, Streptococcus-, and Moraxella-dominant profiles in a symptomatic pediatric population at the ED. However, we could not assess significant associations between microbiome profiles and disease severity measures.

Group 2 innate lymphoid cells (ILC2s) orchestrate protective type 2 immunity and have been implicated in various immune disorders. In the mouse, circulatory inflammatory ILC2s (iILC2s) were identified as a major source of type 2 cytokines. The human equivalent of the iILC2 subset remains unknown. Here, we identify a human inflammatory ILC2 population that resides in inflamed mucosal tissue and is specifically marked by surface CD45RO expression. CD45RO+ ILC2s are derived from resting CD45RA+ ILC2s upon activation by epithelial alarmins such as IL-33 and TSLP, which is tightly linked to STAT5 activation and up-regulation of the IRF4/BATF transcription factors. Transcriptome analysis reveals marked similarities between human CD45RO+ ILC2s and mouse iILC2s. Frequencies of CD45RO+ inflammatory ILC2 are increased in inflamed mucosal tissue and in the circulation of patients with chronic rhinosinusitis or asthma, correlating with disease severity and resistance to corticosteroid therapy. CD45RA-to-CD45RO ILC2 conversion is suppressed by corticosteroids via induction of differentiation toward an immunomodulatory ILC2 phenotype characterized by low type 2 cytokine and high amphiregulin expression. Once converted, however, CD45RO+ ILC2s are resistant to corticosteroids, which is associated with metabolic reprogramming resulting in the activation of detoxification pathways. Our combined data identify CD45RO+ inflammatory ILC2s as a human analog of mouse iILC2s linked to severe type 2 inflammatory disease and therapy resistance.

Background Migrants are not routinely screened for hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV) in the Netherlands. We estimated the prevalence and determined factors associated with HBV, HCV and/or HIV infections among undocumented migrants and uninsured legal residents. Methods In this cross-sectional study, undocumented migrants and uninsured legal residents were recruited at a non governmental organization (NGO), healthcare facility in the Netherlands and were invited to be tested for hepatitis B surface antigen (HBsAg), anti-hepatitis B core antibodies (anti-HBcAb), HCV-RNA, and anti-HIV antibodies or HIV antigen at a local laboratory. Results Of the 1376 patients invited, 784 (57%) participated. Participants originated from Africa (35%), Asia (30%) and North/South America (30%). 451/784 (58%) participants went to the laboratory for testing. Of participants 30% were HBV exposed (anti-HBcAb-positive), with 27% (n = 119/438, 95% CI 23.1% to 31.6%) having resolved HBV infection (HBsAg-negative) and 2.5% (n = 11/438, 95%CI 1.3% to 4.5%, 64% new infection) having chronic HBV infection (HBsAg-positive). Compared to HBV non-exposed, HBV exposed individuals were older (p = 0.034) and more often originated from Africa (p Conclusion Prevalence of chronic HBV, chronic HCV and HIV infections in our study population is higher compared to the Dutch population, thus emphasizing the importance of case finding for these infections through primary care and public health in this specific group of migrants. Screening uptake could be improved by on-site testing.

EUCLIDS consortium., Non-coding genetic variants play an important role in driving susceptibility to complex diseases but their characterization remains challenging. Here, we employed a novel approach to interrogate the genetic risk of such polymorphisms in a more systematic way by targeting specific regulatory regions relevant for the phenotype studied. We applied this method to meningococcal disease susceptibility, using the DNA binding pattern of RELA – a NF-kB subunit, master regulator of the response to infection – under bacterial stimuli in nasopharyngeal epithelial cells. We designed a custom panel to cover these RELA binding sites and used it for targeted sequencing in cases and controls. Variant calling and association analysis were performed followed by validation of candidate polymorphisms by genotyping in three independent cohorts. We identified two new polymorphisms, rs4823231 and rs11913168, showing signs of association with meningococcal disease susceptibility. In addition, using our genomic data as well as publicly available resources, we found evidences for these SNPs to have potential regulatory effects on ATXN10 and LIF genes respectively. The variants and related candidate genes are relevant for infectious diseases and may have important contribution for meningococcal disease pathology. Finally, we described a novel genetic association approach that could be applied to other phenotypes., This work has been partially funded by the European Seventh Framework Programme for Research and Technological Development (FP7) under EUCLIDS Grant Agreement No. 279185. The UK meningococcal cohort was established with support from Meningitis Research Foundation through grants to Imperial College London. The Research from Newcastle partners was supported by the National Institute for Health Research Newcastle Biomedical Research Centre based at Newcastle Hospitals NHS Foundation Trust and Newcastle University. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR or the Department of Health. The ESIGEM Research group activities were supported by grants from Instituto de Salud Carlos III (Proyecto de Investigación en Salud, Acción Estratégica en Salud: proyecto GePEM PI16/01478) (A.S.); Consellería de Sanidade, Xunta de Galicia (RHI07/2-intensificación actividad investigadora, PS09749 and 10PXIB918184PR), Instituto de Salud Carlos III (Intensificación de la actividad investigadora 2007–2016), Convenio de colaboración de investigación (Wyeth España-Fundación IDICHUS 2007–2011), Convenio de colaboración de investigación (Novartis España-Fundación IDICHUS 2010–2011), Fondo de Investigación Sanitaria (FIS; PI070069/PI1000540) del plan nacional de I + D + I and ‘fondos FEDER’ (F.M.T.). The Swiss Pediatric Sepsis study was funded by grants from the Swiss National Science Foundation (342730_153158/1), the Swiss Society of Intensive Care, the Bangerter Foundation, the Vinetum and Borer Foundation, and the Foundation for the Health of Children and Adolescents. The Western Europe Meningococcal Study was supported by grants no 8842, 10112 and 12710 of the Oesterreichische Nationalbank (Austria), grants A3-16.K-8/2008-11 and A3-16.K-8/2006–9 of the Department for Science and Research of the Styrian federal government (Austria) and the non for profit association ‘In Vita’, Graz (Austria).

Cable bacteria are long, multicellular, filamentous bacteria that can conduct electrons over centimeter distances in marine and freshwater sediments. Recent studies indicate that cable bacteria are widely present in many coastal environments, where they exert a major influence on the biogeochemistry of the sediment. Their energy metabolism can be based on the aerobic oxidation of sulfide, and hence to better understand their natural occurrence and distribution, we examined the growth and activity of cable bacteria in relation to bottom water oxygenation. To this end, we conducted laboratory sediment incubations at four different O2 levels in the overlying water (10%, 20%, 40%, and 100% air saturation). The abundance of cable bacteria was determined by fluorescence in situ hybridization, while their activity was assessed via microsensor profiling and geochemical pore‐water analysis. Cable bacteria did not develop in the 10% air saturation O2 incubation but were present and active at all higher O2 levels. These data show that microbial long‐distance electron transport can occur under a wide range of bottom water O2 concentrations. However, the growth rate was notably slower at lower oxygen concentrations, suggesting a reduced metabolic activity of the population when the O2 supply becomes restricted. Finally, in response to lower O2 levels, cable bacteria filaments appear to partially emerge out of the sediment and extend into the overlying water, thus likely enhancing their oxygen supply.

Cable bacteria induce long-distance electron transport in the seafloor and can exert a powerful control on the elemental cycling in marine sediments by creating extreme excursions in porewater pH. Yet, the natural distribution of cable bacteria is still largely unknown, and so their role in coastal biogeochemical cycling remains poorly quantified. Here we show that cable bacteria can be abundant in the sediments of intertidal bivalve reefs, where they strongly influence the pore water geochemistry, resulting in a potentially beneficial interaction between the sulfur oxidizing microbes and biodepositing fauna. Cable bacteria occurred in sediments accumulating within mussel and oyster reefs in the Wadden Sea (The Netherlands), at cumulative filament densities up to 1038 m cm−2. Additionally, cable bacteria were found at moderately high cumulative filament densities (up to 56 m cm−2) in a heavily bioturbated sandy sediment adjacent to the muddy reefs. Microsensor profiling revealed strong sulfide removal and intense acid generation associated with the electrogenic sulfide oxidation metabolism of the cable bacteria. Strongly elevated concentrations of dissolved calcium (up to 35 mM), manganese (up to 250 μM), and iron (up to 700 μM) were observed in the pore waters, consistent with acidity-driven dissolution of calcium carbonates and iron sulfides. This field study provides substantive evidence that cable bacteria exert a decisive control on the cycling of sulfur and carbonate minerals in cohesive coastal sediments, and identifies that the distribution and influence of cable bacteria covers a greater range of natural habitats than previously believed.

Pasteurella multocida hyaluronan synthase (PmHAS) is a bi-functional glycosyltransferase, containing a ß1,3-glucuronyltransferase and ß1,4-N-acetylglucosaminetransferase domain. PmHAS catalyzes the elongation of hyaluronan (HA) through the sequential addition of single monosaccharides to the non-reducing end of the hyaluronan chain. Research is focused on the relation between the length of the HA oligo- saccharide and the single-step elongation ki- netics from HA4 up to HA9. It was found that the turnover number kcat increased with length to maximum values of 11 and 14 s-1 for NAc- and UA-transfer, respectively. Interestingly, the spe- cificity constant kcat/KM increased with polymer length from HA5 to HA7 to a value of 44 mM-1·s-1, indicating an oligosaccharide binding site with increasing specificity towards a heptasaccha- ride at the UA domain. The value of kcat/KM re- mained moderately constant around 8 mM-1·s-1 for HA4, HA6, and HA8, indicating a binding site with significantly lower binding specificity at the NAc domain than at the UA domain. These find- ings are further corroborated by a structural homology model of PmHAS, revealing two dis- tinct sites for binding of oligosaccharides of different sizes, one in each transferase domain. Structural alignment studies between PmHAS and glycosyltransferases of the GT-A fold showed significant similarity in the binding of the UDP-sugars and the orientation of the ac- ceptor substrate. These similarities in substrate orientation in the active site and in essential amino acid residues involved in substrate bind- ing were utilized to localize the two HA oligo- saccharide binding sites.

Most infections are caused by bacteria, many of which are ever-evolving and resistant to nearly all available antibiotics. β-Lactams and glycopeptides are used to combat these infections by inhibiting bacterial cell-wall synthesis. This mechanism remains an interesting target in the search for new antibiotics in light of failed genomic approaches and the limited input of major pharmaceutical companies. Several strategies have enriched the pipeline of bacterial cell-wall inhibitors; examples include combining screening strategies with lesser-explored microbial diversity, or reinventing known scaffolds based on structure-function relationships. Drugs developed using novel strategies will contribute to the arsenal in fight against the continued emergence of bacterial resistance. © 2010 Elsevier Ltd.

The biomass yield on light energy of Dunaliella tertiolecta and Chlorella sorokiniana was investigated in a 1.25- and 2.15-cm light path panel photobioreactor at constant ingoing photon flux density (930 µmol photons m⁻² s⁻¹). At the optimal combination of biomass density and dilution rate, equal biomass yields on light energy were observed for both light paths for both microalgae. The observed biomass yield on light energy appeared to be based on a constant intrinsic biomass yield and a constant maintenance energy requirement per gram biomass. Using the model of Pirt (New Phytol 102:3-37, 1986), a biomass yield on light energy of 0.78 and 0.75 g mol photons⁻¹ and a maintenance requirement of 0.0133 and 0.0068 mol photons g⁻¹ h⁻¹ were found for D. tertiolecta and C. sorokiniana, respectively. The observed yield decreases steeply at low light supply rates, and according to this model, this is related to the increase of the amount of useable light energy diverted to biomass maintenance. With this study, we demonstrated that the observed biomass yield on light in short light path bioreactors at high biomass densities decreases because maintenance requirements are relatively high at these conditions. All our experimental data for the two strains tested could be described by the physiological models of Pirt (New Phytol 102:3-37, 1986). Consequently, for the design of a photobioreactor, we should maintain a relatively high specific light supply rate. A process with high biomass densities and high yields at high light intensities can only be obtained in short light path photobioreactors.

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.

In this work, the dependency of the volumetric hydrogen production rate of ammonium-limited Rhodobacter capsulatus chemostat cultures on their imposed biomass concentration and dilution rate was investigated. A deceleration-stat experiment was performed by lowering the dilution rate from 1.0 d(-1) to zero aimed at a constant biomass concentration of 4.0 g L(-1) at constant incident light intensity. The results displayed a maximal volumetric hydrogen production rate of 0.6 mmol m(-3) s(-1), well below model predictions. Possibly the high cell density limited the average light availability, resulting in a sub-optimal specific hydrogen production rate. To investigate this hypothesis, a gradient-stat experiment was conducted at constant dilution rate of 0.4 d(-1) at constant incident light intensity. The biomass concentration was increased from 0.7 to 4.0 g L(-1) by increasing the influent ammonium concentration. Up to a biomass concentration of 1.5 g L(-1), the volumetric hydrogen production rate of the system increased according to model predictions, after which it started to decline. The results obtained provide strong evidence that the observed decline in volumetric hydrogen production rate at higher biomass concentrations was at least partly caused by a decrease in light availability.

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 7.4 pmol 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.

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.

Objectives: Patients with cystic fibrosis (CF) are frequently colonized by macrolide-resistant Staphylococcus aureus, a result of maintenance macrolide therapy. As transmission of S. aureus between household contacts is common, we examined the prevalence of macrolide-resistant S. aureus colonization in CF patients on maintenance azithromycin therapy and their household contacts and compared this with the S. aureus macrolide resistance prevalence in the community. Patients and methods: Sixty-five CF patients on maintenance macrolide therapy and 194 household contacts were screened for S. aureus colonization by culturing sputa, cough swabs and nasal swabs. Resistance to macrolide, lincosamide and methicillin was determined by disc diffusion tests. The prevalence of macrolide-resistant S. aureus colonization in both groups was compared with figures from a nationwide study into S. aureus carriership and resistance. To assess possible transmission, genotyping of S. aureus was performed using the spa-typing method. Results: Macrolide resistance among CF patients with S. aureus colonization was 69.6%; 75% of these isolates displayed lincosamide resistance too. Among household contacts, macrolide resistance prevalence did not differ significantly from resistance prevalence in the community (9.6% versus 6.3%; P 5 0.358). No methicillin resistance was observed. No identical (macrolide-resistant and -susceptible) S. aureus genotypes were observed between CF patients and their household contacts except for one household, suggesting a probable transmission. Conclusions: No significant increase in macrolide-resistant S. aureus colonization was observed among household contacts of CF patients on long-term azithromycin therapy. Transmission of macrolide-resistant S. aureus could not be proved by genotyping in the majority of households.

There is a need for novel protein sources. Insects are a possible interesting source of protein. They are nutritious in terms of protein (40-75 g/100g dry weight) and minerals. Insect protein is of high quality and has a high digestibility (77-98%) and concentration of essential amino acids (46-96% of the nutritional profile). Also insect cells may be a promising novel source of protein. Choice of cell line, growth conditions and use of the baculovirus expression system opens up possibilities to engineer the nutritional value of the biomass. The technological limits as well as consumer acceptance of insect cell based food remains to be investigated.

The production of acellular pertussis in comparison with whole cell pertussis vaccines demands 5-25 times the amount of Bordetella pertussis' virulence factors, such as Pertussis Toxin (PT), to produce the same number of vaccine doses. An increase in the volumetric productivity by employing fed-batch rather than the currently used batch cultivations of B. pertussis could reduce the cost price of acellular pertussis vaccines. This study defined the conditions that enable fed-batch cultivations at high specific PT production. A solution containing lactate and glutamate was fed to the cultures at various rates. The feed rate and whether or not the fed substrates were completely consumed, significantly influenced cellular metabolism. If lactate was detectable in the culture broth while glutamate was not, poly-hydroxy-butyrate (PHB) was formed. Any PHB present was metabolized when glutamate became detectable again in the culture liquid. At higher lactate and glutamate concentrations, free fatty acids were produced. Though toxic, free fatty acids were not the reason the cultures stopped growing. By choosing appropriate conditions, a cell density of 6.5 g/L dry weight was reached, i.e. a 7-fold increase compared to batch culture. The metabolic mechanisms behind the formation of PHB and fatty acids are discussed, as well as how to increase the cell density further. The PT production stopped at 12 mg/L, well before growth stopped, indicating that regulatory mechanisms of PT production may be involved.

The main bottleneck in scale-up of phototrophic fermentation is the low efficiency of light energy conversion to the desired product, which is caused by an excessive dissipation of light energy to heat. The photoheterotrophic formation of hydrogen from acetate and light energy by the microorganism Rhodobacter capsulatus NCIMB 11773 was chosen as a case study in this work. A light energy balance was set up, in which the total bacterial light energy absorption is split up and attributed to its destinations. These are biomass growth and maintenance, generation of hydrogen and photosynthetic heat dissipation. The constants defined in the light energy balance were determined experimentally using a flat-panel photobioreactor with a 3-cm optical path. An experimental method called D-stat was applied. Continuous cultures were kept in a so-called pseudo steady state, while the dilution rate was reduced slowly and smoothly. The biomass yield and maintenance coefficients of Rhodobacter capsulatus biomass on light energy were determined at 12.4 W/m(2) (400-950 nm) and amounted to 2.58 x 10(-8) +/- 0.04 x 10(-8) kg/J and 102 +/- 3.5 W/kg, respectively. The fraction of the absorbed light energy that was dissipated to heat at 473 W/m(2) depended on the biomass concentration in the reactor and varied between 0.80 and 0.88, as the biomass concentration was increased from 2.0 to 8.0 kg/m(3). The process conditions were estimated at which a 3.7% conversion efficiency of absorbed light energy to produced hydrogen energy should be attainable at 473 W/m(2).

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.

Marine sponges are known to produce an overwhelming array of secondary metabolites with pharmaceutical potential. The technical and economical potential of using marine sponges for large-scale production of these compounds was assessed for two cases: the anticancer molecule halichondrin B from a Lissodendoryx sp., and avarol from Dysidea avara for its antipsoriasis activity. An economic and technical analysis was done for three potential production methods: mariculture, ex situ culture (in tanks), and cell culture. We concluded that avarol produced by mariculture or ex situ culture could become a viable alternative to currently used pharmaceuticals for the treatment of psoriasis. Production of halichondrin B from sponge biomass was found to not be a feasible process, mainly due to the extremely low concentration of the compound in the sponge. Technical feasibility was also analyzed for five alternatives: chemical synthesis, wild harvest, primmorph culture, genetic modification and semi-synthesis. It was concluded that the latter two approaches could prove to be valuable methods for the production of pharmaceuticals, based on chemical structures of secondary metabolites present in trace amounts in marine sponges. © 2005 Wiley Periodicals, Inc.

The amino acid-catalysed retroaldol condensation previously described for citral has been extended to other alpha,beta-unsaturated aldehydes. In the presence of glycine and an elevated pH, six other alpha,beta-unsaturated aldehydes also underwent retroaldol condensation. Crotonaldehyde, as well as its proposed intermediate, aldol, were converted into acetaldehyde. Hexenal, decenal, methyl crotonaldehyde, farnesal and cinnamaldehyde were also converted into acetaldehyde plus butanal, octanal, acetone, geranyl acetone and benzaldehyde, respectively. Subsequently, the effects of temperature, pH, the buffer and substrate concentration on the conversion rate were studied, using the conversion of cinnamaldehyde into the important flavour compound benzaldehyde as a model. Copyright (C) 2004 John Wiley Sons, Ltd.

The leading question of this article is whether it is acceptable, from a moral point of view, to take wild animals that are ill out of their natural habitat and temporarily bring them under human control with the purpose of curing them. To this end the so-called 'seal debate' was examined. In the Netherlands, seals that are lost or ill are rescued and taken into shelters, where they are cured and afterwards reintroduced into their natural environment. Recently, this practice has been criticised because it is thought to interfere with the wildness of the animals and population. In this research, the moral assumptions behind the arguments of both the proponents and opponents of sheltering have been analysed within a morally pluralistic framework. It is concluded that sheltering on too large a scale would be contrary to the efforts of the last few decades to maintain an independent or wild seal population, which means that a certain amount of caution is called for. However, in the current situation there is ...

Este artículo contiene 5 páginas, 1 figura, During the symposium ‘‘Marine Biotechnology: Basics and Applications’’, held 25 February /1 March, 2003 in Matalascan˜as, 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

Insect cells have been cultured for over 30 years, but their application is still hampered by low cell densities in batch fermentations and expensive culture media. With respect to the culture method, the fed-batch culture mode is often found to give the best yields. However, optimization of the feed composition is usually a laborious task. In this report, the successful use of genetic algorithms (GAs) to optimize the growth of insect cells is described. A feed was developed from 11 different medium components, each used at a wide range of concentrations. The feed was optimized within four sets of 20 experiments. The optimized feed was tested in bioreactors and the addition scheme was further improved. The viable-cell density of HzAm1 (Helicoverpa zea) insect cells improved 550% to 19.5 × 106 cells/mL compared to a control fermentation in an optimized commercial medium. No accumulation of waste products was found, and none of the amino acids was depleted. Glucose was depleted, which suggests that even further improvement is possible. We show that GAs are a successful method to optimize a complex fermentation in a relatively short time frame and without the need of detailed information concerning the cellular physiology or metabolism. © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 269–278, 2003.

A novel lyase, combining hydratase and aldolase activity, that converts citral into methylheptenone and acetaldehyde, was purified from spores of Penicillium digitatum. Remarkably, citral lyase activity was induced 118-fold by incubating nongerminating spores with the substrate, citral. This cofactor independent hydratase/aldolase, was purified and found to be a monomeric enzyme of 31 kDa. Citral lyase has a Km of 0.058 mM and a Vmax of 52.6 U·mg-1. Enzyme activity was optimal at 20°C and pH 7.6. The enzyme has a strong preference for the trans isomer of citral (geranial). Citral lyase also converts other α,β-unsaturated aldehydes (farnesal, methyl-crotonaldehyde, decenal and cinnemaldehyde).

Simultaneous production and selective extraction of beta-carotene from living cells of Dunaliella salina in a two-phase system of aqueous and organic phases has been investigated. Solvents with values of log P(octanol), which denotes hydrophobicity of a compound, ranging from 3 to 9 were used as organic phase. Viability and activity of Dunaliella salina in the presence of organic solvents were checked by microscopic observation and photosynthetic oxygen-production-rate measurements, respectively. Extraction ability of different solvents for both beta-carotene and chlorophyll was determined spectrophotometrically. In addition, beta-carotene contents of the cells growing in the aqueous phase and extracted beta-carotene by the different organic phases were quantified by the same method. Results showed that solvents having log P(octanol) > 6 can be considered biocompatible for this alga. Moreover, pigment extraction ability of a solvent is inversely dependent on its log P(octanol) value. By increasing the degenerative hydrophobicity the extraction ability for both chlorophyll and beta-carotene, decreases. However, this decrease is more profound for chlorophyll. Therefore, selective extraction of beta-carotene becomes feasible. Comparison of the total beta-carotene produced in the presence and in the absence of solvents shows that the presence of a second phase of biocompatible solvents in the culture media may induce the beta-carotene production pathway. The beta-carotene productivity per cell in a two-phase system with dodecane was the highest observed. Extraction ability of the biocompatible solvents dodecane, tetradecan, and hexadecane was similar.

Oxygen transfer is for two reasons a major concern in scale-up and process control in industrial application of aerobic fungal solid-state fermentation (SSF): 1) heat production is proportional to oxygen uptake and it is well known that heat removal is one of the main problems in scaled-up fermenters, and 2) oxygen supply to the mycelium on the surface of or inside the substrate particles may be hampered by diffusion limitation. This article gives the first experimental evidence that aerial hyphae are important for fungal respiration in SSF. In cultures of A. oryzae on a wheat-flour model substrate, aerial hyphae contributed up to 75% of the oxygen uptake rate by the fungus. This is due to the fact that A. oryzae forms very abundant aerial mycelium and diffusion of oxygen in the gas-filled pores of the aerial hyphae layer is rapid. It means that diffusion limitation in the densely packed mycelium layer that is formed closer to the substrate surface and that has liquid-filled pores is much less important for A. oryzae than was previously reported for R. oligosporus and C. minitans. It also means that the overall oxygen uptake rate for A. oryzae is much higher than the oxygen uptake rate that can be predicted in the densely packed mycelium layer for R. oligosporus and C. minitans. This would imply that cooling problems become more pronounced. Therefore, it is very important to clarify the physiological role of aerial hyphae in SSF.

Gradients inside substrate particles cannot be prevented in solid-state fermentation. These gradients can have a strong effect on the physiology of the microorganisms but have hitherto received little attention in experimental studies. We report gradients in moisture and glucose content during cultivation of Aspergillus oryzae on membrane-covered wheat-dough slices that were calculated from (1)H-NMR images. We found that moisture gradients in the solid substrate remain small when evaporation is minimized. This is corroborated by predictions of a diffusion model. In contrast, strong glucose gradients developed. Glucose concentrations just below the fungal mat remained low due to high glucose uptake rates, but deeper in the matrix glucose accumulated to very high levels. Integration of the glucose profile gave an average concentration close to the measured average content. On the basis of published data, we expect that the glucose levels in the matrix cause a strong decrease in water activity. The results demonstrate that NMR can play an important role in quantitative analysis of water and glucose gradients at the particle level during solid-state fermentation, which is needed to improve our understanding of the response of fungi to this nonconventional fermentation environment.

The respiration kinetics of Aspergillus oryzae on different varieties of whole wheat kernels were studied. Six wheat varieties were pretreated in two different ways. Five of the six substrates fermented similarly and independently of the pretreatment method. However, pretreatment affected fermentation of one variety of soft wheat (Apollo). T21H-NMR imaging of the water inside the kernels showed a change in water binding inside the kernels when a different pretreatment strategy was used. Differences in free sugar or amino acid content or in kernel stiffness were not significant.

The rate of food particle uptake of the tropical sponge Pseudosuberites aff. andrewsi was studied in relation to particle concentrations and particle size. A range of different concentrations of either the marine microalga Dunaliella tertiolecta (approximately 5-8 microm) or the marine cyanobacterium Synechococcus sp. (approximately 1 microm) was supplied to the sponges. D. tertiolecta had a pronounced effect on the filtration activity of the sponges: at concentrations higher than approximately 4 x 10(5) cells/cm(3), the filtration rates dropped dramatically. Such a clear effect was not found for Synechococcus sp. The results further showed that the maximal amount of food (when expressed in organic carbon) that can be taken up per cubic centimeter of sponge volume per unit of time should in principle be sufficient to enable growth (irrespective of the food particle type). At the maximal food particle concentration that did not affect the filtration rates, the uptake of organic carbon is already highly in excess of the amount of organic carbon that the sponges need to cope with their respiratory demand. Based on these findings, a series of growth experiments was carried out in which the sponges were subjected to a constant concentration of different types of food particles (Synechococcus sp. and the microalgae Chlorella sorokiniana and Nannochloropsis sp). Although initial growth was sometimes observed, continuous growth at a constant rate could not be obtained. It is concluded that qualitative aspects of feeding rather than quantitative aspects are the key to successful in vivo sponge culture.

Hydrogen is a clean energy alternative to fossil fuels. Photosynthetic bacteria produce hydrogen from organic compounds by an anaerobic light-dependent electron transfer process. In the present study hydrogen production by three photosynthetic bacterial strains (Rhodopseudomonas sp., Rhodopseudomonas palustris and a non-identified strain), from four different short-chain organic acids (lactate, malate, acetate and butyrate) was investigated. The effect of light intensity on hydrogen production was also studied by supplying two different light intensities, using acetate as the electron donor. Hydrogen production rates and light efficiencies were compared. Rhodopseudomonas sp. produced the highest volume of H2. This strain reached a maximum H2 production rate of 25 ml H2 l(-1) h(-1), under a light intensity of 680 micromol photons m(-2) s(-1), and a maximum light efficiency of 6.2% under a light intensity of 43 micromol photons m(-2) s(-1). Furthermore, a decrease in acetate concentration from 22 to 11 mM resulted in a decrease in the hydrogen evolved from 214 to 27 ml H2 per vessel.

Pseudomonas putida MC2 is a solvent-tolerant strain that accumulates 3-methylcatechol. In aqueous media, 10 mM of 3-methylcatechol was produced and production was limited by 3-methylcatechol toxicity to the biocatalyst. Production levels increased by introduction of a second, organic phase that provides the substrate toluene and extracts the product from the culture medium. Octanol was shown to be an appropriate second phase with respect to tolerance of the strain for this solvent and with respect to partitioning of both substrate and product. Per unit of overall reactor volume (octanol and water), best results were obtained with 50% (v/v) of octanol: an overall 3-methylcatechol concentration of 25 mM was reached with 96% of the product present in the octanol phase. These product concentrations are much higher than in aqueous media without organic solvent, indicating that biocatalysis in an organic/aqueous two-phase system is an improved set-up for high production levels of 3-methylcatechol.

Inside photobioreactors algae are exposed to light/dark fluctuations. In this study the marine green alga Dunaliella tertiolecta was cultivated under short light/dark cycles of 3/3 s, 94/94 ms and 31/156 ms as found in medium and short light-path reactors. The photon flux density (PFD) during the light period was 440–455 μmol m −2 s −1 and, under the 31/156 ms cycle only, 1025 μmol m −2 s −1 . The photosynthetic efficiency was determined and expressed as the biomass yield on light energy in gram protein produced per mol of photons absorbed. The yield under the 94/94 ms cycle was higher than the yield under continuous light of 440–455 μmol m −2 s −1 . Apparently a light integration effect occurs under the 94/94 ms cycle together with an increase of the photosynthetic efficiency. On the contrary, at the 3/3 s cycle the efficiency decreased. Also under the 31/156 ms cycle, with 1025 μmol m −2 s −1 in the light period, the yield was lower than under continuous light. The results obtained under the 31/156 ms light/dark cycle are discussed with respect to the performance of short light-path flat panel photobioreactors operated at high biomass densities.

To develop a bioreactor for solid-to-solid conversions, the conversion of solid Ca-maleate to solid Ca-D-malate by permeabilized Pseudomonas pseudoalcaligenes was studied. In a bioreactor seeded with product (Ca-D-malate) crystals, growth of Ca-D-malate crystals is the last step in the solid-to-solid conversion and is described here. Crystal growth is described as a transport process followed by surface processes. In contrast to the linear rate law obeyed by the transport process, the surface processes of a crystal-growth process can also obey a parabolic or exponential rate law. Growth of Ca-D-malate crystals from a supersaturated aqueous solution was found to be surface-controlled and obeyed an exponential rate law. Based on this rate law, a kinetic model was developed which describes the decrease in supersaturation due to Ca-D-malate crystal growth as a function of the constituent ions, Ca2 and D-malate2-. The kinetic parameters depended on temperature, but, as expected (surface-controlled), they were hardly affected by the stirring speed

The light regime inside a photobioreactor is characterized by a light gradient with full (sun)light at the light-exposed surface and darkness in the interior of the bioreactor. Consequently, depending on the mixing characteristics, algae will be exposed to certain light/dark cycles. In this study the green alga Chlamydomonas reinhardtii was cultivated under five different light regimes: (1) continuous illumination; (2) a square-wave light/dark cycle with a light fraction (epsilon) of 0.5 and a duration (t(c)) of 6.1 s; (3) epsilon=0.5, t(c)=14.5 s; (4) epsilon=0.5, t(c)=24.3 s and (5) epsilon=0.8, t(c)=15.2 s. The biomass yield on light energy, protein per photons, decreased under light/dark cycles (epsilon=0. 5) in comparison to continuous light (CL), from 0.207 (CL) to 0.117-0.153 g mol(-1) (epsilon=0.5). Concomitantly, the maximal specific photosynthetic activity, oxygen production per protein, decreased from 0.94 (CL) to 0.64-0.66 g g(-1) h(-1) (epsilon=0.5). Also the quantum yield of photochemistry, yield of the conversion of light energy into chemical energy, decreased from 0.47 (CL) to 0. 23 (epsilon=0.5, t(c)=24.3 s). Apparently, C. reinhardtii is not able to maintain a high photosynthetic capacity under medium-duration light/dark cycles and since specific light absorption did not change, light utilization efficiency decreased in comparison to continuous illumination.

The green micro-algae Chlamydomonas reinhardtiiand Dunaliella tertiolecta were cultivated undermedium-duration square-wave light/dark cycles with acycle time of 15 s. These cycles were used to simulatethe light regime experienced by micro-algae inexternally-illuminated (sunlight) air-lift loopbioreactors with internal draft tube. Biomass yieldin relation to light energy was determined as gprotein per mol of photons (400–700 nm). Between 600and 1200 μmol m-2 s-1 the yield at a10/5 s light/dark cycle was equal to the yield atcontinuous illumination. Consequently, provided thatthe liquid circulation time is 15 s, a considerabledark zone seems to be allowed in the interior ofair-lift loop photobioreactors (33% v/v) without lossof light utilization efficiency. However, at a 5/10 slight/dark cycle, corresponding to a 67% v/v darkzone, biomass yield decreased. Furthermore, bothalgae, C. reinhardtii and D. tertiolecta,responded similarly to these cycles with respect tobiomass yield. This was interesting because they werereported to exhibit a different photoacclimationstrategy. Finally, it was demonstrated that D.tertiolecta was much more efficient at low (average)photon flux densities (57–370 μmol m-2s-1) than at high PFDs (> 600 μmol m-2s-1) and it was shown that D. tertiolectawas cultivated at a sub-optimal temperature (20 °C).

Non-mixed and mixed SSF reactors were evaluated for their applicability in large-scale spore production of the biocontrol fungus Coniothyrium minitans. The major problem to overcome in large-scale SSF is heat accumulation. Testing various cooling strategies in large-scale bioreactors would be very expensive and time consuming, therefore lab experiments in combination with mathematical simulations were used instead. The metabolic heat production rate, estimated from the oxygen consumption rate of C. minitans on oats in Erlenmeyer flasks, was about 500 Watt per m(3) bed. Conductive cooling in packed-bed reactors was insufficient to cool large reactor volumes (radius > 0.2 m). The poor thermal conductivity of the bed (lambda(b) = 0.1 W m(-2) K(-1)) resulted in steep radial temperature profiles. Adequate temperature control could be achieved with forced aeration, but concomitant water losses lead to significant shrinkage of the oats (30%) and critically low water activities, even though the bed was assumed to be aerated with water saturated air. Mixed systems, however, allowed heat removal without the need of evaporative cooling. Simulations showed that large volumes could be cooled via the wall at low mixing intensities and small temperature driving forces. Experimental studies showed no detrimental effect of mixing on spore production by C. minitans. The spore production yield in a continuously mixed scraped-drum reactor (0.2 rpm) was 5 x 10(12) spores per kg dry oats after 450 hours. Based on the scale-up potential of the mixed system and the absence of detrimental mixing effects it is believed that a mixed bioreactor is superior to a non-mixed system for large-scale production of C. minitans spores.

Hybridoma cells were grown at steady state under both reductiveand oxidative stress and the intracellular fluxes weredetermined by mass-balancing techniques. By decreasing the dissolved oxygen pressure (pO2) in the bioreactor, the reduced formof nicotinamide adenine nucleotide (NADH) was enhanced relativeto the oxidized form (NAD+). Oxidative stress, as a resultof which the NAP(P)+/NAD(P)H-ratio increases, was generatedby both the enhancement of the pO2 to 100% air saturationand by the addition of the artificial electron acceptorphenazine methosulphate (PMS) to the culture medium. It wasfound that fluxes of dehydrogenase reactions by which NAD(P)H isproduced decreased under hypoxic conditions. For example, thedegradation rates of arginine, isoleucine, lysine and theglutamate dehydrogenase flux were significantly lower at oxygenlimitation, and increased at higher pO2 levels and when PMSwas added to the culture medium. In contrast, the prolinesynthesis reaction, which requires NADPH, decreased under PMSstress. The flux of the NADH-requiring lactate dehydrogenase reaction also strongly decreased from 19 to 3,4 pmol/cell/day,under oxygen limitation and under PMS stress, respectively. Thedata show that metabolic-flux balancing can be used to determinehow mammalian respond to oxidative and reduction stress.

An isolate of Coniothyrium minitans did not sporulate on media to which 0‐05 mm magnesium was added when j-carrageenan was used to solidify the medium. Normal conidiation was observed when a technical grade agar or agar-agar was used as the gelling agent. The j-carrageenan and agars used in this study contained significant amounts of Mg. In the agar media all the Mg present was available to the fungus. By contrast, in the j-carrageenan gel a large portion of the Mg was bound by the gel and not available. Sporulation of C. minitans was only observed on media containing & 0‐17 mm of unbound Mg. The possible role of Mg in the initiation of conidium formation by C. minitans is discussed. Most microbial cultivations in industry are done in submerged culture. For some processes, however, cultivation on solid substrates in the absence of free-flowing water (solid-substrate cultivation) is advantageous. The production of high quantities of Coniothyrium minitans W. A. Campb. conidia, for instance, in stirred-tank reactors has heretofore not been reported. Numerous conidia, however, can easily be obtained when C. minitans is grown on various solid substrates (McQuilken & Whipps, 1995). These conidia are a promising biocontrol agent to treat Sclerotinia sclerotiorum contaminated soils and plants (Whipps & Gerlagh, 1992). The prospects of a biocontrol agent based on C. minitans are promising, provided that an ecient production process is developed. Optimization of the solid substrate with respect to spore formation is essential for the development of a cost-eective biocontrol agent. Quantification of biomass dry weight is important in such optimization studies. Direct gravimetric quantification of biomass is usually not possible when natural solid substrates are used, due to the close association of the microorganism with the substrate. During the development of an alternative model system based on j-carrageenan which facilitates the quantitative recovery of biomass dry matter (Weber, Tramper, & Rinzema, 1999), we found that magnesium is very important for sporulation. The present study evaluates the eect of several minerals in combination with various gelling agents on sporulation of C. minitans. We demonstrate that the amount of freely diusible magnesium strongly aects the spore yield. Furthermore, we demonstrate that the gelling agent used to mimic solid substrate cultivation may produce artefacts in medium optimization studies.

In a model system for solid-substrate cultivation of Rhizopus oligosporus , it was observed that without pH control rapid acidification occurred, which inhibited the biomass production. With some degree of pH control, biomass production was improved significantly. Several buffering agents with different initial pH values were tested to find the optimum pH and the most suitable buffer for this model system. Between pH 4 and 6, the biomass-production rate was not significantly influenced by pH and that citric acid was the most suitable buffer. Addition of 5 g/l tryptone to a standard mineral medium increased both the maximum specific growth rate ( μ max ) and the maximum CO 2 -production rate, and strongly shortened the lag phase.

Explants of the Indo-Pacific sponge Pseudosuberites aff. andrewsi were fed with the microalgae Chlorella sorokiniana and Rhodomonas sp. It was microscopically observed that these algae were ingested and digested by the sponge cells, suggesting that they were consumed by the sponges. The algae were further used for two growth experiments with five explants of P. aff. andrewsi and four explants of P. andrewsi. Growth was measured as the increase in projected body area. The explants showed considerable growth (up to 730% in 54 days for P. aff. andrewsi and up to 680% in 22 days for P. andrewsi), which is much higher than previously reported growth rates for sponges. Growth started after a stationary phase of 5–20 days in which the projected body area did not increase. The growth of P. aff. andrewsi appeared to be linear and was inhibited at the end of the experiment. Two explants of P. andrewsi showed exponential growth instead of linear growth. Hence, no general statements about the growth kinetics of these sponges can be made at this time. However, the high growth rates found in this study suggest a promising future for cultivation of sponges in closed systems.

The specific growth rate of Chlamydomonas reinhardtii and Chlorella sorokiniana decreased under square-wave light/dark cycles of medium duration, 13–87 s, in comparison to continuous illumination. Three experiments were done in three different turbidostats at saturating and sub-saturating light intensities during the light period, 240–630 μmol m− 2 s− 1. Within each experiment the light intensity during the light periods of the intermittent light regimes was equal and this intensity was also applied under continuous illumination. The specific growth rate decreased proportional or more than proportional to the fraction of time the algae were exposed to light; this light fraction ranged from 0.32 to 0.88. We conclude that under these light regimes the chlorophyta C. reinhardtii and C. sorokiniana are not able to store light energy in the light period to sustain growth in the dark period at the same rate as under continuous illumination. C. reinhardtii increased its specific light absorbing surface by increasing its chloropyll-a content under light/dark cycles of 13 s duration and a light fraction of 0.67 at 240 μmol m− 2 s-1; the chloropyll-a content was twice as high under intermittent illumination in comparison to continuous illumination. The combination of a higher specific light absorption together with a lower specific growth rate led to a decrease of the yield of biomass on light energy under intermittent illumination.

Statistically-based experimental designs were used to optimize a chemically defined solid medium for the spore production of Coniothyrium minitans. In the first optimization step the influence of starch, urea, phosphate, magnesium, calcium, thiamin and trace elements on spore production was evaluated using a fractional factorial design. Starch and trace elements influenced spore production positively while urea affected spore production negatively. The other components had no significant influence on spore production. In the second and third steps the concentrations of starch, urea and trace elements were further optimized using central composite designs and response surface analysis. This optimization strategy allowed the spore production to be increased by a factor 7 from 4 x 10(9) to almost 3 x 10(10) spores per Petri dish of 9 cm diameter. Copyright 1999 John Wiley & Sons, Inc.

The use of a dissolvable solid matrix, κ-carrageenan, to quantify biomass grown on solid media was studied. A firm gel was obtained with 2% (w/v) κ-carrageenan and 20 mM K+ which could be easily dissolved in demineralized water. Direct quantification of Coniothyrium minitans biomass grown on this medium was feasible. No effects of the dissolution on the amount of biomass recovered were detected.

As part of development of a continuous process for D-malate production, the stability of maleate hydratase in permeabilized Pseudomonas pseudoalcaligenes was characterized as a function of relevant process conditions. In a system where D-malate is produced from a Ca-maleate suspension, these conditions were temperature, D-malate2-, Ca2+, and biocatalyst concentration. The decrease of maleate-hydratase activity with time was described by first-order irreversible inactivation. The first-order inactivation rate constant increased with temperature between 20°C and 35°C and decreased with D-malate2- concentration between 0 and 50 mM; the temperature dependency increased with D-malate2- concentration. Although seemingly even more attractive with respect to biocatalyst stability, the effects of temperatures below 20°C and D-malate2- concentrations exceeding 50 mM were not determined, as biocatalyst activity at these temperatures is extremely low and the D-malate2- concentration will not exceed 50 mM due to the l...

Some of the structural parameters of starch (e.g. % beta- or gluco-hydrolysis) were influenced by the increase in mass during the hydrolysis reactions (hydrolytic gain). Procedures were derived to correct this apparent % of hydrolysis to actual % of hydrolysis. These analytically derived equations are not only valid for the hydrolysis of starch but also for the hydrolysis of lower molecular weight saccharides (i.e. α-limit dextrin). With a small modification, these equations can be used to correct for hydrolytic gain which occurs during the hydrolysis of other (bio)polymers.

Sporulation of Coniothyrium minitans on 20 g glucose/l or 18 g starch/l at an initial C/N ratio of 10 was the lowest for nitrate and histidine (1 × 109 vs 5–6 × 109 spores/dish). At 100 g glucose/l or 90 g starch/l lower spore numbers as compared to the lower concentrations are obtained for all nitrogen sources tested; only the spore numbers with urea and glycine were equal with either 18 or 90 g starch/l. Spore numbers at 90 g starch/l were all a factor two or more higher than at 100 g glucose/l.

The world's oceans harbour a large diversity of living organisms. As tropical rainforests have been searched for natural drugs, these marine organisms are being screened for useful products, and a number have been found in marine sponges. These are often produced only in trace amounts, and so a large quantity of sponges must be collected to obtain sufficient amounts of the target compounds. Hence, sustainable production of these compounds requires alternatives to harvesting sponge biomass directly from the sea, including the biotechnological production of sponge metabolites.