Jens Jørgen Lønsmann Iversen

On-line estimation of O(2) production, CO(2) uptake, and growth kinetics of microalgal cultures in a gas-tight photobioreactor.

Growth of the green algae Chlamydomonas reinhardtii and Chlorella sp. in batch cultures was investigated in a novel gas-tight photobioreactor, in which CO2, H2, and N2 were titrated into the gas phase to control medium pH, dissolved oxygen partial pressure, and headspace pressure, respectively. The exit gas from the reactor was circulated through a loop of tubing and re-introduced into the culture. CO2 uptake was estimated from the addition of CO2 as acidic titrant and O2 evolution was estimated from titration by H2, which was used to reduce O2 over a Pd catalyst. The photosynthetic quotient, PQ, was estimated as the ratio between O2 evolution and CO2 up-take rates. NH4+, NO2−, or NO3− was the final cell density limiting nutrient. Cultures of both algae were, in general, characterised by a nitrogen sufficient growth phase followed by a nitrogen depleted phase in which starch was the major product. The estimated PQ values were dependent on the level of oxidation of the nitrogen source. The PQ was 1 with NH4+ as the nitrogen source and 1.3 when NO3− was the nitrogen source. In cultures grown on all nitrogen sources, the PQ value approached 1 when the nitrogen source was depleted and starch synthesis became dominant, to further increase towards 1.3 over a period of 3–4 days. This latter increase in PQ, which was indicative of production of reduced compounds like lipids, correlated with a simultaneous increase in the degree of reduction of the biomass. When using the titrations of CO2 and H2 into the reactor headspace to estimate the up-take of CO2, the production of O2, and the PQ, the rate of biomass production could be followed, the stoichiometrical composition of the produced algal biomass could be estimated, and different growth phases could be identified.

Transcriptomic comparison of Aspergillus niger growing on two different sugars reveals coordinated regulation of the secretory pathway

BackgroundThe filamentous fungus, Aspergillus niger, responds to nutrient availability by modulating secretion of various substrate degrading hydrolases. This ability has made it an important organism in industrial production of secreted glycoproteins. The recent publication of the A. niger genome sequence and availability of microarrays allow high resolution studies of transcriptional regulation of basal cellular processes, like those of glycoprotein synthesis and secretion. It is known that the activities of certain secretory pathway enzymes involved N-glycosylation are elevated in response to carbon source induced secretion of the glycoprotein glucoamylase. We have investigated whether carbon source dependent enhancement of protein secretion can lead to upregulation of secretory pathway elements extending beyond those involved in N-glycosylation.ResultsThis study compares the physiology and transcriptome of A. niger growing at the same specific growth rate (0.16 h-1) on xylose or maltose in carbon-limited chemostat cultures. Transcription profiles were obtained using Affymetrix GeneChip analysis of six replicate cultures for each of the two growth-limiting carbon sources. The production rate of extracellular proteins per gram dry mycelium was about three times higher on maltose compared to xylose. The defined culture conditions resulted in high reproducibility, discriminating even low-fold differences in transcription, which is characteristic of genes encoding basal cellular functions. This included elements in the secretory pathway and central metabolic pathways. Increased protein secretion on maltose was accompanied by induced transcription of > 90 genes related to protein secretion. The upregulated genes encode key elements in protein translocation to the endoplasmic reticulum (ER), folding, N-glycosylation, quality control, and vesicle packaging and transport between ER and Golgi. The induction effect of maltose resembles the unfolded protein response (UPR), which results from ER-stress and has previously been defined by treatment with chemicals interfering with folding of glycoproteins or by expression of heterologous proteins.ConclusionWe show that upregulation of secretory pathway genes also occurs in conditions inducing secretion of endogenous glycoproteins – representing a more normal physiological state. Transcriptional regulation of protein synthesis and secretory pathway genes may thus reflect a general mechanism for modulation of secretion capacity in response to the conditional need for extracellular enzymes.

Increased NADPH concentration obtained by metabolic engineering of the pentose phosphate pathway in Aspergillus niger

Many biosynthetic reactions and bioconversions are limited by low availability of NADPH. With the purpose of increasing the NADPH concentration and/or the flux through the pentose phosphate pathway in Aspergillus niger, the genes encoding glucose 6‐phosphate dehydrogenase (gsdA), 6‐phosphogluconate dehydrogenase (gndA) and transketolase (tktA) were cloned and overexpressed in separate strains. Intracellular NADPH concentration was increased two‐ to ninefold as a result of 13‐fold overproduction of 6‐phosphogluconate dehydrogenase. Although overproduction of glucose 6‐phosphate dehydrogenase and transketolase changed the concentration of several metabolites it did not result in increased NADPH concentration. To establish the effects of overexpression of the three genes, wild‐type and overexpressing strains were characterized in detail in exponential and stationary phase of bioreactor cultures containing minimal media, with glucose as the carbon source and ammonium or nitrate as the nitrogen source and final cell density limiting substrate. Enzymes, intermediary metabolites, polyol pools (intra‐ and extracellular), organic acids, growth rates and rate constant of induction of acid production in postexponential phase were measured. None of the modified strains had a changed growth rate. Partial least square regressions showed the correlations between NADPH and up to 40 other variables (concentration of enzymes and metabolites) and it was possible to predict the intracellular NADPH concentration from relatively easily obtainable data (the concentration of enzymes, polyols and oxalate). This prediction might be used in screening for high NADPH levels in engineered strains or mutants of other organisms.

Determination of first order rate constants by natural logarithm of the slope plot exemplified by analysis of Aspergillus niger in batch culture

Finding rate constants from experimental data is often difficult because of offset and noise. A computer program was developed to average experimental data points, reducing the effect of noise, and to produce a loge of slope plot – a plot of the natural logarithm of the slope of a curve – eliminating the effect of any offset. If y-values depend exponentially on x-values the loge of slope plot is rectilinear and the slope is equal to the first order rate constant. Therefore the loge of slope plot provides easy identification of exponential sections of any experimental or calculated data, corresponding rate constants, and small changes in the rate constant as exemplified by analysis of titrant added to a batch culture of Aspergillus niger. The loge of slope plot was easily applicable and superior to conventional methods of analysis of exponential decreasing or increasing data.

The Returns to Education in Entrepreneurship: Heterogeneity and Non-Linearities

The returns to education in self-employment are addressed in four different specifications of the relationship between log income and years of schooling. The specifications range from a standard Mincer equation with a constant percentage increase in income for an additional year of schooling to the most flexible specification with dummy variables for different combinations of years of schooling and fields of study. Based on the more flexible specifications, important non-linearities and heterogeneity in the returns to education in self-employment are found. These results are robust across different estimation methods: OLS, Heckit correction models handling sample selection, and IV dealing with the potential endogeneity of years of schooling. Moreover, the results are robust to the use of different sample years, different definitions of self-employment, and different income measures for the self-employed.