Niels Thomas Eriksen

Production of phycocyanin - a pigment with applications in biology, biotechnology, foods, and medicine

C-phycocyanin (C-PC) is a blue pigment in cyanobacteria, rhodophytes and cryptophytes with fluorescent and antioxidative properties. C-PC is presently extracted from open pond cultures of the cyanobacterium Arthrospira platensis although these cultures are not very productive and open for contaminating organisms. C-PC is considered a healthy ingredient in cyanobacterial-based foods and health foods while its colouring, fluorescent or antioxidant properties are utilised only to a minor extent. However, recent research and developments in C-PC synthesis and functionality have expanded the potential applications of C-PC in biotechnology, diagnostics, foods and medicine: The productivity of C-PC has been increased in heterotrophic, high cell density cultures of the rhodophyte Galdieria sulphuraria that are grown under well-controlled and axenic conditions. C-PC purification protocols based on various chromatographic principles or novel two-phase aqueous extraction methods have expanded in numbers and improved in performance. The functionality of C-PC as a fluorescent dye has been improved by chemical stabilisation of C-PC complexes, while protein engineering has also introduced increased stability and novel biospecific binding sites into C-PC fusion proteins. Finally, our understanding of the physiological functions of C-PC in humans has been improved by a mechanistic hypothesis that links the chemical properties of the phycocyanobilin chromophores of C-PC to the natural antioxidant, bilirubin, and may explain the observed health benefits of C-PC intake. This review outlines how C-PC is produced and utilised and discusses the novel C-PC synthesis procedures and applications.

The technology of microalgal culturing

This review outlines the current status and recent developments in the technology of microalgal culturing in enclosed photobioreactors. Light distribution and mixing are the primary variables that affect productivities of photoautotrophic cultures and have strong impacts on photobioreactor designs. Process monitoring and control, physiological engineering, and heterotrophic microalgae are additional aspects of microalgal culturing, which have gained considerable attention in recent years.

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.

Effects of phosphorous, nitrogen, and carbon limitation on biomass composition in batch and continuous flow cultures of the heterotrophic dinoflagellate Crypthecodinium cohnii

We have used phosphate, nitrogen, or carbon limited batch and continuous flow cultures to study how growth and biochemical composition of the dinoflagellate Crypthecodinium cohnii CCMP 316 is affected by nutrient limitation. Specific contents of phosphorous, proteins, and starch were differently affected by nutrient limitation. The specific phosphorous content in C. cohnii varied 10–20 times depending on phosphate availability in the medium. When phosphate was available it was taken up in excess and stored to be re‐utilized during phosphate limitation. The specific protein content varied twofold. At most conditions, proteins made up 12–15% of the biomass dry weight but when cells were nitrogen limited, the specific protein content was only half this value. Floridean starch was the major cell constituent of C. cohnii accounting for 40–50% of the biomass dry weight. Only during carbon limitation did the specific starch content decrease. In contrast was the specific lipid content almost unaffected by nutrient availability and lipids accounted for 12–15% of the biomass dry weight irrespectively of which nutrient that was limiting. Lipid production does therefore not depend on nutrient limitation in C. cohnii and lipids are produced even by carbon limited cells. Cultures grown under phosphate limitation resulted in formation of cells with maximal specific contents of all the three major cell constituents; starch, lipid, and protein. Biotechnol. Bioeng. 2012; 109:2005–2016.

Mixotrophic continuous flow cultivation of Chlorella protothecoides for lipids.

The oleaginous alga Chlorella protothecoides accumulates lipid in its biomass when grown in nitrogen-restricted conditions. To assess the relationship between nitrogen provision and lipid accumulation and to determine the contribution of photosynthesis in mixotrophic growth, C. protothecoides was grown in mixo- and heterotrophic nitrogen-limited continuous flow cultures. Lipid content increased with decreasing C/N, while biomass yield on glucose was not affected. Continuous production of high lipid levels (57% of biomass) was possible at high C/N (87-94). However, the lipid production rate (2.48 g L(-1) d(-1)) was higher at D=0.84 d(-1) with C/N 37 than at D=0.44 d(-1) and C/N 87 even though the lipid content of the biomass was lower (38%). Photosynthesis contributed to biomass and lipid production in mixotrophic conditions, resulting in 13-38% reduction in CO2 production compared with heterotrophic cultures, demonstrating that photo- and heterotrophic growth occurred simultaneously in the same population.

Sequential secretion of collagenolytic, elastolytic, and keratinolytic proteases in peptide-limited cultures of two Bacillus cereus strains isolated from wool

Aims:  To characterize the secretion of proteolytic activities against keratin, collagen and elastin in liquid cultures of Bacillus cereus IZ‐06b and IZ‐06r isolated from wool.

Purification of the photosynthetic pigment C-phycocyanin from heterotrophic Galdieria sulphuraria

BACKGROUND The phycobiliprotein C-phycocyanin (C-PC) is used in cosmetics, diagnostics and foods and also as a nutraceutical or biopharmaceutical. It is produced in the cyanobacterium Arthrospira platensis grown phototrophically in open cultures. C-PC may alternatively be produced heterotrophically in the unicellular rhodophyte Galdieria sulphuraria at higher productivities and under improved hygienic standards if it can be purified as efficiently as C-PC from A. platensis. RESULTS Ammonium sulfate fractionation, aqueous two-phase extraction, tangential flow ultrafiltration and anion exchange chromatography were evaluated with respect to the purification of C-PC from G. sulphuraria extracts. Galdieria sulphuraria C-PC showed similar properties to those described for cyanobacterial C-PC with respect to separation by all methodologies. The presence of micelles in G. sulphuraria extracts influenced the different procedures. Only chromatography was able to separate C-PC from a second phycobiliprotein, allophycocyanin. CONCLUSION C-PC from heterotrophic G. sulphuraria shows similar properties to cyanobacterial C-PC and can be purified to the same standards, despite initial C-PC concentrations being low and impurity concentrations high in G. sulphuraria extracts.

Quantification of amino acids in fermentation media by isocratic HPLC analysis of their α-hydroxy acid derivatives.

In this paper we describe a novel method for quantification of amino acids. First, α-hydroxy acid derivatives of amino acids were formed after reaction with dinitrogen trioxide by the van Slyke reaction. Second, the α-hydroxy acid derivatives were separated on an Aminex HPX-87H column (Bio-Rad) eluted isocratically with 5 mM H(2)SO(4) and quantified by refractive index detection. We were able to measure the reaction products of 13 of the 20 classical amino acids: glycine, l-alanine, l-valine, l-leucine, l-isoleucine, l-methionine, l-serine, l-threonine, l-asparagine, l-glutamine, l-aspartic acid, l-glutamic acid, and l-proline. We obtained linear relationships between the product peak areas and initial amino acid concentration, whereby the concentrations of these amino acids could be quantified on the basis of the quantification of their products. The method can be used to analyze amino acids in parallel with other small molecules, such as sugars or short chain fatty acids, and was used for parallel quantification of glycine, l-alanine, or l-glutamic acid, and glucose uptake in cultures of the heterotrophic dinoflagellate Crypthecodinium cohnii . The method can also be used to quantify other amines, as demonstrated by detection of Tris (2-amino-2-(hydroxymethyl)propane-1,3-diol).

Growth and proton exchange in recombinant Escherichia coli BL21

Abstract We have quantified the processes responsible for exchange of protons in batch cultures of Escherichia coli BL21 and a recombinant strain growing on glucose or glycerol as carbon substrates and ammonium as nitrogen source. Ammonium uptake was quantitatively the most important process accounting for 70±3 and 94±6% of overall proton production when glucose and glycerol were used as carbon sources, respectively. Formation of bicarbonate accounted for 11±1 and 6±1%, respectively, while acetic acid contributed with 19±2% of overall proton exchange when glucose was the carbon source. The amount of basic titrant added to maintain constant pH corresponded to the sum of the proton generating processes and could be used to estimate biomass concentration and specific growth rate of the cultures. Induction of recombinant protein production did not markedly change the stoichiometry between biomass formation and proton production though the specific growth rate decreased. Because of the conserved relationship between biomass formation and proton production, titrant additions were suited for indirect on-line estimation of biomass concentration and specific growth rate during recombinant protein production processes in E. coli BL21.

Biomass Composition of Blue Mussels, Mytilus edulis, is Affected by Living Site and Species of Ingested Microalgae

We have investigated changes in specific contents of protein, glycogen and lipid, and fatty acids of blue mussels, Mytilus edulis, under different conditions in the field and in laboratory feeding experiments using different microalgae. Specific contents of glycogen and lipid increased in mussels relocated to net bags at a location in Kerteminde Bay (Great Belt, Denmark) in contrast to mussels relocated to a location in Sallingsund (Limfjorden, Denmark). The polyunsaturated fatty acid, eicosapentaenoic acid, reached 3 times higher values in the mussels in Kerteminde Bay. Mussels fed pure cultures of Crypthecodinium cohnii, which is rich in the polyunsaturated fatty acid, docosahexaenoic acid, and glycogen, gained the highest specific contents of this fatty acid and glycogen. Mussels feeding on the most protein rich of the microalgae, Bracteacoccus sp., gained the highest protein contents. The specific glycogen content of the mussels was influenced by their “condition” (body dry weight/shell length ratio) while specific protein and lipid contents were not. Starvation affected mainly the specific glycogen content. These results show that biomass composition of blue mussels is affected by living site and local phytoplankton species and that the fatty acids composition of mussels reflects the content of fatty acids in the diet.