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.

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.

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.

H(2) synthesis from pentoses and biomass in Thermotoga spp.

We have investigated H(2) production on glucose, xylose, arabinose, and glycerol in Thermotoga maritima and T. neapolitana. Both species metabolised all sugars with hydrogen yields of 2.7-3.8 mol mol(-1) sugar. Both pentoses were at least comparable to glucose with respect to their qualities as substrates for hydrogen production, while glycerol was not metabolised by either species. Glycerol was also not metabolised by T. elfii. We also demonstrated that T. neapolitana can use wet oxidised wheat straws, in which most sugars are stored in glycoside polymers, for growth and efficient hydrogen production, while glucose, xylose and arabinose are consumed in parallel.

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).

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.

Hydrogen production in anaerobic and microaerobic Thermotoga neapolitana.

We have tested the hypothesis (Van Ooteghem et al. Appl Biochem Biotechnol 2002 98-100: 177-189) that microaerobic metabolism may increase the yield of H(2) from the thermophilic bacterium Thermotoga neapolitana. In anaerobic conditions, T. neapolitana converted glucose into acetic acid and lactic acid and yielded 2.4 +/- 0.3 mol H(2) mol(-1) glucose. The bacterium tolerated low O(2) partial pressures but the H(2) yield was not improved under microaerobic conditions. Our results indicate that T. neapolitana only produces H(2) by anaerobic metabolism, and that the yield of H(2) can be maximised by minimising the production of lactic acid.

Acid phosphatase production by Aspergillus niger N402A in continuous flow culture.

The production of acid phosphatases (E.C.3.1.3.2, ACPs) by Aspergillus niger N402A is regulated by specific growth rate, as well as phosphate availability and pH, as demonstrated by studies in continuous flow culture. Specific ACP activity was highest when A. niger was grown at pH 6.3 (64+/-8 U g(-1)) or pH 2.8 (99+/-11 U g(-1)), at a dilution rate of 0.07 h(-1) and phosphate concentrations below 0.46 mM. ACP production was growth correlated for specific growth rates between 0.07 and 0.13 h(-1). Four different ACPs, including two phytases, were produced by A. niger N402A. The ACP and the phytase with maximal activities at pH 5.5 were differentially expressed at different culture pH values, with greater production at low pH.