Image Analysis Supported Moss Cell Disruption in Photo-Bioreactors

Abstract

Diverse methods for the disruption of cell entanglements and pellets of the moss Physcomitrella patens were tested in order to improve the homogeneity of suspension cultures. The morphological characterization of the moss was carried out by means of image analysis. Selected morphological parameters were defined and compared to the reduction of the carbon dioxide fixation, and the released pigments after cell disruption. The size control of the moss entanglements based on the rotor stator principle allowed a focused shear stress, avoiding a severe reduction in the photosynthesis. Batch cultures of P. patens in a 30.0-l pilot tubular photo-bioreactor with cell disruption showed no significant variation in growth rate and a delayed cell differentiation, when compared to undisrupted cultures. A highly controlled photoautotrophic culture of P. patens in a scalable photo-bioreactor was established, contributing to the development required for the future use of mosses as producers of relevant heterologous proteins.

Key words

Physcomitrella patens - photo-bioreactor - image analysis - cell disruption - suspension culture - differentiation

References

• 1 Boyd P. J., Hall J., Cove D.. An airlift fermenter for the culture of the moss Physcomitrella patens. . Glime, J. M., ed. Methods in Bryology , Proceedings of the Bryological Methods Workshop Mainz, Hattori Botanical Laboratory, Nichinan. (1988): 41-45
• 2 Cove D. J., Ashton N. W.. The hormonal regulation of gametophytic development in bryophytes. Dyer, A. F. and Duckett, J. G., eds. The Experimental Biology of Bryophytes. London; Academic Press (1984): 177-196
• 3 Decker E., Reski R.. The moss bioreactor.  Current Opinion in Plant Biology. (2004);  7 166-170
• 4 Frank W., Ratnadewi D., Reski R.. Physcomitrella patens is highly tolerant against drought, salt and osmotic stress.  Planta. (2005);  220 384-394
• 5 Gorr G.. Biotechnologische Nutzung von Physcomitrella patens (hedw.) B.S.G. PhD thesis, University of Hamburg. (1999)
• 6 Hohe A., Schween G., Reski R.. Establishment of a semicontinuous bioreactor culture of Physcomitrella patens for mass production of protoplasts.  Acta Horticulturae. (2001);  560 425-428
• 7 Hohe A., Decker E. L., Gorr G., Schween G., Reski R.. Tight control of growth and cell differentiation in photoautotrophically growing moss (Physcomitrella patens) bioreactor cultures.  Plant Cell Reports. (2002);  20 1135-1140
• 8 Hohe A., Reski R.. Optimisation of a bioreactor culture of the moss Physcomitrella patens for mass production of protoplasts.  Plant Science. (2002);  163 69-74
• 9 Holtorf H., Guitton M.-C., Reski R.. Plant functional genomics.  Naturwissenschaften. (2002);  89 235-249
• 10 Huang Y., Rorrer G.. Dynamics of oxygen evolution and biomass production during cultivation of Agardhiella subulata microplantlets in a bubble-column photobioreactor under medium perfusion.  Biotechnology Progress. (2002);  18 62-71
• 11 Knoop B.. Development in bryophytes. Dyer, A. F. and Duckett, J. G., eds. The Experimental Biology of Bryophytes. London; Academic Press (1984): 143-176
• 12 Koprivova A., Stemmer C., Altmann F., Hoffmann A., Kopriva S., Gorr G., Reski R., Decker E.. Targeted knockouts of Physcomitrella patens lacking plant-specific immunogenic N-glycans.  Plant Biotechnology Journal. (2004);  2 517-523
• 13 Lucumi A., Fleck P., Posten C.. Scale down of photobioreactors in the cultivation of moss cells in suspension. Hvoslef-Eide, A. K. and Preil, W., eds. Plant Propagation in Liquid Culture Systems. Dordrecht; Kluver Academic Publishing, in press (2005)
• 14 Perner I.. Scale-Down von Photobioreaktoren. PhD thesis, University of Karlsruhe. (2003)
• 15 Perner I., Posten C., Broneske J.. CFD optimization of a plate photobioreactor used for the cultivation of microalge.  Engineering in Life Sciences. (2003);  3 287-291
• 16 Pinto L. S., Vieira L. M., Pons M. N., Fonseca M. M. R., Menezes J. C.. Morphology and viability analysis of Streptomyces clavuligerus in industrial cultivation systems.  Bioprocess and Biosystems Engineering. (2004);  26 177-184
• 17 Pons M. N., Drouin J. F., Louvel L., Vanhoutte B., Vivier H., Germain P.. Physiological investigations by image analysis.  Journal of Biotechnology. (1998);  65 3-14
• 18 Pons M.-N., Vivier H.. Biomass quantification by image analysis.  Advances in Biochemical Engineering/Biotechnology. (1999);  66 133-184
• 19 Reski R.. Development, genetics and molecular biology of mosses.  Botanica Acta. (1998);  111 1-15
• 20 Schillberg S., Emans N., Fischer R.. Antibody molecular farming in plants and plant cells.  Phytochemistry Reviews. (2002);  1 45-54
• 21 Schween G., Gorr G., Hohe A., Reski R.. Unique tissue-specific cell cycle in Physcomitrella. .  Plant Biology. (2003);  5 50-58
• 22 Schaefer D.. Gene targeting in Physcomitrella patens. .  Current Opinion in Plant Science. (2001);  4 143-150
• 23 Schaefer D., Zrӱd J.-P.. The Moss Physcomitrella patens, Now and Then.  Plant Physiology. (2001);  127 1430-1438
• 24 Vandanjon L., Rossignol N., Jaouen P., Roberts J. M., Quemeneur F.. Effects of shear on two microalgae species. Contribution of pumps and valves in tangential flow filtration systems.  Biotechnology and Bioengineering. (1999);  63 1-9
• 25 Wiedmann W. M.. Wirkungsweise von Rotor-Stator-Dispergiermaschinen. PhD Dissertation, University of Stuttgart. (1975)

Guest Editor: R. Reski