Use of Bioluminometry for Determination of Active Yeast Biomass Immobilized in Ionotropic Hydrogels 1 M. Navra ´ til,* ,2 Z. Do ¨me ´ny,* V. Hronsky ´,* E. S ˇ turdı ´k,* D. S ˇ mogrovic ˇova ´ ,* and P. Gemeiner† *Department of Biochemical Technology, Faculty of Chemical Technology, Slovak University of Technology, Radlinske ´ho 9, SK-81237 Bratislava, Slovak Republic; and †Institute of Chemistry, Slovak Academy of Sciences, Du ´ bravska ´ Cesta 9, SK-84238 Bratislava, Slovak Republic Received April 17, 2000 The technique of bioluminometry was used to deter- mine the biomass concentration of yeast cells immobi- lized in ionotropic hydrogel beads, including alginate, pectate, and -carrageenan. The method uses determi- nation of ATP extracted from viable cells, the concen- tration of which is then expressed as the active bio- mass concentration. Seven yeast strains divided into three categories (brewing, wine-making, and ethanol- producing yeasts) were tested, and different biomass concentrations were determined in all three immobi- lization materials. The described method is character- ized by a good correlation (up to 99%) to classical dry biomass determination. The method is quicker, easier, and not so laborious, providing sufficient determina- tion accuracy, and can be used for a rapid estimation of viable biomass in most biotechnological processes using immobilized living cells. © 2000 Academic Press One of the critical parameters in studying biotech- nological processes using living cells is the quantity of microorganisms. Determination of the active biomass concentration is even more attractive. Substrate up- take, cell growth, and product accumulation are pa- rameters critical for the successful modeling and de- signing of reactors using immobilized cells. All of these parameters depend on the concentration of active bio- mass, so they make it essential for description of im- mobilized cell processes. Bioluminometry offers the feature of rapid active biomass determination. The estimation of biomass concentration is usually carried out by biomass drying. Alternative methods use the determination of concentration of biomass com- ponents, such as proteins, DNA, and NADH expressed as biomass concentration. Although the dry biomass estimation is the most common, it represents both vi- able (active) and nonviable biomass. In addition, the gel-immobilized cells may produce extracellular poly- mers, which may cause overestimation of the activity values in dry biomass measurements (1). The determi- nation of active biomass can be performed by measur- ing the specific oxygen consumption rate (2), but this method is limited only to oxygen-consuming cells. The luminometric method of determination of active biomass concentration is based on measuring the ATP content in cells. ATP serves as a carrier of chemical energy in cells, where available energy is stored in chemical bonds between two final phosphate groups. After cell death, the ATP concentration rapidly de- creases. Because ATP is a good indicator of cell viabil- ity, its concentration is dependent on active biomass amount (2). The concentration of ATP can be conve- niently determined by the bioluminescence method, where ATP is initially extracted from cells and then reacted with luciferin (LH 2 ) 3 in a reaction catalyzed by the enzyme luciferase, while bioluminescent radiation is emitted. The reaction, which takes place, is de- scribed as follows (3): LH 2 + ATP 3 LH 2  AMP + P–P, LH 2  AMP + 1 2 O 2 O ¡ luciferase L  AMP* + H 2 O, L  AMP* 3 L  AMP + h  . 1 This work has been carried out within EU Projects 2/5059/98, 2/4149/97, 1/7347/20, and 1/6252/99. 2 To whom correspondence should be addressed. Fax: ++421-7- 52967085. E-mail: navratil@chelin.chtf.stuba.sk. 3 Abbreviations used: LH 2 , luciferin; RLU, relative light units. 394 0003-2697/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Analytical Biochemistry 284, 394 – 400 (2000) doi:10.1006/abio.2000.4700, available online at http://www.idealibrary.com on