Analysis of the effects of hyperbaric gases on S. cerevisiae cell cycle through a morphological approach M.A.Z. Coelho a, * , J.A.P. Coutinho b , E.C. Ferreira c , M. Mota c , I. Belo c a Departamento de Engenharia Bioquı ´mica, Escola de Quı ´mica/UFRJ, 21949-900 Rio de Janeiro, Brazil b CICECO, Departamento de Quı ´mica, Universidade de Aveiro, Aveiro, Portugal c IBB—Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057 Braga, Portugal Received 27 December 2006; received in revised form 25 April 2007; accepted 5 July 2007 Abstract The effects of hyperbaric gases on the cell cycle of Saccharomyces cerevisiae were studied in batch cultures under pressures between 0.1 and 0.6 MPa and different gas compositions (air, oxygen, nitrogen or carbon dioxide). Classification of S. cerevisiae cells based on their morphology stages was obtained using an automatic image analysis procedure. Information on the distribution of different sub-populations along the cell cycle is reported. A structured morphological model was developed and used to describe the measured data. The results herein reported demonstrate that the bud separation phase is the limiting step in cell duplication. Additionally, the influence of the environmental conditions, specially the oxygen partial pressure, on the START event is reported. Under anaerobic conditions, no significant influence of hyperbaric gases on the cell cycle was verified. # 2007 Elsevier Ltd. All rights reserved. Keywords: Cell cycle; Saccharomyces cerevisiae; Hyperbaric conditions; Digital image analysis; Morphology 1. Introduction Although a general agreement exists that cell growth and division are functionally coordinated, the mechanisms that link these two processes are poorly understood [1]. The basic mechanisms for cell cycle control seem to be similar in eukaryotic organisms and are mainly based on size control, i.e. there is a critical size for DNA replication and cell division [2,3]. Saccharomyces cerevisiae and Schizosaccharomyces pombe are simple yet powerful organisms generally used in studies of eukaryotic cell cycle [4–10]. The cell cycle compromises a succession of discrete events subjected to complex genetic regulations. S. cerevisiae cell division initially involves a protuberance development, called bud, that begins with the STARTevent or S phase related to plaque duplication and separation (Fig. 1). The bud formation is the visible evidence that the cell passes the START event and is followed by nuclear migration at G 2 phase and spindle elongation and nuclear division at M phase. The limiting step for cell cycle progression is related to protein synthesis. The bud detachment occurs along G 1 phase and a new cell is born. Daughter cells are not able to enter in a division process until they have reached a critical size (around 35 mm 3 for haploid wild-type cells in rich medium) and become adult cells [11]. This size control of the cell cycle consists of two components: a ‘sizer’ phase, which is the time for the cell to reach the critical size, followed by a ‘timer’ phase, which is nearly independent of cell size. Thus, cells that are born larger than the critical size have an almost constant cycle time, regardless of their birth size. For cells born below the critical size, the cycle time lengthens as birth sizes decrease due to the influence of the sizer phase [1]. Due to the heterogeneity of S. cerevisiae population, its culture in a bioreactor comprises a large group of cells that are able to bud and also daughter cells, all of them exposed to the same environmental conditions, but carrying distinct metabolic reactions according to its own intrinsic characteristics. Small variations of phenotypical properties among cells may incur in interferences on gene expression, malfunction of a genetic www.elsevier.com/locate/procbio Process Biochemistry 42 (2007) 1378–1383 * Corresponding author. Tel.: +55 21 25627572; fax: +55 21 25627622. E-mail address: mcoelho@dq.ua.pt (M.A.Z. Coelho). 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.07.003