Experimental and statistical analysis of nutritional requirements for the growth of the extremophile Deinococcus geothermalis DSM 11300 Julie Bornot, Cesar Arturo Aceves-Lara, Carole Molina-Jouve, Jean-Louis Uribelarrea, Nathalie Gorret To cite this version: Julie Bornot, Cesar Arturo Aceves-Lara, Carole Molina-Jouve, Jean-Louis Uribelarrea, Nathalie Gor- ret. Experimental and statistical analysis of nutritional requirements for the growth of the ex- tremophile Deinococcus geothermalis DSM 11300. Extremophiles, Springer Verlag, 2014, 18 (6), pp.1009-1021. 10.1007/s00792-014-0671-8. hal-01268947 HAL Id: hal-01268947 https://hal.archives-ouvertes.fr/hal-01268947 Submitted on 11 Mar 2019 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. OATAO is an open access repository that collects the work of Toulouse researchers and makes it freely available over the web where possible This is an author’s version published in: http://oatao.univ-toulouse.fr/22999 Official URL: https://doi.org/10.1007/s00792-014-0671-8 To cite this version: Bornot, Julie and Aceves-Lara, César-Arturo and Molina-Jouve, Carole and Uribelarrea, Jean-Louis and Gorret, Nathalie Experimental and statistical analysis of nutritional requirements for the growth of the extremophile Deinococcus geothermalis DSM 11300. (2014) Extremophiles, 18 (6). 1009-1021. ISSN 1431-0651 Any correspondence concerning this service should be sent to the repository administrator: [email protected] Experimental and statistical analysis of nutritional requirements for the growth of the extremophile Deinococcus geothermalis DSM 11300 Julie Bornot • Ce´sar-Arturo Aceves-Lara • Carole Molina-Jouve • Jean-Louis Uribelarrea • Nathalie Gorret Abstract Few studies concerning the nutritional 72:1074–1082, 2006) was the best basal medium and was requirements of Deinococcus geothermalis DSM 11300 chosen for further studies. A growth rate of 0.03 h-1 and a have been conducted to date. Three defined media com- final OD600nm of 0.55 were obtained, but the growth was positions have been published for the growth of this strain linear. Then, the effects of several medium components on but they were found to be inadequate to achieve growth oxygen uptake and biomass production by Deinococcus without limitation. Furthermore, growth curves, biomass geothermalis DSM 11300 were studied using a respirom- concentration and growth rates were generally not avail- etry-based method, to search for the nutritional limitation. able. Analysis in Principal Components was used in this The results revealed that the whole yeast extract in the work to compare and consequently to highlight the main medium with glucose is necessary to obtain a non-limiting compounds which differ between published chemically growth of Deinococcus geothermalis DSM 11300 at a defined media. When available, biomass concentration, maximum growth rate of 0.64 h-1 at 45 °C. and/or growth rate were superimposed to the PCA analysis. The formulations of the media were collected from existing Keywords Deinococcus geothermalis Á Nutritional literature; media compositions designed for the growth of requirements Á Respirometry-based method Á Principal several strains of Deinococcaceae or Micrococcaceae were Components Analysis included. The results showed that a defined medium adapted from Holland et al. (Appl Microbiol Biotechnol Introduction Communicated by M. da Costa. Deinococcus geothermalis belongs to the Deinococcus– Thermus group. It is a gram-positive and red-pigmented bacterium that forms spherical cells of 1.5–2 lmin diameter. It grows typically as diplococcus or tetracoccus. J. Bornot Á C.-A. Aceves-Lara Á C. Molina-Jouve Á Deinococcus geothermalis was isolated for the first time in & J.-L. Uribelarrea Á N. Gorret ( ) hot springs in Naples, Italy and in Sa˜o Pedro do Sul, Por- Universite´ de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, 31077 Toulouse, France tugal. It was identified at other locations including indus- e-mail: [email protected]; trial paper machines where it can form biofilms (Va¨isa¨nen [email protected] et al. 1998; Kolari et al. 2002; Peltola 2011). ThegenomeofD. geothermalis is composed of a circular J. Bornot Á C.-A. Aceves-Lara Á C. Molina-Jouve Á J.-L. Uribelarrea Á N. Gorret chromosome of 2,467,205 base pairs and two plasmids of INRA, UMR792 Inge´nierie des Syste`mes Biologiques et des 574,127 base pairs and 205,686 base pairs (Makarova et al. Proce´de´s, 31400 Toulouse, France 2007). D. geothermalis can survive and repair its genome even after an exposition to high radiation levels (Ferreira J. Bornot Á C.-A. Aceves-Lara Á C. Molina-Jouve Á J.-L. Uribelarrea Á N. Gorret et al. 1997;Brimetal.2003). Deinococcus geothermalis is a CNRS, UMR5504, 31400 Toulouse, France moderate thermophile with an optimal growth temperature between 45 and 50 °C. It can grow between pH 4.5–8.5 with partners (program DEINOL ISI of OSEO). The strain was -1 an optimal at 6.5 (Ferreira et al. 1997). inoculated into PGY medium broth (peptone 10 g L , -1 -1 D. geothermalis can tolerate high concentrations of solvents glucose 10 g L , yeast extract 5 g L ) and incubated at and reduce a variety of heavy metals which make it a good 37 °C for 24 h. The bacterial cells were then stored at candidate for bioremediation of radioactive wastes and envi- -80 °C with 20 % (v/v) glycerol. ronments. For example, D. geothermalis is able to reduce Fe(III) and Cr(VI) and has been genetically modified to reduce Media and growth conditions Hg(II) (Brim et al. 2003). A strain of D. geothermalis isolated from soil samples (from hot springs areas, Krabi, Thailand) Pre-cultures of D. geothermalis DSM 11300 were carried tolerates high concentrations of various solvents as decane, di- out in a 5 mL tube containing 1.8 mL Complex Medium ethylphtalate, butyl acetate and ethyl acetate and can assimilate Glucose (CMG) incubated at 37 or 45 °C for 24 h on an hydrocarbon solvents as carbon sources (Kongpol et al. 2008). orbital shaker (110 rpm). The Complex Medium Glucose -1 -1 D. geothermalis is commonly cultivated in rich media was prepared by adding 10 g L of glucose, 5 g L of -1 containing at least one complex nutrient source such as yeast yeast extract, and 2 g L of bacto-peptone to the mineral extract, peptone or tryptone (Ferreira et al. 1997;Brimetal. medium (see below). For each inoculum preparation, the 2003). Just a few defined media compositions have been strain was plated on PGY agar medium (PGY medium -1 published for the growth of D. geothermalis.Inthese con- broth supplemented with agar 14 g L ) for 48 h at 37 °C. ditions, D. geothermalis is able to grow on ammonium sul- Only one colony was chosen for each inoculum. Two fate as nitrogen source and glucose, fructose and toluene as successive steps of pre-cultures, 15 and 150 mL for flask carbon sources (Ferreira et al. 1997;Brimetal.2003; experiments or 18 and 180 mL for bioreactor cultures, Kongpol et al. 2008); but they were found to be inadequate were then carried out in CMG medium, in baffled Erlen- for a good growth. The maximum growth rate at 45 °Cwas meyer flasks with an inoculum at 10 % (v/v). Each flask -1 0.12 h (Kongpol et al. 2008). However, no quantitative was incubated 12 h at 37 or 45 °C and 110 rpm. In studies on the nutritional needs are available in literature. Erlenmeyer flask experiments, the pH of the medium was Furthermore, the nutritional requirements of the strain and between 6.5 and 6.8, the optimum pH for the growth of physiological behavior were very difficult to evaluate from Deinococcus geothermalis being 6.5. the published data because of the differences between the Due to technical constraints, the first experiments in compositions of the media and the absence of biomass mea- flasks were conducted at 37 °C. Only four tests in baffled surements and/or growth rates. Therefore, the main objective Erlenmeyer flasks and cultures in bioreactors were made at of this study was to determine the nutritional requirements of 45 °C. It is important to note that temperature positively the reference strain Deinococcus geothermalis DSM 11300 to affects growth rate, but has no effect on the maximal allow new insights into physiology of D. geothermalis.A biomass concentration (Online Resource l). synthetic defined medium is necessary to insure reproducible After centrifugation 5 min at 4000g (Centrifuge 5810R, culture conditions to carry out physiological studies in well- Eppendorf), the pellet was rinsed with physiological saline -1 controlled environmental conditions. solution (9 g L NaCl) before inoculation. The multifactorial statistical method, Principal Component Basal mineral media compositions designed for the Analysis, was carried to compare and highlight the main dif- growth of various bacteria were tested: the mineral medium ferences in composition between media described in literature DM used in this study was adapted from a medium and media tested in flask experiments. Then, Deinococcus developed for the growth of Deinococcus radiodurans geothermalis DSM 11300 was cultivated in a bioreactor on a (Holland et al. 2006). The composition of the DM was basal defined medium deducted from the PCA analyses. designed and provided by Deinol project partners. Unlike Finally, a respirometry-based strategy was chosen to study the the medium described by Holland et al.