BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING A process for the production of ectoine and poly(3-hydroxybutyrate) by Halomonas boliviensis Héctor Guzmán & Doan Van-Thuoc & Javier Martín & Rajni Hatti-Kaul & Jorge Quillaguamán Received: 14 January 2009 / Revised: 23 April 2009 / Accepted: 4 May 2009 / Published online: 23 May 2009 # Springer-Verlag 2009 Abstract The paper reports a study involving the use of Halomonas boliviensis, a moderate halophile, for co- production of compatible solute ectoine and biopolyester poly(3-hydroxybutyrate) (PHB) in a process comprising two fed-batch cultures. Initial investigations on the growth of the organism in a medium with varying NaCl concen- trations showed the highest level of intracellular accumu- lation of ectoine (0.74 g L -1 ) at 10–15% (w/v) NaCl, while at 15% (w/v) NaCl, the presence of hydroxyectoine (50 mg L -1 ) was also noted. On the other hand, the maximum cell dry weight and PHB concentration of 10 and 5.8 g L -1 , respectively, were obtained at 5–7.5% (w/v) NaCl. A process comprising two fed-batch cultivations was developed—the first culture aimed at obtaining high cell mass and the second for achieving high yields of ectoine and PHB. In the first fed-batch culture, H. boliviensis was grown in a medium with 4.5% (w/v) NaCl and sufficient levels of monosodium glutamate, NH 4 + , and PO 4 3- . In the second fed-batch culture, the NaCl concentration was increased to 7.5% (w/v) to trigger ectoine synthesis, while nitrogen and phosphorus sources were fed only during the first 3 h and then stopped to favor PHB accumulation. The process resulted in PHB yield of 68.5 wt.% of cell dry weight and volumetric productivity of about 1 g L -1 h -1 and ectoine concentration, content, and volumetric productivity of 4.3 g L -1 , 7.2 wt.%, and 2.8 g L -1 day -1 , respectively. At salt concentration of 12.5% (w/v) during the second cultivation, the ectoine content was increased to 17 wt.% and produc- tivity to 3.4 g L -1 day -1 . Keywords Moderate halophile . Halomonas boliviensis . Compatible solutes . Poly(3-hydroxybutyrate) . Fed-batch cultivation Introduction Microorganisms that grow in environments with moderate or high salt concentrations are known to produce a variety of metabolites primarily as a means of adaptation to their environment (Oren 2002). Moderate halophilic and hal- otolerant microorganisms are able to grow at high salt concentrations (15–30% w/v) by counteracting the salt stress through intracellular accumulation of osmolytes, also called “compatible solutes”, to achieve osmotic equilib- rium with respect to the salts in the surrounding environment (Kunte et al. 1993; Roberts 2005; Lentzen and Schwarz 2006). Compatible solutes can be zwitterionic, noncharged, or anionic and are represented by various classes of organic compounds including polyols, sugars, amino acids, betaines, ectoines and their derivatives, among others (Roberts 2005; Lentzen and Schwarz 2006). In general, they are usually low molecular weight compounds and are highly water- soluble allowing their accumulation in high concentrations in the cytoplasm. Zero net charge of the compounds is an Appl Microbiol Biotechnol (2009) 84:1069–1077 DOI 10.1007/s00253-009-2036-2 H. Guzmán : D. Van-Thuoc : J. Martín : R. Hatti-Kaul : J. Quillaguamán Department of Biotechnology, Lund University, P.O. Box 124, 221 00 Lund, Sweden H. Guzmán : J. Quillaguamán (*) Centro de Biotecnología, Facultad de Ciencias y Tecnología, Universidad Mayor de San Simón, Cochabamba, Bolivia e-mail: jorgeqs@supernet.com.bo D. Van-Thuoc Department of Micro-biotechnology, Faculty of Agro-Biology, Hanoi National University of Education, 136 Xuan Thuy, Cau Giay, Hanoi, Vietnam