Investigation of factors influencing oxygen content in Halobacterium salinarum growth medium for improved bacteriorhodopsin production

International Journal of Industrial Chemistry https://doi.org/10.1007/s40090-019-0189-0 RESEARCH Investigation of factors influencing oxygen content in Halobacterium salinarum growth medium for improved bacteriorhodopsin production Shadi Rajab1 · Valiollah Babaeipour2 · Sirwan Khanchezar3 · Ghasem Amoabediny4 · Fatemeh Yazdian1 · Mohammad Reza Mofid5 Received: 31 October 2018 / Accepted: 4 June 2019 © The Author(s) 2019 Abstract Improving production of bacteriorhodopsin in the culture medium of Halobacterium salinarum confronts indeterminacy related to culture conditions. Several studies have revealed that high oxygen content increases the growth of Halobacterium salinarum whereas it down-regulates the expression of genes responsible for bacteriorhodopsin production. The focus of this study was to clarify this contradictory role of oxygen in bacteriorhodopsin production and to indirectly regulate the oxygen content of the culture medium at a level that would increase the final concentration of bacteriorhodopsin. Oxygen consumption evaluation showed tha in a typical growth of Halobacterium salinarum at aerobic condition, the decrease in oxygen demand was concurrent with a sharp increase in bacteriorhodopsin production. Further investigation on culture conditions revealed that agitation rate and filling volume had a linear correlation with the cell growth and bacteriorhodopsin production by each cell, however, a two-factor interaction model described the relationship between the culture condition and overall bacteriorhodopsin concentration. It was concluded that although each cell of Halobacterium salinarum produced high amount of bacteriorhodopsin at low turbulence condition, the low yield of biomass production at this condition caused a low overall bacteriorhodopsin concentration. The highest overall bacteriorhodopsin concentration was obtained from high turbulence condition, in which cell numbers were high enough to compensate for low production of bacteriorhodopsin by each cell. Keywords Bacteriorhodopsin · Halobacterium salinarum · Optimization · Oxygen transfer rate Introduction * Valiollah Babaeipour 1 Department of Life Science Engineering, Faculty of New Sciences and Technologies, University of Tehran, Tehran, Iran 2 Faculty of Chemistry and Chemical engineering, Malek Ashtar University of Technology, Tehran, Iran 3 Department of Biotechnology, Chemical Engineering Faculty, Tarbiat Modares University, Tehran, Iran 4 Research Center for New Technologies in Life Science Engineering, University of Tehran, Tehran, Iran 5 Department of Biochemistry, School of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran Switching of metabolic programs is a common way for microorganisms to adapt and survive from environmental changes, e.g., changing the energy-producing pathway in cyanobacteria in response to light–dark cycle [1]. Halobacterium salinarum (H. salinarum) is a bioenergetically flexible organism which can obtain energy from respiration, retinal-based photosynthesis and fermentation under various environmental conditions [2]. This archaeon lives in highly saline environments where osmotic pressure and temperature may be high and oxygen levels might become low [3, 4]. In such situations, aerobic growth is ceased after a while, therefore, fermentation (if there is sufficient amount of arginine in surroundings) and retinal-based photosynthesis would be the main sources of energy supply for this archaeon in its natural habitat [4]. Retinal-based photosynthesis in H. salinarum is performed by bacteriorhodopsin (BR), a retinal-based, non-chlorophyll protein with a proton-pumping function 13 Vol.:(0123456789) International Journal of Industrial Chemistry which is activated by absorption of green light. This protein is located in the purple membrane of H. salinarum and its production increases under anaerobic conditions when the organism needs photosynthesis to provide energy [2, 5–7]. By absorbing light photons, BR undergoes some conformational changes which leads to proton transfer outward from the cell [8, 9]. This proton pumping generates an electrochemical gradient across the cell membrane that is used by ATP synthase to produce ATP [7]. Over the last decades, there has been a growing interest in BR and its photochemical/physical characteristic [10]. BR has recently got a lot of actual and potential applications in its native and mutated form [10]. This protein has been used in variety of fields from biology, medicine, and medical instruments (e.g., in protein structure studies, drug screening, treatment of eye disorders and contact lenses) to information technology and electronics (e.g., in optical data storage, holographic storage, security ink and biosensor transducers) [10–13]. Several researchers have focused on enhancing BR production by H. salinarum with optimization of medium composition and using some culture strategies (e.g., batch or fed-batch operations), however, fewer of them evaluated culture conditions (e.g., aeration, and agitation rate) [14–16]. Unfortunately, favorable conditions for induction of the bop regulon (including the genes responsible for BR production, i.e., brp, bop and bat genes) is unfavorable for H. salinarum growth [17, 18]. This archaeon grows in aerobic condition up to five times faster than photosynthesis condition (low oxygen concentration in presence of light) [2]. However, the bop regulon induced in photosynthesis condition several times higher than aerobic condition [17]. Since oxygen affects the bop regulon expression and H. salinarum growth differently, regulation of oxygen concentration in the culture medium plays an important role in BR production. The temperature, agitation rate, and the filling volume are some factors which modulate oxygen concentration in a liquid medium. In this study, to increase BR concentration, these oxygen-regulating factors were investigated and optimized. Materials and methods Materials All the materials were obtained from the Merck Company (Germany) except that Hy-case was purchased from the Fluka and M nSO4·H2O and DNase 1(DN25) were purchased from the Sigma-Aldrich co. (Germany). 13 Microorganism and media Halobacterium salinarum R1 (collection number: DSM 671) was purchased from the German Culture Collection (DSMZ, Braunschweig, Germany). A medium containing 10 gl−1 yeast extract; 0.5 gl−1 Hycase; 0.2 gl −1 Na 3-citrate. 2 H 2O; 0.5 gl −1 meat extract; 0.5 gl −1 glycerol; 20 gl −1 M gSO 4 ·7H 2 O; 2 gl −1 KCl; 0.05 gl −1 F eSO 4 ·7H 2 O; 0.0002 gl −1 M nSO 4 ·H 2 O and 250 gl−1 NaCl was used as the liquid culture medium in all the experiments. Minerals were autoclaved separately and mixed with other medium components at room temperature to avoid precipitation, then its pH adjusted to 7.3 ± 0.1 aseptically using 1 M NaOH. Growth kinetic, BR measurement and BR productivity For monitoring cell growth, the number of cells was counted using a Neubauer countin (...truncated)