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Physiological Characterization of Sulfolobus Acidocaldarius in a Controlled Bioreactor Environment

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Physiological Characterization of Sulfolobus Acidocaldarius in a Controlled Bioreactor Environment International Journal of Environmental Research and Public Health Article Physiological Characterization of Sulfolobus acidocaldarius in a Controlled Bioreactor Environment Kerstin Rastädter , David Johannes Wurm , Oliver Spadiut and Julian Quehenberger * Research Division Biochemical Engineering, Faculty of Technical Chemistry, Institute of Chemical, Environmental and Bioscience Engineering, TU Wien, 1060 Vienna, Austria; [email protected] (K.R.); [email protected] (D.J.W.); [email protected] (O.S.) * Correspondence: [email protected] Abstract: The crenarchaeal model organism Sulfolobus acidocaldarius is typically cultivated in shake flasks. Although shake flasks represent the state-of-the-art for the cultivation of this microorganism, in these systems crucial process parameters, like pH or substrate availability, are only set initially, but cannot be controlled during the cultivation process. As a result, a thorough characterization of growth parameters under controlled conditions is still missing for S. acidocaldarius. In this study, we conducted chemostat cultivations at 75 ◦C using a growth medium containing L-glutamate and D-glucose as main carbon sources. Different pH values and dilution rates were applied with the goal to physiologically characterize the organism in a controlled bioreactor environment. Under these controlled conditions a pH optimum of 3.0 was determined. Washout of the cells occurred at a dilution rate of 0.097 h−1 and the optimal productivity of biomass was observed at a dilution rate −1 of 0.062 h . While both carbon sources were taken up by S. acidocaldarius concomitantly, a 6.6-fold higher affinity for L-glutamate was shown. When exposed to suboptimal growth conditions, S. Citation: Rastädter, K.; Wurm, D.J.; acidocaldarius reacted with a change in the respiratory behavior and an increased trehalose production Spadiut, O.; Quehenberger, J. rate in addition to a decreased growth rate. Physiological Characterization of Sulfolobus acidocaldarius in a Keywords: Sulfolobus acidocaldarius; characterization; chemostat; dilution rate; substrate affinity; Controlled Bioreactor Environment. trehalose Int. J. Environ. Res. Public Health 2021, 18, 5532. https://doi.org/10.3390/ ijerph18115532 1. Introduction Academic Editor: Paul B. Tchounwou The extremophile Sulfolobus acidocaldarius is a widely used model organism of the phylum Crenarchaeota. Its genome sequence is available [1] and a powerful genetic Received: 18 April 2021 tool box for investigating and engineering gene functions has been developed [2] and is Accepted: 19 May 2021 continuously being improved [3]. The organism thrives in a temperature range of 65 to Published: 21 May 2021 85 ◦C and at a pH ranging from pH 2.0 to 5.5 [4], while under laboratory conditions it is generally cultivated at temperatures of 70 to 75 ◦C and at a pH of 2.25 to 3.5 [5–10]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Most research with this organism is performed in batch cultivations using shake flasks published maps and institutional affil- as culture vessels [5,7,11]. However, a continuous cultivation in a stirred-tank reactor iations. (CSTR) with the possibility to monitor and control pH, temperature and dissolved oxygen content (dO2) is the basis for stable growth conditions, thereby allowing the acquisition of reliable and reproducible process data [12]. Furthermore, providing the substrate in limiting amounts might also represent conditions that resembles the natural habitat of most microorganism more closely than the nutrient enriched system of batch cultivations [13–15]. Copyright: © 2021 by the authors. In chemostat cultures the growth rate, biomass, substrate and product concentration Licensee MDPI, Basel, Switzerland. This article is an open access article remain constant after reaching steady state [16], thus making it a reliable tool for strain distributed under the terms and characterization as shown for a variety of microorganisms [17–19]. Critical parameters for conditions of the Creative Commons bioprocess development, such as the optimal and critical dilution rate, affinity constants Attribution (CC BY) license (https:// for substrates as well as maintenance coefficient, can be readily determined in a CSTR. All creativecommons.org/licenses/by/ these parameters are missing in the current literature of Sulfolobales. 4.0/). Int. J. Environ. Res. Public Health 2021, 18, 5532. https://doi.org/10.3390/ijerph18115532 https://www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2021, 18, 5532 2 of 13 In a CSTR a potent technique to assess the physiological condition of an organism is the monitoring of the respiratory behavior, expressed as the respiratory quotient (RQ). The RQ is the molar ratio of produced CO2 to consumed O2 and depends on the state of oxidation of the metabolized substrate and the utilized catabolic pathways [20]. Higher RQs than the theoretical value for complete oxidation of the respective substrates imply incomplete substrate utilization and potential waste of energy, while lower RQs can signify a change in the carbon flow towards metabolites and hint towards a less efficient utilization of O2. A further way to monitor metabolic stress is the direct measurement of metabolites in the culture supernatant. S. acidocaldarius produces the non-reducing disaccharide trehalose, a compound that can be found in all three domains of life, where it functions as a general protecting agent against unfavorable environmental conditions [21]. In this study, the strain S. acidocaldarius DSM639 was grown in continuous cultiva- tion on a medium containing L-glutamate and D-glucose as main carbon sources and was physiologically characterized regarding its pH optimum, maximum growth rate, spe- cific substrate uptake rates and its stress response indicated by trehalose production and respiratory behavior. 2. Materials and Methods 2.1. Bioreactor Cultivations S. acidocaldarius DSM639, obtained at German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), was continuously cultivated in a DASbox Mini Bioreactor System (Eppendorf AG, Hamburg, Germany) with a working volume of 200 mL per vessel. Four parallel bioreactors, filled with Vienna Defined (VD) Medium [22] with modified carbon source concentrations (2 g/L monosodium glutamate (MSG), 1 g/L D-glucose) were inoculated with a starting OD600 of 0.17, yielding a total batch volume of 150 mL. Feed used during fed-batch and chemostat phases contained a 5-times concentrated VD Medium with modified carbon source concentrations (9.5 g/L MSG, 4.5 g/L D-glucose and a minute amount of 0.5 g/L NZ-amine to rule out any long-term nutrient deficiencies). Fed-batch phase was performed with a continuous feed rate of 3.0 mL/h until a reactor volume of 200 mL was reached. During the following chemostat phase, the feed rate was set to 6.0 mL/h (D = 0.03 h−1). To maintain a constant volume of 200 mL, broth was withdrawn from the reactor via a bleed tube at a fixed height. The reactors were stirred with 600 rpm and supplied with 0.3 vvm (volumes gas flow per working volume per minute; equal to: 3.6 sL/h; liter at standardized conditions, 273.15 K and 1 bar per hour) pressurized air. A constant growth temperature of 75 ◦C was achieved by submerging the reactor vessels in a stirred heated oil bath. pH was measured with an EasyFerm Plus K8 120 electrode (Hamilton, OH, USA) and was adjusted by automatic addition of 4.8% H2SO4. Dissolved oxygen (dO2) was monitored by a VisiFerm DO225 probe (Hamilton, OH, USA). CO2 and O2 concentrations were measured using the DASGIP GA4 exhaust analyzer (Eppendorf AG, Hamburg, Germany). To assess reproducibility three bioreactors of the DASbox Mini Bioreactor System were subjected to the same conditions (75 ◦C, pH 3.0, D = 0.03 h−1). Table1 gives an overview of the experiments performed in this study. Table 1. Experimental plan of this study: chemostat cultivations with a dilution rate (D) of 0.03 h−1 were performed at 5 different pH values. The following set of experiments where the dilution rate was varied from 0.010 h−1 to 0.097 h−1 was then performed at the prior determined pH optimum of 3.0. Fixed Parameter Variable Parameter D 0.03 h−1 pH 2.0, 2.5, 3.0, 3.5, 4.0 −1 −1 −1 −1 −1 −1 −1 −1 −1 pH 3.0 D 0.010 h , 0.021 h , 0.032 h , 0.043 h , 0.053 h , 0.064 h , 0.074 h , 0.084 h , 0.097 h Since the cultivation performed at pH 2.0 in the DASbox Mini Bioreactor System yielded an inexplicably high CO2 yield and consequently a C-balance that significantly Int. J. Environ. Res. Public Health 2021, 18, 5532 3 of 13 exceeded a value of 1.0, this experiment was excluded from the study and was instead repeated in a 2 L Biostat A-plus bioreactor (Sartorius, Goettingen, Germany). The reactor was stirred at 300 rpm and supplied with 0.23 vvm (0.45 sL/h) pressurized air. The pH was measured with an EasyFerm Plus electrode (Hamilton, OH, USA) and controlled via automatic addition of 4.8% H2SO4. CO2 and O2 concentrations in the exhaust gas were measured using a gas analyzing unit (Müller Systems AG, Esslingen, Switzerland). The batch phase was started with 1.5 L and chemostat culturing was done at 2 L and a dilution rate of 0.03 h−1. During fed-batch an exponential feed was applied, starting with 14.8 g/h and a growth rate of 0.035 h−1. The cultivation was controlled using the Lucullus process control system (SecureCell AG, Urdorf, Switzerland). All other process parameters were set as mentioned for the DASbox Mini Bioreactor System. 2.2. Biomass Determination Optical density, OD600, was determined photometrically at 600 nm with a spectropho- tometer against a blank of deionized water (ONDA V-10 PLUS, XS instruments, Carpi, Italy).
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