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Marine-derived Actinomycetes Cultured in Carbon Dioxide Atmosphere and Buffered Media

Rita Lourenço1, 2, Inês Ferreira1, 2, Miriam Presas2, Ana V. Nunes2, Luís C. Branco2, Manuel Nunes Da Ponte2, João Carlos Lima2, Mário S. Diniz1, Susana P. Gaudêncio1 and Zeljko Petrovski2*
  • 1 Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, UCIBIO-REQUIMTE, Department of Chemistry and Department of Life Sciences, Portugal
  • 2 Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, LAQV-REQUIMTE, Department of Chemistry, Portugal

Actinomycetes are Gram-positive bacteria that show variable tolerance to oxygen and range from obligatory aerobic to obligatory anaerobic [1]. In addition, actinomycetes can be cultured as autotrophic organisms consuming carbon monoxide or carbon dioxide and hydrogen [2] and also in media containing ammonium and glucose [3]. Evidence is presented that some strains of actinomycetes, require carbon dioxide for anaerobic growth. Some of these strains are obligate anaerobes to microaerophils, while others appear to be facultative anaerobes. Cultures which are capable of aerobic growth may or may not require carbon dioxide for such growth [4]. Fermentations of glucose in the presence of 14CO2 carried out with strains of actinomycetes under aerobic and anaerobic conditions demonstrate incorporation of radioactive carbon in high percentage in the methylene carbons of succinic acid. When actinomycetes were cultured in the presence of oxygen, 90% of the total radioactivity of the succinic acid was found in the methylene carbons as compared to 62% when it was cultured anaerobically [5]. Carbon dioxide was also needed for aspartic acid synthesis [6]. More recent studies have also shown that relative proportion of fungi, filamentous bacteria, and Gram-positive bacteria in microbial community of soil was increased and the proportion of Gram-negative bacteria decreased by incubation in the high partial pressure of CO2. Heterotrophic CO2 fixation was proven and the main portion of the fixed CO2 (98–99%) was found in extracellular metabolites while only 1% CO2 was incorporated into microbial cell [7]. The bacterial potential for growth under an atmosphere of carbon dioxide is perhaps a viable strategy to mitigate global climate change [8-10]. In this study the marine-derived PTM-420 actinomycete, phylogenetically related to Streptomyces aculeolatus, was used with the main goal of analyzing the influence of an atmosphere saturated with CO2 on its growth and metabolism while hoping to induce sequestration of the gas and possibly to improve its value through the production of new metabolites. The principal objective was to compare the growing differences under standard growing conditions [11] and under increased partial pressure of carbon dioxide (herein 50% and 100 % carbon dioxide atmosphere) and in the presence or absence of salt additives (potassium, sodium and ammonium bicarbonate). The required concentration of bicarbonate salt in the nutrient solution was determined using the formula pH=7.74+log[HCO3-]/p(CO2) [12]. The optimal pH was set to 6.7 as at higher values occurred salt precipitation due to reaction of sea water with bicarbonate resulting in uncontrollable pH oscillations (unbuffered solutions). In the case of ammonium bicarbonate the formula could not be used for accurate pH determination due to ammonium hydrolysis and variation of salt concentration (the salt itself was prepared in situ from autoclaved ammonia and autoclaved nutrient solution). Therefore in this case the initial pH was determined experimentally. The culture was performed in liquid media and compared to standard conditions (medium A1), [11] while the growth monitoring was carried out using Fischer and Sawers, an universally applicable and rapid method for measuring the growth of Streptomyces and other filamentous microorganisms by using methylene blue adsorption-desorption [13]. Growth curves analysis of PTM-420 under the several mentioned conditions revealed that no better growing conditions under 100% and 50% carbon dioxide atmosphere were achieved than under the standard conditions, yet the results showed some significant differences. Thus, the growth rate remains the lowest in case of pure carbon dioxide most probably due to low pH and much higher in case of bicarbonate buffer. The influence of the cation seems to be less noticeable with 100% carbon dioxide atmosphere while under 50% it is clear that Na+ provides much better growing conditions followed by NH4+and then K+. This result is in accordance with the fact that marine obligate actinomycete strains require Na+ for their growth [14]. Response to stress is being evaluated, first the determination of total protein is performed by Bradford method [15] and the stress tests include glutathione S-transferase activity [16], lipid peroxidation [17], catalase activity [18] and HSP70 ELISA immunoassay [19].

Acknowledgements

This study had the financial support of Fundação de Ciência e a Tecnologia (FCT), through the grants: PTDC/QUIQUI/119116/2010, IF/00700/2014, SFRH/BPD/111262/2015, UID/Multi/04378/2013 (UCIBIO), UID/QUI/ 50006/2013 (LAQV) and co-financed by FEDER under PT2020 partnership agreement POCI-01-0145-FEDER-007728 and POCI-01-0145-FEDER-007265.

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Keywords: Marine-derived actinomycetes, Culture under CO2, stress evaluation, Climate Change, Streptomyces aculeolatus

Conference: IMMR'18 | International Meeting on Marine Research 2018, Peniche, Portugal, 5 Jul - 6 Jul, 2018.

Presentation Type: Poster Presentation

Topic: Blue Biotech

Citation: Lourenço R, Ferreira I, Presas M, Nunes AV, Branco LC, Nunes Da Ponte M, Lima J, Diniz MS, Gaudêncio SP and Petrovski Z (2019). Marine-derived Actinomycetes Cultured in Carbon Dioxide Atmosphere and Buffered Media. Front. Mar. Sci. Conference Abstract: IMMR'18 | International Meeting on Marine Research 2018. doi: 10.3389/conf.FMARS.2018.06.00091

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Received: 28 Apr 2018; Published Online: 07 Jan 2019.

* Correspondence: Dr. Zeljko Petrovski, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa, LAQV-REQUIMTE, Department of Chemistry, Almada, Portugal, z.petrovski@fct.unl.pt