Contents lists available at ScienceDirect Fuel journal homepage: www.elsevier.com/locate/fuel Full Length Article Microbial enhanced oil recovery potential of surfactin-producing Bacillus subtilis AB2.0 Vanessa Marques Alvarez a , Carolina Reis Guimarães a,b , Diogo Jurelevicius a , Livia Vieira Araujo de Castilho b , Joab Sampaio de Sousa b , Fabio Faria da Mota c , Denise Maria Guimarães Freire b , Lucy Seldin a, ⁎ a Laboratório de Genética Microbiana, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro (UFRJ), Brazil b Laboratório de Biotecnologia Microbiana, Instituto de Química, UFRJ, Brazil c Laboratório de Biologia Computacional e Sistemas, Instituto Oswaldo Cruz, Rio de Janeiro, Brazil ARTICLE INFO Keywords: Biosurfactant Bacillus subtilis AB2.0 Surfactin Genome MEOR ABSTRACT Bacillus subtilis AB2.0 showed to produce a surface-active compound that was identified by mass spectrometry analysis as a surfactin. Four different isoforms were identified in the protonated form [M+H] + with m/z of 1030.6350, 1044.6511, 1058.6661 and 1072.6848. They were constituted of fatty acids varying from C13 to C16; however, about 70% of the isoforms showed m/z of 1058.6661 and the fatty acid C 15 . Based on the AB2.0 genome sequence, only one gene group related to surfactin biosynthesis was found. Strain AB2.0 produced 99.6 mg/l of surfactin in Tryptic Soy Broth after 48 h. The biosurfactant presented an emulsification activity of 45.7% in this growth condition. It considerably decreased the water surface tension from 72.0 mN/m to 24.7 mN/m and the interfacial tension of n-hexadecane from 30.1 mN/m to 7.5 mN/m, as well as of a medium crude oil from 42.3 mN/m to 6.9 mN/m and of a light crude oil from 36.4 to 3.8 mN/m. Calcite flotation test was conducted to access the wettability reversal of the biosurfactant in high salinity condition similar to that ob- served in an oil production reservoir, and AB2.0 reversed 100% using concentrations as low as 25 ppm of biosurfactant. Oil contaminated sand columns were used to simulate microbial enhanced oil recovery at different temperatures and high salinity. When surfactin from AB2.0 was added to the sand columns, the TPH con- centration in liquid phases recovered was enhanced up to 24× indicating that AB2.0 is an excellent candidate for its application in MEOR in subsurface environments. 1. Introduction Tertiary recovery is an oil exploration technology also known as enhanced oil recovery (EOR) that contemplates a variety of methods that aim to change the mobility of fluids within an oil reservoir. It is used when the internal pressure of the reservoir decreases in a level that neither water nor gas injection is enough to bring the oil to surface. This objective is achieved mainly through modifications in one or more of the parameters that influence the interactions between oil, water and rock: petroleum viscosity, water viscosity, surface tensions between oil / rock and water / rock, interfacial tension between oil / water, the wettability of the rock and the relative permeability of different zones within the reservoir. In this context, microbial enhanced oil recovery (MEOR) comprises the set of tertiary recovery technologies based on the action of microorganisms and / or microbial bioproducts [1–4]. One of the most promising bioproducts for MEOR are the bio- surfactants, a class of amphiphilic or amphipathic molecules which have the property of accumulating on surfaces / interfaces of a system and significantly change the free energy of these surfaces / interfaces [5,6]. In contrast to chemical surfactants, biosurfactants are those synthesized by the metabolism of living organisms. Biosurfactants produced by bacteria, archaea and fungi have been considered as at- tractive alternatives to chemical surfactants due to properties such as: (1) low ecotoxicity, (2) higher biodegradability, (3) greater stability under extreme pH, salinity and temperature, (4) milder production conditions, and (5) possibility of production using renewable raw ma- terials [7–10]. Biosurfactant molecules are synthesized by a large multifunctional non-ribosomal enzyme complex called nonribosomal peptide synthe- tase (NRPS) [11]. In a typical NRPS module, at least three essential https://doi.org/10.1016/j.fuel.2020.117730 Received 18 December 2019; Received in revised form 24 March 2020; Accepted 26 March 2020 ⁎ Corresponding author at: Laboratório de Genética Microbiana, Departamento de Microbiologia Geral, Instituto de Microbiologia Paulo de Góes, Universidade Federal do Rio de Janeiro, Centro de Ciências da Saúde, Bloco I, Ilha do Fundão, CEP 21941.902, Rio de Janeiro, RJ, Brazil. E-mail address: lseldin@micro.ufrj.br (L. Seldin). Fuel 272 (2020) 117730 Available online 02 April 2020 0016-2361/ © 2020 Elsevier Ltd. All rights reserved. T