title: Isolation and characterization of gram-positive biosurfactant-producing halothermophilic bacilli from Iranian petroleum reservoirs.
abstract: BACKGROUND
  Petroleum reservoirs have long been known as the hosts of extremophilic microorganisms. Some of these microorganisms are known for their potential biotechnological applications, particularly production of extra and intracellular polymers and enzymes.
  
  
  OBJECTIVE
  Here, 14 petroleum liquid samples from southern Iranian oil reservoirs were screened for presence of biosurfactant-producing halothermophiles.
  
  
  METHODS
  Mixture of the reservoir fluid samples with a minimal growth medium was incubated under an N2 atmosphere in 40°C; 0.5 mL samples were transferred from the aqueous phase to agar plates after 72 hours of incubation; 100 mL cell cultures were prepared using the MSS-1 (mineral salt solution 1) liquid medium with 5% (w/v) NaCl. The time-course samples were analyzed by recording the absorbance at 600 nm using a spectrophotometer. Incubation was carried out in 40°C with mild shaking in aerobic conditions. Thermotolerance was evaluated by growing the isolates at 40, 50, 60 and 70°C with varying NaCl concentrations of 5% and 10% (w/v). Halotolerance was evaluated using NaCl concentrations of 5%, 10%, 12.5% and 15% (w/v) and incubating them at 40°C under aerobic and anaerobic conditions. Different phenotypic characteristics were evaluated, as outlined in Bergey's manual of determinative bacteriology. Comparing 16S rDNA sequences is one of the most powerful tools for classification of microorganisms.
  
  
  RESULTS
  Among 34 isolates, 10 demonstrated biosurfactant production and growth at temperatures between 40°C and 70°C in saline media containing 5%-15% w/v NaCl. Using partial 16S rDNA sequencing (and amplified ribosomal DNA restriction analysis [ARDRA]) and biochemical tests (API tests 20E and 50 CHB), all the 10 isolates proved to be facultative anaerobic, Gram-positive moderate thermohalophiles of the genus Bacillus (B. thermoglucosidasius, B. thermodenitrificans, B. thermoleovorans, B. stearothermophilus and B. licheniformis), exhibiting surface-active behaviors.
  
  
  CONCLUSIONS
  General patterns include decreasing the thermotolerance with increasing the salt concentrations and also more halotolerance in the aerobic environment compared with anaerobic conditions. The results demonstrated that Iranian petroleum reservoirs enjoy a source of indigenous extremophilic microorganisms with potential applications in microbial enhanced oil recovery and commercial enzyme production.
authors.affiliation: Department of Petroleum Engineering, Colorado School of Mines, Colorado, USA.
authors.forename: Saeed
authors.initials: S
authors.lastname: Zargari
authors.affiliation: Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, IR Iran.
authors.forename: Amin
authors.initials: A
authors.lastname: Ramezani
authors.affiliation: School of Chemical, Oregon State University, Corvallis Oregon, USA.
authors.forename: Sassan
authors.initials: S
authors.lastname: Ostvar
authors.affiliation: Biotechnology Institute, Shiraz University, Shiraz, IR Iran.
authors.forename: Rasool
authors.initials: R
authors.lastname: Rezaei
authors.affiliation: Biotechnology Institute, Shiraz University, Shiraz, IR Iran.
authors.forename: Ali
authors.initials: A
authors.lastname: Niazi
authors.affiliation: Enhanced Oil Recovery Research Center, School of Engineering, Shiraz University, Shiraz, IR Iran.
authors.forename: Shahab
authors.initials: S
authors.lastname: Ayatollahi
keywords: Bacillus; Microorganism; Petroleum