Characterisation of surfactant-expressing bacteria and their potential bioremediation properties from hydrocarbon-contaminated and uncontaminated soils

Student thesis: Doctoral Thesis

Abstract

Bacteria synthesise various surface-active molecules called surfactants with numerous behaviours in air-water and oil-water interfaces and have different biological roles and applications in biotechnology. The goal of this study was to characterise the bacteria isolated from hydrocarbon-contaminated soils in the UK and Nigeria that express biodegradation properties. The specific objectives were to isolate and characterise surfactant-producing bacteria from various soils, and to identify lipase-producing bacteria for possible biodegradation among the surfactant-producing strains. Another goal was to investigate the genes involved in surfactant synthesis and hydrocarbon biodegradation in the strains using bioinformatics analysis. Using Pseudomonas-selective and non-selective nutrient media, 1460 colonies from five locations were isolated and examined for surfactant expression via drop-collapse assay and quantitative tensiometry methods. Sixty isolates were chosen, and their phenotypes were evaluated using a number of growth and behaviour-based tests, as well as their tolerance to various heavy metal concentrations and temperature ranges. Their bioremediation capabilities were determined by their ability utilise diesel as a carbon source, as well as their lipase production potentials. The pH, temperature, and salt tolerance of eight selected lipase-active lysates were studied using enzyme activity profiling. The nature of surfactants, heavy metal tolerance mechanisms, and hydrocarbon-degrading enzymes produced by the bacteria were further investigated using bioinformatics analysis of draft whole genome sequences of the eight strains. Sixty isolates were chosen from a total of 168 identified to express surfactants (24.7 mNm-1 – 26.7 mNm-1), and when examined using Hierarchical cluster analysis, they revealed significant phenotypic variation in six distinct clusters. Eight of the 60 isolates could grow at high temperatures (50 °C), 35 of the 60 isolates used diesel as their sole carbon source, preliminary biodegradation of diesel revealed two isolates B5 and C9 that degraded diesel by 42.75 % and 31.57 % respectively, and the majority of the 60 isolates could tolerate high concentrations of heavy metals such as cadmium (30 mM), chromium (30 mM), cobalt (10 mM), copper (5 mM), lead (20 mM), mercury (5 mM), nickel, and zinc (30 mM) in comparison with published values. Some of the isolates were identified as belonging to the Pseudomonas, Bacillus, and Stenotrophomonas genera including Bacillus subtilis subs subtilis NBRC 13719, Pseudomonas aeruginosa PAO1 and Stenotrophomonas maltophilia k279a using 16S rDNA sequencing and phylogenetic analysis. Specific lipolytic activities ranged from 2.29 – 67.42 U/mg (soluble-fraction lipase) and 8.48 – 130.06 U/mg (membrane-fraction lipase). Results of pH stability and temperature profiles of eight chosen strains demonstrated optimal lipolytic activity at pH 7 and 30 °C, respectively, whereas salt tolerance profiling revealed an expected reduction in lipolytic activity as NaCl concentration increased. AntiSMASH and RAST bioinformatics analyses of the eight draft whole genome sequences revealed NRPS-like clusters (probable surfactant sequences) with srfAC surfactin subunit 2 identified in strain A2, cytochrome P450 sequences (five strains), catechol-1,2/2,3-dioxygenase sequences (seven strains) with Bacillus subtilis VOC family protein identified in strain A2, lipase gene sequences (eight strains), and heavy metal resistance gene sequences including copZ, merR, and cadA possibly conferring zinc (in strain A2), mercury (in strain A7), and cadmium resistance (in strain D11) respectively. Overall, this study found that surfactant production was dependent on hydrocarbon contamination, and there was a positive correlation between hydrocarbon contamination and lipase production. This study may give valuable information and isolates for possible applications in in situ or ex situ bioremediation of hydrocarbon-contaminated soils, particularly in Nigeria, where fewer than 0.2 percent of 5000 polluted sites have records of bioremediation attempts as of 2016.
Date of Award25 Jan 2022
Original languageEnglish
Awarding Institution
  • Abertay University
SponsorsPetroleum Technology Development Fund (PTDF)
SupervisorScott Cameron (Supervisor) & Andrew Spiers (Supervisor)

Keywords

  • Hydrocarbon contamination
  • Surfactants
  • Pseudomonas
  • Bacteria
  • Heavy metals
  • Bacillus
  • Soil

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