Microbial biodegradation of paraffin wax in Malaysian crude oil mediated by degradative enzymes

The deposition of paraffin wax in crude oil is a problem faced by the oil and gas industry during extraction, transportation, and refining of crude oil. Most of the commercialized chemical additives to prevent wax are expensive and toxic. As an environmentally friendly alternative, this study aims to find a novel thermophilic bacterial strain capable of degrading paraffin wax in crude oil to control wax deposition. To achieve this, the biodegradation of crude oil paraffin wax by 11 bacteria isolated from seawater and oil-contaminated soil samples was investigated at 70°C. The bacteria were identified as Geobacillus kaustophilus N3A7, NFA23, DFY1, Geobacillus jurassicus MK7, Geobacillus thermocatenulatus T7, Parageobacillus caldoxylosilyticus DFY3 and AZ72, Anoxybacillus geothermalis D9, Geobacillus stearothermophilus SA36, AD11, and AD24. The GCMS analysis showed that strains N3A7, MK7, DFY1, AD11, and AD24 achieved more than 70% biodegradation efficiency of crude oil in a short period (3 days). Notably, most of the strains could completely degrade C37–C40 and increase the ratio of C14–C18, especially during the initial 2 days incubation. In addition, the degradation of crude oil also resulted in changes in the pH of the medium. The degradation of crude oil is associated with the production of degradative enzymes such as alkane monooxygenase, alcohol dehydrogenase, lipase, and esterase. Among the 11 strains, the highest activities of alkane monooxygenase were recorded in strain AD24. A comparatively higher overall alcohol dehydrogenase, lipase, and esterase activities were observed in strains N3A7, MK7, DFY1, AD11, and AD24. Thus, there is a potential to use these strains in oil reservoirs, crude oil processing, and recovery to control wax deposition. Their ability to withstand high temperature and produce degradative enzymes for long-chain hydrocarbon degradation led to an increase in the short-chain hydrocarbon ratio, and subsequently, improving the quality of the oi

Newly isolated alkane hydroxylase and lipase producing Geobacillus and Anoxybacillus species involved in crude oil degradation

Isolation and studies of novel, crude oil biodegrading thermophilic strains may provide a wider knowledge in understanding their role in petroleum degradation. In this study, the screening of ten new thermophilic strains revealed that all strains were alkane hydroxylase producers and seven of them produced lipase concurrently. Three best strains were characterized and identified through 16S rRNA sequence analysis as Geobacillus sp. D4, Geobacillus sp. D7, and Anoxybacillus geothermalis D9 with GenBank accession numbers MK615934.1, MK615935.1, and MK615936.1, respectively. Gas chromatography (GC) analysis showed that all three strains were able to breakdown various compounds in crude oil such as alkanes, toxic poly-aromatic hydrocarbons (PAHs), organosulfur, carboxylic acids, alkene, resins, organosilicon, alcohol, organochlorine, and ester. For the first time, alkane hydroxylase and lipase activity as well as crude oil degradation by A. geothermalis species were reported. Geobacillus sp. D7 is the best alkane degrader followed by A. geothermalis D9 and Geobacillus sp. D4 with 17.3%, 13.1%, and 12.1% biodegradation efficiency (BE%), respectively. The potential of thermophiles isolated can be explored further for bioremediation of sites polluted by petroleum and oil spills

Main structural targets for engineering lipase substrate specificity

Microbial lipases represent one of the most important groups of biotechnological biocatalysts. However, the high-level production of lipases requires an understanding of the molecular mechanisms of gene expression, folding, and secretion processes. Stable, selective, and productive lipase is essential for modern chemical industries, as most lipases cannot work in different process conditions. However, the screening and isolation of a new lipase with desired and specific properties would be time consuming, and costly, so researchers typically modify an available lipase with a certain potential for minimizing cost. Improving enzyme properties is associated with altering the enzymatic structure by changing one or several amino acids in the protein sequence. This review detailed the main sources, classification, structural properties, and mutagenic approaches, such as rational design (site direct mutagenesis, iterative saturation mutagenesis) and direct evolution (error prone PCR, DNA shuffling), for achieving modification goals. Here, both techniques were reviewed, with different results for lipase engineering, with a particular focus on improving or changing lipase specificity. Changing the amino acid sequences of the binding pocket or lid region of the lipase led to remarkable enzyme substrate specificity and enantioselectivity improvement. Site-directed mutagenesis is one of the appropriate methods to alter the enzyme sequence, as compared to random mutagenesis, such as error-prone PCR. This contribution has summarized and evaluated several experimental studies on modifying the substrate specificity of lipase