Enhanced Biotransformation of Fluoranthene by Intertidally Derived Cunninghamella elegans under Biofilm-Based and Niche-Mimicking Conditions

Abstract

The aims of the investigation were to ascertain if surface attachment of Cunninghamella elegans and niche intertidal conditions provided in a bioreactor influenced biotransformation of fluoranthene by C. elegans. A newly designed polymethylmethacrylate (PMMA) conico-cylindrical flask (CCF) holding eight equidistantly spaced rectangular strips mounted radially on a circular disc allowed comparison of fluoranthene biotransformation between CCFs with a hydrophobic surface (PMMA-CCF) and a hydrophilic glass surface (GS-CCF) and a 500-ml Erlenmeyer flask (EF). Fluoranthene biotransformation was higher by 22-fold, biofilm growth was higher by 3-fold, and cytochrome P450 gene expression was higher by 2.1-fold when C. elegans was cultivated with 2% inoculum as biofilm culture in PMMA-CCF compared to planktonic culture in EF. Biotransformation was enhanced by 7-fold with 10% inoculum. The temporal pattern of biofilm progression based on three-channel fluorescence detection by confocal laser scanning microscopy demonstrated well-developed, stable biofilm with greater colocalization of fluoranthene within extracellular polymeric substances and filaments of the biofilm grown on PMMA in contrast to a glass surface. A bioreactor with discs rotating at 2 revolutions per day affording 6-hourly emersion and immersion mimicked the niche intertidal habitat of C. elegans and supported biofilm formation and transformation of fluoranthene. The amount of transformed metabolite was 3.5-fold, biofilm growth was 3-fold, and cytochrome P450 gene expression was 1.9-fold higher in the process mimicking the intertidal conditions than in a submerged process without disc rotation. In the CCF and reactor, where biofilm formation was comparatively greater, higher concentration of exopolysaccharides allowed increased mobilization of fluoranthene within the biofilm with consequential higher gene expression leading to enhanced volumetric productivity.

The CYPome of the model xenobiotic-biotransforming fungus Cunninghamella elegans

“…elegans was grown in the latter medium the biotransformation of the drug flurbiprofen was very poor in comparison to cells grown in SDB, which was further evidence that CYP5313D1 is an enzyme involved in xenobiotic metabolism in the fungus. Other researchers 15–17 previously investigated CYP expression in Cunninghamella spp. by RT-PCR, using primers based on the conserved haem-binding region of cytochromes P450.…”

“…elegans cultures were grown for 72 h and the biomass (200 mg) was collected aseptically using sterile tweezers after separation from the supernatant by centrifugation (Eppendorf 5810 centrifuge). The biomass was then flash frozen with liquid nitrogen and ground into a powder using a pestle and mortar as described previously 17,36 . The crushed biomass was resuspended immediately in 300 μl of MicroBead Solution from the DNeasy UltraClean Microbial Kit (Qiagen) and processed using the steps outlined in the instruction manual.…”

“…elegans and its expression demonstrated by northern blot 14 . Expression of the cpr and cyp509A1 has been measured under different culturing conditions by other researchers 15–17 and these studies have shown their up-regulation in the presence of xenobiotic compounds. However, the absence of a defined function of the proteins is unsatisfactory, in particular given the central role of CYPs in phase-I metabolism and their value as biocatalysts.…”

Monitoring of biofilms grown on differentially structured metallic surfaces using confocal laser scanning microscopy

“…Data were exported in HTML format and statistically analyzed using Origin 2016G. ImageJ with Volume Viewer 1.31 plugin (https://imagej.nih.gov/ij/plugins/volume-viewer.html) was used for processing of generated image stacks .…”

“… reviewed the ecological roles and biotechnological applications of intertidal microbial biofilms like production of antimicrobial or antifouling compounds, exopolysaccharides, pigments, vitamins, and biotechnologically important enzymes. Immobilization of cells in the form of biofilms can be beneficial for various bioprocesses such as wastewater treatment , environmental bioremediation or microbial fuel cells . It has been discovered that the surface topography of the substratum also plays an important role in microbial cell attachment to the surface .…”