A novel multi-enzyme preparation produced from Aspergillus niger using biodegradable waste: a possible option to combat heterogeneous biofilms

Abstract

Extracellular polymeric substance (EPS) produced by the microorganisms provides protection and stability to them when they are encased within biofilms. Heterogeneous polysaccharides form a major constituent of the EPS and are crucial for the formation and integrity of the biofilms/slime. Thus, breakdown of polysaccharides might help in dispersion of biofilms from abiotic surfaces. In the present study we isolated a fungus, Aspergillus niger APS, capable of concurrently producing a cocktail of carbohydrases and optimized the conditions for higher yields of all the enzymes by one variable at a time (OVAT) approach. The optimization studies resulted in 1.5 to 12 fold augmentation in the enzyme yields using biodegradable waste. Further, keeping in view the heterogeneous nature of polysaccharides in biofilm matrix, the in-house produced enzyme cocktail was used for the dispersal of biofilms formed by Salmonella enterica serovar Typhi, Escherichia coli and Staphylococcus aureus. Treatment with enzyme preparation caused 90.23 ± 4.0, 82.64 ± 5.0 and 76.32 ± 5.0% reduction of the biofilms formed by these organisms respectively which was also evidenced by Field emission scanning electron microscopy (FESEM) revealing the loss of biofilm architecture. Interestingly, the enzyme cocktail could also remove viscous slime formed under natural conditions in the kitchen drainage pipe (KDP). To the best of our knowledge, this is the first report on biotreatment of abiotic surfaces for removal of biofilms/slime formed under natural conditions. The study thus indicates the prospects of using multiple carbohydrases as an anti-biofouling agent on abiotic surfaces like equipments as well as implants/prostheses and pipelines.

Cocktail of carbohydrases from Aspergillus niger: an economical and eco-friendly option for biofilm clearance from biopolymer surfaces

“…Earlier we had reported enzyme optimization by classical one variable at a time (OVAT) approach (Kaur et al 2020a ). For the application of enzymes for biofilm dispersal, the yields of all the enzymes were further enhanced by evaluation of interactions between the most important variables promoting enzyme production by response surface methodology (RSM) studies.…”

“…The in-house produced enzyme cocktail (Kaur et al 2020a ) was obtained from cell free supernatant of A. niger APS, isolated from soil, (GenBank accession number MN559364; deposited in MTCC with accession number 12975), comprised of cellulases, hemicellulases, pectinase, amylases, and alginate lyase. The enzyme preparation was filter-sterilized using 0.22 µm syringe filters before use.…”

“…The biofilms were developed in sterile polystyrene flat-bottomed microtitre plates by the method described by Stepanović et al ( 2000 ) and reported by us earlier (Kaur et al 2020a ). Briefly, the wells of a sterile polystyrene microplate were filled with 230 μl of medium (Luria broth for E. coli and P. aeruginosa biofilms and Tryptic soy broth for the development S. aureus ).…”

“…Dentin discs without enzymatic treatment served as controls. After the treatment, the test and control discs were processed for FESEM analysis as described by us earlier (Kaur et al 2020a ). Briefly, the test and the control discs were fixed with 2.5% glutaraldehyde for 2 h. Following fixation, glutaraldehyde was removed by washing thrice with 0.1 M PBS (pH 7.2).…”

“…Various agents including enzymes, antibiofilm peptides, and dispersal molecules that target the biofilm architecture have been found to be promising as anti-biofilm agents (Fleming and Rumbaugh 2017 ). Recently, we reported the use of an enzyme consortium obtained from Aspergillus niger , a soil isolate, for the clearance of biofouling and black gunk developed on the surface of kitchen drainage pipes (Kaur et al 2020a ). In continuation, in the present study, yet another application of the same enzyme consortium i.e.…”