Determination of the biofilm production capacities and characteristics of members belonging to Bacillaceae family.

The biofilm characteristics of many endospore-forming bacilli, especially the thermophiles are still unclear. In this study, a detailed identification and description of biofilm production characteristics of totally 145 isolates and reference strains belonging to Bacillaceae family, displaying thermophilic (n = 115), facultative thermophilic (n = 24) and mesophilic (n = 6) growth from genera Anoxybacillus, Geobacillus, Thermolongibacillus, Aeribacillus, Brevibacillus, Paenibacillus and Bacillus were presented. The incubation temperatures were adjusted to 37, 45 and 55-65 °C for mesophiles, facultative thermophiles, and thermophiles, respectively. The bacilli were evaluated based on their colony morphotypes on Congo red (CR) agar, their complex exopolysaccharide production on calcofluor supplemented tryptic soy agar, and as well as their pellicle formation at the liquid-air surface in tryptic soy broth cultures. Their biofilm production capabilities were also tested on abiotic surfaces of both polystyrene and stainless steel by crystal violet binding assay and viable biofilm cell enumerations, respectively. As a result, the biofilm production capacities of Bacillaceae members from genera to species level, the effects of osmolarity, temperature, incubation time and abiotic surfaces on biofilm formation as well as the CR morphotypes associated with the biofilm production were able to reveal in a wide group of bacilli. Besides, general enrichment-inoculation approaches and methodologies were also offered, which allow and facilitate the screening and determining the biofilm producing endospore forming bacilli.

Biofilm characteristics and evaluation of the sanitation procedures of thermophilic Aeribacillus pallidus E334 biofilms.

The ability of Aeribacillus pallidus E334 to produce pellicle and form a biofilm was studied. Optimal biofilm formation occurred at 60 °C, pH 7.5 and 1.5% NaCl. Extra polymeric substances (EPS) were composed of proteins and eDNA (21.4 kb). E334 formed biofilm on many surfaces, but mostly preferred polypropylene and glass. Using CLSM analysis, the network-like structure of the EPS was observed. The A. pallidus biofilm had a novel eDNA content. DNaseI susceptibility (86.8% removal) of eDNA revealed its importance in mature biofilms, but the purified eDNA was resistant to DNaseI, probably due to its extended folding outside the matrix. Among 15 cleaning agents, biofilms could be removed with alkaline protease and sodium dodecyl sulphate (SDS). The removal of cells from polypropylene and biomass on glass was achieved with combined SDS/alkaline protease treatment. Strong A. pallidus biofilms could cause risks for industrial processes and abiotic surfaces must be taken into consideration in terms of sanitation procedures.