Biofilm development is a multiplex procedure that is influenced by many factors, which include growth condition, stress and surface attributes (Kostakioti et al.2013). Microbial biofilms are captivating areas to study because of their wide varieties in nature, importance in infection and use in bioremediation and industries. Advances in molecular and biochemical methods have helped in improving our comprehension of biofilm structure and functions (Velmourougane et al.2017). Exploiting naturally occurring- and in-situ biofilm devolvement are promising ways for future advances in agricultural field as they can possibly give different advantages like increase crop production, protection of the economically important plants from insects and improvement of the crop variety (Franklin et al.2015).Investigations uncovered the intricate conditions experienced in biofilms and during infection create extraordinary heterogeneity inside the populace (Bisht et.al 2019).
Regarding the regulation of biofilm formation, it was reported that in case of Bacillus subtilis, glucose repressed biofilm development through the catabolic control protein CcpA (Stanley et al.2003). On the other hand, interesting and contrasting reports showed that addition of glucose with NaCl in Tryptic Soy broth (TSB) altogether favoured more biofilm formation in Bacillus cereus cells compared to the addition of glucose or glycerol or NaCl in growth media (Kwon et al.2017). Many studies suggest that the biofilm develops in restricted supplement of nutrients or in the presence of any sort of stress i.e. antibiotic, high salt or a corrosive substance like acid (Khatoon et al.2018).It is also reported that the nutrient accessibility affects adherence, biofilm arrangement and synthesis of EPS (Xiao et al.2017).
Recent studies revealed the biofilm forming abilities the Bacillus thuringiensis isolates invitro and on plant surfaces (Verplaetse et al.2015). B. thuringiensis biofilms on plant surfaces thus can help the plant by protecting it from the pathogens like agricultural pests. It is also reported that Bacillus thuringiensis spores have a higher hydrophobicity, conferring them a higher adhesive potential to diverse materials (Wiencek et al.1990 ). Interesting findings in recent times revealed that Bacillus thuringiensis cells undergoes differentiations and represent a mixed population of heterogenous cells under stressed conditions (Verplaetse et al.2015).
Previous study from our laboratory revealed that addition of higher concentrations of glucose in growth media could induce restricted swarming motility due to impaired flagellation in Bacillus thuringiensis KPWP1 on nutrient agar plates (Roy et al.2010). This finding had raised the question whether the restricted motility in presence of glucose can trigger the biofilm forming ability in Bacillus thuringiensis KPWP1 cells? The present study was therefore to explore whether restricted motility induced by glucose can regulate the adherence property, production of EPS components, thus biofilm formation ability in Bacillus thuringiensis KPWP1. The results from this present study clearly indicate that the presence of added glucose in nutrient-rich media induces biofilm formation (Fig. 1B) by KPWP1 as a result of increase in EPS production (Fig. 6) and cell surface hydrophobicity (Fig. 4) and such induced biofilm formation is glucose specific as addition of galactose or arabinose in growth media could not induce any significant biofilm formation by KPWP1(Fig. 1D).
The increased biofilm formation, in presence of glucose, was manifested by the increased thickness (Fig. 2A), bio-volume (Fig. 2B), and biomass (Fig. 2C). Moreover, it was observed that the skewness (Fig. 2D) which reflects the intracellular void spaces and the kurtosis values (Fig. 2E) which expresses the adherence of microscopic organisms in KPWP1 biofilm also increased with the increased glucose concentrations in growth media.
It is reported that EPSs add to the mass and 3-D structure of the biofilm framework . The present study revealed that the EPS components i.e. polysaccharides, proteins, lipids and eDNA concentrations increased (Fig. 6A, 6B and 6C) in KPWP1 biofilms with the increase in glucose concentration in growth media. In the 48-h grown biofilms, EPS contents - sugar, proteins lipid and eDNA were altogether higher in the presence of higher concentrations of glucose than that of control condition and supports the observed increase in physical appearance of EPS in SEM images of KPWP1 biofilms grown in different glucose concentrations (Fig. 3). Furthermore, it is also indicative that the EPSs formed in presence of 1 and 2% glucose helps in the adherence of the bacteria to the surface (Fig. 6). Collectively, the present study uncovered the effect of glucose on biofilm arrangement of KPWP1 cells. Glucose prompts the adherence property of the Bacillus thuringiensis KPWP1 cells and induces more EPS production resulting in more biofilm formation.
The results of the present study signify that restricted motility in Bacillus thuringiensis KPWP1 induced by glucose helps the bacterial cells to form biofilm, thus give a major premise to a more elaborate investigation of the in-vivo biofilm formation by this insecticidal bacterium Bacillus thuringiensis on plant surfaces, particularly in response to glucose.