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. 2021 Oct 10;9(10):2127.
doi: 10.3390/microorganisms9102127.

New Exopolysaccharides Produced by Bacillus licheniformis 24 Display Substrate-Dependent Content and Antioxidant Activity

Penka Petrova  1 , Alexander Arsov  1 , Ivan Ivanov  1 , Lidia Tsigoriyna  2 , Kaloyan Petrov  2
Affiliations

New Exopolysaccharides Produced by Bacillus licheniformis 24 Display Substrate-Dependent Content and Antioxidant Activity

Penka Petrova et al. Microorganisms. .

Abstract

Bacillus licheniformis is a soil bacterium with many industrial applications. In addition to enzymes, platform chemicals, antibiotics and phytohormones, the species produces exopolysaccharides (EPSs) of various biological activities. This study revealed that Bulgarian isolate B. licheniformis 24 produced EPSs consisting of galactose, glucose and mannose with substrate-dependent ratio. From glucose, B. licheniformis 24 secreted EPS1, consisting of 54% galactose, 39% glucose and 7% mannose. From fructose, the strain formed EPS2, containing 51% glucose, 30% mannose and 19% galactose. Batch cultivation in flasks yielded 2.2-2.6 g/L EPS1 and 1.90-2.11 g/L EPS2. Four to five times higher yields of EPS were obtained from both substrates during batch and fed-batch processes in a fermenter at 37.8 °C, pH 6.2 and aeration 3.68 vvm. The batch process with 200 g/L of starting substrates received 9.64 g/L EPS1 and 6.29 g/L EPS2, reaching maximum values at the 33rd and 24th h, respectively. Fed-batch fermentation resulted in the highest yields, 12.61 g/L EPS1 and 7.03 g/L EPS2. In all processes, EPSs were produced only in the exponential growth phase. Both EPSs exhibited antioxidant activity, but EPS2 was much more potent in this regard, reaching 811 μM Vitamin C Equivalent Antioxidant Capacity (versus 135 μM for EPS1). EPS1 displayed antibacterial activity against a non-O1 strain of Vibrio cholerae.

Keywords: Bacillus licheniformis; antibacterial activity; antioxidant activity; batch; exopolysaccharide; fed-batch.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Colonies of B. licheniformis 24 on Luria–Bertani (LB) agar medium.
Figure 2
Figure 2
Time profiles of EPS production by B. licheniformis 24 during fermentation in flasks. (a) EPS production from glucose or fructose with initial concentration 50 g/L; (b) EPS production from glucose or fructose with initial concentration 100 g/L. The mean values from three independent cultivations are presented.
Figure 3
Figure 3
HPLC profiles of monosaccharide content of acid-hydrolysed EPSs produced by B. licheniformis 24. The sugars were separated by Aminex HPX-87P column and detected by RI detector. (a) Sugar profile of EPS1 produced from glucose; (b) Sugar profile of EPS2 produced from fructose; (c) Referent sugar standards.
Figure 4
Figure 4
Time profiles of biomass formation and EPS production by B. licheniformis 24 during batch and fed-batch fermentation in fermenter Biostat® Aplus, working volume 1 L. (a) EPS1 production during batch fermentation of glucose; (b) EPS2 production during batch fermentation of fructose; (c) EPS1 production during fed-batch fermentation of glucose; (d) EPS2 production during fed-batch fermentation of fructose. The starting sugar concentrations were 200 g/L; sterile glucose or fructose was added during the fed-processes—125 mL glucose (700 g/L) and 210 mL fructose (870 g/L). The mean values from two independent batch and fed-batch processes are presented.
Figure 5
Figure 5
Antioxidant activity of EPS1 and EPS2 from B. licheniformis 24. (a) ABTS reduction measured in VCEAC (μM); (b) total antioxidant activity in eBQC values; (c) superoxide scavenging measured in % SOD activity inhibition. All measurements were performed in triplicates.
Figure 6
Figure 6
Antimicrobial activity of EPS from B. licheniformis 24 against Vibrio cholerae non-O1 strain. (a) EPS1, 10 mg/mL; (b) EPS1, 5 mg/mL; (c) EPS1, 1 mg/mL; (d) EPS2, 10 mg/mL; (e) EPS2, 5 mg/mL; (f) EPS2, 1 mg/mL.
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