Salinity Stress Enhances the Antioxidant Capacity of Bacillus and Planococcus Species Isolated From Saline Lake Environment

Abstract

This study aims at exploiting salinity stress as an innovative, simple, and cheap method to enhance the production of antioxidant metabolites and enzymes from bacteria for potential application as functional additives to foods and pharmaceuticals. We investigated the physiological and biochemical responses of four bacterial isolates, which exhibited high tolerance to 20% NaCl (wt/vol), out of 27 bacterial strains isolated from Aushazia Lake, Qassim region, Saudi Arabia. The phylogenetic analysis of the 16S rRNA genes of these four isolates indicated that strains ST1 and ST2 belong to genus Bacillus, whereas strains ST3 and ST4 belong to genus Planococcus. Salinity stress differentially induced oxidative damage, where strains ST3 and ST4 showed increased lipid peroxidation, lipoxygenase, and xanthine oxidase levels. Consequently, high antioxidant contents were produced to control oxidative stress, particularly in ST3 and ST4. These two Planococcus strains showed increased glutathione cycle, phenols, flavonoids, antioxidant capacity, catalase, and/or superoxide dismutase (SOD). Interestingly, the production of glutathione by Planococcus strains was some thousand folds greater than by higher plants. On the other hand, the induction of antioxidants in ST1 and ST2 was restricted to phenols, flavonoids, peroxidase, glutaredoxin, and/or SOD. The hierarchical analysis also supported strain-specific responses. This is the first report that exploited salinity stress for promoting the production of antioxidants from bacterial isolates, which can be utilized as postbiotics for promising applications in foods and pharmaceuticals.

Keywords: Bacillus; Planococcus; antioxidants; salinity stress; stress markers.

FIGURE 1

Neighbor-joining phylogenetic tree showing the relationships between the salt-tolerant Bacillus strains from the present study (ST1 and ST2) and the closely related species. All ambiguous positions were removed for each sequence pair (pairwise deletion option). Escherichia coli ATCC 11775 was used as the out–group.

FIGURE 2

Neighbor-joining phylogenetic tree showing the relationships between the salt-tolerant Planococcus strains from the present study (ST3 and ST4) and the closely related species. All ambiguous positions were removed for each sequence pair (pairwise deletion option). Bacillus subtilis AB8 was used as the out–group.

FIGURE 3

The oxidative damage markers of four salt-tolerant bacterial isolates (ST1, ST2, ST3, and ST4) in terms of (A) lipid peroxidation (MDA), (B) lipoxygenase activity, and (C) xanthine oxidase activity under control and salinity stress conditions. Data are represented by the mean of at least three replicates, and error bars represent the standard error. Different small letters (a, b, c…) above bars indicate significant differences between means at p < 0.05.

FIGURE 4

The overall antioxidant capacity of four salt-tolerant bacterial isolates (ST1, ST2, ST3, and ST4) in terms of (A) FRAP, (B) DPPH %, and (C) superoxide scavenging (SOS) under control and salinity stress conditions. Data are represented by the mean of at least three replicates, and error bars represent the standard error. Different small letters (a, b, c…) above bars indicate significant differences between means at p < 0.05.

FIGURE 5

The antioxidant metabolites of four salt-tolerant bacterial isolates (ST1, ST2, ST3, and ST4) in terms of (A) flavonoids, (B) polyphenols, (C) reduced GSH, (D) oxidized GSH (GSSG), and (E) total GSH per gram of bacterial cell weight under control and salinity stress conditions. Data are represented by the mean of at least three replicates, and error bars represent the standard error. Different small letters (a, b, c…) above bars indicate significant differences between means at p < 0.05.

FIGURE 6

The antioxidant enzyme activities that directly scavenge ROS of four salt-tolerant bacterial isolates (ST1, ST2, ST3, and ST4) including (A) CAT, (B) SOD, (C) GPX, and (D) POX under control and salinity stress conditions. Data are represented by the mean of at least three replicates, and error bars represent the standard error. Different small letters (a, b, c…) above bars indicate significant differences between means at p < 0.05.

FIGURE 7

The antioxidant enzyme activities that have reducing activities to the antioxidant metabolites of four salt-tolerant bacterial isolates (ST1, ST2, ST3, and ST4) including (A) GR, (B) Grx, and (C) TRD, as well as the detoxification enzyme (D) GST activity under control and salinity stress conditions. Data are represented by the mean of at least three replicates, and error bars represent the standard error. Different small letters (a, b, c…) above bars indicate significant differences between means at p < 0.05.

FIGURE 8

Strain-specific responses of four salt-tolerant bacterial isolates (ST1, ST2, ST3, and ST4) to the stress induced by high salt concentration at both physiological and biochemical levels shown by the hierarchical graph. The measured parameters represented by damage markers, overall antioxidant capacity, antioxidant metabolites, and antioxidant enzymes are grouped into three main clusters based on their response to high salt stress.