Biopolymer production by halotolerant bacteria isolated from Caatinga biome

Abstract

Saline environments are extreme habitats with a high diversity of microorganisms source of a myriad of biomolecules. These microorganisms are assigned as extremophiles recognized to be producers of new natural compounds, which can be synthesized by helping to survive under harshness and extreme conditions. In Brazil, in the saline and semi-arid region of Areia Branca (Caatinga biome), halotolerant bacteria (able to growth at high NaCl concentrations) were isolated from rhizosphere of native plants Blutaparon portulacoides and Spergularia sp. and their biopolymer production was studied. A total of 25 bacterial isolates were identified at genus level based on 16S rRNA gene sequence analysis. Isolates were mainly Gram-positive bacteria from Bacillaceae, Staphylococcaceae, Microbacteriaceae, and Bacillales XII incertae sedis families, affiliates to Bacillus, Staphylococcus, Curtobacterium, and Exiguobacterium genera, respectively. One of the Gram-negative isolates was identified as member of the Pseudomonadaceae family, genus Pseudomonas. All the identified strains were halotolerant bacteria with optimum growth at 0.6-2.0 M salt concentrations. Assays for biopolymer production showed that the halotolerant strains are a rich source of compounds as polyhydroxyalkanoates (PHA), biodegradable biopolymer, such as poly(3-hydroxybutyrate) (PHB) produced from low-cost substrates, and exopolysaccharides (EPS), such as hyaluronic acid (HA), metabolite of great interest to the cosmetic and pharmaceutical industry. Also, eight bacterial EPS extracts showed immunostimulatory activity, promising results that can be used in biomedical applications. Overall, our findings demonstrate that these biomolecules can be produced in culture medium with 0.6-2.0 M NaCl concentrations, relevant feature to avoid costly production processes. This is the first report of biopolymer-producing bacteria from a saline region of Caatinga biome that showed important biological activities.

Keywords: Biopolymers; Caatinga biome; Exopolysaccharides (EPS); Halotolerant microorganisms; Hyaluronic acid (HA); Immunostimulatory activity; Polyhydroxyalkanoates (PHA).

Fig. 1

Phylogenetic tree based on 16S rRNA gene sequences of halotolerant bacterial isolates from the rhizosphere of Spergularia sp. (a), and B. portulacoides (b). Sequence accession numbers are given in brackets. The tree was constructed using neighbor-joining method. Blastopirellula marina was used as an outgroup

Fig. 2

Distribution of the relative abundance of the bacterial genera isolated from the rhizosphere of Spergularia sp. and B. portulacoides

Fig. 3

NaCl tolerance profile of halotolerant bacterial isolates from the rhizosphere of Spergularia sp. and B. portulacoides

Fig. 4

Accumulation profile of PHA [P(3HB)] by isolates Sac33 and Sac34 in MSM with NaCl and supplement with glucose, xylose and glycerol. B. filamentosus Sac33 (a) and B. filamentosus Sac34 (b). Average and standard deviation of triplicate cultivation after 72 h

Fig. 5

Evaluation of immunostimulatory activity by the Griess test. The figure shows the nitrite levels in the supernatant of murine macrophages (RAW264.7). Cells were exposed to sterile saline (negative control), 100 ng/mL lipopolysaccharide from Escherichia coli (LPS) (positive control), and the Caatinga bacterial exopolysaccharides (Sac) after 48 hours of incubation. The obtained absorbance values were transformed into nitrite concentration by interpolation and then evaluated by ANOVA followed by Tukey's post-test comparing all the samples. a = p <0,05 when compared with C-; and b = p <0,05 when compared with LPS. a) Assays carried out with strains belonged to B. paralicheniformis (Sac1, Sac7, Sac9 and Sac16) , b) B. paralicheniformis (Sac18 and Sac22) and B. haynesii (Sac45), c) B. paralicheniformis (Sac51 and Sac64) and C. oceanosedimentum (Sac54)

Fig. 6

Relative concentration of hyaluronic acid (%) obtained from halotolerant strains. Positive control (dashed line-100%), Streptococcus equi subsp. zooepidemicus ATCC 39920. Average and standard deviation of triplicate cultivation after 7 days