Genomic analysis and potential polyhydroxybutyrate (PHB) production from Bacillus strains isolated from extreme environments in Mexico

Abstract

Background: Plastic pollution is a significant environmental problem caused by its high resistance to degradation. One potential solution is polyhydroxybutyrate (PHB), a microbial biodegradable polymer. Mexico has great uncovered microbial diversity with high potential for biotechnological applications. The best polymer producers tend to be isolated from environments that require survival adaptations from microorganisms, the high-producing Bacillus cereus strain saba.zh comes from refinery wastewater, the costs of production have been a limiting factor for biopolymer production, and one of the focuses of interest has been finding novel strains with better production or singular traits that help in industrial processes.

Results: The isolates were taxonomically classified as Bacillus cereus MSF4 and Bacillus inaquosorum MSD1 from Mina, Nuevo Leon; B. cereus S07C; and Paenibacillis dendritiformis from the active volcano "El Chichonal" on Chiapas. The strains had growth temperatures ranging from 35 to 50 °C and pH tolerance values ranging from 3 to 9. The best PHB-producing strain, B. cereus MSF4, produced 0.43 g/kg PHB on orange peels, followed by B. inaquosorum MSD1 at 0.40 g/kg, B. cereus S07C at 0.23 g/kg and P. dendritiformis at 0.26 g/kg.

Conclusions: The findings of this study affirm the potential of the Mexican isolated strains as PHB-producing organisms, enabling further studies to test their viability as industrial producers. The ability of P. dendritiformis and B. inaquosorum to synthetize PHB was also confirmed by the observations made providing novel evidence to consider these species as potential producers.

Keywords: Bacillus i naquosorum; Bacillus cereus; Paenibacillus dendritiformis; Dry cell weight; Genome; Phylogeny; Polyhydroxybutyrate (PHB); YteA.

Fig. 1

Nucleotide sequence of the PhaC gene. The aligned sequence used to design the forward primer is highlighted in blue, the sequence used for the reverse primer is highlighted in green, and the conserved sequence is highlighted in red

Fig. 2

Sudan Black colonial staining; (A) MSF4 strain with black streaks; (B) MSD1 strain with streak remnants over the whole Petri dish; (C) S07C strain with light-stained streaks; (D) S07D strain with heavily stained colonies washed out over the culture; and (E) negative control E. coli strain with white streaks

Fig. 3

PHB produced as a percentage of the cell dry weight (CDW) of the strains and total g/kg of PHB produced when orange peel was used as the substrate [10].

Fig. 4

Constructed synteny plot featuring two reference genomes, B. cereus FORC_047 (1) and B. cereus ATCC 14,579 (2), B. cereus S07C (3), and Bacillus cereus MSF4 (4), and two NCBI genomes, B. cereus J2 (5) and B. cereus 4 N (6)

Fig. 5

Constructed synteny: Featuring the reference genome of B. inaquosorum A65.1 (1), Bacillus inaquosorum MSD1 (2), B. inaquosorum KCTC13429 (3), B. inaquosorum lba001 (4) and B. inaquosorum S13 (5)

Fig. 6

Constructed synteny: Featuring the reference genomes of P. dendritiformis J27TS7 (1), Paenibacillus dendritiformis S07D (2) and P. dendritiformis 2022ck (3)

Fig. 7

Circular view of the genomes of (A) B. cereus strain MSF4, (B) B. inaquosorum strain MSD1, (C) B. cereus strain S07C and (D) P. dendritiformis S07D, with all the annotated genes and their functional clusters highlighted by color

Fig. 8

(A) B. cereus MSF4 PHB synthesis genes and comparison with other class IV PHB producers in the BV-BCR database. Strain MSF4 presented six PHB synthase genes related to butanoate metabolism: PHB synthase PhaC, repressor PhaR, Acetoacetyl-CoA reductase PhaB, Phasin protein PhaP, (R)-specific enoyl-CoA hydratase PhaJ, and repressor PhaQ, which are arranged in the same way as those in the cereus group. P. megaterium is part of a different subgroup of Class IV PHAs, but unlike Classes I, II and III, the figure shows all the genes of the PHB-producing cluster. (B) Maximum likelihood phylogenetic tree comparing the polyhydroxybutyrate synthase gene PhaC of each of the four classes. All Bacillus were grouped in class IV, Haloferax in group III, Pseudomonas in group II and Cupriavidus in group I

Fig. 9

Maximum likelihood phylogenetic tree (A) comparing the sporulation control gene YteA, which is highly conserved among endospore-forming bacteria. Individual species from the outgroups are paired with completely conserved sequences, while different species are separated from each other. (B) The second tree compares the 16 S ribosomal subunits of various Bacillus strains, with Clostridium as the outgroup. This marker is the standard reference for phylogenies, but for Bacillus, the distinction between species is very narrow and ends with an almost flat line on the branches

Fig. 10

PhaR metabolic interactions. PhaR regulates the polyhydroxybutyrate operon and is induced by interactions from the citric acid cycle. phbB represents acetoacetyl-CoA reductase, phaC represents poly-beta-hydroxybutyrate polymerase, odhA represents 2-oxoglutarate dehydrogenase, and phaP PhaP represents protein surface-bidding protein. odhA represents the most significant interaction outside the PhaC operon and is highlighted in blue inside the citric acid cycle Figure [26]