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.

Background

One of the most important industries on the planet is the production of petrochemical-based plastics. This material has changed the outlook of modern life, as it is an inexpensive material. The demand is projected to reach 840 million tons by 2025 [1]. This represents a considerable environmental challenge, as synthetic plastics are highly resistant to biological and environmental degradation and can accumulate in the environment for hundreds of years [2].

Polyhydroxyalkanoate (PHA) polyesters are a group of biologically produced polymers composed of hydroxylated fatty acid monomer chains ranging from 300 to 35,000. Each monomer has an R chain that is usually an alkyl, with a melting temperature of up to 175 °C and a degradation time in the range of several weeks; thus, biodegradable thermoplastics represent a promising alternative to traditional plastics [3]. The most common compound of the PHA group is polyhydroxybutyric acid (PHB), a biopolymer produced by bacteria in response to excess carbon sources and limited nitrogen and minerals. It protects cells against stress conditions and can be considered a cell survival mechanism. PhaC synthase is the key enzyme in polyhydroxyalkanoate (PHA) production. The synthases can be classified into four classes. Class I consists of a single subunit (PhaC) that mainly produces short-chain-length monomers and was first discovered in Ralstonia. Class II also consists of a single PhaC subunit but mainly synthetizes medium-chain-length monomers found in Pseudomonas aeruginosa. Class III consists of two subunits, PhaC and PhaE, which are found in Allochromatium vinosum. Class IV consists of the subunits PhaC and PhaR, the former being the catalytic subunit and the latter being theorized as responsible for being a regulatory subunit; however, its exact function remains unknown [4], and it is found in Bacillus [5], with the highest dry cell weight (DCW)% of PHB, 91.48%, reported in B. cereus NDRMN001 [6]. Identifying novel PHB-producing strains is an essential part of efforts to develop more cost-effective production protocols. Bacteria isolated from extreme environments often have adaptable metabolisms robust enough to survive bioreactor conditions with a reduced risk of viability loss. Comparative genomics between these environmental isolates and established PHB-producing strains represents a valuable tool for identifying new potential PHB producers. Comparative genomics between isolated environmental strains and well-known PHB-producing strains is a valuable tool for identifying potential producers of this polymer.

The PHB metabolic production pathway is activated as a survival mechanism and is composed of three enzymes: β-keto thiolase, encoded by the phaA gene, which catalyzes two molecules of acetyl-CoA into one molecule of acetoacetyl-CoA; NADPH-dependent acetoacetyl-CoA dehydrogenase, encoded by the phaB gene; and P(3HB) synthase, encoded by the phaC gene, which is the specific enzyme for this pathway that polymerizes (R)-3-hydroxybutyryl-CoA monomers [7, 8]. Class IV PHB synthesis genes in Bacillus can be divided into two subgroups. The megaterium cluster consists of two operons, phaR-phaB-phaC and phaP-phaQ, in opposite directions. The cereus group also has two operons in opposite directions, along with the phaJ gene as part of the phaP-phaQ-phaJ operon. PhaJ is a gene encoding R-specific enoyl-CoA hydratase, which is proposed to contribute to the monomer supply from β-oxidation. Additionally, cereus synthase produces a broader range of monomers and has wider substrate specificity than B. megaterium does [9].

Mexico ranks fifth among megadiverse countries and has many ecosystems where microorganisms of biotechnological interest can be isolated, such as deserts, jungles, crop fields, and refineries, which provide the opportunity to be used for industrial processes where resistance to alkalinity or acidity, high temperatures, and low nutritional requirements, among other harsh conditions, are common [10]. Mina, Nuevo Leon, is a desert area in the northern part of Mexico. El Chichonal, an active volcano with a central lake that can reach boiling temperatures, is situated in the southern state of Chiapas. This zone provides a new area for potential novel strain, as volcanic areas are not widely reported isolation areas for PHB producers despite the harsh conditions present that provide a good potential combination of factors for a robust organism that is resilient to bioreactor conditions, which combines tolerance for poorly available nutrients, higher-than-average temperatures, wide pH variations and exposure to high concentrations of volcanic gases. Mexico is also a major agricultural producer that provides inexpensive potential substrates for the production of PHB, including rapeseed cake, wheat bran, and orange peel, which have been tested and confirmed as PHB inducers [11].

The present study aims to isolate and characterize Bacillus strains obtained from dry soil in Mina, Nuevo Leon, and El Chichonal, Chiapas, to identify and characterize the ability of these bacterial strains to produce PHB with a focus on comparative genomics which may reveal genetic adaptations that support industrial viability. This goes beyond the typical focus on production levels and provides a deeper understanding of traits that enhance resilience in bioreactor conditions. This study also examines PHB production on orange peel, a potential solid substrate with agro-industrial origins seeking an approach that reduces production costs and supports sustainable practices by repurposing waste materials.

Results

Morphological and biochemical identification of selected strains

The gram-positive motile rods were identified in cultures in which the B. anthracis colonies were sterilized. The isolates observed via microscopy shared the morphological and biochemical characteristics of Bacillus species described in Bergey’s Manual [12], with four outliers that were tolerant to temperatures up to 50 °C and alkaline pH 9. The four strains coined “MSD1” and “MSF4” were isolated from desert soil from Mina, Nuevo Leon, and “S07D” and “S07C”, a consortium isolated from a microbial mat obtained from a volcanic lake from El Chichonal. Specifically, the consortium from the volcano along with the Mina soil isolate “MSD1” showed a temperature tolerance of up to 50 °C and the ability to be reactivated after being exposed to 60 °C, whereas both volcanic strains along with the Mina strain “MSF4” also managed to grow at pH 9, from which S07D retained high stress tolerance when separated. Four of the 17 bacteria isolated were selected to test their PHB-producing ability.

PCR identification

For the chosen target for PCR identification, we used the conserved domain of the PhaC gene to identify possible PHB-producing strains. PhaC gene sequences were downloaded from the NCBI nucleotide database for primer design [13], and alignment allowed us to visualize the high-conservation region among all the aligned strains (Fig. 1). The four selected strains tested positive.

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

Sudan black test for PHB production

PHB production was verified by Sudan black staining, this tinction technique can also indicate the production of polyhydroxyvalerate (PHV), lipids, fatty acids and proteins, which is why previous screening with PCR was necessary. The predicted productive strains displayed dark coloration, with producer strains displaying stark black coloration, in contrast to the colorless streak of E. coli used as a negative control. All the tested strains were PB producers. (Fig. 2).

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

PHB production data

PHB production variability between the strains was tested after 48 h [14] of growing on 17 g of orange peel as a substrate to induce PHB production for 48 h. Chloroform extraction resulted in maximum PHB production per gram of orange on MSF4 at 0.43 g/kg, whereas the highest percentage of PHB per dry cell weight was observed for strain S07C at 7.9% (Fig. 3).

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].

Genome comparison.

The sequenced genomes were preliminarily identified via the BLAST alignment tool and aligned with reference assemblies from NCBI, B. cereus FORC_047 and ATCC 14,579 for the MSF4 and S07C strains, and P. dendritiformis J27TS7 for S07D and B. inaquosorum A65.1 were aligned with MSD1. The contigs aligned with the Mauve tool confirmed high similarity between the B. cereus genomes isolated from Nuevo Leon and the reference genomes, with strain 4 N [15] being the exception, as shown in Fig. 4. The rearrangements for B. inaquosorum A65.1, strain MSD1 and strain KCTC 13,429 presented high synteny. (Fig. 5)P. dendritiformis presented high similarity with the strain 2022ck, whereas J27TS7 presented differences in the arrangement of the genome. (Fig. 6)

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)

Genome annotation identified the Mina strain “MSF4” as Bacillus cereus and the “MSD1” strain as Bacillus inaquosorum, the “El Chichonal” strain “S07C” was identified as B. cereus, and the strain “S07D” was identified as Paenibacillus dendritiformis. “MSF4” had a genome size of 5096 kb, with a GC content of 35%. Similarly, S07C had the same GC% but a smaller genome at 4990 kb, both of which were found to contain genes essential for butanoate metabolism, including PhaC, which aligns with the cereus group within the Bacillus genus. S07D has the highest GC% at 53% and the largest genome size at 6956 kb. MSD1 displayed a smaller genome of 4272 kb, with a GC content of 43%, indicating a closer relationship to the subtilis group. The isolated strains were compared with strains obtained from NCBI that had the highest average nucleotide identity (ANI) as a comparison point. (Table 1 ). The four genomes were assembled and annotated (Fig. 7), and the information included specialized genes found in the databases of their respective organisms, the genome of B. cereus MSF4 and B. inaquosorum MSD1  was compared to other PHB producers reported on literature (Table 2); of particular interest, the full PHB synthesis machinery of B. cereus was also observed and compared with that of other PHB-producing Bacillus spp. (Fig. 8).

Table 1 Genome features of isolated strains compared with the reference genomes

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

Species phylogenetic identification

Phylogenetic analysis of the 16 S, PhaC and YteA genes revealed clear distinctions between our strains and established Bacillus species, corroborating the results of the biochemical and genetic identification. PhaC alignment was performed utilizing representative species for each of the four classes of PHB synthase groups and the resulting maximum likelihood tree with the four classes in separated branches, confirming the place of our sequenced genomes among Class IV Bacillus polyhydroxybutyrate producers (Fig. 8). YteA was utilized as a genetic marker because it is a specific gene for endospore-forming bacteria with high conservation [16]. The resulting tree satisfactorily separated different Bacillus species and grouped into members of the same genus, with branches divided according to the Bacillus and Clostridium genera, and the former further divided into the cereus group and the subtilis group, with two additional separated branches for Priestella megaterium and Paenibacillus spp. “MSF4” and “S07C” are closely related to B. cereus, “MSD1” pairs on the B. inaquosorum branch, a bacterium formerly classified as B. subtilis subspecies, and “S07D” pairs with P. dendritiformis; as a reference and point of comparison, a 16 S tree was generated in the same way, revealing problems in distinguishing among the groups of bacteria in the cereus group. (Fig. 9)

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

Table 2 Genome characteristics of 11 Bacillus spp. and related strains

The metabolic pathways identified in the sequenced genome included the complete machinery for the synthesis of polyhydroxybutyrate. B. cereus MSF4, S07C and ATCC14579 were compared. The PhaR repressor regulates this activity, and its strongest interaction outside the PHB genes is with the odhA part of the oxoglutarate dehydrogenase complex, whose activity is highly correlated with citric acid cycle activity [25]. Without experimental data, the genomic information used to predict the interactions between PHB metabolism genes includes the PHB-granule surface-bidding protein PhaP (Fig. 10).

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]

Discussion

Bacillus species are known for their adaptability in extreme environments, especially in conditions with multiple stress factors, due to their ability to produce endospores. Endospore production has a high energy cost; under mesophilic conditions with a single stress factor, the percentage of Firmicutes present in the environment, including Bacillus, is relatively low. In terms of the total population, places with multiple stress conditions provide the highest proportion of spore-forming bacteria [27]. The strains from Mina B. cereus MSF4 and the Chichonal strains B. cereus S07C and P. dendritiformis S07D can be useful because of their unusually high alkaline tolerance, indicating broader environmental adaptability than typically observed within the pH range of 6–8 for Bacillus species. This makes the tested strains potentially better suited for bioreactor conditions, reducing the risk of viability loss [12]. Bacillus inaquosorum in particular also offers particular advantages as a PHB producer compared to previously reported bacteria. One notable strength of tis species first isolated from the Mojave Desert is its capacity to tolerate desiccation [28]. This trait is particularly valuable in solid-state fermentation systems, which are low-cost and require minimal water usage compared to traditional liquid fermentation. Solid-state fermentation systems, which utilize agro-industrial residues as substrates, can significantly reduce production costs, and the ability of B. inaquosorum to thrive in these conditions makes it well-suited for this type of production.

Compared with our other strain, P. dendritiformis S07 D, which had an exponential phase at the seventh growing hour, followed by a short stationary phase before declining around to hour 22, reports from Paenibacillus describe it as a species with quick growth times [29], which is consistent with what we observed in this work; quick growth times and high stress tolerance are two desirable factors for a metabolite producer.

The PHB synthesis process is traditionally attributed to carbon source mobilization and storage for further nutritional needs. Other physiological cell functions of PHB provide protection mechanisms against protein aggregation, reactive oxygen free radicals, and heat shock [30], as corroborated by the observed bacteria, as all the PHB-producing strains tested in this work were isolated from hostile environments where resistance to abiotic stress is needed, particularly heat shock resistance, as both the desert and the volcano were exposed to high temperatures. The strains isolated from Mina Nuevo León generally grew at the upper end of the temperature range of Bacillus spp. and were able to grow at 45 °C.

The strains B. cereus S07C and P. dendritiformis S07 D isolated from “El Chichonal” along with the B. cereus strain MSF4 from Mina had higher tolerances to alkaline conditions than did most of its members, which are already known for their high adaptability to a wide array of conditions [30]. In addition, the ecological distribution of Bacillus is strongly determined by ecological adaptations, in addition to other adaptations, including heat shock, osmotic shock by salts and different ranges of acidic and alkaline pH resistance [31], enabling the discovery of the environmental adaptations of P. dendritiformis S07D. The metabolic adaptability of these strains under stress conditions such as limited nutrients and variable temperatures indicates a robust physiological profile that may enhance PHB yield in non-optimal environments. This robustness not only lowers maintenance requirements but also potentially increases production efficiency by reducing the need for strictly controlled conditions. As result, these organisms could be a promising candidates for industrial-scale PHB production, particularly in economically viable setups where resilience and adaptability are key factors. Strain S07C of B. cereus in contrast had a slower growth rate under most conditions, suggesting that it was particularly adaptable to its microbial mat community, which opens the possibility of using both bacteria as consortium instead of pure cultures.

Among the seventeen isolated strains, seven tested positive for PhaC via PCR (41%) with the designed oligonucleotides, and a statistical report by Hassan et al. [7] revealed approximately 74 producers among 153 isolated bacteria (48%), indicating that approximately half of the wild-type bacteria can be PHB producers among cultivable organisms of the Bacillus genus. Hassan author along with Singh, et al. [5] reported that B. subtilis and B. megaterium are PHB producers; however, the NCBI database reports only the cereus group sequence of PhaC, although the functional region, in theory, should be preserved to maintain protein functionality. Paenibacillus dendritiformis is a bacterial species that has not been previously characterized as a potential PHB producer until this research; to our knowledge, one study reported it to be PhaC positive, but no further evaluations were made [32].

PHB production ability was detected in the strains from the two sampling locations, as confirmed by Sudan black staining on minimal medium agar when the strains were growing under stressful conditions (Fig. 2). The variation in the amount of PHB production between strains was significant even among members of the same species. The PHB production values obtained in this study were relatively high in the tested strains grown on orange peels, with 0.43 g/kg of PHB at the highest value for B. cereus MSF4 isolated from desert soil (Fig. 3), compared with the reported value of 0.025 g/L of strain OK2 isolated from commercial fermented soybean natto. According to the reports of Sukan et al. 2014, B. subtilis OK2, even when nitrogen starvation on the medium is a well-documented influencing factor on the PHB synthesis pathway, a certain amount of nitrogen is needed, pure orange peel is low in nitrogen, making it relatively unoptimized. The two strains of the same species had different outputs despite the same substrate being utilized. Most studies highlight producer strains grown on liquid media, including Bacillus subtilis NG220, which was isolated from sugarcane fields and was observed to accumulate 51.8% (w/w) of PHB via sugar industry wastewater supplemented with maltose and ammonium sulfate; Bacillus cereus SE-1, with an accumulation of 40% (w/w); and Bacillus sp. CS-605, with an accumulation of 33% (w/w) collected from a garbage dumping site and a lake, both of which were grown on minimal media supplemented with 1% dextrose [33]. and B. cereus NDRMN001, which were isolated from the soil of a polluted lake, with a reported PHB% production of 91.48% (w/w) when grown on selective media [6]. While those represent a higher PHB production compared to our organisms liquid substrates tend to be more expensive than solid-state fermentation alternatives because of the greater volume and water needed for submerged bioreactors [34], other potential advantages of using agro-industrial subproducts include improved cytocompatibility and biodegradability [35]. This finding also reinforces the great variability observed in the amount of PHB produced between strains. The strains isolated from environments with adverse conditions tended to have higher PHB production, with MSF4 from deserts being a better producer than OK2 and the highest overall producer coming from contaminated soil; in all cases, soil-isolated bacteria were observed to outproduce their liquid-born environment counterparts across the board. Stress sources rarely occur during isolation, heat often results in water scarcity, and the organisms that adapt to parallel or sequential threats are naturally selected to survive [36], as PHB confers protection to multiple stressors; at the same time, it should be overproduced in locations that warrant it as a survival mechanism. Protection from ultraviolet light, in particular, can be a determining factor for why isolated soil strains tend to be better producers than isolated liquid strains and are exposed to more direct sunlight.

Genome analysis from the DNA extracted from the Mina, Nuevo León, MSF4 strain, showed an average nucleotide identity of 99% to the B. cereus strain J2, identifying the species of MSF4 strain as B. cereus, likewise the S07C strain, isolated from the volcano had an ANI of 97% with B. cereus strain ATCC 14,579, other identified species include MSD1 having an ANI of 98.97 with B. inaquosorum A65 and S07D with an ANI of 98% with P. dendrtitiformis 2022CK-00834, all formed appropriately similar synteny plots (Figs. 4, 5 and 6) The presence of single copies of PhaC, PhaB, and the repressor PhaR downstream was confirmed in the genome of B. cereus strains, the group of genes phaQ, phaP, and phaJ were also found upstream forming two operons on both B. cereus MSF4 and B. cereus S07C(Figs. 7 and 8), these genes on that same configuration are present in on most Bacillus cereus sensu lato species like B. mycoides, Bacillus thuringiensis, and Bacillus anthracis with the same configuration, while Priestella megaterium another species know to have PHB producing strains like NBRC15308, lacks the phaJ gene [9].

The phylogenetic tree generated via the sporulation control gene YteA (Fig. 9), which is a highly conserved gene in sporulation bacteria [16], generated well-defined branches for the cereus, priestella, subtilis and inaquosorum branches. PhaC additionally divides all the species into subgroups representing the PhaC class of the PhaC operon [37]. The gram-positive endospore species types of PhaC are Class IV for Bacillus, Class III for Haloferax, Class II for Pseudomonas and Class I for the golden standard of industrial production Ralstonia, which can be highly variable between strains of the same species. Despite being the usual way to perform phylogenetic identification, as attested by multiple authors cited in this paper, the 16 s marker proved to be ineffective in identifying individual species from the cereus group in this work owing to the extreme conservation levels between them resulting in a branch without observable separation between the different species under it, which has been noted by some authors, such as Okinaka in 2016, who reported that the group Bacillus cereus sensu lato, which includes B. anthracis, B. thuringiensis and other less known members such as B. mycoides, initially had enough similarity on the basis of early molecular approaches in which a taxonomic reclassification was discussed [38]. In contrast to the B. cereus group, the MSD1 strains of B. inaquosorum and the S07D strain of P. dendritiformis reached more than 98% ANI with respect to their respective reference strains and were separated into their respective branches on both the YteA and 16 S trees.

PHB operon comparison revealed a close relationship between PhaR and the citric acid cycle, and no direct interaction with nitrogen metabolism was found, with no direct utilization of nitrogen found in the function of the immediate PHA cluster genes. The effect of nitrogen was previously reported by Kumar and collaborators in 2009 [39], with this genomic study observing the interconnected pathway in silico and its interactions involved in the process reinforcing the hypothesis about the relationship between route activation and nitrogen starvation, which suggests an indirect mechanism regulated outside the studied operon (Fig. 10).

Conclusions

Four fully characterized PHB-producing strains were isolated, and PCR and genome sequencing confirmed that they encoded enzymes for PHB production. The strains were taxonomically identified by biochemical and molecular protocols as Bacillus cereus (MSF4 and S07C), Bacillus inaquosorum (MSD1) or Paenibacillus dendritiformis (S07D). The specifically designed oligonucleotides proved to be useful as a rapid test to detect possible novel PHB-producing strains. B. inaquosorum and P. dendritiformis may be of particular future interest, as these species are rarely reported in PHB production studies utilizing data collected from the annotated genome and proteome metabolic pathways. All the selected strains were PHB producers with the capacity to utilize the agro-industrial waste of orange as a substrate. Some limitations and opportunities for future research into our isolated strains include optimizing the culture media to increase PHB production, the utilization of different PHB extraction methods utilizing more ecologically friendly solvents along with measures of both the productivity and purity of the extracted polymer, and industrial scaling tests at the laboratory bioreactor level if the developed protocol and productivity show promise.

Materials and methods

Isolation from collected samples

Samples were collected from extremophilic environments found in desert soil from Mina and volcanic environmental samples from El Chichonal. The samples were diluted in 10 ml aliquots to achieve ^10–4 dilutions. The 10^− 3 and 10^− 4 dilutions were subsequently spread on blood agar media and incubated at 37 °C for 24 h. Bacteria that did not undergo hemolysis were subjected to a motility test on semisolid Dibico Luria Bertani (LB) agar plates at 37 °C to screen for possible Bacillus anthracis strains to discard samples with this hazardous bacterium. B. anthracis samples were identified by isolating hemolytic and/or motile bacteria via blood agar plates and semisolid agar cultures, respectively, which are characterized by a combination of a lack of hemolysis and a lack of motility. All the obtained isolates were incubated and observed under a microscope at 100x with Gram staining to ensure the isolation of gram-positive bacilli. Standard biochemical tests for bacterial identification, including acid production with different carbon sources, temperature ranges, pH tolerances and gelatin hydrolysis, have also been applied as basic identification methods [12].

Selection of PHB-producing strains

PHB production was assessed in triplicate via PHB-inducing media consisting of 2.5 g/L yeast extract, 2 g/L peptone, 1.25 g/L NaCl and 15 g/L glucose, which act as low-nutrient media with an abundance of carbon sources, as reported by Martinez-Herrera et al. [37]. Sudan Black B staining was used to differentiate PHB-producing colonies grown on agar plates [40].

PHB production

For PHB production from agro-industrial material, four sets of 17 g of orange peel pretreated with distilled water at 70 °C were shaken at 100 rpm for 2 h as a solid substrate and then autoclaved at 121 °C for 20 min. The substrate was inoculated with an equivalent of 30% volume/mass of liquid LB medium from each strain and incubated at 37 °C for 72 h [10]. The resulting biomass was collected, centrifuged and subsequently dried at 80 °C overnight, and the dried cells were used for PHB extraction.

PHB extraction

The harvested dry cells were treated with chloroform according to the extraction method. Chloroform was added to the dried cell biomass obtained from the previously centrifuged cultures, incubated at 70 °C for 10 min, and then centrifuged at 5000× g for 10 min. The resulting solution was separated and collected, and the chloroform was allowed to evaporate [39]. The PHB yield was calculated via the following equation: PHB (%) = dry PHB weight (g/L) × 100/dry cell weight (g/L).

DNA extraction

The strains were cultured in LB media and then processed for DNA extraction. Biomass was suspended in Tris-EDTA (TE) buffer, treated with proteinase K, and subjected to phenol/chloroform extraction. The DNA was then precipitated with ethanol and ammonium acetate, washed, and resuspended in TE buffer for quality assessment via a NanoDrop spectrophotometer [41].

PhaC primer design and PCR gene detection

PhaC gene sequences were sourced from the NCBI nucleotide database for primer design [13]. Oligonucleotides were designed on the basis of consensus sequences identified via alignment tools, including BioEdit, the NEB Tm calculator, and the NCBI Primer Design Tool [42]. PCR amplification and agarose gel electrophoresis were employed to detect gene amplification, with specific markers used for comparison [31]. DNA from PhaC-positive strains was sent for sequencing: isolates from Mina were sent to the Laboratory Cinvestav Irapuato, and Chichonal isolates were sent to Cinvestav Merida. The sequencing was performed via a MiSeq Illumina sequencer [43].

Assembly and annotation of selected genomes

Genome sequence quality was evaluated via the FastQC bioinformatics tool on the Galaxy platform [44], the assembly was performed via the SPAdes and Unicycler algorithms provided by the BV-BRC platform [45], and the annotation was performed via the RASTtk program provided on the same platform [46]. Contigs were compared against NCBI database sequences to identify similarities and differences.

Phylogenetic tree generation

The assembled genomes were analyzed alongside sequences from the NCBI and BV-BRC databases and subsequently aligned via the maximum likelihood algorithm in the bioinformatics software MEGA1.1 [47] Three genetic markers, the YteA sporulation control gene, the PHB production gene PhaC, and the 16 S RNA marker, were used to construct the phylogenetic trees. This analysis facilitated the identification of species relationships and evolutionary lineages.

Metabolic pathway description

Genome mining was employed to elucidate pathways influencing PhaC metabolism, with a focus on butanoate metabolism and related carbon. Stress response pathways were identified via databases and tools such as the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) [48].