The economics of polyhydroxyalkanoate production have long been the central obstacle to commercialization, and the choice of carbon substrate accounts for a substantial share of total fermentation cost. Sugar beet molasses, a viscous byproduct of beet sugar refining, is an attractive feedstock because it is already concentrated in fermentable sugars, requires little additional processing before use, and is produced in large quantities in sugar-processing regions at low cost. Unlike purified glucose or sucrose, molasses carries residual minerals, vitamins, and nitrogenous compounds that can support microbial growth, which reduces the need for expensive supplemental nutrients. The Egyptian research team’s decision to screen twenty-nine local bacterial isolates against this substrate reflects a practical screening philosophy: rather than engineering a model organism to tolerate cheap feedstocks, they searched for organisms already adapted to them. This approach has precedent in the PHA literature, where Bacillus-lineage bacteria isolated from soil, rhizosphere, and industrial environments have repeatedly shown tolerance to crude sugar streams and the ability to accumulate significant polymer reserves under nutrient-imbalanced conditions.

The taxonomic identity of the winning isolate deserves attention. The strain SNS1 was assigned to Priestia megaterium, a member of the large-celled Bacillus group that was reclassified into the genus Priestia following the genome-based reorganization of the family Bacillaceae. Prior to this reclassification, many of these organisms appeared in older literature as Bacillus megaterium, and a considerable body of PHA research was conducted on strains now understood to belong to this broader clade. Priestia megaterium has a long history as a biotechnological workhorse, valued for its large cell size, plasmid compatibility, and capacity to accumulate intracellular storage granules. Its ability to convert molasses sugars into poly(3-hydroxybutyrate) aligns with the metabolic profile of the group: these bacteria deploy the canonical PHA biosynthesis pathway, in which two acetyl-CoA molecules are condensed to acetoacetyl-CoA and then reduced to the hydroxybutyrate monomer that is polymerized into granules visible under the microscope. The accumulation of these granules serves as a carbon and energy sink when nitrogen or phosphorus becomes limiting, which is precisely the physiological state that fermentation engineers aim to induce.

The optimization strategy reported in the study illustrates why statistical experimental design has become standard practice in bioprocess development. Traditional one-factor-at-a-time experiments examine each variable in isolation, but fermentation outputs such as PHA yield emerge from interactions among carbon concentration, nitrogen availability, pH, temperature, agitation, and cultivation time. The Plackett-Burman design, a fractional factorial screening method, allows many variables to be tested simultaneously with a minimal number of runs, identifying which factors exert statistically significant influence on the response. In this case, the design converged on a specific combination: six percent sugar beet molasses as the carbon source, ammonium oxalate at half a gram per liter as the nitrogen source, an alkaline pH of 8.5, incubation at 37 degrees Celsius, agitation at 200 revolutions per minute, and a 24-hour cultivation period. The resulting polymer concentration of 4.79 grams per liter is notable for the short cultivation window. Many published PHA fermentations require 48 to 72 hours to reach comparable titers, so a productive 24-hour process reduces energy consumption per batch and increases the theoretical throughput of existing equipment.

The choice of ammonium oxalate as the nitrogen source is an interesting detail. Nitrogen limitation is the classical trigger for PHA accumulation, because when nitrogen becomes scarce, protein synthesis and cell division slow down while excess carbon is diverted into storage polymers. However, the identity of the nitrogen source matters: some nitrogen salts alter medium pH during consumption, and oxalate salts are relatively inexpensive and benign compared with more commonly used ammonium sulfate or ammonium chloride. The alkaline setpoint of pH 8.5 is also worth noting, since many PHA-producing bacteria prefer neutral to slightly alkaline conditions, and pH control at the upper end of the tolerance range can suppress contaminating organisms in non-sterile or minimally sterilized processes, an advantage when the goal is low-cost production.

Chemical confirmation of the polymer’s identity relied on a complementary suite of analytical techniques, each providing independent evidence. Fourier-transform infrared spectroscopy detects the characteristic ester carbonyl absorption near 1720 wavenumbers and the C-O-C stretching bands that define polyester backbones. Proton and carbon-13 nuclear magnetic resonance spectroscopy resolve the specific chemical environments of the methyl, methylene, and methine protons and carbons of the 3-hydroxybutyrate repeat unit, distinguishing P3HB from copolymers containing longer-chain hydroxyalkanoate monomers. Gas chromatography-mass spectrometry, typically performed on the methanolysis products of the polymer, quantifies the monomer composition with high specificity. When all four techniques agree, as they did here, the conclusion that the molasses-grown polymer is the homopolymer P3HB is well supported. This matters commercially because homopolymer P3HB has different material properties from the copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate): P3HB is stiffer and more brittle, with a higher melting temperature, while copolymers are more flexible and easier to process by melt techniques such as extrusion and injection molding.

The thermal analyses reported in the study reinforce the structural assignment. Differential scanning calorimetry measures the melting temperature and the heat of fusion, values that for P3HB fall in a well-characterized range near 175 degrees Celsius for the melting peak, and the observed thermal transitions consistent with pure P3HB indicate that the molasses substrate did not introduce unusual monomer impurities into the polymer chain. Thermogravimetric analysis, which tracks mass loss as the sample is heated, provides information on thermal stability and degradation onset, parameters that determine whether a polymer can survive melt processing without significant chain scission. Together, these measurements give processors a first indication of how the material would behave in industrial equipment.

Perhaps the most unexpected finding is the ultraviolet-shielding behavior of the purified polymer sheet. The researchers measured an ultraviolet-shielding factor of 15.23, corresponding to roughly 35 percent absorption of incident ultraviolet light. This property is not commonly reported for neat P3HB films, and its origin may lie in the polymer’s aromatic-free but carbonyl-rich structure, which absorbs weakly in the UV-B and UV-C regions, or in residual chromophores carried over from the molasses substrate or introduced during extraction. The authors frame this as a potential sun-protective application, drawing on the established spectrophotometric methods used to determine sun protection factors in cosmetic science. While a shielding factor of 15 is modest compared with formulated sunscreens, which routinely achieve factors of 30 or higher through combinations of organic and inorganic filters, the finding suggests that PHA films or coatings could contribute ultraviolet attenuation in packaging, agricultural films, or composite materials without added synthetic UV absorbers. The reference to ocular ultraviolet protection in the cited literature hints that eyewear or lens coatings might also be contemplated, though such applications would require extensive additional validation.

Placed in a broader context, the study contributes to a growing body of work demonstrating that Priestia and related Bacillaceae can produce PHA from agricultural and food-industry residues. Previous reports from overlapping research groups have documented PHA production from sugarcane molasses, cheese whey, glycerol, and vinasse, each substrate carrying its own sugar profile and inhibitor load. Sugar beet molasses differs from cane molasses in having a higher sucrose content and lower levels of certain phenolic inhibitors, which may explain why beet molasses supports efficient growth with minimal pretreatment. The economic logic is compelling: molasses is often priced at a fraction of the cost of refined sugars, and diverting it to bioplastic production avoids competition with food uses while giving sugar mills an additional revenue stream for a byproduct that might otherwise be sold at low value as animal feed.

Several challenges remain before such a process could scale. Recovery of PHA from bacterial cells typically requires cell disruption and solvent extraction, both of which carry cost and environmental burdens unless greener solvents or enzymatic lysis methods are employed. The mechanical properties of P3HB, particularly its brittleness and narrow processing window, may require blending or plasticization for consumer applications. Consistency of molasses composition between harvests and factories could introduce variability in fermentation performance, demanding adaptive process control. And the ultraviolet-shielding claim, while intriguing, rests on a single measurement that would need replication across film thicknesses, formulations, and aging conditions. Nevertheless, the combination of a cheap substrate, a fast fermentation, a well-characterized polymer product, and an unexpected functional property makes this work a useful data point in the ongoing effort to bring microbial bioplastics closer to economic viability, and it underscores the value of systematically screening local microbial biodiversity for organisms suited to regional waste streams.

Subject of Research: …

Article Title: Optimized production of polyhydroxyalkanoates from Priestia megaterium using sugar beet molasses: a cost-effective approach for eco-friendly bioplastics

Article References: Optimized production of polyhydroxyalkanoates from Priestia megaterium using sugar beet molasses: a cost-effective approach for eco-friendly bioplastics. (n.d.). https://doi.org/10.1007/s11356-026-38285-8

Image Credits: AI Generated

DOI: 10.1007/s11356-026-38285-8

Keywords: Optimized, production, polyhydroxyalkanoates, Priestia, megaterium, sugar, beet, molasses, cost-effective, approach, eco-friendly, bioplastics

In research published in BMC Genomics, Tomasz Grzyb, Małgorzata Wlaźlak and Justyna Szulc took six bacterial strains isolated from soils in post-maize cultivation fields and subjected them to a battery of genome-based species assignment methods. Their targets belonged to the order Caryophanales, a group that includes the enormously consequential genera Bacillus, Priestia and Paenibacillus, organisms used in agriculture, industry and biotechnology, and close relatives of dangerous pathogens. What they found was a taxonomic landscape riddled with contradiction: species boundaries that shift depending on which metric you trust, genomes that intermix across supposedly distinct species names, and a widely used identity threshold that may be significantly miscalibrated.

The team compared several complementary approaches. Average Nucleotide Identity, or ANI, was computed with two different tools, FastANI and skani, which estimate the overall similarity between two genomes by aligning shared DNA sequence. Digital DNA-DNA hybridization, dDDH, a computational descendant of the wet-laboratory hybridization experiments that once defined bacterial species, was calculated using formula 2. The researchers also employed tetranucleotide Z-score distance, TZMD, which measures differences in short-word DNA composition, and single-copy gene phylogenomics, reconstructing evolutionary trees from sets of genes present exactly once in each genome. Crucially, rather than comparing each strain against only its nearest named neighbors, the authors performed comprehensive pairwise comparisons among all available RefSeq genomes at both complete and chromosome assembly levels for each taxonomic neighborhood, an unusually thorough sweep designed to reveal the true structure of variation around each strain.

For two of the six strains, the answer came back clean. Both could be assigned without ambiguity, one to Bacillus subtilis and one to Bacillus licheniformis, with every ANI comparison against their respective species exceeding the conventional 95 percent threshold and with clear phylogenomic separation from neighboring taxa. These cases show that the classical framework still works when species boundaries are genuinely well separated. The complications began with the remaining four strains, each of which landed in a different taxonomic minefield.

Strain Bac2 fell within the Operational Group Bacillus amyloliquefaciens, and here the analysis documented what the authors call fundamental boundary inconsistency. ANI values measured between recognized species within this group exceeded the ANI values measured among genomes assigned to Bacillus amyloliquefaciens itself. In other words, genomes bearing different species names were more similar to each other than genomes carrying the same name, a direct inversion of what a coherent species concept requires. Single-copy gene phylogenomics confirmed the chaos, revealing extensive intermixing of named species across the tree. A name assigned under these conditions conveys little about evolutionary relatedness.

Strain zielonkawy was assigned to the Bacillus cereus sensu stricto genomospecies, but its placement highlighted a familiar and stubborn problem: the deep intermixing of Bacillus cereus sensu stricto and Bacillus thuringiensis. These two names describe bacteria with dramatically different ecological roles, one an opportunistic pathogen and the other an insecticidal biocontrol agent, yet their genomes remain so entangled that no genomic metric reliably separates them. The new data add one more well-documented instance to a debate that has persisted since whole genomes first became available.

The two remaining strains, assigned to Priestia megaterium and Paenibacillus amylolyticus, produced perhaps the most novel findings. Both ANI multi-comparison analysis and single-copy gene phylogenomics suggested possible species intermixing or mislabelling within the public reference databases themselves. To the authors’ knowledge, this is the first study to quantitatively document species delimitation problems between Priestia megaterium and Priestia aryabhattai, and between Paenibacillus amylolyticus and Paenibacillus xylanexedens. The Paenibacillus analysis carried the caveat of a small available sample size, but the Priestia result points to a quietly widespread issue: reference databases, which thousands of labs treat as ground truth, may contain genomes whose species labels do not survive close genomic scrutiny.

Beyond the individual assignments, the study delivers a quantitative contribution to the methodology of bacterial taxonomy itself. Concordance analysis between FastANI and dDDH formula 2 revealed a non-linear relationship, well described by a quadratic fit with an R-squared of 0.991. From this relationship, the researchers derived a striking number: the traditional 70 percent dDDH species threshold, inherited from the pre-genomic era of DNA reassociation experiments, corresponds not to the conventionally assumed 95 percent ANI but to approximately 96.16 percent ANI. In practical terms, dDDH formula 2 is the more conservative of the two metrics, meaning that genomes judged to be the same species by the 95 percent ANI rule could still fail the dDDH test. Laboratories relying on ANI alone may be lumping together organisms that a stricter standard would split.

The comparison between alignment-free and tree-based approaches added further nuance. Correlations between skani genomic distances and single-copy gene phylogenomic patristic distances, the branch-length distances separating genomes on the reconstructed trees, were high across all datasets, with R-squared values ranging from 0.900 to 0.997. Overall genomic structure, in other words, is consistent between methods. But Spearman rank correlations, while still strong, were consistently lower, at 0.780 to 0.916, indicating that the precise identity of a genome’s closest neighbors can shift depending on whether you measure raw sequence similarity or reconstructed evolutionary distance. For taxonomists deciding whether a strain belongs to one species or its nearest rival, that rank-order disagreement is exactly where decisions get made.

As a constructive response, the authors propose a complementary diagnostic tool: within- and between-species ANI multi-comparison analysis paired with PERMANOVA, a non-parametric statistical test for differences among groups, followed by post-hoc pairwise testing. The idea is to treat species boundaries not as a single threshold but as a statistical question: do the ANI distributions within a named species differ significantly from the distributions between it and its relatives? Applied alongside single-copy gene phylogenomics, this framework could flag boundary inconsistencies that single-threshold assignment silently passes over, giving taxonomists an explicit, reproducible way to test whether a species name still carves nature at its joints.

The broader significance of the work extends well beyond six soil isolates from Polish maize fields. The Caryophanales taxa examined here anchor industries from probiotics to pest control and include the closest relatives of the anthrax bacillus. If species boundaries in these groups are inconsistent, then everything built on top of those names, from safety assessments of biocontrol strains to regulatory definitions of pathogenic species, inherits the uncertainty. The study also lands amid an ongoing, sometimes contentious community effort to redefine prokaryotic species entirely, with competing proposals for genome-based circumscriptions. By showing that even gold-standard tools disagree at the margins, and that the canonical thresholds themselves encode hidden conservatism, the Lodz team’s results argue for pluralism: no single number can settle species questions in difficult groups, but a disciplined combination of metrics, statistics and phylogenetics can at least make the disagreements visible, quantifiable and, ultimately, resolvable.

Subject of Research: Genome-based species delimitation and taxonomic boundary evaluation in Caryophanales bacteria using ANI, dDDH and phylogenomics

Article Title: Challenges in Caryophanales species delimitation: comparative evaluation of genomic similarity metrics and phylogenomics based on six environmental strains

Article References: Grzyb, T., Wlaźlak, M., & Szulc, J. (2026). Challenges in Caryophanales species delimitation: comparative evaluation of genomic similarity metrics and phylogenomics based on six environmental strains. BMC Genomics. https://doi.org/10.1186/s12864-026-13320-7

Image Credits: AI Generated

DOI: 10.1186/s12864-026-13320-7

Keywords: Caryophanales, species delimitation, phylogenomics, Average Nucleotide Identity, digital DNA-DNA hybridization, Bacillus subtilis group, Bacillus cereus group, Priestia, Paenibacillus, bacterial taxonomy, BMC Genomics, environmental strains