The pivotal discovery is that crystalline nanorod formation is not an intrinsic enzymatic property of the bacterium itself but an emergent property of its biofilm. In a carefully designed three-state experiment, the researchers exposed biofilm-associated cells, free-swimming planktonic cells, and cells stripped of their extracellular polymeric substance (EPS) matrix to 2 millimolar sodium selenite under identical conditions. Only the intact biofilms produced precipitates ranging from dark red to metallic grey, the classic visual signature of the amorphous-to-trigonal transition. X-ray diffraction confirmed the observation with striking clarity: biofilm-derived samples displayed sharp, well-resolved reflections at 2-theta values of approximately 23.5, 29.7, and 43.6 degrees, assigned to the (100), (011), and (110) crystallographic planes of trigonal selenium. The planktonic condition showed only weak trigonal signals superimposed on dominant amorphous scattering, while the EPS-depleted condition produced essentially no detectable crystalline phase at all. Because all three states retained the metabolic capacity to reduce selenite, the conclusion is unambiguous: the EPS matrix is required to direct the structural form of the reduction product, not the reduction itself.

The speed of the transformation makes the finding even more remarkable. Thermal annealing of amorphous selenium into the trigonal phase normally requires temperatures above 100 degrees Celsius sustained over hours to days. The biofilm condition achieved sharp, intense trigonal diffraction patterns after just 168 hours at 28 degrees Celsius with no thermal treatment whatsoever, implying that the EPS matrix substantially lowers the kinetic barrier to trigonal nucleation and effectively functions as a crystallization catalyst operating at ambient temperature. This is, according to the authors, the first reported demonstration of ambient-temperature, EPS-driven crystallization of trigonal selenium by a bacterial biofilm system, mechanistically distinct from all previously described thermally driven recrystallization routes.

High-resolution imaging and electron diffraction added atomic-scale confirmation. HAADF-STEM microscopy revealed that biofilm-associated cells generate a high density of elongated nanostructures, 250 to 500 nanometres in length, distributed mainly in the extracellular space surrounding cell clusters. Selected-area electron diffraction of individual nanorods produced discrete diffraction spots rather than diffuse rings, indicating single-crystal or highly textured material indexed to the (101) and (111) planes of trigonal selenium. Critically, the (100) reflection, which corresponds to the lateral facets of the helical selenium chain axis, was detected exclusively in biofilm-derived nanorods. In the trigonal selenium crystal system, helical chains run parallel to the c-axis, the natural elongation direction of the crystal. The exclusive presence of this reflection in the biofilm condition indicates that nanorod elongation occurs with a high degree of orientational order and requires the organized template that only an intact EPS matrix provides. Planktonic and EPS-depleted particles, lacking this template, remained isotropic and randomly oriented, explaining their spherical morphology.

Fourier-transform infrared spectroscopy illuminated the molecular interactions underlying this templating. Exposure to selenite produced pronounced shifts in the amide I and amide II bands of EPS-associated proteins, indicating conformational changes consistent with selenium coordination to amino acid side chains, particularly cysteine thiols and lysine amines. Perturbations in the polysaccharide-dominated regions, including carboxylate and hydroxyl stretching frequencies, pointed to direct electrostatic coordination of selenite oxyanions to these metal-binding functional groups. The researchers propose a division of labour within the matrix: polysaccharide components capture and concentrate selenium ions, while proteinaceous components, notably flagellin filaments, provide the structural axis that imposes crystallographic directionality on nucleation and elongation. Flagellin from other bacteria has previously been shown to template one-dimensional silica nanotubes and gold surfaces, lending independent plausibility to this model.

But an intact biofilm is only useful if it survives the very conditions it is meant to remediate. Heavy metals such as nickel, cadmium, and copper are known to suppress biofilm formation in most bacteria by disrupting quorum-sensing signalling, the chemical communication system that coordinates EPS production. Here BII-R7 revealed a second extraordinary trait. While comparator strains suffered biofilm reductions of 60 to 84 percent under metal stress, BII-R7’s biofilm biomass actually increased by nearly 22 percent in the presence of 2 millimolar nickel, a metal-stimulated enhancement that runs counter to the general trend among environmental isolates. Comparative gene screening supplied a genomic explanation: BII-R7 is the only strain in the comparison that lacks both rpfF and smoR, the two central regulators of the Stenotrophomonas diffusible signal factor quorum-sensing system. By bypassing the signalling pathway that metals sabotage, BII-R7 maintains its EPS-producing biofilm precisely where other organisms lose it, preserving the scaffold on which crystalline nanorod formation depends.

To probe the deeper genetic basis of this dual capacity, the team re-sequenced the BII-R7 genome, producing a far higher-quality assembly than the previous draft, and compared it against 37 other Stenotrophomonas genomes. The pangenome analysis revealed an open structure with a small core genome of just 670 genes and an expansive cloud genome of over 21,500 gene clusters, a signature of frequent horizontal gene transfer. BII-R7 stood out dramatically, harbouring more than 2,300 genes found in no other strain, many showing sequence homology with diverse soil and aquatic bacteria, consistent with long-term residence in geochemically complex clay formations. Among these exclusive genes, the researchers identified a suite of metal resistance determinants absent from every comparator: copF and copB encoding dual copper efflux mechanisms, czcA for broad-spectrum divalent cation extrusion, a NodT-family RND efflux lipoprotein, and an exclusive molybdate ABC transporter that supports the molybdenum-dependent enzymatic pathway implicated in selenium oxyanion reduction.

Functional validation confirmed that these genomic predictions translate into real phenotypes. Under copper stress, BII-R7 maintained growth comparable to untreated controls at 2 millimolar copper, while all three comparator strains showed roughly 50 percent growth suppression. Quantitative real-time PCR demonstrated that the exclusive resistance genes are actively transcribed and metal-responsive: copF was induced 8.6-fold and copB 3.4-fold under copper stress, while czcA surged 42.2-fold under nickel stress. Two exclusive EPS-remodelling proteins emerged as the strongest candidates for the nanorod-templating phenotype itself. The first, a Wzy-family polysaccharide ligase, possesses a divergent extracellular loop enriched in twelve acidic residues, three histidines, and two cysteines, established ligands for selenium oxyanions, predicted to generate a polysaccharide scaffold with a charge distribution and metal-coordination geometry unlike any other strain. The second, a secreted GH92-family alpha-mannosidase that is transcriptionally induced under selenium stress, is predicted to trim mannosyl branches from the maturing matrix, linearizing EPS fibrils into parallel nano-channels of uniform geometry ideal for templating anisotropic crystal growth.

The authors are careful to frame their genomic framework as hypothesis-generating rather than definitively causal, noting that targeted knockout and complementation experiments are the immediate next step, alongside biochemical characterization of the EPS itself and quantitative crystallography of nanorod yields. Even so, the practical implications are considerable. In bioremediation, trigonal nanorods settle and filter far more efficiently than amorphous nanospheres, improving the operational performance of bioreactor systems treating mining drainage and industrial effluents. Previous work has already demonstrated up to 70 percent selenite removal from contaminated water using BII-R7 immobilized in alginate hydrogels, and promoting biofilm development within such matrices is predicted to enhance removal further while enabling recovery of crystalline product. In green nanotechnology, the biofilm route offers a scalable, low-energy alternative to conventional synthesis that demands high temperatures, toxic reductants, or organic solvents. Crystalline selenium nanostructures also show superior antitumoral and antimicrobial activity relative to their amorphous counterparts. With its non-pathogenic status, exclusive genetic toolkit, and demonstrated compatibility with immobilization formats, BII-R7 offers a coherent blueprint: preserve the biofilm, and the biofilm builds the crystal.

Subject of Research: EPS-templated biosynthesis of crystalline selenium nanorods and multimetal resistance in Stenotrophomonas bentonitica BII-R7

Article Title: Genomic Insights Into the Multimetal Resilience and Biofilm‐Templated Nanorod Biosynthesis of Stenotrophomonas bentonitica BII‐R7: Bioremediation and Green Nanotechnology Implications

Article References: Perez‐Muelas, E., Ruiz‐Fresneda, M. A., Lazuen‐Lopez, G., Lopez‐Perez, T., Eddaoudi‐Lakraichi, F., Bakkali, M., & Merroun, M. L. (2026). Genomic Insights Into the Multimetal Resilience and Biofilm‐Templated Nanorod Biosynthesis of Stenotrophomonas bentonitica BII‐R7: Bioremediation and Green Nanotechnology Implications. Microbial Biotechnology, 19(9), Article e70422. https://doi.org/10.1111/1751-7915.70422

Image Credits: AI Generated

DOI: 10.1111/1751-7915.70422

Keywords: Stenotrophomonas bentonitica, selenium nanoparticles, bioremediation, biofilm, extracellular polymeric substances, pangenomics, green nanotechnology, heavy metal resistance, trigonal selenium, horizontal gene transfer, quorum sensing, biomineralization