Analysis of biocide transport limitation in an artificial biofilm system

An alginate gel bead artificial biofilm system was used to assay biofilm susceptibility to four biocides and to analyse the extent to which each agent penetrated the biofilm. Chlorine, glutaraldehyde, an isothiazolone, and a quaternary ammonium compound were tested on alginate-entrapped Enterobacter aerogenes in gel beads ranging from 1·8 to 6 mm in diameter. Gel-entrapped bacteria were less susceptible to all four antimicrobial agents than were planktonic micro-organisms. The degree of kill measured in artificial biofilm gel beads depended on the size of the gel bead and the cell density at which it was loaded. Disinfection efficacy decreased as gel bead radius or cell density increased. The manifest dependence of biofilm disinfection efficacy on the physical properties of the artificial biofilm (radius and cell density) suggests the impingement of transport limitation of biocide transport into the biofilm. A previously developed theory of biocide reaction and diffusion in biofilm was tested by calculating an appropriate Thiele modulus. In accordance with the theory, the efficacy of all four biocides decreased, albeit noisily, as the Thiele modulus exceeded 1. This result demonstrates that transport limitation can impact antimicrobial performance against biofilms not only of oxidizing biocides but also of non-oxidizing agents.     

Oxygen diffusivity in gel beads containing viable cells

This article proposes a simple steady-state method for measuring the effective diffusion coefficient of oxygen (D(e)) in gel beads entrapping viable cells. We applied this method to the measurement of D(e) in Ca- and Ba-alginate gel beads entrapping Saccharomyces cerevisiae and Pseudomonas ovalis. The diffusivity of oxygen through gel beads containing viable cells was measured within an accuracy of +/-7% and found not to be influenced by cell density (0-30 g/L gel), cell type, and cell viability in gel beads. The oxygen diffusivity in the Ca-alginate gel beads was superior to that of the Ba-alginate gel beads, and the D(e) in the Ca-alginate gel beads nearly equalled the molecular diffusion coefficient in the liquid containing the gel beads. The oxygen concentration profile in a single Ca-alginate gel bead was calculated and compared to the distribution of mycelia of Aspergillus awamori grown in that gel bead. This procedure indicated that the oxygen concentration profile is useful for the estimation of the thickness of the cell layer in a gel bead. Numerical investigation revealed that high effectiveness factors, greater than 0.8, could be obtained using microgel beads with a radius of 0.25 mm.     

Survival of Bifidobacterium longum Immobilized in Calcium Alginate Beads in Simulated Gastric Juices and Bile Salt Solution

Bifidobacterium longum KCTC 3128 and HLC 3742 were independently immobilized (entrapped) in calcium alginate beads containing 2, 3, and 4% sodium alginate. When the bifidobacteria entrapped in calcium alginate beads were exposed to simulated gastric juices and a bile salt solution, the death rate of the cells in the beads decreased proportionally with an increase in both the alginate gel concentration and bead size. The initial cell numbers in the beads affected the numbers of survivors after exposure to these solutions; however, the death rates of the viable cells were not affected. Accordingly, a mathematical model was formulated which expressed the influences of several parameters (gel concentration, bead size, and initial cell numbers) on the survival of entrapped bifidobacteria after sequential exposure to simulated gastric juices followed by a bile salt solution. The model proposed in this paper may be useful for estimating the survival of bifidobacteria in beads and establishing optimal entrapment conditions.     

A laboratory hot tub model for disinfectant efficacy evaluation

This paper describes a novel laboratory hot tub (LHT) apparatus and associated standard operating procedure (SOP) designed to reproduce the key biological, chemical, and engineering parameters associated with recreational and therapeutic hot tubs. Efficacy, as measured quantitatively by log reduction values, was determined against both biofilm and planktonic bacteria. When the LHT was run according to the SOP, with no antimicrobial treatment, a consistent level of bacterial contamination occurred. The means of log10 viable cell densities (+/- the repeatability standard deviation of log densities) were 7.2 (+/-0.31) for the bulk water (density in units of cfu ml-1), 5.3 (+/-0.56) for the coupons (density in units of cfu cm-2), and 6.6 (+/-0.50) for the filters (density in units of cfu cm-2). When control and chlorine treated LHTs were run in parallel, the log reduction increased significantly with chlorine concentration for samples of planktonic bacteria in the bulk water (p=0.016), biofilm bacteria on the coupons (p=0.09) and biofilm bacteria on the filter (p=0.005), indicating that the method was sensitive to chlorine concentration. The method also displayed sensitivity by differentiating between chlorine and bromine treatments; in every case, chlorine produced a greater log reduction than did the same concentration of bromine. The model and SOP were shown to be rugged with respect to slight changes in fluid mixing intensity, water chemistry (saturation index), inoculum size, and organic loading. The LHT and associated SOP provide a reliable second tier in a three-tiered testing process, in which the first tier is a suspension test and the final tier is a field test.     

Biofilm formation on the surface of ceramic tiles

The aim of the study was to investigate the formation of biofilm on the surface of ceramic tiles, widely present in public and private buildings, using six parallel flow chambers. Our flow system was conceived and made to compare biofilm results by parallel distributed rectangular tiles. The tiles, divided into two identical A and B sections, were placed within the flow chambers. Biofilm formation was performed after 72 h and was quantified by viable counts of bacteria. Average viable counts ranged from 1.1x10(7) to 7.3x10(7) cfu cm(-2) and from 1.1x10(7) to 5.8x10(7) cfu cm(-2) respectively for biofilm A and B sections. As statistical analysis does not show significant differences, we can conclude that biofilms obtained were so similar to each other that they confirmed the system reproducibility. Our next step will be to use our system to study Legionella pneumophila and to evaluate the efficacy of antibacterial agents.     

Role of dose concentration in biocide efficacy against Pseudomonas aeruginosa biofilms

Pseudomonas aeruginosa entrapped in alginate gel beads to form artificial biofilms resisted killing by chlorine, glutaraldehyde, 2,2-dibromo-3-nitrilopropionamide (DBNPA), and an alkyl dimethyl benzyl ammonium compound (ADBAC). The degree of resistance was quantified by a resistance factor that compared killing times for biofilm and planktonic cells in response to the same concentration of antimicrobial agent. Resistance factors averaged 120 for chlorine, 34 for glutaraldehyde, 29 for DBNPA, and 1900 for ADBAC. In every case, resistance factors decreased with increasing concentration of the antimicrobial agent. An independent analysis of the concentration dependence of the apparent rates of killing of planktonic and biofilm bacteria showed that elevating the treatment concentration increased bacterial killing more in the biofilm than it did in a suspension culture. Calculation of a transport modulus comparing the rates of biocide reaction and diffusion suggested that at least part of the biofilm resistance to chlorine, glutaraldehdye, and DBNPA could be attributed to incomplete or slow penetration of these agents into the biofilm. Time-kill curves were nonlinear for biofilm bacteria in some cases. The shapes of these curves implicated retarded antimicrobial penetration for chlorine and glutaraldehyde and the presence of a tolerant subpopulation for DBNPA and ADBAC. The results indicate that treating biofilms with a concentrated dose of biocide is more effective than using prolonged doses of a lower concentration. Journal of Industrial Microbiology & Biotechnology (2002) 29, 10–15 doi:10.1038/sj.jim.7000256 Received 29 October 2001/ Accepted in revised form 18 March 2002     

Batch fermentation with entrapped growing cells ofLactobacillus casei

Summary Growing cells ofLactobacillus casei were entrapped in-carrageenan/locust bean gum (LBG) (2:1 or 2.75%:0.25% w/w respectively) mixed gel beads (two ranges of diameter: 0.5–1.0 and 1.0–2.0 mm) to fermentLactobacillus Selection (LBS) medium and produce biomass. The results showed significant influence of initial cell loading of the beads and bead size on the fermentation rate. The highest cell release rates were obtained with 2.75%:0.25%-carrageenan/LBG small diameter gel beads. However, 17 h fermentation of LBS medium with immobilized cells resulted in substantial softening of the gel matrix, prohibiting reuse of immobilized biocatalysts as inoculum in subsequent batch fermentation. A dynamic shear rheological study showed that the gel weakness was related to chemical interactions with the medium. Results indicated that part of the matrix-stabilizing K⁺ ions diffused back to the medium. Stabilization of the gel was obtained by adding potassium ions to the LBS medium;L. casei growth was not altered by this supplementation. Fermentation of LBS medium supplemented with KCl byL. casei showed higher cell counts in the broth medium with immobilized cells than with free cells, reaching 10¹⁰ cells/ml after about 10 h with entrapped cells in 0.5–1.0 mm diameter beads and 17 h with free cells. Counts in the gel beads after fermentation were higher than 10¹¹ cells/ml and bead integrity was maintained throughout fermentation.     

Transport and consumption rate of O2 in alginate gel beads entrapping hepatocytes

Experiments carried out with hepatocytes entrapped in alginate gel particles with a cell density of about 10⁷ cells ml^–1 showed an oxygen consumption rate of about 2.2×10^–16 mol cell^–1 s^–1. Under these conditions, no inner anoxic core is present in 1.8 mm diameter beads. From these data, the maximum bead size consistent with non-critical O₂ concentration at the bead center has been evaluated for different cell densities and O₂ concentrations in the medium.     

Further analysis of transcytosis of free polypeptides and polypeptide-coated nanobeads in rabbit nasal mucosa.

In rabbit nasal mucosa, free polypeptides and polypeptide-coated nanospheres are actively absorbed by the M cells present in specialized areas of the epithelium. Because polypeptide-coated nanosphere transport was abolished in the presence of free polypeptides, free polypeptides and polypeptide-coated nanospheres are shown here to compete. Fluxes of polypeptide-coated nanospheres with 356, 490, and 548 nm diameters have been compared. BSA-coated beads were poorly transported, at the same rate, when bead diameters were 356 or 490 nm [net flux of approximately 2-2.5 x 10(6) nanospheres (nan). cm(-2) x h(-1)]; however, their net transport largely increased toward a value of 25 x 10(6) nan. cm(-2) x h(-1) at a diameter of 548 nm. Insulin-coated beads displayed a net flux that was significantly higher than BSA-coated beads but equally were transported at the same rate (net flux of approximately 8.0 x 10(6) nan. cm(-2) x h(-1)) at diameters of 356 or 490 nm; once again, their net flux significantly increased toward a value of 25 x 10(6) nan. cm(-2) x h(-1), if the bead diameter was 548 nm. Insulin plus anti-insulin IgG-coated 490-nm-diameter beads displayed a very high net flux, although not yet saturating (approximately 60 x 10(6) nan. cm(-2) x h(-1)); however, a significantly lower saturated net flux (once again approximately 25 x 10(6) nan. cm(-2) x h(-1)) was shown with 548-nm-diameter beads. In conclusion, 1) in the range of 356-490 nm diameter, net transport was independent of bead diameter and, conversely, largely dependent on the coating polypeptides, and 2) at 548 nm diameter, nanospheres tended to be transferred at similar rates independently of coating kind and the maximal net transport capacity of the mucosa was reduced. The suspension viscosity largely increased with 548-nm polypeptide-coated nanospheres; this fact is hypothetically proposed to be the cause of these events.     

Decolorization of azo dye using PVA-immobilized microorganisms

A microbial consortium having a high capacity for rapid decolorization of azo dye (RED RBN) was immobilized by a phosphorylated polyvinyl alcohol (PVA) gel. The immobilized-cell beads exhibited a color removal capability of 75%, even at a high concentration of RED RBN (500 mg l(-1)) within 12 h using flask culture. The continuous operation was conducted at a hydraulic retention time (HRT) of 5-20 h in which the dye loading rate ranged from 240 to 60 mg dye h(-1). A removal efficiency exceeding 90% was obtained at the HRT higher than 10 h. No recognizable destruction of bead appearance was observed in the 6-month operation. Examination of the mechanism of the decolorization process by cell beads indicated that it proceeded primarily by biological decolorization associated with partial adsorption of the dye onto the entrapped cells and gel matrix. Microscopic observation revealed that the microbial consortium contained in the gel beads was at least made up of three kinds of bacterial species. From the economical viewpoint, alternative cheaper nitrogen sources such as fish meal, soybean meal, pharmamedia and vita yeast powder were examined.     

Artificial biofilm model – a useful tool for biofilm research

For biofilm studies, artificial models can be very helpful in studying processes in hydrogels of defined composition and structure. Two different types of artificial biofilm models were developed. Homogeneous agarose beads (50–500 μm diameter) and porous beads (260 μm mean diameter) containing pores with diameters from 10 to 80 μm (28 μm on average) allowed the embedding of cells, particles and typical biofilm matrix components such as proteins and polysaccharides. The characterisation of the matrix structures and of the distribution of microorganisms was performed by confocal laser scanning microscopy. The physiological condition of the embedded bacteria was examined by redox activity (CTC-assay) and membrane integrity (Molecular Probes LIVE/DEAD-Kit). Approximately 35% of the immobilised cells (Pseudomonas aeruginosa SG81) were damaged due to the elevated temperature required for the embedding process. It was shown that the surviving cells were able to multiply when provided with nutrients. In the case of homogeneous agarose beads, cell growth only occurred near the bead surface, while substrate limitation prevented growth of more deeply embedded cells. In the porous hydrogel, cell division was observed across the entire matrix due to better mass transport. It could be shown that embedding in the artificial gel matrix provided protection of immobilized cells against toxic substances such as sodium hypochlorite (0.5 mg/l, 30 min) in comparison to suspended cells, as observed in other immobilized systems. Thus, the model is suited to simulate important biofilm matrix properties. Received: 21 December 1999 / Received revision: 7 March 2000 / Accepted: 10 March 2000     

Spectrophotometric quantification of lactic bacteria in alginate and control of cell release with chitosan coating

Lactococcus lactis ssp. cremoris was entrapped within a Ca-alginate matrix, and an in situ spectrophotometric method for monitoring cell population in calcium alginate beads described. The intracapsular cell population can be estimated by measuring the optical density of beads containing cells, using cell-free beads as reference, or by measuring absorbance of a liquified bead suspension. Alginate beads, and beads coated with chitosan type I, II, and I and II mixtures, were examined for cell release. Lower viscosity chitosan (type I) coatings reduced cell release by a factor of 100 from10⁵ cfu ml⁻¹ to 10³ cfu ml⁻¹ after 6 h of fermentation. Reuse of chitosan I coated alginate beads also showed a reduction in cell release by a factor of 100. Cell loading and initial cell growth within the beads greatly affected cell release. Reducing the initial cell release would lower the overall levels of cell release throughout the fermentation. Compared to non-immobilized cultures, a 20–40% reduction in the lactic acid production rate was observed for alginate beads and chitosan I coated alginate beads, respectively. This reduction can be compensated for by increasing the intracapsular cell loading during immobilization, or before the onset of fermentation.     

Survival of Bifidobacterium longum immobilized in calcium alginate beads in simulated gastric juices and bile salt solution

Bifidobacterium longum KCTC 3128 and HLC 3742 were independently immobilized (entrapped) in calcium alginate beads containing 2, 3, and 4% sodium alginate. When the bifidobacteria entrapped in calcium alginate beads were exposed to simulated gastric juices and a bile salt solution, the death rate of the cells in the beads decreased proportionally with an increase in both the alginate gel concentration and bead size. The initial cell numbers in the beads affected the numbers of survivors after exposure to these solutions; however, the death rates of the viable cells were not affected. Accordingly, a mathematical model was formulated which expressed the influences of several parameters (gel concentration, bead size, and initial cell numbers) on the survival of entrapped bifidobacteria after sequential exposure to simulated gastric juices followed by a bile salt solution. The model proposed in this paper may be useful for estimating the survival of bifidobacteria in beads and establishing optimal entrapment conditions.     

Growth and morphology of thermophilic dairy starters in alginate beads

The aim of this research was to produce concentrated biomasses of thermophilic lactic starters using immobilized cell technology (ICT). Fermentations were carried out in milk using pH control with cells microentrapped in alginate beads. In the ICT fermentations, beads represented 17% of the weight. Some assays were carried out with free cells without pH control, in order to compare the ICT populations with those of classical starters. With Streptococcus thermophilus, overall populations in the fermentor were similar, but maximum bead population for (8.2 x 10(9) cfu/g beads) was 13 times higher than that obtained in a traditional starter (4.9 x 10(8) cfu/ml). For both Lactobacillus helveticus strains studied, immobilized-cell populations were about 3 x 10(9) cfu/g beads. Production of immobilized Lb. bulgaricus 210R strain was not possible, since no increases in viable counts occurred in beads. Therefore, production of concentrated cell suspension in alginate beads was more effective for S. thermophilus. Photomicrographs of cells in alginate beads demonstrated that, while the morphology of S. thermophilus remained unchanged during the ICT fermentation, immobilized cells of Lb. helveticus appeared wider. In addition, cells of Lb. bulgaricus were curved and elongated. These morphological changes would also impair the growth of immobilized lactobacilli.     

Role of RpoS and AlgT in Pseudomonas aeruginosa biofilm resistance to hydrogen peroxide and monochloramine

The role of two sigma factors, AlgT and RpoS, in mediating Pseudomonas aeruginosa biofilm resistance to hydrogen peroxide and monochloramine was investigated. Two knock out mutant strains, SS24 (rpoS-) and PAO6852 (algT-), were compared with a wild type, PAO1, in their susceptibility to monochloramine and hydrogen peroxide. When grown as biofilms on alginate gel beads (mean untreated areal cell density 3.7 +/- 0.27 log cfu cm-2) or on glass slides (mean untreated areal cell density 7.6 +/- 0.9 log cfu cm-2), wild type bacteria exhibited reduced susceptibility to both antimicrobial agents in comparison with suspended cells. On alginate gel beads, all strains were equally resistant to monochloramine. rpoS- and algT- gel bead biofilms of 24-hour-old were more susceptible to hydrogen peroxide disinfection than were biofilms formed by PAO1. Biofilm disinfection rate coefficients for the two mutant strains were statistically indistinguishable from planktonic disinfection rate coefficients, indicating complete loss of biofilm resistance. While 48-hour-old algT- biofilm cells became resistant to hydrogen peroxide, 48-hour-old rpoS- biofilm cells remained highly susceptible. With the thicker biofilms formed on glass coupons, all strains were equally resistant to both hydrogen peroxide and monochloramine. It is concluded that while RpoS and AlgT may play a transient role in protecting thin biofilms from hydrogen peroxide, these sigma factors do not mediate resistance to monochloramine and do not contribute significantly to the hydrogen peroxide resistance of thick biofilms.     

Batch fermentations with a mixed culture of lactic acid bacteria immobilized separately in κ-carrageenan locust bean gum gel beads

Summary Streptococcus salivarius subsp. thermophilus and Lactobacillus delbrueckii subsp. Bulgaricus were immobilized separately in -carrageenan-locust bean gum gel beads. The beads were prepared by a dispersion process in a two-phase system (water in oil) and two ranges of bead diameter selected by sieving (0.5–1.0 mm and 1.0–2.0 mm). Fermentations with the two strains were conducted in bench bioreactors in a supplemented whey permeate medium. Free and entrapped cells (two ranges of bead diameter and two levels of initial bead cell load) were grown in mixed culture, and carbohydrate utilization, acid production and cell growth or cell release rate measured. Fermentation rates were influenced by bead diameter and initial cell load of the beads. Beads with high initial cell density increased fermentation rates compared to low cell density beads or free cells. Smaller diameter beads (0.5–1.0 mm) showed a stable tendency (not statistically significant p _a > 0.05) towards higher cell release rates, lactose utilization, galactose accumulation and lactic acid production than did larger diameter beads (1.0–2.0 mm). Immobilization of S. salivarius subsp. thermophilus and L. delbrueckii subsp. bulgaricus in separate beads did not seem to affect protocooperation during batch fermentation, and allowed for high cell release rates into the medium.     

Entrapment of lipid vesicles and membrane protein-lipid vesicles in gel bead pores

Phospholipid vesicles were entrapped in gel beads of Sepharose 6B and Sephacryl S-1000 during vesicle preparation by dialysis. Egg-yolk phospholipids solubilized with cholate or octyl glucoside were dialysed together with gel beads for 2.5 days in a flat dialysis bag. Some vesicles were formed in gel bead pores and vesicles of sufficient size became trapped. Red cell membrane protein-phospholipid vesicles could be immobilized in the same way. Non-trapped vesicles were carefully removed by chromatographic procedures and by centrifugation. The amount of entrapped vesicles increased with the initial lipid concentration and was dependent on the relative sizes of vesicles and gel pores. The largest amount of trapped vesicles, corresponding to 9.5 mumol of phospholipids per ml gel, was achieved when Sepharose 6B gel beads were dialysed with cholate-solubilized lipids at a concentration of 50 mM. In this case the vesicles had an average diameter of 60 nm and an internal volume of 15 microliters/ml gel. The amount of vesicles trapped in Sephacryl S-1000 gel beads upon dialysis under the same conditions was smaller: 2.2 mumol of phospholipids per ml gel. Probably most of the gel pores were too large to trap such vesicles. Larger vesicles, with an average diameter of 230 nm, were entrapped in the Sephacryl S-1000 matrix in an amount corresponding to 3.0 mumol phospholipids per ml gel upon dialysis of the gel beads and octyl glucoside-solubilized lipids at a concentration of 20 mM. The internal volume of these vesicles was 22 microliters/ml gel. The yield of immobilized phospholipids was up to 19%. The entrapped vesicles were somewhat unstable: 9% of the phospholipids were released during 9 days of storage at 4 degrees C. By the dialysis entrapment method vesicles can be immobilized in the gel beads without using hydrophobic ligands or covalent coupling.     

Immobilized growing lactic acid bacteria with κ-carrageenan — locust bean gum gel

Summary A cell entrapment process using -carrageenan — locust bean gum gel is presented. Streptococcus thermophilus, Lactobacillus bulgaricus and S. lactis were immobilized in small gel beads (0.5–1.0 mm and 1.0–2.0 mm diameter) and fermentations in bench bioreactors were conducted. Viability of entrapped cells, lactose utilization, lactic acid production and cell release rates were measured during fermentation. The procedure was effective for S. thermophilus, L. bulgaricus and S. lactis, and the viability of these bacteria remained very high throughout entrapment steps and subsequent storage. Bead diameter influenced the fermentation rate: smaller beads (0.5–1.0 mm) permitted an increase in release rates, lactose utilization and acid production by entrapped cells, approximating values attained with free cells.     

Diffusion of lactose in kappa-carrageenan/locust bean gum gel beads with or without entrapped growing lactic acid bacteria

Effective diffusion coefficients (De) of lactose in kappa-carrageenan (2.75% wt/wt)/locust bean gum (0.25% wt/wt) (LBG) gel beads (1.5-2.0-mm diameter)with or without entrapped lactic acid bacteria (LAB) were determined at 40 degrees C. The effects of lactose concentration, bacteria strain (Streptococcus salivarius subsp. thermophilus and Lactobacillus casei subsp. casei) and cell content at various steps of the fermentation process (after immobilization, pre-incubation of the beads and successive fermentations) were measured on De as a first step for process modelling. Results were obtained from transiend concentration changes n well-stirred lactose solutions in which the beads were suspended. A mathematical model of unsteady-state diffusion in a sphere was used, and De was obtained from the best fit of the experimental data. Diffusivity of lactose in cell-tree beads was significantly lower than in pure water mainly because of the obstruction effect of the polymer chains and the hydration region. Furthermore, effective diffusivity and equilibrium partition factor were independent of lactose concentration in the range from 12.5 to 50 g/L. No significant difference was found for De (effective diffusivity) and Kp (partition) coefficients between beads entrapping S. thermophilus (approximately 5 x 10(9) CFU/mL) and cell-free beads. On the other hand higher cell counts obtained with L. casei (close to 1.8 x 10(11) CFU/mL) increased mass transfer resistance resulting in lower effective diffusivities and Kp. Finally, the effects of the type of bacteria and their distribution in the beads on the diffusivity were also discussed.     

Immobilization of recombinant nanobiofiber CS3 fimbriae onto alginate beads for improvement of cadmium biosorption

A cell surface display system with metalbinding properties was previously developed using CS3 fimbriae, which are hollow tubes 20 nm-thick and 2 nm in diameter. In this study, hybrid CS3 pili were separated from recombinant Escherichia coli and entrapped in calcium alginate gel beads in order to improve their stabilization and also adsorption of heavy metals. The surface morphology of the gel beads containing pili was investigated by scanning electron microscopy (SEM). Immunofluorescence microscopy was employed to confirm the attachment of nanobiofibers to the alginate beads. The effects of three variables (sodium alginate concentration, protein to alginate mass ratio, and bead size) at two levels each on Cd²⁺ biosorption efficiency were investigated by full factorial experimental design. A second-order polynomial equation modeled the design space for the process response of cadmium removal capacity. The optimal values of the factors were obtained as follows: 1% sodium alginate concentration, 0.25 protein to alginate mass ratio, and a 6 mm bead size. Under these conditions, Cd²⁺ was adsorbed at 45.45 mg/g to the nanobiofiber. The results indicate that the immobilized recombinant hybrid CS3 pili may be an appropriate biosorbent for removal of heavy metals from polluted aquatic environments.