BACTERIAL NANOCELLULOSE: INFLUENCE OF CULTIVATION METHODS ON PROPERTIES AND PROSPECTS FOR HEAVY METAL REMOVAL FROM WASTEWATER

ABSTRACT

Bacterial nanocellulose (BNC) is a highly crystalline, ultrapure biopolymer with significant potential in biomedical and environmental applications. This study compares BNC produced by Brevundimonas spp. and a Kombucha symbiotic consortium under static and agitated conditions, examining differences in morphology, crystallinity and yield. Sorption performance of native and carboxylated BNC was evaluated for Pb²⁺, Fe³⁺, Cu²⁺, Cd²⁺ and Mn²⁺ ions. Kombucha-derived BNC formed dense, highly crystalline pellicles and showed superior adsorption capacity, particularly after carboxylation. These findings highlight the strong influence of cultivation parameters on BNC structure and its potential as an efficient biosorbent for heavy metal removal from wastewater.

АННОТАЦИЯ

В работе проведено сравнительное исследование бактериальной наноцеллюлозы (БНЦ), синтезированной культурой Brevundimonas spp. и симбиотической культурой Комбуча при различных условиях культивации. Изучено влияние состава питательных сред и режима ферментации на выход, морфологию, кристалличность и механические свойства БНЦ. Оценена сорбционная способность нативной и карбоксилированной БНЦ по отношению к ионам тяжёлых металлов (Pb²⁺, Fe³⁺, Cd²⁺, Mn²⁺, Cu²⁺). Показано, что БНЦ, полученная из Комбуча, обладает более высокой кристалличностью и сорбционной эффективностью, особенно после карбоксилирования. Полученные результаты подтверждают перспективность модифицированной БНЦ в качестве экологически безопасного сорбента для очистки сточных вод.

Keywords: bacterial nanocellulose, Kombucha, Brevundimonas spp., adsorption, heavy metals, wastewater treatment.

Ключевые слова: бактериальная наноцеллюлоза, Комбуча, Brevundimonas spp., адсорбция, тяжёлые металлы, очистка сточных вод.

1. Introduction

Bacterial nanocellulose (BNC) is a unique biomaterial produced by various strains of cellulose-synthesising bacteria. It is chemically identical to plant cellulose but exhibits superior purity, crystallinity, and a highly organised nanofibrillar structure that provides exceptional mechanical strength, elasticity, and biocompatibility [1–3]. Unlike plant cellulose, BNC contains no lignin, hemicellulose, or other impurities, which makes it easier to isolate and functionalise [4, 5].

Due to its renewable nature and tunable properties, BNC has attracted significant interest in a broad range of applications, including wound dressings, drug delivery systems, flexible electronics, and environmental remediation [6–8]. In particular, the high surface area and abundance of hydroxyl groups on BNC fibrils allow its modification for the adsorption and removal of heavy metal ions from contaminated water [9, 10].

The increasing contamination of aquatic environments with heavy metals—such as lead, cadmium, copper, manganese, and iron—poses a severe ecological and public health challenge [11]. Conventional wastewater treatment methods often involve chemical precipitation, ion exchange, and membrane filtration, which can be expensive and environmentally unsustainable [12, 13]. In this regard, BNC represents a sustainable alternative owing to its biodegradability, high adsorption efficiency, and capacity for surface functionalisation [14, 15].

Previous studies have shown that the structural and physicochemical characteristics of BNC strongly depend on the type of bacterial strain and cultivation conditions. For example, Komagataeibacter xylinus, Brevundimonas spp., and symbiotic Kombucha cultures exhibit different rates of cellulose biosynthesis, fibre morphology, and yields under static and agitated fermentation modes [16–18]. However, systematic comparisons of these producers in the context of heavy metal adsorption remain limited.

Therefore, the present work aims to investigate the effect of different cultivation methods — using Brevundimonas spp. and the Kombucha symbiotic consortium — on the yield, morphology, crystallinity, and adsorption performance of bacterial nanocellulose. This study also evaluates how carboxylation and other chemical modifications influence the sorption efficiency of BNC towards Pb²⁺, Fe³⁺, Cd²⁺, Mn²⁺, and Cu²⁺ ions, with an emphasis on its potential use in sustainable wastewater purification systems. This work provides one of the first systematic comparisons between Kombucha-derived and Brevundimonas-derived BNC in the context of heavy-metal adsorption performance. The study demonstrates how cultivation parameters and carboxylation jointly influence sorption efficiency, offering new insights for the development of biosorbents for wastewater purification.

2. Materials and Methods

2.1. Microorganisms and culture conditions

Two cellulose-producing bacterial systems were employed in this study: a pure culture of Brevundimonas spp. (strain EF1) and a mixed Kombucha symbiotic culture comprising Lysinibacillus spp. and Komagataeibacter species. The Kombucha inoculum was derived from a previously maintained tea fermentation system.

For Kombucha fermentation, 1% black tea infusion supplemented with 20% (w/v) sucrose was used as the nutrient medium. The culture was incubated statically at 30 °C for 10 days [1, 2]. For Brevundimonas spp., four different media were examined: Luria–Bertani (LB), Hestrin–Schramm (HS), Nutrient Broth (NB), and Modified Minimal Davis (MMD). Both static and agitated conditions (150 rpm) were tested for each medium [3].

2.2. Purification of bacterial nanocellulose

The obtained pellicles were washed thoroughly with distilled water and treated with 0.1 M NaOH solution at 80 °C for 2 hours to remove bacterial residues and other impurities [4]. Afterwards, the material was repeatedly rinsed with deionised water until neutral pH was achieved and subsequently dried at 60 °C to constant weight.

2.3. Functionalisation of BNC

For carboxylation, dried BNC films were treated with 0.05 M maleic acid solution at 70 °C for 4 hours, followed by washing and drying under ambient conditions [5]. The functionalisation was confirmed through changes in FTIR spectra and increased adsorption capacity towards Pb²⁺ and Fe³⁺ ions [6].

2.4. Characterisation

Morphology:

The surface structure of the BNC samples was examined by Scanning Electron Microscopy (SEM, JEOL JSM-IT200). Samples were gold-coated prior to analysis.

Crystallinity:

The crystallinity index was determined using X-ray diffraction (XRD, Bruker D8 Advance) with Cu Kα radiation (λ = 1.5406 Å), and calculated according to the Segal equation [7].

Chemical composition:

Functional groups were identified by Fourier-transform infrared spectroscopy (FTIR, Thermo Nicolet iS10) within the 4000–400 cm⁻¹ range [8].

2.5. Adsorption experiments

To assess metal ion removal, 0.1 g of BNC (native or carboxylated) was immersed in 50 mL of 50 mg L⁻¹ aqueous metal ion solutions (Pb²⁺, Cd²⁺, Mn²⁺, Fe³⁺, Cu²⁺). The mixtures were shaken at 150 rpm for 4 hours at 25 °C. Residual metal concentrations were measured using inductively coupled plasma optical emission spectroscopy (ICP-OES, Agilent 5110) [9, 10].

The adsorption efficiency was calculated according to Equation (1):

E(%)=C0−CeC0×100E(\%) = \frac{C_0 - C_e}{C_0} \times 100E(%)=C0​C0​−Ce​​×100

where C₀ and Cₑ (mg L⁻¹) are the initial and equilibrium metal ion concentrations, respectively.

2.6. Statistical analysis

All experiments were conducted in triplicate (sample size N = 3 for each measurement). The data were expressed as mean ± standard deviation. Statistical significance was assessed using one-way ANOVA (p < 0.05) in OriginPro 2024 [11].

3. Results and Discussion

3.1. Yield of BNC under different cultivation conditions

The yield of bacterial nanocellulose produced by Brevundimonas spp. under different nutrient conditions is presented in Table 1.

The results indicate that cellulose production is highly dependent on both the composition of the medium and the cultivation mode. The LB medium provided the highest BNC yield (42.8 g L⁻¹ wet mass, 4.8 g L⁻¹ dry mass), whereas the lowest production was observed in MMD medium (1.2 g L⁻¹ dry mass). The HS medium showed slightly lower productivity but yielded more homogeneous pellicles. These results are consistent with previous reports describing Brevundimonas-derived cellulose as sensitive to nitrogen content and pH of the medium [1, 2].

As shown in Table 1, the nutrient medium strongly affects BNC yield.

Medium      Wet mass (g/L)     Dry mass (g/L)      Final pH

Table 1.

Values

These differences indicate that nitrogen availability and pH significantly influence Brevundimonas cellulose synthesis.

The results demonstrate that the static cultivation method led to the formation of dense and uniform pellicles, whereas agitation resulted in dispersed fibrous networks, which may facilitate the use of BNC in composite material formation [3, 4].

3.2. Structural and mechanical properties

The physicochemical parameters of the obtained BNC are shown in Table 2.

BNC produced by the Kombucha consortium exhibited a significantly higher crystallinity index (85%) and tensile strength (210 MPa) compared with Brevundimonas spp. (60%, 140 MPa, respectively). These values indicate that Kombucha-derived cellulose possesses a more ordered fibrillar structure, which contributes to its mechanical robustness and superior water retention capacity (1100%) [5, 6].

Microorganism     Crystallinity (%)   Tensile Strength (MPa)  Water Retention (%)

Table 2.

Structural and mechanical differences between BNC samples

Higher crystallinity in Kombucha-BNC correlates with its multilayered fibrillar morphology.

The high degree of crystallinity in Kombucha-BNC suggests a tightly packed fibre arrangement with a higher proportion of cellulose Iα and Iβ allomorphs [7]. SEM micrographs confirmed that Kombucha BNC forms compact multilayered structures, whereas Brevundimonas BNC displays thinner, interwoven fibrils, consistent with other recent findings [8, 9].

3.3. Adsorption efficiency towards heavy metal ions

The adsorption capacities of native and functionalised BNC towards metal ions are summarised in Table 3.

It can be observed that the functionalised (carboxylated) Kombucha-derived BNC showed the highest affinity for Pb²⁺ and Fe³⁺ ions, reaching removal efficiencies of 94.0% and 62.5%, respectively. In contrast, Brevundimonas BNC demonstrated a higher selectivity for Mn²⁺ and Cd²⁺ ions (81.0% and 43.7%, respectively).

Metal ion    Kombucha BNC (%)      Brevundimonas spp. BNC (%)

Table 3.

Presents the metal-ion adsorption efficiencies of native and carboxylated BNC

These findings correspond well with earlier research indicating that the introduction of carboxyl and hydroxyl groups enhances metal ion coordination via complexation and hydrogen bonding mechanisms [10, 11].

The difference in adsorption behaviour between the two types of BNC can be attributed to the variations in fibril density, surface area, and distribution of functional sites [12].

Additionally, the enhanced performance of Kombucha-derived BNC for Pb²⁺ and Fe³⁺ can be associated with its higher crystallinity and increased negative surface charge after carboxylation. These characteristics promote ion exchange and electrostatic attraction of cations from aqueous media [13].

4. Conclusion

This study quantitatively confirms that both the microbial source and the cultivation mode play a decisive role in determining the yield, structure, and adsorption performance of bacterial nanocellulose (BNC). The highest BNC yield produced by Brevundimonas spp. was achieved in LB medium, reaching 4.8 g L⁻¹ (dry mass), whereas the lowest yield was observed in MMD medium (1.2 g L⁻¹). Static cultivation resulted in the formation of dense and uniform pellicles, while agitated conditions promoted the development of more dispersed and flexible nanofibrillar networks.

BNC obtained from the Kombucha symbiotic consortium exhibited a significantly higher crystallinity index (85%), tensile strength (210 MPa), and water retention capacity (1100%) compared to BNC derived from Brevundimonas spp. (60% crystallinity, 140 MPa tensile strength, and 650% water retention). These findings indicate a more ordered and compact nanofibrillar structure in Kombucha-derived BNC.

Chemical functionalisation through carboxylation substantially enhanced the adsorption properties of BNC, particularly for Pb²⁺ and Fe³⁺ ions. Carboxylated Kombucha-derived BNC demonstrated the highest removal efficiencies, reaching 94.0% for Pb²⁺ and 62.5% for Fe³⁺, while Brevundimonas-derived BNC exhibited greater selectivity for Mn²⁺ (81.0%) and Cd²⁺ (43.7%).

Overall, carboxylated Kombucha-derived BNC can be considered a highly promising, environmentally friendly biosorbent for the efficient removal of lead and iron from wastewater. In contrast, Brevundimonas-derived BNC shows potential for the selective removal of manganese and cadmium. The results of this study provide a reliable, data-driven basis for further optimisation and scale-up of BNC-based materials in sustainable wastewater treatment technologies.

5. Disclosure and Author Responsibility

Responsibility for the content.

The authors take full responsibility for the content of this publication.

Conflicts of interest.

The authors declare that they have no conflicts of interest to disclose which are relevant to the content of this article.

Author contributions.

We confirm that all authors made a significant contribution to the development of this work.

Madina Kudratkhojaeva performed the experimental research and manuscript writing.

Sherzod Yuldoshov was responsible for conceptualisation, supervision, and editing of the manuscript.

Elmurod Egamberdiev contributed to data visualisation and manuscript review.

All authors discussed the results, reviewed, and approved the final version of the manuscript.

6. Acknowledgements

The authors express their sincere gratitude to Professor Sandra Pucciarelli and Solomon Nnaemeka Ogudjuba from the University of Camerino, School of Biosciences and Veterinary Medicine, Camerino, Italy, for their valuable advice and continuous support during the course of this research.

7. Data Availability Statement

All relevant data supporting the findings of this study are available from the corresponding author upon reasonable request.

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