A new application of bacterial cellulose in textiles and fashion: using Kombucha-derived biofilm to remove dye from polluted water

TL;DR

This study explores using Kombucha-derived bacterial cellulose to remove synthetic dyes from polluted water, offering a sustainable solution for the fashion industry.

Contribution

The study introduces bacterial cellulose as a novel material for dye removal in textile wastewater treatment.

Findings

Kombucha-derived biofilms reduced dye color intensity by over 79% for acid blue and 63% for reactive navy.

Biofilms developed in black tea with active microbes showed the most effective dye removal.

The microbial consortium in Kombucha may help address color pollution in dyestuff wastewater.

Abstract

The fashion and textile industries face mounting pressure to adopt sustainable practices due to their environmental impacts, including waste generation and water pollution from dyeing. Bacterial cellulose, a renewable, biodegradable material produced via microbial fermentation, offers a promising solution. While bacterial cellulose has been explored as a sustainable textile material in fashion apparel, this study introduces its potential for removing synthetic dyes from dyehouse wastewater. Dyeing processes produce wastewater contaminated with synthetic dyes, which are toxic, persistent, and bio accumulative, posing ecological risks. Bacterial cellulose’s nanofibrillar structure makes it effective for capturing liquid contaminants through chemical bonding and physical trapping. Using a microbial consortium (Kombucha), bacterial biofilms were developed over 30 days in either black tea and sugar or Hestrin and Schramm medium. They were then immersed in dye-polluted water. Kombucha-derived bacterial cellulose biofilms reduced dye colour intensity by over 79% for acid blue and 63% for reactive navy, with the most effective results from biofilms developed in black tea containing active microbes. Results indicate that the microbial consortium in the Kombucha-derived pellicle may have a role in removing colour pollution from dyestuff wastewater, thereby presenting a sustainable pathway for addressing key environmental challenges in fashion and textile industries.

Figures

Dyestuff concentration (ppm) acid blue. Dotted line shows measured concentration of Initial dyestuff prior to inoculation (16.6 ppm). Error bars = standard deviation, *n* = 3.

Dyestuff concentration (ppm) reactive navy. Dotted line shows measured concentration of initial dyestuff prior to inoculation (13.0 ppm). Error bars = standard deviation, *n* = 3.

Hue and intensity change of (a) acid blue and (b) reactive navy dye solution after Biofilm Inoculation. Images produced using ImageJ Software ([www.imagej.net](http://www.imagej.net)).

Colour (Hue & intensity) change of biofilm inocula removed from (a) acid blue and (b) reactive navy dye solution. Images produced using ImageJ Software ([www.imagej.net](http://www.imagej.net)).

Acid blue solution absorbance vs. concentration at 597 nm. ε = 0.01318, R^2^ = 0.9770.

Reactive navy solution absorbance vs. concentration at 583 nm. ε = 0.02416, R^2^ = 0.9881.

Full text

Introduction

Fibres used in the textile industry can be categorized into three main types: natural cellulosic (e.g., cotton, linen), natural protein (e.g. wool, silk) and synthetic (including modified fibres -e.g. polyester, nylon, acrylic, acetate, viscose)^1^. Most textiles require colour to be imparted on the fibres and the techniques and chemicals used to undertake this process are varied^2,3^. Many end-uses need a mixture of fibre types in a fabric; thus, a mixture of dyestuffs and auxiliary chemicals may be used in any one process to achieve the desired output.

The process of dyeing and finishing textiles demands large volumes of water, with estimates suggesting 120–220 L per kilo of fabric^4^, generating vast quantities of highly polluted liquid waste or effluent^1,2,5^. This waste contains a range of organic and inorganic chemicals in both dissolved and suspended solid form, such as sizing agents, finishing agents, inhibitors, salts, chlorine compounds and phosphates alongside the actual colour^1,2,6–8^. The composition of the effluent is highly dependent on the specific manufacturing process, and it is not uncommon to see large fluctuations in pH, colour, salinity, temperature, biochemical oxygen demand (BOD) and chemical oxygen demand (COD), which are all strictly measured before the liquid is deemed safe for discharge^1,3^. Discharge of effluent into natural waterways, untreated to a sufficient standard, can cause irreversible damage to aquatic life and potable water^8,9^.

Whilst traditional dyestuffs from natural sources are still used, most modern dyehouses use synthetic dyestuffs^10^. Cao et al.^11^ estimate 280,000 tonnes of these dyes are discharged as waste worldwide on an annual basis. They are categorized according to their chemical structure (e.g., azo, anthraquinone, sulphur) and mode of application (e.g., reactive, basic, disperse, acid)^12,13^.

Dyes have been modified to improve ease of application^8^ and to resist breakdown by exposure to sunlight, water, soap, bleaching and perspiration to improve the colour’s longevity, performance, and customer satisfaction of the product^14,15^. Dyestuffs displaying these properties are complex structures with a high molecular weight and therefore a high biodegradability index^2,16^. For example, anthraquinone – based dyes have an aromatic molecular structure and are more resistant to breakdown in nature, whilst some metal dyes are based on chromium and release carcinogenic chromium into the environment upon breakdown^17^. Whilst there is a general trend in the industry to seek alternatives to some of the more toxic dyes and chemicals (with some already banned from use), the amount of dyestuff that is not fixed to the textile during the dyeing process remains an ongoing challenge, as this is discharged in effluent.

The textile industry is evolving, with new technologies to address the significant issue of coloured effluent^18,19^. Studies indicate that 15–50% of dyestuffs used in textile production are not fixed to fibers and are discharged as waste^20,21^. Estimates suggest that around 280,000 tons of textile dyes are discharged globally each year^22^. Even at concentrations below 1 ppm, dye residues can be highly visible and degrade the aesthetic quality of water^17,23^. Although colour may not always be the most toxic component, it can indicate water pollution and affect gas solubility^8^. Additionally, some dyes can break down into harmful by-products when exposed to sunlight and water^24^. As global standards tighten, the adoption of effective color removal and wastewater treatment technologies becomes increasingly important^23^.

Traditional wastewater treatments can be expensive and inefficient in the removal of many coloured dyestuffs from effluent^17,25^, possibly due to the complexity and diversity of the molecular structures that make up dyes used in the modern industry^26^. Physical (e.g. flotation, flocculation, precipitation), chemical (e.g. ion exchange, oxidation, activated carbon) and biological methods (e.g. biosorption, enzymatic degradation) have been explored with varying degrees of success^14,21,27–31^. However, the costs of the treatments are frequently prohibitive, and no single technique alone has been completely effective.

Microbiological approaches are considered an emerging field in wastewater treatment, and several bacterial and/or fungal species can efficiently break down a dye^32^. It is considered that the microbes in consortia adapt their metabolic activities depending on the surrounding conditions^33^. Wu^28^ suggests that when conditions are favourable, the mix of microorganisms can exhibit enhanced performance (compared to single species inocula) in terms of adsorption and degradation of contaminants and therefore can prove useful in the removal of pollution in wastewater. The consortium is often consolidated by extra polymeric substances (EPS), usually made up of proteins and carbohydrates known as biofilm. Sheng^34^ suggests that the content and physical structure of the EPS is critical in terms of the adsorption ability, surface characteristics and formation of the microbial community. Sharma^35^ discusses that the effluent treatment performance is enhanced by the microbes being considered ‘immobile’ due to the EPS structure, and that this can allow the consortium to be used for multiple treatment cycles, thus, reducing the cost of the process.

Bacterial cellulose (BC) has been explored in water treatment applications as an adsorbent and nano-filter due to its high strength, surface area, and nanofibrillar structure^36^. BC can be produced as a biofilm from a microbial consortium, such a Kombucha. Kombucha is known as a symbiotic culture of bacteria and yeast (SCOBY). It is formed as a thick, gelatinous biofilm on the surface of liquids during a fermentation process which is often used to produce drinks purported to support human health^37–39^. Some of the bacterial species found in the Kombucha biofilm (e.g. Komagataeibacter xylinus) produce nanofibrils of BC and it is these nanofibrils, the microbial and yeast communities and the EPS present in the Kombucha of particular interest in this study. However, no evidence could be found in the existing literature to suggest that biofilms developed from Kombucha have been investigated as colour removers for dyehouse effluent.

This paper presents the findings of an exploratory study to test the potential effectiveness of a BC biofilm developed from a Kombucha inoculum in the capture of colour from dyehouse liquid waste.

Results

Colour reduction in dye solutions

Analysis of optical density measurements for both dye solutions indicated that there were no significant differences in dyestuff concentration across incubation times (Figs. 1 and 2). Therefore, the analysis focused on the modes of dye solution treatment rather than the treatment durations.

Fig. 1. Dyestuff concentration (ppm) acid blue. Dotted line shows measured concentration of Initial dyestuff prior to inoculation (16.6 ppm). Error bars = standard deviation, n = 3.

Fig. 2. Dyestuff concentration (ppm) reactive navy. Dotted line shows measured concentration of initial dyestuff prior to inoculation (13.0 ppm). Error bars = standard deviation, n = 3.

Acid blue

Acid Blue dye solution inoculated with biofilms developed in tea broth (tea biofilms) that had not been treated with NaOH visually displayed the greatest reduction of colour intensity and hue, as illustrated by the analysis of the photographed solutions using Image J software (Fig. 3). As these biofilms had not been treated with NaOH, they were assumed to contain active microbial communities. This visual reduction in colour was supported by the measurements of dyestuff concentration using the spectrophotometer (Fig. 1). A significant difference (p < 0.0001) was measured between the optical density of the solutions inoculated with untreated tea biofilms compared to all other inocula types; less colour remained in these solutions. The reduction in optical density (and therefore reduction in colour intensity) ranged from 79% in tea (dry) after 7 days to 61% in tea (wet) after 56 days.

In contrast to the tea biofilm inocula, the biofilms developed in H&S liquid medium (H&S biofilms) did not reduce colour in the Acid Blue dye solutions to the same degree, highlighted by both optical density measurements (Fig. 2) and visual observations (Fig. 3). However, H&S (dry) showed a shade change to a green / brown hue (Fig. 3), speculated to be due to microbial cell activity on the surface of the biofilm.

Fig. 3. Hue and intensity change of (a) acid blue and (b) reactive navy dye solution after Biofilm Inoculation. Images produced using ImageJ Software (www.imagej.net).

Reactive navy

The colour of Reactive Navy dye solutions visually changed when inoculated with different biofilm types (Fig. 3). As observed with Acid Blue, treatment with biofilms developed in tea was more effective in colour removal from solutions than treatment with H&S derived biofilms. The highest Reactive Navy colour reduction both visually (Fig. 3) and according to optical density measurements (Fig. 2) was achieved in solutions inoculated with tea biofilms not treated with NaOH (63.5% - tea (dry) − 42 days).

Colour reduction by biofilm type

Tea biofilms used as inocula were significantly more effective (p < 0.0001) at reducing colour intensity of dye solutions than those developed in H&S media, whilst biofilms treated with NaOH were significantly less effective (p > 0.05) at reducing colour intensity than those left untreated. For example, in Acid Blue, tea (dry) biofilms reduced colour by 79% compared to tea (dry NaOH) 15%; in Reactive Navy the maximum reduction was achieved by tea (dry), 63.5%, whilst tea (dry NaOH) achieved 29.5% (Figs. 1 and 2).

Biofilm colour uptake

After removal from the dye solution, as the biofilms dried, they became wrinkled (i.e. did not have a flat surface) therefore collection of light absorbance data using a spectrophotometer was not possible. Images of the biofilms were processed using ImageJ software and the results presented in Fig. 4.

Fig. 4. Colour (Hue & intensity) change of biofilm inocula removed from (a) acid blue and (b) reactive navy dye solution. Images produced using ImageJ Software (www.imagej.net).

Reactive Navy and Acid Blue tea biofilms were darkest in appearance, supporting the findings previously discussed of the reduction in colour of the dye solutions. Except for H&S (dry) biofilms, colour adsorption was observed in all biofilms submerged in Reactive Navy dye solution.

In general, biofilms were more heavily stained with colour from the Reactive Navy dye solution compared to those submerged in the Acid Blue solution, in contrast with the measurements of dye remaining in the liquids when the biofilms were removed (colour intensity was less in Acid Blue solution compared to Reactive Navy solution after biofilm treatment).

Discussion

There have been various studies evaluating the effects of both bacterial and fungal species on the reduction of colour intensity in dye solutions.

Using single species of bacteria in suspension, colour intensity reductions of 100%, 87% and 76% have been reported for Reactive Red 22 (Escherichia coli)^40^, Reactive Golden Yellow (Brevibacillus laterosporus)^41^ and Reactive Black 5 (Aeromonas hydrophilia)^42^ respectively. Other studies have found some success using single fungal species as colour solution treatments with colour reduction rates reported of up to 100% on Acid Blue (Candida tropicalis)^43^ and Reactive Blue (Trichosporon akiyoshidainum)^44^. Further studies have evaluated the effects of microbial consortia, with reports of reductions of 94%^45^ and 100%^46^ on Acid Red and Reactive Blue respectively.

Whilst this study demonstrated a smaller reduction in colour intensity than those mentioned in literature surveyed, most existing studies focus on reactive dyes, with relatively fewer reporting effects on other dye classes. No evidence could be found in the literature of a single strain bacterial approach to reducing the colour of acid dye. This could be due to the prevalence of reactive dyes in the modern textile industry, but it is suggested that this could also be due to the ability of microbial species to break down the different dye molecules found in other dye classes.

However, an important distinction between the literature stated above and this study is that the treatments were applied in suspension and not as part of a biofilm, thus only the effect of the microbial degradation was considered. Evidence of a biofilm approach to dye effluent treatment using microbial consortia is limited. Agrawal et al.^26^ reported 81% colour reduction in effluent using a biofilm developed on a bed of porous brick pieces when applied to a variety of acid and reactive dyestuffs. However, the microbial consortium used was collected from the indigenous microbial populations surrounding the dyehouses from which the effluent was discharged. The biofilm used in this study (Kombucha) did not need to be specifically developed, it is already freely available as both a food stuff and a method for drinks fermentation. Additionally, it is widely reported that fermentation of Kombucha can be considered a continuous process; that is the biofilm can be produced as an inexhaustible supply if the growth media is replenished with sufficient liquid and carbon source^47,48^, thus providing an inexpensive route for wastewater treatment.

Another important feature of this study was the effect of the cellulosic fibres within the biofilm on the capture of colour from the dye solutions. In the dyeing of cellulosic fibres, dye uptake is generally improved by the ‘wetting out’ or pre swelling of the fibres before the dye is introduced because this increases the surface area of the fibre and improves diffusion of the dye into the fibre structure^49,50^. NaOH is commonly used in this process (this is referred to as mercerization when used on cotton fibre^51^. Additionally, NaOH is used to improve the adherence of reactive dye molecules to the cellulose fibre by activating cellulosic -OH groups and acting as a catalyst to enhance covalent bond formation. Therefore, it was anticipated that treating the biofilms with NaOH would improve their uptake of reactive dye by making the cellulosic nanofibres more receptive to the dye molecules. However, in this study, treatment of the biofilms with NaOH resulted in significantly less colour reduction in the solutions, indicating a poorer uptake of dyestuff by biofilms treated with NaOH. It has been suggested that treatment with NaOH could affect the molecular structure of cellulose by increasing its crystalline area^52,53^ and thus reduce its ability to take up dye, which could explain the differences observed. Nevertheless, the primary use of NaOH in this study was to lyse any residual bacterial cells within the biofilm, thus the effects of a BC biofilm without a residual bacterial community (NaOH treated) could be compared to one with an active community (untreated). Thus, the putative absence of viable microbes (due to the NaOH treatment) may have had some effect on the ability of the NaOH treated biofilms to reduce colour intensity in the dye solution.

Additionally, there was no significant difference between the colour reduction of Acid Blue and Reactive Navy dye solutions when treated with tea biofilms (wet or dry) across incubation times. It was expected that there would be a greater effect on Reactive Navy dye solutions (as explained above). However, as the effects of the biofilm treatment were similar in both dye types, it is suggested that alongside chemical bonding to the cellulose fibres, there could be other mechanisms responsible for colour reduction. This suggests that Kombucha derived BC biofilms could be capable of offering a treatment option for a variety of dye classes often found in dyehouse effluents.

One such mechanism could be the trapping of the dye molecules within the nanofibrillar matrix as suggested by previous studies^36,54^. Additionally, it is postulated that this nanofibrillar matrix could offer an increased surface area for dye molecule capture, providing an alternative to conventional adsorbent approaches. Whilst this could prove effective for acid dyes (that are unable to form chemical bonds with the cellulosic fibre), this could also be a method of capturing hydrolysed reactive dye molecules that are unable to bond with cellulose after reacting with water in the dye solution. Hydrolysed reactive dyes are an identified source of colour in dyehouse effluent, so this mechanism of capture could prove effective in pollution treatment^55^.

The other mechanism of colour reduction could be that of microbial activity. However, Ali^56^ and Mishra^57^ agree that hue change can occur due to microbial breakdown of the dye structure and whilst this leads to colour change, it does not necessarily lead to loss of colour intensity and could even produce toxic by-products. Whilst microbial metabolism was not measured as part of this study, its effect cannot be disregarded as greater reduction in colour intensity was noted in biofilms not treated with NaOH and therefore considered to contain active microbes. The enhanced performance of untreated biofilms noted in this study underscores the potential importance of microbial activity in colour removal.

However, previous studies have remarked that it is the combination of microbial and physical approaches (e.g. adsorption) that have been found to be most effective at colour removal from effluent^58–60^.

The dyes in this study were of anthraquinone (Reactive Navy) and azo (Acid Blue) molecular structure, both of which have shown a degree of resistance to breakdown by microbial processes in the natural environment^2,17^. Nevertheless, in other studies, it has been noted that Pseudomonas sp. and Candida sp. are effective in colour reduction of reactive dye solution. Pseudomonas sp. reduced colour intensity by 80–99% in Reactive Red solution^61,62^ and Candida sp. reduced colour intensity by 90% in Reactive Blue solution^63^. Pseudomonas sp. and Candida sp. were both identified in the communities of bacteria present in the Kombucha SCOBY used in this study^64^. However, the effects may be strain-specific: the work should be repeated with pure cultures of appropriate test species. In short, a change in hue, rather than a reduction in colour intensity is not useful for effluent treatment, regardless of possible molecular breakdown, as colour intensity reduction is critical before liquid waste can be discharged into the environment. Thus, the kombucha pellicle consortium is of more interest than suspended cells in terms of dye removal/metabolism.

The findings suggest that it is a combination of the BC biofilm developed in tea and the active microbial community that has the highest efficacy in colour reduction in dye solution in thecases of both Acid Blue and Reactive Navy. In accordance with these results, Suvilampi^32^ states that the most effective colour reduction occurs when a variety of bacterial and fungal species are used to treat dye solutions. Additionally, this concurs with the findings of Wang et al.^65^, Hayat et al.^59^ and Masi et al.^60^ who all agree a biological approach to the removal of colour from effluent is most effective when combined with other approaches, such as adsorption.

Overall, this preliminary study presents the potential advantages in the use of Kombucha derived BC biofilms for sustainable effluent treatment. The biofilms can be produced continuously and inexpensively in black tea using readily available components, eliminating the need for complex material manufacture or specialized microbial culture facilities. The potential of dual functionality – combining the nanofibrillar matrix adsorption functions with those of an active mixed microbial community – makes these biofilms a realistic proposition for the treatment of coloured wastewater from dyehouses.

Conclusions

The textile and fashion industries are under pressure to address the issues of pollution due to dyehouse effluent emmissions using sustainable approaches. Whilst there are methods currently in use to treat coloured liquid pollution from dyehouses, most are either expensive and not 100% effective or not produced from sustainable sources. This study evaluated the effectiveness of BC biofilms developed using Kombucha SCOBY inocula in black tea and H&S media for their ability to reduce colour intensity in single dye solutions.

The study found that biofilms developed in tea (and not treated with NaOH thus containing an active microbial community) achieved the highest reductions, lowering colour intensity of Reactive Navy and Acid Blue dye solutions by approximately 30–35% over the incubation period.

In contrast the biofilms developed in H&S medium and treated with NaOH (therefore lacking active microbes), reduced colour intensity by only 10–15%. These results indicate that both the microbial activity and the nanofibrillar structure of the biofilm contribute to dye removal. Whilst these reductions are lower than those reported for single species bacterial treatments in suspension (up to 100% for some reactive dyes), the use of biofilms cultivated in black tea is particularly promising for wastewater treatment containing mixed dye types. Kombucha biofilms can be produced sustainably and continuously using inexpensive and widely available materials without the need for specialized microbial cultures. The similar efficacy observed for Acid Blue and Reactive Navy suggests that the biofilm can act on multiple dye classes, likely through a combination of adsorption into the cellulose matrix and microbial metabolic activity.

A limitation of this study is the use of single dye solutions in distilled water, rather than complex multi component dyehouse effluent. It is recommended that future work should assess the effectiveness of the Kombucha SCOBY biofilms under industry relevant conditions and explore strain-specific influences within the microbial consortium to optimise colour removal.

Nevertheless, this study indicates that BC biofilms with active microbial communities offer a practical and sustainable approach for reducing coloured dye pollution in textile effluents.

Methods

Inoculum preparation

To prepare the stock inoculum, a commercially available 200 g Kombucha SCOBY starter in 100 mL of green tea was purchased from a web-based company ‘Happy Kombucha’ (https://happyKombucha.co.uk/). This starter was stored in its original tea broth, in ambient conditions as advised by the supplier (room temperature 22 °C + / − 2 °C, on a bench top). Whilst the exact composition of this tea broth was not known, it had a base of green tea. The microbial composition of the Kombucha starter has previously been sequenced and reported^64^. 250 mL Hestrin and Schramm medium was prepared (Table 1) and 100 g of the Kombucha SCOBY starter added and incubated to provide a standardised inoculum. A lid was loosely placed on the pot. This pot was incubated at 30 °C for 15 days after which, any solid biofilm was removed from the pot by filtration and discarded: and the remaining filtrate (broth) was used immediately as inoculum. This process was repeated to produce sufficient Kombucha liquid inoculum required for the study.

Table 1. Media to support biofilm growth.Type of mediumPreparationCommentsBlack teaTea broth was prepared by steeping 1 teabag (‘Yorkshire’ brand black tea) in 1 L boilingwater for 15 min (approx. 3 g / l tea). The bagwas removed, and 100 g glucoseadded (10%)After preparation, each liquid was decantedinto separate sterile 500 mL Duran bottles andsterilised in an autoclave for 10 min @ 115 °C(the lower temperature was used to reducerisk of glucose caramelisation)Hestrin & Schramm2% Glucose, 0.5% Bactopeptone, 0.5%Yeast extract added to 1 L distilled,deionised water

To prepare the tea and H&S biofilms required for the study, 200 mL medium was decanted into a 500 mL sterile pot (4 pots each of black tea with sugar and H&S (Table 1)) and inoculated with 20 mL of standard Kombucha liquid inoculum. The lids of the pots were loosely attached, and the pots placed in a 30 °C incubator for 30 days. After the incubation period, biofilms had developed on the surface of the sterile media and were removed from the pots.

Once removed, one biofilm per growth medium was treated by either:

• Cutting into 1 g pieces and stored in sterile water until needed (untreated control).
• Removing moisture by pressing the biofilm between sheets of filter paper. The biofilm was then cut into pieces weighing 1 g each. The pieces were placed on a petri dish and dried in a 60 °C fan oven for 24 h. The dried pieces were stored in a petri dish in a desiccator until needed (dry).
• Placing the biofilm in a conical flask, containing 300 mL 0.1 M NaOH and heated to 80 °C in a water bath for one hour. The flask was then removed from the water bath, allowed to cool and the biofilm removed. The surface liquid of the biofilm was removed by pressing the biofilm between sheets of filter paper. The biofilms were then cut into pieces weighing 1 g each. The pieces were placed on petri dishes and dried in a 60 °C fan oven for 24 h. The dried pieces were stored in a petri dish in a desiccator until needed (NaOH dry).
• Placing the biofilm in a conical flask, containing 300 mL 0.1 M NaOH. The conical flask was placed in a water bath and heated to 80 °C for one hour. The flask was removed from the water bath, allowed to cool and the biofilm removed. The biofilm was cut into 1 g pieces and stored in sterile water until needed (NaOH wet).

This resulted in four sets of prepared biofilms per growth medium, with 3 replicates per permutation.

Dye solution preparation

To create the dye solutions for testing with the Kombucha BC biofilm and liquid inocula, 3 L of dye solution per dye (Procion Reactive Navy MX (Reactive Blue 4) and Acid Brilliant Blue (Acid Blue 9) were prepared by dissolving 20 ppm powdered dyestuff in distilled water. The absorbance of the dye solution was measured (597 nm for Acid Blue and 583 nm for Reactive Navy) using a Jenway 6305 spectrophotometer (Fischer Scientific, Leicestershire, UK). A 20ppm dye concentration was used because it produced absorbance values in a linear reliable range of the spectrophotometer. A calibration curve was prepared for each dye to confirm Beer-Lambert linearity, as detailed in section  “Sample evaluation”.

15 mL of dyestuff was decanted into universal tubes (total volume 20 mL) and the lids tightly screwed on until ready for use. Three pots per inoculum type per sample timepoint were prepared (a total of 120 pots per dye type).

Inoculation

Each universal tube of dyestuff was inoculated with either 1 g biofilm or 1 mL of reserved growth medium, as described in Table 2. The lids of the universal tubes were replaced loosely to support the aerobic activity of the microbial consortium. Each sample was replicated in triplicate. Five sets of triplicate samples were prepared per dyestuff type and placed in a 30 °C incubator, in the dark to prevent any dye degradation due to UV light. One set per dyestuff was removed after one, two, four, six, and eight weeks to assess the effect of both the inoculum and incubation time on the colour intensity of the liquid.

Table 2. Details of inoculum preparation.InoculumGrowth mediumPreparationReferenceSolid biofilmH&SSurface liquid removed anddried at 60 °C for 24 h andstored in a desiccatorH&S (dry)Placed in distilled waterH&S (wet)Treated with NaOH (0.1 M)for 1 h at 80 °C. Dried at60 °C for 24 h and storedin a desiccatorH&S (NaOH dry)Treated with NaOH (0.1 M)for 1 h at 80 °C. Placedin distilled waterH&S (NaOHwet)Black tea with sugarSurface liquid removed anddried at 60 °C for 24 h andstored in a desiccatorTea (dry)Placed in distilled waterTea (wet)Treated with NaOH (0.1 M)for 1 h at 80 °C. Dried at60 °C for 24 h and storedin a desiccatorTea (NaOH dry)Treated with NaOH (0.1 M)for 1 h at 80 °C. Placedin distilled waterTea (NaOH wet)Liquid mediumH&SDecanted from growth potH&S liquidBlack tea with sugarDecanted from growth potTea liquid

Sample evaluation

At the end of incubation period the wet biofilms were removed and placed with sterile forceps onto a petri dish, photographed (Samsung 12 MP camera with LED flash at 1.5 x optical zoom), visual observations (hue and colour intensity) were recorded, and the biofilms allowed to dry in ambient conditions until constant weight (24 h).

The remaining liquid in each of the universal pots was photographed and visual observations recorded. Aliquots were placed into a cuvette and optical density (path length = 1 cm) measured on a Jenway 6305 spectrophotometer (Fischer Scientific, Leicestershire, UK) at 597 nm for Acid Blue and at 583 nm for Reactive Navy dye samples. Each measurement was performed in duplicate, from opposite sides of the cuvette. The Beer - Lambert law was used to translate absorbance data into concentration (parts per million) dyestuff remaining in the dye solution as follows:

\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$C\,=\,A/\varepsilon b$$\end{document}

Where: A = absorbance; ε = molar absorptivity; b = length of light path; C = dye.

concentration.

For this spectrophotometer model, the light path length was 1 cm. The molar absorptivity (ε) had been previously calculated by plotting the measured absorptivity against a range of known concentration of each dyestuff and calculating the gradient of the slopes. R^2^ indicates ‘goodness of fit’ of the linear slope to the plotted points. In both cases, this is close to 1, indicating the slope is an accurate representation of the trend of the plotted measurements (Figs. 5 and 6).

Fig. 5. Acid blue solution absorbance vs. concentration at 597 nm. ε = 0.01318, R^2^ = 0.9770.

Fig. 6. Reactive navy solution absorbance vs. concentration at 583 nm. ε = 0.02416, R^2^ = 0.9881.

Percentage reduction of colour intensity was calculated by comparing the optical density measurement of the untreated dye solution at the start of the experiment with the optical density of the treated dye solution at each timepoint sampled as follows:

\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\mathrm{Colourreduction}}\% {\text{ }}={\text{ Opticaldensity TDS}}/{\mathrm{OpticaldensityUDS}}$$\end{document}

Where: TDS = treated dye solution; UDS = untreated dye solution.

The data collected was analysed using ANOVA methods to evaluate the statistical significance of any differences observed.

The photographs of the biofilms and liquids were processed and analysed using Image J open-source software visual analysis tool (www.imagej.net) to facilitate visual comparison of hue changes.