Abstract
A bacterial consortium was used to assess the potential of bioremediation for treating textile wastewater and evaluating the phytotoxicity effects of biotreated wastewater using tomato seed germination and seedling growth. The optimum conditions for bioremediation were achieved by varying the concentration of inoculum, temperature, incubation time and concentration of textile wastewater for maximum decolorization were 8 mL inoculum, 37 °C, 4 days incubation time, and 25 to 50 mL of the textile wastewater. The optimized treatment was implemented in a laboratory-scale bioreactor, resulting in substantial improvement in the measured wastewater-quality parameters. The COD removal reached approximately 86%, while TSS removal exceeded 80%. The untreated textile wastewater (TW) resulted in a decline in tomato seed germination and seedling development, whereas biotreated wastewater (Bt-W) improved growth performance. The percentage of germination in the control, TW, and Bt-W was 94%, 81%, and 90%, respectively. The seedling vigor index was highest in Bt-W (676.80), compared with the control (655.18) and TW (567.0). Under laboratory scale conditions, bacterial consortium-based biotreatment caused reduction of wastewater related phytotoxic effects and potential future development of reused wastewater from textile effluents. The approach supports the United Nations Sustainable Development Goals.
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Sustainable Bioremediation of Textile Wastewater Using Bacterial Consortia and Its Reuse Potential for Irrigation
Anjala Ayoub,a, Sikander Ali,a,* Muhammad Usman Ahmad,a Ayaz Ali Khan,b Thorya Abdulrahman Fallatah,c Shereen Magdy Korany,d Wejdan T. Alsaggaf,e Fahad Al-Asmari f and Syed Shujait Ali,g,*
A bacterial consortium was used to assess the potential of bioremediation for treating textile wastewater and evaluating the phytotoxicity effects of biotreated wastewater using tomato seed germination and seedling growth. The optimum conditions for bioremediation were achieved by varying the concentration of inoculum, temperature, incubation time and concentration of textile wastewater for maximum decolorization were 8 mL inoculum, 37 °C, 4 days incubation time, and 25 to 50 mL of the textile wastewater. The optimized treatment was implemented in a laboratory-scale bioreactor, resulting in substantial improvement in the measured wastewater-quality parameters. The COD removal reached approximately 86%, while TSS removal exceeded 80%. The untreated textile wastewater (TW) resulted in a decline in tomato seed germination and seedling development, whereas biotreated wastewater (Bt-W) improved growth performance. The percentage of germination in the control, TW, and Bt-W was 94%, 81%, and 90%, respectively. The seedling vigor index was highest in Bt-W (676.80), compared with the control (655.18) and TW (567.0). Under laboratory scale conditions, bacterial consortium-based biotreatment caused reduction of wastewater related phytotoxic effects and potential future development of reused wastewater from textile effluents. The approach supports the United Nations Sustainable Development Goals.
DOI: 10.15376/biores.21.4.10689-10719
Keywords: Bioremediation; Textile effluents; Decolorization; Bacterial consortia; Biotreated water
Contact information: a: Department of Microbiology, Ikram-Ul-Haq Institute of Ind. Biotechnology (IIB), GC University Lahore, Pakistan; b: Department of Biotechnology, University of Malakand Chakdara Dir Lower, Khyber Pakhtunkhwa Pakistan; c: Department of Biological Sciences, College of Science, University of Jeddah, 21959, Jeddah, Saudi Arabia; d: Department of Biology, College of Science, Princess Nourah bint Abdulrahman University, P.O. Box 84428, Riyadh 11671, Saudi Arabia; e: Department of Chemistry, Faculty of Science, King Abdulaziz University, P. O. Box 80203, Jeddah, 21589, Saudi Arabia; f: Department of Food and Nutrition Sciences, College of Agricultural and Food Sciences, King Faisal University, Al Ahsa, Saudi Arabia; g: Biodiversity Genomics Unit, University of Tabuk, 71491, Tabuk Saudi Arabia;
* Corresponding authors: [email protected]; [email protected]; [email protected]
INTRODUCTION
Biological treatment is one of the most recognised and widely applied methods for wastewater management in municipal and industrial practices. Activated sludge systems are one of the more widely used biological wastewater treatment technologies and rely on a diverse microbial community to degrade organic pollutants under aerobic conditions (Metcalf and Eddy 2014). Anaerobic digestion is also commonly used, such as sludge stabilization and biogas production for resource recovery (Liu et al. 2020). Such processes are well-known and highlight the importance of microbial communities in real wastewater treatment systems, such as in organic matter degradation and resource recovery (Drewnowski et al. 2019).
However, industrial wastewater streams such as textile effluents remain challenging to treat due to their complex composition, which contains synthetic dyes, salts, heavy metals, and other persistent contaminants (Mohanty and Kumar 2021; Rai et al. 2024). Anthraquinone dyes can be degraded more effectively by microbial monocultures and developed consortia through the production of specific enzymes. While traditional biological treatment methods can be used to remove organic pollution, the removal of certain contaminants and associated toxicity may not be possible under specific wastewater conditions and operational parameters (Rai et al. 2024; Elnabi et al. 2025). Therefore, rather than replacing conventional biological treatment systems, targeted microbial approaches, including the application of selected bacterial consortia, are being investigated as complementary strategies to enhance treatment performance and reduce wastewater related toxicity (Elnabi et al., 2025). The present study does not aim to replace established wastewater treatment technologies but evaluates whether a selected indigenous bacterial consortia can improve measured textile wastewater quality characteristics and reduce phytotoxic effects under laboratory-scale conditions. Indigenous bacterial consortia may provide additional treatment potential because microorganisms adapted to contaminated environments can possess synergistic metabolic capabilities that support the transformation of complex pollutants (Kumar et al. 2025).
Biological wastewater treatment systems are already based on the concept of microbial enrichment, with biomass retention and acclimation. In aerobic activated sludge systems, the continuous recirculation of microbial-rich sludge allows the development of diverse microbial communities which progressively adjust to the characteristics of the influent and operational conditions. Microbial community structure, stability, and functional adaptation to wastewater composition are closely linked to the effectiveness of activated sludge treatment (Chen et al. 2017; Dottorini et al. 2023). Likewise, anaerobic systems, such as sludge-bed reactors, depend on the presence of retained microbial biomass and structured sludge communities which enable the development of stable consortia and anaerobic degradation pathways, including syntrophic metabolism and methanogenesis (Duan et al. 2021; Wu et al. 2021). These proven treatment methods have shown that acclimation of the microorganisms and selection of the microbial community are key concepts in the successful operation of wastewater treatment processes.
Intentional bioaugmentation with selected microorganisms differs from the natural development of microbial communities in conventional reactors. Purposeful inoculation involves the intentional addition of microorganisms that possess a distinct functional capability that can improve treatment of specific contaminants or speed the adaptation process in the face of difficult conditions, whereas activated sludge and anaerobic systems rely on the ability of the microbial community to adapt over time by maintaining biomass for a extended solids retention periods.
Nevertheless, microbial community composition remains dynamic and is influenced by influent properties, ecological interactions, and reactor operating conditions (Dottorini et al. 2023; Duan et al. 2021). Thus, the potential value of the selected bacterial consortia is not to replace the existing biological treatment systems, but to act as a complement to the existing microbial process for specific functions, especially in complex industrial wastewater streams such as textile effluents.
Phytotoxicity assays are frequently carried out with tomato seeds as bioindicators because of their sensitivity (Baruah et al. 2019). The tomato plant, Solanum lycopersicum, is a widely used bioindicator plant in phytotoxicity testing because of its low tolerance to environmental contaminants and the fast rate of germination. Seeds can be used as a good reproducible indicator of toxicity, as small fluctuations in concentrations of pollutants can lead to large variations in germination, root elongation and seedling growth (Gautam et al. 2015). Tomatoes also have agricultural significance, and the results of the wastewater toxicity or remediation strategies can be directly applied to crop production and food safety. Moreover, it has been seen that the use of tomato seeds to test treated effluents can indicate better germination and vigour of the seedlings, which is a good indicator to measure the efficiency of bioremediation (Baruah et al. 2019). They are easy to handle in laboratory conditions, have a fast growth rate, and have well characterised physiology making them suitable for controlled experiments using bioreactors and pollutant degradation studies (Kumar et al. 2017). Untreated textile effluents can cause a reduction in the germination rate and growth of seedlings because of the presence of dyes and toxic substances that can inhibit seed germination and seedling growth (Rehman et al. 2008; Khan and Malik 2018). However, the effluents generated from the biotreated wastes usually show low toxicity, and at times improve the germination rate and seedling vigour, owing to the good degradation and reduction of hazardous substances by the biological activity in the treated effluents (Alrowais et al. 2023).
While naturally occurring, microbial communities are used in conventional wastewater treatment systems, the addition of selected microorganisms can offer further advantages for certain wastewater treatment problems. Bioaugmentation is the intentional addition of selected microbial strains or mixed cultures to increase the degradation of specific contaminants or to increase the effectiveness of the treatment (Herrero and Stuckey 2015). The bacterial consortium was not used as an alternative to existing biological treatment technologies, but rather as a targeted approach, under optimized laboratory conditions, in the present study. Environmental conditions such as inoculum concentration, temperature, treatment time, and wastewater concentration were optimized to facilitate the activity of the consortium, as environmental factors can have a significant impact on the survival, adaptation, and efficiency of the consortium in the removal of pollutants during the bioaugmentation process (Chettri et al. 2024). Thus, the aim of this study was to investigate whether a selected indigenous bacterial consortium can improve the performance of the textile wastewater treatment beyond the general microbial activities and reduce the phytotoxicity of the wastewater.
The present study aimed to (i) optimize selected bacterial consortia isolated from textile wastewater; (ii) apply bioremediation using designed bioreactor system with textile effluents; (iii) evaluate the changes in the key physicochemical parameters prior to and after bioremediation; (iv) measure the percentage of reduction of metals (Ni, Cu, Cr, Zn, Co, Mn, Pb, Cd) in textile wastewater; and (v) conduct a comparative study of seed germination and growth of seeds in control (tap water) conditions, untreated (TW) and biotreated wastewater (Bt-W). The above goals were designed to gain insights into the potential sustainable use of microbial consortiums for environmentally friendly and potentially scalable treatment of textile effluents (Singh et al. 2021). The objective was to evaluate changes in measured wastewater-quality parameters and contaminant concentrations following treatment and to assess the phytotoxic response of untreated and biotreated wastewater using tomato seed germination and seedling growth.
EXPERIMENTAL
Materials and Methods
Sample collection
Textile wastewater samples were collected in pre-cleaned and sterilized containers from seven distinct textile industrial sites located along the 12 km Lahore-Sheikhupura-Faisalabad Rd, Kot Abdul Malik in Pakistan. For the development of bacterial consortia BC-23, the strains with the highest decolorization percentages were selected from 67 isolates. The consortium comprised one indigenous isolate of Bacillus subtilis (JF70263.1) and three strains of Bacillus cereus with accession numbers of MG563677.1, OP572138.1, and LC052668.1, respectively. This was used as inoculum for the bioremediation of textile wastewater.
Optimization of Bioremediation Parameters
The optimization experiments were performed sequentially rather than independently and included inoculum concentration, temperature, time duration, and concentration of TW. The reason for optimization trials was to determine the most appropriate conditions meant for active bioremediation, which was assessed by decolorization efficiency before scaling up the activity in the bioreactor (Jimoh et al. 2025).
Bioreactor Design
A laboratory-scale batch bioreactor made of stainless steel, with a total capacity of 25 L, was used for the experiment. Approximately 15.5 L was maintained as the working volume, while the remaining capacity served as headspace above the liquid surface during batch operation. The cylindrical bioreactor was equipped with an electrically driven mechanical stirrer to ensure continuous mixing and promote uniform distribution of reactor contents throughout the wastewater matrix (Zaburko et al. 2021). The lower section of the vessel allowed settlement of biomass and particulate material, while a sampling outlet located above this region was used for collection of biotreated wastewater (Bt-W). Mechanical agitation was maintained continuously throughout the 8-day treatment period using an electrically driven stirrer to ensure adequate mixing of the reactor contents with the agitation rate estimated to be approximately 100 to 200 rpm. Temperature was maintained between 37 and 39 °C using an external heat source, which corresponds to a suitable range for mesophilic microbial activity (Meena et al. 2022). The system was operated as a non-aerated batch treatment under mixed conditions rather than as a strictly anaerobic process. Although the reactor contained a lower biomass settlement region, it was not operated as an upflow anaerobic sludge blanket (UASB) reactor because no continuous influent or effluent flow was maintained. The reactor was used to evaluate short-term bioremediation performance under controlled laboratory conditions. The pH was monitored using a pH probe and maintained within the range of 7.5 to 8.0 throughout the treatment period.
Inoculum Preparation and Bioreactor Operation
Initially, each strain was cultured on separate NA (nutrient agar) plates and incubated for 24 h at 37 °C to attain active colonies. The prepared nutrient broth media was distributed into twelve 500 mL conical flasks (three flasks per bacterial isolate). Each flask was inoculated with a loopful of specific bacterial strain and incubated for 6 h at 37 °C under shaking at 130 to 150 rpm. Broth cultures were mixed thoroughly to form a homogeneous mixture of bacterial consortium and were subsequently incubated at 37 °C for 24 h to promote microbial activity and consortium stability. Next, the developed consortium of 3.5 L and was added into the 12 L of TW in bioreactor. The system was operated under controlled conditions to facilitate bioremediation. This approach was consistent with previous findings (Desai et al. 2009).
Each laboratory scale bioreactor experiment was conducted as one independent batch treatment. The biotreated wastewater obtained from this single eight-day batch was subsequently used for physicochemical characterization, heavy-metal analysis, and phytotoxicity assessment. Accordingly, repeated measurements performed on aliquots of this treated wastewater, where applicable, represent analytical or technical replicates rather than independent biological replicates of the bioreactor treatment.
Bacterial Inactivation Under Acetic Acid Stress
Bacterial inactivation was done by subjecting the Bt-W to acetic acid stress which was added at a concentration of 0.05 mL/L, and the pH of the medium was noted. The biotreated effluent bearing acetic acid was consequently cultured for 24 h on nutrient agar plates to evaluate the colony-forming ability (CFA) of the bacterial consortium (Gravemann et al. 2018), the plates were examined for bacterial growth to determine the effectiveness of acetic acid treatment in reducing or eradicating viable bacterial colonies (Gomaa 2012; Hua et al. 2024).
Preparation TW and Bt-W for Seed Germination
Three types of water were used in the germination and seedling growth experiment: tap water, untreated textile wastewater, and biotreated textile wastewater. Tap water was used as the control treatment, while untreated textile wastewater (TW) and biotreated wastewater (Bt-W) were used as the experimental treatments. Before application, both TW and Bt-W were diluted by preparing a 70:30 mixture, consisting of 70% wastewater and 30% clean water. The use of different wastewater concentrations is commonly applied in textile wastewater phytotoxicity studies to evaluate plant responses under controlled exposure levels and to compare the effects on seed germination, radicle, and shoot growth, and biomass production (Das et al. 2019). Seed germination and seedling growth responses were later assessed using germination and phytotoxicity-related indices, following the general approach of seed-based phytotoxicity bioassays and germination index assessment (Kong et al. 2023). The seed germination experiment was performed using three replicates for each treatment group (tap water, untreated textile wastewater, and biotreated wastewater).
Experimental Design for Germination
The tomato seeds were first washed with dH2O to remove dust and dirt from the surface of the seed and then dipped in 2% sodium hypochlorite (NaOCl) solution for 30 minutes (George et al. 2008). This treatment was found to be effective in killing fungi, bacteria, and other contaminants on the seed coat (Cassells 2012). The seeds were then washed with sterile dH2O to remove all traces of NaOCl solution to ensure that no chemicals are present to affect seed germination (Bhojwani and Razdan 1986). Sterilized seeds (100 each) were placed in containers lined with filter paper and moistened periodically with tap water, textile wastewater, or biotreated water as needed to keep the paper moist throughout the germination (Bharti et al. 2018). The seedlings of different days were used to measure the radicle and plumule length to check the performance of the treatments. At the end of the experiment, germination parameters including plumule and radicle length were recorded and compared (Zouagri et al. 2020). Each treatment was conducted in triplicate (n = 3) to account for biological variation among seedlings. Radicle and plumule measurements were expressed as mean ± standard deviation (SD) based on replicate observations.
Physicochemical Characterization of TW and Bt-W
Characterization of the TW and Bt-W for various parameters such as temperature, color, pH, electric conductivity (EC), chemical oxygen demand (COD), biological oxygen demand (BOD), total dissolved solids (TDS), and total suspended solids (TSS) was conducted before and after the bioremediation by bacterial consortium (Bashir et al. 2023). Physicochemical parameters were measured using samples obtained before and after the single bioreactor treatment. Each analysis was performed in triplicate (n = 3) and represents analytical replicates of the same wastewater sample rather than independent biological replicates of the bioreactor treatment.
Statistical Analysis
Statistical analysis was performed using SPSS, version 24. Descriptive statistics were used to summarize the experimental observations. Radicle and plumule lengths were expressed as mean ± standard deviation (SD) based on three replicate observations (n = 3), as reported in Tables 2 and 3. Other treatment outcomes, including physicochemical parameters, heavy-metal concentrations, percentage reductions, germination counts, and derived germination indices, were evaluated descriptively using the observed values and calculated treatment-response measures. Because the laboratory-scale bioreactor treatment was conducted as a single independent batch, inferential statistical testing was not applied to comparisons between untreated and biotreated wastewater. Therefore, no significance threshold was applied to these single-batch comparisons, and statistical significance was not inferred from the observed differences.
RESULTS AND DISCUSSION
Optimization of Bioremediation Conditions for TW Decolorization
Bacterial consortia were subjected to optimization by the concentration of inoculum, temperature, incubation period, and TW concentration to increase bioremediation efficiency (Fig. 1). The decolorization efficiency increased gradually up to an inoculum concentration of 8 mL, at which point the highest decolorization response was observed. It indicated a balance of a suitable level of microbial biomass for effective activity, and the accuracy of the findings was further confirmed by the increasing trend followed by a slight decline, which is consistent with the findings of El‐Bendary et al. (2023). Temperature also strongly affected microbial activity. Decolorization efficiency increased from 30 °C to 37 °C, reaching a maximum of 49% at 37 °C , before decreasing to 43% at 60 °C, which is quite consistent with studies done earlier (Gomez et al. 2020). The observed mesophilic optimum supports microbial physiology, whereby the enzyme systems operate optimally at moderate temperatures. At higher temperatures, the enzymatic systems have been found to become partially denatured and inactive (Paissoni et al. 2024). This classical trend further supports reproducibility of the results. Decolorization was highly influenced by the incubation period. Efficiency rose gradually between day 1 and day 4, then sharply dropped on day 5 and leveled off at 48% (Ikram et al. 2022a). This trend may reflect microbial growth dynamics, with the highest decolorization response occurring during the earlier treatment period and declining at longer incubation times, potentially because of nutrient limitation and the accumulation of metabolic products (Fig. 1). Closeness to known microbial kinetics increases the reliability of the findings (Ikram et al. 2022b). The quantity of wastewater was negatively correlated with the degradation efficiency. Optimal decolorization of 18% was achieved at 25 mL, with a progressive decline in decolorization as the volume increased to 150 mL with 6% decolorization. This is consistent with pollutant overload, oxygen limitation, and recalcitrance effects documented for textile effluents (Zafar et al. 2022). This observation supports the reliability of the results and highlights important practical implications, such as the need for pretreatment or dilution to enhance treatment efficiency. Overall, the systematic variation of parameters and the consistency of the observed trends reinforce the validity of the findings. Finally, the most suitable conditions were 8 mL inoculum, 37 °C, 4 days of incubation, and a range of 25 to 50 mL wastewater concentration to attain maximum decolorization in an experimental flask.
Monitoring Bioreactor and Bacterial Growth
The bioreactor system was continuously monitored to regulate microbial activity during the biotreatment process. For bioreactor operation, 3.5 L of inoculum was prepared with 875 mL per strain distributed in 12 conical flasks (three flasks per bacterial isolate) of 500 mL. The system contained a total liquid volume of 15.5 L, including 12 L of TW and 3.5 L of inoculum.
The pH was maintained between 7.5 and 8, and continuous stirring was carried out for a period of 8 days (Fig. 2). Although maximum decolorization was achieved after approximately four days during flask-scale optimization (Fig. 1C), the laboratory-scale bioreactor experiment was conducted for eight days. The optimization experiment was performed using a lower wastewater volume and a different experimental configuration compared with the subsequent bioreactor treatment. The larger working volume and bioreactor configuration may affect microbial contact, mixing conditions, and biotreatment kinetics during scale-up (Garcia-Ochoa and Gomez 2009).
Acetic acid is used as a control agent against undesirable bacterial growth, and it is in line with the studies indicating that organic acids would have an inhibitory effect on the bacterial metabolism and its capacity (Park et al. 2021). The experimental results showed that growth of the bacteria was greatly inhibited in the presence of acetic acid. All bacterial isolates in the consortium, however, were still viable under normal operating conditions, indicating their suitability and stability for bioremediation applications (Sun et al. 2021). When the isolates were treated with acetic acid, however, the acids were not found to be able to withstand the harsh environment, which shows the inhibitory effect of acetic acid on microbial growth (Huang et al. 2011).
Fig. 1. Sequential optimization of treatment parameters affecting decolorization efficiency, including inoculum concentration, temperature, incubation period, and wastewater concentration: (A) Effect of inoculum concentration; (B) Effect of temperature; (C) Effect of incubation period; and (D) Effect of TW concentration
Fig. 2. Schematic diagram of the laboratory-scale bioreactor
Physicochemical Characterization of Untreated and Biotreated Water
Rate of fermentation
The physicochemical parameters measured before and after treatment were used to evaluate the performance of the bioremediation process. A marked decrease was observed in EC, TDS, TSS, BOD₅, and COD following biotreatment, indicating an overall reduction in the measured pollution indicators (Fig. 3). The reduction in the level of COD was around 85 to 87%, indicating substantial reduction in the oxidizable load of the effluent (Sun et al. 2020). However, a reduction in COD does not always indicate biodegradation or complete mineralization, as it included parts that may not be biodegradable. In addition to biological transformation, a reduction of COD can also occur due to adsorption to biomass, precipitation, sedimentation, or biomass-associated removal (Wei et al. 2019). Therefore, the relative contribution of these processes and complete mineralization to CO₂ and water cannot be established from COD measurements alone (Lacalamita et al. 2024; Fertal et al. 2025). In the same way, TSS removal exceeded 80%. This indicates that the suspended solids were reduced effectively. Conversely, EC and TDS showed moderate reductions indicating the persistence of dissolved inorganic salts (Fig. 4).
Overall, these findings suggested the effectiveness of microbial consortium in enhancing the quality of wastewater. The textile wastewater quality analysis both before and after biotreatment is effectively demonstrated through Figs. 3 to 5. The untreated textile wastewater (TW) exhibited elevated EC, TDS, TSS, BOD₅, and COD, indicating a substantial pollution load (Fig. 3), which is consistent with the characteristics commonly reported for textile-industry effluents (Yaseen and Scholz 2019; Wang et al. 2022). These parameters decreased considerably following biological treatment, together with observed reductions in color and odor, indicating an overall improvement in the measured wastewater-quality characteristics (Holkar et al. 2016; Ceretta et al. 2020). However, the analyses presented in the current manuscript do not establish the specific mechanisms responsible for these changes.
Fig. 3. Comparative physicochemical characteristics of untreated textile wastewater (TW) and biotreated textile wastewater (Bt-W)
Fig. 4. Percentage reduction of selected wastewater quality parameters following biotreatment. The figure provides a comparative assessment of treatment efficiency, while the corresponding measured values before and after treatment are presented in Fig. 3.
Fig. 5. Comparison of wastewater quality parameters with NEQS standards before and after biotreatment
Comparative Assessment of Textile Water
Metal concentrations in untreated (TW) and biotreated wastewater (Bt-W) samples were determined using inductively coupled plasma optical emission spectrometry (ICP-OES). Each sample was analysed in duplicate (n = 2), and the results were expressed as mean ± standard deviation (SD) where applicable. Since the bioreactor treatment was conducted as a single independent batch, these replicate measurements represent analytical variation of the same wastewater samples rather than independent biological replicates of the treatment process. Comparison with the prescribed NEQS limits suggests the efficacy of bioremediation in dropping metal contamination. Untreated TW showed higher Cu, Co, Mn, and Cd levels, supporting the presence of high concentrations of heavy metals commonly found in textile effluents. The bacterial consortium treatment notably reduced the concentration of most metals, showing effective bioremediation potential of the consortium in textile wastewater management.
The concentrations of some metals in the aqueous phase decreased following the reported biotreatment, but the extent of the decrease varied from one metal to another (Fig. 6). Cu showed one of the strongest treatment responses, while Ni, Zn, Mn, Cd, and Co also decreased to varying degrees. This trend of removal of elements is consistent with previous studies showing that microbial consortia do not remove all metals at the same rate. For instance Sannasi et al. (2010) reported variable removal of Cu, Pb, Cd, and Ni using an environmental bacterial consortium, while Qurbani et al. (2025) reported different removal efficiencies for Cu, Zn and Ni using a mixture of Pseudomonas putida and Pasteurella aerogenes. These variations may reflect differences in metal speciation, initial concentration, pH, competing ions, biomass concentration, microbial composition, contact time, and availability of metal binding sites (Igiri et al. 2018; Razzak et al. 2022; Qattan 2025). The observed decreases represent reductions in measured aqueous metal concentrations rather than evidence of a specific removal mechanism, but rather a decrease in the measured aqueous metal concentration. Unlike organic contaminants, which may undergo biological degradation and transformation, metals cannot be biodegraded; however, microorganisms may immobilize, accumulate, precipitate, transform or redistribute metals into different dissolved, particulate and biomass-associated fractions (Igiri et al. 2018; Medfu et al. 2020). The possible processes that can contribute to the observed reductions are passive biosorption onto the functional groups of cell walls and extracellular polymeric substances, biomass-associated accumulation, biologically influenced precipitation, redox-mediated changes in metal speciation, and sedimentation of metal-bearing biomass (Razzak et al. 2022; Qattan 2025). The present results, however, do not allow conclusions regarding the relative contribution of these processes because metal speciation, biomass-bound metals, sludge composition and mass balances were not investigated.
The residual Ni, Mn, Cd, and Co concentrations after the treatment showed that there is a potential to further optimize treatment performance where concentrations are still above the applicable regulatory limits, whereas the concentration of Zn was not above the applicable regulatory limit. The incomplete or varying removal of metals may be due to competition for binding sites, difference in metal speciation, and microbial responses in the complex wastewater matrix (Igiri et al. 2018; Qasem et al. 2021). The impact of pH, biomass concentration, treatment time, mixing conditions, and other operational parameters should be studied in the future to understand their role on the efficiency of metal removal. Complementary processes, such as adsorption, chemical precipitation, ion exchange, membrane separation, or electrochemical treatment, may also be considered where lower residual metal concentrations are required (Barakat 2011; Qasem et al. 2021). Cr and Pb were not detected in either biotreated or untreated wastewater. The lack of detection does not necessarily mean that they are not present, as concentrations below the analytical sensitivity of the method cannot be ruled out. Concentrations and relative abundances of metals in textile effluents can also be quite different depending on the type of fibers being used, dyes/pigments being used, auxiliary chemicals and the manufacturing process. (Yaseen and Scholz 2019; Velusamy et al. 2021). Therefore, the absence of detection of Cr and Pb in the biotreated wastewater should not be attributed to treatment because these metals were also not detected in untreated TW; their non-detection may instead reflect low initial concentrations, production-specific inputs, or analytical sensitivity.
Fig. 6. Comparison of heavy metal concentrations in TW and Bt-W with NEQS permissible limits
Germination and Growth Performance
Radicle development varied among treatments and over time (Figs. 7 to 9). Bt-W showed greater early radicle growth, but this pattern was not sustained. TW produced relatively long radicles at some time points but did not result in better plumule development or overall germination performance (Tables 2 and 3; Fig. 9). This comparison indicates that root elongation alone is not sufficient to represent overall seedling performance. Root and shoot responses to abiotic stress can differ, as plants may alter root-system architecture and biomass distribution under resource limitations and other environmental constraints (Comas et al. 2013; Karlova et al. 2021; Shoaib et al. 2022). For example, maize primary roots have been shown to continue elongating at water potentials that inhibit shoot growth (Sharp et al. 1988). The comparatively greater radicle elongation observed under TW may therefore reflect differential root and shoot sensitivity or a stress-associated allocation response rather than a general stimulation of seedling growth (Sharp et al. 2004; Karlova et al. 2021). The treatments were more clearly differentiated by plumule development. Bt-W showed greater plumule growth, reaching approximately 5.0 to 6.0 cm on days 6 to 7 and remaining slightly above the control on day 18. TW, on the other hand, produced shorter plumules during the later growth period, indicating a contrasting root and shoot growth response (Table 3; Fig. 9).
Similar studies indicate that the effects of textile wastewater on seedling development depend on effluent concentration, composition, and plant species. High concentrations of textile effluent have been associated with reductions in germination, root and shoot development, biomass, and physiological performance, whereas lower concentrations may sometimes stimulate individual growth parameters (Khan et al. 2011; Rahman et al. 2018; Kurmi et al. 2023). These concentration-dependent responses may help explain why TW produced comparatively high radicle lengths at individual observations while still restricting plumule development and overall seedling performance. Radicle elongation, plumule elongation, and germination are complementary phytotoxicity endpoints and may differ in their sensitivity to wastewater constituents (Priac et al. 2017; Khelil et al. 2022). Although root elongation is frequently used as a sensitive endpoint because roots are in direct contact with the test solution, interpretation based on root length alone may overlook adverse effects on shoot development and overall seedling vigour (Priac et al. 2017). The relatively high TW radicle values should therefore be interpreted together with plumule length, germination percentage, and seedling-vigour indices (Tables 4 and 5).
Fig. 7. Germination progression of tomato seeds under different water treatments. Panels (a, b, c) represent seeds on the 3rd day; (d, e, f) on the 4th day; (g, h, i) on the 5th day; (j, k, l) on the 6th day; and (m, n, o) on the 7th day. Seeds were grown using tap water, textile wastewater, and biotreated water, highlighting variations in germination performance over time.
The more balanced radicle and plumule development observed under Bt-W is consistent with reduced phytotoxic effects following treatment. Untreated wastewater was found to have phytotoxic effects on seed germination and seedling growth, while bacterial treatment was found to decrease the observed phytotoxic effects (Fendri et al. 2013). Ravindran et al. (2016) also found that wastewater treatment resulted in better germination and early growth responses.
Fig. 8. Germination progression and seedling growth of radicle and plumule across 3rd, 4th, 5th, 6th, 7th, and 18th day
These studies were performed with domestic wastewater instead of textile wastewater, but they corroborate the general finding that biological treatment can decrease the phytotoxicity of wastewater. This interpretation is supported by Akshaya Vidhya et al. (2022), who found that the germination rate and root and shoot growth of Vigna radiata were better in the biologically treated synthetic azo-dye wastewater than in the untreated wastewater. These findings further emphasize that radicle, plumule, and germination responses should be interpreted collectively because individual seedling endpoints and plant species may differ in their sensitivity to wastewater constituents (Teixeira et al. 2024).
Following these root and shoot responses, germination performance also showed a clear descriptive pattern among the treatments. The control maintained the highest final germination, with 94 of 100 seeds germinating by day 18, followed closely by Bt-W with 90 seeds, whereas TW reached 81 seeds (Table 1).
Table 1. Germination Count of Tomato Seeds Grown in Tap Water (C), Textile Wastewater (TW), and Biotreated Textile Water (Bt-W)
Table 2. Radicle Length (cm) under Different Treatments Over Time (mean ± SD, n = 3 replicate observations)
Table 3. Plumule Length (cm) under Different Treatments Over Time (mean ± SD, n = 3 replicate observations)
The relatively close germination response of Bt-W to the control, together with its greater plumule development, suggests that biotreatment reduced the inhibitory effects associated with untreated wastewater. Comparable studies have reported improved germination and reduced phytotoxicity following biological treatment of textile- or dye-containing wastewater (Sonu et al. 2021; Selim et al. 2021; Akshaya Vidhya et al. 2022). When germination counts are considered together with the seedling-growth responses, Bt-W showed a more balanced overall response than TW (Tables 1–5; Fig. 9).
Fig. 9. Effect of TW and Bt-W on seedling development over time: (left) radicle length and (right) plumule length compared to control
Table 4. Germination and Seedling Growth Indices, Formulas, Explanations, and References Used To Evaluate Seed Germination Performance, Seedling Vigor, Stress Tolerance, and Phytotoxicity Under Control and Treatment Conditions
Table 5. Comparative Germination Parameters and Stress-Tolerance Indices of Seeds Exposed to C, TW, and Bt-W
