Abstract
Water pollution is a burning issue that can originate from both urbanization and industrialization. This study aimed to evaluate the industrial wastewater collected from Hayatabad Industrial Estate and to use indigenous bacteria, Pseudomonas aeruginosa and Enterobacter aerogenes for bioremediation. The water samples collected were analyzed for physicochemical parameters and microbial pollution. To analyze the pollution removal efficiency by indigenous bacterial species, a pot experiment was performed for 14 days. Before and after experiment, the water samples were analyzed for trace metal concentration by Atomic Absorption Spectroscopy. The biochemical and molecular analysis confirmed the presence of two bacterial species (P. aeruginosa and E. aerogenes). The industrial wastewater treated with these isolated bacterial species showed significantly decreased level of electrical conductivity (42.33–86.45%), dissolved oxygen (16.35–63.37%), biological oxygen demand (33.33–80.62%), chemical oxygen demand (00-83.52%), total suspended solids (00–80%), and total dissolved solids (0.00-54.93%). The P. aeruginosa removal efficiency for Cu, Cd, and Pb was ranging 77.58–82.35%, 19.67-50%, and 20.40–91.66%, respectively. Similarly, the E. aerogenes removed Cu, Cd, and Pb in the range of 47.05–60.61%, 54.55–62.29%, and 85.21–91.6%, respectively. Phytotoxicity results revealed that the wastewater treated with both P. aeruginosa and E. aerogenes gives better Triticum sp. % germination rate, leaf length, and root and shoot weight. The highest plant % germination was showed by treated P. aeruginosa in control (100%), followed by E. aerogenes in control (100%). The t- test analysis showed the concentration of trace metals (TM) in industrial wastewater was significantly reduced (p ≤ 0.05) by bacterio-remediation. The study concluded that both bacterial species are active in the removal of pollution and TM from the wastewater.
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Introduction
Industrial wastewater (IWW) is a significant environmental concern due to its complex composition and high levels of pollutants, including trace metals (TMs), organic matter, and other toxic substances1. Industries such as textiles, pharmaceuticals, paper, and chemicals generate vast amounts of effluents that, if untreated, pose severe risks to aquatic ecosystems and human health2. The untreated discharge of IWW into rivers, lakes, and groundwater adversely affect the water quality and the biodiversity of aquatic life3. One of the major environmental challenges posed by IWW is TM contamination, particularly from elements like copper (Cu), cadmium (Cd), and lead (Pb)4. These TMs are non-biodegradable and can accumulate in the environment, posing long-term toxic effects on ecosystems and human health5. Prolonged exposure to these metals, even in trace amounts, can lead to different diseases like cancer, organ failure, and neurological disorders6. In the context of Pakistan, IWW from industrial estates such as Hayatabad Industrial Estate (HIE) and Gadoon Industrial Estate (GIE) has been found to contain high levels of TMs, exceeding the permissible limits set by the Pakistan Environmental Protection Agency7. Effective treatment and management of IWW are, therefore, crucial to mitigate its environmental impacts8.
Several physicochemical and biological techniques have been developed to treat IWW, ranging from chemical precipitation and ion exchange to advanced oxidation processes and membrane filtration9. However, these conventional methods often suffer from high operational costs, secondary pollution, and inefficiency in removing trace amounts of TMs10,11. As a result, biological treatment methods, such as bioremediation, have gained attention due to their cost-effectiveness, sustainability, and eco-friendly nature12,13. Bioremediation, the process of using microorganisms to degrade, detoxify, or transform hazardous substances into less harmful products, has emerged as a promising alternative for wastewater (WW) treatment2. This approach leverages the natural metabolic processes of microorganisms to remove contaminants, including TMs, from WW14. Bacterial species such as Pseudomonas aeruginosa and Enterobacter aerogenes have demonstrated high potential for the bioremediation of IWW, particularly in removing TMs and improving water quality through physicochemical adjustments15,16. Pseudomonas aeruginosa is a gram-negative, rod-shaped bacterium known for its versatile metabolic capabilities and ability to thrive in diverse environments, including polluted waters17,18. It has been widely studied for its TM tolerance and ability to reduce the level of chemical oxygen demand (COD), biological oxygen demand (BOD), and total suspended solids (TSS)19. A previous study showed that P. aeruginosa can significantly reduce concentrations of TMs in IWW, making it a valuable candidate for bioremediation20. Similarly, Enterobacter aerogenes, another gram-negative bacterium, is known for its role in WW treatment, particularly in degrading organic pollutants and reducing TM concentrations21. Like P. aeruginosa, E. aerogenes has been used in various bioremediation studies to treat WW containing a range of pollutants due to its metabolic flexibility and resistance to toxic environments22. Recent research has highlighted its ability to improve the physicochemical properties of WW by reducing pH, COD, BOD, and total dissolved solids (TDS) levels, making it an effective organism for WW treatment applications23,24. In addition to contaminating aquatic environments, IWW can have severe phytotoxic effects, hindering the germination and growth of plants25,26.
The adsorption of TMs like Cu2+, Cd2+, and Pb2+ by indigenous bacteria such as Pseudomonas aeruginosa and Enterobacter aerogenes involves several steps that aid in removing pollutants from wastewater. Initially, the metal ions diffuse toward the bacterial surface and interact with functional groups like carboxyl, amino, and phosphate groups, facilitating both physical and chemical adsorption. This step is critical as it allows the bacteria to bind metal ions, forming complexes that prevent further contamination27. Extracellular polymeric substances produced by the bacteria enhance this biosorption process by providing additional binding sites. This further increases the efficiency of metal uptake as the ions become entrapped in these substances28. Subsequently, the metal ions may diffuse into the bacterial cell or be actively transported across the cell membrane, where they undergo detoxification. Some bacterial species also influence the surrounding environment by producing sulfides or altering the pH, which can lead to metal precipitation or the formation of metal hydroxides29,30. Ion exchange is another mechanism where metal ions replace lighter cations, such as Na⁺ or Ca²⁺, on the bacterial surface, leading to higher adsorption efficiency. Eventually, once all binding sites are occupied, an equilibrium is reached, halting further metal uptake31. This multi-step process highlights the effectiveness of using bacterial biosorbents for the eco-friendly remediation of industrial wastewater32,33.
Several studies have explored the potential of bacterial species for the bioremediation of IWW. For instance, it was investigated that the bioremediation of IWW from HIE using bacterial strains and found that Pseudomonas species exhibited significant removal of TMs and improvement in water quality parameters34. Bashir et al.35 demonstrated the effectiveness of an algal-bacterial consortium in treating WW by lowering pollutant loads and enhancing the water’s physicochemical properties. Despite these advances, gaps remain in understanding the comparative efficiency of different bacterial species under varying pollution loads and environmental conditions36. Moreover, while much research has focused on individual bacterial strains, limited information exists on the combined or synergistic effects of multiple microbial species in the bioremediation process14. Additionally, most bioremediation studies have focused on laboratory-scale experiments, and there is a need for field-based research to assess the practical feasibility of microbial treatments in real-world IWW systems37. A novel approach has been introduced in this investigation to address the IWW contamination through the use of Pseudomonas aeruginosa and Enterobacter aerogenes, two bacterial species with bioremediation potential. While both microorganisms have been studied individually for their ability to remove TMs and improve water quality, the comparative efficiency of these bacteria under identical controlled conditions has not been extensively explored, particularly in the context of IWW from regions like HIE. This investigation offers a unique perspective on the adaptability and effectiveness of local bacterial species by isolating indigenous strains from the contaminated site and testing them under real-world conditions. Furthermore, the study advances the understanding of phytotoxicity following bioremediation, an area that remains underexplored in WW treatment research. While most studies focus on removing pollutants from water, the impact of treated WW on plant health is crucial for ecological safety. By incorporating Triticum sp. germination tests, this research provides essential insights into post-treatment ecological impacts, highlighting the broader environmental implications of bacterial bioremediation techniques38. The field relevance of this research is another key contribution. Most bioremediation studies remain confined to laboratory settings, limiting their practical application39. This study bridges the gap by simulating field-relevant conditions through controlled pot experiments, making the results more applicable to real-world WW treatment challenges. Given the rising industrialization in regions like Pakistan, the findings from this research are critical for developing sustainable and cost-effective WW management solutions, ultimately contributing to environmental conservation and public health protection40. The study assesses the physicochemical characteristics of IWW, including pH, electrical conductivity (EC), dissolved oxygen (DO), BOD, COD, TSS, and TDS. It isolates and identifies bacterial strains from the IWW, evaluates the bioremediation efficiency of Pseudomonas aeruginosa and Enterobacter aerogenes in removing TMs and improving water quality, and conducts phytotoxicity tests to assess the ecological safety of treated WW using Triticum sp. germination. Through these investigations, the research contributes to the growing body of knowledge on sustainable WW management techniques. The Fig. 1A and B revealed the mechanisms of action in bioremediation of TM through plants and microbes.
Materials and methods
Collection and analysis of industrial wastewater
The IWW was collected from the main drain of HIE (supplementary information Fig. S1). The water samples were directly collected into Tarson polypropylene bottles. From each sample collected, an aliquot was preserved by the addition of conc. HNO3 (0.1% v/v) as to avert the variable metal oxidation and the other aliquot was preserved in icebox without any addition of acid for analyzing different water quality parameters41. The Hayatabad Industrial Estate in Peshawar, Pakistan, is situated at approximately 33.98917°N latitude and 71.42252°E longitude. The effluents in the main drain of HIE are composed of effluents released from various industries such as the match industry, rubber, steel, plastic, paint, and pharmaceutical industry42. The suspended solid materials, including soil, small sediments, and sand particles were eliminated. The IWW samples were then examined before experimentation for various physiochemical parameters like pH, TDS, temperature, COD, electrical conductivity (EC), TSS, dissolved oxygen (DO), BOD, and TMs (Cd, Cu, and Pb). The quantification of TMs was performed using Atomic Absorption Spectroscopy (AAS) in the Centralized Resource Laboratory (CRL), University of Peshawar17,43.
pH and electric conductivity (EC)
The pH and EC of the water samples were analysed using a pH meter (Model: PH110 Hong Kong, China) and EC meter (InoLab, Mexico City, Mexico), respectively35.
Biological oxygen demand (BOD)
For the initial DO (dissolved oxygen), a water sample (200 mL) was taken in a titration flask, and 1 mL each of concentrated H2SO4, alkali iodide and MnSO4 was added. Then, a few drops of the indicator (starch) were added. In the burette, the solution of Na2S2O3 was taken. The Na2S2O3 in the water sample was titrated until disappearance of the colour. In BOD bottles, 20 mL water samples were taken to store the samples for 5 days in an incubator at 20 °C. The same titration process was performed after 5 days for the final DO value. The difference between the initial and final DO is the BOD of the water sample35.
DOi = initial DO., DOf = final DO at 20 °C after 5 days of incubation.
Chemical oxygen demand (COD)
COD is the measure of water and WW quality. First of all, the sample blanks were prepared, and 2 mL of each sample was taken through a micropipette and poured in the COD reagent vial available in the market. The vial was inverted several times to mix it properly. The vial became hot during mixing. For blank preparation, the 2 mL of deionized water was poured into another COD reagent vial and mixed properly. The samples were then ready for digestion. Both the vials were placed into the COD reactor for digestion. The temperature was set at 150 °C for 2 h. After digestion, the samples were cooled. The blank vial was inserted into the adapter slowly with minimum shaking and placed into the spectrophotometer. The blank vial reading was noted as zero. The blank vial was removed from the adaptor, and the sample vial was inserted into the adaptor. The reading that appeared on the spectrophotometer was noted35.
Total suspended solids (TSS)
Whatman No. 42 filter paper was dried in oven at 101 °C and then was cooled and weighed. On this filter paper, a 10 mL water sample was filtered and kept again in an oven for drying at 101 °C and then was cooled and weighed. The difference in initial weight (Wi) and final weight (Wf) of the filter paper was the value of TSS35.
Wi = filter paper’s initial weight, Wf = filter paper’s final weight,
Total dissolved solids (TDS)
In the oven, a clean crucible was placed at 105 °C. Using a desiccator, the crucible was cooled and weighed. For evaporation, the filtered 10 mL water sample in the crucible was kept at 105 °C in the oven. Then, after cooling, the crucible was weighed in the desiccator. The difference in initial (Wi) and final crucible weight (Wf) was TDS35.
Wi = Crucible’s initial weight, Wf = Crucible’s final weight.
Trace metal analysis
The water samples were prepared for TM analysis. A few drops of nitric acid (HNO3) were added to each water sample (50mL) for preservation. The prepared water samples were assessed for Cu, Cd, and Pb concentrations by AAS (AAS-700 PerkinElmer: Norwalk, CT, USA). For physicochemical parameters like pH and temperature EC, BOD, and COD the water samples were analyzed using the standard17. The pH and EC were analysed using pH meter (Lutron Electronic Enterprise Co Ltd., Model PH-208, Taiwan) and EC meter (InoLab, Cond Level 1, Germany), respectively35.
Media used
Nutrient agar
Nutrient Agar media was used in the microbial isolation in this research project. It is general-purpose media that supports various types of microbes and is composed of all those nutrients that help in the growth of bacteria. Nutrient agar powder was dissolved in the distilled water, stirred in the media continuously, and shaken well. Then the media was sterilized using an autoclave for 15 min at 121ºC. It was then allowed to cool for some time, poured into the Petri plates, and allowed get solidify44.
Here 28 g/1000 represents the standard preparation ratio for nutrient agar powder, where 28 g of nutrient agar is dissolved in 1000 milliliters (1 L) of distilled water, 50 ml is the volume of nutrient agar solution to prepare and 1.5 g is the calculated amount of nutrient agar powder required to prepare 50 milliliters of the solution.
MacConkey agar
MacConkey agar media was used as a differential media for the identification of bacteria. It differentiates between lactose non-fermenting and fermenting bacteria. MacConkey agar powder was dissolved in the distilled water and stirred vigorously so all the media dissolved in the water. The media was then kept in the autoclave for 15 min at 121 °C. It was cooled and poured into petri plates and left to solidify45.
Here 52 g/1000 is the standard preparation ratio for MacConkey agar powder, which specifies that 52 g of MacConkey agar powder is required to make 1000 milliliters (1 L) of the medium, 50 ml is the specific volume of MacConkey agar solution and 2.6 g is the calculated amount of MacConkey agar powder required to prepare 50 milliliters of the solution.
Serial dilution and streak plating technique
The IWW (2000 mL) sample was centrifuged at 2000 rpm for five minutes, the supernatant was removed and the remaining sample was treated for microbial analysis44. Each sample was properly shaken and divided into two parts to detect microbes and then was properly labeled for experimental work. A serial dilution method was used to isolate and identify the pure bacterial colony from the WW. Test tubes were taken and washed properly then autoclaved at 121 °C for 15 min. After autoclaving, all the test tubes were labeled in proper sequence such as T1, T2, T3, T4, T5, T6, T7, T8, and T9. The diluents (WW) were poured into each tube by picking 1mL from the first tube through a micropipette and pouring it into the second tube, the process was repeated till the 9th test tube. Test tubes T7, T8, and T9 were selected for further processing and characterization. The process of serial dilution was done to obtain a pure colony from the water sample44. A streak plate technique was used to isolate bacterial species from the WW. A platinum wire was used. One loopful of inoculum was transferred on the surface of the well-dried petri plate. The inoculated sample was taken on a wire loop, spread to the periphery, and then distributed over the plate by streaking on various segments of the plate. On incubation, after 24 h the separated colonies were obtained at the last series of the streak for further identification45.
Bacterial identification
Morphological characteristics
The shape (circular, irregular, and rod-shaped), size (small, medium, large), pigmentation (color of colonies), Opacity (opaque or translucent), and Elevation (flat, raised, and convex) of colonies were observed. The standard Gram staining method was used for the identification of bacterial isolates by microscopy to differentiate between gram-negative and gram-positive bacteria44.
Biochemical test for bacterial identification
The bacteria (gram-negative or gram-positive) were identified by performing various tests including, Oxidase, Catalase, Citrate, Urease, Indole, Glucose, and Gelatinase. All the tests were performed using the API 20 E kit. Catalase and Oxidase tests were performed separately on slides by a technique adapted by Jaffali et al.46. An API 20 E kit (BIOMERIEUX) was used for the bacterial characterization of selected bacteria47. A bacterial suspension was prepared by picking a selected microbial colony from a Petri plate. Sterile syringes were used to fill the bacterial suspension and were labeled properly. A small amount of water droplets were added to the kit tray to maintain humidity. The strips were placed on the tray in a little inclined manner. Further, the bacterial suspension was added to each cup evenly to avoid bubble formation in the cups. The kit was covered via lid and placed in an incubator for 24 h at 37 °C. After 24 h, various metabolic activities produced color in each cup. For the test Indole, Voges–Proskauer (VP), and Tryptophan deaminase (TDA) reagents were used and allowed for a few seconds to observe the color change. All the readings were noted as positive (+) or negative (–) and the particular species were identified47.
Molecular identification of the isolates through 16 S rRNA sequencing and phylogenetic analysis
Pure bacterial cells (grown in nutrient agar medium) were harvested and sent to the Beijing Genomics Institute Mainland China (BGI) (https://en.genomics.cn/ for 16 S rRNA sequencing. Total DNA was extracted from the isolates (SM3 and SM8) and purity was verified by using 1% agarose gel. The DNA was amplified for 16 S rRNA sequencing by using primers Forward 27 F—5′-AGAGTTTGATCMTGGCTCAG-3′ and Reverse 1492R– 5′-CGGTTACCTTGTTACGACTT-3′48. The contents of the 25 µL PCR reaction mixture was: 1.25 µL of 27 F primer, 1.25 µL of 1492R primer, 5 µL of nuclease-free water, 12.5 µL of PromTaq®Green Master Mix Promega, and 5 µL of template DNA. The PCR profile was as follows: 5 min of initial denaturation at 94 °C, 60 s of denaturation at 94 °C in 35 cycles, 45 s of annealing at 53 °C for 35 cycles, and 2 min of elongation at 68 °C in 35 cycles reaction. The reaction was finally extended for 10 min at 72 °C and then kept at 4 °C. The amplified product was visualized using 1% agarose gel and purified by Wizard ® PCR Preps (Promega, New England) and ABI 3730XL automated DNA sequences was used to obtain readings.
Chromas software 2.6.6 was used to examine the chromatograms that were acquired from BGI (http://technelysium.com.au/wp/chromas/) to eliminate the poor-quality readings for additional improvement. The sequences were compared for similarity with already reported sequences in GenBank based on identity ranking (> 97%) and E-values (0.0) by submitting to BLASTN (National Center for Biotechnology Information; http://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequences from current study (SM3 and SM8) were submitted for accession number to GenBank. Molecular Evolutionary Genetics Analysis software 10 (MEGA version 10: http://www.megasoftware.net was used to analyze the bacterial isolates’ phylogenetic networks. A reference sequence was used for sequence alignment, and phylogenetic trees were constructed using neighbor-joining techniques with 1000 bootstrap values set to the default option48.
Selection and transplantation of microbes
In this study, two bacterial isolates SM3 (Pseudomonas aeruginosa) and SM8 (Enterobacter aerogenes) were isolated from the WW of HIE. Using serial dilution, gram staining, microscopic examination, and culture techniques, a large number of SM3 and SM8 were isolated from the WW and were further selected for pot experimentation. The purpose behind the selection of these species is their ability to accumulate TMs, high tolerance towards TM toxicity, and fast growth.
The water samples (tap water as control and WW as treatment water) were autoclaved at 121 °C for 15 min. The desired bacteria were then inoculated and assimilated into the water samples for fourteen days at room temperature. After fourteen days water samples were collected and were further analyzed in the laboratory.
Experimental design
A pot experiment was carried out to assess the TM removal efficiency of bacterial species individually. A pot experiment is a controlled experimental setup typically conducted in containers (pots) to study the effects of specific treatments on plants, soil, or microorganisms under standardized conditions. Four containers were utilized that were thoroughly rinsed using double 10% diluted nitric acid and deionized distilled water (DDW). Two control containers, labeled CTPA (Control Treatment for Pseudomonas aeruginosa (SM3)) and CTEA (Control Treatment for Enterobacter aerogenes (SM8)), were used for tap water samples. Two additional containers, labeled PA (Treatment for P. aeruginosa (SM3)) and EA (Treatment for E. aerogenes (SM8)), served as experimental groups for wastewater samples. Using a sterile wire loop, an inoculum of each specie one by one was picked from the culture plates, diluted in 5 mL of water, and added to the appropriately labeled containers. The treatment containers were provided with WW (500mL) while the control containers contained clean tap water (500 mL). All the containers were placed at room temperature (32 °C) for fourteen days. After 14 days of the experiment, water samples were collected from each container and analyzed for TMs (Cu, Cd, and Pb) and physiochemical characteristics. Each experiment was conducted in triplicate to ensure the accuracy and reproducibility of the results.
Water sampling and analysis
Water samples collected from each container (CTPA, CTEA, PA, and EA) at the end of the experimentation were further analyzed in the laboratory. All water samples collected from each container were analyzed in triplicate to account for variations and enhance reliability by the procedure as mention in section “Collection and analysis of industrial wastewater”.
Removal efficiency (%)
Removal efficiency is a quantitative measure used to evaluate the effectiveness of the bacterial isolates in removing TM from IWW. Bio removal efficiency (%) was calculated by following the formula adopted by Shamshad et al.49.
Where R represents the removal percentage, Ci and Cf are initial and final TM level in water samples, respectively.
Phytotoxicity assay
The species Triticum aestivum was utilized for phytotoxicity evaluation. The cultivation of plant species was done in clay soil 2 kg per container (25 cm in diameter) under natural environmental conditions following triplicate for each treatment. A total of five seeds per container were cultivated. The first container was irrigated with sterilized untreated IWW as a positive control (UW), and the other four containers were irrigated separately with WW treated by the bacterial species (CTPA, PA, CTEA, EA). Before cultivation, all the water samples were autoclaved. The plants were continuously irrigated after every 3rd day. After one month of growth, the plants from each pot were obtained and washed with DDW, separated into leaves, roots, and shoots, and weighted and oven-dried at 80 °C for 24 h for dry weight measurements. The plant was measured for shoot and root dry and fresh weight and leaf length17. The experiment was conducted in triplicate for each treatment to assess the phytotoxicity of Triticum sp.
Statistical analysis
The results of the study were presented and analyzed on Microsoft Excel and SPSS (version 22.0). The data for various parameters including before and after treatment values were compared by t-test with two-tailed p-values using SPSS software.
Results
Physicochemical characteristics of wastewater
The pH observed for the tap water and IWW samples was 7.4 and 8.39, respectively. The highest pH values were obtained for IWW, while the lowest was for tap water. The pH values observed for both samples were within the maximum permissible limit (6–10) (MPL) set by the Pakistan Environmental Protection Agency (Pak-EPA, 2008). The detailed results of the pollution load of tap water and IWW are given in Table 1. The EC values noted for tap water and IWW were 0.489 and 2.134 mS/cm, respectively. The EC highest value was recorded in IWW (Table 1). The DO values recorded for tap and IWW were 324 and 172 mg/L, respectively. The DO highest value was observed in the tap water sample (Table 1). In the current study, the BOD values recorded for tap water and IWW were 03 and 129 mg/L, respectively. The BOD highest value was observed for IWW (Table 1). The COD value recorded for tap water and IWW were 05 and 862 mg/L, respectively. The COD highest value was observed in IWW (Table 1). The TSS values recorded for tap water and IWW were 02 and 308 mg/L, respectively. The highest TSS value was observed in IWW sample (Table 1). The TDS values determined for tap water and IWW were 272 and 912 mg/L, respectively. The highest TSS value was observed in IWW sample (Table 1).
Bacteriological study of WW
Isolation of bacteria
A total of 18 strains of bacteria were obtained from WW (SM1–SM18). Two bacterial isolates SM3 and SM8 were selected for further analysis and bioremediation of TMs due to their abundance in IWW. The description of the morphological characteristics of the desired species (SM3 and SM8) is given in the Table 2. The agar plates (Nutrient and MacConkey) were observed for shape, arrangement, and colony characteristics of the isolates (supplementary information Fig. S2). The SM3 strain showed mucoid, opaque and flat colonies on the nutrient agar media plate while colorless colonies were obtained on the MacConkey agar plate. The SM3 was observed to be lactose non-fermenting bacteria. The other bacterial strain SM8 appeared as white, smooth, and circular colonies on the nutrient agar media plate whereas, on the MacConkey agar plate media, mucoid, pink colonies were observed and observed as lactose fermenting bacteria (Table 2).
Gram staining and biochemical identification of selected bacteria
Based on the gram staining technique both the isolates SM3 and SM8 were confirmed as gram negative as pink-colored and rod-shaped species were obtained (supplementary information Fig. S4). The biochemical analysis results are shown in Table 3. Positive for Oxidase (supplementary information Fig. S3), Catalase (supplementary information Fig. S3), Citrase, Glucose, H2S, Gelatinase and Mannose tests and negative for Urease and Indole tests identified isolate SM3 as P. aeruginosa. On the other hand, positive for Catalase (supplementary information Fig. S3), Citrase, Glucose and Mannose tests and negative for Oxidase (supplementary information Fig. S3), Urease, Indole and H2S tests refer to E. aerogenes (SM8) (supplementary information Fig. S4).
Molecular identification of isolates
16 S rRNA sequencing was used to determine the isolates’ molecular identity. BLAST NCBI was initially used to identify the isolates, and they were then deposited in GenBank for accession number (PQ772640 and PQ772641). The phylogenetic analysis was carried out against the reference sequences to further clarify the molecular identity of our isolates (Table 4). The phylogenetic tree construction using the neighbor-joining method by MEGA10 showed clustering of our isolates SM3 with reference sequence of Pseudomonas aeruginosa (JF911361, MW661177 and KJ872834) (Fig. 2) and SM8 with reference sequence of Enterobacter aerogenes (AJ251468 and AB244467) (Fig. 3) confirming the molecular identity and supporting of biochemical results.
Phylogenetic analysis of Pseudomonas aeruginosa through Neighbor joining (NJ) methods with MEGA 10 using 1000 bootstraps. This analysis involved 10 nucleotide sequences from different Pseudomonas species. The current study sequence was displayed with red dot. Our isolate SM3 clustered with Pseudomonas aeruginosa in the same clade confirms molecular similarity.
Phylogenetic analysis of Enterobacter aerogenes through Neighbor joining (NJ) methods with MEGA 10 using 1000 bootstraps. This analysis involved 13 nucleotide sequences from different Enterobacter species. The current study sequence was displayed with red dot. Our isolate SM8 clustered with Enterobacter aerogenes in the same clade confirms molecular similarity.
Comparative analysis of the removal efficiency (%) of bacteria for control and treatment
Pseudomonas aeruginosa
To analyze the removal efficiency of P. aeruginosa at lower and higher pollution loads, the species was cultured in tap and IWW, respectively. The results showed that the efficiency of pollution removal of P. aeruginosa was higher in treatment as compared to the control (Table 5). The study revealed that P. aeruginosa can be best used for the remediation of highly polluted water (Fig. 4).
At the final stages of the pot experiment the values of EC, pH, COD, DO, TSS, BOD, and TDS in water samples (CTPA, PA) were noted in the range of 0.282–1.076 mS/cm, 7.03–7.47, 4-276 mg/L, 108–271 mg/L, 2.0–113 mg/L, 2.0–34 mg/L and 161–411 mg/L, respectively (Table 3). The removal efficiency of physiochemical parameters observed in control (CTPA) for pH, COD, EC, DO, TSS, BOD and TDS were 5.0, 20, 42.22, 16.35, 0, 33.3, 40.80%, respectively. The removal efficiency for P. aeruginosa was also observed in treatment (PA) as pH (10.96%), EC (49.57%), DO (37.20%), BOD (73.64%), COD (67.98%), TSS (63.31%) and TDS (54.93%) (Fig. 4). The finding revealed that the removal efficiency of P. aeruginosa was higher in treatment (PA) as compared to control (CTPA). The study revealed that P. aeruginosa can be best used for the remediation of highly polluted water (Fig. 4).
Enterobacter aerogenes
The mean concentrations of EC, pH, COD, DO, TSS, BOD, and TDS in water samples for E. aerogenes at final stages of the pot experiment were noted in the range of 0.106–0.289 mS/cm, 6.69–7.31, 5.0–142 mg/L, 63–145 mg/L, 1.0–138 mg/L, 1.0–25 mg/L and 272–912 mg/L, respectively (Table 3). The removal efficiency of pollution determined in control (CTEA) for pH, COD, EC, DO, TSS, BOD and TDS was 9.59, 0.0, 78.32, 55.24, 80, 66.66, 0.0%, respectively. The removal efficiency for E. aerogenes was investigated in treatment (EA) as pH (12.87%), EC (86.45%), DO (63.37%), BOD (80.62%), COD (83.52%), TSS (55.19%) and TDS (0.0%) (Fig. 5) The findings of the study revealed that the removal efficiency of E. aerogenes was higher in treatment (EA) as compared to the control excluding TSS removal efficiency. The study revealed that E. aerogenes can be best utilized for the treatment of highly polluted water (Fig. 5).
Effect of bacteria on trace metals
Copper (Cu)
Cu concentration observed in the water samples for the initial level and final levels ranged from 0.017 to 0.843 and 0.011 to 0.403 mg/L, respectively (Table 6). The highest Cu values were determined in the treatment (AN and EA) in initial water samples while in EA for final water samples. The study observed 47.05 to 82.35% reduction in the final water samples. The Cu bio removal efficiency observed for CTPA, PA, CTEA, and PA from the water were 82.35, 77.58, 47.05, and 60.61%, respectively (Fig. 6). The highest bio removal efficiency for Cu was observed in CTPA while the lowest value was observed in CTEA. The t-test analysis (Table 6) showed that the bacterial species significantly reduced (p ≤ 0.05) Cu from water samples.
Cadmium (Cd)
Cd concentration observed in the water samples for the initial level and final levels ranged from 0.011 to 0.61 mg/L and 0.005 to 0.49 mg/L, respectively (Table 6). The highest values were recorded of Cd in the PA and EA in initial water samples while in PA for final water samples. The study observed a 19.67 to 62.29% reduction in the final water samples. The removal efficiency (R) observed for CTPA, PA, CTEA, and EA of Cd from the water were 50, 19, 67, 54.55, and 62.29%, respectively (Fig. 7). The highest removal efficiency for Cd was observed in EA while the lowest value was observed in PA. The t-test analysis (Table 6) showed that the bacterial species significantly reduced (P ≤ 0.05) Cd from water samples.
Lead (Pb)
Pb concentration observed in the water samples for the initial level and final levels ranged from 0.012 to 1.42 mg/L and 0.001 to 1.13 mg/L, respectively (Table 6). The highest values were recorded of Pb in the PA and EA in initial water samples while in PA for final water samples. The study observed a 20.40 to 91.66% reduction in the final water samples. The Pb removal efficiency (R) observed for CTPA, PA, CTEA, and EA from the water were 90.66, 20.40, 91.6, and 85.21%, respectively (Fig. 8). The highest removal efficiency for Pb was observed in CTEA while the lowest value was observed in PA (Fig. 8). The t-test analysis (Table 6) showed that the bacterial species significantly reduced (P ≤ 0.05) Pb from water samples.
Phytotoxicity assay
A phytotoxicity assay was conducted to evaluate the toxicity of the untreated (UW) and treated IWW samples. The seeds of Triticum sp. irrigated with untreated IWW revealed 60% germination. Similarly, other containers having treated water such as CTPA (tap water treated with Pseudomonas aeruginosa), PA (WW treated with Pseudomonas aeruginosa), CTEA (tap water treated with Enterobacter aerogenes), and EA (WW treated with Enterobacter aerogenes) showed 100, 60, 100 and 80% germination, respectively, as shown in Fig. 9A. The CTPA and CTEA showed highest germination rate while the lowest germination was showed by UW and PA. The results showed that the highest leaf length was observed in the plants irrigated with CTPA (16.3 cm) and CTEA (15.9 cm) followed by EA (10.2 cm) and then PA (7.8 cm). Conversely, the lowest leaf length (7.1 cm) was found in the plant irrigated with untreated water sample (UW) as shown in Fig. 9B. For the weight of fresh shoots, a significant difference (p = 0.05) among different treatment containers was recorded. The highest fresh shoot weight was found in the plant of CTPA (14.9 g) followed by the plant irrigated in CTEA (13.9 g), while the lowest value was observed in the plant water with PA (6.9 g) (Fig. 10). Regarding the shoot fresh weight, the irrigation of Triticum sp. with untreated or treated WW improved the shoot fresh weight as compared to UW (Fig. 10). The appliance of treatments in the plant irrigation caused an enhancement in shoot dry weight comparing UW, while the highest shoot dry weight in the CTPA was 4.3 g compared with 2.50 g for the UW (Fig. 10). Though, in terms of root dry weight, significant variations were not observed among the different treatments. However the highest value was found in the plant of CTEA (2.6 g) (Fig. 11).
Discussion
The physicochemical characteristics of IWW revealed significant differences when compared to tap water, highlighting the pollution load in IWW samples. The observed pH values for IWW were higher than for tap water, suggesting that the IWW is slightly more alkaline. While both samples fall within the permissible limits set by Pak-EPA (6–10), the higher pH values for IWW indicate potential impacts on aquatic ecosystems and treatment processes. This variation could be attributed to different industrial activities, effluent composition, or seasonal changes at the time of sample collection. Notably, these pH values deviate from those reported by Khan et al.17 and Bashir et al.35, who found slightly acidic to neutral pH levels in IWW from the HIE. In the current study, the initial pH of the WW samples was identified as a critical factor influencing the adsorption efficiency of metal pollutants. Initial pH significantly affects the adsorption process by altering the ionization state of both the metal ions and the functional groups on the biosorbent. The pH levels at the start of the adsorption process can impact the solubility and speciation of metal ions, with lower pH values increasing solubility and enhancing initial adsorption rates due to the higher availability of metal ions50. The initial pH values in the study ranged from 5.0 to 7.0, which is common for many WW treatments. However, while the initial pH provides a starting point for understanding adsorption behavior, it is recognized that equilibrium pH offers a more accurate measure of the biosorbent’s performance over the entire process35. This is because the interaction between the biosorbent and metal ions during the adsorption process can alter the pH, influencing final adsorption capacity.
The adsorption process is highly pH-dependent, as it influences both the surface charge of the adsorbent and the ionization state of the adsorbate51. In this study, while initial pH values were used to evaluate adsorption, future experiments should focus on equilibrium pH for more comprehensive insights. Equilibrium pH reflects the final conditions after the interaction between the adsorbent and metal ions, offering a clearer understanding of the process’s efficiency52. Additionally, the presence of coexisting ions in actual WW poses another challenge for adsorption efficiency. In real-world scenarios, WW often contains a mixture of various TMs and other ions, which can compete with target metal ions for available adsorption sites3. These competing ions can block active binding sites or form complexes with trace metals, reducing the adsorption efficiency of the biosorbent53. While the current model demonstrated high removal efficiencies under controlled conditions with single and multi-metal systems, its direct applicability to real WW treatment is limited due to these complexities. Future research should address these challenges by incorporating coexisting ion effects in real WW samples and developing more sophisticated models that account for ion competition and interference54. This could involve optimizing biosorbent dosage, adjusting operational conditions, or developing biosorbents with higher specificity for target TM. Moreover, equilibrium models such as Langmuir and Freundlich isotherms should be applied to predict adsorption behavior more accurately under real WW conditions.
The higher EC value for IWW compared to tap water (0.489 mS/cm) reflects the increased presence of dissolved ions, often associated with industrial discharges. These results is in agreement with the findings of Khan et al.42, although studies from other regions, such as Morocco39, report even higher EC values due to differing effluent compositions. This indicates variability in the types and quantities of dissolved solids, highlighting the need for tailored treatment approaches based on specific industrial activities. The DO levels were significantly lower in IWW than in tap water, consistent with expectations for industrial effluents. Reduced DO in IWW points to higher organic loads, which demand oxygen for decomposition and can lead to oxygen depletion in receiving water bodies, thus harming aquatic life. These results are in agreement with Khan et al.42, underscoring the adverse impact of untreated industrial discharges on aquatic ecosystems. The BOD of IWW was substantially higher than that of tap water, indicating a TM load in the effluent. This result, although lower than values observed in other studies39, still represents significant organic pollution. The lower BOD values observed in studies like Jayathilake et al.55. could reflect differences in effluent composition or treatment technologies used. The elevated BOD underscores the necessity for efficient biological treatment processes to reduce the organic load before discharge into the environment. The COD levels in IWW were also much higher than in tap water, reflecting the presence of both biodegradable and non-biodegradable organic matter. This value exceeds those reported by Khan et al.34, suggesting variability in effluent sources and potentially incomplete treatment at the industrial estate. Such high COD values highlight the need for advanced treatment technologies, such as oxidation processes, to effectively reduce the total organic load. The TSS in IWW were significantly higher than in tap water, indicative of particulate matter from industrial discharges. The elevated TSS values align with studies by Adam et al.37, though variations in reported values across different studies reflect the diverse nature of industrial effluents. The high TSS levels can lead to sedimentation and clogging of waterways, affecting water quality and aquatic habitats. The TDS in IWW were also markedly higher than in tap water, signifying increased dissolved inorganic and organic substances. While these values fall below the extremely high values reported in some studies55, they still indicate a substantial pollution load, with potential implications for water quality and treatment costs. Variations in TDS across different studies may stem from differences in industrial practices, effluent management, and geographic location.
The isolation and identification of bacterial strains from IWW is a crucial step in understanding their potential for bioremediation. The process of isolating bacteria from IWW has been extensively reported in previous studies. Similar work was conducted by Kishor et al.56, who isolated bacteria for the detoxification and degradation of pollutants such as Congo red dye. The current study’s approach is agreeing with this by targeting strains capable of removing TMs from highly polluted WW. Other researchers, such as Sher et al.57. , have also isolated TM-resistant bacterial strains like Micrococcus luteus for arsenic bioremediation from IWW, highlighting the significance of isolating bacteria with specific capabilities for pollutant removal. The bacterial isolates SM3 and SM8 displayed distinct morphological and colony characteristics, which helped in their preliminary identification. This finding is in agreement with previous studies where Pseudomonas species have been widely isolated from WW due to their strong ability to survive and thrive in contaminated environments58. Enterobacter species are often reported in WW studies due to their proficiency in degrading organic pollutants and their potential for bioremediation21. The biochemical identification tests provided additional confirmation of the species. P. aeruginosa (SM3) tested positive for oxidase, catalase, citrate, glucose, H₂S, gelatinase, and mannose, while being negative for urease and indole. This biochemical profile is characteristic of Pseudomonas aeruginosa, which is well-known for its ability to metabolize a wide range of substrates and thrive in challenging environments, such as IWW59. On the other hand, E. aerogenes (SM8) tested positive for catalase, citrate, glucose, and mannose, while being negative for oxidase, urease, indole, and H₂S. This biochemical pattern is consistent with the traits of Enterobacter aerogenes, a species frequently associated with the degradation of organic pollutants and bioremediation of TMs60. The absence of H₂S production, alongside the positive reactions for catalase and citrate, further agreeing with previous studies that have isolated and identified Enterobacter aerogenes for similar environmental applications. The findings of this study are in agreement with several previous studies that have isolated and characterized bacterial strains for WW bioremediation. Fathy et al.61 isolated bacteria for phosphate bioremediation from IWW, showing that the use of isolated bacteria can significantly reduce pollution levels. Similarly, Shomar et al.36 highlighted the importance of molecular bacteriology in WW treatment, emphasizing the role of bacterial isolates in improving water quality through the degradation of pollutants. The isolation and identification techniques used in the current study reflect a well-established approach for selecting effective bacterial species for bioremediation.
The treatment using Pseudomonas aeruginosa (PA) exhibited superior removal efficiencies across most parameters compared to the control (CTPA). Specifically, the removal of BOD, COD, and TSS demonstrates the capacity of P. aeruginosa to degrade organic and suspended pollutants in highly contaminated WW. These findings are supported by studies such as Haq et al.22, who observed significant reductions in TDS (87%) and COD (68%) when using Pseudomonas putida for bioremediation of paper industry WW. Bera and Tank62 similarly reported substantial decreases in BOD (85%) and COD (90%) with Pseudomonas stutzeri. This suggests that P. aeruginosa is highly efficient at breaking down organic compounds and suspended solids, making it a strong candidate for bioremediation applications in industrial effluents. The removal efficiency for DO was also high (37.20%), suggesting improved oxygenation levels in treated water, which is crucial for aquatic life. The relatively lower pH removal efficiency (10.96%) could be due to the buffer capacity of IWW, which resists changes in pH. However, even small adjustments in pH can significantly impact water quality, contributing to the overall improvement of WW characteristics. The performance of P. aeruginosa in reducing pollution loads demonstrates its resilience and adaptability in treating highly polluted environments. Similarly, Enterobacter aerogenes (EA) exhibited high removal efficiency for several parameters, with BOD (80.62%), COD (83.52%), and DO (63.37%) removal being particularly notable. These results are consistent with the findings of Sharma et al.21, who observed a 54.8% reduction in BOD and a 61.1% reduction in COD when using Enterobacter spp. for textile WW treatment. In comparison to Pseudomonas aeruginosa, E. aerogenes demonstrated a higher overall removal efficiency, especially in COD and BOD reduction, indicating its strong ability to degrade organic pollutants. The EC removal efficiency of E. aerogenes (86.45%) was also significantly higher than that of Pseudomonas aeruginosa (49.57%), suggesting that E. aerogenes is more effective at reducing ion concentrations in WW. This aligns with the results of Rajalakshmi et al.63, who reported an 87% COD reduction and 80% TDS reduction from cheese processing WW using E. cloacae. The comparatively lower removal efficiency of TSS (55.19%) and TDS (0.0%) in E. aerogenes treatments indicates that while the bacterium is effective at reducing organic load, it may be less capable of removing suspended solids and dissolved inorganic substances. The comparison between the two bacterial species shows that while both are effective at pollutant removal, Enterobacter aerogenes demonstrated a higher overall efficiency, especially in reducing COD, BOD, and EC. On the other hand, Pseudomonas aeruginosa performed better in reducing TSS, making it potentially more suitable for WW with a high concentration of suspended solids. These differences highlight the importance of selecting the right bacterial species based on the specific characteristics of the WW being treated. Studies such as Onuoha et al.64 and Ratna and Kumar24 have shown similar variations in bacterial performance depending on WW composition. This suggests that a combination of both bacterial species could be explored for a more comprehensive remediation strategy, leveraging the strengths of each organism for optimal pollutant removal. The bioremediation potential of Pseudomonas aeruginosa and Enterobacter aerogenes for treating IWW has been clearly demonstrated in this study. Both species effectively reduced key pollutants, with E. aerogenes showing higher overall efficiency. These results align with previous findings in the literature, further supporting the role of bacteria in sustainable WW treatment strategies. Future studies could explore the synergistic effects of combining these bacterial species or applying them in different IWW contexts to maximize remediation outcomes.
The effectiveness of bacterial species for the bioremediation of TMs in IWW was evaluated by monitoring the reduction in concentrations of Cu, Cd, and Pb after treatment with specific bacterial strains. The results demonstrate the potential of Pseudomonas aeruginosa (PA) and Enterobacter aerogenes (EA) to remove significant amounts of TMs from WW, supporting their potential as viable options for bioremediation applications. The highest initial Cu concentration was observed in the PA and EA treatments, while the EA treatment also showed the highest final concentration of Cu. The bioremediation efficiency for Cu removal ranged from 47.05 to 82.35%, with the highest efficiency observed in the P. aeruginosa treatment (CTPA), which achieved an 82.35% reduction, and the lowest efficiency observed in Enterobacter aerogenes treatment (CTEA), which resulted in a 47.05% reduction. These findings are consistent with previous research. For example, Mubashar et al.65 recorded a 72% reduction in Cu concentration using Chlorella vulgaris and Enterobacter strains, while An et al.66 demonstrated an impressive 94.92% Cu removal efficiency using immobilized Pseudomonas hibiscicola. This variation in efficiency can be attributed to the specific bacterial strains and the environmental conditions in the WW. The t-test analysis further confirmed that the bacterial treatments significantly reduced Cu concentrations (p ≤ 0.05). The high Cu removal efficiency observed in Pseudomonas aeruginosa reflects the strong biosorption and bioaccumulation capabilities of this species, which has been widely reported in the literature for TM remediation. Cadmium concentrations in the water samples were reduced from an initial range of 0.011 to 0.61 mg/L to a final range of 0.005 to 0.49 mg/L, with removal efficiencies ranging from 19.67 to 62.29%. The highest efficiency was observed in the EA treatment (62.29%), while the lowest efficiency was observed in the PA treatment (19.67%). The t-test analysis confirmed that both bacterial species significantly reduced Cd concentrations in the water samples (p ≤ 0.05). Previous studies also support the efficacy of Pseudomonas aeruginosa and Enterobacter aerogenes in Cd removal. For example, Ugboma et al.67 reported a 77.59% Cd reduction using Pseudomonas aeruginosa, while Mubashar et al.63 recorded a remarkable 93% Cd removal using Chlorella vulgaris and Enterobacter. The biosorption efficiency of Enterobacter strains in Cd removal is particularly well-documented, as demonstrated by Yaqoob et al.68, who observed a 94.20% Cd removal efficiency in a bioremediation study. The lower bioremediation efficiency observed for Pseudomonas aeruginosa in this study may be attributed to the initial concentration of Cd in the water samples or variations in environmental conditions, which can influence bacterial metabolism and biosorption capabilities. The concentration of Pb in the water samples decreased significantly, from an initial range of 0.012 to 1.42 mg/L to a final range of 0.001 to 1.13 mg/L. Pb removal efficiencies ranged from 20.40 to 91.66%, with the highest efficiency observed in the P. aeruginosa treatment (CTEA), which achieved a 91.66% reduction, and the lowest efficiency observed in the PA treatment (20.40%). The t-test analysis confirmed that the bacterial species significantly reduced Pb concentrations in the water samples (p ≤ 0.05). These results align with the findings of previous studies. For instance, Ratna and Kumar24 reported a 73.06% reduction in Pb using Pseudomonas aeruginosa, while Mubashar et al.65 observed a 79% Pb reduction using Enterobacter. Yaqoob et al.68 recorded an even higher Pb removal efficiency of 97.34%, using potato waste as a bioremediation substrate. The high removal efficiency of Pb by Pseudomonas aeruginosa in this study can be attributed to the strong biosorption capabilities of this bacterial species. Additionally, the capacity of Enterobacter aerogenes to remove significant amounts of Pb also supports its potential for use in bioremediation processes targeting Pb-contaminated WW. Overall, the results of this study demonstrate that both Pseudomonas aeruginosa and Enterobacter aerogenes are highly effective in removing Cu, Cd, and Pb from IWW, with significant reductions in TM concentrations observed across all treatments. The bioremediation efficiency of these bacterial strains is comparable to previously reported studies, and their potential for large-scale bioremediation applications is promising. While both bacterial strains exhibited excellent bioremediation potential, the variation in removal efficiencies across different TMs and bacterial treatments suggests that the success of bioremediation efforts is influenced by several factors, including the bacterial species, initial TM concentrations, and environmental conditions. Further research is recommended to optimize these parameters for improved bioremediation outcomes.
The phytotoxicity assay conducted with Triticum sp. to evaluate the effects of untreated (UW) and treated IWW on germination, leaf length, shoot and root weights showed significant variations in plant response depending on the treatment applied. The results provide important insights into the toxicity reduction and water quality improvement after bacterial treatment of IWW. The germination rate of Triticum sp. varied notably between untreated and treated water samples. Seeds irrigated with UW showed a 60% germination rate, indicating moderate phytotoxicity of untreated WW. On the other hand, containers treated with Pseudomonas aeruginosa (CTPA and PA) and Enterobacter aerogenes (CTEA and EA) exhibited enhanced germination rates, with CTPA and CTEA showing 100% germination, indicating that the bacterial treatments substantially mitigated the toxic effects of the WW. The 100% germination in CTPA and CTEA suggests that Pseudomonas aeruginosa and Enterobacter aerogenes effectively reduced harmful contaminants in the WW, making it conducive for plant growth. This is further supported by other studies where bioremediation treatments enhanced seed germination and plant growth by detoxifying WW69. The reduction in germination to 60% in PA treatment, equivalent to UW, indicates that P. aeruginosa-treated WW still retained some phytotoxicity, potentially due to incomplete removal of contaminants. A similar trend was observed in leaf length, where plants irrigated with CTPA and CTEA-treated water exhibited the longest leaves at 16.3 cm and 15.9 cm, respectively, compared to just 7.1 cm for plants irrigated with untreated IWW. These findings suggest that plants exposed to UW experienced stress or toxicity, inhibiting growth and leaf development, which correlates with other studies where untreated IWW resulted in stunted plant growth68. Plants irrigated with treated water from EA also showed improved leaf length (10.2 cm), although not as pronounced as the CTPA and CTEA treatments. The shorter leaf length in the PA-treated group (7.8 cm) could be attributed to residual contaminants that may have persisted despite bacterial treatment. This highlights the importance of selecting effective bacterial strains for bioremediation to ensure adequate reduction of phytotoxic agents in IWW. The shoot fresh weight varied significantly across treatments, with the highest weight observed in plants irrigated with CTPA (14.9 g) and CTEA (13.9 g), followed by EA (8.4 g), and the lowest weight in PA-treated plants (6.9 g). Plants irrigated with UW had a considerably lower shoot weight (6.1 g), indicating that UW inhibits biomass production due to the presence of toxic TM or other pollutants. The enhancement in shoot weight after treatment with Pseudomonas aeruginosa and Enterobacter aerogenes indicates the effectiveness of these strains in detoxifying the water and providing an improved growth environment for the plants. The observed increase in root fresh weight, particularly in CTEA and EA treatments, further underscores the positive impact of bacterial treatment on plant development. However, the limited improvement in root weight in the PA treatment suggests that Pseudomonas aeruginosa may not be as effective as Enterobacter aerogenes in mitigating phytotoxicity, consistent with the lower germination rate and shorter leaf length. Significant differences were observed in shoot dry weight, with plants irrigated with CTPA-treated water achieving the highest dry weight (4.3 g) compared to the untreated control (2.5 g). This substantial increase in dry biomass reflects the enhanced growth conditions provided by the CTPA treatment, which facilitated better nutrient absorption and water retention, likely due to the reduction of toxic elements such as TMs70. Similarly, CTEA-treated water resulted in higher shoot dry weight (3.9 g), reinforcing the effectiveness of Enterobacter aerogenes in reducing phytotoxicity. Interestingly, there was no significant variation in root dry weight across treatments, although the highest value was recorded in the CTEA treatment (2.6 g). This suggests that while the treatments effectively promoted shoot growth, their impact on root development was less pronounced. This might be due to differences in how each bacterial species interacts with the root system and the availability of nutrients in the treated water. The phytotoxicity assay revealed that bacterial treatment of IWW using Pseudomonas aeruginosa and Enterobacter aerogenes significantly reduced the toxic effects of the WW on Triticum sp., leading to improved germination, leaf length, and shoot biomass. The most effective treatments were CTPA and CTEA, which outperformed other treatments in all key plant growth parameters. These results emphasize the potential of these bacterial strains for effective bioremediation of IWW, reducing its phytotoxicity and promoting healthier plant growth. Further research could focus on optimizing the conditions for bacterial treatment to maximize bioremediation efficiency and ensure minimal residual phytotoxicity in treated water.
The presence of organic content in IWW can significantly impact the effectiveness of bioremediation processes. Organic matter, often consisting of complex compounds, can serve as a nutrient source for microorganisms, potentially enhancing their growth and activity71. This can be beneficial for the overall bioremediation process, as a more robust microbial community can improve the degradation of contaminants. However, high concentrations of organic content may also lead to competition for metal binding sites or interfere with the bioavailability of trace metals, potentially reducing the efficiency of metal removal72. For instance, organic compounds can form complexes with trace metals, making them less available for microbial uptake and removal2. Furthermore, the presence of excessive organic matter can lead to the formation of sludge and affect the operational stability of bioreactors73. These factors highlight the need for careful management of organic content in WW treatment processes to optimize bioremediation outcomes. Future studies should explore strategies to mitigate these limitations, such as pre-treatment methods to reduce organic load or the development of specialized microbial strains capable of handling high organic content74. In this study, Pseudomonas aeruginosa and Enterobacter aerogenes were evaluated for their effectiveness in the bioremediation of IWW, focusing on the removal of trace metals (Cu, Cd, Pb). The presence of multiple TMs in IWW presents a more complex scenario compared to single-metal systems, as competition between metal ions for active sites on the biosorbent affects the overall adsorption capacity. The general trend observed in our study was Pb > Cd > Cu in removal efficiency, indicating that Pb ions had the highest affinity for the biosorbent, followed by Cd and Cu. This is consistent with previous studies that have reported the selective affinity of biosorbents in multi-metal environments44,75.
In terms of operational costs, the majority of materials used in this study, including the bacterial strains (Pseudomonas aeruginosa and Enterobacter aerogenes), WW samples, and laboratory equipment, were provided free of cost. The only direct expense was for the pots used in the experiment, which cost approximately $10. Since no specialized equipment or reagents were required, the overall material costs were minimal. For scaling up the process, operational costs would include labor and energy expenses associated with managing bioreactors, including temperature and aeration control. Although heavy metal analysis (e.g., atomic absorption spectroscopy) was free for this study, typical costs can be significant, approximately $30 per sample. Bioremediation, as demonstrated in this study, offers a cost-effective alternative to traditional methods like chemical precipitation, ion exchange, or membrane filtration, which often involve high costs for chemicals, energy, and infrastructure. Additionally, bioremediation produces minimal hazardous waste, primarily limited to treated water and bacterial biomass. Disposal of excess biomass is relatively straightforward and less costly than the disposal of sludge from conventional treatment processes. This makes bioremediation a more environmentally and economically favorable alternative. Future cost analyses for scaling up this bioremediation approach should take into account labor, energy, and reactor maintenance. However, initial results suggest that the method could provide significant savings compared to conventional treatments, making it a feasible and cost-effective solution for treating IWW.
Practical applications and future perspectives
Our study introduces significant advancements in the bioremediation of IWW through the use of indigenous bacterial species Pseudomonas aeruginosa and Enterobacter aerogenes. Unlike previous research, which often relies on synthetic chemicals or less effective biological agents, our approach leverages these native bacteria for their exceptional capabilities in removing trace metals such as Pb, Cd, and Cu. This research is novel in its integrated use of microbial and phytotoxicity assessments, providing a comprehensive solution for multi-metal pollution that improves water quality and environmental safety more effectively than existing methods. The findings of this study open several avenues for future research. Further investigations could explore the scalability of the bioremediation process for larger industrial applications and assess its effectiveness in diverse types of WW. Research could also focus on optimizing the conditions under which these bacterial species perform best, evaluating their long-term stability and performance in real-world settings, and exploring the potential synergistic effects of combining these bacteria with other bioremediation techniques or genetic modifications to enhance their efficacy. The practical applications of our work are substantial. The bioremediation method developed can be employed in various industrial sectors to treat WW contaminated with TM, providing a cost-effective and environmentally friendly alternative to traditional treatment methods. This approach is particularly relevant for industries such as mining, manufacturing, and electroplating, where heavy metal pollution is prevalent. Furthermore, our method minimizes the need for harmful chemicals and reduces the environmental footprint of WW treatment by ensuring that TM are removed without generating hazardous byproducts. Effective removal of toxic metals from IWW has direct implications for public health. By improving the quality of water released into natural water bodies, this method helps to prevent the contamination of drinking water sources and agricultural lands, thereby reducing the risks of heavy metal exposure to humans and wildlife. Additionally, the successful integration of microbial and phytotoxicity assessments allows for the safe reuse of treated water in agricultural practices, supporting sustainable resource management and water conservation efforts.
Conclusion
This study demonstrates a significant advancement in the treatment of industrial wastewater (IWW) through the bioremediation potential of indigenous bacterial species, Pseudomonas aeruginosa and Enterobacter aerogenes. These bacterial strains effectively reduced pollution levels in wastewater from Hayatabad Industrial Estate (HIE), where parameters such as pH, total dissolved solids (TDS), and copper (Cu) were within permissible limits, while TDS, chemical oxygen demand (COD), biological oxygen demand (BOD), and trace metal concentrations (lead [Pb] and cadmium [Cd]) exceeded the thresholds set by the Pakistan Environmental Protection Agency (Pak-EPA, 2008). Among the tested bacterial species, P. aeruginosa exhibited the highest Cu removal efficiency (up to 82.35%), while E. aerogenes was more effective in removing Cd and Pb, following the trend Pb > Cd > Cu. These findings validate the hypothesis that indigenous bacteria can outperform conventional treatment methods and demonstrate their resilience in metal-stressed environments. While bioremediation offers an eco-friendly and cost-effective approach to wastewater treatment, integrating hybrid technologies—such as microbial fuel cells (MFCs), biochar-assisted bioremediation, phytoremediation, and advanced oxidation processes (AOPs)—could further enhance pollutant removal efficiency. Compared to conventional physicochemical treatment methods, which often involve high energy consumption, chemical sludge generation, and operational costs, the proposed bioremediation approach is more sustainable. However, coupling bioremediation with nanotechnology-based adsorbents or electrochemical methods may optimize metal recovery and improve overall treatment efficiency.
Data availability
All data are fully available and can be found within the manuscript or in the Supporting Information file. There are no legal restrictions in this regard. Sequences generated from the current study has been available from NCBI GenBank with accession numbers https://www.ncbi.nlm.nih.gov/nuccore/PQ772640 and https://www.ncbi.nlm.nih.gov/nuccore/PQ772641.
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Acknowledgements
The authors would like to extend their sincere appreciation to the Researchers Supporting Project number (RSP2025R165), King Saud University, Riyadh, Saudi Arabia. The authors would also like to extend their sincere appreciation to the Central Resources Laboratory (CRL) Peshawar and Microbiology Research Lab (MRL) Abasyn University Peshawar Pakistan.
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The authors would like to extend their sincere appreciation to the Researchers Supporting Project number (RSP2025R165), King Saud University, Riyadh, Saudi Arabia.
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Conceptualization by Sara Khan., Amin Ullah and Saba Mazhar Shah; methodology by Sara Khan. and Amin Ullah; software by Sara Khan; validation by Bushra Rehman., Semih Yilmaz and Ramzan Ali.; resources by Amin Ullah and Ramzan Ali; data curation by Nadia BiBi., Saba Mazhar Shah and Ramzan Ali; writing—original draft preparation was prepared by Saba Mazhar Shah., Sara Khan and Amin Ullah; writing—review and editing by Qurban Ali., Amin Ullah., and Nadia Bibi; visualization by Nadia Bibi and Semih Yilmaz; supervision, Amin Ullah and Sara Khan; project administration, Semih Yilmaz, Daoud Ali and Amin Ullah.; All authors have read and agreed to the published version of the manuscript.
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Shah, S.M., Khan, S., Bibi, N. et al. Indigenous bacteria as potential agents for trace metal remediation in industrial wastewater. Sci Rep 15, 13141 (2025). https://doi.org/10.1038/s41598-025-97711-y
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DOI: https://doi.org/10.1038/s41598-025-97711-y