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Article

Development of Bacterial Augmented Floating Treatment Wetlands System (FTWs) for Eco-Friendly Degradation of Malachite Green Dye in Water

by
Sania Sahreen
and
Hamid Mukhtar
*
Institute of Industrial Biotechnology, Government College University, Lahore 54000, Pakistan
*
Author to whom correspondence should be addressed.
Sustainability 2023, 15(5), 4541; https://0-doi-org.brum.beds.ac.uk/10.3390/su15054541
Submission received: 30 November 2022 / Revised: 20 February 2023 / Accepted: 22 February 2023 / Published: 3 March 2023
(This article belongs to the Special Issue Sustainable Approach for Clean Environment through Phytoremediation)
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Abstract

:
Industrial revolution has intensified water pollution due to the indiscriminate discharge of untreated industrial effluents into water bodies, posing a serious threat to the whole ecosystem. Recently, the floating treatment wetlands system (FTWs) technique has been used as one of the most innovative, cost-effective and environment-friendly option for wastewater treatment. The present study is aimed at investigating the Malachite green (MG) dye decolorizing potential of Eichhornia crassipes (water hyacinth) through the development of a bacterial augmented FTWs. To an artificial FTWs, vegetated with E. crassipes and bioaugmented with Pseudomonas putida and Pseudomonas sp., synthetically prepared MG dye enriched wastewater was added. Among all the treatments developed, floating wetlands treatment T2 (consisting of dye, E. crassipes and Pseudomonas putida) performed the best in decolorizing the dye and in reducing values of electrical conductivity (EC), pH and total dissolved solids (TDS) of the treated water. Bacterial inoculation proved fruitful in assisting the increased MG dye decolorization in partnership with E. crassipes and also helped in aquatic plant growth promotion. MG dye toxicity effects were studied through phytotoxicity assay using FTWs treated water on Pisum sativum seeds, and satisfactory results were obtained. From experimental results, it can be seen that Eichhornia crassipes with bacterial inoculation have a strong ability to degrade and decolorize MG dye in textile effluents. We conclude that the plant-microbial assisted FTWs technique can be a unique approach to remediate the textile dye effluents before their release into water bodies.

Graphical Abstract

1. Introduction

Industrial revolution has intensified water pollution due to the indiscriminate discharge of untreated industrial effluents into water bodies. Across the world, many developing countries discharge their industrial effluents directly into water bodies without any prior treatment, resulting in greasy, oily water formation in water bodies [1]. These countries also use water from contaminated waterbodies for crop irrigation. Approximately, 50 countries in the world use untreated or poorly treated industrial wastewater for irrigation of 20 million hectares of land [2]. Dyeing industries produce contaminant-full wastewater containing nitrates, nitrites, anions (CO32−, HCO3− and Cl−), cations (Na, Mg, Ca and K) and toxic heavy metals (Cu, Cd, Hg, Co, Ni, Pb, Cr and Fe) in addition to different dyes [3]. Industrially discharged effluents have high pH, temperature, COD (chemical oxygen demand), BOD (biological oxygen demand) and color. It is also turbid, smells strong and contains loads of toxic chemicals. Discharge of industrial effluents without treatment in water bodies lowers water’s DO (dissolved oxygen) levels and has a negative effect on many fauna and flora posing a serious threat for aquatic ecosystem sustainability [2].
Triphenylmethane (TPM) dyes are intense, brightly colored synthetic dyes with their applications in textile industry. Around 30–40% consumption of this dye has been reported in the textile industry for dyeing of wool, cotton and silk fabrics [4]. Malachite green (MG) is a water-soluble, cationic-green crystalline dye that belongs to the triphenylmethane dye category (Table 1) [5]. It is used in the textile industry for dyeing purposes, and later this colored textile effluent is discharged into water bodies without any prior treatment. MG dye also has its applications in the fish industry where it is used for treating parasitic, fungal and bacterial infections [6,7]. MG enriched discharged textile water contains phenols and amines that are difficult to degrade, which is a threat to aquatic fauna and flora as it hinders the photosynthetic process of aquatic life forms [8]. This dye has also been reported as a multi organ toxin dye in humans upon its inhalation and ingestion [9,10]. It affects skin, bones, eyes, liver, lungs, spleen, heart, kidney, brain and nervous system. It can also cause respiratory toxicity, chromosomal fractures, mutagenesis and carcinogenesis, etc. [5,6]. Because of these, the removal of malachite green dye from polluted water bodies is greatly needed.
Traditional wastewater treatment methods are unable to degrade all the aromatic ring structure-based water pollutants. Thus, for complete degradation of dye effluents, various physico-chemical methods, such as membrane bioreactors, floatation, chemical oxidation, adsorption, and coagulation, are used in combination. These techniques are a source of secondary pollution as they produce toxic byproducts and large amounts of sludge [4]. They also have other requirements, including engineering skills, labor administration, high operational cost, etc. [11]. In contrast to this, biological methods are ecofriendly and can degrade pollutants completely [12]. Recent advances in ecological engineering have given birth to the floating treatment wetlands system (FTWs) technique for polluted wastewater treatment [11]. Use of the floating treatment wetlands system (FTWs) vegetated with aquatic plants for wastewater treatment has become popular nowadays as an efficient, cheap and cost-effective approach in comparison to other traditional wastewater treatment methods [13,14,15].
Many researchers have employed FTWs in their studies for effective wastewater treatment, i.e., acid mine drainage wastewater [16], sewage effluents [17], stormwater [18], industrial wastewater [19] and municipal wastewater [20,21]. However, all these studies reported poor wastewater treatment due to the weak metabolic capabilities of the used plants [22]. As a result of this, addition of bacteria (capable of pollutant degradation and plant growth promotion) as an additional approach was suggested by Shehzadi et al. [23] and Saleem et al. [24]. Hence, vegetated plants in the FTWS were bioaugmented with bacteria to form a plant bacteria partnership where inoculated bacteria not only helped plants in wastewater treatment but also assisted in plants’ growth through their plant growth-promoting traits [25,26]. Many researchers have obtained excellent results from employing a plant bacteria partnership approach in FTWs for treatment of activated sludge, textile effluents, pesticide-contaminated soil and petroleum hydrocarbons contaminated soil [27,28,29,30] as well as for remediation of various types of wastewaters [31,32,33].
Aquatic plants like Lemna sp., Spirodela sp., Pistia stratiotes and Eichhornia crassipes have been reported for their potential in domestic wastewater treatment. Numerous studies, including Gamage et al. [34], Saha et al. [35] and Ansari et al. [36], have used E. crassipes for wastewater treatment due to its amazing phytoremediation potential. Dias et al. [37] used Eichhornia crassipes, along with three other aquatic plants Spirodela sp., Pistia stratiotes and Lemna sp., for effluent treatment from Mussuré stream (Brazil). In their study, E. crassipes removed wastewater color up to 95%, biochemical oxygen demand (BOD) and turbidity by 53% and 83%, respectively. Similar to this, Qamar et al. [38] and Tusief et al. [39] have also successfully used E. crassipes vegetated FTWs bioaugmented with Bacillus cereus and Bacillus subtilis for H2O2 and Blue reactive dye polluted textile water treatment. All these studies highlighted the phytoremediation ability of E. crassipes aquatic plant for satisfactory wastewater treatment and its later safe release into water bodies.
Eichhornia crassipes is an indigenous aquatic plant of Lahore, Pakistan and hence was selected to conduct this study. So far, the potential of Eichhornia crassipes (water hyacinth) for ecofriendly degradation of Malachite green dye though the establishment of the FTWs has not yet been explored much. The present study is probably the first attempt to study environment friendly degradation of Malachite green dye in water through establishment of bacterial augmented floating treatment wetlands system. Therefore, in the present study, the FTWs vegetated with Eichhornia crassipes were bioaugmented with two MG dye decolorizing and plant growth-promoting bacteria. These bacteria were isolated from aquatic plants and were also screened for their plant growth-promoting traits. Following that, parameters that might affect FTWs’ performance were also studied. Lastly, the potential of E. crassipes with bacterial inoculation for MG dye degradation was also studied for effective wastewater treatment.

2. Materials and Methods

2.1. Sampling Sites

Aquatic plants used in the study were collected from various sites of Lahore, Pakistan as sampling sites lie between 31°15′–1°45′ N and 74°01′–74°39′ E (see Supplementary Information File Section 2.1 for more information). The sampling area has a semi-arid climate, 24.8-inch average annual rainfall, 53% humidity and a year-round, average temperature between 46 °F and 103 °F (see Figure S1).

2.2. Collection of Aquatic Plants

Fresh and juvenile aquatic plants, i.e., Nymphaea sp., Azolla filiculoides, Pistia stratiotes, Hydrocotyle vulgaris, Eichhornia crassipes, Lemna minor and Hydrilla verticillata from the botanical garden of Government College University Lahore as well as locally available Spirodela polyrhiza, Lemna minor and Wolffia arrhiza in Lahore (Atta Baksh road 1&2, i.e., roadside stream and residential area plot covered with rainy water, turned muddy for years now, respectively), were handpicked in polythene bags for immediate transfer to the laboratory (see Supplementary Information File Section 2.2 for more information). At the lab, all plants were washed with tap water and then with distilled water to ensure the complete removal of dirt or soil present on them.

2.3. Isolation of Bacteria

Collected aquatic plants were cut into small pieces (1–2 inch size) and added to 10 mL of sterile distilled water in 50 mL conical tubes for isolation of bacteria. Due to small size, both duckweed and Azolla plants were cut into their respective fronds and roots. All plant samples were sonicated with a 0.5 s on-and-off interval for 1 min to detach bacteria associated with the plant body. All samples were serially diluted, and 0.1 mL of dilution 10−4, 10−5 and 10−6 was added on nutrient agar plates, which were then incubated at 30 °C for 24–48 h [40]. Bacterial colonies, which morphologically differ from each other, were selected and purified for further study.

2.4. Screening of Bacterial Isolates for MG Dye Decolorization

Selected bacterial isolates were then screened for Malachite green (MG) dye decolorization on nutrient agar plates supplemented with 100 ppm of MG dye. All plates were incubated at 30 °C for 24–48 h, and later bacterial growth on them was observed. Additionally, flask screening was also performed to quantitatively determine MG dye decolorization potential of all bacterial isolates. All isolates were then inoculated in 50 mL nutrient broth medium (augmented with 100 ppm dye) and incubated at 30 °C. Culture broth was centrifuged for 10 min at 6000 rpm, and supernatant was used for optical density (O.D) measurement at 618 nm through UV/Vis spectrophotometer [4,41,42]. MG dye decolorization percentage was calculated by the following equation:
Dye   decolorization   ( % ) = A 0 − At A 0 × 100    
where A0 = Initial absorbance of the medium and At = Absorbance of medium at time t.

2.5. Screening of Bacterial Isolates for Plant Growth-Promoting (PGP) Traits

Bacterial isolates were further screened for plant growth-promoting (PGP) traits, such as phosphate solubilization, indole acetic acid production (IAA), nitrogen-fixation, HCN production and potassium solubilization, present in them. The phosphate solubilizing ability of isolated bacteria was tested on Pikovskaya (PVK) agar plates containing insoluble tri-calcium phosphate, according to the method described by Gupta et al. [43]. For indole acetic acid production, isolates were cultured in nutrient broth amended with 0.1% DL tryptophan, following the method of Loper and Schroth [44] and Gordon and Weber [45]. The nitrogen-fixing ability of isolates was tested according to the method of Pérez-Rodriguez et al. [46] for detection of pellicle formation, indicating the ability of the isolate to fix nitrogen. For screening of HCN-producing bacteria, the method of Lorck [47] was employed, and a change in filter paper color was observed. Rajawat et al.’s [48] unmodified method was used for the determination of potassium solubilizing bacteria on Aleksandrov medium plates.

2.6. Morphological and Biochemical Characterization

Based on MG dye decolorization and PGP screening results, two bacterial isolates were selected and subjected to morphological and biochemical testing. Isolates were morphologically characterized based on colony characters that appeared on a 24 h old nutrient agar plate, followed by Gram staining, to observe their cell morphology. Biochemical tests, including the Catalase and Oxidase tests, were performed. On the basis of Gram stain and Oxidase test results, Biomerieux API 20 NE Kits were used for genus level identification of selected bacterial isolates.

2.7. Preparation of Floating Treatment Wetlands System (FTWs)

2.7.1. Lab Synthesis of MG Dye Enriched Industrial Wastewater

Synthetically prepared Malachite green (MG) dye enriched wastewater was prepared in 100 ppm dye concentration (see Supplementary Information File Section 2.7.1 for detailed wastewater composition.

2.7.2. Macrophytes Used

Eichhornia crassipes (water hyacinth) was selected for the development of the FTWs in the current study due to being an indigenous aquatic flora of Lahore, Pakistan. It was obtained from the botanical garden associated with Government College University, Lahore, located at 31°33′23.7″ N and 74°19′41.9″ E (see Supplementary Information File Section 2.2 for more information).

2.7.3. Bacterial Inoculum Preparation

Two of the selected bacterial strains were used for inoculum preparation in FTWs. A bacterial inoculum was developed by inoculating a loopful of bacterial isolate from a fresh agar slant into an Erlenmeyer flask containing 25 mL of sterilized nutrient broth and incubated at 30 °C in a shaking incubator. After 24 h of incubation, culture broths were centrifuged and cell pellets were re-suspended in physiological saline (0.87% NaCl at pH 7) [49]. For each bacterial inoculum, optical density was set at 0.9 (600 nm) using a UV/Vis spectrophotometer.

2.8. Designing and Experimental Setup of FTWs

A glass aquarium with the capacity of six liters was used to develop floating treatment wetlands system vegetated with Eichhornia crassipes (three plants of equal weight). Each aquarium was filled up to 3 L with synthetically prepared MG enriched wastewater containing 100 mL of bacterial inoculum (3.3%) and Hoagland solution, according to the experimental design. The experiments were run in the following treatment designs:
  • Control 1 = Only dye;
  • Control 2 = Freshwater and plants;
  • T1 = Dye and plants;
  • T2 = Dye, plants and bacteria (F1);
  • T3 = Dye, plants and bacteria (F2).

2.9. Optimization Studies for Dye Degradation in FTWs

The effect of factors, such as treatment time (24, 48, 72 and 96 h), initial dye concentration (50, 100, 150 and 200 ppm) and pH (6, 6.5, 7, 7.5 and 8) on MG dye decolorization in FTWs by bacterial augmented Eichhornia crassipes, were studied. For optimization of MG dye exposure time, pH and initial dye concentration, study was carried out at pH 8 with 100 ppm of MG dye concentration. All treatments were run for 48 h and incubated at room temperature unless stated otherwise.

2.10. Study of Physiochemical Parameters of Wastewater in FTWs

Using results from optimization studies from each treatment, water samples were collected and used for analysis of water quality parameters, such as electrical conductivity (EC), total dissolved solids (TDS), pH and MG dye decolorization (%) at 24, 48, 72 and 96 h of FTWs development (see Supplementary Information File Section 2.10 for more information). Freshwater was added in each glass aquarium up to 3 L to recover the evaporation loss.

2.11. Enumeration of Bacterial Survival in Water

The bacterial inoculum prepared above was added to the glass aquarium, and bacterial count in wastewater was calculated at 24 and 96 h of FTWs development through plate count method. Water samples from the glass aquarium were serially diluted, and 10−3 dilution was spread on nutrient agar plates for CFU/mL count at the start and end of the experiment.

2.12. Plant Growth Monitoring Study

Plant weight, stalk and root length were measured at the end of the experiment to study the effect of bacterial inoculation and MG dye toxicity on plant growth. Plant weight was measured with the help of a digital weighing balance. For root and stalk length measurement, a measuring scale was used.

2.13. Chlorophyll and Carotenoids Contents

For chlorophyll and carotenoids content estimation, Hussain et al.’s [50] method was adopted. Briefly, a 50 mg leaf piece was homogenized in 10 mL acetone (80%) and then left in the dark (for 1 h) for pigment extraction at 4 °C. It was then centrifuged, and supernatant was used for chlorophyll a and b estimation at 650 nm and 665 nm by UV/Vis spectrophotometer. Aliquots of the same supernatant were mixed with 15 mL of dimethyl ether and 5 mL of NaOH (1 M) and were used for carotenoid content estimation at 450 nm using UV/Vis spectrophotometer.

2.14. Phytotoxicity Assay

Phytotoxicity assay was performed to study MG dye toxicity effect on Pisum sativum seeds before and after treatment in FTWs. Simply, all seeds were first surface sterilized using 0.01% sodium hypochlorite for 1 to 2 min, then rinsed in distilled water twice or thrice and later placed on wet tissues set in autoclaved petri dishes. Each plate contains a total of 30 seeds and was semi-sealed to prevent moisture loss. For five days, 2 mL of tap water, synthetically prepared MG wastewater (100 ppm dye) and treated water were sprayed on tested seeds. After 5 days, the plates were observed, and results of the percentage of seed germination, plumule and radicle lengths were noted [51,52].

3. Results

3.1. Isolation and Screening of MG Dye Decolorizing Bacteria

A total of 40 epiphytic bacterial isolates, which differed from each other in their colony characters, were isolated on nutrient agar plates from aquatic plants and later purified for further study. Among them, 14 bacterial isolates showed the ability to decolorize Malachite green (MG) dye on nutrient agar-amended MG plates. From there, six further isolates (F1, F2, F4, F7, F10 and F12), showing the strongest ability to decolorize and degrade MG dye, were selected for further testing (Figure 1).

3.2. Assessment of Bacterial Isolates for Plant Growth-Promoting (PGP) Traits

The selected epiphytic bacterial isolates were assessed for PGP traits, such as phosphate solubilization, IAA production, nitrogen fixation, HCN production and potassium solubilization activity. Screening results of PGP results are given in Table 2. None of the isolates were positive for potassium solubilization, and no isolates except F4 were positive for phosphate solubilization. All isolates showed weak to strong IAA production in the test medium, except the F4 isolate which was unable to produce IAA in the medium. Out of all 6 bacterial isolates tested for nitrogen fixation ability, only F10 was able to fix nitrogen in the medium. All isolates except F7 were HCN producers in the medium plate.

3.3. Bacterial Isolates Selected for Further Study

Based on highest MG dye decolorizing ability and PGP traits, bacterial isolates F1 and F2 were chosen for further study. Bacterial isolate F1 was isolated from the roots of Spirodela polyrhiza obtained from Attabaksh road 2, i.e., the residential area plot covered with rainy water, turned muddy for years now, and similarly, bacterial isolate F2 was isolated from the roots of Hydrocotyle vulgaris aquatic plant obtained from GCU botanical garden.

3.4. Identification of Selected Bacterial Isolates

Based on morphological colony characters that appeared on nutrient agar plates after 24 h of plate incubation and API 20 NE biochemical test results, bacterial isolate F1 was identified as Pseudomonas putida, and isolate F2 was identified as Pseudomonas sp. (Table 3 and Table 4).

3.5. Optimization Studies for MG Dye Decolorization in FTWs

Optimization factors, such as treatment time (24, 48, 72 and 96 h), initial dye concentration (50, 100, 150 and 200 ppm) and pH (6, 6.5, 7, 7.5 and 8) on MG dye decolorization in FTWs, were studied. The graphs from Figure 2, Figure 3 and Figure 4 show the effect of treatment time, pH and initial dye concentration on MG dye decolorization by aquatic plants and inoculated bacterial isolates in floating treatment wetlands system (FTWs).
Study findings revealed that treatment time has a significant correlation with dye decolorization percentage where an increase in treatment time resulted in enhanced dye decolorization. Initially at 24 h, treatment T1 (consisting of dye and Eichhornia crassipes), treatment T2 (consisting of dye, Eichhornia crassipes and Pseudomonas putida) and treatment T3 (consisting of dye, Eichhornia crassipes and Pseudomonas sp.) decolorized dye up to 53.5 ± 1.68%, 60.41 ± 1.5% and 56.57 ± 1.03%, respectively. However, when treatment time reached 96 h, an increased dye decolorization was observed by all three treatments T1, T2 and T3, i.e., 93.68 ± 1.5%, 97.24 ± 1.43% and 95.3 ± 1.73%, respectively. Out of all three treatments developed, treatment T2 consisting of dye, Eichhornia crassipes and Pseudomonas putida decolorized the highest percentage of dye (Figure 2).
pH 8 was found to be the most suitable pH for effective dye decolorization in FTWs, where highest dye decolorization results were obtained by all three designed treatments. Not much difference in percentage dye decolorization was observed at different pH by designed treatment T1 (consisting of dye and Eichhornia crassipes), T2 (consisting of dye, Eichhornia crassipes and Pseudomonas putida) and T3 (consisting of dye, Eichhornia crassipes and Pseudomonas sp.), and dye decolorization results ranged between pH 6 to pH 8. Treatment T1, T2 and T3 decolorized dye from 82.67 ± 2.65%, 90.99 ± 1.90% and 86.04 ± 1.5% at pH 6 to 86.59 ± 1.91%, 94.84 ± 1.86% and 89.79 ± 1.89%, respectively, at pH 8. Maximum dye decolorization results were provided by treatment T2 comprised of dye, Eichhornia crassipes and Pseudomonas putida (Figure 3).
A significant effect of initial dye concentration was observed on percentage dye decolorization. At low dye concentration, all treatments worked well and decolorized high percentages of dye from the water. However, at high concentrations of dye, a decline pattern in percentage dye decolorization was observed. Initially, at 50 ppm of dye concentration, treatment T1 (containing dye and Eichhornia crassipes), treatment T2 (containing dye, Eichhornia crassipes and Pseudomonas putida) and treatment T3 (containing dye, Eichhornia crassipes and Pseudomonas sp.) decolorized dye up to 88.81 ± 1.68%, 95.39 ± 1.51% and 92.59 ± 1.03%, respectively. However, when dye concentration was gradually increased to 200 ppm, a decline pattern in percentage dye decolorization was observed by all three treatments T1, T2 and T3, i.e., 34.77 ± 1.55%, 49.62 ± 1.43% and 37.4 ± 1.73%, respectively. Out of all three treatments, treatment T2 consisting of dye, Eichhornia crassipes and Pseudomonas putida decolorized the highest percentage of dye at all provided concentrations of dye in water (Figure 4).

3.6. Physicochemical Parameters of Treated Wastewater

FTWs designed in our study had effectively reduced values of electrical conductivity (EC), total dissolved solids (TDS) and pH in the treated wastewater in addition to the increased values of percentage dye decolorization. Designed treatments T2 and T3 inoculated with bacteria performed better than un-inoculated treatment T1. Out of all three designed treatments, treatment T2 consisting of dye, Eichhornia crassipes and Pseudomonas putida performed well for all the tested wastewater quality parameters, i.e., EC, pH, TDS and dye decolorization.
A direct relation between treatment days and dye decolorization was found. Initially on day 1, low values of percentage dye decolorization were seen, but with an increase in treatment days, a remarkable increase in percentage dye decolorization was also observed (Figure 5). In treatment T1 containing dye and Eichhornia crassipes, significant dye decolorization percentages were observed. However, with bacterial inoculation, an increase in percentage dye decolorization was found in both treatment T2 consisting of dye, Eichhornia crassipes and Pseudomonas putida and treatment T3 consisting of dye, Eichhornia crassipes and Pseudomonas sp. Nonetheless, the highest percentage of dye decolorization, i.e., 91.58 ± 1.5%, was found in the case of floating wetlands designed treatment T2 (consisting of dye, Eichhornia crassipes and Pseudomonas putida).
For all designed floating wetlands treatment, an inverse relation was seen between treatment time and electrical conductivity (EC). Starting from day 1 to day 4, a significant decrease in designed FTWs was observed in EC values. The highest reduction in EC values, i.e., 1.52 ± 0.02 to 0.475 ± 0.02 mS/cm, was observed in the case of treatment T2 (consisting of dye, Eichhornia crassipes and Pseudomonas putida). Other treatments like treatment T1 (consisting of dye and Eichhornia crassipes) lowered down EC values from 1.516 ± 0.03 to 0.605 ± 0.01 mS/cm and treatment T3 (consisting of dye, Eichhornia crassipes and Pseudomonas sp.) from 1.523 ± 0.02 to 0.565 ± 0.03 mS/cm, respectively (Figure 6).
A pattern similar to electrical conductivity was also observed in the case of total dissolved solids (TDS) analysis. It was found that it had an inverse relation with treatment days, and the maximum decrease in TDS values was found on treatment day 4 (Figure 7). The highest decrease was observed in case of treatment T2 (consisting of dye, Eichhornia crassipes and Pseudomonas putida), i.e., 0.486 ± 0.01 from 0.951 ± 0.006 (g/L), followed by treatment T3 (consisting of dye, Eichhornia crassipes and Pseudomonas sp.) and treatment T1 (consisting of dye and Eichhornia crassipes).
In all designed treatments, a shift in pH from basic to neutral condition was observed. In case of treatment T1 (containing dye, Eichhornia crassipes), T2 (containing dye, Eichhornia crassipes and Pseudomonas putida) and T3 (containing dye, Eichhornia crassipes and Pseudomonas sp.), pH moved from 8.0 ± 0.1 to 7.48 ± 0.06, 7.32 ± 0.07 and 7.34 ± 0.06, respectively. Again, the highest decrease in the values of pH was observed in the case of treatment T2 consisting of dye, Eichhornia crassipes and Pseudomonas putida (Figure 8).
Out of three treatment systems developed, treatment T2 consisting of dye, plant and Pseudomonas putida performed the best in terms of EC, TDS, pH and dye removal from water. A table where removal efficiencies of all three treatments in terms of EC, TDS and dye removal has been given below (Table 5). From the table, it is evident that treatments with bacterial inoculation gave better results than treatment T1 with no bacterial inoculation.

3.7. Enumeration of Bacterial Survival in Water

With the help of plate count method, bacterial survival in treatment T2 (containing dye, Eichhornia crassipes and Pseudomonas putida) and T3 (containing dye, Eichhornia crassipes and Pseudomonas sp.) was observed. In both treatments, inoculated bacteria showed persisted survival in the treated wastewater. However, compared to day 1, a decrease in bacterial count was observed on day 4 (Table 6) (see Supplementary Information File Section 3.7 for more information).

3.8. Plant Growth Monitoring Study

The effect of bacterial inoculation and dye toxicity on plant growth and development was monitored. From the study results, it was found that plants grown in tap water only (Control 2) showed maximum growth. However, plants grown in dye water inoculated with bacteria showed better growth than un-inoculated plants grown in wastewater. Compared to all FTWs-designed treatments, maximum plant growth was observed in Control 2 where plants were grown in tap water only. The second-best plant growth results were found in FTWs treatment T2 (containing dye, Eichhornia crassipes and Pseudomonas putida) (Table 7). These results confirm the beneficial role and effect of bacterial inoculation on plant growth in treatments, despite the dye-induced toxicity.

3.9. Chlorophyll and Carotenoids Content

Effect of dye toxicity on aquatic plant’s photosynthetic pigments, i.e., chlorophyll a, chlorophyll b and carotenoids content was also studied. From study results, compared to initial pigment content, a decrease in pigment content was found in plants (Figure 9).

3.10. Phytotoxicity Assessment of Treated Wastewater

Textile dyes pose a serious threat to the environment, and the direct dumping of textile effluents in water bodies is a serious global concern. To determine toxicity effects of MG dye treated in FTWs, seed germination assay was used. Significant values of percentage seed germination, plumule and radicle lengths were observed on Pisum sativum seeds treated with wastewater as compared to synthetically prepared MG-enriched industrial wastewater (Table 8). Phytotoxicity results suggest that floating wetlands treatments designed in the current study successfully reduced the toxic nature of MG dye and its degraded metabolites.

4. Discussion

In the present study, dye degrading and plant growth-promoting bacteria were isolated from aquatic plants and were inoculated in the floating treatment wetlands system (FTWs) for efficient treatment of Malachite green (MG) enriched industrial wastewater. Study results indicated the remarkable ability of designed FTWs vegetated with Eichhornia crassipes and inoculated with bacteria for MG dye decolorization and decreased pH, EC and TDS values to significant levels. The performance of FTWs indicates the potential role of aquatic plants and bacteria in treating pollutant-contaminated water.
Available studies indicate that, similar to terrestrial plants, aquatic plant are also a great source for the isolation of significant bacteria. In many previously published studies, aquatic plants have been used for isolation of bacteria as reported by Yoneda et al. [40] and Tanaka et al. [53]. They used Lythrum anceps, Phragmites australis and duckweed for isolation of bacteria. Many bacteria such as Ochrobactrum pseudogrignonense strain GGUPV1 [54], Enterobacter spp. CV-S1 and Enterobacter spp. CM-S1 [55], Stenotrophomonas maltophilia [4], Pseudomonas putida [56] and Bacillus cereus strain KM201428 [57] are reported in literature for their MG dye decolorizing potential. In a study on MG dye decolorization using Stenotrophomonas maltophilia, Alaya et al. [4] were able to achieve 99% dye decolorization with 100 ppm of MG dye concentration in the medium.
Just as terrestrial plants, a large variety of both epiphytic and endophytic microbes colonize aquatic plants. And upon their inoculation in FTWs, they efficiently help aquatic plants in enhanced wastewater treatment [26]. Selected bacterial isolates, i.e., Pseudomonas putida and Pseudomonas sp., were inoculated in treatment T2 and T3 in the current study because both have a high dye decolorizing potential (82% for Pseudomonas putida and 70% for Pseudomonas sp.) and had plant growth promoting (PGP) traits in them, too. Many studies have reported that interactions between aquatic plants and bioaugmented bacteria are beneficial in terms of helping plants in contaminants removal from water as well as in promoting the growth of wetland plants [11,58]. PGP bacteria help plants in fighting against phytopathogenic effects of other microbes (indirectly) or through secretion, production and synthesis of plant growth important nutrient, such as P, N, K, Zn, etc. (directly) [59,60,61]. They also help plants in fixing nitrogen; secreting growth hormones, such as abscisic acid, gibberellins and indole-3-acetic acid (IAA); or producing siderophore and deaminase activity (1-aminocyclopropane-1-carboxylate) [46].
In FTWs optimization studies, pH 8 was found to be the best pH for highest percentage dye decolorization. Ekanayake et al. [62], Kagalkar et al. [63] and Anjana and Thanga [64] have used E. crassipes and Pistia sp. in their study and reported pH 8 as the most suitable pH for dye decolorization. It is because, above this pH, wetlands plants are unable to bear the harsh environment of untreated textile wastewater and start showing wilting symptoms. Another important factor for increased dye decolorization in FTWs is treatment time. In their study, Ekanayake et al. [62] reported 100% dye decolorization by E. crassipes in 48 h and by P. stratiotes in 84 h (initially P. stratiotes was only able to decolorize 60–70% dye in 48 h). These results indicate that treatment time has a significantly positive effect on increased dye decolorization by aquatic plants. Initial dye concentration also has an effect on percentage dye decolorization in FTWs as reported by Muthunarayanan et al. [65] in their study. They worked on red RB (95%) and black B dye (99%) decolorization by E. crassipes plants. They found that at low concentrations of dye, aquatic plants worked more efficiently. Similar results have also been reported by Ekanayake et al. [62] and Kagalkar et al. [66], where they found that initial dye concentrations negatively affected dye decolorization.
Many phytoremediation studies, similar to this study, had used common aquatic plants, such as Eichhornia crassipes [34], Pistia sp. [67], Salvinia sp. [64], Lemna sp. [68], Chrysopogon zizanioides [69], Phragmites australis [70] and Typha sp. [71] in their work. For use of aquatic plants in phytoremediation studies, factors like high plant growth rate, easy maintenance, pollutants tolerance and high pollutants uptake capacity, easy handling and local availability are most considered factors [72].
In current study, we have used floating treatment wetlands system vegetated with Eichhornia crassipes and bioaugmented with bacterial inoculation as many studies have reported the significant role bacteria play in dye removal and degradation in partnership with the plants [11,73]. Our study suggests that treatment T2 (consisting of dye, Eichhornia crassipes and Pseudomonas putida) was the best designed treatment as it provided maximum MG dye decolorization, i.e., 91.58 ± 1.5% compared to treatment T1 (consists of dye and Eichhornia crassipes only) where MG dye decolorization percentage was 82.71 ± 1.6%. Results similar to this study have been reported by Nawaz et al. [74] for textile effluent treatment (Bemaplex Black DRKP Bezma dye), where they used a bacterial consortium of Pseudomonas indoloxydans, Rhodococcus sp. and Acinetobacter junii along with aquatic plants, i.e., Phragmites australis. They were able to successfully decolorize dye (500 ppm concentration) up to 85% after a period of 20 days. The nature of dye and aquatic plants used in the study can be a reason of high percentage dye decolorization in our study.
In all three designed treatments, a decrease in values of TDS, EC and pH was found. According to some researchers, plant roots release organic acids in water which then shift water pH [22,75]. Another reason reported in literature is that, due to the combined action of plant and bacteria, biotransformation of dye occurs, causing water pH to move from basic to neutral [32]. A decrease in EC values might be attributed to the binding of water pollutants with plants roots as well as plants’ nutrient uptake from water resulting in reduced EC values in treatment water [49]. Physical and biological processes of FTWs are responsible for decreasing levels of TDS in water. The suspended water particles were trapped by plants’ bacteria (residing in their roots) where they were either precipitated at the bottom of FTWs or absorbed by bacterial populations after their degradation [76,77].
Due to the assisting role of inoculated bacteria in phytoremediation studies, bacterial survival in treated wastewater is very important for efficient performance of FTWs [23,75,78]. In both treatment T2 and T3 (consist of dye, plant and bacteria), a persistence in bacterial survival was observed. Inoculated bacteria colonized in plant roots after adhering to them and helped vegetated plant in increased dye decolorization and better plant growth through their PGP activities [11]. Similar study findings have also been reported by Ijaz et al. [79] and Rehman et al. [33], where inoculated bacteria helped in pollutant removal regardless of decline in their count. Most of the bacteria preferably colonize plants roots, which can explain why a decline in bacterial survival was observed in water. Bacterial isolates used in this study were originally isolated from plants roots where they might have an adaptive survival mechanism [74]. For efficient working of FTWs, periodic bacterial inoculation is a way to deal with the problem of bacterial decline in water [80,81].
Toxic wastewater pollutants not only have a negative effect on plant growth but also affect floating wetlands wastewater treatment efficiency. Other than control 2, in all three treatments, MG dye enriched wastewater had significantly affected plant growth parameters (weight, root and stalk length) [82]. Compared to uninoculated treatment T1 (consist of dye and Eichhornia crassipes only), in both bacterial inoculated treatment T2 (consist of dye, Eichhornia crassipes and Pseudomonas putida) and T3 (consist of dye, Eichhornia crassipes and Pseudomonas sp.), highest plant weight, root and stalk length only second to control 2 (consists of fresh water and Eichhornia crassipes) was observed. Bacterial inoculation helped vegetated plant through their plant growth-promoting traits [83,84]. Study results showed that inoculated bacteria in FTWs not only helped aquatic plants in improved dye decolorization percentages with low values of EC, TDS and pH but also provided their assistance in increased plant weight, root and stalk lengths.
The beneficial role of bacterial inoculation in FTWs has been reported in many studies [11,33,58,74,85]. Afzal et al. [86] have reported rhizospheric bacteria for their pollutants’ degrading ability. A decline in plant photosynthetic pigment content was observed in a current study that might be due to the inhibitory effects of the dye on plant enzymes responsible for chlorophyll synthesis in them. A decrease in chlorophyll content has also been reported by Anjana and Thanga [64] in their study on E. crassipes, Pistia sp., and Salvinia sp. when these plants were exposed to Congo red, dark blue and direct black dyes.
Effect of dye toxicity was observed on Pisum sativum seeds, and poor growth of seeds was found when they were grown in synthetically prepared MG-enriched industrial wastewater. Among all three designed floating wetlands treatments, treatment T2 (consists of dye, Eichhornia crassipes and Pseudomonas putida) showed best results for seed germination, plumule and radicle length as its results were close to results of seeds grown in tap water. Malachite green dye and its products are not only phytotoxic but also an environmental hazard as they are usually disposed-off in water with no prior treatment. In their study on MG dye, Alaya et al. [4] used seedlings of sorghum and finger millet to study dye phytotoxicity effects. Their findings are very similar to our study where degraded MG dye products showed satisfactory results for seed germination, and plumule and radicle length. Although both MG and its degraded products are phytotoxic, compared to MG dye, its degraded products are less toxic [54,62].
The floating treatment wetlands system is a promising, environment-friendly and cost-effective technology for the effective treatment of industrial wastewater. It has minimal cost of construction, operation and maintenance as all necessary materials for its construction and establishment are locally and commercially available. Additionally, its installation does not require any special skills and hence has become a practical method in developing countries for various types of wastewater treatment [23,87,88]. In a study on 18 municipalities of Jaén province, southern Europe, published by Pajares et al. [89], it was found that the average estimated cost per m3 of treated water in wastewater treatment facilities is EUR 0.31. Furthermore, as per the Directive 91/271/CEE criteria established, this cost was found to be varying between EUR 0.30 per m3 and EUR 0.88 per m3. In the same year, probably the first-ever report on the field establishment of FTWs with all the associated costs was published by Afzal et al. [90], where their team of scientists installed a FTW system in Faisalabad, Pakistan in 2018 for wastewater treatment. Just after a year of its installation, FTWs treated water met all water quality standards with an operational cost of USD 0.0026 per m3 of treated water. The results of this study were not only of great importance for Pakistan but also for other developing countries, too, which cannot afford costly wastewater treatment technologies. To further emphasize the low-cost benefit of FTWs installation for wastewater treatment, another study reported in 2021 that cost of constructed wetlands is approximately 50–90% less than traditional wastewater treatment facilities [91].
Water pollutants, such as dyes containing toxic heavy metals and chemicals in them, are a great source of phytotoxicity, thereby inhibiting aquatic plants’ growth in water. In the current study, potential of E. crassipes with and without bacterial inoculation was tested by developing floating treatment wetland systems for treatment of Malachite green enriched industrial wastewater. Bacteria present in the FTWs not only helped in wastewater treatment but also helped in decreasing the biotic and abiotic stresses of the plants and thus improved plant growth.

5. Conclusions

Study findings revealed that Eichhornia crassipes, bioaugmented with bacterial strains, has a strong potential in MG dye degradation. E. crassipes vegetated floating treatments efficiently lower down values of electrical conductivity (EC), pH and total dissolved solids (TDs) as well as provided increased dye decolorization percentages. Inoculated bacteria helped Eichhornia crassipes in MG dye removal from water with improved plant growth compared to plants grown in uninoculated treatments. Study results indicated that all the treatments inoculated with bacteria performed better than treatments without bacterial inoculation. Hence, in reference to study findings, we conclude that FTWs operated with bacterial inoculation worked remarkably in wastewater treatment. FTWs is a promising, environment friendly and cost-effective technology for effective treatment of dye enriched industrial wastewater. This approach can be applied to existing water bodies or purposely built ponds for an effective and efficient pollutant removal from them. Nevertheless, further research is needed on the careful and objective specific bacteria and plants selection, process optimization and necessary steps to implement this technology at a larger scale.

Supplementary Materials

The following supporting information can be downloaded at: https://0-www-mdpi-com.brum.beds.ac.uk/article/10.3390/su15054541/s1. Supplementary Information File: All necessary information has been added according to its respective main document sections (Figure S1).

Author Contributions

Conceptualization, S.S.; methodology, S.S.; validation, H.M.; formal analysis, S.S.; investigation, S.S.; resources, H.M; data curation, S.S.; writing—original draft preparation, S.S.; writing—review and editing, H.M.; supervision, H.M.; project administration, H.M. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. MG dye decolorization (%) by bacterial isolates through flask screening. Based on the results of flask screening, top six MG dye decolorizing bacteria i.e., F1, F2, F4, F7, F10 and F12 were selected for further study. Each value is a mean of three replicates, and error bars represent the standard deviation.
Figure 1. MG dye decolorization (%) by bacterial isolates through flask screening. Based on the results of flask screening, top six MG dye decolorizing bacteria i.e., F1, F2, F4, F7, F10 and F12 were selected for further study. Each value is a mean of three replicates, and error bars represent the standard deviation.
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Figure 2. Effect of different treatment time on percentage dye decolorization by Eichhornia crassipes vegetated FTWs. Symbol key: T1 = Dye and plants, T2 = Dye, plants and Pseudomonas putida, T3 = Dye, plants and Pseudomonas sp. Each value is a mean of three replicates, and error bars represent the standard deviation.
Figure 2. Effect of different treatment time on percentage dye decolorization by Eichhornia crassipes vegetated FTWs. Symbol key: T1 = Dye and plants, T2 = Dye, plants and Pseudomonas putida, T3 = Dye, plants and Pseudomonas sp. Each value is a mean of three replicates, and error bars represent the standard deviation.
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Figure 3. Effect of different pH on percentage dye decolorization by Eichhornia crassipes vegetated FTWs. Symbol key: T1 = Dye and plant, T2 = Dye, plant and Pseudomonas putida, T3 = Dye, plant and Pseudomonas sp. Each value is a mean of three replicates, and error bars represent the standard deviation.
Figure 3. Effect of different pH on percentage dye decolorization by Eichhornia crassipes vegetated FTWs. Symbol key: T1 = Dye and plant, T2 = Dye, plant and Pseudomonas putida, T3 = Dye, plant and Pseudomonas sp. Each value is a mean of three replicates, and error bars represent the standard deviation.
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Figure 4. Effect of initial dye concentrations on percentage dye decolorization by Eichhornia crassipes vegetated FTWs. Symbol key: T1 = Dye and plant, T2 = Dye, plant and Pseudomonas putida, T3 = Dye, plant and Pseudomonas sp. Each value is a mean of three replicates, and error bars represent the standard deviation.
Figure 4. Effect of initial dye concentrations on percentage dye decolorization by Eichhornia crassipes vegetated FTWs. Symbol key: T1 = Dye and plant, T2 = Dye, plant and Pseudomonas putida, T3 = Dye, plant and Pseudomonas sp. Each value is a mean of three replicates, and error bars represent the standard deviation.
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Figure 5. Effect of Eichhornia crassipes vegetated floating treatment wetlands on percentage dye decolorization. Symbol key: T1 = Dye and plant, T2 = Dye, plant and Pseudomonas putida, T3 = Dye, plant and Pseudomonas sp. Each value is a mean of three replicates, and error bars represent the standard deviation.
Figure 5. Effect of Eichhornia crassipes vegetated floating treatment wetlands on percentage dye decolorization. Symbol key: T1 = Dye and plant, T2 = Dye, plant and Pseudomonas putida, T3 = Dye, plant and Pseudomonas sp. Each value is a mean of three replicates, and error bars represent the standard deviation.
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Figure 6. Effect of Eichhornia crassipes vegetated floating treatment wetlands on electrical conductivity (EC). Symbol key: T1 = Dye and plant, T2 = Dye, plant and Pseudomonas putida, T3 = Dye, plant and Pseudomonas sp. Each value is a mean of three replicates, and error bars represent the standard deviation.
Figure 6. Effect of Eichhornia crassipes vegetated floating treatment wetlands on electrical conductivity (EC). Symbol key: T1 = Dye and plant, T2 = Dye, plant and Pseudomonas putida, T3 = Dye, plant and Pseudomonas sp. Each value is a mean of three replicates, and error bars represent the standard deviation.
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Figure 7. Effect of Eichhornia crassipes vegetated floating treatment wetlands on total dissolved solids (TDS). Symbol key: T1 = Dye and plant, T2 = Dye, plant and Pseudomonas putida, T3 = Dye, plant and Pseudomonas sp. Each value is a mean of three replicates, and error bars represent the standard deviation.
Figure 7. Effect of Eichhornia crassipes vegetated floating treatment wetlands on total dissolved solids (TDS). Symbol key: T1 = Dye and plant, T2 = Dye, plant and Pseudomonas putida, T3 = Dye, plant and Pseudomonas sp. Each value is a mean of three replicates, and error bars represent the standard deviation.
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Figure 8. Effect of Eichhornia crassipes vegetated floating treatment wetlands on pH. Symbol key: T1 = Dye and plant, T2 = Dye, plant and Pseudomonas putida, T3 = Dye, plant and Pseudomonas sp. Each value is a mean of three replicates, and error bars represent the standard deviation.
Figure 8. Effect of Eichhornia crassipes vegetated floating treatment wetlands on pH. Symbol key: T1 = Dye and plant, T2 = Dye, plant and Pseudomonas putida, T3 = Dye, plant and Pseudomonas sp. Each value is a mean of three replicates, and error bars represent the standard deviation.
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Figure 9. Comparison of photosynthetic pigments in control and test plants. Symbol key: E-initial (conc. in Eichhornia crassipes before dye exposure), E-final (conc. in Eichhornia crassipes after dye exposure). Each value is a mean of three replicates, and error bars represent the standard deviation.
Figure 9. Comparison of photosynthetic pigments in control and test plants. Symbol key: E-initial (conc. in Eichhornia crassipes before dye exposure), E-final (conc. in Eichhornia crassipes after dye exposure). Each value is a mean of three replicates, and error bars represent the standard deviation.
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Table
Table 1. Chemical structure and other properties of Malachite green dye.
Table 1. Chemical structure and other properties of Malachite green dye.
PropertiesMalachite Green Dye
Chemical structureSustainability 15 04541 i001
Chemical formulaC23H25ClN2
Molecular weight364.9 g/mol
Maximum strength618 nm
Water solubility6. 104 mg/dm3
Chemical SafetySustainability 15 04541 i002
Corrosive   Irritant
GHS SignalDanger
GHS Hazard StatementsH302 (100%): Harmful if swallowed [Warning Acute toxicity, oral]
H318 (100%): Causes serious eye damage [Danger Serious eye damage/eye irritation]
Other namesAniline green, Victoria green B, Basic green 4 (BG4), Diamond green B
Table
Table 2. Summary of PGP traits in selected bacterial isolates.
Table 2. Summary of PGP traits in selected bacterial isolates.
PGP TraitsBacterial Isolates
F1F2F4F7F10F12
Phosphate solubilization−−+−−−
IAA production++−+++++
N2 fixation−−−−+−
HCN production+++++−++++
Potassium solubilization−−−−−−
Symbol key: IAA= Weak producer (+), Strong producer (++); HCN= Weak producer (+), Moderate producer (++), Strong producer (+++); “+” = Positive isolate, “−” = Negative isolate.
Table
Table 3. Typical morphological characteristics of two bacterial isolates.
Table 3. Typical morphological characteristics of two bacterial isolates.
Colony CharactersBacterial Isolates
F1F2
FormRoundRound
MarginsEntireEntire
ElevationsConvexConvex
SizeModerateSmall
TextureSmoothSmooth
AppearanceShinyShiny
ColorCreamy whiteCreamy white
Optical propertyOpaqueOpaque
Gram stainGram negativeGram negative
Cell shapeShort rodsShort rods
Table
Table 4. Biochemical test results of bacterial isolates based on API 20 NE.
Table 4. Biochemical test results of bacterial isolates based on API 20 NE.
Test NameBacterial Isolates
F1F2
Nitrate reduction (NO3)−−
Indole production (TRP)−−
Glucose fermentation (GLU)−−
Arginine dihydrolase production (ADH)−−
Urease production (URE)−−
Β-glucosidase hydrolysis (ESC)−−
Protease hydrolysis (GEL)−−
B-galactosidase production (PNPG)−−
Assimilation of
Glucose (GLU)++
Arabinose (ARA)−−
Mannose (MNE)++
Mannitol (MAN)−+
N-Acetyl-Glucosamine (NAG)−−
Maltose (MAL)−−
Potassium Gluconate (GNT)++
Capric acid (CAP)++
Adipic acid (ADI)−−
Malate (MLT)++
Trisodium Citrate (CIT)−+
PhenylAcetic acid (PAC)+−
Cytochrome oxidase production (OX)++
API Codes & organismPseudomonas putida (42456)Pseudomonas sp. (46455)
Symbol key: “+” = Positive isolate, “−” = Negative isolate.
Table
Table 5. Removal efficiencies of treatments developed with and without bacterial inoculation for MG dye, electrical conductivity (EC) and total dissolved solids (TDS).
Table 5. Removal efficiencies of treatments developed with and without bacterial inoculation for MG dye, electrical conductivity (EC) and total dissolved solids (TDS).
TreatmentsDye RemovalECTDS
T182.7160.0940.1
T291.5868.7548.89
T387.3962.945.84
Symbol key: T1 = Dye and plant, T2 = Dye, plant and Pseudomonas putida, T3 = Dye, plant and Pseudomonas sp.
Table
Table 6. Bacterial survival in FTWs treated wastewater.
Table 6. Bacterial survival in FTWs treated wastewater.
Floating Wetlands Treatment Day 1
(CFU)/mL × 103)		Day 4
(CFU)/mL × 103)
T2219 ± 1.52211 ± 5.00
T3219 ± 1.00210 ± 4.00
Symbol key: T2 = Dye, plant and Pseudomonas putida, T3 = Dye, plant and Pseudomonas sp. Each value is a mean of three replicates, and error bars represent the standard deviation.
Table
Table 7. Aquatic plant weight, stalk and root lengths observed in designed treatments.
Table 7. Aquatic plant weight, stalk and root lengths observed in designed treatments.
TreatmentsTotal Weight (g)Stalk Length (cm)Root Length (cm)
Control 2139.43 ± 0.3732.02 ± 0.0120.08 ± 0.75
T1113.51 ± 0.4425.13 ± 0.1111.26 ± 0.11
T2127.19 ± 0.1626.04 ± 0.0415.06 ± 0.05
T3121.45 ± 0.3922.06 ± 0.0513.64 ± 0.56
Symbol key: Control 2 = Freshwater and plant, T1 = Dye and plant, T2 = Dye, plant and Pseudomonas putida, T3 = Dye, plant and Pseudomonas sp. Each value is a mean of three replicates, and error bars represent the standard deviation.
Table
Table 8. Phytotoxicity assessment of the wastewater after treatment process.
Table 8. Phytotoxicity assessment of the wastewater after treatment process.
TreatmentsGermination (%)Plumule (cm)Radicle (cm)
Tap water99.70 ± 0.522.40 ± 0.011.57 ± 0.06
Synthetic wastewater (MG dye)30.73 ± 0.580.77 ± 0.060.49 ± 0.01
T192.53 ± 0.152.37 ± 0.111.15 ± 0.01
T296.57 ± 0.212.36 ± 0.051.20 ± 0.01
T394.43 ± 0.062.37 ± 0.031.17 ± 0.02
Symbol key: T1 = Dye and plant, T2 = Dye, plant and Pseudomonas putida, T3 = Dye, plant and Pseudomonas sp. Each value is a mean of three replicates, and error bars represent the standard deviation.
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Sahreen, S.; Mukhtar, H. Development of Bacterial Augmented Floating Treatment Wetlands System (FTWs) for Eco-Friendly Degradation of Malachite Green Dye in Water. Sustainability 2023, 15, 4541. https://0-doi-org.brum.beds.ac.uk/10.3390/su15054541

AMA Style

Sahreen S, Mukhtar H. Development of Bacterial Augmented Floating Treatment Wetlands System (FTWs) for Eco-Friendly Degradation of Malachite Green Dye in Water. Sustainability. 2023; 15(5):4541. https://0-doi-org.brum.beds.ac.uk/10.3390/su15054541

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Sahreen, Sania, and Hamid Mukhtar. 2023. "Development of Bacterial Augmented Floating Treatment Wetlands System (FTWs) for Eco-Friendly Degradation of Malachite Green Dye in Water" Sustainability 15, no. 5: 4541. https://0-doi-org.brum.beds.ac.uk/10.3390/su15054541

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