The evaluation of bacterial-augmented floating treatment wetlands for concomitant removal of phenol and chromium from contaminated water

Abstract Contamination of aquatic ecosystems with organic and inorganic contaminants is a global threat due to their hazardous effects on the environment and human health. Floating treatment wetland (FTW) technology is a cost-effective and sustainable alternative to existing treatment approaches. It consists of a buoyant mat in which wetland plants can grow and develop their roots in a suspended manner and can be implemented to treat stormwater, municipal wastewater, and industrial effluents. Here we explored the potential of bacterial-augmented FTWs for the concurrent remediation of phenol and hexavalent chromium (Cr6+) contaminated water and evaluated treated water toxicity using Triticum aestivum L. (wheat) as a test plant. The FTWs carrying Phragmites australis L. (common reed) were inoculated with a consortium of four bacterial strains (Burkholderia phytofirmans PsJN, Acinetobacter lwofii ACRH76, Pseudomonas aeruginosa PJRS20, Bacillus sp. PJRS25) and evaluated for their potential to simultaneously remove phenol and chromium (Cr) from contaminated water. Results revealed that the FTWs efficiently improved water quality by removing phenol (86%) and Cr (80%), with combined use of P. australis and bacterial consortium after 50 days. The phytotoxicity assay demonstrated that the germination of wheat seed (96%) was significantly higher where bacterial-augmented FTWs treated water was used compared to untreated water. This pilot-scale study highlights that the combined application of wetland plants and bacterial consortium in FTWs is a promising approach for concomitant abatement of phenol and Cr from contaminated water, especially for developing countries like Pakistan where the application of advanced and expensive technologies is limited. NOVELTY STATEMENT This pilot-scale research provides new interventions and information required for establishing a large-scale remediation framework for the effective, sustainable and eco-friendly remediation of phenol and Cr co-contaminated aquatic ecosystems, using bacterial augmented floating wetlands technology (FTWs).


Introduction
Industrialization and urbanization led to the over-exploitation of natural water and soil resources globally, and especially in developing countries.Despite getting advantages from these rapid advancements, the planet, Earth, is getting harmed to an irreversible extent (Ali et al. 2023;Gayathiri et al. 2022).Untreated wastewater containing various mixed contaminants is released into the aquatic and terrestrial environments and causes potential risks to the food chain (Batra et al. 2022;Younas et al. 2023).A huge percentage of industrial wastewater is dumped into the water bodies and land, which makes them the most contaminated and damaged component of the biosphere.Industrial effluents typically consist of organic (e.g., crude oil, phenols, aromatic compounds) and inorganic (such as heavy metals) contaminants.Among the mixed toxic substances, chromium (Cr) and phenol are reported to be simultaneously present in industrial wastewater (Bhattacharya et al. 2015), which are discharged directly into the water without prior treatment (Guo et al. 2021;Shah et al. 2022).
Chromium is a toxic metal existing in trivalent (Cr 3þ ) and hexavalent (Cr 6þ ) forms (Younas et al. 2022).It is widely used in the tanning industry (Tripathi et al. 2022).
The toxicity of both forms of Cr is related to their oxidation state, solubility, and bioavailability (Nowicka 2022).Cr 6þ is more toxic, mutagenic, and carcinogenic than Cr 3þ ; the former is reported to cause shortness of breath, skin burns, neurological and gastrointestinal effects, abdominal pain, vomiting and hemorrhage when ingested in high quantities by humans or animals (Yasir et al. 2021).The recommended limits for Cr concentration in well water/groundwater is 0.5 mg/L (Ullah et al. 2022).Under natural conditions, plant's Cr content is <1 lg/g.Toxicity of Cr has been reported in plant nutrient solution 0.5-5 mg/L and in soil 5-100 mg/g (Kapoor et al. 2022).Above this concentration, Cr inhibits plant growth, creates nutrient imbalance and affects biochemically important processes in plants.Further to the above, Cr is responsible for interfering with DNA replication in microbes, which leads to mutation and altering the enzyme structures (Hossini et al. 2022).In the case of phenol (an organic contaminant), it is a toxic aromatic compound that is used in the production of phenolic resins, nylon, and other synthetic fibers (Supreeth 2022).Like Cr, it can cause serious health hazards to humans, such as skin irritation, reproductive, and developmental damage in humans (Garg et al. 2022).Hence, the US Environmental Protection Agency (USEPA) has set a safe concentration of phenol in wastewater at 0.1 mg/L (United States Environmental Protection Agency) (EPA 2023).
Various chemical and mechanical techniques such as precipitation, coagulation/flocculation, and screening are commonly employed in wastewater treatment, but they have certain limitations.Such as, these physico-chemical approaches require significant energy inputs, which lead to increased operational costs (Kumar et al. 2022).The floating treatment wetlands (FTWs) turned out to be the most promising solution for remediation of both organic and inorganic contaminants remediation (Singh et al. 2022).Floating treatment wetlands technology has gained much attention because it is a cost-effective, environmentally friendly, esthetically pleasing, and effective water treatment approach (Rehman et al. 2019).The setup allows maximum root contact with wastewater for effective Cr and phenol removal through various mechanisms such as phytoextraction, phytotransformation, phytostabilization, rhizofiltration and phytovolatilization (Sinha et al. 2009;Sharma et al. 2021).Moreover, plant roots in floating mats provide a habitat for the microorganisms to survive either aerobically or anaerobically, resulting in biofilm (Shahid et al. 2020).Several remediation strategies are employed using bacteria such as, biosorption, bioaccumulation, biotransformation, and bioleaching to convert Cr 6þ to Cr 3þ ; the later being less toxic and immobile (Shahid et al. 2020).To detoxify phenol, bacteria may possess phenol hydroxylase enzymes which breaks down the phenol to catechol followed by further degradation to intermediates such as muconic acid and fumarate, thus entering central metabolic pathways for energy generation.Thus, beneficial association between plants and microbes facilitates the efficient removal of toxic chemicals/substances and contributes to the overall purification of wastewater in FTWs (Shahid et al. 2020).
While remediation of complex Cr and phenol co-contaminated water has not been explored previously, this study investigated the potential of FTWs planted with Phragmites australis L. (common reed) along with bacterial co-cultures for the concomitant elimination of phenol and Cr from contaminated water.Equally important, the efficiency of FTWs for wastewater treatment was also evaluated through a phytotoxicity bioassay of the treated wastewater, using Triticum aesativum as a test plant.Phragmites australis L. (Common reed), a halophytic grass, was chosen to develop FTWs because of its well-known ability to survive in the presence of various contaminants (Shi et al. 2018;Saleem et al. 2019;Younas et al. 2022).

Determination of minimal inhibitory concentrations (MICs)
Resistance against phenol and chromate (Cr 6þ ) was examined separately (Panneerselvam et al. 2013;Poi et al. 2017).Bacterial strains were aseptically inoculated on minimal salt medium (MSM) agar plates containing 10 to 100 mg/L of Cr 6þ and incubated at 37 C for 72 h.While plates of MSM comprising 100 to 1,500 mg/L phenol were spot-inoculated with selected bacterial strains and incubated at 37 C for 72 h (Darma et al. 2020).

In vitro compatibility among selected bacterial strains
The compatibility of selected bacterial strains was studied by co-culturing them on MSM agar medium.Co-inoculated strains were streaked perpendicularly and plates were subjected to incubation at 37 C for 24 h and observed for zone of inhibition.After compatibility test, bacterial strains were cultivated separately in MSM broth at 37 C for 24 h followed by culture standardization with 0.5 McFarland standard.Bacterial cells were harvested by centrifugation and re-suspended in sterile 0.9% NaCl solution.After re-suspension, bacterial strains were equally mixed in 1:1:1:1 ratio to formulate bacterial consortium (Fatima et al. 2018).

Simultaneous removal of phenol and chromium by bacterial consortium
For the concomitant removal of phenol and Cr, selected bacterial consortium (1%) was inoculated in 250 mL Erlenmeyer flasks containing 100 mL MSM broth with phenol or Cr concentrations (phenol/Cr: 100/5, 300/15, 500/25, 700/35, 900/45 and 1,100/55 mg/L) (Chandrasekaran et al. 2018).All the flasks along with control were incubated in a shaking incubator for 7 days at 37 C and 120 rpm.The FTW experiment was performed in triplicate and percentage removal of Cr and phenol was calculated as follows:

Development of FTWs
Fifteen FTWs microcosms were developed using a polystyrene sheet as a mat.The sheet was cut in a circular shape; each mat was bored to create a hole to insert five healthy seedlings of P. australis.The mats with seedlings were placed over the water tanks having 20 L tap water.The plants were allowed to develop their roots in the tap water for one month, the tap water was replaced with phenol (500 mg/L) and Cr (25 mg/L) co-contaminated water.Each treatment was run in triplicate in natural environmental conditions at the National Institute for Biotechnology and Genetic Engineering (NIBGE) in Faisalabad, Pakistan.Control (C1): fresh water (without phenol and Cr) having FTWs Control (C2): water contaminated with phenol (500 mg/L) and Cr (25 mg/L) without FTWs Treatment 1 (T1): water contaminated with phenol (500 mg/L) and Cr (25 mg/L) and FTWs Treatment 2 (T2): water contaminated with phenol (500 mg/L), Cr (25 mg/L), FTWs and bacterial consortium Treatment 3 (T3): water contaminated with phenol (500 mg/L), Cr (25 mg/L) and bacterial consortium

Analysis of residual concentration of phenol and Cr in water
Treated water samples were collected for 50 days at 10 days intervals as reported earlier (Afzal et al. 2014).The residual concentration of phenol in treated water was detected spectrophotometrically. Water samples (25 mL) were taken and ammonium hydroxide (NH 4 OH; 0.5 mL) solution was added to it before analysis.The pH was adjusted immediately to 7.9 ± 0.1 with phosphate buffer; 0.5 mL 4-amino antipyrine (APP) solution and one mL potassium ferricyanide (K 3 Fe (CN) 6) solution were added into it and mixed well.After 15 min, absorbance was recorded at 500 nm using an UV-Vs Spectrophotometer (Shimadzu, Japan, CECIL CE7200) and readings were compared with standard phenol.
Water samples were collected to analyze Cr 6þ removal by 1,5-diphenylcarbazide (DPC) method as previously described by (Lace et al. 2019).Briefly, 10 mL sample was added in test tubes, followed by a few drops of 3 M H 2 SO 4 and 0.5 mL DPC.The absorbance of the mixture was taken at 540 nm using an UV-Vis Spectrophotometer.

Plant growth
Plants were harvested after 50 days of growth in FTWs.Plant roots were washed carefully with tap water, followed by rinsing in deionized water.Roots and shoots were cut and their length and biomass were recorded for each treatment.The plant samples were oven-dried at 65 C for three days and dry biomass was recorded as well (Hwang et al. 2020).

Evaluation of toxicity of treated water
Phytotoxicity bioassay was performed to asses the efficacy of bacterial augmented FTWs treated wastewater using Triticum aestivum L. (wheat) seeds.Firstly, all seeds were surface sterilized with 0.01% sodium hypochlorite for 1 to 2 min, then rinsed two or three times in distilled water.The seeds (30) were placed in 80 mm diameter petri plates containing agricultural uncontaminated soil (300 g).For 7 days, 3 mL tap water, phenol and Cr contaminated water and bacterial augmented FTWs treated water were sprinkled on seeds.The percentage of seed germination was recorded after 7 days (L opez-Luna et al. 2009).

Statistical analysis
Data were analyzed using the SPSS software package (SPSS Inc., Chicago, IL, USA) and analysis of variance was applied following Duncan's multiple range test (MRT) to estimate significant variances between treatments.

Determination of phenol and chromium resistance
Two strains, Ps.JN and ACRH76, exhibited maximum growth in the presence of phenol at 1,100 mg/L.The A1 and CYRH21 showed resistance against phenol upto 500 mg/L (Table S1).Moreover, PJRS20 and PJRS25 were capable to grow in the presence of Cr upto 60 mg/L, while PJS11, HU33 and HU38 showed maximum resistance up to 40 mg/ L (Table S2).
The bacterial strains exhibiting maximum resistance were checked for their compatibility to formulate bacterial consortium.Bacterial strains, namely Ps.JN, ACRH76, PJRS20 and PJRS25, were able to grow on MSM agar plates as no clearing zone was observed.

Microbial growth and simultaneous removal of phenol and chromium
Bacterial consortium was able to grow at 500 and 25 mg/L phenol and Cr concentrations, respectively.The maximum Cr decrease at 25 mg/L concentration was 36% while for phenol it was 51% at 500 mg/L (Figure 1).Phenol and Cr reduction was not observed in sterile MSM controls.

Plant growth monitoring study
The effect of bacterial inoculation on roots, shoots and biomass of P. australis was recorded at the end of the experiment (Table 1).In phenol-Cr co-contaminated water, bacterial inoculation significantly increased root length (14%) and shoot length (9%) of P. australis.It produced lower amount of biomass compared to the plants in polluted water (Table 2), although inoculation of bacterial consortium enhanced plant growth and hence biomass.

Removal of phenol and Cr by FTWs
The elimination of phenol and Cr in different FTWs is presented in Figure 2. The initial concentration of phenol in contaminated water was 500 mg/L and for Cr it was 25 mg/L.A minimal reduction in the concentrations of phenol and Cr was detected in the treatments, where bacterial consortium was inoculated without vegetation (T3).In T3, 61% phenol and 44% Cr concentration were reduced compared to control.For vegetated reactors, removal percentage (67% phenol and 60% Cr) was slightly high (T1), albeit maximum removal (86% phenol and 80% Cr) was found in the vegetated reactors with bacterial consortia (T2).

Phytotoxicity bioassay
Phytotoxicity bioassay was performed to determine the detoxification level of treated water.Results showed that there was more germination of seeds (96%) exposed to the water treated in FTWs augmented with the bacterial consortium (T2).Minimum seed germination (30%) was observed by the seeds exposed to untreated phenol-and Cr-contaminated water.

Discussion
Environmental contaminants, either organic or inorganic, present a grave challenge to the development of a sustainable ecosystem.Industries tend to produce tons of pollutants, thus releasing them to the water and soil environments, which not only damage the ecosystem, but also pose a serious long lasting threat due to their persistent nature (Singh et al. 2021).In this study, combined application of selected bacterial consortium (Ps.JN, ACRH76, PJRS20 and PJRS25) and P. australis were chosen to assess simultaneous removal of Cr and phenol from polluted water because of their reported resistance and growth in the presence of these environmental pollutants.
For the selection of phenol resistant bacterial strains, MIC was performed for PsJN, ACRH76, A1, and CYRH21.The results showed that PsJN and ACRH76, resisted phenol up to a concentration of 1,300 mg/L while A1 and CYRH21 showed growth till 1,100 mg/L (Table S1).Several studies have focused on the catabolic genes of gram-negative bacteria such as Pseudomonas, Burkholderia, Acinetobacter, and Sphingomonas (Gao et al. 2017;Tian et al. 2017).They carry dioxygenase and catechol 2, 3-dioxygenase genes which are essential for the removal of a variety of toxicants in polluted sites (Murphy et al. 2023).
In the case of Cr þ6 resistant bacterial strains, MIC was performed for PJRS20, PJRS25, HU33 and HU38.The results revealed that PJRS20 and PJRS25 resisted Cr 6þ up to 100 mg/L while HU33 and HU38, showed growth up to 80 mg/L (Table S2).Resistance to Cr could be due to the efflux pumps taking up Cr 6þ and reducing it to less toxic form (Cr 3þ ) in the presence of Cr reductase enzyme.Hence, on the basis of MIC, PsJN, ACRH76, PJRS20, and PJRS25 were chosen for a compatibility test, formulation of bacterial consortium and simultaneous removal of phenol and Cr.
In MSMs, bacterial consortium simultaneously reduced the phenol and Cr concentrations by 51% and 36% respectively compared with values of controls (Figure 1).Bhattacharya et al. (2014) reported efficient concomitant removal of phenol and Cr using Acinetobacter sp.B9.Initially, the water was contaminated with phenol (47 mg/L) and Cr 6þ (16 mg/L).The complete elimination of phenol and 87% reduction of Cr 6þ were observed, displaying the proficiency of the bacterial strain for probable application in  Plants and microbes are well known to reduce, detoxify, and degrade environmental pollutants.But, the main disadvantage is their slow removal process (Priyadarshanee and Das 2021).However, the combined usage of microbes and plants have been proven as cost-effective and efficient method (Ancona et al. 2022;Raklami et al. 2022;Yaashikaa et al. 2022).In pilot-scale study, the treatment T1 (water contaminated with phenol (500 mg/L) and Cr (25 mg/L) and FTWs) led to reduce 67% phenol and 60% Cr, while treatment T3 (water contaminated with phenol (500 mg/L), Cr (25 mg/L) and bacterial consortium) was successful in removing 61% phenol and 44% Cr.The Treatment T2 (water contaminated with phenol (500 mg/L), Cr (25 mg/L) FTW and bacterial consortium) exhibited 86% and 80% removal of phenol and Cr, respectively.Saleem et al. (2019) reported that the removal rate of phenol (96%) was significantly high in the treatment where vegetation and bacterial consortium was used as compared with the individual partners, i.e., plants (66%) and bacteria (61%) separately.Sharma et al. (2021) reported that FTWs vegetated with Eichhornia crassipes (water hyacinth) can remove 98.83% of Cr from the tannery effluent.When the plants and bacteria are used in combination, plants release chemicals and nutrients resulting in chemotaxis while bacteria produce essential enzymes and metabolites like dioxygenases, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, Indole 3acetic acid (IAA) etc. enabling plants to survive in pollutant rich environment (Danish et al. 2019;Del Carmen et al. 2020).
The controlled application of pretreated wastewater in horticulture or main water bodies is a common practice in many countries (Chojnacka et al. 2020;Kumar and Goyal, 2020).Seed germination of wheat was assessed to determine the efficiency of FTWs treated wastewater.The results revealed that maximum seed germinations were recorded in T2 (vegetation þ bacteria) as compared to T1 (vegetation only) and T3 (bacteria only) treated wastewater.This is because of the maximum removal of phenol and Cr from water by the combined application of bacterial consortium and FTWs.These results are in compliance with the study conducted by Phoungthong et al. (2016).
Hence, the selected bacterial consortium along with constructed FTW in our study proved to be an effective strategy to treat co-contaminated water, which also allows the use of treated wastewater for agricultural purposes (Magwaza et al. 2020;Oliveira et al. 2021).The study also paves the way to conduct similar field experiments and evaluate the real-time success in long-term processes.

Conclusions
The findings from this study reveal that bacterial augmented Phragmites australis in FTWs represents a potent solution for simultaneous removal of phenol and Cr from contaminated water and can be an alternative approach for conventional wastewater treatment.Inoculated bacteria (PsJN, ACRH76, PJRS20 and PJRS25) helped P. australis in the removal of phenol (86%) and Cr (80%) from water with improved plant biomass compared to plants grown in uninoculated treatments.Hence, we suggest that bacterial augmented FTWs is a promising, environmentally-friendly and cost-effective solution for effective treatment of organic and inorganic contaminants.As this is a pilot-scale study, further research is required to assess the impact of selected bacterial consortium, wetland plant and optimum conditions to implement this set-up at a large-scale where co-contamination of organic and inorganic pollutants prevails in realworld scenario.

Figure 1 .
Figure 1.Representaion of simultaneous removal of Phenol and Cr by selected bacterial consortium (PsJN, ACRH76, PJRS20 and PJRS25) showing a maximum 51% of phenol and 36% of Cr removal.

Table 1 .
Effect of bacterial consortium on the growth of Phragmites australis.Treatment Initial day Final day (50) Root length (cm) Shoot length (cm) Root length (cm) Shoot length (cm) Control 1 19 a (1.2) 27 a (2.5) 34 b (3.1) 58 b (3.6) T1 18 a (1.1) 26 a (2.4) 29 a (2.6) 40 a (3.6)T2 16 a (1.0) 28 a (2.6) 40 c (3.5) 65 c (3.9) Control 1: Treatment containing fresh water (without phenol and Cr) and vegetated with P. australis; T1: Treatment containing phenol and Cr contaminated water and vegetated with P. australis; T2: Treatment containing phenol and Cr contaminated water, selected bacterial consortium and vegetated with P. australis Each value is the mean of three replicates; means in the same column followed by different letters are statastically different at a 5% level of significance; standard deviations are presented in parentheses.industrial contamination control.The strain ACRH76 is also an identified bacterium from genus Acinetobacter and results in phenol and Cr removal from the water.Yasir et al. (2021) observed that the use of Burkholderia sp.led to simultaneous elimination of chlorinated biphenyls and Cr þ6 which complies with our results, as PsJN was identified as Burkholderia phytofirmans.
Treatment containing fresh water (without phenol and Cr) and vegetated with P. australis; T1: Treatment containing phenol and Cr contaminated water and vegetated with P. australis; T2: Treatment containing phenol and Cr contaminated water, bacterial consortium and vegetated with P. australis.Each value is the mean of three replicates; means in the same column followed by different letters are statastically different at a 5% level of significance; standard deviations are presented in parentheses.

Figure 2 .
Figure 2. Simultaneous removal of phenol and Cr by selected bacterial consortium (PsJN, ACRH76, PJRS20, and PJRS25) and FTWs in pilot-scale study.C: phenol and Cr contaminated water, T1: phenol and Cr-contaminated water þ FTW, T2: phenol and Cr contaminated water þ FTW and bacteria, T3: phenol and Cr contaminated water þ bacteria.The error bars represent the standard errors.

Table 2 .
Effect of bacterial consortium on the biomass of Phragmites australis.