Promoting Water Sustainability through Degradation of 4-Nitrophenols by Spirodela polyrrhiza and Rhodococcus sp. Strain PKR-1 Association

The removal of chemicals from water sources that are harmful to humans and the environment can contribute to improving water quality. Biological treatment methods, such as bioaugmentation are an environmentally sustainable approach for pollutant removal. The 4-nitrophenol is the most hazardous nitrophenol chemical pollutant. In this study, a laboratory investigation was conducted on a flask scale to evaluate the rhizoaugmentation of 4-nitrophenol-polluted water. This was achieved by employing Rhodococcus sp. strain PKR-1, which was reintroduced into the roots of Spirodela polyrhiza. The selected strains were inoculated into the root at the rate of 104 to 106 colony-forming units (CFU) per plant. At high levels exhibited stability across five consecutive two-day degradation cycles, and full elimination of 4-nitrophenol was accomplished within these five repeated cycles. Therefore, the introduction of degraders into the root systems of aquatic plants has proven to be a successful method for treating effluents or aquatic resources contaminated with 4-nitrophenol.


Introduction
In today's world, water resources face a pressing challenge due to water contamination.The presence of different nitrophenol isomers in water resources presents a significant risk to human health and ecosystems.It is evident that this issue has direct implications for SDG 6, which strives to ensure clean water and sanitation for all [1].The presence of nitrophenol chemicals, such as those found in wood preservatives, insecticides, explosive industrial goods, and dyes, is very high in aquatic environments [2,3].Due to the potential hazards that 4-nitrophenol (4-NP) presents to both human beings and aquatic organisms, the United States Environmental Protection Agency has designated 4nitrophenol as a priority pollutant [4].Numerous approaches have been investigated to ensure the safety of both human beings and aquatic species in relation to nitrophenols, taking into account the corresponding financial implications and energy utilization.Within the realm of biological treatment methods, phytoremediation is widely regarded as an economically viable and environmentally sustainable approach for the elimination of various pollutants from aquatic environments [5,6,7].Nevertheless, the efficacy of phytoremediation is hindered by various variables, including the low speed of the process and the limited capacity of microorganisms in the rhizosphere to survive and degrade contaminants in polluted water sources or contaminated areas.
Bioaugmentation has emerged as a highly promising technique in the field of phytoremediation due to its ability to effectively mitigate the risks and challenges associated with the dispersal of microorganisms [8].The primary emphasis of many remedial technologies is on the dissipation of pollutants, sometimes overlooking the sustainability of pollutant removal and the potential ecological consequences arising from the introduction of microorganisms to the environment, particularly to indigenous microorganisms [5,8].Biodegradation within the rhizosphere can be enhanced by the presence of oxygen and the release of organic exudates from plant roots.The phenomenon referred to as the "rhizosphere effect" involves the creation of a microenvironment by plants that facilitates the enhanced activity of microorganisms in decomposing pollutants.
To date, the environmental effects of potential solutions have been largely overlooked by decisionmakers.Even though many studies have shown that synthetic surfactants and aromatic compounds degrade at a much faster rate in the rhizosphere of S. polyrrhiza [9,10,11], the effect of rhizoaugmentation on the long-term stability of 4-Nitrophenol degradation in polluted water is still poorly understood.The purpose of this research was to examine the efficacy of rhizoaugmentation by treating 4-Nitrophenol-contaminated water in flask-scale sequencing batch reactors (SBRs) for five cycles with S. polyrrhiza and Rhododcoccus sp.Strain PKR-1, bacteria isolated from S. polyrrhiza.

Methodology 2.1. Plant and Chemicals
To ensure the absence of bacteria in S. polyrrhiza, the plants underwent a sterilization process consisting of a 1-minute wash in 70% ethanol followed by a 5-minute wash in a sodium hypochlorite solution containing 5% available chlorine.The plants were then rinsed twice with autoclaved deionized water and subsequently germinated in a sterile modified Hoagland nutrient medium, as previously described by Kristanti et al. [9].The bacteria-free plants were maintained in a sterile Hoagland solution in an incubation chamber (28 ͦ C, 10,000 lux, 16 h (light): 8 h (dark).A water sample of secondary effluent from a sewage treatment plant from a local factory was used in SBR experiments.4-NP was purchased from Sigma Aldrich.

Microorganism Strain and Growth Condition
4-nitrophenol degrading bacteria strain PKR-1 isolated from S. polyrhiza roots [9,10] was used in this study.Strain PKR-1 was cultured in basal salts medium [9] containing 4-NP as the sole carbon source (4NP-BSM).Agar was added with the concentration of 1.5% (w/v).

Inoculation of Strain PKR-1 on S. polyrrhiza
Strain PKR-1 was cultivated on a rotary shaker at 28 ͦ C and 150 rpm.To recover late exponential phase cells, it was centrifuged (4500 × g at 4 ͦ C for 20 min) and washed with BSM.After resuspending strain PKR-1 cells in 20 mL sterile Hoagland solution, their OD600 was 0.03.Plants without bacteria were immersed for 10 min in bacterial suspension and twice washed with sterile Hoagland solution.

4-Nitrophenol degradation by plant and rhizobacteria associations
Using Erlenmeyer flasks with a capacity of 300 mL and a concentration of 10 mg L -1 of Hoagland solution, two different testing groups were set up.In the first group, a total of 10 plants from the plants-PKR-1 link were planted in an experimental flask.The second group, which was called the "control," was made up of strain PKR-1 in a sterile Hoagland solution with 4-NP added, but no plants.The flasks were incubated at 28°C, 8000 lux, 16:8 h.Every day for 4 days, the activity of removing 4-NP was tracked, and samples of the number of cells from strain PKR-1 were taken at 0, 2, and 4 days.

SBR experiment
We tested the efficacy of inoculating plant roots with 4-NP degrading bacteria through a series of SBR experiments in flasks (2 days reaction time each cycle, 5 cycles) using water contaminated with various types of 4-nitrophenol (4-NP).To create 4NP-contaminated water, we dissolved 10 mg L -1 of 4NP into either sterile Hoagland solution, river water, or a secondary effluent sample.Two test systems were set up in each Erlenmeyer flasks of 300 mL, with 100 mL of the 4-NP-polluted water.Ten plants-PKR-1 were grown in water contaminated with 4-NP in 28 °C (16:8 h light-dark).After 2 days, 10 plants were moved to a new flask with water contaminated with 4-NP and kept in the incubator at the same temperature and humidity.Each 2-day cycle was repeated five times in triplicate.The 4-NP concentration was monitored daily.

Analytical procedures
The water samples were centrifuged at 9600 × g at 4 ͦ C for 10 minutes.The 4-NP concentration of the resultant supernatant was then determined using high-performance liquid chromatography (HPLC) equipped with a UV-vis detector and a Shim-pack VP-ODS column (150 mm 4.6 mm, 5 mm; Shimadzu).The mobile phase was acetonitrile, water, and acetic acid (500:498:2, vol/vol/vol), and the flow rate was 1 mL min -1 ; the wavelength employed for detection was 280 nm.Bacterial cells were isolated from bulk water and root-surface samples to track microbial abundance [11].The results are presented as CFU per flask, calculated as follows: Bulk water: (CFU per mL) × (total volume of bulk water in the flask) Rhizosphere: (CFU per plant) × (total number of plants in the flask)

Statistical analysis
Results from triplicate studies were presented as mean values with standard deviations (±95% confidence interval).Significant differences were identified using Student's t-test (p < 0.05).

4-NP Degradation in the presence of S. polyrrhiza
Fig. 1 shows strain PKR-1 colonizing sterile S. polyrhiza root surfaces and degrading 4-NP.After inoculation, strain PKR-1 cells attached to S. polyrhiza roots and released the inoculation strains into the Hoagland solution while maintaining root populations.The populations of inoculated strains on the roots increased until the end of the experiment.The association degraded 4-NP in 3 days.In the control test, the cell numbers were 1.7× 10 7 CFU per flask after 4 days, indicating population levels 10 to 100 times larger than before inoculation.There were no significant differences (p < 0.05) in flask cell counts with or without S. polyrrhiza.

Sustainable 4-NP removal by sequencing batch reactors (SBR) using S. polyrrhiza-strain association
To assess the potential of S. polyrrhiza-strain PKR-1 association for the treatment of aquatic water and waste water polluted with 4-NP, flask-scale SBR experiments (2 days of reaction time/cycle; 5 cycles; 100 mL of water polluted with 10 mg/L of 4-NP; 10 plants of S. polyrrhiza-strains PKR-1 association) were conducted.4-NP removal by SBR using S. polyrrhiza-strains PKR-1 association (test A), and S. polyrrhiza alone (test B) are shown in Figure 1.
In Hoagland solution test system of A, faster 4-NP removal was always observed in comparison to test system B, and complete 4-NP removal was observed within 2 d of incubation period (Figure 2A).Rapid adaptation of S. polyrrhiza-strains PKR-1 association was suggested since the 4-NP removal rate was continually repeated until 5-cycles batch experiment.In Hoagland solution test system B, 4-NP removal gradually increased to a certain extent with repeated cycles.4-NP removal in river water and WWTP test system A and B were found to be the same removal tendency from Hoagland test system (Figure 2B, and 2C).However, 4-NP were able to completely remove from test system B after cycles.Uninoculated S. polyrhiza may have removed 4-NP by degrading indigenous bacteria in the secondary effluent sample and by itself.In this investigation, indigenous bacteria could also remove 4-NP after exposure to 4-NP.(3.9±0.3)×10 7 a Results (mean±95% confidence interval); b not tested The performance of the two test systems is shown in Table 1.Results showed that 4-NP removal rates were attained in S. polyrrhiza-strains PKR-1 association (test A) than S. polyrrhiza alone (test B).The removal rates of 4-NP test A were found higher than those of test B. In the Hoagland solution system, the removal rate of 4-NP in a range of 1.367-1.469mg/L/d, could be achieved in test A, and those values were higher compared to those in test B; 0.425-1.303mg/L/d of 4-NP, were removed IOP Publishing doi:10.1088/1755-1315/1324/1/0121016 during repeated batch experiments.Both tests were found to be increased during the repeated batch experiments.These results indicated the strains PKR-1 utilize 4-NP as their carbon source.All NPs removal rates increased in test B over the repeated cycles experiment.The 4-NP removal rate tendency of WWTP test systems was the same as in the case of river water systems.These results indicated that both introduced and indigenous 4-NP degrading bacteria were able to enhance 4-NP removal rates.
Table 2 shows the total culturable, strain PKR-1 distribution and change in bulk water and rhizosphere microcosm during SBR tests.On the first day of inoculation, strain PKR-1 adhered to roots at 2.1 × 10 4 CFU per plant.Strain PKR-1 was released from the roots into the Hoagland Solution after 1 day while root populations were maintained.S. polyrrhiza was able to accumulate strain PKR-1 since its cell counts increased by 10-1000 from the original injection and lasted for five cycles.The same phenomena as in the river water and WWTP system, strain PKR-1 increased to 100 cell numbers on day 6 and slightly increased in rhizosphere fraction in the presence and absence of introduced bacteria, suggesting 4-NP-degrading bacteria can adhere to root surfaces and that indigenous bacteria did not inhibit them.

Growth promotion during SBR experiments
During the long-term SBR experiments, we noticed that the number of fronds markedly increased in the presence of indigenous bacteria (river water and secondary effluent water of WTTP) (Table 3).The number of S. polyrrhiza in the river water test A and test B increased by 2.5-and 1.5-fold at the end of the experimental period, respectively.Similarly, the increased number of S. polyrrhiza in the WWTP test A and test B increased by 2.7-and 1.7-fold at the end of the experiment, respectively.The results indicate that indigenous bacteria in river water and secondary effluent water of WWTP promoted the growth of S. polyrrhiza.Moreover, interestingly, relationships between strains PKR-1 and indigenous bacteria in river water and secondary effluent water of WWTP promoted S. polyrrhiza growth more strongly.

Conclusions
The degradation capacity of the S. polyrrhiza-bacteria symbiosis towards 4-NP exhibited a significant enhancement upon exposure to 4-NP, accompanied by a noteworthy augmentation in the population of culturable bacteria capable of degrading 4-NP.The simultaneous removal of 4-NP from polluted water by the association of S. polyrrhiza and Rhodococcus sp.strain PKR-1 was observed, and the consortium exhibited the ability to be reused multiple times without any degrading activity loss.There was a significant rise in the population of culturable Rhodococcus sp.strain PKR-1 and the abundance of S. polyrrhiza.This suggests a mutually advantageous symbiotic relationship between Rhodococcus sp.strain PKR-1 and S. polyrrhiza.The inclusion of indigenous bacteria in the system did not yield any discernible competition effect.Therefore, the implementation of rhizoaugmentation, a technique

Table 1 .
Removal rate of 4-NP during SBR experiments using S. polyrrhiza-strains PKR-1 association (test A) and S. polyrrhiza alone (test B)

Table 2 .
Distribution and change of total culturable, Rhodococcus sp.PKR-1 in bulk water and rhizosphere in test A (plant-strain PKR-1 association) and test B (plant alone)