Removal of Reactive Yellow 86 from Synthetic Wastewater in Lab-Scale Constructed Wetlands Planted with Cattail and Papyrus

Abstract

Synthetic wastewater was treated in lab-scale constructed wetlands (CWs) in sequencing batch mode to evaluate roles of aquatic plants for removing an azo dye: Reactive Yellow 86 (RY86). Under hydraulic retention time (HRT) of 5 days, removal by unplanted CWs was less than 20% for RY86 of 10–50 mg/L. The CWs planted with cattail and papyrus demonstrated RY86 removal of 50–68% and 73–84%, respectively. For wastewater containing 50 mg/L RY86, removal in the unplanted CW was <12%, even under a 15 day HRT, although it was 22–71% in CWs planted with cattail and 34–81% in CWs planted with cattail, with increasing values under HRTs of 1 day to 15 days. Both cattail and papyrus grew well, extending their roots in the CWs for 90 days. RY86-decolorizing microorganisms were detected in CW effluent. Overall, RY86 removal was positively correlated with evapotranspiration in the CWs, indicating the plant uptake as the main removal mechanism. Papyrus and cattail, especially the former, are suitable plants for CWs intended to treat RY86-containing wastewater.

1. Introduction

Many countries such as Indonesia [1,2], Malaysia [2], Bangladesh [3], and China [4] have thriving textile industries. Reactive dyes dominate the dye market because of their low cost, simple dyeing processes, and improved fastness attributable to their reaction with cellulosic fibers [5]. Textile industries generate large amounts of wastewater that contains unfixed residual dyes. Direct discharge of textile wastewater into receiving water bodies causes severe environmental pollution because of its intense color and toxicity. Several methods such as chemical adsorption [6], photodegradation [7], and ozonation [8,9] have been assessed to address difficulties related to contamination of wastewater by effluent [6,7,8,9]. Nevertheless, these methods entail high investment costs and operational difficulties. Because of the complex aromatic structure and stability of azo dyes, conventional biological treatment methods are inadequate for dye removal [2,6].

Recently, many studies have examined the application of constructed wetlands (CWs) for cost-effective treatment of dye wastewater [10,11,12,13,14]. Treatment in CWs specifically emphasizes dye adsorption by soil, degradation by microbial activity, and phytoaccumulation by plant uptake. In particular, the role of plants in CWs has attracted great interest. Dogdu and Yalcuk [13] reported greater than 90% color removal and cleavage of azo bonds of Yellow 2G by vertical flow CWs planted with cattails (Typha angustifolia L.) and Canna indica. Noonpui and Thiravetyan [11] reported that Reactive Red 2 (RR2, MW = 615), with smaller molecular size, was taken up more easily than Reactive Red 120 (MW = 1469) or Reactive Red 141 (MW = 1775), with larger molecular size, by burhead (Echinodorus cordifolius L.) under soil-free conditions. By contrast, Hussen and Scholz [14] reported that the presence of common reed (Phragmites australis) did not affect removal of Acid Blue 113 or Basic Red 46.

Our research group has surveyed CWs installed to treat Batik wastewater in Pekalongan city, Indonesia [1]. Cattail, papyrus, and heloconia were planted in the CWs, but the role of the plant in dye removal was unclear [1]. This study specifically examines the roles of aquatic plants in CWs for removing Reactive Yellow 86 (RY86), commonly used for dyeing textiles including garments, fabrics, and home textiles, from synthetic wastewater. Reportedly, RY86 is degraded by photolysis using multi-structured Fe₂O₃ nanoparticles as catalysts [15,16,17], the photo-Fenton process [18], electrochemical treatment [19], and laccase released by Bacillus safensis [20]. As far as we know, no report of the relevant literature describes RY86 removal by CWs. For this study, a 90 day experiment was conducted to remove RY86 from wastewater using lab-scale CWs planted with cattail and papyrus.

2. Materials and Methods

2.1. Synthetic Wastewater

The synthetic wastewater used for this study contained 10–50 mg/L RY86 (C₁₈H₁₄Cl₂N₈Na₂O₉S₂, MW = 667.357; MP Biomedicals, Santa Ana, CA, USA), 21.8 mg/L K₂HPO₄, 8.5 mg/L KH₂PO₄, 44.6 mg/L Na₂HPO₄·12H₂O, 1.7 mg/L NH₄Cl, 22.5 mg/L MgSO₄·7H₂O, 27.5 mg/L CaCl₂, and 0.25 mg/L FeCl₃·6H₂O in 1 L deionized water. Potato dextrose agar medium (Nihon Pharmaceutical Co., Ltd., Chuo-ku, Japan) and R2A agar medium (Nihon Pharmaceutical Co., Ltd.) were used for screening RY86-degrading microorganisms in the CWs.

2.2. Lab-Scale CWs

Lab-scale CWs were set up in a greenhouse at the Biwako Kusatsu Campus of Ritsumeikan University in Kusatsu City, Shiga Prefecture, Japan, as shown in Figure 1. Each CW consisted of a plastic container (L 10.7 cm × W 8.8 cm × H 25.0 cm) filled with 1.3 kg of gravel (3–5 mm) with a porosity of 37.5%. A cock with a tube was equipped at the bottom of the container for discharging treated water. The CWs were planted with oriental cattail Typha orientalis (height 88 cm, 3 plants) (Cattail-CW) or dwarf papyrus Cyperus isocladus (height 71 cm, 3 plants) (Papyrus-CW), or were left unplanted (Unplanted CW). Before planting in the CWs, roots were washed gently using tap water to remove the original culture soil. All CWs were prepared in duplicate. The plants, which had been purchased from Tojaku Engei Co., Ltd. (Joyo, Kyoto, Japan), were grown in tap water in the CWs for 1 week before wastewater treatment.

2.3. Operation of CWs

Synthetic wastewater was treated in the CWs in a sequencing batch mode. The operational conditions are presented in

Table 1. Operating conditions of constructed wetlands in sequencing batch mode for treating synthetic wastewater containing RY86.

Runs	Period (2022)	RY86 (mg/L)	Influent (mL/Batch)	HRT (days)
1–3	12 September–26 September	10	300	5
4–6	26 September–11 October	20	200
7–9	17 October–1 November	30
10–12	1 November–16 November	40
13–15	16 November–26 November	50
16	26 November–28 November			3
17	29 November–30 November			1
18	30 November–10 December			10
19	10 December–25 December			15

. On the first day, wastewater of 300 or 200 mL (influent) was poured into the CWs. After the defined number of days, the treated water (effluent) was drained from in each CW. Then, fresh wastewater (300 mL or 200 mL) was poured again to the CWs. This operation was repeated for all CWs at every defined interval.

During 7 September–26 November 2022, the RY86 concentration in wastewater was increased gradually from 10 mg/L to 50 mg/L with HRT of 5 days. During 29 November to 25 December, the HRT was varied from 1 day to 15 days with 50 mg/L RY86 in wastewater.

2.4. Screening RY86-Decolorzing Microorganisms

For screening microorganisms with RY86-decolorizng ability, microorganisms in the effluent of the CWs were cultivated using the potato dextrose agar medium and R2A agar medium, containing and 50 mg/L RY86. It was assumed that some microorganisms on the gravel and in the rhizosphere were contained in the effluent. An appropriately diluted effluent sample of 0.1 mL was applied to these plate media. The potato dextrose agar plates and the R2A agar plates were incubated at 28 °C for 1 day under aerobic conditions and under anaerobic conditions in anaerobic chambers (Anero Pack Kenki; Mitsubishi Gas Chemical Co. Inc., Tokyo, Japan).

2.5. Analytical Procedures

The radiation and air temperature in the greenhouse were recorded by a recorder (TR-74Ui; T&D Corporation, Matsumoto, Japan). Dissolved oxygen (DO) was measured using a multimeter (HQ30d) and a probe (LDO10101; Hach Co., Loveland, CO, USA). The absorbance spectrum was measured using a UV-visible light spectrophotometer (UV–2600; Shimadzu Corp., Kyoto, Japan). A calibration curve was obtained by measuring the absorbance of a RY86 solution with defined concentrations at 416 nm wavelength [15,16,17].

Evaporation (%) in unplanted CWs and evapotranspiration (%) in the planted CWs for each run were determined by the following equation:

Evaporation/Evapotranspiration (%) = 100 × (V_i − V_e)/V_i,

(1)

The mass-based removal efficiency of RY86 in the CW was calculated using the following equation:

Removal (%) = 100 × (C_iV_i − C_eV_e)/C_iV_i,

(2)

Therein, C represents the RY86 concentration (mg/L). V stands for the wastewater volume (L/batch). Subscripts i and e respectively denote influent and effluent.

3. Results

3.1. Effects of RY86 Concentration on CW Effluent Quality

During runs 1–15 conducted from 12 September through 30 November 2022, R86-containing wastewater was treated in the CWs in the sequencing batch mode under HRT of 5 days. Figure 2 shows the air temperature, evapotranspiration ratio, and the effluent RY86 concentration.

During runs 1–3 at temperatures higher than 25 °C in September, evaporation of wastewater in the unplanted CW was 10–35%, resulting in the effluent RY86 concentration of 12–15 mg/L, higher than 10 mg/L in wastewater. Evapotranspiration in the planted CWs was 30–50%. However, the effluent RY86 concentration was 6.6–7.7 mg/L in the Cattail-CW and 4.2–4.9 mg/L in the Papyrus-CW, indicating a marked degree of removal of RY86 by the presence of the plants. The effluent DO concentrations were 4.2–7.3 mg/L in the unplanted CW, 3.8–4.8 mg/L in the Cattail–CW, and 5.9–6.3 mg/L in the papyrus-CW.

For runs 4–6, evaporation/evapotranspiration was 6–15% in the unplanted CW and 26–62% in the planted CWs. The RY86 concentration in wastewater at 20 mg/L was lowered to 16.8–21.0 mg/L in the unplanted CW, 9.1–13.1 mg/L in the Cattail-CW, and 5.1–8.5 mg/L in the Papyrus-CW. For Runs 7–9, evaporation/evapotranspiration was 8–21% in the unplanted CW and 30–47% in the planted CWs. The RY86 concentration in wastewater at 30 mg/L was lowered to 26.3–29.2 mg/L in the unplanted CW, 14.6–19.0 mg/L in the Cattail-CW, and 9.8–11.0 mg/L in the Papyrus-CW. For runs 10–12, evaporation/evapotranspiration was 8–13% in the unplanted CW and 17–46% in the planted CWs.

The RY86 concentration in wastewater at 40 mg/L was lowered to 36.5–39.9 mg/L in the unplanted CW, 21.2–28.6 mg/L in the Cattail-CW, and 10.9–13.4 mg/L in the Papyrus-CW.

For runs 13–15 at 12–15 °C in November, evaporation/evapotranspiration was only 5–9% in the unplanted CW and 15–32% in the planted CWs. The RY86 concentration was set as 50 mg/L in wastewater, and it was 45.5–47.9 mg/L in the unplanted CW, 29.4–35.3 mg/L in the Cattail-CW, and 14.5–18.5 mg/L in the Papyrus-CW. The effluent DO concentration was 9.2–9.7 mg/L in the unplanted CW, 5.4–6.6 mg/L in the cattail-CW, and 7.6–8.3 mg/L in the Papyrus-CW, suggesting DO consumption by respiration of the plants and microorganisms in the rhizosphere.

Figure 3 shows the typical absorbance spectra of wastewater containing 50 mg/L RY86 and its treated water in run 15. The absorbance of wastewater peaked at 416 nm. This peak value of effluent was markedly lower in the Cattail-CW and the Papyrus-CW. However, little or no decrease in the peak was observed in the unplanted CW. Apparent peak shift was not observed in the effluent samples. The main removal mechanism for RY86 in the CWs was regarded as adsorption to the roots and uptake by the plants.

Relations between the RY86 concentration in wastewater and its mass removal for runs 1–15 at HRT of 5 days are summarized in Figure 4. The RY86 removal in the unplanted CW was less than 20% for 10–50 mg/L RY86. The removal values in the Cattail-CW and the Papyrus-CW were, respectively, 50–68% and 73–84%. The slight decrease in the removal with increase in the RY86 concentration was probably attributable to the effect of decreasing temperature.

3.2. Effects of HRT on CW Effluent Quality

In runs 16–19 from 26 November through 25 December 2022, wastewater containing 50 mg/L RY86 was treated in the CWs under HRTs of 3, 1, 10, and 15 days. The air temperature, evapotranspiration ratio, and the effluent RY86 concentrations for these runs are also presented in Figure 2. The above-ground parts of the plants withered in winter, but the roots remained. Root elongation of cattail and papyrus after run 19 in the CWs was also depicted in Figure 1. The roots and stem of both cattail and papyrus were colored yellow, indicating that RY86 was attached on and uptaken by the plants.

For run 16 at 13 °C, the evaporation/evapotranspiration ratio was only 2% in the unplanted CW and 6–9% in the planted CWs with a 3 day HRT. The RY86 concentration was 47.8 mg/L in the unplanted CW, 35.5 mg/L in the Cattail-CW, and 26.7 mg/L in the Papyrus-CW. For run 17 at 16 °C, the evaporation was only 4% in the unplanted CW; evapotranspiration in the planted CWs was 1–4% with HRT of 1 day. The RY86 concentration was 48.2 mg/L in the unplanted CW, 40.5 mg/L in the Cattail-CW, and 33.2 mg/L in the Papyrus-CW. For run 18 at 9 °C, the evaporation ratio was 10% in the unplanted CW; evapotranspiration in the planted CWs was 25–30% with a 10 day HRT. The RY86 concentration was 49.2 mg/L in the unplanted CW, 33.3 mg/L in the Cattail-CW, and 22.0 mg/L in the Papyrus-CW. Finally, for run 19 at 6 °C, the evaporation/evapotranspiration was 13% in the unplanted CW and 52–55% in the planted CWs with a 15 day HRT. The RY86 concentration was 50.0 mg/L in the unplanted CW, 30.8 mg/L in the Cattail-CW, and 21.7 mg/L in the Papyrus-CW.

Effects of HRT on RY86 removal in the CWs for 50 mg/L RY86 are summarized in Figure 5. The RY86 removal was less than 12% in the unplanted CW, even with a 15 day HRT. With increased HRT from 1 day to 15 days, the RY86 removal increased from 22% to 71% in the Cattail-CW and 34% to 81% in the Papyrus-CW.

3.3. RY86-Degrading Microorganisms

RY86-decolorizing microorganisms were screened from effluent samples of run 14 of the CWs. Microbial colonies of 10²–10³ CFU/mL appeared on the agar plates containing RY86 of 50 mg/L under aerobic and anaerobic conditions, as shown in Figure 6. The yellow color of RY86 faded around some colonies derived from effluent samples of the unplanted CW, the Cattail-CW, and the Papyrus-CW, especially aerobically grown on the potato dextrose agar plates, indicating enzymes secreted outside the cells decolorized the dye.

4. Discussion

This study examined the roles of plants in CWs for removing RY86. CWs planted with cattail and papyrus demonstrated much higher removal for RY86 than the unplanted CW. The gravel packed in the CWs adsorbed only a small amount of RY86 in wastewater. RY86 is hardly degraded by photolysis under usual conditions [15,16,17]. Reportedly, the presence of common reed did not affect the removal of dyes [14]. However, the results obtained in this study indicate the importance of selecting plants for treating dye-containing wastewater. Cattail and papyrus were actually planted in CWs in Pekalongan city for treating Batik wastewater [1]. The RY86 removal in the planted CWs increased with HRT from 1 day to 15 days. Earlier studies also presumed HRT of 1.5–15 days in CWs for the treatment of textile wastewater [12,15].

The relation between the evapotranspiration of wastewater and the RY86 removal in the CWs is summarized in Figure 7. Root development is related to the efficiency of uptake of water, nutrients, and RY86. Although a large amount of evapotranspiration occurs in the CWs with long HRTs, few studies have evaluated its effects on dye uptake by plants. For evaporation of 2.5–22.5%, RY86 removal in the unplanted CW was less than 23%. With increased evapotranspiration, the RY86 removal increased drastically, suggesting that RY86 was uptaken by the plants. Over the same range of the evapotranspiration, the Papyrus-CW showed higher RY86 removal than that of Cattail-CW. The maximum RY86 removal values in the Cattail-CW and the Papyrus-CW were, respectively, 83% and 91%, with evapotranspiration of 62% and 47%. Greater efficiency of C. papyrus was also reported for treating municipal wastewater than that of P. australis [21] and for removing heavy metals and enteric bacteria than that of broad leaf cattail Typha latifolia [22]. Reportedly, RR2 (MW = 615) with a small molecular size was taken up easily by burhead [11]. With molecular size as small as that of RR2, RY86 (MW = 667.37) might be easily uptaken by cattail and papyrus. Detoxification pattern of xenobiotic molecule depends upon the plant species [23]. Detailed information about inherent pathways of the plants to metabolize the toxicants is lacking [24]. Further studies of accumulation, degradation, and toxic effects of RY86 in the plant tissues must be conducted to elucidate the fate of the dye in the CWs. The mineralization efficiency for RY86 in the CWs remained unknown.

The higher removal for RY86 than the evapotranspiration ratio (Figure 7) suggests that the dye was removed by degradation by microorganisms in addition to the plant uptake (Figure 6). Root exudates of plants can grow microorganisms in their rhizosphere. Azo dyes can be degraded by azo reductases for the reducing azo bond (-N=N-) by anaerobic/anoxic bacteria [25] and lignin peroxidase, laccases, and peroxidases of fungi [26,27]. However, few studies described decolorization specific for RY86 by microorganisms [20]. Reportedly, B. safensis isolated from earthworm gut can degrade RY86 [18]. Phylogenetic identification and characterization of RY86-decolorizing microorganisms found in this study (Figure 6) will be helpful for further elucidation of decolorization mechanisms in the CWs. The biodegradation pathway of RY86 by microorganisms should be elucidated because toxic aromatic amines can be accumulated in the azo-dye degradation process [28,29].

The organic matter in wastewater used for this study was only RY86, which is hardly biodegradable. Addition of easily and slowly degradable organic matter might enhance the growth of dye-degrading microorganisms. Reportedly, application of rice agricultural waste to CW with inoculation of a dye-degrading Psychrobacter alimentarius strain and planted with Presaria barbata can enhance removal of Reactive Black 5 [14]. Such agricultural wastes are expected to be suitable for developing habitats for bacteria, fungi, earthworms, and plants. When indigenous microorganisms are unable to degrade the dye, bioaugmentation is an effective approach to enhance CW performance. Further studies must be conducted for identification and characterization of RY86-degrading microorganisms in the CWs.

5. Conclusions

CWs planted with cattail and papyrus demonstrated much higher removal for RY86 than the unplanted CW. The RY86 removal in the planted CWs increased with HRT. Gravel in the CWs contributed slightly to the RY86 removal from wastewater by adsorption. RY86-decolorizing microorganisms were detected in the CW effluent. Concomitantly with increased evapotranspiration, the RY86 removal increased drastically, indicating the main removal mechanism for RY86 was plant uptake in the CWs. Papyrus and cattails, especially the former, are suitable plants for application to CWs treating RY86-containing wastewater. These finding will contribute to establish design and operation of CWs for cos-effective treatment of dye wastewater.