Removal of an anionic azo dye direct black 19 from water using white mustard seed ( Semen sinapis ) protein as a natural coagulant

In this study, standard jar tests were conducted using white mustard seed protein (WMSP) as a natural coagulant to remove direct black 19 (DB-19) from its aqueous solution. Comparative coagulation tests were performed using commercial polyaluminum chloride (PAC). The results showed that DB-19 removal by WMSP increased with increasing settling time and reached the maximum removal at 180 min. The DB-19 removal descended from 98.4 to 46.2% as pH increased from 4 to 10. The most effective temperature for DB-19 removal was 25 C. The removal of DB-19 was weakened by the presence of Na 2 S 2 O 4 . Overall, WMSP was more ef ﬁ cient than PAC for DB-19 removal in all experiments except at pH 4 and 5. The mechanism of the removal of DB-19 by WMSP could be attributed to adsorption and charge neutralization processes.


INTRODUCTION
Globally, the textile industry is one of the largest fresh water consumers which consequently generates large amounts of wastewater. According to the data of the China statistical yearbook on the environment, the textile industry Among the techniques, coagulation/flocculation has many advantages such as cost effectiveness, convenience in operation, low energy consumption, and no generation of harmful and toxic intermediates (Shi et al. ; Verma et al. a).
Currently, the commercial coagulants used for water treatment consist of two major groups, namely, inorganic coagulants and organic polymers. Though both of them are playing a very important role in water treatment, many drawbacks arise in water treatment, such as inefficiency at low temperature, changes of pH, corrosion of instruments, and generation of a huge amount of non-biodegradable sludge and so on. Furthermore, their residuals in water can cause adverse impacts on living beings. For example, studies revealed that aluminum is neurotoxic and can cause pathogenesis of Alzheimer's disease (Flaten ; Polizzi et al. ). Also, the residual monomers of polyacrylamide (PAM) present in treated water have been proven to be neurotoxic and carcinogenic (Šciban et al. ). Hence, the development of eco-friendly coagulant has been a major topic of research in water treatment.
Plant-based coagulants, including mustard seed, Jatropha curcas seed, guar gum, copra, Cactus latifaria, Prosopis juliflora seed, common bean, chitosan, orange waste and so on, have been used to remove a large variety of pollutants including turbidity, algae, dyes, humic acid, bacteria and metals etc. from water due to their advantages such as abundant source, high efficiency, low sludge production, biodegradability, and nontoxicity (Pritcharda For instance, Ndabigengesere et al. () reported that the active agents in aqueous Moringa extracts were cationic proteins with a molecular weight of 13 kDa and an isoelectric point between 10 and 11. The optimal dosage of shelled Moringa oleifera seed was almost the same as alum, whereas purified proteins were more effective than alum for turbid water treatment. Furthermore, the innocuous coagulant did not affect the pH and conductivity of treated water with four or five times less volume of chemical sludge created compared to alum.
Recently, Boulaadjoul et al. () reported that Moringa oleifera seed powder was used to enhance the primary treatment of paper mill effluent. The results indicated that the turbidity and COD abatements were 96.02 and 97.28% using Moringa oleifera seed powder as coagulant, whereas the respective removals of turbidity and COD by alum were 97.1 and 92.67%, indicating that Moringa oleifera is a very efficient natural coagulant for the treatment of paper mill effluent. Betatache et al. () reported that the optimum dosage of prickly pear cactus was 0.4 g/kg for sewage sludge conditioning, which was much more efficient than three polyelectrolytes, namely, 0.8 g/kg for Chimfloc C4346, 80 g/kg for FeCl 3 and 60 g/kg for Al 2 (SO 4 ) 3 .
In recent research, mustard seed proteins with the molecular weight of approximately 6.5 and 9 kDa have been proven to be more efficient to remove turbidity from pond water than Moringa oleifera seed proteins (Bodlund et al.

).
Although mustard seed has more advantages in wider distribution, abundance, and availability at low price compared with Moringa oleifera seed, the only application of the mustard protein for water treatment obtained from the published literature was to remove the turbidity, which limits its versatility in water treatment. Hence, our study is aimed at investigating the potential of mustard seed protein as a natural coagulant for the removal of dye from its aqueous solution.
In this study, direct black 19 (DB-19, Figure 1) was selected as a target pollutant to test the coagulation ability of white mustard seed protein (WMSP) as it is a widely used anionic dye, especially in some Asian countries (Shi et al. ). Meanwhile, both this dye and its reduction product have been proven to be mutagenic (Joachim et al. ).
A commercial coagulant, polyaluminum chloride (PAC), that is most widely used for water treatment in China was selected to carry out the same experiment as a comparison to evaluate the coagulating performance of WMSP. The removal effects with respect to pH, settling time, temperature, coagulant dosage and influence of inorganic salts presence was investigated.

MATERIALS AND METHODS
Preparation of coagulants and dye-containing wastewater White mustard seed (Figure 2), a traditional Chinese medicine used widely to resolve phlegm and dispel colds (vocabulary of traditional Chinese medical science), was purchased from a local pharmacy in Zhengzhou, China.
The WMSP was extracted in the same way as described in our previous study (Tie et al. ). The only difference from the previous method was that the dialysis tube with a molecular weight cut-off of 3,500 Da was used in this study. The WMSP content was measured at 596 nm (UVmini-1240, Shimadzu, Japan) using bovine serum albumin as the standard (Zhou & Chen ). The PAC solution at the same concentration was synthesized by adding PAC into deionized water.
The DB-19 solution used in this study was prepared using the same method described in our previous study (Tie et al. ). The pH was adjusted to different values in the range of 4 to 9 using 0.1 M HCl and NaOH solution.

Jar tests
The jar tests were carried out using standard jar test equip-   655 nm. The removal rate of DB-19 was calculated by the following equation: where C 0 and C e are the initial and final concentrations of DB-19 in solution (mg L À1 ), respectively.

Characterization
Fourier transform infrared spectroscopy (FTIR, Thermo Scientific NicoletiS10, USA) and X-ray photoelectron spectroscopy (XPS, Thermo Scientific ESCALAB 250Xi, USA) were used to characterize WMSP, DB-19, and the reaction products between WMSP and DB-19.  The DB-19 removals at different pH using two coagulants are shown in Figure 4 for the two coagulants.  In the case of the same tests using PAC as the coagulant, the removal efficiency was lower at each dosage. Therefore, WMSP was shown to be more efficient for DB-19 removal than PAC in the experiment.

RESULTS AND DISCUSSION
Effect of temperature on DB-19 removal Figure 6 shows the effect of temperature on DB-19 removal in the range of 10-45 C. It can be seen that the removal rate varied at 31.7-43.7% for PAC and 70.0-83.2% for WMSP, respectively. At each temperature set for the experiment, WMSP performed better than PAC for DB-19 removal.
The optimal temperature for DB-19 removal by both of the two coagulants was 25 C, and the corresponding removal rates were 43.7 and 83.2% for PAC and WMSP, respectively.  The test results indicated that WMSP was more efficient than PAC at all selected temperatures.

Effect of the presence of inorganic salt on DB-19
removal Sodium sulfate is widely adopted to improve dyeing performance and fixation property by enhancing the dye molecules transfer from the solution to cotton fibers when direct dyes are used (Teixeira et al. ). Hence, the effect of Na 2 SO 4 on DB-19 removal was investigated. Figure 7 shows that, overall, Na 2 SO 4 weakened the DB-19 removal for both coagulants, whereas WMSP still outperformed PAC in the presence of Na 2 SO 4 .

Mechanism of DB-19 removal by WMSP
The effect of pH on the flocculation is critical to explore the coagulation mechanism (Teixeira et al. ). Figure 8 shows the effect of pH on WMSP content. The lowest WMSP content occurred at pH 9.0, indicating the isoelectric point of WMSP was around pH 9.0 due to the fact that protein as an amphoteric compound has the least solubility at the isoelectric point (Zhao et al. ). Hence, WMSP was positively charged by adsorbing H þ on its amino-groups in the pH range of -9 set for the experiment.     where D and W denote DB-19 and WMSP, respectively.
The decrease of residual DB-19 content with descending pH shown in Figure 3 can be explained by the mechanism mentioned above. The adsorption and charge neutralization between WMSP and DB-19 was weakened, resulting in higher residual DB-19 content since WMSP molecular was negatively charged at pH 10 higher than its isoelectric point of pH 9. However, the reaction was reinforced by the increase of positive charge of WMSP with decreasing pH below its isoelectric point to create lower DB-19 residual.

CONCLUSIONS
This study describes an attempt of removal of the anionic dye DB-19 from water using proteins extracted from white mustard seed as a natural coagulant. The results indicate that the coagulation efficiency of WMSP was better than PAC at the same dosage for all other experiments except at pH 4 and 5. The main mechanism of the removal of DB-19 by WMSP could be adsorption and charge neutralization.
To our knowledge, it is the first report on removal of an anionic dye using WMSP as a natural coagulant, and the results showed that WMSP had excellent coagulation ability to remove DB-19.