Removal efficiency of MIEX® pretreatment on typical proteins and amino acids derived from Microcystis aeruginosa

Abstract

Dissolved organic nitrogen (DON) is now considered as one of the most important precursors of nitrogenous disinfection byproducts (N-DBPs), and algal cells are the main source of DON in eutrophic water sources. In this study, typical forms of DON in Microcystis aeruginosa cells were analyzed and the removal efficiency of magnetic ion exchange resin (MIEX®) treatment on typical forms of DON and the factors influencing it were also investigated. About 185 proteins and 13 amino acids were found in the intracellular organic matter (IOM) of M. aeruginosa cells. The isoelectric points (PIs) of these proteins were mostly acidic, and phycocyanin was the principal protein with a concentration of about 0.673 mg L⁻¹. In addition, high levels of aspartic acid, histidine, tryptophan, and asparagine were also noted, which presented higher potential to produce N-DBPs during the chlorination process. The removal efficiencies of MIEX® on various amino acids and phycocyanin were quite different primarily because of their different PIs and molecular properties. The pH was noted to be the main factor affecting the removal efficiency, and common inorganic anions also had obvious influence on the removal of amino acids and phycocyanin the following order: SO₄²⁻ > Cl⁻ > HCO₃⁻. The proteins in the IOM were effectively removed by MIEX® and only 50 proteins were found in the spectrum of two-dimensional electrophoresis. Thus, coagulation could be significantly enhanced by MIEX® pretreatment, which is one of the good alternatives to control typical DON in IOM.

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1. Introduction

Algal bloom is one of the problems faced by waterworks, especially those that use eutrophic water as the water source. The rapid growth of algal cells not only seriously affects the normal operation of the drinking water treatment process, but also deteriorates the quality of water.¹ With the detection of nitrogenous disinfection byproducts (N-DBPs), dissolved organic nitrogen (DON) has currently become a hot spot in the field of water treatment.^2,3 Algal organic matter (AOM) is an important source of DON in water. As DON includes a large spectrum of natural compounds such as free and hydrolysable amino acids, chlorophyll, and amino sugars, as well as synthetic compounds such as pesticides (e.g. atrazine),⁴ determination of the types and content of DON in raw water is important. Microcystis aeruginosa is one of the most common algae associated with algae blooms and has been widely studied in recent years.^5,6 In a previous study, Pivokonsky reported a noticeable increase in the concentration of proteins during the cultivation of M. aeruginosa, with the proportion of proteins in the intracellular organic matter (IOM) amounting up to 29.1% in the stationary phase of growth.⁷ Furthermore, the main categories of proteins in the IOM of M. aeruginosa cells were investigated in our earlier study.⁸ However, to the best of our knowledge, the other compositions have not yet been examined.

One of the options to control the level of N-DBPs in the effluent is to remove DON, the main precursor of N-DBPs, before chlorination. However, the DON removal efficiency of conventional drinking water treatment processes commonly used at present is limited (generally <20–30%),⁹ and biological pretreatment and biological activated carbon treatment process are likely to lead to an increase in the level of DON in water.¹⁰ Hence, there is a need to develop new methods for better removal of DON.

Magnetic ion exchange resin (MIEX®) was developed by Orica Watercare and South Australia Water Corporation, and has been found to exhibit good removal efficiency on natural organic matter (NOM), significantly enhance coagulation, and reduce the amount of DBPs when employed as a pretreatment process.^11,12 When compared with conventional anion exchange resin, MIEX® presents two particular features. First, MIEX® is a strong base anion resin with chloride as the exchangeable ion.¹³ When compared with conventional resins, the average size of the MIEX® resin particles is 2–5 times smaller, and hence, its unit volume has a much larger surface area, leading to rapid ion exchange kinetic.¹⁴ Second, the MIEX® has magnetized iron oxide incorporated into the polymer matrix. The magnetic component aids in agglomeration and produces high settlement rate.¹⁵ A new magnetic ion exchange resin had been developed based on the MIEX®, which exhibited better removal of humic acids and oxytetracycline.^16,17 However, only few studies have been conducted on DON removal by using ion exchange resins. The study of Aryal reported the performance of MIEX® treatment and biological activated carbon to remove DON from the secondary wastewater effluent (SWWE).¹⁸ No study on the removal of specific types of DON in algae-containing water sources by MIEX® was found. The categories of N-containing organics varied with the water source and exhibited diverse properties, which affected the removal performance significantly. Moreover, it should be noted that only MIEX® is currently manufactured in large scale.

Therefore, in the present study, the typical categories and characteristics of DON in M. aeruginosa cells were first analyzed, and the removal efficiency of MIEX® on the typical DON was examined. In addition, the factors influencing MIEX® treatment and the effects of MIEX® pretreatment on coagulation were also discussed.

2. Materials and methods

2.1 Materials

All the chemicals used in this study were of guaranteed or higher reagent grade and were purchased from Sinopharm Chemical Reagent Co. (Shanghai, China). Unless otherwise stated, all the chemicals were used directly without further purification.

M. aeruginosa, which is a common algal species in eutrophic surface water, was purchased from the Institute of Hydrobiology, Chinese Academy of Sciences, China. Axenic cultures were prepared in batch mode in 1 L conical flasks with BG11 medium. The conical flasks were placed in an incubator and the algal cells were cultured at 25 °C with illumination of 5000 L× provided for 14 h every day. The live algal suspensions were harvested between 15 and 28 days and diluted with ultrapure water to prepare the IOM samples. The method for the preparation of IOM samples has been described in our previous study.¹⁹

The standard stock solutions of amino acids and proteins were prepared by dissolving an aliquot of amino acids or proteins in 1 L of Milli-Q water to a concentration of 1000 mg L⁻¹. The solutions for the experiments were prepared from the stock solution to the desired concentration by successive dilutions.

The MIEX® was obtained from the Chinese agent of Orica Watercare (Victoria, Australia). Before use, the resins were repeatedly washed to remove impurities and stored in Milli-Q water. The MIEX® was used according to the method reported by Ding et al.²⁰

2.2 Experimental methods

MIEX® pretreatment: certain dosage of MIEX® was added to 1000 ml of the sample in a circular jar and agitated using a shaking apparatus at 150 rpm for a specific period of time. Subsequently, the solid fraction was removed by filtration through a 0.45 μm cellulose filter membrane, and DON and protein concentrations were determined.

2.3 Analytical methods

DON was determined as the difference between the total dissolved nitrogen (TDN) and sum of the total inorganic nitrogen (TIN) as follows (eqn (1)).

DON (mg L⁻¹) = TN − (NH₃ − N + NO₂ − N + NO₃ − N) (1)

Three-dimensional excitation-emission-matrix (EEM) fluorescence spectrophotometer (Hitachi F-4500 fluorescence spectrometer, Japan) was used to ascertain the composition of IOM based on the procedures developed by Chen et al.²¹ In addition, the composition of IOM was also determined by two-dimensional electrophoresis (2-DE) using a commercially available electrophoresis unit (GE Ettan DALT II system, GE, USA) according to published procedures.^22,23 Although Coomassie Brilliant Blue method is commonly used to determine the concentration of soluble proteins, it is not adequate for comprehensive investigation of the evolution of proteins. Based on 2-DE, tandem mass tags (TMT) was used to identify the categories of the main proteins. For the analysis of amino acids, precolumn derivatization by O-phthalaldehyde (OPA) was performed using HPLC (1260-LC, Agilent, USA) with a ZO RBA X X DB-C18 column (3.0 mm × 250 mm × 5 μm) and a fluorescence detector (λ_ex/λ_em = 337/454 nm).²⁴

3. Results and discussion

3.1 Identification of the typical DON in the IOM of M. aeruginosa

Algae are the main source of DON in natural waters, and proteins and amino acids are the principal components of DON from algae. As M. aeruginosa is one of the most common algae found during blue algae outbreaks,^5,6 in the present study, we analyzed the types and contents of proteins and amino acids in M. aeruginosa.

3.1.1 Proteins in the IOM of M. aeruginosa. The distribution of proteins in the IOM of M. aeruginosa determined using 2-DE is shown in Fig. 1.

Fig. 1 The spectrum of 2-DE of IOM (c_DON = 1 mg L⁻¹) (a) before and (b) after MIEX® treatment.

About 185 protein spots were found in the spectra. The molecular weight (MW) of the principal proteins was 30–80 kDa and most of the proteins were located in the acidic end, indicating that their isoelectric points (PIs) were mostly acidic.⁸ The results of the TMT analysis revealed similar types of proteins in the same IOM sample. The concentrations of 15 proteins were found to be higher than 0.066 mg L⁻¹.⁸ In particular, phycocyanin, including apo-α-phycocyanin and apo-β-phycocyanin, exhibited a concentration of 0.673 mg L⁻¹. Although the total concentration of the dissolved proteins was about 7.22 mg L⁻¹ based on the Coomassie Brilliant Blue method, phycocyanin was found to be the main protein in the IOM and may influence the coagulation performance because the proteins in IOM have been confirmed to be the primaryinhibitorsofcoagulation.²⁵ In addition, phycocyanin also occurs in cyanobacteria, red algae, and some dinoflagellates,²⁶ and therefore, was chosen as the typical protein in the algal cells.

3.1.2 Amino acids in the IOM samples. The amino acid concentrations determined in the IOM samples are shown in Table 1, along with some results of previous studies for comparison.^27,28

Table 1 Concentration of amino acids in the IOM of M. aeruginosa

Categories of amino acids	IOM^27 (μg N mg^−1 DOC)	Dianchi^28 (g per 100 g cells)	This study (μg N mg^−1 DOC)	Categories of amino acids	IOM^27 (μg N mg^−1 DOC)	Dianchi^28 (g per 100 g cells)	This study (μg N mg^−1 DOC)
Histidine (His)	1.5294	0.42	1.4976	Tyrosine (Tyr)	1.4117	1.47	1.3799
Lysine (Lys)	2.8235	1.36	2.6833	Proline (Pro)	0.0500	1.14	—
Arginine (Arg)	3.4118	2.09	3.4015	Methionine (Met)	0.1176	0.33	—
Valine (Val)	0.5882	2.22	0.5697	Cystine (Cys)	1.4118	0.31	1.4211
Glutamine (Gln)	1.1765	—	—	Phenylalanine (Phe)	1.7647	1.52	1.7749
Glutamate (Glu)	—	5.40	1.1880	Isoleucine (Ile)	0.8235	1.94	0.8196
Serine (Ser)	1.4118	1.79	1.4221	Leucine (Leu)	0.3529	3.09	0.3518
Glycine (Gly)	2.4706	1.64	2.4679	Threonine (Thr)	—	1.65	—
Alanine (Ala)	1.4117	3.28	1.4209	Aspartic acid (Asp)	—	3.12	—

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About 13 amino acids, covering the main categories of basic amino acids, were detected in the IOM samples. With regard to the concentrations of the amino acids, significant differences were observed among the categories. The maximum and minimum concentrations of the amino acids were 3.4015 and 0.3518 μg N mg⁻¹ DOC, respectively. Although these findings are similar to those reported by Fang et al.,²⁷ they contradict the results obtained in a study on algae cells in Dianchi Lake,²⁸ which may be owing to the categories of algae and unit of concentration used. Although the dominant algae in the eutrophic water was primarily M. aeruginosa (accounting for 95% of the total algae), the difference in the growth period and presence of other algae in the sample may have caused the variation in the amino acid categories and concentrations. Fang et al.²⁷ reported that the concentrations of the measured free amino acids and aliphatic amines only accounted for 11.3% of the TON in IOM. Together with their characteristics and concentrations, it can be concluded that the interference by the amino acids in IOM on coagulation and other unit process is insignificant. However, the results of previous studies on DBPs' precursors showed that amino acids were one of the main precursors of DBPs and had significant effect on the chlorination process. The R-group of amino acids has been noted to be a key factor in generating DBPs. For instance, tryptophan, tyrosine, phenylalanine, and histidine have great potential to produce trihalomethanes; aspartic acid, histidine, tryptophan, and asparagine have significant potential to produce haloacetic acids (HAAs); asparagine, glutamate, phenylalanine, and tyrosine can easily produce haloacetonitrile; and asparagines, histidine, threonine, and tryptophan can easily produce chloropicrin, 1,1-dichloro-2-propanone, and 1,1,1-trichloro-2-propanone.^29–31 In addition, proteins and other N-containing organics have the tendency to transform into amino acids during the water treatment process through hydrolysis or oxidation.

As proteins and amino acids are the important constituents of DON in water with excess algal cells and pose great threat to water quality and normal operation of waterworks, their effective removal is necessary. Based on the concentration of the amino acid categories as well as the impact on water quality, phycocyanin and four amino acids (histidine, glutamate, phenylalanine, and asparagine) were selected as the target compounds in the present study.

3.2 Removal efficiency of MIEX® pretreatment on proteins and amino acids

3.2.1 Removal efficiency of MIEX® pretreatment. Fig. 2 shows the removal efficiency of MIEX® pretreatment on phycocyanin and the four amino acids at different reaction periods. As shown in Fig. 2a, the removal of phycocyanin was rapid, reaching semi-equilibrium in less than 20 min, similar to that noted in previous studies.^11,32 As the particle size of MIEX® was about 200 μm and little pores existed in the particle, the exchange groups were mainly distributed on the surface of the resin. As a result, the exchange reaction promptly occurred without the limitation of pore-diffusion process. With regard to the two indices (phycocyanin and DON) used to express the concentration of phycocyanin, the removal rate of DON was lower than that of phycocyanin, because the purity of phycocyanin used in the present study was relatively low and contained other N-containing organics. This difference between the removal rates of phycocyanin and DON indicated that the phycocyanin or proteins in the sample were readily removed, when compared with other N-containing organics. Moreover, the PIs of most of the proteins were below 7 and presented negative charge, which facilitated the exchange reaction with MIEX® resin.

Fig. 2 Removal rates of phycocyanin (a) and the four amino acids (b) by MIEX® treatment (MIEX® dosage = 10 ml L⁻¹).

The removal efficiency of MIEX® treatment on the four amino acids presented striking difference. Glutamate was effectively removed, with about 70% glutamate being removed in the first 10 min. The glutamate removal rate was higher than phycocyanin removal rate owing to the lower MW of glutamate (147 vs. ∼30 000 kDa). In contrast, the removal rates of histidine, phenylalanine, and asparagine under the same condition were poor owing to the differences in their PIs. The PIs of glutamic acid, histidine, phenylalanine, and asparagines were 3.22, 7.59, 5.48, and 5.41, respectively. As the pH of the water used in the experiment was 6.5, histidine was positively charged and the other three amino acids (glutamic acids, phenylalanine, and asparagine) were negatively charged. Therefore, the exchange between histidine and the –Cl group of the MIEX® resin failed to occur and the three amino acids could only be removed with discrepant effects based on their different molecular properties. The classification and charge of the amino acids based on the difference between their R-group are shown in Table 2.

Table 2 Classification and charge of the amino acids³²

Nonpolar amino acid (hydrophobic amino acid)			Polar amino acid (hydrophilic amino acid)
			Uncharged amino acids		Positively charged amino acids (basic amino acid)	Negatively charged amino acids (acidic amino acid)
Alanine (Ala)	Isoleucine (Ile)	Tryptophan (Trp)	Tyrosine (Tyr)	Cysteine (Cys)	Lysine (Lys)	Asparagine (Asp)
Valine (Val)	Proline (Pro)	Methionine (Met)	Serine (Ser)	Asparagine (Asn)	Arginine (Arg)	Glutamate (Glu)
Leucine (Leu)	Phenylalanine (Phe)	Glycine (Gly)	Threonine (Thr)	Glutamine (Gln)	Histidine (His)

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Phenylalanine belongs to hydrophobic amino acids, with side chain containing highly hydrophobic benzene ring, whereas the other three amino acids are hydrophilic with different structures. Glutamate is an acidic amino acid with side chain containing a carboxyl group; histidine is a basic amino acid with side chain containing an amino group; and asparagine is a neutral amino acid without any amino and carboxyl group side chain, except for the R-group. This difference caused variation in the removal efficiency of MIEX® treatment on various categories of amino acids.

3.2.2 Main factors influencing the removal efficiency of MIEX® treatment. There are many factors that may affect the removal efficiency of MIEX® treatment on proteins and amino acids, and several key factors, such as MIEX® dosage, pH, and inorganic anions were analyzed in this study.
3.2.2.1 MIEX® dosage. Fig. 3 shows the effects of MIEX® dosage on the removal efficiency of MIEX® treatment on phycocyanin and the four amino acids.

Fig. 3 Effect of MIEX® dosage on the removal rates (reaction time = 30 min).

Similar to the results obtained in previous studies on NOM and AOM,^11,18 the removal rates of phycocyanin and glutamate increased with the increase in MIEX® dosage. Following certain initial concentration, the high resin volume provided more exchange points resulting in the removal of more compounds accordingly. However, an increase in the MIEX® dosage showed little effects on the removal of asparagine and phenylalanine, and no effect on the removal of histidine owing the differences in their structures as mentioned earlier.

3.2.2.2 pH. The pH of raw water with high content of algal cells is substantially neutral or alkaline; for example, the pH of Taihu Lake varies from 7.5 to 8.5 with the quantity of algal cells.¹⁹ As the PIs of different amino acids and proteins vary, their charges in the actual raw water also differ. Fig. 4 shows the effect of pH on the removal of phycocyanin and the four amino acids by MIEX® treatment.

Fig. 4 Effect of pH on the removal rates of phycocyanin and the four amino acids by MIEX® treatment (MIEX® dosage = 10 ml L⁻¹, reaction time = 30 min).

When the pH increased from 7 to 11, the removal rates of phycocyanin and the four amino acids evidently improved. As the PIs of these amino acids were close to the pH of raw water, they were not negatively charged or their electronegativity was low. However, with the increasing pH, the amino acids may become negatively charged or their electronegativity would enhance, with the ion exchange reactions occurring more easily. Thus, it can be concluded that the pH of the solution is an important factor influencing the removal of amino acids and proteins. Nevertheless, the alkaline agent needed for the purpose of increasing the pH should be considered from the point of operation cost.

3.2.2.3 Inorganic anions. Raw water is a complex of organics and ions, containing not only various organic matter, but also inorganic anions such as SO₄²⁻, Cl⁻, and HCO₃⁻. These anions compete with amino acids and proteins and affect their removal from water. Fig. 5(a) and (b) shows the effects of inorganic anions on the removal efficiency of MIEX® treatment on phycocyanin and glutamate.

Fig. 5 Effect of inorganic anions on the removal rates of phycocyanin (a) and glutamate (b) (MIEX® dosage = 10 ml L⁻¹, reaction time = 30 min).

It can be observed from the figure that SO₄²⁻ had a strong influence on the removal of glutamate and phycocyanin by MIEX®, and its influence increased with its increasing concentration. In contrast, although Cl⁻ and HCO₃⁻ also had negative effects on the removal of glutamate and phycocyanin by MIEX®, the effects were significantly weaker than those exerted by SO₄²⁻. The presence of Cl⁻ reversed the exchange equilibrium of the ions, ultimately affecting the removal of glutamate and phycocyanin, whereas HCO₃⁻ competed with glutamate and phycocyanin, making the water alkaline and enhancing the negative charge of glutamate and phycocyanin, exerting the weakest effect. Thus, at the same concentration, the major anions decreased the removal rates of amino acids and phycocyanin in the following order: SO₄²⁻ > Cl⁻ > HCO₃⁻.

4. Discussion

MIEX® pretreatment showed excellent removal efficiency on phycocyanin and the four amino acids in water, and its performance with respect to removal of DON, proteins, and typical amino acids in IOM was studied, and the results are shown in Fig. 6.

Fig. 6 Removal of DON in IOM by MIEX® pretreatment (DON = 1 mg L⁻¹; MIEX dosage = 15 ml L⁻¹).

As illustrated in the figure, MIEX® pretreatment effectively removed proteins, and the removal rate was about 55% for a dosage of 15 ml L⁻¹. Most of the proteins in the IOM were significantly negatively charged and easily reacted with the exchange group of MIEX®, because they were mostly acidic with PIs lower than 7 (Fig. 1a). As shown in Fig. 1b, numerous protein points disappeared after MIEX® pretreatment, with only about 60 points detected mostly at the low MW positions, indicating that a large proportion of the proteins in the IOM was removed during MIEX® treatment. Similar results were also obtained in fluorescence EEM, as shown in Fig. 7.

Fig. 7 Fluorescence EEMs of the IOM samples before (a) and after (b) MIEX® pretreatment (MIEX® dosage = 15 ml L⁻¹).

Based on previous studies,^21,33 the position of E_x–E_m of region I (E_x/E_m: 220–270/280–330) and region II (E_x/E_m: 220–270/330–380) corresponded to aromatic proteins such as tyrosine and tryptophan-related compounds, respectively. Region III (E_x/E_m: 220–270/380–550), region IV (E_x/E_m: 270–440/280–380), and region V (E_x/E_m: 270–440/380–550) corresponded to fulvic-acid-like compounds, soluble microbial products (SMPs), and humic-acid-like compounds, respectively. On comparing Fig. 7a and b, it can be noted that the positions of the fluorescence peaks have not changed, indicating that the entire course of reaction did not produce other types of organic matter; however, a decrease in the fluorescence intensity suggested that the fluorescence groups were removed by MIEX® and the extent of decrease in the fluorescent intensity demonstrated the removal effects on the proteins or other substances.

With regard to the amino acids, the removal effects obviously differed among the different categories. Some amino acids, such as glutamate, could be effectively removed owing to its negatively charged and suitable molecular structure. However, other amino acids could not be removed successfully owing to their higher PIs and molecular properties.

It should be noted that the removal rate of DON was about 60%, similar to the results in Aryal's study¹⁸ despite of the distinct water quality (IOM in the algae cells vs. SWWE). However, the removal rate was lower than that of proteins and particular amino acids. The lower removal rate of DON may be owing to the numerous categories of DON in IOM, with some DON (such as the residues in Fig. 1b and 7b) that could not be removed or for which the removal effects were poor. Besides, as the initial concentrations of phycocyanin, glutamate, and DON in the IOM were different, their removal rate may also significantly vary. The great difference among the removal performance of the various DON compositions indicated the necessary of study to the specific nitrogen-containing organics. The overall DON removal results could not reflect the control performance of the N-DBPs for the great diversity on the contribution of the DON compositions to the N-DBPs. The poor removal of MIEX® treatment to the histidine, phenylalanine, and asparagine posed a potential threat to the safety of drinking water. In future, other methods for the removal of DON in the IOM should be explored. Furthermore, as the eutrophic raw water was weakly alkaline with pH higher than 8, most of the proteins and amino acids were negatively charged, which favored the removal process of MIEX® pretreatment.

5. Conclusion

1. Proteins and amino acids are the important components of DON in water containing excess algal cells, and phycocyanin is the most representative protein, which is one of the sources of amino acids in water. The amino acids have great potential to generate N-DBPs.

2. The removal rates of different kinds of amino acids and proteins are quite diverse, mainly because their PIs and charges vary at the same pH. Therefore, pH is the main factor affecting the removal rates, and when pH increased from 7 to 11, the removal rate significantly improved.

3. Common inorganic anions in water containing excess algal cells have significant influence on the removal of amino acids and phycocyanin. At the same concentration, the major anions decreased the removal rates of amino acids and phycocyanin in the following order: SO₄²⁻ > Cl⁻ > HCO₃⁻.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (51378174, 51438006), the Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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