Extraction of extracellular polymeric substances (EPS) from a newly isolated self-flocculating microalga Neocystis mucosa SX with different methods

Algal Research 40 (2019) 101479

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Extraction of extracellular polymeric substances (EPS) from a newly isolated self-flocculating microalga Neocystis mucosa SX with different methods ⁎

Junping Lv , Fei Zhao, Jia Feng, Qi Liu, Fangru Nan, Shulian Xie

T

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School of Life Science, Shanxi University, Taiyuan, 030006, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Microalgae Extracellular polymeric substances Cation exchange resin Heating treatment Neocystis mucosa SX

Extracellular polymeric substances (EPS) played an important role in flocculation of microalgae. Nevertheless, no universal method was reported for EPS extraction from microalgae. In the study, a microalga with high selfflocculating efficiency was isolated and identified as Neocystis mucosa SX. Then the efficiency of extracting EPS from the microalga using cation exchange resin (CER), heating, EDTA, acidic treatment and alkaline treatment were compared. Both CER and heating treatment extracted more EPS with rich chemical groups than other three methods. Moreover, it was favorable for EPS extraction by prolonging treatment time for CER method and raising temperature for heating treatment. Nevertheless, long treatment time for CER method and high temperature for heating treatment resulted in severe cell lysis through the analysis of DNA content, dehydrogenase activity (DHA) and the cell membrane integrity. Based on both EPS extraction efficiency and cell viability, the CER extraction for 6 h could be applied for the actual study.

1. Introduction Extracellular polymeric substances (EPS), which are widely studied in microorganisms and activated sludge, are metabolic products accumulating outside of cells and in the interior of microbial aggregates [1]. EPS are composed of various organic substances. Carbohydrates and proteins are usually found to be the two main components of EPS [2,3]. Microalgae are a group of plants widely existed in nature. They can absorb nutrients (mainly nitrogen, phosphorus and organic matter) from wastewater and assimilate them into biomass as raw materials for production of feed, biodiesel and so on [4]. Therefore, a large amount of researches on microalgae-based wastewater treatment have been widely carried out in recent years [5]. It is interesting that some microalgae, such as Chlorella vulgaris, Chlorococcum sp. GD, Ettlia texensis and Scenedesmus obliquus, have excellent self-flocculating ability, which enables them to separate effectively from wastewater without extra equipment or addition of costly flocculants to cells [6–9]. Along with the deepening of research, some results show that the excellent flocculating ability of self-flocculating microalgae is closely related to the secretion of EPS [8,9]. Accordingly, the role of EPS in microalgal harvesting from wastewater has become an important concern. To qualitatively and quantitatively analyze characteristics of EPS and elucidate the relationship between EPS and flocculating, it is very important to extract EPS from microalgae cells. An ideal EPS extraction

⁎

method should have high EPS extraction efficiency. At the same time, the method should avoid damage to cells as much as possible to prevent the influence of leakage of intracellular substances on the property and content of EPS. At present, extraction methods commonly used in the EPS study of activated sludge and bacterial pure culture have been gradually used for the extraction of microalgae EPS. For example, Takahashi et al. [10] extracted EPS from a marine benthic diatom Navicula jeffreyi with six methods of extraction with distilled water, cation exchange resin (CER), artificial seawater of half salinity and extractions after pretreatment with gluteraldehyde by three methods: water, CER water and CER buffer. The study establishes the best method with high yields of EPS and minimum cell lysis. Nevertheless, the study is aimed at diatoms, which have distinct cellular structures with green microalgae. In view of the superiority of the use of green microalgae in wastewater treatment [11], it is not clear whether these methods are applicable to extract EPS of green microalgae. Of course, some methods, such as high speed centrifugation, physical agitation, sonication, base treatment, heating, etc., are also used to extract EPS of green microalgae [8,9,12,13]. However, there is no relevant assessment of whether cells are lysed in these studies. Moreover, some methods, such as EDTA method and acidic treatment method, have been widely used for EPS extraction of activated sludge [1]. Nonetheless, the feasibility of these methods for EPS extraction of green microalgae has not been evaluated so far. Therefore, in order to objectively and truly

Corresponding authors. E-mail addresses: [email protected] (J. Lv), [email protected] (S. Xie).

https://doi.org/10.1016/j.algal.2019.101479 Received 13 November 2018; Received in revised form 7 March 2019; Accepted 24 March 2019 2211-9264/ © 2019 Elsevier B.V. All rights reserved.

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reflect the characteristics of EPS, it is important to find some methods with high extraction efficiency of EPS and small damage to green microalgae cells. In the present study, a green microalga was isolated from soil of Shanxi Province, China, and exhibits excellent self-flocculating property. To extract EPS from cells for further studies on the flocculating mechanism of microalgae, five EPS extraction methods of EDTA, CER, heating, acidic treatment and base treatment are evaluated in both EPS extraction efficiency and cell viability.

Then the mixture was stirred at 300 rpm for 3, 6, and 9 h, respectively, at 25 °C [17]. For EDTA method, 2% (v/v) EDTA was added to the pellet. After that, mix the suspension and let it stand at 4 °C for 3 h [18]. For heating method, the suspension was heated to 45 °C for 1 h and 60 °C for 0.5 h, respectively [9]. For acidic treatment method, 1 mol/L H2SO4 was added to the suspension to reach a pH of 2.5. The suspension was then stirred for 3 h [19]. For alkaline treatment, the suspension was supplemented with 1 mol/L NaOH to reach a pH of 12.5 and stirred for 3 h [20]. For all above extraction methods, the treated samples were centrifuged at 10000 rpm for 10 min. The supernatant was then filtered with 0.45 μm acetate cellulose membranes. Finally, the extractant reagent in the EPS solution was removed by membrane dialysis (3500 Da) for 24 h at 4 °C [18]. All experiments were repeated four times in the study.

2. Material and methods 2.1. Microalgal strain and cultivation conditions The microalga was isolated from soil of Pangquangou National Nature Reserve (37°54′N, 111°23′E), Shanxi Province, China via BG11 medium plates [14]. Then the microalga was cultivated in BG11 liquid medium for identification of strain, determination of flocculation capacity and extraction of EPS. The strain was deposited in Freshwater Algae Culture Collection at the Institute of Hydrobiology (FACHB-collection) with a number of FACHB-2328. To identify the microalga, the genomic DNA of microalgal cells was extracted by a commercial plant DNA extraction kit (Sangon Biotech, Shanghai, China). For polymerase chain reaction (PCR) amplification of the internal transcribed spacer region (ITS), primers A and primers B were used [15]. PCR amplifications were performed in accordance to the method of Feng et al. [16]. Then the phylogenetic tree was constructed from the aligned gene sequences using neighbor-joining (NJ) method as described by Feng et al. [16]. The nucleotide sequence of ITS rDNA region from the isolated microalga was deposited in the nucleotide sequence database (GenBank). Other nucleotide sequences of ITS rDNA region used in this paper were also deposited in GenBank. The details on accession number were listed in Table S1.

2.4. Quantitative and qualitative analysis of EPS 2.4.1. The content of proteins and carbohydrates The content of proteins was measured by the Bradford assay method [21] using BSA as the standard. The carbohydrates content was measured by the Anthrone method [22] with glucose as the standard.

2.4.2. Fourier transform infrared (FTIR) spectroscopy EPS extracts were placed in a refrigerator for 24 h at −80 °C. The frozen EPS were completely dried to a powder via a vacuum freeze drier (SCIENTZ-18 N, Ningbo Scientz Biotechnology Co., Ltd., China). After that, EPS samples were mixed with KBr a ratio of 1:100 and then homogenized in an agate grinder. The mixture was compressed and analyzed on a Nicolet iS50 FTIR spectrometer (Thermo Fisher Scientific, Inc.) in a spectral range of 4000–400 cm −1.

2.4.3. Excitation-emission matrix (EEM) fluorescence spectroscopy EEM spectra of EPS extracts measured with F-280 fluorescence spectrophotometer (Gangdong, China) were performed in the protocol described by Lv et al. [9]. Spectra were collected with subsequent scanning of emission spectra from 250 to 550 nm at 1 nm increments by varying the excitation wavelength from 200 to 450 nm at 10 nm increments. Excitation and emission slits were maintained at 5 nm and the scanning speed was set at 1200 nm/min. The voltage of the photomultiplier tube (PMT) was set to 700 V. The software Origin 8.0 was employed for handling EEM data.

2.2. Flocculating ability test The flocculating ability of microalgae was analyzed according to Lv et al. [9] with some modifications. After 10 d of cultivation in BG11 medium, 25 mL mixture of the microalga was distributed in 25 mL cylindrical glass tubes, and followed by gently mixing for 1 min at room temperature. An aliquot of the mixture was withdrawn at a height of two-thirds from glass tubes when the mixture was settled for 30, 60, 120 and 180 min, respectively. After that, the optical density (OD) of above aliquots was measured at 694 nm. The flocculating ability was calculated according to the equation as following:

2.4.4. High performance liquid chromatography (HPLC) The monomer composition of the carbohydrates from EPS was analyzed using HPLC. In brief, EPS solution with addition of ethanol was placed overnight at 4 °C. The precipitate was collected by centrifugation at 10000 g for 10 min and lyophilized as the carbohydrates extract. Carbohydrates extract was subsequently hydrolyzed with 2 mol/L trifluoroacetic acid (TFA) at 120 °C for 2 h in a sealed tube. After that, the excess TFA was removed by codistillation with methanol. Then the carbohydrates hydrolysate was derivatized. The derivatized carbohydrates hydrolysate adjusted pH to 7.0 with 0.3 mol/L HCl was extracted with chloroform three times and the aqueous phase was filtered through a 0.45 μm microporous filters. 20 μL of the resulted solution was injected into Unitary C18 column (250 mm × 4.6 mm id, 5 μm, Acchrom, China). Mobile phase A was potassium dihydrogen phosphate buffer (50 mM, pH 6.7). Mobile phase B was 100% acetonitrile. The flow rate of mobile phase was 1.0 mL/min. The column temperature was 30 °C. The monomer of carbohydrates from EPS was identified by comparison with reference monosaccharides (glucose, mannose, galactose, xylose, arabinose, fucose, rhamnose, galacturonic acid and glucuronic acid).

Flocculating ability = (A − B)/A ∗ 100% where A and B were the optical density (OD694) of the aliquot before and after flocculation. 2.3. Extraction protocols of EPS EPS consist of soluble EPS and bound EPS [1]. The extraction method of soluble EPS was relatively fixed, that is, centrifugation, and the extraction method of combined EPS was diversified [1]. Therefore, the extract method of bound EPS was the focus of attention in this study. The detailed process was as follows. Microalgal suspension was centrifuged at 5000 rpm for 5 min after 10 d of cultivation. The pellet was then washed with deionized water and centrifuged at 5000 rpm for 5 min. After that, the washed pellet was resuspended to deionized water. In the present study, EDTA, CER, heating, acidic treatment and alkaline treatment were used to extract EPS from the microalga according to the procedure from former literatures with some modifications. For CER method, the CER (versatile strong acid, sodium type) was added to the suspension with a dose of 70 g/g dry microalgal biomass. 2

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2.5. Analysis on viability of microalgal cells 2.5.1. The content of deoxyribonucleic acid (DNA) in EPS The DNA content in EPS was measured by the diphenylamine colorimetric method [23] using 2-Dexoy-D-Ribose as the standard. 2.5.2. Determination of dehydrogenase activity (DHA) Before and after EPS extraction, the determination of DHA of microalgal cells was assessed using the commercial kit from Beijing Solarbio Technology Co., Ltd. (Beijing, China). 2.5.3. Flow cytometry analysis Microalgal cells before and after EPS extraction were suspended in fresh phosphate buffer sodium (PBS, 50 mM, pH 7.4). Then cells were stained by propidium iodide (PI, Sigma-Aldrich, USA) with a final concentration of 30 μmol/L. Cell membrane integrity was analyzed by flow cytometer (Accuri C6; BD Accuri Cytometers, Ann Arbor, MI, USA). Detailed procedures were according to Xiao et al. [24].

Fig. 2. The flocculating ability of N. mucosa SX with different settlement time. Error bars represent standard deviation for each group of data (n = 4). Significant differences (p < 0.05) between different treatments are represented by different letters.

2.6. Statistical analysis Neocystis genus. According to the phylogenetic analysis, it was reasonable to identify the microalga as Neocystis mucosa SX (Fig. 1).

All data were represented as the mean ± standard deviation (SD; n = 4). The differences among different treatments were analyzed by one-way ANOVA via SPSS software (version 19.0). p < 0.05 was considered for statistical significance.

3.2. The self-flocculating efficiency of N. mucosa SX

3. Results

An important feature of N. mucosa SX was that it had good selfflocculating ability. As depicted in Fig. 2, the flocculating efficiency of N. mucosa SX was 63.2% after 0.5 h of settling. Then it significantly increased to 68.4% and 79.7%, respectively, after 1 h and 2 h of settling (p < 0.05). When N. mucosa SX was settled for 3 h, the flocculating efficiency was up to 93.6% (p < 0.05).

3.1. Identification of microalgal strain One microalga was isolated from soil of Shanxi Province, China. To identify the taxonomic position of the microalga, the NJ tree based on ITS sequences was constructed from individually aligned datasets comprising sequences of the microalga and others. It was revealed that the microalga exhibited a close phylogenic relationship with the

Fig. 1. The phylogenetic tree of the isolated microalga based on NJ analysis of the ITS gene. 3

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Fig. 3. The proteins and carbohydrates content of EPS extracted from N. mucosa SX by different methods. Error bars represent standard deviation for each group of data (n = 4). Significant differences (p < 0.05) between different treatments are represented by different letters.

extract more substances with different chemical properties, compared to other methods. In the present study, FTIR analysis was also explored to unravel the chemical structure of EPS extracted from N. mucosa SX with different methods. The details were shown in Table 1. Six peaks were found in FTIR spectra. The position and number of FTIR peaks for EPS extracted from different methods were different. For CER 9 h, EDTA and heating at 60 °C for 0.5 h, five characteristic bands could be noticed: a peak at 3303–3407 cm−1 related to stretching vibration of OH and NeH vibrations, a peak at 1623–1652 cm−1 related to stretching vibration of C]O and CeN, a peak at 1350–1397 cm−1 related to symmetric stretching vibration of COO−, a peak at 1077–1090 cm−1 related to stretching vibration C-O-C and a peak at 751–990 cm−1 related to “Fingerprint” zone. With the CER extraction time shortened to 6 h and 3 h, only three and two peaks belonged to above five emerged. Similarly, the number of peaks decreased when EPS was extracted with heating at 45 °C for 1 h. For EPS extraction with acidic treatment, a peak at 2956 cm−1 related to asymmetric stretching vibration of CH2 and CH3 emerged, which didn't emerge in EPS extracted with other methods. For EPS extraction with alkaline treatment, only two peaks emerged. It indicated that CER, EDTA, acidic treatment and heating could extract more substances with different chemical characteristics, compared to alkaline treatment. For CER and heating method, increasing the contact time of cells with CER and increasing the temperature of heat treatment was beneficial to get more information. To summarize, EPS extraction methods had obvious effects on chemical characteristics of EPS from the microalga. Moreover, the monomer composition of the carbohydrates from EPS was also analyzed using HPLC. The carbohydrates extracted by CER, acidic, alkaline treatment and heating consisted of glucose, mannose, galactose and arabinose with a molecular ratio of 42:13:2:1. For EDTA method, carbohydrates consisted of glucose, mannose and xylose with a molecular ratio of 15:9:1.

3.3. Quantities and chemical characteristics of EPS extracted from N. mucosa SX with different methods 3.3.1. EPS quantities of N. mucosa SX extracted with different methods Fig. 3 described the amounts of EPS extracted from N. mucosa SX with different methods. EPS were mainly composed of carbohydrates and proteins. As shown in Fig. 3a, the content of proteins among different EPS extraction methods ranged from 0.78 to 23.5 mg/g dry weight of biomass. There were significant differences on the content of proteins among different EPS extraction methods (p < 0.05). The order of proteins content extracted from N. mucosa SX with different methods was CER 9 h > CER 6 h and alkaline treatment > heating at 60 °C for 0.5 h and heating at 45 °C for 1 h > CER 3 h, EDTA and acidic treatment. The content of carbohydrates among different EPS extraction methods ranged from 1.02 to 103 mg/g dry weight of biomass (Fig. 3b). Similarly, there were also significant differences on the content of carbohydrates among different EPS extraction methods (p < 0.05). The order of carbohydrates content extracted from N. mucosa SX with different methods was CER 9 h > CER 6 h and heating at 60 °C for 0.5 h > CER 3 h > EDTA, acidic treatment, alkaline treatment and heating at 45 °C for 1 h. It was clear that both CER and heating methods extracted more EPS than other methods. It was also found that it was beneficial to extract EPS via increasing the contact time of cells with CER and increasing the temperature of heat treatment.

3.3.2. Chemical characteristics of EPS extracted from N. mucosa SX with different methods In addition to the amounts of EPS, the chemical characteristics were also important features of EPS. In the present study, EEM spectroscopy was applied for characterizing EPS. Each EEM could give spectral information on the chemical compositions of EPS extracted from N. mucosa SX with different methods. As depicted in Fig. 4, there were some differences on the chemical compositions of EPS extracted with different methods. One or two peaks were found in each EEM spectrum. Peak B was located at Ex/Em of 290 to 310 nm/350 to 373 nm, and Peak C was located at Ex/Em of 340 to 360 nm/400 to 450 nm. For EPS extracted by heating at 60 °C for 0.5 h, heating at 45 °C for 1 h, CER 6 h and CER 9 h, both peak B and peak C were detected. For EPS extracted by alkaline treatment, only peak B was detected. Only peak C emerged in EPS extracted by acidic treatment and CER 3 h. No obvious peak was found in EPS extracted by EDTA. In addition, the EEM spectrum also reflected the fluorescence intensity of each peak. It was obvious that the order of total fluorescence intensity of EPS extracted from N. mucosa SX with different methods was CER 9 h > CER 6 h > heating at 60 °C for 0.5 h > alkaline treatment > heating at 45 °C for 1 h > CER 3 h and acidic treatment. All results above indicated that CER and heating could

3.4. Cells viability of N. mucosa SX 3.4.1. DNA content of EPS extracted from N. mucosa SX with different methods The DNA content of EPS extracted from N. mucosa SX with different methods was different (Fig. 5). The DNA content was highest when EPS of N. mucosa SX were extracted by CER for 9 h. When EPS of N. mucosa SX were extracted by heating at 60 °C for 0.5 h, high content of DNA was detected. Small amounts of DNA were released into EPS extracted by CER 3 h, CER 6 h, EDTA, acidic treatment, alkaline treatment and heating at 45 °C for 1 h, which was significant lower than that extracted by other methods (p < 0.05).

4

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Fig. 4. EEM fluorescence spectra of EPS extracted from N. mucosa SX by different methods.

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Table 1 Main functional groups and absorbance observed from IR-spectra of EPS extracted by different methods. Wave number (cm−1) in former literatures

Vibration type

3300–3500

Stretching vibration of OH and NeH vibrations Asymmetric stretching vibration of CH2 and CH3 Stretching vibration of C]O and CeN (amide I) symmetric stretching vibration of COO− Stretching vibration C–O–C “Fingerprint” zone

2900–3000

1637–1660

1360–1450 900–1200 < 1000

Functional type

OH into Polymeric compounds

Proteins (peptidic bond)

References

Wave number (cm−1) in this study

CER 3h

CER 6h

CER 9h

EDTA

acidic treatment

alkaline treatment

45 °C 1h

60 °C 0.5 h

[25]

33033407

−

+

+

+

+

+

+

+

[25]

2956

−

−

−

−

+

−

−

−

[26]

16231652

+

+

+

+

+

−

−

+

[27]

13501397 10771090 751990

−

−

+

+

+

+

+

+

−

−

+

+

−

−

+

+

+

+

+

+

−

+

+

[28] Polysaccharides Phosphate or sulphur functional groups

EPS extraction methods

[26]

+

+: the emergence of functional groups; −: the inexistence of functional groups.

Fig. 5. The DNA content of EPS extracted from N. mucosa SX by different methods. Error bars represent standard deviation for each group of data (n = 4). Significant differences (p < 0.05) between different treatments are represented by different letters.

Fig. 6. The DHA of N. mucosa SX after EPS extracted by different methods. Error bars represent standard deviation for each group of data (n = 4). Significant differences (p < 0.05) between different treatments are represented by different letters.

3.4.2. DHA of N. mucosa SX after EPS extraction with different methods The DHA of N. mucosa SX after EPS extraction with different methods was evaluated as shown in Fig. 6. The DHA of N. mucosa SX was significantly inhibited after EPS extraction with different methods (p < 0.05). Moreover, there were some differences on DHA of N. mucosa SX after EPS extraction with different methods. After EPS extraction by CER 9 h, the DHA of N. mucosa SX was the lowest, which was followed by EPS extraction by heating at 60 °C for 0.5 h. The DHA of N. mucosa SX after EPS extraction by CER 3 h, CER 6 h and EDTA was at the same level, which was significantly higher than that of N. mucosa SX after EPS extraction by acidic treatment, alkaline treatment and heating at 45 °C for 1 h.

respectively, when EPS was extracted by CER for 9 h, acidic treatment and heating at 60 °C for 0.5 h. The proportion of damaged cells ranged from 0.38% to 0.72% with EPS extraction via CER for 3 h, CER for 6 h, EDTA and heating at 45 °C for 1 h.

4. Discussion EPS were important to explain the mechanism on self-flocculation of microalgae [8,9]. Up to now, there was no universal method to extract EPS from microalgae. Methods on EPS extraction from sludge and microorganisms were always referred to extract EPS from microalgae [8–10]. However, there were still a lot of problems. How efficient were these EPS extraction methods used for the extraction of EPS from microalgae? Whether the EPS extraction method had damage to microalgal cells? These problems were not very clear in the extraction of microalgal EPS.

3.4.3. Cell membrane integrity of N. mucosa SX after EPS extraction with different methods Compared to the control, the cell membrane integrity of N. mucosa SX was significantly affected by the process of EPS extraction (Fig. 7). The cell membrane integrity differed after EPS extraction with different methods. The highest proportion of damaged N. mucosa SX cells was 10.74% when EPS was extracted by alkaline treatment. The proportion of damaged N. mucosa SX cells was 3.68%, 1.36% and 1.05%, 6

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other chemical extraction protocols. Our finding was consistent to this result. However, the EPS content extracted from sludge by the EDTA method was obviously not the lowest in other reports [18]. It was speculated that the differences in extraction efficiency might derive from differences in biological characteristics of samples. There were also small amounts of EPS (6.6 mg/g dry weight of biomass) extracted by acidic treatment. It was consistent to former reports showing that H2SO4 was not an efficient extractant for EPS from Rhodopseudomonas acidophila [33]. EPS content of N. mucosa SX extracted by alkaline treatment was low, compared to former literatures [18,34]. One possible reason was that only a small amount of NaOH was added to microalgal solution in the study. It was also found that more carbohydrates were extracted than proteins via alkaline treatment. The tendency was similar to the ratio of proteins to carbohydrates of sludges in former studies [18,34]. Based on the above analysis, we found that the extraction efficiency of EPS was related to extraction methods. Relative high content of EPS could be extracted by CER method. However, the absolute content of EPS was indeed low in this study, compared to cases described by Delattre et al. [3]. When the microalga was cultivated in BG11 supplemented with 4 g/L glucose, the EPS content extracted by CER for 6 h was 94.62 mg/g dry weight of microalgal biomass. It was 1.66 times higher than that obtained in BG11 only. It was clear that EPS content was related to culture parameters [3]. In the present study, the aim of EPS extraction was to further investigate the flocculating mechanism of microalgae. As described by former literatures [6,7], structure characteristics (such as hydroxyl and carboxyl groups) of EPS were closely related to flocculation. EEM and FTIR had been widely used to analyze characteristics related to flocculation in EPS [6,7,35]. Therefore, we analyzed EPS composition by EEM and FTIR to evaluate whether these EPS extraction methods could reflect the characteristics related to flocculation in EPS. According to EEM spectroscopy, both Peak B (Ex/Em: 290–310 nm/ 350–373 nm) and Peak C (Ex/Em: 340–360 nm/400–450 nm) were found in EEM spectra. Peak B was described as protein-like peak, which was associated with the aromatic amino acids and tryptophan [36]. Peaks C was attributed to the presence of humic acid-like substances [36,37]. The two peaks had been widely reported to be found in EPS from sludge [36,38]. The peak B was also found in EPS of Chlorococcum sp. GD and P. kessleri TY [9]. Peak C was firstly reported in EPS of microalgae. Tryptophan was a hydrophobic amino acid, which was beneficial to promote the formation of aerobic granule and the flocculation of microalgae [9,39]. Therefore, the presence of tryptophan protein-like substances was one of the reasons for the self-flocculation of N. mucosa SX as shown in Fig. 2. In this study, it was clearly found that the location and fluorescence intensity of peaks were affected by extraction methods. The phenomenon was consistent with former reports showing the differences on the number and intensity of peaks from EPS of sludge and biofilm extracted by different methods [40,41]. In the study, the total number and intensity of peaks were the highest for CER 6 h and 9 h. Moreover, the order of total fluorescence intensity extracted from N. mucosa SX with different methods was almost consistent with the order of proteins content of EPS extracted from N. mucosa SX with different methods. It meant that proteins content correlated with intensities of EEM peaks when EEM was used for quantitative analysis as proposed by Sheng and Yu [36]. EPS of N. mucosa SX extracted with different methods was analyzed by the FTIR spectrometer. As described by Badireddy et al. [42], the peak at 1640 cm−1 was related to the C]O stretching vibration of βsheets in secondary protein structures, which was beneficial to bioflocculation of activated sludge microorganisms [42]. The peak at approximately 3303–3407 cm−1 was mainly assigned to hydroxyl groups [25]. The region between 1350 and 1397 cm−1 corresponded to the symmetric stretching vibration COO– [27]. The characteristic band around 1077–1099 cm−1 was attributed to the stretch of C-O-C as the presence of carbohydrates [28]. The major functional groups such as

Fig. 7. The proportion of damaged cells of N. mucosa SX after EPS extracted by different methods. Error bars represent standard deviation for each group of data (n = 4). Significant differences (p < 0.05) between different treatments are represented by different letters.

4.1. Extraction method efficiency based on EPS content and chemical characteristics In the process of EPS extraction by CER, the resin could be readily removed from EPS solution and not affect subsequent analysis. More importantly, the method exhibited high efficiency [1]. Therefore, CER had been widely used to extract EPS from sludge and was regarded as one of the best candidates [1]. Unlike the wide application of CER for EPS extraction from sludge, the method was almost not used for the extraction of microalgal EPS except for a marine benthic diatom N. jeffreyi and a diatom-dominated intertidal mudflat [10,29]. Especially for EPS extraction from N. jeffreyi, authors recommended extraction with CER, compared to other methods [10]. Taking into account differences on microalgal cell structure between diatoms and green microalgae, it was essential to evaluate the EPS extraction efficiency of CER from green microalgae. In the present study, extraction with CER had high content of proteins and carbohydrates. Especially for N. mucosa SX treated by CER for 6 h and 9 h, the proteins content was 6.64 and 23.5 mg/g dry weight of biomass and the carbohydrates content was 50.4 and 103 mg/g dry weight of biomass. The yield of EPS of N. mucosa SX extracted by CER was comparable to that of sludge extracted with the same method [18]. It was obvious that the CER method was feasible for EPS extraction from green microalgae. Prolonging the contact time of cells and CER enabled CER to remove more divalent cations in EPS and thus caused EPS to fall apart and release [1]. The molecular movement was enhanced in the process of heating, which accelerated the release and dissolution of EPS [1]. Therefore, the heating extract method had been widely used to extract EPS from sludge [30,31]. Currently, the method had been also used to extract EPS from a diatom N. jeffreyi and two green microalgae Chlorococcum sp. and Parachlorella kessleri [9,10]. Compared to other EPS extraction methods, the heating extracted the highest amount of EPS from a diatom N. jeffreyi [10]. It was also extracted 82.04 mg/g dry weight biomass of carbohydrates from Chlorococcum sp. [9]. It indicated that the heating extract method could be used to extract EPS from microalgae. In this study, it was found that heating at 60 °C for 0.5 h was effective for EPS extraction (especially for carbohydrates), which was comparable to that of Chlorococcum sp. in former literatures [9]. Therefore, our results proved again that the heating extract method was effective for microalgal EPS extraction. In the present study, there were small amounts of EPS extracted by EDTA. D'Abzac et al. [32] reported that EDTA extracts yielded the lowest quantities of EPS from anaerobic granular sludge, compared to 7

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5. Conclusions

hydroxyl and carboxyl groups in the polysaccharide might serve as binding sites during flocculation process [6,7]. Based on above reasons, it was speculated that these chemical groups in EPS of N. mucosa SX played an important role in its flocculation as shown in Fig. 2. Moreover, it was found that more chemical groups were found when the extraction time was increased for CER method and the treatment temperature was enhanced for heating method. Combined with the data on EPS content, it was indicated that both CER and heating methods not only extracted more EPS than other methods but also extracted more substances of different chemical properties. Therefore, the two methods could be used to extract EPS for qualitative and quantitative analysis. Although EPS content extracted by EDTA and acidic treatment was low, both EPS contained rich chemical groups. Therefore, it was suggested that the EPS extracted by the two methods could be used for qualitative analysis. Alkaline treatment could neither extract more EPS nor get more chemical groups. It was believed that this method was not suitable for the extraction of EPS from microalgae. Of course, more detailed biochemical nature was beneficial to further clarify the characteristics of EPS. Therefore, HPLC was used to analyze the monomer composition of the carbohydrates from EPS. In this study, the main monomer compositions of the carbohydrates were glucose and mannose. It was consistent to previous reports showing that both glucose and mannose were predominant sugar monomers of EPS for self-flocculating microalgae C. vulgaris and S. obliquus [6,7]. Nevertheless, some monomer compositions of the carbohydrates extracted by EDTA method were different from those of the carbohydrates extracted by other methods. It was probably due to different extraction methods.

Based on the above analysis, it indicated that both CER and heating treatment extracted more EPS with rich chemical groups than other three methods (EDTA, acidic treatment and alkaline treatment) from a newly isolated self-flocculating microalga N. mucosa SX. The efficiency of EPS extraction by CER and heating treatment was dependent on treatment time and temperature. Although long treatment time (9 h) for CER method and high temperature (60 °C) for heating method yielded more EPS, it resulted in severe cell lysis. Comprehensive analysis, the CER extraction for 6 h was regarded as a good choice for microalgal EPS extraction. Conflict of interest The authors declare that they have no conflict of interest. Authors' contributions Junping Lv and Shulian Xie conceived and designed the experiments. Fei Zhao and Junping Lv conducted the laboratory experiments. Jia Feng, Qi Liu, Fangru Nan and Xudong Liu analyzed and interpreted the results. Shulian Xie reviewed the manuscript. All authors approved the final manuscript. Acknowledgments This research project was financed by the Natural Science Foundation of China (No. 31700310), the Key Scientific Development Project of Shanxi Province, China (No. FT-2014-01-15), the Social Development Foundation of Shanxi, China (No. 201603D321008 and No. 201803D31020) and the Fund for Shanxi “1331 Project”. We were also very grateful for the test platform for FTIR analysis provided by Scientific Instrument Center of Shanxi University. We were also very grateful to Xuemei Qin and Ke Li for their technical support in the analysis of the monomer composition of the carbohydrates.

4.2. The response of microalgal viability to EPS extraction by different methods Cell lysis might occur when EPS were extracted. Plenty of studies had taken nucleic acid content of EPS as an indicator of cell lysis [1]. A high level of nucleic acids after EPS extraction usually indicated severe cell lysis [1]. In this study, the highest content of DNA was released for EPS extraction by CER for 9 h. CER was commonly a good choice to extract EPS from sludge because small quantities of DNA were released [18,32]. In above studies, the contact time between CER and cells was only 1 h, which was far less than the time in this study. As described by Frolund et al. [17], a long extraction time (e.g., longer than 12 h) was needed for effective EPS extraction from sludge. However, it resulted in cell lysis. It was speculated that the highest DNA of EPS extracted by CER was closely related to the contact time. In this study, when the contact time reduced to 6 h and 3 h, the DNA content significantly decreased. Compared to other methods, heating at 60 °C for 0.5 h also resulted in serious cell lysis and thus large quantities of DNA were released into EPS. As described by McSwain et al. [43], the heat extraction produced a higher protein and polysaccharide content of sludge from cell lysis, which was again proved in this experiment. When the temperature was reduced to 45 °C, the DNA content significantly decreased. Besides DNA content, DHA also reflected cells viability. It was clearly found that DHA of cells after EPS extraction by CER 9 h and heating at 60 °C for 0.5 h significantly decreased, which was consistent to the result of DNA content. Damaged cell membranes emitted red fluorescence upon excitation stained by PI. In this study, the cell membrane integrity of N. mucosa SX was significantly affected by EPS extraction via CER 9 h and heating at 60 °C for 0.5 h. The result again proved that CER 9 h and heating at 60 °C for 0.5 h had severe effects on cells viability. Interestingly, the highest proportion of damaged N. mucosa SX cells was 10.74% when EPS was extracted by alkaline treatment. The result was in agreement with former reports showing that 11% of cells in biofilm and 9% of cells in sludge were dead by alkaline treatment [44]. It indicated that the method was not beneficial to EPS extraction from microalgae.

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