Environ Sci Pollut Res (2014) 21:3946–3954 DOI 10.1007/s11356-013-2114-y

RESEARCH ARTICLE

Isolation and characterization of dissolved organic matter fractions from antialgal products of Microcystis aeruginosa Yun Kong & Liang Zhu & Pei Zou & Jiaoqin Qi & Qi Yang & Liming Song & Xiangyang Xu

Received: 20 May 2013 / Accepted: 29 August 2013 / Published online: 30 November 2013 # Springer-Verlag Berlin Heidelberg 2013

Abstract An antialgal bacterium, Streptomyces sp. HJC-D1, was applied for the biodegradation of cyanobacterium Microcystis aeruginosa, and the isolation and characterization of dissolved organic matter (DOM) fractions in antialgal products were studied. Results showed the the growth of M. aeruginosa was significantly inhibited by the cell-free filtrate of Streptomyces sp. HJC-D1 with the growth inhibition of 86± 7 %. The antialgal products were divided using resin adsorbents into the hydrophilic fraction (HPI), hydrophobic acid (HPO-A), transphilic acid (TPI-A), hydrophobic neutral and transphilic neutral, and then the five fractions were analyzed by the 3-D fluorescence spectroscopy, gel permeation chromatography, and Fourier transform infrared spectroscopy. The results indicated that the HPI component was the most abundant DOM fraction in the antialgal products, and its concentration was increased with the increase of cell-free filtrate concentration. The fluorescence peak location and intensity analysis showed that the protein-, Responsible editor: Céline Guéguen Y. Kong : L. Zhu (*) : J. Qi : Q. Yang : X. Xu Department of Environmental Engineering, Zhejiang University, Hangzhou, Zhejiang 310058, China e-mail: [email protected] Y. Kong e-mail: [email protected] P. Zou Hubei Longyin Environmental Protection Science & Technology Co., Ltd., Chibi, Hubei 437300, China Y. Kong : J. Qi : L. Song Yixing Urban Supervision & Inspection Administration of Product Quality, National Supervision & Inspection Center of Environmental Protection Equipment Quality (Jiangsu, preparation), Yixing, Jiangsu 214205, China X. Xu (*) ZJU-UWA Joint Centre in Integrated Water Management and Protection, Hangzhou, Zhejiang 310058, China e-mail: [email protected]

fulvic-, and humic-like substances were dominant in the HPI, HPO-A, and TPI-A fractions, and intensities of the relevant fluorescence peaks were stronger in the experimental groups than those of the control groups. It was also found that the number-average molecular weight of DOM fractions ranged from 245 to 1,452 g mol−1, and thereinto organic acids such as HPO-A and TPI-A exhibited lower molecular weights. Keywords Antialgal products . Streptomyces sp. HJC-D1 . Microcystis aeruginosa . Dissolved organic matter (DOM) . Isolation

Introduction In recent years, harmful algal blooms (HABs) have been occurring frequently in various lakes and reservoirs (Lewis et al. 2011; Otten et al. 2012), and the associated algogenic organic matter (AOM) during the algae growth causes the serious water pollution and threatens ecological security (Hitzfeld et al. 2000; Qin et al. 2006; Tang et al. 2012). Researches indicated that AOM consists of the extracellular organic matter (EOM) via metabolic excretion and intracellular organic matter (IOM) produced by the autolysis of cells (Fang et al. 2010a; Henderson et al. 2008; Li et al. 2012; Pivokonsky et al. 2006). One of the major problems for the drinking water supply is the release of AOM in natural waters, and its presence affects water quality with the index of dissolved organic matter (DOM) (Henderson et al. 2008; Hyung and Kim 2008; Philippe et al. 2010; Wang et al. 2010). Taking cyanobacteria as an example, the organic substances produced during the growth of cyanobacteria include a wide range of compounds such as the oligosaccharides, polysaccharides, proteins, peptides, nucleic acids, lipids, and small molecules (Hnatukova et al. 2011; Pivokonsky et al. 2006), of which proteins are the major component and precursors of trihalomethanes (THMs) (Fang et al. 2010b; Kanokkantapong

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et al. 2006; Mosteo et al. 2009; Yang et al. 2011). The increases of toxic cyanobacteria and excreted metabolic substances cause a series of problems for the drinking water treatment in the world (Cai and Benjamin 2011; Campinas and Rosa 2010; Henderson et al. 2010; Hyung and Kim 2008; Wang et al. 2010; Zhang et al. 2011). As is well known, microorganisms in the aquatic environment play an important role in the elimination of eutrophication. In the recent years, previous studies have reported that toxic cyanobacteria could be inhibited or degraded by microorganisms such as bacteria (Choi et al. 2005; Jia et al. 2010; Kong et al. 2013b), phytoplankton (Philippe et al. 2010), and algae (Zhang et al. 2009b). Based on numerous laboratory studies (Choi et al. 2005; Jia et al. 2010; Kong et al. 2013b; Lovejoy et al. 1998; Su et al. 2007), bacteria are considered to be the major contributor of phytoplankton growth inhibition or nutrient substance removal. Previous studies have also indicated that the mechanism of algae biodegradation is through the direct or indirect attack of bacteria (Kong et al. 2013b; Lovejoy et al. 1998), and the antialgal products have a significant effect on the natural water quality. However, research on the compositions of antialgal products and their possible risk is lacking in theoretical foundation. In recent years, information on organic matter character has been achieved by fluorescence excitation– emission matrices (EEMs) and infrared spectroscopy (FTIR) (Chen et al. 2003; Fang et al. 2010a; Li et al. 2012; Maruyama et al. 2001; Wang et al. 2009), especially on protein and humic/fulvic-like substances in DOM and sewage effluent (Henderson et al. 2008, 2010; Philippe et al. 2010). Therefore, it may reveal that the AOM produced during the antialgal process plays an important role in the safety of natural water. In the present study, the characteristics of Microcystis aeruginosa (the model species of cyanophytes commonly found in eutrophic waters) exposed to the antialgal bacterium Streptomyces sp. HJC-D1 were investigated by monitoring the concentration of chlorophyll a (Chl a), and the compositions and character of antialgal products were investigated by the technologies of EEMs, gel permeation chromatography (GPC), and FTIR. It’s hoped to demonstrate the microbial antialgal mechanism and evaluate the quality risk of antialgal microorganism application.

Materials and methods Target algae, microorganism, and their culture conditions M. aeruginosa FACHB-905 was purchased from the Freshwater Algae Culture Collection of the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China). Before being used as inoculant, it was cultured for 7 days to reach the exponential growth phase, and the culture conditions were as follows: sterilized BG11 medium (Jia et al.

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2010; Rippka et al. 1979), 2,000 lx white light, and light/dark= 14 h/10 h, 25±1 °C. The strain Streptomyces sp. HJC-D1 used in this study was isolated from a eutrophication pond in Hangzhou, China, and showed excellent antialgal activity (Kong et al. 2013a, b). The Streptomyces sp. HJC-D1 was maintained at 4 °C in Gause’s synthetic agar medium (Huang et al. 2008), and fermentation liquid was prepared by incubating the seed culture at 28 °C with a shaking speed of 150 rpm for 72 h (the cell density was nearly 5.45×105 cells mL−1). The Streptomyces sp. HJC-D1 cell-free filtrate was obtained as follows before use: The fermentation liquid was centrifuged at 10,000×g for 10 min and then filtered through a 0.22-μm cellulose acetate membrane to acquire a cell-free filtrate. The cell-free filtrate was subsequently inoculated into M. aeruginosa culture for cyanobactericidal activity tests. Antialgal activity test of Streptomyces sp. HJC-D1 on M. aeruginosa The effect of antialgal efficiency was studied by adding Streptomyces sp. HJC-D1 cell-free filtrate with ratios of 0, 1, 3, 5, and 10 % (v/v) in 500-mL sterilized conical beakers with 225-mL BG11 medium containing M. aeruginosa cells, brought to a final volume of 250 mL by addition of Gause’s synthetic medium. A negative control was made by adding 25 mL Gause’s medium into 225 mL cyanobacterial solution. The growth inhibition experiments of M. aeruginosa were done in aseptic condition. All the controls and treatments were replicated three times, and the arithmetical means (±SD) were obtained and used as the final results. Isolation and collection of antialgal products For the gel permeation chromatography, Fourier transform infrared spectroscopy, and 3-D fluorescence spectroscopy analysis of antialgal products, the growth inhibition experiment was studied by adding 10 % (v /v ) of Streptomyces sp. HJC-D1 cell-free filtrate. After 8 days of incubation, the antialgal products were divided using Amberlite XAD-8/ XAD-4 resins into five fractions: hydrophilic fraction (HPI), hydrophobic acid (HPO-A), transphilic acid (TPI-A), hydrophobic neutral (HPO-N), and transphilic neutral (TPI-N) (Wei et al. 2008). The fractionation of AOM was performed by following the procedure modified from Wei et al. (2008) and Zhang et al. (2009a). AOM samples filtered through 0.45 μm GF/F membrane (Whatman) were acidified to pH 2.0 with 0.1 or 1.0 M HCl and passed through XAD-8 and XAD-4 resins at a flow rate of 10 bed volumes h−1, and HPI was the effluent from XAD-4. The eluate from XAD-8 and XAD-4 resins with 0.1 M NaOH at a flow rate of 2 bed volumes h−1 was defined as HPO-A and TPI-A, respectively; HPO-N and TPI-N were desorbed from the XAD resins using methanol, and methanol

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was subsequently removed by using rotary evaporation; the resin isolates were lyophilized for other determinations. For the analysis of carbohydrates and proteins, EOM and IOM were separated from the algal suspensions or algal cells as described by Fang et al. (2010a) and Liu et al. (2011). The algal solution was centrifuged for 10 min at a speed of 12,000 rpm. The supernatant was collected and filtered through a GF/F membrane and then was referred to as the EOM solution. The pellets of algal cells were washed with 20 mL Milli-Q water (Milli-Q Biocel), followed by two cycles of centrifugation and supernatant removal, and then resuspended in the same volume of Milli-Q water and subjected to sonication (Sonics 800 W/50 Hz, USA) in an ice bath with the amplitude of 100 % for one hundred 5-s periods separated by 5-s intervals. The suspension was centrifuged for 10 min at 12,000 rpm and followed by filtration through a GF/F membrane. The filtrate was hereafter referred to as the IOM solution. The EOM and IOM were collected to assess the concentration of total dissolved organic carbon (DOCT) and nonprotein (carbohydrates) (DOCNP) organic matter, and the DOCP of the protein portion was calculated according to the Hnatukova et al. (2011). Analytical methods

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in series. The flow rate was 0.8 mL min−1, and the injection volume was 50 μL, using a mobile phase of 0.1 M sodium nitrate. The weight-average MW was calculated by the software Baseline, Waters. Fourier transform infrared spectroscopy analysis FTIR analysis was used to characterize the major functional groups of organic matters and to predict the major components (Kong et al. 2013a; Zhang et al. 2009a). AOM fractions obtained through freeze-drying were analyzed for the structural and chemical characteristics. KBr was mixed with the fractions at a ratio of about 100:1, and the FTIR spectra of the mixture were obtained by scanning with FTIR-8900 spectrometer (Shimadzu, Japan). 3-D excitation–emission matrix fluorescence spectroscopy The 3-D EEM fluorescence spectra were recorded on a LS-55 fluorescence spectrophotometer (PerkinElmer, USA). Threedimensional spectra were obtained by measuring the emission spectra ranging from 300 to 550 nm repeatedly and at the excitation wavelengths from 200 to 400 nm. The excitation and emission slits were maintained at 10 nm, and the scanning speed was set at 1,200 nm min−1 (Kong et al. 2013a).

Determination of algicidal activity The concentration of Chl a was determined by spectrophotometric method using 90 % acetone extraction (APHA 1998). The growth inhibition of M. aeruginosa was calculated according to the Zhang et al. (2009b). Organic carbon analysis DOC was measured with a TOC analyzer (TOC-180 VCPH, Shimadzu, Japan). All samples were filtered through a 0.45-μm GF/F membrane. All measurements were conducted in triplicate, and errors were less than 2 %. Molecular size distribution analysis The number-average molecular weight (Mn), weight-average molecular weight (Mw), and polydispersity index (PDI) of different fractions in antialgal products were measured by GPC (Zheng et al. 2012). The standard samples employed for calibration were narrow molecular weight (MW) distribution polyethylene glycol (PEG, MW 310,000, 82,500, 44,000, 25,300, 4,000, and 2,000 Da) obtained from Waters Corporation (Belford, USA) (Kong et al. 2013a). The chromatographic system was equipped with a Waters 515 HPLC pump (Waters) and Waters 2410 differential refractive index detector. The column set included the Ultrahydrogel guard columns Ultrahydrogel TM500 and Ultrahydrogel TM120 connected

Results and discussion Effect of Streptomyces sp. on the growth of M. aeruginosa Five different concentrations of cell-free filtrate produced by Streptomyces sp. (0, 1, 3, 5, and 10 %, v/v) were prepared to evaluate the ability to inhibit the growth of M. aeruginosa. In all experiments, the initial Chl a concentration of M. aeruginosa cells was controlled at 0.3517±0.1154 mg L−1 in BG11 medium. It was found that Chl a concentration was plotted as a function of the reaction time, and curves with similar shapes were observed (Fig. 1a). After the incubation for 2 days, the growth inhibition of Chl a was increased to 21±2, 34±2, 39±4, and 38±2 %, respectively (Fig. 1). The growth of M. aeruginosa was significantly inhibited when it was exposed to the cell-free filtrate for 4 days at the addition ratio of cell-free filtrate concentration of more than 3 %. Finally, the growth inhibition increased from 58±5 to 86±7 % after 8 days as the cell-free filtrate concentration increased from 3 to 10 % (Fig. 1b). It is generally recognized that algicidal agents produced by bacteria are a promising and environmentally friendly way to control HABs (Lovejoy et al. 1998). Several studies have revealed the existence of bacteria that are capable of inhibiting or degrading algal blooms in marine and freshwater environments (Jia et al. 2010; Kong et al.

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3949 60

a

CK

2.0 1%

3%

5%

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1.6

45

DOC (mg/L)

-1

Chlorophyll a (mg L )

CK %

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0.8

30

0.4

15 0.0 0

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8

Time (d)

0

b

HPI

100

Growth inhibition (%)

1%

3%

5%

HPO-A

TPI-A

HPO-N

TPI-N

Fig. 2 DOC concentration of different fractions isolated from degradation products

10 %

80

60

40

20

0 2

4

6

8

Time(d) Fig. 1 Biodegradation of M. aeruginosa by Streptomyces sp. cell-free filtrate. a Concentration of Chl a; b growth inhibition of M. aeruginosa

2013b). These algicidal bacteria increased in abundance concurrently with the decline of algal blooms, suggesting that they could control the algal bloom. The study confirmed that the antialgal bacterium Streptomyces sp. HJCD1 significantly inhibited the growth of cyanobacterium M. aeruginosa. Isolation of antialgal products The antialgal products were divided into five fractions using XAD-8/XAD-4 resins, and the concentrations of each fraction in terms of DOC were shown in Fig. 2. For all the samples collected from the control and experimental groups, the HPI component was found to be the most dominant fraction and showed a positive correlation with the addition of cell-free filtrate, accounting for 61±6, 49±11, 73±4, 74±5, and 74± 6 % of the total DOM, respectively. At the same time, the variation of the TPO-A and HPO-N fractions was similar to that of the HPI fraction. The HPO-A fraction in the systems with a different addition ratio of cell-free filtrate was 21±1, 30±7, 16±3, 11±0, and 6±0 %, respectively. Results

showed that the changes of HPO-A fraction were different from the other fractions. As the hydrophilic fraction is less readily removed by traditional flocculation, sedimentation, and filtration than the hydrophobic fraction, it would decrease the water treatment efficiency (Li et al. 2012). Hence, the characteristics of the DOM fractions in antialgal products were determined in this paper. HPI was found to be the most dominant fraction and accounting for 61±6 and 74±6 % of the total DOM for the control and experimental group respectively, and HPO-A fraction was the second component. Her et al. (2004) studied the cyanobacteria AOM character and found the HPI and HPO occupied 57 and 26 %, respectively. The results also showed that the major composition of AOM was HPI, which belonged to the hydrophilic fraction. However, the AOM fraction distribution varied substantially depending on the types of algae and different growth phase. It was found that the HPO fraction was 11, 15, and 30 % in the stationary phase and 12, 18, and 29 % in the exponential phase for the AOM of Chlorella vulgaris, Asterionella formosa, and M. aeruginosa, respectively (Henderson et al. 2008). MW distribution of antialgal products The MW distribution of macromolecules in antialgal products was investigated using GPC, and the results were shown in Table 1. The Mn of the five fractions was 1,430, 245, 256, 1,452, and 1,020 Da, with a PDI value of 1.037, 1.057, 1.021, 1.388, and 1.025, respectively. The results of the PDI value indicated that the antialgal products had a narrow MW distribution. Additionally, there was no significant difference of macromolecules with MW about 1,000 Da in the DOM fractions from antialgal products.

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Table 1 Molecular weight and polydispersity of different fractions from antialgal products Mn (Da)

Mw (Da)

PDI (Mw/Mn)

1,430 245 256 1,452

1,484 259 262 2,061

1.037 1.057 1.021 1.388

TPI-N

1,020

1,046

1.025

T%

HPI HPO-A TPI-A HPO-N

In this study, organic acids HPO-A and TPI-A exhibited lower MW. Similar results for MW distribution were reported previously, showing that the low-MW matter in the ranges of 60 kDa (Pivokonsky et al. 2006) and >800 kDa (mostly phycocyanin and carbohydrates) (Henderson et al. 2008, 2010), and the results that IOM consisted of more high-MW organic nitrogen substances than low-MW substances were also found in previous studies (Fang et al. 2010a; Li et al. 2012). Results demonstrated that the existence of low-MW substances was common, and the fact that high-MW substances could not be detected in this study was probably due to the size exclusion limits of the column used. The detection might be slightly affected by BG11 cultivation media as the RID-10A was a nonselective detector (Fang et al. 2010b; Li et al. 2012). FTIR analysis of different AOM fractions In order to understand the spectroscopy characteristics of major functional groups of the AOM organic matters in antialgal products, FTIR analysis was also conducted. Figure 3 showed the FTIR spectra of the five DOM fractions from the experimental group (10 %, v/v). Results showed that the HPI and HPO-A fractions were similar in spectra with four distinctive adsorption bands. A broad region of adsorption around a peak at 2,960 cm−1 revealed the presence of aliphatic chains with a small shoulder (CH asymmetric stretching in CH2 and CH3); the adsorption band of 1,651 cm−1 was attributed to the stretching of the O–H bond in hydroxyl functional groups primarily associated with the peptide carbonyls, and a peak at 1,506 cm−1 might be due to the stretching of C–H bonds; these two peaks (1,700–1,600 and 1,550–1,500 cm−1) were unique to the protein secondary structure as namely amides I and II; the last from 900 to 1,200 cm−1 was very broad and could be attributed to carbohydrates of the C–C bond, O-alky group of the CO bond, or the OH bond in alcohols and carboxylic acids. Compared with HPI and HPO-A fractions, the other three fractions had the same four

80 60 40 20 80 60 40 20 80 60 40 20 80 60 40 20 80 60 40 20 0 4000

HPI 2358 2960

669

864

1651 1506 1384

1141 835

HPO-A 2358 1384 1141

800

TPI-A

2358

686

902 864

HPO-N 1031

2358 2468

518 881

TPI-N 2468

2358

1780 835 1099

1670 1440

3500

3000

2500

2000

1500

1000

500

Wavenumber (cm-1) Fig. 3 FTIR spectra of different fractions from antialgal products

adsorption bands except the band at 2,960 cm−1; in addition to the distinctive adsorption band at 1,440 cm−1 which resulted in the mixed absorption of OH bond, CH bond, and weaker symmetrical stretching bands of carboxylate anions, TPI-N showed a relatively weak band at 864 cm−1 corresponding to the ester carbonyl; TPI-A had quite low peaks, whereas the adsorption band at 1,031 cm−1 could be corresponding to carbohydrates and polysaccharide-like substances. Hydrophobic and hydrophilic organics were similar with their FTIR spectra of aromatics and carbohydrates (Leenheer 1981; Zhang et al. 2009a). These compounds were probably metabolites of the coculture algae, and the same results were also observed in municipal wastewater and drinking water treatment plants (Pernet-Coudrier et al. 2011; Wang et al. 2009). In addition, a broad peak at 1,031 cm−1 exhibited the character of carbohydrates or carbohydrate-like substances, which showed that carbohydrates were present in the AOM. The results observed were consistent with those published by Her et al. (2004). 3-D EEM fluorescence spectroscopy of different AOM fractions EEM was used to characterize different AOM fractions. The fluorescence in the current study was designated according to Chen et al. (2003): peak A was humic-like acid with Ex of 300–370 nm and Em of 400–500 nm; peak C with Ex of 230– 260 and 320–350 nm and exhibited maximums at Em wavelength of 420–450 nm, peak T was tryptophan like or protein

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relevant fluorescence peaks were stronger in the experimental groups than in control groups. EEM was a common qualitative analysis method for determining protein-, humic-, or fulvic acid-like substances of drinking water, soluble microbial products, and extracellular polymeric substances in biological water treatment (Tang et al. 2010; Wang et al. 2009), and this method had been applied for the determination of algae such as the cyanobacterium M. aeruginosa (Fang et al. 2010a; Li et al. 2012), diatom Nitzschia , and green algae Scenedesmus quadricauda (Henderson et al. 2008). The EEM results of AOM fractions were similar to those obtained from M. aeruginosa in previous studies, indicating that tryptophan-like fluorescence was detected; moreover, additional fluorescence was also detected in locations attributable to humic/fulvic-like substances, and the high percentage of the hydrophilic fraction was mainly protein-like compounds (Her et al. 2004).

like which exhibited Ex at 220275 nm and Em wavelength of 340350 nm. The fluorescence EEM spectra of the results showed that different AOM fractions exhibited different peaks (Fig. 4). It is commonly acknowledged that protein- and humic-like substances are dominant in algal EOM (Fang et al. 2010a). Although the fluorescence peak position of AOM fractions for the control and experimental groups was nearly the same, the fluorescence peak intensities for the experimental groups were much stronger than that of the control group (Table 2), indicating that there were more protein-like, fulvic-like, and humic-like substances in the antialgal products, and these fluorescence substances should be from the antialgal of M. aeruginosa. The fluorescence characteristics including the peak location and fluorescence intensity showed that the protein-, fulvic-, and humic-like substances were dominant in the HPI, HPO-A, and TPI-A fraction, and intensities of the

400

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Peak C

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300

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200 300

Ex(nm)

225.0

360

Peak A

Ex(nm)

Ex(nm)

380

112.5

360

Ex(nm)

Fig. 4 Fluorescence spectroscopy of the AOM fractions (a, c, and e are HPI, HPO-A, and TPI-A in the control group; b, d, and f are HPI, HPO-A, and TPI-A in the experimental group)

200 300

350

400

450

Em(nm)

500

550

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Table 2 EEM peaks for the AOM fractions in the control and experimental groups (10 %, v/v)

CK

AOM fraction

Peak

Ex (nm)/ Em (nm)

Intensity

Substance

HPI

A C T A C A C T A

330/425 260/440 300/335 300/405 230/405 300/405 230/400 220/305 330/440

127.61 105.42 32.48 38.82 75.28 19.00 35.99 29.54 263.46

Humic-like acid Fulvic-like acid Protein-like Humic-like acid Fulvic-like acid Humic-like acid Fulvic-like acid Protein-like Humic-like acid

C T A C A C T

260/455 280/435 320/430 230/425 310/405 230/410 220/310

238.36 188.82 106.86 204.92 31.32 60.60 24.09

Fulvic-like acid Protein-like Humic-like acid Fulvic-like acid Humic-like acid Fulvic-like acid Protein-like

HPO-A

TPI-A 10 %

HPI

HPO-A

TPI-A

Carbohydrates and proteins of EOM and IOM As carbohydrates and proteins were two basic indicators to reflect the physiological state of cells, it could be inferred that these organic matters might be secreted as the algae exposed to xenobiotics (Henderson et al. 2008). The concentrations of proteins and carbohydrates in terms of DOCP and DOCNP from EOM and IOM are shown in Table 3. With the cell-free filtrate concentrations from 0 to 10 % (v/v), the DOCP of antialgal products was decreased from 96.7±9.3 to 10.8±2.6 mg L−1 in the EOM and from 40.0±6.4 to 6.2±2.2 mg L−1 in the IOM on day 8, respectively, while the DOCNP was varied with values from 40.3±3.8 to 18.6±1.3 mg L−1 in the EOM and from 72.7±3.5 to 13.8±4.0 mg L−1 in the IOM. The proportion of DOCNP was decreased from 68±11∼92±5 to 29±3∼74±6 % in the EOM and 85±13∼97±16 to 60±1∼69±1 % in the IOM,

which demonstrated that carbohydrates and polysaccharide-like substances were predominant in antialgal products; in addition, DOCP and DOCNP showed a significant decrease with the increase of cell-free filtrate concentration. Hence, it was DOCNP concentration that was responsible for the variability observed in DOCT, which supported the observations in the current study (“3-D EEM fluorescence spectroscopy of different AOM fractions”). In a previous study, it was found that the protein was approximately 60 % of DOCT in the AOM, and the nonprotein materials were 40 % (Hnatukova et al. 2011). In this study, the recovery of carbohydrates across the fractionation procedure varied from 72 to 82 % at the exponential phase and 93 to 104 % at the stationary phase. Pivokonsky et al. (2006) evaluated the production and composition of AOM produced by M. aeruginosa; the ratio of DOCP comprised up to 31 % of the DOCT at the stationary phase in EOM, and the ratio of DOCP was 66 % as a part of IOM (Pivokonsky et al. 2006). It might be assumed that increases in DOM hydrophilic fraction were related to the decrease in the EOM carbohydrates (DOCNP). Generally, blue–green algae have more protein contents (41–69 %) than diatoms (12–50 %), while diatoms appear to accumulate more lipids in the cells (5–43 %) than the blue–green algae and green algae (2–30 %) (Henderson et al. 2008). In the present study, the composition of antialgal products was influenced by the interaction of the algal cells and the cell-free filtrate produced by Streptomyces sp., and the high concentrations of DOCP and DOCNP in EOM could be due to the cell-free filtrate or Gause’s synthetic medium. This observation probably results in DOM predominantly consisting of carbohydrates and polysaccharide-like substances, as a result of the antialgal products of M. aeruginosa. As M. aeruginosa cells were cultured, the concentrations of proteins (DOCP) in EOM and IOM increased obviously (Pivokonsky et al. 2006), and this could explain the results of EOM and IOM in this study. In view of the obtained results, it was concluded that the growth of M. aeruginosa was significantly inhibited by Streptomyces sp. HJC-D1 cell-free filtrate (10 %, v/v), and

Table 3 DOCP and DOCNP of EOM and IOM from degradation products EOM

IOM

DOCP

CK 1% 3% 5% 10 %

DOCNP

DOCP

DOCNP

0 day

8 days

0 day

8 days

0 day

8 days

0 day

8 days

59.2±6.0 121.2±9.6 134.1±7.3 83.9±8.5 51.1±5.9

96.7±9.3 31.2±2.9 19.5±12.5 13.3±2.6 10.8±2.6

666.3±35.5 528.1±37.2 418.2±32.6 348.4±34.4 106.9±17.1

40.3±3.8 30.9±2.4 37.0±12.5 34.9±3.0 18.6±1.3

1.5±0.9 1.5±0.1 1.4±1.3 1.5±1.2 1.0±0.9

40.0±6.4 34.3±5.8 29.9±2.1 16.8±0.8 6.2±2.2

12.1±1.9 10.6±1.6 10.8±1.8 9.1±0.8 7.7±0.7

72.7±3.5 76.2±0.5 49.5±11.4 24.8±0.6 13.8±4.0

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the growth inhibition reached 86±7 %. HPI was found to be the most dominant fraction, and the percentage was about 61±6∼74±6 % in antialgal products. The fluorescence characteristics of different fractions showed that the protein-like, fulvic-like acid and humic-like substances were dominant in the HPI, HPO-A, and TPI-A fractions. The number-average MW of DOM fractions ranged from 245 to 1,452 g mol−1, and thereinto organic acids such as HPO-A and TPI-A exhibited lower MW. The HPI and HPO-A materials were similar in spectra with four distinctive adsorption bands, and the adsorption band at 1,031 cm−1 for TPI-A corresponded to carbohydrates and polysaccharide-like substances. Overall, the results demonstrated the potential of the antialgal bacterium Streptomyces sp. HJC-D1 for inhibiting the cyanobacterium M. aeruginosa, and the spectroscopic characterization of the antialgal products showed that carbohydrates and polysaccharide-like substances were predominant in antialgal products. Acknowledgments This work was funded by the National Key Technologies Research and Development Program of China (no. 2006BAJ08B01/2012BAJ25B07).

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Isolation and characterization of dissolved organic matter fractions from antialgal products of Microcystis aeruginosa.

An antialgal bacterium, Streptomyces sp. HJC-D1, was applied for the biodegradation of cyanobacterium Microcystis aeruginosa, and the isolation and ch...
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