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Flocculation mechanism of the actinomycete Streptomyces sp. hsn06 on Chlorella vulgaris
Accepted Manuscript Flocculation mechanism of the actinomycete Streptomyces sp. hsn06 on Chlorella vulgaris Yi Li, Yanting Xu, Tianling Zheng, Hailei Wang PII: DOI: Reference:
S0960-8524(17)30667-3 http://dx.doi.org/10.1016/j.biortech.2017.05.028 BITE 18054
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
30 March 2017 3 May 2017 4 May 2017
Please cite this article as: Li, Y., Xu, Y., Zheng, T., Wang, H., Flocculation mechanism of the actinomycete Streptomyces sp. hsn06 on Chlorella vulgaris, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/ j.biortech.2017.05.028
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Flocculation mechanism of the actinomycete Streptomyces sp. hsn06 on Chlorella vulgaris Yi Li1, Yanting Xu1, Tianling Zheng2 and Hailei Wang1* 1
College of Life Sciences, Henan Normal University, Xinxiang 453007, China
2
State Key Laboratory of Marine Environmental Science, School of Life Sciences,
Xiamen University, Xiamen 361005, China *Corresponding author: Tel.: +86 3733326340; Fax: +86 3733326916. E-mail address: [email protected] (H. Wang)
Abstract In this study, an actinomycete Streptomyces sp. hsn06 with the ability to harvest Chlorella vulgaris biomass was used to investigate the flocculation mechanism. Streptomyces sp. hsn06 exhibited flocculation activity on algal cells through mycelial pellets with adding calcium. Calcium was determined to promote flocculation activity of mycelial pellets as a bridge binding with mycelial pellets and algal cells, which implied that calcium bridging is the main flocculation mechanism for mycelial pellets. Characteristics of flocculation activity confirmed proteins in mycelial pellets involved in flocculation procedure. The morphology and structure of mycelial pellets also caused dramatic effects on flocculation activity of mycelial pellets. According to the results, Streptomyces sp. hsn06 can be used as a novel flocculating microbial resource for high-efficiency harvesting of microalgae biomass.
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Keywords Streptomyces sp. hsn06; Chlorella vulgaris; Mycelial pellets; Flocculation mechanism
1. Introduction Energy crisis is threatening human existence because of the excessive exploitation and the environment damage (Fong et al., 2016). Microalgal biofuels are very importance to alleviate energy crisis and in favor of environmental protection, which have caused increasing attention (Wijffels and Barbosa, 2010). However, the development of microalgae industrialization is mainly limited by algal cultivation and harvesting of algal biomass (Maeda et al., 2016). Recently, microalgal cultivation obtains effective improvement based on optimization of photobioreactor, breeding of high oil microalgae, and genetic engineering modification (Vandamme et al., 2013). Meanwhile, harvesting of microalgae biomass becomes the greatest bottleneck for microalgal development (Weschler et al., 2014). To harvest microalgae biomass, various methods including addition of chemical flocculants, physical flocculation, and bioflocculation have been implemented (Zhang et al., 2010). Chemical flocculants were always used to sewage treatment, mining and other industrial flocculation, however, chemical flocculant would pose threat to algal biomass, once performed to harvest algal biomass (Rwehumbiza et al., 2012). The physical flocculation can avoid the pollution of algae biomass, however, its high cost limits the application on harvesting algal biomass (Cerff et al., 2012). Compared with these methods, bioflocculation especially microbial flocculation is considered to be 2
one of the most promising method (Powell and Hill, 2013). Many types of microorganisms including bacteria and fungi with flocculation activity on microalgae biomass have been isolated (Kim et al., 2011; Yin et al., 2014), however, flocculating actinomycetes are relatively unexplored. The flocculation mechanism of flocculating actinomycetes on microalgae biomass was also barely reported. Microbiol flocculation mechanisms have been proposed a lot of hypothesizes, charge neutralization and ions bridging of which are generally accepted (Ries and Meyers, 1968). Charge neutralization eliminates electrostatic repulsion between cells, and particles will coagulate or flocculate (Yang et al., 2013). Ions bridging requires that the ions simultaneously bind to different particles to form a bridge between them, this bridge brings the particles together and causes flocculation (Biggs et al., 2000). In this study, Streptomyces sp. hsn06 which belongs to a large genus of actinomycetes was isolated and firstly confirmed with the ability to harvest Chlorella vulgaris (C. vulgaris) biomass. The flocculation activity of strain hsn06 on C. vulgaris was investigated, flocculation mechanism was determined, the effect of flocculation conditions on flocculation activity were confirmed, and the flocculation procedure was observed. The objectives of this study were to explore new type of flocculating microorganism, and investigate the flocculation mechanism, which supply a novel choice to harvest microalgae biomass.
2. Materials and methods
2.1 Algal culture 3
The algae C. vulgaris was supplied by Freshwater Algae Culture Collection at the Institute of Hydrobiology (Wuhan, China). The C. vulgaris strain was cultivated in BG-11 medium (Hanna and Marcin, 2001) under a 12:12 h light-dark cycle with a light intensity of 50 mol photons m-2 s-1 at 25 °C. 2.2 Isolation and identification of flocculation actinomycete The activated sludge samples were used to isolate flocculating actinomycetes, and then serially diluted (10-fold) using sterile distilled water, and 0.1 mL aliquots of each dilution were spread onto Gauserime synthetic agar medium (Soluble starch, 20 g; KNO3, 1 g; K2HPO4·3H2O, 0.5 g; MgSO4·7H2O, 0.5 g; NaCl, 0.5 g; FeSO4·7H2O, 0.01 g; agar, 20 g in 1 L distilled water; pH 7.4-7.6) followed by incubation for 7 days at 28 °C. Individual colonies of distinct morphology were further purified three times, and inoculated in flocculation medium (glucose, 10 g; NaCl, 24 g; NH4Cl, 1 g; MgSO47H2O, 0.5 g; yeast extract, 0.6 g; K2HPO43H2O, 6.5 g and KH2PO4, 2 g in 1 L distilled water) to determine the flocculation efficiency, and the strain hsn06 with high flocculation activity on C. vulgaris was isolated. Strain hsn06 was cultured in Gauserime synthetic medium for 2 days at 28 °C with shaking at 120 rpm. Mycelium were obtained and resuspended in STE solution buffer (Tris-HCl with the pH 8.0, 20 mM; EDTA, 25mM; NaCl, 75 mM) containing lysozyme with final concentration of 1 gL-1. Proteins were removed by phenol-chloroform-isoamyl alcohol (25:24:1) extraction. After isopropanol precipitation, DNA was resuspended in sterile water (50 mL) and stored at -20 °C until further analysis. The 16S rRNA gene sequence of strain hsn06 was amplified 4
using PCR with primers 27F and 1492R (Delong, 1992). Purification of the PCR product was cloned into vector pMD19-T and sequenced. Sequences of related taxa were obtained from the GenBank database and EZBioCloud server (http://www.ezbiocloud.net/identify). Phylogenetic analysis was performed using MEGA version 4 (Kumar et al., 2007) after multiple alignment of data by ClustalX (version 1.83). Evolutionary distances and clustering were constructed using the neighbor-joining method (Saitou and Nei, 1987), and were evaluated using bootstrap values based on 1000 replications. 2.3 Flocculation activity of strain hsn06 Strain hsn06 was cultured in flocculation medium and Gauserime synthetic medium for 2 days at 28 °C with shaking at 120 rpm, respectively. Both of the two culture media could promote strain hsn06 to form mycelial pellets, and then mycelial pellets were added into C. vulgaris cultures to investigate flocculation activity. The normal growth algae were set as control. Final concentration of 5 mM CaCl2 was added in every treatment groups and control, followed by mixing at 120 rpm, and then the mixing was stopped to let the mixture settle at 25 °C. After the flocculation of the algal cells, an aliquot of the culture in treatment groups and control was pipetted from a height of two-thirds from the bottom for the evaluation of the flocculation effect using a spectrometer. All the treatment groups and control group were carried out in triplicate. The flocculation activity was calculated according to the equation 1: (1) 5
where A and B are the absorbency (OD) at 680 nm of the microalgal culture in control and treatment groups, respectively. To investigate the effect of different additive amounts on the flocculation activity, mycelial pellets which cultured in flocculation medium and Gauserime synthetic medium with concentrations of 0.005, 0.015 and 0.03 gmL-1 were added into C. vulgaris cultures, respectively. Control group was normal growth algae. All the treatment groups and control groups were added with final concentration of 5 mM CaCl2 and carried out in triplicate. The flocculation activity of each treatment groups was determined according to equation 1. 2.4 Effect of calcium on flocculation activity 0.015 gmL-1 of mycelial pellets after cultured in flocculation medium were added into C. vulgaris cultures with or without adding final concentration of 5 mM CaCl2, to investigate the effect of calcium on flocculation activity. Control groups were normal growth algae with or without adding same concentration of CaCl2. Mycelial pellets with adding different concentrations (0.1, 0.3, 0.5, 1, 2, 3, 4, and 5 mM) of CaCl2 were tested as cationic coagulants for the flocculation of C. vulgaris culture. Control groups were normal growth algae with adding same concentrations of CaCl2. The flocculation activities were calculated according to the equation 1. All the treatment groups and control groups were carried out in triplicate. 2.5 Calcium binding assay Calcium binding was evaluated by measuring the concentration of the remaining calcium after the addition of mycelial pellets which were cultured in flocculation 6
medium and Gauserime synthetic medium or algal cells. Mycelial pellets and algal cells were harvested by centrifugation at 3,000g for 5 min, and then washed twice by sterile deionized water. To investigate the effect of binding time on calcium binding efficiency, final concentration of 5 mM CaCl2 was added to cells in pH 10 deionized water, followed by shaking at 120 rpm for different times including 10, 60, 120, and 180 min at room temperature. After binding time determination, the effect of calcium concentrations on calcium binding efficiency were evaluated by adding known concentrations of CaCl2 (final concentrations of 0.1, 0.3, 0.5, 0.7, 1, 2, 3, 4 and 5 mM) to cells in pH 10 deionized water, followed by shaking at 120 rpm at room temperature. Mycelial pellets with adding CaCl2 were treatment groups as well as adding CaCl2 to algal cells as control. Cells were then removed by centrifugation at 10,000 g for 3 min. The calcium concentration in the supernatant was measured using the Calcium Assay Kit (Nanjing Jiancheng Bioengineering Institute, China). The kit was adapted for use in a 96-well format and measured in a Spectramax M5 plate reader. The readout for the assay was at an absorbance of 610 nm. Concentration of calcium standard solution was 2.5 mM, and deionized water was added as control group. Concentration of the remaining calcium could be calculated according to the kit’s Operation Manual from Nanjing Jiancheng Bioengineering Institute, China. To further investigate whether the decline of calcium concentration could influence the flocculation activity during the flocculation procedure, different concentrations (1, 3, and 5 gL-1) of sodium carbonate were added into algal cultures after adding final concentration of 5 mM CaCl2. The control groups were normal growth algae with 7
adding final concentration of 5 mM CaCl2 and same concentrations of sodium carbonate. All the treatment groups and control groups were carried out in triplicate. 2.6 Characteristics of the flocculation activity To determine the effect of different temperatures on flocculation activity, flocculation activity was investigated after mycelial pellets treated under different temperatures. 0.015 gmL-1 of mycelial pellets were incubated at 40, 60, 80, 99 and 121 °C for 2 h, respectively. Then, the treated mycelial pellets were added into 10 mL of algal culture after cooling to room temperature. Control group was normal growth algae. The pH stability of flocculation activity was tested using acid and alkali treatments on mycelial pellets. The mycelial pellets were added into solutions at pH 3 and 12 for 2 h, and then the treated mycelial pellets were used to determine flocculation efficiency. Mycelial pellets of strain hsn06 were digested with 100 mgL-1 proteinase K in sterile BG11 at pH 7.4 for 2 h at room temperature, and then washed twice with sterile BG11 to remove the proteinase K and then tested the flocculation activity. 0.015 gmL-1 of mycelial pellets were inoculated with adding 3 gL-1 of lysozyme for 2 h at room temperature, and then washed twice with sterile BG11 to remove the proteinase K and then tested the flocculation activity. Inhibition of flocculation activity by sodium dodecyl sulfate (SDS) was tested by adding SDS into algal culture with a final concentration of 1 mM. 0.015 gmL-1 of mycelial pellets were added into algal culture with adding SDS. Control group was normal growth algae with adding 1mM SDS. Mycelial pellets of strain hsn06 were loaded into dialysis bags with molecular intercept values of 8 kD and dialyzed in sterile distilled 8
water for 48 h to perform the dialysis treatment. All the treatment groups and control group were carried out in triplicate. Final concentration of 5 mM CaCl2 was added in every treatment groups and controls, followed by mixing at 120 rpm at room temperature. All the treatment groups and control groups were carried out in triplicate. The flocculation activity of each treatment groups was determined according to equation 1. 2.7 Microscopy observation Microscopic observation using a fluorescence microscope was performed to investigate the interaction between the algal cells and mycelium, and compare the mycelial morphology of mycelial pellets which were cultured in flocculation medium and Gauserime synthetic medium, respectively. Fluorescence microscopy was performed after fluorescent staining. The mycelium-algae complex and two pieces of mycelium which cultured in flocculation medium and Gauserime synthetic medium were picked up and put on each glass slide, and stained with Calcofluor White Stain (0.1 %) (Sigma-Aldrich) and 10 % potassium hydroxide for 1 min in the dark. Covered with the clean cover slip and examined under a fluorescence microscope (Olympus BX63, Chiyoda-ku, Tokyo, Japan), equipped with a epifluorescent filter. The Calcofluor White stained mycelium and algal chlorophyll were excited with a 355- and 488-nm argon laser, respectively. The signal for the Calcofluor White-stained mycelium and chlorophyll autofluorescence were visualized using 440 and 600-nm long-pass filters, respectively. 2.8 Statistics 9
All data were presented as mean ± standard error and were evaluated using one-way analysis of variance followed by the least significant difference test, with p<0.01 and p<0.05 (Origin 8.5 for Windows).
3. Results and discussion 3.1 Characterization and identification of strain A total of 12 bacterial strains, 6 fungal strains, and 7 actinomycetes strains were isolated from active sludge samples. Active sludge contains various microbial community and supplies organic and inorganic nutrients which always deems as the source of flocculation microorganism (Biggs and Lant, 2000). Among of them, strain hsn06 exhibited high flocculation activity on C. vulgaris through flocculation experiments. A nearly full-length 16S rRNA gene sequence (1429 nt) of strain hsn06 was determined, 16S rRNA gene sequence comparisons showed that strain hsn06 (GenBank accession number KY774315) was a member of the genus Streptomyces, sharing highest similarity (98.74 %) with Streptomyces parvulus NBRC 13193 (AB184326). The phylogenetic analysis of the strain hsn06 based on the 16S rRNA gene sequence indicated that this strain formed a distinct lineage with species Streptomyces parvulus NBRC 13193 (AB184326) (Fig. 1). Colonies of strain hsn06 on Gauserime synthetic agar medium were shiny, light yellow, circular with regular, dry edges and were 1-2 mm in diameter after 2 days incubation at 28 °C (Fig. S1a). The aerial mycelia of strain hsn06 were more thicker and deeper-color than substrate mycelium ((Fig. S1b). After the spores were inoculated into liquid medium, the hyphae grew quickly and eventually formed little pellets (Fig. S1c). Therefore, 10
according to the 16S rRNA gene sequence and morphological characteristics, strain hsn06 was a species of the genus Streptomyces, for which the name Streptomyces sp. hsn06 is proposed. The genus Streptomyces are mycelial multicellular soil actinomycetes (Bentley et al., 2002), which can produce many kinds of important secondary metabolites, as well as the source of most antibiotics (Ikeda et al., 2003). There have been reported that Streptomyces showed algicidal activity on algal cells (Zhang et al., 2014; Zhang et al., 2016) and also confirmed that Streptomyces could secrete bioflocculant (Nwodo et al., 2014; Shimofuruya et al., 1996). However, there have not been reported that the mycelial pellets of genus Streptomyces have flocculation activity on microalgal cells. Therefore, this study firstly evidence that genus Streptomyces with flocculation activity on C. vulgaris biomass, and investigate the flocculation mechanism. 3.2 Determination of flocculation activity The mycelial pellets of strain hsn06 after cultured in different culture media showed greatly different flocculation activity. As shown in Fig. S2a, the flocculation activity of mycelial pellets after cultured in flocculation medium reached high flocculation efficiency, and a large number of algal cells gathered around and inside the mycelium (Fig. S2a inset). However, the mycelial pellets after cultured in Gauserime synthetic medium did not show any flocculation activity on C. vulgaris cells, which was significantly (p<0.01) lower than that cultured in flocculation medium. Starch as the main component in Gauserime synthetic medium was more expensive than glucose which was the main component in flocculation medium, therefore, strain hsn06 which cultured in flocculation medium showed higher flocculation activity and lower cost than that cultured in Gauserime synthetic medium. Mycelial pellets of strain hsn06 showed high flocculation activity on algal cells with 11
an direct flocculation procedure, this is different from that most reported bioflocculants produced by Streptomyces (Nwodo et al., 2012; Zhang et al., 2013), which belonged to indirect flocculation activity. Therefore, the flocculation activity of Streptomyces sp. hsn06 on algal cells is a unique and scarcity mode, which need to further explore in the future. To determine suitable addition amount of mycelial pellets, different concentrations of mycelial pellets were added into algal culture (Fig. S2b). All the different concentrations of mycelial pellets after cultured in Gauserime synthetic medium showed obvious (p<0.01) low flocculation activity, however, different concentrations of mycelial pellets after cultured in flocculation medium showed different flocculation efficiency. 0.015 gmL-1 of mycelial pellets reached highest flocculation activity (77.5 %), which was significantly (p<0.01) higher than that of other concentrations of mycelial pellets, and was 1.57 times (p<0.01) and 1.32 times (p<0.01) those of 0.005 gmL-1 and 0.03 gmL-1. Therefore, concentration of 0.015 gmL-1 was the suitable addition amount for mycelial pellets to flocculate C. vulgaris biomass. Mycelial pellets after cultured in flocculation medium showed high flocculation activity, however, mycelial pellets after cultured in Gauserime synthetic medium lost flocculation activity, which implied that different culture media caused some differences during the formation of mycelial pellets. Therefore, mycelial pellets after cultured in the two different culture media were used to perform experiments at the same time to compare the distinctions and determine the flocculation mechanism. Mycelial pellets of Streptomyces sp. hsn06 showed high flocculation activity on algal cells with the biosafety, and the lipid of actinomycetes mycelium could improve the algal lipid yield, which is a good choice for harvesting microalgal biomass.
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3.3 Effect of calcium on flocculation activity Mycelial pellets showed flocculation activity on algal cells with adding final concentration of 5 mM CaCl2. To investigate effect of calcium on flocculation activity, flocculation activity was performed with and without adding CaCl2. As shown in Fig. S3a, mycelial pellets showed high flocculation activity on algal cells with adding calcium, however, mycelial pellets almost lost flocculation activity without adding calcium, which was only 0.009-fold compared to mycelial pellets with adding calcium. Therefore, calcium plays a vital role in flocculation activity of mycelial pellets. To further determine the effect of calcium on flocculation activity, different concentrations (0.1, 0.3, 0.5, 1, 2, 3, 4, and 5 mM) of calcium were added into algal cultures alongside with mycelial pellets (Fig. S3b). Mycelial pellets with adding low concentrations of calcium could not exhibit high flocculation activity, and high flocculation activity showed up since the concentration of calcium reached more than 1 mM, which flocculation activity were significantly (p<0.01) higher than that of low concentrations of calcium. The flocculation activity of mycelial pellets increased along with the increase of calcium, which indicated that mycelial pellets cannot show flocculation activity without calcium. Metal ions such as Ca2+, Fe3+, and Mg2+ always added into algal cultures as coagulants to significantly improve flocculation efficiency. Li et al. (2016) have reported that bioflocculant from Shinella albus xn-1 could be used to harvest C. vulgaris with adding metal ions as coagulants. Lei et al. (2015) added Ca2+ to increase the flocculation efficiency of bioflocculant for flotation of C. vulgaris. However, flocculating microorganism have also been reported to show flocculation activity without adding metal ions. Wan et al. (2013) have reported that the increasing concentrations of metal ions does not influence flocculation activity, and no metal ion was required by the bioflocculant to flocculate algal cells. Microbial 13
flocculation mechanism mainly belonged to charge neutralization and ions bridging (Vandamme et al., 2013). Charge neutralization can eliminate electrostatic repulsion between algal cells and microorganism with or without adding metal ions, however, ions bridging needs ions bound to algal cells and microorganism as a bridge (Powell and Hill, 2014). Therefore, to further determine flocculation mechanism of mycelial pellets, calcium binding assay should be performed. 3.4 Determination of calcium binding ability The calcium binding ability of mycelial pellets can further determine the flocculation mechanism, therefore calcium binding assay of mycelial pellets after cultured in flocculation medium and Gauserime synthetic medium were investigated, respectively. As shown in Fig. 2a, calcium binding ability of mycelial pellets with adding final concentration of 5 mM CaCl2 was treated by different times. Within 10 min of treatment time, calcium binding ability of mycelial pellets showed no difference between different culture media. However, the calcium binding ability of mycelial pellets after cultured in flocculation medium was significantly (p<0.01) higher than that in Gauserime synthetic medium within the 60 min of treatment time. When processing time increased to 120 min, there also showed no difference between different culture media. Within 180 min of treatment time, calcium binding ability of mycelial pellets showed great difference between different culture media, mycelial pellets after cultured in Gauserime synthetic medium showed significantly lower ability than that in flocculation medium, which implied that mycelial pellets after cultured in flocculation medium show high calcium binding ability, as well as high flocculation activity.
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To investigate calcium binding ability of mycelial pellets with different concentrations of CaCl2, different concentrations of calcium were added in mycelial pellets after cultured in different culture media (Fig. 2b). The mycelial pellets after cultured in Gauserime synthetic medium did not show any calcium binding ability until the concentration of CaCl2 exceeded 0.7 mM. Meanwhile, the mycelial pellets after cultured in flocculation medium could exhibit calcium binding ability when the concentration of CaCl2 was 0.1 mM, which indicated that mycelial pellets after cultured in flocculation medium possess more sensitive calcium binding ability. When the concentration of CaCl2 was lower than 3 mM, the mycelial pellets after cultured in different culture media did not show any obvious differences. However, the mycelial pellets after cultured in flocculation medium showed greatly (p<0.01) higher calcium binding ability than that in Gauserime synthetic medium with adding concentrations of 4 mM and 5 mM CaCl2. The mycelial pellets after cultured in flocculation medium showed high flocculation activity, and exhibited more higher calcium binding ability than mycelial pellets after cultured in Gauserime synthetic medium without flocculation activity. Therefore, calcium binding ability of mycelial pellets is crucial to flocculation activity, calcium plays the role of a bridge to connect mycelial pellets and microalgal cells, which indicated that ions bridging is mainly flocculation mechanism of mycelial pellets on C. vulgaris cells. Powell and Hill (2014) investigated the flocculation mechanism of Bacillus sp. Strain RP1137 on Nannochloropsis oceanica IMET1 through calcium binding assay, and the results showed that the cells still aggregated without addition of calcium, and the algal cells did not tightly bind calcium at their cell surface, which indicated that aggregation likely occurs via charge neutralization.
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To determine again the important of calcium to flocculation activity, the experiments with adding different concentrations of sodium carbonate to competitive bind calcium against mycelial pellets were performed. As shown in Fig. S4, flocculation activity of mycelial pellets declined with adding different concentrations of sodium carbonate, compared to positive control. The flocculation activities of mycelial pellets after adding sodium carbonate were significantly lower than that of control, which were 0.90 times (p<0.05), 0.64 times (p<0.01), and 0.69 times (p<0.01) those of the control with adding concentrations of 1, 3, and 5 gL-1. Sodium carbonate can bind calcium to form precipitation as well as decline of the calcium bind amount of mycelial pellets, and flocculation activity subsequently decreased, therefore, calcium bridging likely plays an important role in flocculation mechanism of mycelial pellets. 3.5 Characteristics of flocculation activity To determine the characteristics of flocculation activity of mycelial pellets on C. vulgaris cells, different treatments have been implemented on mycelial pellets. As shown in Fig. 3a, flocculation activities of mycelial pellets after treated under different temperatures were investigated. The flocculation activities of mycelial pellets after treated under different temperatures were significantly (p<0.01) decreased, compared to control. The flocculation rates of mycelial pellets after heating under 40, 60, 80, and 99 °C were 52.4, 60.1, 62.3, and 31.6 % of the control, respectively. The mycelial pellets after heating under 121 °C lost flocculation activity totally. The results indicated that mycelial pellets exhibit flocculation activity without thermal stability. In general, proteins will lost biological activity under high temperature condition. Sun et al. (2015) have reported that flocculation activity of 16
bioflocculant decreased after treated under 100 °C, which evidenced that the proteins contributed to the flocculation activity of bioflocculant. Proteins denaturation will also be caused after acid and alkali treatments, therefore, flocculation activity of mycelial pellets after treated by acid and alkali were determined (Fig. S5a). The flocculation activities of mycelial pellets after treated by acid and alkali were significantly (p<0.01) lower than positive control. Acid treatment induced complete inactivation of flocculation activity, and flocculation rate of mycelial pellets was 0.17 times (p<0.01) those of the control after alkali treatment, which implied that flocculation activity of mycelial pellets exhibited low pH stability. Tan et al. (2012) investigated the effect of heat-alkaline treatment on protein degradation, which indicated proteins were totally hydrolyzed at pH 13. However, proteins do not always lose activity under strong acid and alkaline treatment. Chen et al. (2016) have reported that the acid and alkali treatments could significantly increase the protein yield. Therefore, whether proteins participate in flocculation procedure of mycelial pellets need to further investigate. Proteins are also sensitive to sodium dodecyl sulfate (SDS) (Kwc and Sathe, 2002) and protease (Baytshtok et al., 2015), which can seriously damage secondary structure of proteins and cause protein denaturation. To determine the effect of SDS and protease on the flocculation activity, the SDS and protease treatment were performed. As shown in Fig. 3b, the flocculation activities of mycelial pellets after treated by SDS and protease were significantly (p<0.01) lower than that of the positive control. SDS and protease treatment could cause proteins denaturation as well as greatly decline of flocculation activity, which indicated proteins also contribute enormously to flocculation activity of mycelial pellets. To determine effect of the intact mycelial structure on flocculation activity, mycelial pellets after treated by lysozyme were used to investigate the flocculation activity (Fig. 3b). The results 17
showed that flocculation activity of mycelial pellets after treated by lysozyme was significantly (p<0.01) lower than that of control. Lysozyme can damage 1, 4-β-D-glucoside bonds and decompose insoluble polysaccharide in cell wall of mycelial pellets into soluble glycopeptide, thus destroy the cellular structure (Matthews, 1995). Mycelial structure was damaged by lysozyme as well as decline of flocculation activity, which indicated that the intact mycelial structure is vital to flocculation activity of mycelial pellets. To determine the molecular weight range of flocculation proteins in mycelial pellets, dialysis treatment was performed. As shown in Fig. S5b, the flocculation activity of mycelial pellets after dialysis treatment was significantly (p<0.01) lower than that of the positive control, which implied that the surface proteins could be dialyzed from the dialysis bag with a molecular intercept value of 8 kDa, and the molecular weight range of surface proteins was lower than 8 kDa. Proteins with small molecular weight maybe involve in flocculation procedure and contribute into flocculation activity. 3.6 Determination of flocculation procedure To determine the flocculation procedure of mycelial pellets on C. vulgaris cells, interactions between mycelium and algal cells were investigated under fluorescence microscope (Fig. S6). As shown in Fig. S6 a-d, the mycelium-microalgae complex with different flocculating times were sampled and observed. At the beginning of flocculation phenomenon (Fig. S6a), algal cells were only found aggregating around the surrounding of mycelial pellets, and cannot be found inside of mycelial pellets. However, algal cells gradually appeared into the inside of mycelial pellets with the increase of flocculating time, and twined by dense mycelial network structures (Fig. S6 b-c). With the increase of treatment time, the number of algal cells inside of mycelial pellets was increasing (Fig. S6d), suggesting that flocculation procedure of 18
mycelial pellets on algal cells is from external to internal, and can flocculate algal cells through internal complex mycelium with efficient utilization. According to Fig. 3b, intact mycelial structure is important to flocculation activity of mycelial pellets, and algal cells could be found inside of mycelial pellets. Therefore, the mycelial distinctions of mycelial pellets after cultured in flocculation medium and Gauserime synthetic medium were necessary to determine. As shown in Fig. S6 e-f, mycelium of mycelial pellets after cultured in flocculation medium and Gauserime synthetic medium were investigated under fluorescence microscope. The mycelial morphology in mycelial pellets after cultured between different culture media exhibited great differences, mycelia of mycelial pellets after cultured in flocculation medium were more thick than that of mycelial pellets after cultured in Gauserime synthetic medium. However, the mycelia of mycelial pellets after cultured in Gauserime synthetic medium were more dense than that of mycelial pellets after cultured in flocculation medium. All the results indicated that the thick mycelia of mycelial pellets maybe bind more algal cells, and the relatively sparse mycelial structure can promise algal cells to easily access inside, thus helping to achieve high flocculation activity of mycelial pellets after cultured in flocculation medium. The distinctions between mycelial pellets after cultured in different culture media caused complete different flocculation activity, which indicated that the morphology and structure of mycelial pellets is also the critical factor to flocculation activity.
4. Conclusions This study firstly confirmed that an actinomycete hsn06, which belonged to genus Streptomyces with high flocculation activity on C. vulgaris biomass through the 19
mycelial pellets with adding calcium ions. Calcium was crucial to flocculation activity, and determined that mycelial pellets bind calcium during flocculation procedure, which indicated calcium bridging is the main flocculation mechanism. Characteristics of flocculation activity could evidence proteins of mycelial pellets also contributed to flocculation activity. Finally, the unique morphology and structure of mycelial pellets were helpful to harvest algal biomass. Determination of flocculation mechanism can promise actinomycetes to be better application on microalgae biomass harvesting.
Acknowledgements: This work is supported by national science foundation of China (No. 51008119, 41576109), Doctoral Scientific Research Start-up Foundation of Henan Normal University (5101049170160) and the Key Scientific Research Programs of Henan Education Department (17A610002).
Reference 1. Baytshtok, V., Baker, T.A., Sauer, R.T., 2015. Assaying the kinetics of protein denaturation catalyzed by AAA+ unfolding machines and proteases. P. Natl. Acad. Sci. USA. 112(17), 5377. 2. Bentley, S.D., Chater, K.F., Cerdeñotárraga, A.M., Challis, G.L., Thomson, N.R., James, K.D., Harris, D.E., Quail, M.A., Kieser, H., Harper, D., 2002. Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417(6885), 141-147.
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3. Biggs, C.A., Lant, P.A., 2000. Activated sludge flocculation: on-line determination of floc size and the effect of shear. Water Res. 34(9), 2542-2550. 4. Biggs, S., Habgood, M., Jameson, G.J., Yan, Y.D., 2000. Aggregate structures formed via a bridging flocculation mechanism. Chem. Eng. J. 80(1), 13-22. 5. Cerff, M., Morweiser, M., Dillschneider, R., Michel, A., Menzel, K., Posten, C., 2012. Harvesting fresh water and marine algae by magnetic separation: screening of separation parameters and high gradient magnetic filtration. Bioresource Technol. 118(8), 289-295. 6. Chen, D., Song, J., Yang, H., Xiong, S., Liu, Y., Liu, R., 2016. Effects of acid and alkali treatment on the properties of proteins recovered from whole gutted grass carp (Ctenopharyngodon idellus) using isoelectric solubilization/precipitation. J. Food Quality 39(6), 707-713. 7. Delong, E.F., 1992. Archaea in coastal marine environments. P. Natl. Acad. Sci. USA. 89(12), 5685-5689. 8. Fong, S., Teo, E., Ng, L.F., Chen, C.B., Lakshmanan, L.N., Tsoi, S.Y., Moore, P.K., Inoue, T., Halliwell, B., Gruber, J., 2016. Energy crisis precedes global metabolic failure in a novel Caenorhabditis elegans Alzheimer Disease model. Sci. Rep. 6, 33781. 9. Hanna, M., Marcin, P., 2001. Stability of cyanotoxins, microcystin-LR, microcystin-RR and nodularin in seawater and BG-11 medium of different salinity. Oceanologia 43(3), 329-339. 10. Ikeda, H., Ishikawa, J., Hanamoto, A., Shinose, M., Kikuchi, H., Shiba, T., Sakaki, 21
Y., Hattori, M., Omacrmura, S., 2003. Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis. Nat. Biotechnol. 21(5), 526-531. 11. Kim, D.G., La, H.J., Ahn, C.Y., Park, Y.H., Oh, H.M., 2011. Harvest of Scenedesmus sp. with bioflocculant and reuse of culture medium for subsequent high-density cultures. Bioresource Technol. 102(3), 3163-3168. 12. Kumar, S., Tamura, K., Jakobsen, I.B., Nei, M., 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24(24), 1596-1599. 13. Kwc, S.T., Sathe, S.K., 2002. Effects of sodium dodecyl sulfate, guanidine hydrochloride, urea, and heat on denaturation of sulfur rich protein in soybeans (Glycine max L.). J. Food Biochem. 25(6), 483-492. 14. Lei, X., Yao, C., Shao, Z., Chen, Z., Yi, L., Hong, Z., Zhang, J., Wei, Z., Zheng, T., 2015. Effective harvesting of the microalgae Chlorella vulgaris via flocculation-flotation with bioflocculant. Bioresource Technol. 198, 922-925. 15. Li, Y., Xu, Y., Liu, L., Jiang, X., Zhang, K., Zheng, T., Wang, H., 2016. First evidence of bioflocculant from Shinella albus with flocculation activity on harvesting of Chlorella vulgaris biomass. Bioresource Technol. 218, 807-815. 16. Maeda, Y., Tateishi, T., Niwa, Y., Muto, M., Yoshino, T., Kisailus, D., Tanaka, T., 2016. Peptide-mediated microalgae harvesting method for efficient biofuel production. Biotechnology for Biofuels, 9(1), 10. 17. Matthews, B.W., 1995. Studies on protein stability with T4 lysozyme. Adv. 22
Protein Chem. 46, 249-278. 18. Nwodo, U.U., Agunbiade, M.O., Ezekiel, G., Mabinya, L.V., Okoh, A.I., 2012. A freshwater Streptomyces, isolated from Tyume River, produces a predominantly extracellular glycoprotein bioflocculant. Int. J. Mol. Sci. 13(7), 8679-8695. 19. Nwodo, U.U., Green, E., Mabinya, L.V., Okaiyeto, K., Rumbold, K., Obi, L.C., Okoh, A.I., 2014. Bioflocculant production by a consortium of Streptomyces and Cellulomonas species and media optimization via surface response model. Colloid. Surface. B 116(2), 257-264. 20. Powell, R.J., Hill, R.T., 2014. Mechanism of algal aggregation by Bacillus sp. strain RP1137. Appl. Environ. Microb. 80(13), 4042-4050. 21. Powell, R.J., Hill, R.T., 2013. Rapid aggregation of biofuel-producing algae by the bacterium Bacillus sp. Strain RP1137. Appl. Environ. Microb. 79(19), 6093-101. 22. Ries, H.E., Meyers, B.L., 1968. Flocculation mechanism: charge neutralization and bridging. Science 160(3835), 1449-1450. 23. Rwehumbiza, V.M., Harrison, R., Thomsen, L., 2012. Alum-induced flocculation of preconcentrated Nannochloropsis salina: residual aluminium in the biomass, FAMEs and its effects on microalgae growth upon media recycling. Chem. Eng. J. 200(16), 168-175. 24. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4(4), 406-425. 25. Shimofuruya, H., Koide, A., Shirota, K., Tsuji, T., Nakamura, M., Suzuk, J., 1996. The production of flocculating substance(s) by Streptomyces griseus. Biosci. 23
Biotech. Bioch. 60(3), 498-500. 26. Sun, P., Hui, C., Bai, N., Yang, S., Wan, L., Zhang, Q., Zhao, Y.H., 2015. Revealing the characteristics of a novel bioflocculant and its flocculation performance in Microcystis aeruginosa removal. Sci. Rep. 5, 17465. 27. Tan, R., Miyanaga, K., Uy, D., Tanji, Y., 2012. Effect of heat-alkaline treatment as a pretreatment method on volatile fatty acid production and protein degradation in excess sludge, pure proteins and pure cultures. Bioresource Technol. 118(8), 390-398. 28. Vandamme, D., Foubert, I., Muylaert, K., 2013. Flocculation as a low-cost method for harvesting microalgae for bulk biomass production. Trends Biotechnol. 31(4), 233. 29. Wan, C., Zhao, X.Q., Guo, S.L., Asraful, A.M., Bai, F.W., 2013. Bioflocculant production from Solibacillus silvestris W01 and its application in cost-effective harvest of marine microalga Nannochloropsis oceanica by flocculation. Bioresource Technol. 135(3), 207-212. 30. Weschler, M.K., Barr, W.J., Harper, W.F., Landis, A.E., 2014. Process energy comparison for the production and harvesting of algal biomass as a biofuel feedstock. Bioresource Technol. 153(153C), 108-115. 31. Wijffels, R.H., Barbosa, M.J., 2010. An outlook on microalgal biofuels. Science 329(5993), 796-799. 32. Yang, Z., Yang, H., Jiang, Z., Cai, T., Li, H., Li, H., Li, A., Cheng, R., 2013. Flocculation of both anionic and cationic dyes in aqueous solutions by the 24
amphoteric grafting flocculant carboxymethyl chitosan-graft-polyacrylamide. J. Hazard. Mater. 255(254-255C), 36-45. 33. Yin, Y.J., Tian, Z.M., Tang, W., Li, L., Song, L.Y., Mcelmurry, S.P., 2014. Production and characterization of high efficiency bioflocculant isolated from Klebsiella sp. ZZ-3. Bioresource Technol. 171C, 336-342. 34. Zhang, B., Cai, G., Wang, H., Li, D., Yang, X., An, X., Zheng, X., Tian, Y., Zheng, W., Zheng, T., 2014. Streptomyces alboflavus RPS and its novel and high algicidal activity against harmful algal bloom species Phaeocystis globosa. Plos One 9(3), e92907. 35. Zhang, B.H., Ding, Z.G., Li, H.Q., Mou, X.Z., Zhang, Y.Q., Yang, J.Y., Zhou, E.M., Li, W.J., 2016. Algicidal activity of metabolites from Streptomyces eurocidicus JXJ-0089 and their effects in the physiology of Microcystis. Appl. Environ. Microb. 82(17), 5132-5143. 36. Zhang, C.L., Cui, Y.N., Wang, Y., 2013. Bioflocculants produced by Gram-positive Bacillus xn12 and Streptomyces xn17 for swine wastewater application. Chem. Biochem. Eng. Q. 27(2), 245-250. 37. Zhang, X., Hu, Q., Sommerfeld, M., Puruhito, E., Chen, Y., 2010. Harvesting algal biomass for biofuels using ultrafiltration membranes. Bioresource Technol. 101(14), 5297-5304.
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Figure captions: Fig. 1 Neighbour-joining tree showing the phylogenetic positions of strain hsn06T and representatives of some other related taxa, based on 16S rRNA gene sequences. Bootstrap values (expressed as percentages of 1000 replications) are shown at branch points; only values >50 % are shown. Bar, 0.0005 nt substitution rate (Knuc) units. Fig. 2 Calcium binding assay of mycelial pellets after cultured in flocculation medium and Gauserime synthetic medium with different flocculating times (a), different concentrations of calcium (b). All error bars indicate the SE of the three biological replicates. *Represents a statistically significant difference at p<0.05 compared with positive control; **represents a statistically significant difference of p<0.01. Fig. 3 Effect of different temperatures (a), and SDS, proteinase K digestion, lysozyme treatment (b) on flocculation activity. All error bars indicate the SE of the three biological replicates. **represents a statistically significant difference of p<0.01 compared with positive control.
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Flocculation mechanism of the actinomycete Streptomyces sp. hsn06 on Chlorella vulgaris Yi Li1, 2, Yanting Xu1, Tianling Zheng2 and Hailei Wang1* 1
College of Life Sciences, Henan Normal University, Xinxiang 453007, China
2
State Key Laboratory of Marine Environmental Science, School of Life Sciences,
Xiamen University, Xiamen 361005, China *Corresponding author: Tel.: +86 3733326340; Fax: +86 3733326916. E-mail address: [email protected] (H. Wang)
Highlights: 1. Streptomyces was firstly used to harvest Chlorella vulgaris biomass; 2. Calcium bridging is the main flocculation mechanism; 3. Proteins in mycelial pellets involved in flocculation procedure; 4. Mycelial morphology and structure caused dramatic effects on flocculation activity.
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S0960-8524(17)30667-3 http://dx.doi.org/10.1016/j.biortech.2017.05.028 BITE 18054
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
30 March 2017 3 May 2017 4 May 2017
Please cite this article as: Li, Y., Xu, Y., Zheng, T., Wang, H., Flocculation mechanism of the actinomycete Streptomyces sp. hsn06 on Chlorella vulgaris, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/ j.biortech.2017.05.028
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Flocculation mechanism of the actinomycete Streptomyces sp. hsn06 on Chlorella vulgaris Yi Li1, Yanting Xu1, Tianling Zheng2 and Hailei Wang1* 1
College of Life Sciences, Henan Normal University, Xinxiang 453007, China
2
State Key Laboratory of Marine Environmental Science, School of Life Sciences,
Xiamen University, Xiamen 361005, China *Corresponding author: Tel.: +86 3733326340; Fax: +86 3733326916. E-mail address: [email protected] (H. Wang)
Abstract In this study, an actinomycete Streptomyces sp. hsn06 with the ability to harvest Chlorella vulgaris biomass was used to investigate the flocculation mechanism. Streptomyces sp. hsn06 exhibited flocculation activity on algal cells through mycelial pellets with adding calcium. Calcium was determined to promote flocculation activity of mycelial pellets as a bridge binding with mycelial pellets and algal cells, which implied that calcium bridging is the main flocculation mechanism for mycelial pellets. Characteristics of flocculation activity confirmed proteins in mycelial pellets involved in flocculation procedure. The morphology and structure of mycelial pellets also caused dramatic effects on flocculation activity of mycelial pellets. According to the results, Streptomyces sp. hsn06 can be used as a novel flocculating microbial resource for high-efficiency harvesting of microalgae biomass.
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Keywords Streptomyces sp. hsn06; Chlorella vulgaris; Mycelial pellets; Flocculation mechanism
1. Introduction Energy crisis is threatening human existence because of the excessive exploitation and the environment damage (Fong et al., 2016). Microalgal biofuels are very importance to alleviate energy crisis and in favor of environmental protection, which have caused increasing attention (Wijffels and Barbosa, 2010). However, the development of microalgae industrialization is mainly limited by algal cultivation and harvesting of algal biomass (Maeda et al., 2016). Recently, microalgal cultivation obtains effective improvement based on optimization of photobioreactor, breeding of high oil microalgae, and genetic engineering modification (Vandamme et al., 2013). Meanwhile, harvesting of microalgae biomass becomes the greatest bottleneck for microalgal development (Weschler et al., 2014). To harvest microalgae biomass, various methods including addition of chemical flocculants, physical flocculation, and bioflocculation have been implemented (Zhang et al., 2010). Chemical flocculants were always used to sewage treatment, mining and other industrial flocculation, however, chemical flocculant would pose threat to algal biomass, once performed to harvest algal biomass (Rwehumbiza et al., 2012). The physical flocculation can avoid the pollution of algae biomass, however, its high cost limits the application on harvesting algal biomass (Cerff et al., 2012). Compared with these methods, bioflocculation especially microbial flocculation is considered to be 2
one of the most promising method (Powell and Hill, 2013). Many types of microorganisms including bacteria and fungi with flocculation activity on microalgae biomass have been isolated (Kim et al., 2011; Yin et al., 2014), however, flocculating actinomycetes are relatively unexplored. The flocculation mechanism of flocculating actinomycetes on microalgae biomass was also barely reported. Microbiol flocculation mechanisms have been proposed a lot of hypothesizes, charge neutralization and ions bridging of which are generally accepted (Ries and Meyers, 1968). Charge neutralization eliminates electrostatic repulsion between cells, and particles will coagulate or flocculate (Yang et al., 2013). Ions bridging requires that the ions simultaneously bind to different particles to form a bridge between them, this bridge brings the particles together and causes flocculation (Biggs et al., 2000). In this study, Streptomyces sp. hsn06 which belongs to a large genus of actinomycetes was isolated and firstly confirmed with the ability to harvest Chlorella vulgaris (C. vulgaris) biomass. The flocculation activity of strain hsn06 on C. vulgaris was investigated, flocculation mechanism was determined, the effect of flocculation conditions on flocculation activity were confirmed, and the flocculation procedure was observed. The objectives of this study were to explore new type of flocculating microorganism, and investigate the flocculation mechanism, which supply a novel choice to harvest microalgae biomass.
2. Materials and methods
2.1 Algal culture 3
The algae C. vulgaris was supplied by Freshwater Algae Culture Collection at the Institute of Hydrobiology (Wuhan, China). The C. vulgaris strain was cultivated in BG-11 medium (Hanna and Marcin, 2001) under a 12:12 h light-dark cycle with a light intensity of 50 mol photons m-2 s-1 at 25 °C. 2.2 Isolation and identification of flocculation actinomycete The activated sludge samples were used to isolate flocculating actinomycetes, and then serially diluted (10-fold) using sterile distilled water, and 0.1 mL aliquots of each dilution were spread onto Gauserime synthetic agar medium (Soluble starch, 20 g; KNO3, 1 g; K2HPO4·3H2O, 0.5 g; MgSO4·7H2O, 0.5 g; NaCl, 0.5 g; FeSO4·7H2O, 0.01 g; agar, 20 g in 1 L distilled water; pH 7.4-7.6) followed by incubation for 7 days at 28 °C. Individual colonies of distinct morphology were further purified three times, and inoculated in flocculation medium (glucose, 10 g; NaCl, 24 g; NH4Cl, 1 g; MgSO47H2O, 0.5 g; yeast extract, 0.6 g; K2HPO43H2O, 6.5 g and KH2PO4, 2 g in 1 L distilled water) to determine the flocculation efficiency, and the strain hsn06 with high flocculation activity on C. vulgaris was isolated. Strain hsn06 was cultured in Gauserime synthetic medium for 2 days at 28 °C with shaking at 120 rpm. Mycelium were obtained and resuspended in STE solution buffer (Tris-HCl with the pH 8.0, 20 mM; EDTA, 25mM; NaCl, 75 mM) containing lysozyme with final concentration of 1 gL-1. Proteins were removed by phenol-chloroform-isoamyl alcohol (25:24:1) extraction. After isopropanol precipitation, DNA was resuspended in sterile water (50 mL) and stored at -20 °C until further analysis. The 16S rRNA gene sequence of strain hsn06 was amplified 4
using PCR with primers 27F and 1492R (Delong, 1992). Purification of the PCR product was cloned into vector pMD19-T and sequenced. Sequences of related taxa were obtained from the GenBank database and EZBioCloud server (http://www.ezbiocloud.net/identify). Phylogenetic analysis was performed using MEGA version 4 (Kumar et al., 2007) after multiple alignment of data by ClustalX (version 1.83). Evolutionary distances and clustering were constructed using the neighbor-joining method (Saitou and Nei, 1987), and were evaluated using bootstrap values based on 1000 replications. 2.3 Flocculation activity of strain hsn06 Strain hsn06 was cultured in flocculation medium and Gauserime synthetic medium for 2 days at 28 °C with shaking at 120 rpm, respectively. Both of the two culture media could promote strain hsn06 to form mycelial pellets, and then mycelial pellets were added into C. vulgaris cultures to investigate flocculation activity. The normal growth algae were set as control. Final concentration of 5 mM CaCl2 was added in every treatment groups and control, followed by mixing at 120 rpm, and then the mixing was stopped to let the mixture settle at 25 °C. After the flocculation of the algal cells, an aliquot of the culture in treatment groups and control was pipetted from a height of two-thirds from the bottom for the evaluation of the flocculation effect using a spectrometer. All the treatment groups and control group were carried out in triplicate. The flocculation activity was calculated according to the equation 1: (1) 5
where A and B are the absorbency (OD) at 680 nm of the microalgal culture in control and treatment groups, respectively. To investigate the effect of different additive amounts on the flocculation activity, mycelial pellets which cultured in flocculation medium and Gauserime synthetic medium with concentrations of 0.005, 0.015 and 0.03 gmL-1 were added into C. vulgaris cultures, respectively. Control group was normal growth algae. All the treatment groups and control groups were added with final concentration of 5 mM CaCl2 and carried out in triplicate. The flocculation activity of each treatment groups was determined according to equation 1. 2.4 Effect of calcium on flocculation activity 0.015 gmL-1 of mycelial pellets after cultured in flocculation medium were added into C. vulgaris cultures with or without adding final concentration of 5 mM CaCl2, to investigate the effect of calcium on flocculation activity. Control groups were normal growth algae with or without adding same concentration of CaCl2. Mycelial pellets with adding different concentrations (0.1, 0.3, 0.5, 1, 2, 3, 4, and 5 mM) of CaCl2 were tested as cationic coagulants for the flocculation of C. vulgaris culture. Control groups were normal growth algae with adding same concentrations of CaCl2. The flocculation activities were calculated according to the equation 1. All the treatment groups and control groups were carried out in triplicate. 2.5 Calcium binding assay Calcium binding was evaluated by measuring the concentration of the remaining calcium after the addition of mycelial pellets which were cultured in flocculation 6
medium and Gauserime synthetic medium or algal cells. Mycelial pellets and algal cells were harvested by centrifugation at 3,000g for 5 min, and then washed twice by sterile deionized water. To investigate the effect of binding time on calcium binding efficiency, final concentration of 5 mM CaCl2 was added to cells in pH 10 deionized water, followed by shaking at 120 rpm for different times including 10, 60, 120, and 180 min at room temperature. After binding time determination, the effect of calcium concentrations on calcium binding efficiency were evaluated by adding known concentrations of CaCl2 (final concentrations of 0.1, 0.3, 0.5, 0.7, 1, 2, 3, 4 and 5 mM) to cells in pH 10 deionized water, followed by shaking at 120 rpm at room temperature. Mycelial pellets with adding CaCl2 were treatment groups as well as adding CaCl2 to algal cells as control. Cells were then removed by centrifugation at 10,000 g for 3 min. The calcium concentration in the supernatant was measured using the Calcium Assay Kit (Nanjing Jiancheng Bioengineering Institute, China). The kit was adapted for use in a 96-well format and measured in a Spectramax M5 plate reader. The readout for the assay was at an absorbance of 610 nm. Concentration of calcium standard solution was 2.5 mM, and deionized water was added as control group. Concentration of the remaining calcium could be calculated according to the kit’s Operation Manual from Nanjing Jiancheng Bioengineering Institute, China. To further investigate whether the decline of calcium concentration could influence the flocculation activity during the flocculation procedure, different concentrations (1, 3, and 5 gL-1) of sodium carbonate were added into algal cultures after adding final concentration of 5 mM CaCl2. The control groups were normal growth algae with 7
adding final concentration of 5 mM CaCl2 and same concentrations of sodium carbonate. All the treatment groups and control groups were carried out in triplicate. 2.6 Characteristics of the flocculation activity To determine the effect of different temperatures on flocculation activity, flocculation activity was investigated after mycelial pellets treated under different temperatures. 0.015 gmL-1 of mycelial pellets were incubated at 40, 60, 80, 99 and 121 °C for 2 h, respectively. Then, the treated mycelial pellets were added into 10 mL of algal culture after cooling to room temperature. Control group was normal growth algae. The pH stability of flocculation activity was tested using acid and alkali treatments on mycelial pellets. The mycelial pellets were added into solutions at pH 3 and 12 for 2 h, and then the treated mycelial pellets were used to determine flocculation efficiency. Mycelial pellets of strain hsn06 were digested with 100 mgL-1 proteinase K in sterile BG11 at pH 7.4 for 2 h at room temperature, and then washed twice with sterile BG11 to remove the proteinase K and then tested the flocculation activity. 0.015 gmL-1 of mycelial pellets were inoculated with adding 3 gL-1 of lysozyme for 2 h at room temperature, and then washed twice with sterile BG11 to remove the proteinase K and then tested the flocculation activity. Inhibition of flocculation activity by sodium dodecyl sulfate (SDS) was tested by adding SDS into algal culture with a final concentration of 1 mM. 0.015 gmL-1 of mycelial pellets were added into algal culture with adding SDS. Control group was normal growth algae with adding 1mM SDS. Mycelial pellets of strain hsn06 were loaded into dialysis bags with molecular intercept values of 8 kD and dialyzed in sterile distilled 8
water for 48 h to perform the dialysis treatment. All the treatment groups and control group were carried out in triplicate. Final concentration of 5 mM CaCl2 was added in every treatment groups and controls, followed by mixing at 120 rpm at room temperature. All the treatment groups and control groups were carried out in triplicate. The flocculation activity of each treatment groups was determined according to equation 1. 2.7 Microscopy observation Microscopic observation using a fluorescence microscope was performed to investigate the interaction between the algal cells and mycelium, and compare the mycelial morphology of mycelial pellets which were cultured in flocculation medium and Gauserime synthetic medium, respectively. Fluorescence microscopy was performed after fluorescent staining. The mycelium-algae complex and two pieces of mycelium which cultured in flocculation medium and Gauserime synthetic medium were picked up and put on each glass slide, and stained with Calcofluor White Stain (0.1 %) (Sigma-Aldrich) and 10 % potassium hydroxide for 1 min in the dark. Covered with the clean cover slip and examined under a fluorescence microscope (Olympus BX63, Chiyoda-ku, Tokyo, Japan), equipped with a epifluorescent filter. The Calcofluor White stained mycelium and algal chlorophyll were excited with a 355- and 488-nm argon laser, respectively. The signal for the Calcofluor White-stained mycelium and chlorophyll autofluorescence were visualized using 440 and 600-nm long-pass filters, respectively. 2.8 Statistics 9
All data were presented as mean ± standard error and were evaluated using one-way analysis of variance followed by the least significant difference test, with p<0.01 and p<0.05 (Origin 8.5 for Windows).
3. Results and discussion 3.1 Characterization and identification of strain A total of 12 bacterial strains, 6 fungal strains, and 7 actinomycetes strains were isolated from active sludge samples. Active sludge contains various microbial community and supplies organic and inorganic nutrients which always deems as the source of flocculation microorganism (Biggs and Lant, 2000). Among of them, strain hsn06 exhibited high flocculation activity on C. vulgaris through flocculation experiments. A nearly full-length 16S rRNA gene sequence (1429 nt) of strain hsn06 was determined, 16S rRNA gene sequence comparisons showed that strain hsn06 (GenBank accession number KY774315) was a member of the genus Streptomyces, sharing highest similarity (98.74 %) with Streptomyces parvulus NBRC 13193 (AB184326). The phylogenetic analysis of the strain hsn06 based on the 16S rRNA gene sequence indicated that this strain formed a distinct lineage with species Streptomyces parvulus NBRC 13193 (AB184326) (Fig. 1). Colonies of strain hsn06 on Gauserime synthetic agar medium were shiny, light yellow, circular with regular, dry edges and were 1-2 mm in diameter after 2 days incubation at 28 °C (Fig. S1a). The aerial mycelia of strain hsn06 were more thicker and deeper-color than substrate mycelium ((Fig. S1b). After the spores were inoculated into liquid medium, the hyphae grew quickly and eventually formed little pellets (Fig. S1c). Therefore, 10
according to the 16S rRNA gene sequence and morphological characteristics, strain hsn06 was a species of the genus Streptomyces, for which the name Streptomyces sp. hsn06 is proposed. The genus Streptomyces are mycelial multicellular soil actinomycetes (Bentley et al., 2002), which can produce many kinds of important secondary metabolites, as well as the source of most antibiotics (Ikeda et al., 2003). There have been reported that Streptomyces showed algicidal activity on algal cells (Zhang et al., 2014; Zhang et al., 2016) and also confirmed that Streptomyces could secrete bioflocculant (Nwodo et al., 2014; Shimofuruya et al., 1996). However, there have not been reported that the mycelial pellets of genus Streptomyces have flocculation activity on microalgal cells. Therefore, this study firstly evidence that genus Streptomyces with flocculation activity on C. vulgaris biomass, and investigate the flocculation mechanism. 3.2 Determination of flocculation activity The mycelial pellets of strain hsn06 after cultured in different culture media showed greatly different flocculation activity. As shown in Fig. S2a, the flocculation activity of mycelial pellets after cultured in flocculation medium reached high flocculation efficiency, and a large number of algal cells gathered around and inside the mycelium (Fig. S2a inset). However, the mycelial pellets after cultured in Gauserime synthetic medium did not show any flocculation activity on C. vulgaris cells, which was significantly (p<0.01) lower than that cultured in flocculation medium. Starch as the main component in Gauserime synthetic medium was more expensive than glucose which was the main component in flocculation medium, therefore, strain hsn06 which cultured in flocculation medium showed higher flocculation activity and lower cost than that cultured in Gauserime synthetic medium. Mycelial pellets of strain hsn06 showed high flocculation activity on algal cells with 11
an direct flocculation procedure, this is different from that most reported bioflocculants produced by Streptomyces (Nwodo et al., 2012; Zhang et al., 2013), which belonged to indirect flocculation activity. Therefore, the flocculation activity of Streptomyces sp. hsn06 on algal cells is a unique and scarcity mode, which need to further explore in the future. To determine suitable addition amount of mycelial pellets, different concentrations of mycelial pellets were added into algal culture (Fig. S2b). All the different concentrations of mycelial pellets after cultured in Gauserime synthetic medium showed obvious (p<0.01) low flocculation activity, however, different concentrations of mycelial pellets after cultured in flocculation medium showed different flocculation efficiency. 0.015 gmL-1 of mycelial pellets reached highest flocculation activity (77.5 %), which was significantly (p<0.01) higher than that of other concentrations of mycelial pellets, and was 1.57 times (p<0.01) and 1.32 times (p<0.01) those of 0.005 gmL-1 and 0.03 gmL-1. Therefore, concentration of 0.015 gmL-1 was the suitable addition amount for mycelial pellets to flocculate C. vulgaris biomass. Mycelial pellets after cultured in flocculation medium showed high flocculation activity, however, mycelial pellets after cultured in Gauserime synthetic medium lost flocculation activity, which implied that different culture media caused some differences during the formation of mycelial pellets. Therefore, mycelial pellets after cultured in the two different culture media were used to perform experiments at the same time to compare the distinctions and determine the flocculation mechanism. Mycelial pellets of Streptomyces sp. hsn06 showed high flocculation activity on algal cells with the biosafety, and the lipid of actinomycetes mycelium could improve the algal lipid yield, which is a good choice for harvesting microalgal biomass.
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3.3 Effect of calcium on flocculation activity Mycelial pellets showed flocculation activity on algal cells with adding final concentration of 5 mM CaCl2. To investigate effect of calcium on flocculation activity, flocculation activity was performed with and without adding CaCl2. As shown in Fig. S3a, mycelial pellets showed high flocculation activity on algal cells with adding calcium, however, mycelial pellets almost lost flocculation activity without adding calcium, which was only 0.009-fold compared to mycelial pellets with adding calcium. Therefore, calcium plays a vital role in flocculation activity of mycelial pellets. To further determine the effect of calcium on flocculation activity, different concentrations (0.1, 0.3, 0.5, 1, 2, 3, 4, and 5 mM) of calcium were added into algal cultures alongside with mycelial pellets (Fig. S3b). Mycelial pellets with adding low concentrations of calcium could not exhibit high flocculation activity, and high flocculation activity showed up since the concentration of calcium reached more than 1 mM, which flocculation activity were significantly (p<0.01) higher than that of low concentrations of calcium. The flocculation activity of mycelial pellets increased along with the increase of calcium, which indicated that mycelial pellets cannot show flocculation activity without calcium. Metal ions such as Ca2+, Fe3+, and Mg2+ always added into algal cultures as coagulants to significantly improve flocculation efficiency. Li et al. (2016) have reported that bioflocculant from Shinella albus xn-1 could be used to harvest C. vulgaris with adding metal ions as coagulants. Lei et al. (2015) added Ca2+ to increase the flocculation efficiency of bioflocculant for flotation of C. vulgaris. However, flocculating microorganism have also been reported to show flocculation activity without adding metal ions. Wan et al. (2013) have reported that the increasing concentrations of metal ions does not influence flocculation activity, and no metal ion was required by the bioflocculant to flocculate algal cells. Microbial 13
flocculation mechanism mainly belonged to charge neutralization and ions bridging (Vandamme et al., 2013). Charge neutralization can eliminate electrostatic repulsion between algal cells and microorganism with or without adding metal ions, however, ions bridging needs ions bound to algal cells and microorganism as a bridge (Powell and Hill, 2014). Therefore, to further determine flocculation mechanism of mycelial pellets, calcium binding assay should be performed. 3.4 Determination of calcium binding ability The calcium binding ability of mycelial pellets can further determine the flocculation mechanism, therefore calcium binding assay of mycelial pellets after cultured in flocculation medium and Gauserime synthetic medium were investigated, respectively. As shown in Fig. 2a, calcium binding ability of mycelial pellets with adding final concentration of 5 mM CaCl2 was treated by different times. Within 10 min of treatment time, calcium binding ability of mycelial pellets showed no difference between different culture media. However, the calcium binding ability of mycelial pellets after cultured in flocculation medium was significantly (p<0.01) higher than that in Gauserime synthetic medium within the 60 min of treatment time. When processing time increased to 120 min, there also showed no difference between different culture media. Within 180 min of treatment time, calcium binding ability of mycelial pellets showed great difference between different culture media, mycelial pellets after cultured in Gauserime synthetic medium showed significantly lower ability than that in flocculation medium, which implied that mycelial pellets after cultured in flocculation medium show high calcium binding ability, as well as high flocculation activity.
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To investigate calcium binding ability of mycelial pellets with different concentrations of CaCl2, different concentrations of calcium were added in mycelial pellets after cultured in different culture media (Fig. 2b). The mycelial pellets after cultured in Gauserime synthetic medium did not show any calcium binding ability until the concentration of CaCl2 exceeded 0.7 mM. Meanwhile, the mycelial pellets after cultured in flocculation medium could exhibit calcium binding ability when the concentration of CaCl2 was 0.1 mM, which indicated that mycelial pellets after cultured in flocculation medium possess more sensitive calcium binding ability. When the concentration of CaCl2 was lower than 3 mM, the mycelial pellets after cultured in different culture media did not show any obvious differences. However, the mycelial pellets after cultured in flocculation medium showed greatly (p<0.01) higher calcium binding ability than that in Gauserime synthetic medium with adding concentrations of 4 mM and 5 mM CaCl2. The mycelial pellets after cultured in flocculation medium showed high flocculation activity, and exhibited more higher calcium binding ability than mycelial pellets after cultured in Gauserime synthetic medium without flocculation activity. Therefore, calcium binding ability of mycelial pellets is crucial to flocculation activity, calcium plays the role of a bridge to connect mycelial pellets and microalgal cells, which indicated that ions bridging is mainly flocculation mechanism of mycelial pellets on C. vulgaris cells. Powell and Hill (2014) investigated the flocculation mechanism of Bacillus sp. Strain RP1137 on Nannochloropsis oceanica IMET1 through calcium binding assay, and the results showed that the cells still aggregated without addition of calcium, and the algal cells did not tightly bind calcium at their cell surface, which indicated that aggregation likely occurs via charge neutralization.
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To determine again the important of calcium to flocculation activity, the experiments with adding different concentrations of sodium carbonate to competitive bind calcium against mycelial pellets were performed. As shown in Fig. S4, flocculation activity of mycelial pellets declined with adding different concentrations of sodium carbonate, compared to positive control. The flocculation activities of mycelial pellets after adding sodium carbonate were significantly lower than that of control, which were 0.90 times (p<0.05), 0.64 times (p<0.01), and 0.69 times (p<0.01) those of the control with adding concentrations of 1, 3, and 5 gL-1. Sodium carbonate can bind calcium to form precipitation as well as decline of the calcium bind amount of mycelial pellets, and flocculation activity subsequently decreased, therefore, calcium bridging likely plays an important role in flocculation mechanism of mycelial pellets. 3.5 Characteristics of flocculation activity To determine the characteristics of flocculation activity of mycelial pellets on C. vulgaris cells, different treatments have been implemented on mycelial pellets. As shown in Fig. 3a, flocculation activities of mycelial pellets after treated under different temperatures were investigated. The flocculation activities of mycelial pellets after treated under different temperatures were significantly (p<0.01) decreased, compared to control. The flocculation rates of mycelial pellets after heating under 40, 60, 80, and 99 °C were 52.4, 60.1, 62.3, and 31.6 % of the control, respectively. The mycelial pellets after heating under 121 °C lost flocculation activity totally. The results indicated that mycelial pellets exhibit flocculation activity without thermal stability. In general, proteins will lost biological activity under high temperature condition. Sun et al. (2015) have reported that flocculation activity of 16
bioflocculant decreased after treated under 100 °C, which evidenced that the proteins contributed to the flocculation activity of bioflocculant. Proteins denaturation will also be caused after acid and alkali treatments, therefore, flocculation activity of mycelial pellets after treated by acid and alkali were determined (Fig. S5a). The flocculation activities of mycelial pellets after treated by acid and alkali were significantly (p<0.01) lower than positive control. Acid treatment induced complete inactivation of flocculation activity, and flocculation rate of mycelial pellets was 0.17 times (p<0.01) those of the control after alkali treatment, which implied that flocculation activity of mycelial pellets exhibited low pH stability. Tan et al. (2012) investigated the effect of heat-alkaline treatment on protein degradation, which indicated proteins were totally hydrolyzed at pH 13. However, proteins do not always lose activity under strong acid and alkaline treatment. Chen et al. (2016) have reported that the acid and alkali treatments could significantly increase the protein yield. Therefore, whether proteins participate in flocculation procedure of mycelial pellets need to further investigate. Proteins are also sensitive to sodium dodecyl sulfate (SDS) (Kwc and Sathe, 2002) and protease (Baytshtok et al., 2015), which can seriously damage secondary structure of proteins and cause protein denaturation. To determine the effect of SDS and protease on the flocculation activity, the SDS and protease treatment were performed. As shown in Fig. 3b, the flocculation activities of mycelial pellets after treated by SDS and protease were significantly (p<0.01) lower than that of the positive control. SDS and protease treatment could cause proteins denaturation as well as greatly decline of flocculation activity, which indicated proteins also contribute enormously to flocculation activity of mycelial pellets. To determine effect of the intact mycelial structure on flocculation activity, mycelial pellets after treated by lysozyme were used to investigate the flocculation activity (Fig. 3b). The results 17
showed that flocculation activity of mycelial pellets after treated by lysozyme was significantly (p<0.01) lower than that of control. Lysozyme can damage 1, 4-β-D-glucoside bonds and decompose insoluble polysaccharide in cell wall of mycelial pellets into soluble glycopeptide, thus destroy the cellular structure (Matthews, 1995). Mycelial structure was damaged by lysozyme as well as decline of flocculation activity, which indicated that the intact mycelial structure is vital to flocculation activity of mycelial pellets. To determine the molecular weight range of flocculation proteins in mycelial pellets, dialysis treatment was performed. As shown in Fig. S5b, the flocculation activity of mycelial pellets after dialysis treatment was significantly (p<0.01) lower than that of the positive control, which implied that the surface proteins could be dialyzed from the dialysis bag with a molecular intercept value of 8 kDa, and the molecular weight range of surface proteins was lower than 8 kDa. Proteins with small molecular weight maybe involve in flocculation procedure and contribute into flocculation activity. 3.6 Determination of flocculation procedure To determine the flocculation procedure of mycelial pellets on C. vulgaris cells, interactions between mycelium and algal cells were investigated under fluorescence microscope (Fig. S6). As shown in Fig. S6 a-d, the mycelium-microalgae complex with different flocculating times were sampled and observed. At the beginning of flocculation phenomenon (Fig. S6a), algal cells were only found aggregating around the surrounding of mycelial pellets, and cannot be found inside of mycelial pellets. However, algal cells gradually appeared into the inside of mycelial pellets with the increase of flocculating time, and twined by dense mycelial network structures (Fig. S6 b-c). With the increase of treatment time, the number of algal cells inside of mycelial pellets was increasing (Fig. S6d), suggesting that flocculation procedure of 18
mycelial pellets on algal cells is from external to internal, and can flocculate algal cells through internal complex mycelium with efficient utilization. According to Fig. 3b, intact mycelial structure is important to flocculation activity of mycelial pellets, and algal cells could be found inside of mycelial pellets. Therefore, the mycelial distinctions of mycelial pellets after cultured in flocculation medium and Gauserime synthetic medium were necessary to determine. As shown in Fig. S6 e-f, mycelium of mycelial pellets after cultured in flocculation medium and Gauserime synthetic medium were investigated under fluorescence microscope. The mycelial morphology in mycelial pellets after cultured between different culture media exhibited great differences, mycelia of mycelial pellets after cultured in flocculation medium were more thick than that of mycelial pellets after cultured in Gauserime synthetic medium. However, the mycelia of mycelial pellets after cultured in Gauserime synthetic medium were more dense than that of mycelial pellets after cultured in flocculation medium. All the results indicated that the thick mycelia of mycelial pellets maybe bind more algal cells, and the relatively sparse mycelial structure can promise algal cells to easily access inside, thus helping to achieve high flocculation activity of mycelial pellets after cultured in flocculation medium. The distinctions between mycelial pellets after cultured in different culture media caused complete different flocculation activity, which indicated that the morphology and structure of mycelial pellets is also the critical factor to flocculation activity.
4. Conclusions This study firstly confirmed that an actinomycete hsn06, which belonged to genus Streptomyces with high flocculation activity on C. vulgaris biomass through the 19
mycelial pellets with adding calcium ions. Calcium was crucial to flocculation activity, and determined that mycelial pellets bind calcium during flocculation procedure, which indicated calcium bridging is the main flocculation mechanism. Characteristics of flocculation activity could evidence proteins of mycelial pellets also contributed to flocculation activity. Finally, the unique morphology and structure of mycelial pellets were helpful to harvest algal biomass. Determination of flocculation mechanism can promise actinomycetes to be better application on microalgae biomass harvesting.
Acknowledgements: This work is supported by national science foundation of China (No. 51008119, 41576109), Doctoral Scientific Research Start-up Foundation of Henan Normal University (5101049170160) and the Key Scientific Research Programs of Henan Education Department (17A610002).
Reference 1. Baytshtok, V., Baker, T.A., Sauer, R.T., 2015. Assaying the kinetics of protein denaturation catalyzed by AAA+ unfolding machines and proteases. P. Natl. Acad. Sci. USA. 112(17), 5377. 2. Bentley, S.D., Chater, K.F., Cerdeñotárraga, A.M., Challis, G.L., Thomson, N.R., James, K.D., Harris, D.E., Quail, M.A., Kieser, H., Harper, D., 2002. Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417(6885), 141-147.
20
3. Biggs, C.A., Lant, P.A., 2000. Activated sludge flocculation: on-line determination of floc size and the effect of shear. Water Res. 34(9), 2542-2550. 4. Biggs, S., Habgood, M., Jameson, G.J., Yan, Y.D., 2000. Aggregate structures formed via a bridging flocculation mechanism. Chem. Eng. J. 80(1), 13-22. 5. Cerff, M., Morweiser, M., Dillschneider, R., Michel, A., Menzel, K., Posten, C., 2012. Harvesting fresh water and marine algae by magnetic separation: screening of separation parameters and high gradient magnetic filtration. Bioresource Technol. 118(8), 289-295. 6. Chen, D., Song, J., Yang, H., Xiong, S., Liu, Y., Liu, R., 2016. Effects of acid and alkali treatment on the properties of proteins recovered from whole gutted grass carp (Ctenopharyngodon idellus) using isoelectric solubilization/precipitation. J. Food Quality 39(6), 707-713. 7. Delong, E.F., 1992. Archaea in coastal marine environments. P. Natl. Acad. Sci. USA. 89(12), 5685-5689. 8. Fong, S., Teo, E., Ng, L.F., Chen, C.B., Lakshmanan, L.N., Tsoi, S.Y., Moore, P.K., Inoue, T., Halliwell, B., Gruber, J., 2016. Energy crisis precedes global metabolic failure in a novel Caenorhabditis elegans Alzheimer Disease model. Sci. Rep. 6, 33781. 9. Hanna, M., Marcin, P., 2001. Stability of cyanotoxins, microcystin-LR, microcystin-RR and nodularin in seawater and BG-11 medium of different salinity. Oceanologia 43(3), 329-339. 10. Ikeda, H., Ishikawa, J., Hanamoto, A., Shinose, M., Kikuchi, H., Shiba, T., Sakaki, 21
Y., Hattori, M., Omacrmura, S., 2003. Complete genome sequence and comparative analysis of the industrial microorganism Streptomyces avermitilis. Nat. Biotechnol. 21(5), 526-531. 11. Kim, D.G., La, H.J., Ahn, C.Y., Park, Y.H., Oh, H.M., 2011. Harvest of Scenedesmus sp. with bioflocculant and reuse of culture medium for subsequent high-density cultures. Bioresource Technol. 102(3), 3163-3168. 12. Kumar, S., Tamura, K., Jakobsen, I.B., Nei, M., 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24(24), 1596-1599. 13. Kwc, S.T., Sathe, S.K., 2002. Effects of sodium dodecyl sulfate, guanidine hydrochloride, urea, and heat on denaturation of sulfur rich protein in soybeans (Glycine max L.). J. Food Biochem. 25(6), 483-492. 14. Lei, X., Yao, C., Shao, Z., Chen, Z., Yi, L., Hong, Z., Zhang, J., Wei, Z., Zheng, T., 2015. Effective harvesting of the microalgae Chlorella vulgaris via flocculation-flotation with bioflocculant. Bioresource Technol. 198, 922-925. 15. Li, Y., Xu, Y., Liu, L., Jiang, X., Zhang, K., Zheng, T., Wang, H., 2016. First evidence of bioflocculant from Shinella albus with flocculation activity on harvesting of Chlorella vulgaris biomass. Bioresource Technol. 218, 807-815. 16. Maeda, Y., Tateishi, T., Niwa, Y., Muto, M., Yoshino, T., Kisailus, D., Tanaka, T., 2016. Peptide-mediated microalgae harvesting method for efficient biofuel production. Biotechnology for Biofuels, 9(1), 10. 17. Matthews, B.W., 1995. Studies on protein stability with T4 lysozyme. Adv. 22
Protein Chem. 46, 249-278. 18. Nwodo, U.U., Agunbiade, M.O., Ezekiel, G., Mabinya, L.V., Okoh, A.I., 2012. A freshwater Streptomyces, isolated from Tyume River, produces a predominantly extracellular glycoprotein bioflocculant. Int. J. Mol. Sci. 13(7), 8679-8695. 19. Nwodo, U.U., Green, E., Mabinya, L.V., Okaiyeto, K., Rumbold, K., Obi, L.C., Okoh, A.I., 2014. Bioflocculant production by a consortium of Streptomyces and Cellulomonas species and media optimization via surface response model. Colloid. Surface. B 116(2), 257-264. 20. Powell, R.J., Hill, R.T., 2014. Mechanism of algal aggregation by Bacillus sp. strain RP1137. Appl. Environ. Microb. 80(13), 4042-4050. 21. Powell, R.J., Hill, R.T., 2013. Rapid aggregation of biofuel-producing algae by the bacterium Bacillus sp. Strain RP1137. Appl. Environ. Microb. 79(19), 6093-101. 22. Ries, H.E., Meyers, B.L., 1968. Flocculation mechanism: charge neutralization and bridging. Science 160(3835), 1449-1450. 23. Rwehumbiza, V.M., Harrison, R., Thomsen, L., 2012. Alum-induced flocculation of preconcentrated Nannochloropsis salina: residual aluminium in the biomass, FAMEs and its effects on microalgae growth upon media recycling. Chem. Eng. J. 200(16), 168-175. 24. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4(4), 406-425. 25. Shimofuruya, H., Koide, A., Shirota, K., Tsuji, T., Nakamura, M., Suzuk, J., 1996. The production of flocculating substance(s) by Streptomyces griseus. Biosci. 23
Biotech. Bioch. 60(3), 498-500. 26. Sun, P., Hui, C., Bai, N., Yang, S., Wan, L., Zhang, Q., Zhao, Y.H., 2015. Revealing the characteristics of a novel bioflocculant and its flocculation performance in Microcystis aeruginosa removal. Sci. Rep. 5, 17465. 27. Tan, R., Miyanaga, K., Uy, D., Tanji, Y., 2012. Effect of heat-alkaline treatment as a pretreatment method on volatile fatty acid production and protein degradation in excess sludge, pure proteins and pure cultures. Bioresource Technol. 118(8), 390-398. 28. Vandamme, D., Foubert, I., Muylaert, K., 2013. Flocculation as a low-cost method for harvesting microalgae for bulk biomass production. Trends Biotechnol. 31(4), 233. 29. Wan, C., Zhao, X.Q., Guo, S.L., Asraful, A.M., Bai, F.W., 2013. Bioflocculant production from Solibacillus silvestris W01 and its application in cost-effective harvest of marine microalga Nannochloropsis oceanica by flocculation. Bioresource Technol. 135(3), 207-212. 30. Weschler, M.K., Barr, W.J., Harper, W.F., Landis, A.E., 2014. Process energy comparison for the production and harvesting of algal biomass as a biofuel feedstock. Bioresource Technol. 153(153C), 108-115. 31. Wijffels, R.H., Barbosa, M.J., 2010. An outlook on microalgal biofuels. Science 329(5993), 796-799. 32. Yang, Z., Yang, H., Jiang, Z., Cai, T., Li, H., Li, H., Li, A., Cheng, R., 2013. Flocculation of both anionic and cationic dyes in aqueous solutions by the 24
amphoteric grafting flocculant carboxymethyl chitosan-graft-polyacrylamide. J. Hazard. Mater. 255(254-255C), 36-45. 33. Yin, Y.J., Tian, Z.M., Tang, W., Li, L., Song, L.Y., Mcelmurry, S.P., 2014. Production and characterization of high efficiency bioflocculant isolated from Klebsiella sp. ZZ-3. Bioresource Technol. 171C, 336-342. 34. Zhang, B., Cai, G., Wang, H., Li, D., Yang, X., An, X., Zheng, X., Tian, Y., Zheng, W., Zheng, T., 2014. Streptomyces alboflavus RPS and its novel and high algicidal activity against harmful algal bloom species Phaeocystis globosa. Plos One 9(3), e92907. 35. Zhang, B.H., Ding, Z.G., Li, H.Q., Mou, X.Z., Zhang, Y.Q., Yang, J.Y., Zhou, E.M., Li, W.J., 2016. Algicidal activity of metabolites from Streptomyces eurocidicus JXJ-0089 and their effects in the physiology of Microcystis. Appl. Environ. Microb. 82(17), 5132-5143. 36. Zhang, C.L., Cui, Y.N., Wang, Y., 2013. Bioflocculants produced by Gram-positive Bacillus xn12 and Streptomyces xn17 for swine wastewater application. Chem. Biochem. Eng. Q. 27(2), 245-250. 37. Zhang, X., Hu, Q., Sommerfeld, M., Puruhito, E., Chen, Y., 2010. Harvesting algal biomass for biofuels using ultrafiltration membranes. Bioresource Technol. 101(14), 5297-5304.
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Figure captions: Fig. 1 Neighbour-joining tree showing the phylogenetic positions of strain hsn06T and representatives of some other related taxa, based on 16S rRNA gene sequences. Bootstrap values (expressed as percentages of 1000 replications) are shown at branch points; only values >50 % are shown. Bar, 0.0005 nt substitution rate (Knuc) units. Fig. 2 Calcium binding assay of mycelial pellets after cultured in flocculation medium and Gauserime synthetic medium with different flocculating times (a), different concentrations of calcium (b). All error bars indicate the SE of the three biological replicates. *Represents a statistically significant difference at p<0.05 compared with positive control; **represents a statistically significant difference of p<0.01. Fig. 3 Effect of different temperatures (a), and SDS, proteinase K digestion, lysozyme treatment (b) on flocculation activity. All error bars indicate the SE of the three biological replicates. **represents a statistically significant difference of p<0.01 compared with positive control.
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Flocculation mechanism of the actinomycete Streptomyces sp. hsn06 on Chlorella vulgaris Yi Li1, 2, Yanting Xu1, Tianling Zheng2 and Hailei Wang1* 1
College of Life Sciences, Henan Normal University, Xinxiang 453007, China
2
State Key Laboratory of Marine Environmental Science, School of Life Sciences,
Xiamen University, Xiamen 361005, China *Corresponding author: Tel.: +86 3733326340; Fax: +86 3733326916. E-mail address: [email protected] (H. Wang)
Highlights: 1. Streptomyces was firstly used to harvest Chlorella vulgaris biomass; 2. Calcium bridging is the main flocculation mechanism; 3. Proteins in mycelial pellets involved in flocculation procedure; 4. Mycelial morphology and structure caused dramatic effects on flocculation activity.
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