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Bacterial community enhances flocculation efficiency of Ettlia sp. by altering extracellular polymeric substances profile
Bioresource Technology 281 (2019) 56–65
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Bacterial community enhances flocculation efficiency of Ettlia sp. by altering extracellular polymeric substances profile
T
Chau Hai Thai Vua,b, Seong-Jun Chuna,b, Seong-Hyun Seoa,c, Yingshun Cuia, Chi-Yong Ahna,b, ⁎ Hee-Mock Oha,b, a
Cell Factory Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea Department of Environmental Biotechnology, KRIBB School of Biotechnology, Korea University of Science & Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 34113, Republic of Korea c Department of Life Science, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul 04763, Republic of Korea b
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Microalgae Bacteria Extracellular polymeric substances (EPS) Ettlia sp. YC001 Flocculation
This study examined the effects of a bacterial community and extracellular polymeric substances (EPS) on Ettlia sp. flocculation. The growth rate, flocculation efficiency (FE), bacterial community, and EPS profile of axenic and xenic Ettlia cultures were monitored during 46 days of cultivation. For the xenic culture, with a great abundance of growth-promoting and flocculation-inducing bacteria, the biomass density was 18.75% higher and its FE reached 100% in the mid-stationary phase. Moreover, microscopic observation and a quantitative analysis of the EPS revealed the exclusive presence of long filamentous EPS and more compact structure in the xenic Ettlia culture, possibly explaining its better FE. Notwithstanding, for the axenic culture, despite a lower biomass density and reduced abundance of EPS, its FE reached 92.54% in the mid-stationary phase. Thus, the role of the bacterial community was found to be supportive rather than vital for the high settleability of the self-flocculating Ettlia microalgal culture.
1. Introduction The advantages of microalgae have already been recognized in a wide variety of applications (Pulz and Gross, 2004). In the case of
⁎
biodiesel, when compared with terrestrial plants, microalgae grow about 10–15 times faster with approximately 10–20 times higher productivity (Wan et al., 2015; Wang et al., 2016). The use of microalgae to produce pharmaceutical and other high-value products by
Corresponding author at: Cell Factory Research Center, KRIBB, 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea. E-mail address: [email protected] (H.-M. Oh).
https://doi.org/10.1016/j.biortech.2019.02.062 Received 11 December 2018; Received in revised form 11 February 2019; Accepted 12 February 2019 Available online 13 February 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
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triplicate, unless otherwise mentioned.
manipulating the cultivation conditions is also very promising. However, due to the small size of microalgae, their identical density with water, and the diluted concentrations of conventional suspension cultivation systems (0.5–10 g/L), the harvesting process—which accounts for roughly 20–30% of the total production cost—still remains the bottleneck for microalgal biotechnology (Chisti, 2007; Wan et al., 2015). Currently, centrifugation, filtration, flotation, and gravity sedimentation are the main harvesting techniques, among which the most popular ones are centrifugation and sedimentation. Flocculation before harvesting, which can be induced by physical, chemical or bio-based flocculation have been reported to save a significant amount of time and energy (Vandamme et al., 2013). However, some of these flocculation processes might contaminate the biomass and are not environment friendly or economically feasible (Wan et al., 2015). Currently, the idea of using self-flocculating microalgal species for harvesting the non-flocculating oleaginous cells has achieved great attention (Salim et al., 2012). Possible application in the field of water bloom treatment might also be promising, although no lab-scale research has been conducted yet. Till now, only a few self-flocculating microalgae species, such as Chlorella vulgaris JSC-7, Scenedesmus obliquus AS-6-1, Ettlia texensis, Ankistrodesmus falcatus, Tetraselmis suecica, and Chlorococcum sp. GD have been reported (Alam et al., 2014; Guo et al., 2013; Lv et al., 2016; Salim et al., 2014, 2013, 2012). Researches have implied the role of released extracellular polymeric substances (EPS) or algal organic matter (AOM) in the abovementioned autoflocculating microalgal species; however, culture axenicity has been overlooked. Without the precise acknowledgment of bacterial role, the auto-flocculation behavior or the real producer of the bioactive EPS might be misleading (Vu et al., 2018). Accordingly, this study examined whether the bacterial community or EPS play a vital role in the previously detected strong settling properties of Ettlia sp. YC001 (La et al., 2016). EPS can be categorized into soluble (S-) and cell surface bound (B-), and the latter further divided into outer loosely bound (LB-) and inner tightly bound (TB-) layers (Sheng et al., 2010). Since the B-EPS accumulate on the cell surface and directly change cell surface characteristics—such as surface hydrophobicity, charge density, and binding sites—only this fraction was studied in this research. The next generation sequencing (NGS) method was used to investigate the bacterial communities of Ettlia culture during different growth phases. The B-EPS of bacteria-free and xenic microalgal cultures were extracted and their compositions investigated. Thereafter, flocculation efficiency (FE) of the two Ettlia cultures were measured and explained based on the bacterial community and EPS profile.
2.2. Microalgal biomass density determination The growth curve was determined by monitoring the biomass density (g/L) based on the dry cell weight (DCW). Due to their floc forming characteristic, the cultures were well shaken before taking the aliquots. The samples were then filtered using an overnight-dried and preweighed GF/C grade glass microfiber filter (Whatman plc, United Kingdom). Thereafter, the filters were dried overnight in an oven at 105 °C until reaching a constant weight. All the measurements were performed in triplicate. 2.3. SEM sample preparation To confirm the axenicity and observe the EPS matrix, the Ettlia cell suspension was initially fixed in a pH 7.2 phosphate-buffered (0.1 M) mixture solution of 2.5% paraformaldehyde and glutaraldehyde for 2 h, and then postfixed in a 1% OsO4 solution with the same buffer for 1 h. The samples were then dehydrated in EtOH, and the solution replaced with isoamyl acetate. Thereafter, the samples were dried at the critical point in liquid CO2 and sputtered with gold using a Polaron SC502 sputter coater (Quorum Technologies Ltd., United Kingdom). The samples were observed using a scanning electron microscope, Quanta™ 250 FEG (FEI company, USA), and cell size was analyzed using the ImageJ measure analysis tool (National Institute of Health, USA). 2.4. Bacterial community analysis For the bacterial community analysis, 2 mL of the xenic Ettlia cell suspension was filtered through sterilized 3.0 μm and 0.22 μm Isopore™ polycarbonate membrane filters (Merck Millipore, Germany) to harvest all the attached and free-living bacteria, respectively. The membrane filters were stored at −80 °C until performing the DNA extraction. The DNA extraction, amplification, and sequencing was conducted as previously described (Chun et al., 2018). The bacterial 16S rRNA gene sequences and accompanying metadata have already been deposited in the Sequence Read Archive (SRA) of the NCBI under project number PRJNA506495. The statistical analyses and graphics were conducted using the R program (https://www.r-project.org). To assess the structural dissimilarities in the xenic Ettlia bacterial community at different growth phases, a multi-response permutation procedure was performed based on a Bray-Curtis distance matrix. Plus, to visualize the results, nonmetric multidimensional scaling (NMDS) was conducted using the vegan package (Oksanen et al., 2013). The bacterial relative abundance at the order level was transformed to a log10 scale, while the shifts in the bacterial community were presented as a heatmap using the gplots package (Warnes et al., 2016). The heatmap analysis only included bacterial OTUs with a maximum relative abundance higher than 1%, and the remaining OTUs were pooled together and presented as “Other bacteria”.
2. Materials and methods 2.1. Microalgal strain and cultivation conditions The microalgal Ettlia sp. YC001 strain (KCTC 12109BP) was obtained from the Korean Collection for Type Cultures (KCTC) at the Korea Research Institute of Bioscience and Biotechnology. A xenic seed culture was maintained in the steady state as previously described (Seo et al., 2017). An axenic culture was then isolated and confirmed using several enriched agar media and scanning electron microscopic (SEM) observation (Vu et al., 2018). For easy detection of contamination during culture maintenance, the axenic culture was stored in a solid agar plate. The axenic and xenic Ettlia seed cultures were inoculated into 1-L Erlenmeyer flasks containing 500 mL of a BG-11 medium at an initial density of 106 cell/mL, and then cultivated in a shaking incubator (100 rpm) at a constant temperature of 25 ± 1 °C with a supply of 5% CO2 at flow rate of 0.1 v/v/m and under continuous illumination of 100 μmol photons/m2/s. Aliquots of the cell suspension were harvested at different growth phases to determine the growth curve and for other analyses. All the experiments were conducted in
2.5. Ettlia sp. cell suspension physical properties The Ettlia cell suspension ξ-potential and FE were monitored at day 0 (initial phase), day 10 (mid-exponential phase), day 26 (mid-stationary phase), and day 46 (decline phase). An aliquot of the microalgal biomass was harvested at each stage and its ξ-potential measured in folded capillary cells using a Zetasizer Nano ZS (Malvern Panalytical, United Kingdom). For the FE, 100 mL of the cell suspension was poured into in a graduated cylinder up to a height of 12 cm and then left for free settlement for a period of 30 min. Aliquots were then taken at a height of 7.5 cm from the cylinder bottom at the initial (t = 0 min) and final (t = 30 min) time point to determine the biomass density. Due to the 57
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floc-forming nature of Ettlia, the DCW was used instead of the optical density. The FE was calculated using the following equation:
B Flocculation efficiency (%) = ⎛1 − ⎞ × 100 A⎠ ⎝
(1)
where A and B are the DCW at the beginning and after a settling time of 30 min, respectively.
2.6. EPS extraction, purification, and chemical analysis The LB- and TB-EPS fractions were separately extracted using a modified method from Li and Yang (2007). A microalgal pellet was harvested by centrifugation at 4,000 × g for 10 min. The nitrogen and carbon concentrations in the supernatant were determined using a TOC analyzer multi N/C® 3100 (Analytik Jena, Germany). Meanwhile, the cell pellet was re-suspended in a 50 °C NaCl solution (0.02 M) and readily vortexed for 1 min at 3,000 rpm to extract the LB-fraction. The LB-EPS was retrieved by centrifugation at 10,000 × g for 30 min and filtration through a 0.45 μm filter. Cell pellet was then re-suspended in the NaCl solution, heated for 30 min at 60 °C in a water bath, and the TB-EPS fraction retrieved in the similar manner to that explained above for the LB-EPS fraction. The cell pellet was freeze-dried to measure the DCW for normalization. The EPS solutions were further purified using 1 kDa MWCO prewetted dialysis tubing (Spectrum Laboratories, Inc., USA) against DI water (renewed twice daily) at 4 °C for 2 days. The EPS solutions were then re-concentrated using a Spectra/Gel® absorbent (Spectrum Laboratories, Inc., USA) according to manufacturer’s instructions. Thereafter, the solutions were aliquoted and stored at −20 °C for further analysis. The EPS content was indirectly measured using a TOC analyzer multi N/C® 3100 (Analytik Jena, Germany). The compositional protein (EPSP) and carbohydrate (EPSC) contents were determined using the methods of Lowry and Anthrone with bovine serum albumin and glucose used as the respective standards (Jermyn, 1975; Waterborg, 2009).
Fig. 1. Changes in biomass density of axenic and xenic Ettlia cultures under supply of 5% CO2. Data represent means of three biological replicates ± SD.
3. Results and discussion 3.1. Ettlia sp. cultures: growth and SEM observation The growth of axenic and xenic Ettlia cultures were quite identical in the first 9 cultivation days with a short lag phase of approximately 5 days, followed by 11 days of an exponential growth phase (Fig. 1). The biomass content in the axenic and xenic cultures reached a maximum of 2.40 ± 0.45 g/L and 2.85 ± 0.70 g/L, respectively, at the beginning of the stationary phase. This 18.75% increase in biomass density was well-agreed with some previous researches in diatom and microalgae cultures. When co-culturing Scenedesmus obliquus with different single or mixed bacterial cultures, the biomass density of S. obliquus was significantly increased by 3.5–24.8% (Wang et al., 2015). Similar increase in growth rate and cell density was obtained with different diatom cultures (Punctastriata sp., Achnanthes minutissima, Phaeodactylum tricornutum UTEX646, etc.) when co-cultured with several bacterial cultures (Bruckner et al., 2011; Grossart, 1999). Some diatom strains even showed positive growth with the addition of bacterial-cell free medium at a low amount as 0.1% (v/v). This growth stimulating phenomenon can be explained by (1) creating a favorable growth environment for microalgae; (2) providing their microalgal partner with essential macro- or micro-nutrients; (3) generating phytohormones, chelators or antibiotics (Vu et al., 2018; Wang et al., 2016). In the SEM micrographs, the Ettlia cells from the initial phase exhibited a spherical shape of 7.0–8.0 μm. The axenic culture showed no presence of bacteria and no or very little evidence of an EPS matrix (SEM micrographs are provided in Supplementary materials). Conversely, the xenic culture showed the presence of 1.5–4.0 μm bacillus bacteria from which interconnecting filamentous EPS matrices were observed. Moreover, the Ettlia cell surface showed evidence of both long and thin filamentous EPS matrices and the smaller, dust-like EPS matrices. As the cultures aged, the two Ettlia cultures started to show some different signs of aggregation/flocculation (SEM micrographs are provided in Supplementary materials). No obvious interconnecting network was detected in the axenic Ettlia culture except for the increased amount of dust-like EPS on the cell surfaces. However, a significantly increased amount of filamentous EPS, which was greatly expanded, entirely filling the gaps and firmly connecting two adjacent Ettlia cells, was discovered in the xenic culture. Dust-like EPS matrices were also detected in this culture, however, the particle size and amount seemed to be far lesser than in the axenic culture. Although the origin of these EPS matrices remain unknown, it is
2.7. Protein purification and SDS-PAGE analysis The extracted extracellular protein was precipitated by adding 3 times the volume of ice-cold acetone, mixed thoroughly, and kept overnight at −20 °C. The protein pellet was then recovered by centrifugation at 22,000 × g at 4 °C for 15 min, followed by drying in a vacuum evaporator for 15 min. Next, the protein pellets were re-suspended in a mixture of a rehydration buffer (urea 7 M, thiourea 2 M, CHAPS 4%, Tris 10 mM) and 2 × Laemli buffer (1:1, v/v) to a final protein concentration of 10 μg/μl. The protein solution was then heated at 90 °C for 5 min, briefly centrifuged at a high speed, and the supernatant was immediately used for SDS-PAGE analysis. The protein bands were separated in a 6–18% Tris-Glycine gradient gel, using Triple color protein marker (10–245 kDa, BioFACT, Korea), and visualized using a Pierce® silver stain kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Relative abundance of bands in each lane was quantified using the ImageJ gels analysis tool (National Institute of Health, USA) and presented as a heatmap using the gplots package in the R program (Section 2.4).
2.8. Statistical analysis The statistical analysis of the variables between the axenic and xenic Ettlia cultures was conducted using Student’s t-test. Differences were regarded as statistically significant when p < 0.05. Pearson’s correlation analysis was used to measure the association strength between the variables (Excel 2016, Microsoft Corporation, USA). 58
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Supplementary materials table, where the relatively higher abundance of the attached community agrees well with their adhesion behavior to cell and detrital surfaces (Williams et al., 2013). Growth-promoting bacteria stimulate the growth of their microalgal partner by various means. Bacteria belonging to the Rhizobiales order are known to fix nitrogen and have enhanced the growth of several microalgal cultures (Kim et al., 2014; Wang et al., 2015). They also stimulate microalgal growth by producing the indole-3-acetic acid phytohormone (Do Nascimento et al., 2013). The presence of such bacteria was possibly responsible for the stable soluble nitrogen concentration in the xenic culture when compared to the control and axenic cultures (Supplementary materials). Another rhizobacterium found in the xenic Ettlia culture was Brevundimonas vesicularis, which belongs to the Caulobacterales order (accounted for the highest abundance of 31.83% and 23.96% in the attached and free-living bacterial community, respectively, in the exponential growth phase, Supplementary materials table). Although the growth promoting mechanism of B. vesicularis remains unknown, it was reported to enhance the growth of Chlorella ellipsoidea approximately three times when compared with the axenic control sample (Park et al., 2008). Therefore, the noticeably high abundance of B. vesicularis in the exponential growth phase may partially explain the abovementioned outcompeted growth of the xenic Ettlia culture over the bacteria-free culture in this stage. Furthermore, Pseudomonas spp. from the order of Pseudomonadales are known for their ability to secrete multiple anti-fungal metabolites that protect their microalgal partner from a wide range of fungal pathogens (Bloemberg and Lugtenberg, 2001). The xenic culture also exhibited high amounts of bacteria previously reported to induce flocculation (Salehizadeh and Shojaosadati, 2001). The bioflocculants produced by bacteria belonging to the Bacillus, Paenibacillus (Bacillales order), and Flavobacterium (Flavobacteriales order) genera have been reported to increase the flocculation efficiency of several green algae cultures (Oh et al., 2001; Salehizadeh and Shojaosadati, 2001). In contrast, the bacteria of the Sphingobacteriales order, rather than their metabolites, have been shown to be vital in the flocculation of C. vulgaris culture (Lee et al., 2013). Acidovorax spp. were the dominant species of the Burkholderiales order found in the current bacterial community (Supplementary materials), and are frequently abundant in activated sludge and form copious flocs in a broth culture (Heijstra et al., 2009).
suggested that the dust-like EPS originated from the microalgae, whereas the filamentous EPS were produced by either the associated bacterial community or the Ettlia itself induced by its bacterial partners. Identical EPS structures were also observed in an Ettlia texensis culture although the axenicity of the culture was unstated (Salim et al., 2014). The authors have reported that the short EPS matrices seemed to patch the cell surfaces yet they seemed too short to visually bind with other E. texensis cells, while the filamentous EPS was greatly extended and bound with other microalgal cells. To our knowledge, our SEM micrographs were the first ones that clearly discriminate the EPS structure of the pure and mixed Ettlia sp. culture. In addition, the cell surface of the xenic culture showed deep and well-defined crinkles, while the cell surface of the axenic culture was rather smooth. A similar phenomenon was also observed in the co-culture of Chlorella ellipsoidea with growthpromoting Brevundimonas sp. bacteria (Park et al., 2008). Possibly, the change of cell surface morphology was in response to an inter-kingdom signal between the microalgae and the bacteria; where the crinkles provided a better attachment for the associated bacteria (Amin et al., 2012). 3.2. Bacterial community analysis The bacterial community composition was investigated in each Ettlia growth phase using an Illumina MiSeq sequencing approach. A total of 393,475 sequence reads were obtained after trimming lowquality sequences. The obtained sequences were then divided into 136 operational taxonomic units (OTUs) based on a pairwise sequence identity cutoff of 99%. The OTUs were then further classified into a total of 15 classes, 32 orders, and 69 genera. The NMDS plot in Fig. 2A separates the OTUs into four distinct groups, indicating the strong grouping of bacterial communities within the experiment replications as well as the community distinction at different cultivation times. An ANOSIM test confirmed the statistical significance of the clustering (R = 0.851, P < 0.001). Moreover, the proximity of the day 0 and day 26 clusters implied a relatively conserved bacterial community across the Ettlia generations, as the initial microalgal suspension was directly retrieved from the cell suspension in its stationary phase in the chemostat. Fig. 2B shows the shift in the relative abundance of the bacterial community during the 46-day cultivation. The Proteobacteria phylum and Sphingobacteriia class were the most dominant, followed by the Actinobacteria, Bacilli, and Flavobacteriia classes. Further analysis of the microbial taxonomic composition showed that 27 of the 136 OTUs were shared by all the samples and over 99% of the sequences belonged to the top 30 OTUs in each sample. In the top 30 OTUs, bacteria belonging to the Flavobacteriia, Bacilli, Proteobacteria, and Sphingobacteriia classes appeared in all the samples. These bacteria are well-known as organic matter decomposing (Flavobacteriales), growth promoting (Rhizobiales, Caulobacteriales, Pseudomonadales), and flocculation inducing (Bacillales, Burkholderiales, Sphingobacteriales, Flavobacteriales). Thus, the presence of these functional bacteria was speculated to play an important role in the higher biomass content and FE in the xenic culture. Supplementary materials present the phylogenetic tree of the 30 most abundant OTUs during the course of the cultivation. The most well-known means of interaction between microalgae and bacteria partners is nutrient exchange. Autotrophic phytoplankton synthesize organic compounds and the photosynthates are then spontaneously released to the surroundings as metabolic by-products or in response to inter-kingdom signals (Vu et al., 2018). While small molecular weight biopolymers (600–800 Da and less) can be directly transferred through bacterial porin channels, larger polymers should be first degraded. The TonB-dependent transporter systems in the major degrader Flavobacteria allow the direct uptake of macromolecules that are too large to diffuse via porins, and then degrade them to smaller polymers that can later be utilized by other heterotrophs (Williams et al., 2013). The Flavobacteriales community abundance is listed in the
3.3. Physical properties of Ettlia cell suspension As shown in Fig. 3, the two Ettlia cultures exhibited very similar trends as regards their physical properties. During the first 10 cultivation days, the ξ-potential (Fig. 3A) dropped rapidly from −22.58 ± 3.43 mV and −28.38 ± 3.83 mV to approximately −13.83 ± 2.14 mV and −12.68 ± 1.20 mV in the middle of the exponential growth phase for the axenic and xenic Ettlia cultures, respectively. The ξ-potential then remained almost unchanged for the rest of the cultivation period. The FE (Fig. 3B) and cultivation time were strongly correlated (Pearson correlation coefficient r > 0.87), where the FE for the xenic culture (69.2–100%) was actually higher than that for the bacteria-free culture (50.0–99.2%), although the differences were not statistically significant. Discrepancies in the sedimentation behaviors of the two cultures were visually detectable in the Supplementary materials, with a distinctly larger floc size and clearer supernatant in the xenic culture and greenish tint in the axenic supernatant. Bacteria might play a decisive role in the flocculation of certain microalgal species (Lee et al., 2013). Therefore, there is a necessity to clearly define the axenicity of the studied cultures to prevent any potential misleading results (Vu et al., 2018). The relatively high FE of the axenic culture has confirmed the self-flocculating properties of Ettlia sp. YC001. Flocculation of microalgae can result from one or a combination of the following mechanisms (Vandamme et al., 2013). Charge 59
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Fig. 2. Analysis of bacterial community structure in xenic Ettlia culture. (A) Non-metric multidimensional scaling (NMDS) ordination plot of Bray-Curtis community dissimilarities based on 16S rRNA gene sequences collected at different cultivation times (D0: initial phase; D10: mid-exponential phase; D26: stationary phase; and D46: decline phase). Small golden circles represent OTUs. (B) Heatmap shows attached (A1–3) and free-living (F1–3) bacterial community structure at order level according to cultivation time. All analyses used three biological replicates.
underlying cause, as EPS have been reported to have a lower negative charge than the native cell surface (Liu et al., 2004; Rao et al., 2018). By lessening the electrostatic repulsion between two adjacent cells, the cells can approach close enough so the EPS matrix can patch or bridge them together. In the axenic culture, with the closely attached cells and the presence of only short, dust-like EPS matrices, electrostatic patching seemed to be the involved mechanism as being suggested by Alam et al. (2016). Conversely, in Ettlia xenic culture, with a large network of cells that were compactly bridged by the obvious filamentous EPS matrices, bridging was the main flocculation mechanism. Electrostatic patching by the dust-like EPS might also play a minor role in this culture (Alam et al., 2016). Therefore, the co-existence of bacterial community, along with the presence of unique filamentous EPS and the additional strong bridging flocculation mechanism, was the major cause of the difference
neutralization or an electrostatic patch is related to the overall or local alteration, respectively, of the microalgal cell surface charge by the adsorption of charged polymers, thereby reducing the repulsion force between adjacent cells. In a bridging mechanism, extended charged polymers simultaneously bind to the surfaces of adjacent particles and “bridge” them together, while sweeping flocculation is the co-precipitation or entrapment of microalgal cells with or within metal precipitates. However, previous studies had suggested that only the first three mentioned mechanisms were important in bio-flocculation process (Alam et al., 2016). In the current study, during the initial cultivation, charge neutralization or an electrostatic patch seemed to play an important role when the increase in the microalgal suspension FE was coincident with a decrease in the negative charge of the cell surface. The production of EPS during the initial stage may have been the
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stimulate the production of EPS from their partners, consuming/modifying them, or even producing their own EPS to induce specific surface attachment. EPS abundance and composition can vary under the impacts of both internal and external factors, such as microbial species, physical stage, nutrients, stress, and especially by the interaction with other species (Sheng et al., 2010). The total EPS amount was independent of the medium C/N ratio whereas EPSP and EPSC were reported to have the respective negative and positive relationship (Sheng et al., 2010; Ye et al., 2011). This is in accordance with the results of the axenic Ettlia batch culture, where inorganic carbon source was continuously provided while the nitrogen source is limited (Supplementary materials). The results of the xenic Ettlia culture, however, were slightly different when compared with the previous literature. This might be due to the dynamics of the bacterial community (Liu et al., 2004). It was discussed that depending on their mutual relationship in the coculture, the EPS abundance and composition can either be increased or decreased (Grossart, 1999; Wang et al., 2015). The axenic Ettlia culture has a relatively high LB-EPS percentage of 26.01–33.02% (Supplementary materials table). A lower LB-proportion in the xenic Ettlia culture (8.98–19.08%) implied the more compact EPS structure as previously it has been suggested that TB-EPS is a well-defined and dense layer firmly connects with the cell surfaces, while LBEPS is a loose structure without an obvious edge (Lin et al., 2014; Sheng et al., 2010). It seemed that the intertwined filamentous EPS structure was seemingly more successful in maintaining the EPS integrity. The increase in the relative percentage of LB-EPS according to the cultivation time likely indicated structural deterioration or cell lysis. Indeed, a high amount of LB-EPS has been reported to have an unfavorable effect on cell flocculation (Li and Yang, 2007). The structure of LB-EPS is not only more diffused, they also tend to be more porous and more loosely connected. Therefore, cells connected by this type of attachment tend to have weaker structure and smaller aggregate size. This result further elucidates the superior FE of the xenic Ettlia culture and explains the greenish tint due to the suspended cells in the supernatant of the axenic culture (Supplementary materials). The predominance of the EPSP fraction in Ettlia exopolymers has also been reported in the previous work of Salim et al. (2014). Due to the reports that most EPSP are hydrophobic and cell surface hydrophobicity is the triggering force for bioflocculation, EPSP fraction has often been reported as vital in maintaining the structure and flocculation ability of aggregates (Basuvaraj et al., 2015; Liu et al., 2004; Qu et al., 2012). Notwithstanding, due to their hydrophilic nature, carbohydrates tend to attract more fluid from the environment, thereby increasing the water content and porosity, and subsequently loosening the linkage strength between adjacent cells (Basuvaraj et al., 2015; Sheng et al., 2010). Therefore, a high content of EPSC, especially in the LB-layer is believed to be detrimental for flocculation. Literature reviews have revealed the connection between relatively high EPSP/EPSC ratio with the high flocculation and settleability in activated sludge (Supplementary materials table). High proportion of EPSP has also seemed to be involved in the intrinsic self-flocculating characteristic of E. texensis and Chlorococcum sp. GD, as well as the colonial growth characteristic of some microalgae/cyanobacteria (Lv et al., 2016, 2018; Salim et al., 2013; Wang et al., 2014). Consequently, a high EPSP/EPSC ratio of both axenic and xenic Ettlia culture, especially at the primary interaction surface of LB-EPS, might explain the self-flocculating characteristic of the Ettlia sp.
Fig. 3. Changes in Ettlia cell suspension ξ-potential (A) and flocculation efficiency (B) during cultivation period. Statistical analysis between axenic and xenic cultures was performed using unpaired Student t-test, where (*) denotes statistical significance (p < 0.05). Data represent means of three biological replicates ± SD.
in the FE between the two cultures. 3.4. Extracellular polymeric substances: quantitative analysis Despite the unique EPS structures discovered previously, there was no statistical difference in the EPS abundance between the axenic and xenic Ettlia cultures. The variation in the EPS profile of the two cultures (Fig. 4) followed a similar trend. In general, the total B-EPS content in the xenic Ettlia was 82–17% higher than that in the pure culture, and an identical trend was also observed for the TB-fraction (Fig. 4A). In the axenic culture, despite a modest content, EPS were steadily produced with a consistent increase throughout the cultivation (Supplementary materials table, r = 0.99). Conversely, there was no significant change in the EPS amount at the p < 0.05 level in the xenic culture. The axenic culture seemed to have a higher proportion of the easily extractable LBfraction, with the absolute abundance roughly 1.5 times higher than that in the xenic culture, although the difference was not statistically significant. During the 46-day cultivation, the EPSP content showed a gradual drop in both cultures, while the EPSC profile showed a similar tendency to the EPS (Fig. 4B and C). In addition, the relatively high mass ratio between EPSP and EPSC (Fig. 4D) has implied the predominance of EPSP in the Ettlia EPS. Microbes can release EPS either actively or passively as the products of metabolic activities and cell lysis. Autotrophic microorganisms can release some of their photosynthates into the phycosphere to attract beneficial bacteria or initiate partner attachment. Bacteria can also interact with EPS in diverse ways by secreting special molecules to
3.5. Extracellular protein: SDS-PAGE analysis SDS-PAGE was conducted for a deeper analysis of the EPSP component in the LB- (Fig. 5A) and TB-fractions (Fig. 5B) of the native and bacteria-associated Ettlia cultures. Both gels repeatedly showed similar protein bands at a high MW range of 180–245 kDa, although the TBEPSP bands intensity were much darker. A thick band also developed exclusively around 135 kDa during the mid-exponential growth phase. 61
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Fig. 4. Changes in EPS content (A) and EPS composition (B–D) of axenic and xenic Ettlia cultures in different growth phases. Statistical analysis was performed using unpaired Student t-test, where (*) denotes statistical significance (p < 0.05). Data represent means of three biological replicates ± SD.
the protein bands with the highest relative abundance. For the LB-EPS, the protein bands with the highest relative abundance appeared in the 63–75 kDa range during the mid-exponential growth phase and then gradually moved to a lower MW range of 48–63 and 35–48 kDa as the cultivation continued. The TB-proteins with the highest relative
Most of the LB-EPSP bands were located in the range of 25–100 kDa with a uniquely thick band appearing around 75 kDa at day 10. The protein bands in the TB-EPSP gel were mainly located in the lower range of 20–48 kDa at day 10, then additional bands appeared in the range of 11–20 kDa at day 26 and 46. Of interest was also the size transition of 62
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Fig. 5. Analysis of compositional (A) LB- and (B) TB-EPSP by SDS-PAGE in different growth phases (D10: mid-exponential phase; D26: stationary phase; and D46: decline phase). Three sample lanes on left were collected from axenic Ettlia (A-), while three lanes on right were from xenic Ettlia (X-). Corresponding heatmap represents relative abundance of proteins from different size fractions. Relative percentage of proteins is color-coded as shown in scale.
appeared in one culture (bands annotated with arrows). While SDS-PAGE profiles of activated sludge have been frequently reported, the current study would seem to be the first SDS-PAGE profile for an isolated microalgae culture at different growth stages (Zhang
abundance were located in the MW range of 35–48 kDa and remained unchanged in the xenic culture, whereas the axenic culture showed a transition to a lower range of 25–35 kDa. Most proteins were found to appear in both the axenic and the xenic cultures, yet certain bands only 63
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et al., 2007; Zhu et al., 2015). When compared with the profiles of activated sludge samples, the bands in the Ettlia EPSP profile were less smeared and more well-defined, probably due to their “purer” constitution and/or the utilization of a more sensitive silver staining protocol. As a result, more noteworthy conclusions could be drawn. There are currently two hypotheses about the origin of LB-EPS: (1) they belong to a higher polymer size fraction that is more prone to be extracted under mild conditions or (2) they result from the shedding of TB-EPS after certain degradation over time (Basuvaraj et al., 2015). Thus, the distinctive higher protein size of EPSP in this study confirmed the accuracy of the first hypothesis and explained why LB-EPS are normally described to have a more diffused and protruded structure. In addition, the dynamic transition of the proteins to a smaller size, especially in the more exposed LB-layer, indicated signs of polymeric degradation by an extracellular enzyme or some kind of transformation in the EPS structure.
and Type IV pili mediate surface attachment by Acidovorax temperans. Antonie Van Leeuwenhoek 95, 343–349. Jermyn, M., 1975. Increasing the sensitivity of the anthrone method for carbohydrate. Anal. Biochem. 68, 332–335. Kim, B.-H., Ramanan, R., Cho, D.-H., Oh, H.-M., Kim, H.-S., 2014. Role of Rhizobium, a plant growth promoting bacterium, in enhancing algal biomass through mutualistic interaction. Biomass Bioenergy 69, 95–105. La, H.-J., Seo, S.-H., Lee, J.-Y., Lee, C.S., Kim, B.-H., Srivastava, A., Han, M.-S., Oh, H.-M., 2016. Improved mixing efficiency and biomass productivity of Ettlia sp. in co-cultivation system with loaches. Algal Res. 17, 300–307. Lee, J., Cho, D.-H., Ramanan, R., Kim, B.-H., Oh, H.-M., Kim, H.-S., 2013. Microalgaeassociated bacteria play a key role in the flocculation of Chlorella vulgaris. Bioresour. Technol. 131, 195–201. Li, X.Y., Yang, S.F., 2007. Influence of loosely bound extracellular polymeric substances (EPS) on the flocculation, sedimentation and dewaterability of activated sludge. Water Res. 41, 1022–1030. Lin, H., Zhang, M., Wang, F., Meng, F., Liao, B.-Q., Hong, H., Chen, J., Gao, W., 2014. A critical review of extracellular polymeric substances (EPSs) in membrane bioreactors: characteristics, roles in membrane fouling and control strategies. J. Membr. Sci. 460, 110–125. Liu, Y.-Q., Liu, Y., Tay, J.-H., 2004. The effects of extracellular polymeric substances on the formation and stability of biogranules. Appl. Microbiol. Biotechnol. 65, 143–148. Lv, J., Guo, J., Feng, J., Liu, Q., Xie, S., 2016. A comparative study on flocculating ability and growth potential of two microalgae in simulated secondary effluent. Bioresour. Technol. 205, 111–117. Lv, J., Wang, X., Liu, W., Feng, J., Liu, Q., Nan, F., Jiao, X., Xie, S., 2018. The performance of a self-flocculating microalga Chlorococcum sp. 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Characterization of dissolved extracellular organic matter (dEOM) and bound extracellular organic matter (bEOM) of Microcystis aeruginosa and their impacts on UF membrane fouling. Water Res. 46, 2881–2890. Rao, N.R.H., Yap, R., Whittaker, M., Stuetz, R.M., Jefferson, B., Peirson, W.L., Granville, A.M., Henderson, R.K., 2018. The role of algal organic matter in the separation of algae and cyanobacteria using the novel “Posi”-Dissolved air flotation process. Water Res. 130, 20–30. Salehizadeh, H., Shojaosadati, S., 2001. Extracellular biopolymeric flocculants: recent trends and biotechnological importance. Biotechnol. Adv. 19, 371–385. Salim, S., Kosterink, N., Wacka, N.T., Vermuë, M., Wijffels, R., 2014. Mechanism behind autoflocculation of unicellular green microalgae Ettlia texensis. J. Biotechnol. 174, 34–38. Salim, S., Shi, Z., Vermuë, M., Wijffels, R., 2013. Effect of growth phase on harvesting characteristics, autoflocculation and lipid content of Ettlia texensis for microalgal biodiesel production. Bioresour. Technol. 138, 214–221. Salim, S., Vermuë, M., Wijffels, R., 2012. Ratio between autoflocculating and target microalgae affects the energy-efficient harvesting by bio-flocculation. Bioresour. Technol. 118, 49–55. Seo, S.-H., Ha, J.-S., Yoo, C., Srivastava, A., Ahn, C.-Y., Cho, D.-H., La, H.-J., Han, M.-S., Oh, H.-M., 2017. Light intensity as major factor to maximize biomass and lipid productivity of Ettlia sp. in CO2-controlled photoautotrophic chemostat. Bioresour. Technol. 244, 621–628. Sheng, G.-P., Yu, H.-Q., Li, X.-Y., 2010. Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: a review. Biotechnol. Adv. 28, 882–894. Vandamme, D., Foubert, I., Muylaert, K., 2013. Flocculation as a low-cost method for harvesting microalgae for bulk biomass production. Trends Biotechnol. 31, 233–239. Vu, C.H.T., Lee, H.-G., Chang, Y.K., Oh, H.-M., 2018. Axenic cultures for microalgal biotechnology: establishment, assessment, maintenance, and applications. Biotechnol. Adv. 36, 380–396. Wan, C., Alam, M.A., Zhao, X.-Q., Zhang, X.-Y., Guo, S.-L., Ho, S.-H., Chang, J.-S., Bai, F.W., 2015. Current progress and future prospect of microalgal biomass harvest using various flocculation technologies. Bioresour. Technol. 184, 251–257. Wang, H., Hill, R.T., Zheng, T., Hu, X., Wang, B., 2016. Effects of bacterial communities on biofuel-producing microalgae: stimulation, inhibition and harvesting. Crit. Rev. Biotechnol. 36, 341–352. Wang, M., Kuo-Dahab, W.C., Dolan, S., Park, C., 2014. Kinetics of nutrient removal and expression of extracellular polymeric substances of the microalgae, Chlorella sp. and Micractinium sp., in wastewater treatment. Bioresour. Technol. 154, 131–137. Wang, R., Xue, S., Zhang, D., Zhang, Q., Wen, S., Kong, D., Yan, C., Cong, W., 2015. Construction and characteristics of artificial consortia of Scenedesmus obliquus-bacteria for S. obliquus growth and lipid production. Algal Res. 12, 436–445. Warnes, M.G.R., Bolker, B., Bonebakker, L., Gentleman, R. 2016. Package ‘gplots’: Various R Programming Tools for Plotting Data, pp. 24–34. Waterborg, J.H., 2009. The Lowry method for protein quantitation. In: Walker, J.M. (Ed.), The Protein Protocols Handbook. Humana Press, Totowa, NJ, pp. 7–10.
4. Conclusions Self-flocculating Ettlia sp. possesses a distinctly high EPSP/EPSC. A bacterial community is not mandatory, yet a facilitating factor for Ettlia sp. flocculation. In this study, the flocculation of the axenic Ettlia occurred via a patching mechanism mediated by small, dust-like EPS particles. The exclusive long filamentous EPS structure established by the bacterial community further increased the flocculation efficiency via an additional bridging mechanism. The bacterial community also strengthened the aggregate integrity by maintaining a more compact EPS structure. Acknowledgements This work was supported by the Advanced Biomass R&D Center (ABC), a Global Frontier Program funded by the Korean Ministry of Science and ICT (2010-0029723). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.02.062. References Alam, M.A., Vandamme, D., Chun, W., Zhao, X., Foubert, I., Wang, Z., Muylaert, K., Yuan, Z., 2016. Bioflocculation as an innovative harvesting strategy for microalgae. Rev. Environ. Sci. Biotechnol. 15, 573–583. Alam, M.A., Wan, C., Guo, S.-L., Zhao, X.-Q., Huang, Z.-Y., Yang, Y.-L., Chang, J.-S., Bai, F.-W., 2014. Characterization of the flocculating agent from the spontaneously flocculating microalga Chlorella vulgaris JSC-7. J. Biosci. Bioeng. 118, 29–33. Amin, S.A., Parker, M.S., Armbrust, E.V., 2012. Interactions between diatoms and bacteria. Microbiol. Mol. Biol. Rev. 76, 667–684. Basuvaraj, M., Fein, J., Liss, S.N., 2015. Protein and polysaccharide content of tightly and loosely bound extracellular polymeric substances and the development of a granular activated sludge floc. Water Res. 82, 104–117. Bloemberg, G.V., Lugtenberg, B.J.J., 2001. Molecular basis of plant growth promotion and biocontrol by rhizobacteria. Curr. Opin. Plant Biol. 4, 343–350. Bruckner, C.G., Rehm, C., Grossart, H.-P., Kroth, P.G., 2011. Growth and release of extracellular organic compounds by benthic diatoms depend on interactions with bacteria. Environ. Microbiol. 13, 1052–1063. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294–306. Chun, S.-J., Cui, Y., Ahn, C.-Y., Oh, H.-M., 2018. Improving water quality using settleable microalga Ettlia sp. and the bacterial community in freshwater recirculating aquaculture system of Danio rerio. Water Res. 135, 112–121. Do Nascimento, M., Dublan, M.d.l.A., Ortiz-Marquez, J.C.F., Curatti, L., 2013. High lipid productivity of an Ankistrodesmus-Rhizobium artificial consortium. Bioresour. Technol. 146, 400–407. Grossart, H.-P., 1999. Interactions between marine bacteria and axenic diatoms (Cylindrotheca fusiformis, Nitzschia laevis, and Thalassiosira weissflogii) incubated under various conditions in the lab. Aquat. Microb. Ecol. 19, 1–11. Guo, S.-L., Zhao, X.-Q., Wan, C., Huang, Z.-Y., Yang, Y.-L., Alam, M.A., Ho, S.-H., Bai, F.W., Chang, J.-S., 2013. Characterization of flocculating agent from the self-flocculating microalga Scenedesmus obliquus AS-6-1 for efficient biomass harvest. Bioresour. Technol. 145, 285–289. Heijstra, B.D., Pichler, F.B., Liang, Q., Blaza, R.G., Turner, S.J., 2009. Extracellular DNA
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37–43. Zhang, L., Feng, X., Zhu, N., Chen, J., 2007. Role of extracellular protein in the formation and stability of aerobic granules. Enzyme Microb. Technol. 41, 551–557. Zhu, L., Zhou, J., Lv, M., Yu, H., Zhao, H., Xu, X., 2015. Specific component comparison of extracellular polymeric substances (EPS) in flocs and granular sludge using EEM and SDS-PAGE. Chemosphere 121, 26–32.
Williams, T.J., Wilkins, D., Long, E., Evans, F., DeMaere, M.Z., Raftery, M.J., Cavicchioli, R., 2013. The role of planktonic Flavobacteria in processing algal organic matter in coastal East Antarctica revealed using metagenomics and metaproteomics. Environ. Microbiol. 15, 1302–1317. Ye, F., Ye, Y., Li, Y., 2011. Effect of C/N ratio on extracellular polymeric substances (EPS) and physicochemical properties of activated sludge flocs. J. Hazard. Mater. 188,
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Bacterial community enhances flocculation efficiency of Ettlia sp. by altering extracellular polymeric substances profile
T
Chau Hai Thai Vua,b, Seong-Jun Chuna,b, Seong-Hyun Seoa,c, Yingshun Cuia, Chi-Yong Ahna,b, ⁎ Hee-Mock Oha,b, a
Cell Factory Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea Department of Environmental Biotechnology, KRIBB School of Biotechnology, Korea University of Science & Technology (UST), 217 Gajeong-ro, Yuseong-gu, Daejeon 34113, Republic of Korea c Department of Life Science, Hanyang University, 222 Wangsimni-ro, Seongdong-gu, Seoul 04763, Republic of Korea b
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Microalgae Bacteria Extracellular polymeric substances (EPS) Ettlia sp. YC001 Flocculation
This study examined the effects of a bacterial community and extracellular polymeric substances (EPS) on Ettlia sp. flocculation. The growth rate, flocculation efficiency (FE), bacterial community, and EPS profile of axenic and xenic Ettlia cultures were monitored during 46 days of cultivation. For the xenic culture, with a great abundance of growth-promoting and flocculation-inducing bacteria, the biomass density was 18.75% higher and its FE reached 100% in the mid-stationary phase. Moreover, microscopic observation and a quantitative analysis of the EPS revealed the exclusive presence of long filamentous EPS and more compact structure in the xenic Ettlia culture, possibly explaining its better FE. Notwithstanding, for the axenic culture, despite a lower biomass density and reduced abundance of EPS, its FE reached 92.54% in the mid-stationary phase. Thus, the role of the bacterial community was found to be supportive rather than vital for the high settleability of the self-flocculating Ettlia microalgal culture.
1. Introduction The advantages of microalgae have already been recognized in a wide variety of applications (Pulz and Gross, 2004). In the case of
⁎
biodiesel, when compared with terrestrial plants, microalgae grow about 10–15 times faster with approximately 10–20 times higher productivity (Wan et al., 2015; Wang et al., 2016). The use of microalgae to produce pharmaceutical and other high-value products by
Corresponding author at: Cell Factory Research Center, KRIBB, 125 Gwahak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea. E-mail address: [email protected] (H.-M. Oh).
https://doi.org/10.1016/j.biortech.2019.02.062 Received 11 December 2018; Received in revised form 11 February 2019; Accepted 12 February 2019 Available online 13 February 2019 0960-8524/ © 2019 Elsevier Ltd. All rights reserved.
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triplicate, unless otherwise mentioned.
manipulating the cultivation conditions is also very promising. However, due to the small size of microalgae, their identical density with water, and the diluted concentrations of conventional suspension cultivation systems (0.5–10 g/L), the harvesting process—which accounts for roughly 20–30% of the total production cost—still remains the bottleneck for microalgal biotechnology (Chisti, 2007; Wan et al., 2015). Currently, centrifugation, filtration, flotation, and gravity sedimentation are the main harvesting techniques, among which the most popular ones are centrifugation and sedimentation. Flocculation before harvesting, which can be induced by physical, chemical or bio-based flocculation have been reported to save a significant amount of time and energy (Vandamme et al., 2013). However, some of these flocculation processes might contaminate the biomass and are not environment friendly or economically feasible (Wan et al., 2015). Currently, the idea of using self-flocculating microalgal species for harvesting the non-flocculating oleaginous cells has achieved great attention (Salim et al., 2012). Possible application in the field of water bloom treatment might also be promising, although no lab-scale research has been conducted yet. Till now, only a few self-flocculating microalgae species, such as Chlorella vulgaris JSC-7, Scenedesmus obliquus AS-6-1, Ettlia texensis, Ankistrodesmus falcatus, Tetraselmis suecica, and Chlorococcum sp. GD have been reported (Alam et al., 2014; Guo et al., 2013; Lv et al., 2016; Salim et al., 2014, 2013, 2012). Researches have implied the role of released extracellular polymeric substances (EPS) or algal organic matter (AOM) in the abovementioned autoflocculating microalgal species; however, culture axenicity has been overlooked. Without the precise acknowledgment of bacterial role, the auto-flocculation behavior or the real producer of the bioactive EPS might be misleading (Vu et al., 2018). Accordingly, this study examined whether the bacterial community or EPS play a vital role in the previously detected strong settling properties of Ettlia sp. YC001 (La et al., 2016). EPS can be categorized into soluble (S-) and cell surface bound (B-), and the latter further divided into outer loosely bound (LB-) and inner tightly bound (TB-) layers (Sheng et al., 2010). Since the B-EPS accumulate on the cell surface and directly change cell surface characteristics—such as surface hydrophobicity, charge density, and binding sites—only this fraction was studied in this research. The next generation sequencing (NGS) method was used to investigate the bacterial communities of Ettlia culture during different growth phases. The B-EPS of bacteria-free and xenic microalgal cultures were extracted and their compositions investigated. Thereafter, flocculation efficiency (FE) of the two Ettlia cultures were measured and explained based on the bacterial community and EPS profile.
2.2. Microalgal biomass density determination The growth curve was determined by monitoring the biomass density (g/L) based on the dry cell weight (DCW). Due to their floc forming characteristic, the cultures were well shaken before taking the aliquots. The samples were then filtered using an overnight-dried and preweighed GF/C grade glass microfiber filter (Whatman plc, United Kingdom). Thereafter, the filters were dried overnight in an oven at 105 °C until reaching a constant weight. All the measurements were performed in triplicate. 2.3. SEM sample preparation To confirm the axenicity and observe the EPS matrix, the Ettlia cell suspension was initially fixed in a pH 7.2 phosphate-buffered (0.1 M) mixture solution of 2.5% paraformaldehyde and glutaraldehyde for 2 h, and then postfixed in a 1% OsO4 solution with the same buffer for 1 h. The samples were then dehydrated in EtOH, and the solution replaced with isoamyl acetate. Thereafter, the samples were dried at the critical point in liquid CO2 and sputtered with gold using a Polaron SC502 sputter coater (Quorum Technologies Ltd., United Kingdom). The samples were observed using a scanning electron microscope, Quanta™ 250 FEG (FEI company, USA), and cell size was analyzed using the ImageJ measure analysis tool (National Institute of Health, USA). 2.4. Bacterial community analysis For the bacterial community analysis, 2 mL of the xenic Ettlia cell suspension was filtered through sterilized 3.0 μm and 0.22 μm Isopore™ polycarbonate membrane filters (Merck Millipore, Germany) to harvest all the attached and free-living bacteria, respectively. The membrane filters were stored at −80 °C until performing the DNA extraction. The DNA extraction, amplification, and sequencing was conducted as previously described (Chun et al., 2018). The bacterial 16S rRNA gene sequences and accompanying metadata have already been deposited in the Sequence Read Archive (SRA) of the NCBI under project number PRJNA506495. The statistical analyses and graphics were conducted using the R program (https://www.r-project.org). To assess the structural dissimilarities in the xenic Ettlia bacterial community at different growth phases, a multi-response permutation procedure was performed based on a Bray-Curtis distance matrix. Plus, to visualize the results, nonmetric multidimensional scaling (NMDS) was conducted using the vegan package (Oksanen et al., 2013). The bacterial relative abundance at the order level was transformed to a log10 scale, while the shifts in the bacterial community were presented as a heatmap using the gplots package (Warnes et al., 2016). The heatmap analysis only included bacterial OTUs with a maximum relative abundance higher than 1%, and the remaining OTUs were pooled together and presented as “Other bacteria”.
2. Materials and methods 2.1. Microalgal strain and cultivation conditions The microalgal Ettlia sp. YC001 strain (KCTC 12109BP) was obtained from the Korean Collection for Type Cultures (KCTC) at the Korea Research Institute of Bioscience and Biotechnology. A xenic seed culture was maintained in the steady state as previously described (Seo et al., 2017). An axenic culture was then isolated and confirmed using several enriched agar media and scanning electron microscopic (SEM) observation (Vu et al., 2018). For easy detection of contamination during culture maintenance, the axenic culture was stored in a solid agar plate. The axenic and xenic Ettlia seed cultures were inoculated into 1-L Erlenmeyer flasks containing 500 mL of a BG-11 medium at an initial density of 106 cell/mL, and then cultivated in a shaking incubator (100 rpm) at a constant temperature of 25 ± 1 °C with a supply of 5% CO2 at flow rate of 0.1 v/v/m and under continuous illumination of 100 μmol photons/m2/s. Aliquots of the cell suspension were harvested at different growth phases to determine the growth curve and for other analyses. All the experiments were conducted in
2.5. Ettlia sp. cell suspension physical properties The Ettlia cell suspension ξ-potential and FE were monitored at day 0 (initial phase), day 10 (mid-exponential phase), day 26 (mid-stationary phase), and day 46 (decline phase). An aliquot of the microalgal biomass was harvested at each stage and its ξ-potential measured in folded capillary cells using a Zetasizer Nano ZS (Malvern Panalytical, United Kingdom). For the FE, 100 mL of the cell suspension was poured into in a graduated cylinder up to a height of 12 cm and then left for free settlement for a period of 30 min. Aliquots were then taken at a height of 7.5 cm from the cylinder bottom at the initial (t = 0 min) and final (t = 30 min) time point to determine the biomass density. Due to the 57
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floc-forming nature of Ettlia, the DCW was used instead of the optical density. The FE was calculated using the following equation:
B Flocculation efficiency (%) = ⎛1 − ⎞ × 100 A⎠ ⎝
(1)
where A and B are the DCW at the beginning and after a settling time of 30 min, respectively.
2.6. EPS extraction, purification, and chemical analysis The LB- and TB-EPS fractions were separately extracted using a modified method from Li and Yang (2007). A microalgal pellet was harvested by centrifugation at 4,000 × g for 10 min. The nitrogen and carbon concentrations in the supernatant were determined using a TOC analyzer multi N/C® 3100 (Analytik Jena, Germany). Meanwhile, the cell pellet was re-suspended in a 50 °C NaCl solution (0.02 M) and readily vortexed for 1 min at 3,000 rpm to extract the LB-fraction. The LB-EPS was retrieved by centrifugation at 10,000 × g for 30 min and filtration through a 0.45 μm filter. Cell pellet was then re-suspended in the NaCl solution, heated for 30 min at 60 °C in a water bath, and the TB-EPS fraction retrieved in the similar manner to that explained above for the LB-EPS fraction. The cell pellet was freeze-dried to measure the DCW for normalization. The EPS solutions were further purified using 1 kDa MWCO prewetted dialysis tubing (Spectrum Laboratories, Inc., USA) against DI water (renewed twice daily) at 4 °C for 2 days. The EPS solutions were then re-concentrated using a Spectra/Gel® absorbent (Spectrum Laboratories, Inc., USA) according to manufacturer’s instructions. Thereafter, the solutions were aliquoted and stored at −20 °C for further analysis. The EPS content was indirectly measured using a TOC analyzer multi N/C® 3100 (Analytik Jena, Germany). The compositional protein (EPSP) and carbohydrate (EPSC) contents were determined using the methods of Lowry and Anthrone with bovine serum albumin and glucose used as the respective standards (Jermyn, 1975; Waterborg, 2009).
Fig. 1. Changes in biomass density of axenic and xenic Ettlia cultures under supply of 5% CO2. Data represent means of three biological replicates ± SD.
3. Results and discussion 3.1. Ettlia sp. cultures: growth and SEM observation The growth of axenic and xenic Ettlia cultures were quite identical in the first 9 cultivation days with a short lag phase of approximately 5 days, followed by 11 days of an exponential growth phase (Fig. 1). The biomass content in the axenic and xenic cultures reached a maximum of 2.40 ± 0.45 g/L and 2.85 ± 0.70 g/L, respectively, at the beginning of the stationary phase. This 18.75% increase in biomass density was well-agreed with some previous researches in diatom and microalgae cultures. When co-culturing Scenedesmus obliquus with different single or mixed bacterial cultures, the biomass density of S. obliquus was significantly increased by 3.5–24.8% (Wang et al., 2015). Similar increase in growth rate and cell density was obtained with different diatom cultures (Punctastriata sp., Achnanthes minutissima, Phaeodactylum tricornutum UTEX646, etc.) when co-cultured with several bacterial cultures (Bruckner et al., 2011; Grossart, 1999). Some diatom strains even showed positive growth with the addition of bacterial-cell free medium at a low amount as 0.1% (v/v). This growth stimulating phenomenon can be explained by (1) creating a favorable growth environment for microalgae; (2) providing their microalgal partner with essential macro- or micro-nutrients; (3) generating phytohormones, chelators or antibiotics (Vu et al., 2018; Wang et al., 2016). In the SEM micrographs, the Ettlia cells from the initial phase exhibited a spherical shape of 7.0–8.0 μm. The axenic culture showed no presence of bacteria and no or very little evidence of an EPS matrix (SEM micrographs are provided in Supplementary materials). Conversely, the xenic culture showed the presence of 1.5–4.0 μm bacillus bacteria from which interconnecting filamentous EPS matrices were observed. Moreover, the Ettlia cell surface showed evidence of both long and thin filamentous EPS matrices and the smaller, dust-like EPS matrices. As the cultures aged, the two Ettlia cultures started to show some different signs of aggregation/flocculation (SEM micrographs are provided in Supplementary materials). No obvious interconnecting network was detected in the axenic Ettlia culture except for the increased amount of dust-like EPS on the cell surfaces. However, a significantly increased amount of filamentous EPS, which was greatly expanded, entirely filling the gaps and firmly connecting two adjacent Ettlia cells, was discovered in the xenic culture. Dust-like EPS matrices were also detected in this culture, however, the particle size and amount seemed to be far lesser than in the axenic culture. Although the origin of these EPS matrices remain unknown, it is
2.7. Protein purification and SDS-PAGE analysis The extracted extracellular protein was precipitated by adding 3 times the volume of ice-cold acetone, mixed thoroughly, and kept overnight at −20 °C. The protein pellet was then recovered by centrifugation at 22,000 × g at 4 °C for 15 min, followed by drying in a vacuum evaporator for 15 min. Next, the protein pellets were re-suspended in a mixture of a rehydration buffer (urea 7 M, thiourea 2 M, CHAPS 4%, Tris 10 mM) and 2 × Laemli buffer (1:1, v/v) to a final protein concentration of 10 μg/μl. The protein solution was then heated at 90 °C for 5 min, briefly centrifuged at a high speed, and the supernatant was immediately used for SDS-PAGE analysis. The protein bands were separated in a 6–18% Tris-Glycine gradient gel, using Triple color protein marker (10–245 kDa, BioFACT, Korea), and visualized using a Pierce® silver stain kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Relative abundance of bands in each lane was quantified using the ImageJ gels analysis tool (National Institute of Health, USA) and presented as a heatmap using the gplots package in the R program (Section 2.4).
2.8. Statistical analysis The statistical analysis of the variables between the axenic and xenic Ettlia cultures was conducted using Student’s t-test. Differences were regarded as statistically significant when p < 0.05. Pearson’s correlation analysis was used to measure the association strength between the variables (Excel 2016, Microsoft Corporation, USA). 58
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Supplementary materials table, where the relatively higher abundance of the attached community agrees well with their adhesion behavior to cell and detrital surfaces (Williams et al., 2013). Growth-promoting bacteria stimulate the growth of their microalgal partner by various means. Bacteria belonging to the Rhizobiales order are known to fix nitrogen and have enhanced the growth of several microalgal cultures (Kim et al., 2014; Wang et al., 2015). They also stimulate microalgal growth by producing the indole-3-acetic acid phytohormone (Do Nascimento et al., 2013). The presence of such bacteria was possibly responsible for the stable soluble nitrogen concentration in the xenic culture when compared to the control and axenic cultures (Supplementary materials). Another rhizobacterium found in the xenic Ettlia culture was Brevundimonas vesicularis, which belongs to the Caulobacterales order (accounted for the highest abundance of 31.83% and 23.96% in the attached and free-living bacterial community, respectively, in the exponential growth phase, Supplementary materials table). Although the growth promoting mechanism of B. vesicularis remains unknown, it was reported to enhance the growth of Chlorella ellipsoidea approximately three times when compared with the axenic control sample (Park et al., 2008). Therefore, the noticeably high abundance of B. vesicularis in the exponential growth phase may partially explain the abovementioned outcompeted growth of the xenic Ettlia culture over the bacteria-free culture in this stage. Furthermore, Pseudomonas spp. from the order of Pseudomonadales are known for their ability to secrete multiple anti-fungal metabolites that protect their microalgal partner from a wide range of fungal pathogens (Bloemberg and Lugtenberg, 2001). The xenic culture also exhibited high amounts of bacteria previously reported to induce flocculation (Salehizadeh and Shojaosadati, 2001). The bioflocculants produced by bacteria belonging to the Bacillus, Paenibacillus (Bacillales order), and Flavobacterium (Flavobacteriales order) genera have been reported to increase the flocculation efficiency of several green algae cultures (Oh et al., 2001; Salehizadeh and Shojaosadati, 2001). In contrast, the bacteria of the Sphingobacteriales order, rather than their metabolites, have been shown to be vital in the flocculation of C. vulgaris culture (Lee et al., 2013). Acidovorax spp. were the dominant species of the Burkholderiales order found in the current bacterial community (Supplementary materials), and are frequently abundant in activated sludge and form copious flocs in a broth culture (Heijstra et al., 2009).
suggested that the dust-like EPS originated from the microalgae, whereas the filamentous EPS were produced by either the associated bacterial community or the Ettlia itself induced by its bacterial partners. Identical EPS structures were also observed in an Ettlia texensis culture although the axenicity of the culture was unstated (Salim et al., 2014). The authors have reported that the short EPS matrices seemed to patch the cell surfaces yet they seemed too short to visually bind with other E. texensis cells, while the filamentous EPS was greatly extended and bound with other microalgal cells. To our knowledge, our SEM micrographs were the first ones that clearly discriminate the EPS structure of the pure and mixed Ettlia sp. culture. In addition, the cell surface of the xenic culture showed deep and well-defined crinkles, while the cell surface of the axenic culture was rather smooth. A similar phenomenon was also observed in the co-culture of Chlorella ellipsoidea with growthpromoting Brevundimonas sp. bacteria (Park et al., 2008). Possibly, the change of cell surface morphology was in response to an inter-kingdom signal between the microalgae and the bacteria; where the crinkles provided a better attachment for the associated bacteria (Amin et al., 2012). 3.2. Bacterial community analysis The bacterial community composition was investigated in each Ettlia growth phase using an Illumina MiSeq sequencing approach. A total of 393,475 sequence reads were obtained after trimming lowquality sequences. The obtained sequences were then divided into 136 operational taxonomic units (OTUs) based on a pairwise sequence identity cutoff of 99%. The OTUs were then further classified into a total of 15 classes, 32 orders, and 69 genera. The NMDS plot in Fig. 2A separates the OTUs into four distinct groups, indicating the strong grouping of bacterial communities within the experiment replications as well as the community distinction at different cultivation times. An ANOSIM test confirmed the statistical significance of the clustering (R = 0.851, P < 0.001). Moreover, the proximity of the day 0 and day 26 clusters implied a relatively conserved bacterial community across the Ettlia generations, as the initial microalgal suspension was directly retrieved from the cell suspension in its stationary phase in the chemostat. Fig. 2B shows the shift in the relative abundance of the bacterial community during the 46-day cultivation. The Proteobacteria phylum and Sphingobacteriia class were the most dominant, followed by the Actinobacteria, Bacilli, and Flavobacteriia classes. Further analysis of the microbial taxonomic composition showed that 27 of the 136 OTUs were shared by all the samples and over 99% of the sequences belonged to the top 30 OTUs in each sample. In the top 30 OTUs, bacteria belonging to the Flavobacteriia, Bacilli, Proteobacteria, and Sphingobacteriia classes appeared in all the samples. These bacteria are well-known as organic matter decomposing (Flavobacteriales), growth promoting (Rhizobiales, Caulobacteriales, Pseudomonadales), and flocculation inducing (Bacillales, Burkholderiales, Sphingobacteriales, Flavobacteriales). Thus, the presence of these functional bacteria was speculated to play an important role in the higher biomass content and FE in the xenic culture. Supplementary materials present the phylogenetic tree of the 30 most abundant OTUs during the course of the cultivation. The most well-known means of interaction between microalgae and bacteria partners is nutrient exchange. Autotrophic phytoplankton synthesize organic compounds and the photosynthates are then spontaneously released to the surroundings as metabolic by-products or in response to inter-kingdom signals (Vu et al., 2018). While small molecular weight biopolymers (600–800 Da and less) can be directly transferred through bacterial porin channels, larger polymers should be first degraded. The TonB-dependent transporter systems in the major degrader Flavobacteria allow the direct uptake of macromolecules that are too large to diffuse via porins, and then degrade them to smaller polymers that can later be utilized by other heterotrophs (Williams et al., 2013). The Flavobacteriales community abundance is listed in the
3.3. Physical properties of Ettlia cell suspension As shown in Fig. 3, the two Ettlia cultures exhibited very similar trends as regards their physical properties. During the first 10 cultivation days, the ξ-potential (Fig. 3A) dropped rapidly from −22.58 ± 3.43 mV and −28.38 ± 3.83 mV to approximately −13.83 ± 2.14 mV and −12.68 ± 1.20 mV in the middle of the exponential growth phase for the axenic and xenic Ettlia cultures, respectively. The ξ-potential then remained almost unchanged for the rest of the cultivation period. The FE (Fig. 3B) and cultivation time were strongly correlated (Pearson correlation coefficient r > 0.87), where the FE for the xenic culture (69.2–100%) was actually higher than that for the bacteria-free culture (50.0–99.2%), although the differences were not statistically significant. Discrepancies in the sedimentation behaviors of the two cultures were visually detectable in the Supplementary materials, with a distinctly larger floc size and clearer supernatant in the xenic culture and greenish tint in the axenic supernatant. Bacteria might play a decisive role in the flocculation of certain microalgal species (Lee et al., 2013). Therefore, there is a necessity to clearly define the axenicity of the studied cultures to prevent any potential misleading results (Vu et al., 2018). The relatively high FE of the axenic culture has confirmed the self-flocculating properties of Ettlia sp. YC001. Flocculation of microalgae can result from one or a combination of the following mechanisms (Vandamme et al., 2013). Charge 59
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Fig. 2. Analysis of bacterial community structure in xenic Ettlia culture. (A) Non-metric multidimensional scaling (NMDS) ordination plot of Bray-Curtis community dissimilarities based on 16S rRNA gene sequences collected at different cultivation times (D0: initial phase; D10: mid-exponential phase; D26: stationary phase; and D46: decline phase). Small golden circles represent OTUs. (B) Heatmap shows attached (A1–3) and free-living (F1–3) bacterial community structure at order level according to cultivation time. All analyses used three biological replicates.
underlying cause, as EPS have been reported to have a lower negative charge than the native cell surface (Liu et al., 2004; Rao et al., 2018). By lessening the electrostatic repulsion between two adjacent cells, the cells can approach close enough so the EPS matrix can patch or bridge them together. In the axenic culture, with the closely attached cells and the presence of only short, dust-like EPS matrices, electrostatic patching seemed to be the involved mechanism as being suggested by Alam et al. (2016). Conversely, in Ettlia xenic culture, with a large network of cells that were compactly bridged by the obvious filamentous EPS matrices, bridging was the main flocculation mechanism. Electrostatic patching by the dust-like EPS might also play a minor role in this culture (Alam et al., 2016). Therefore, the co-existence of bacterial community, along with the presence of unique filamentous EPS and the additional strong bridging flocculation mechanism, was the major cause of the difference
neutralization or an electrostatic patch is related to the overall or local alteration, respectively, of the microalgal cell surface charge by the adsorption of charged polymers, thereby reducing the repulsion force between adjacent cells. In a bridging mechanism, extended charged polymers simultaneously bind to the surfaces of adjacent particles and “bridge” them together, while sweeping flocculation is the co-precipitation or entrapment of microalgal cells with or within metal precipitates. However, previous studies had suggested that only the first three mentioned mechanisms were important in bio-flocculation process (Alam et al., 2016). In the current study, during the initial cultivation, charge neutralization or an electrostatic patch seemed to play an important role when the increase in the microalgal suspension FE was coincident with a decrease in the negative charge of the cell surface. The production of EPS during the initial stage may have been the
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stimulate the production of EPS from their partners, consuming/modifying them, or even producing their own EPS to induce specific surface attachment. EPS abundance and composition can vary under the impacts of both internal and external factors, such as microbial species, physical stage, nutrients, stress, and especially by the interaction with other species (Sheng et al., 2010). The total EPS amount was independent of the medium C/N ratio whereas EPSP and EPSC were reported to have the respective negative and positive relationship (Sheng et al., 2010; Ye et al., 2011). This is in accordance with the results of the axenic Ettlia batch culture, where inorganic carbon source was continuously provided while the nitrogen source is limited (Supplementary materials). The results of the xenic Ettlia culture, however, were slightly different when compared with the previous literature. This might be due to the dynamics of the bacterial community (Liu et al., 2004). It was discussed that depending on their mutual relationship in the coculture, the EPS abundance and composition can either be increased or decreased (Grossart, 1999; Wang et al., 2015). The axenic Ettlia culture has a relatively high LB-EPS percentage of 26.01–33.02% (Supplementary materials table). A lower LB-proportion in the xenic Ettlia culture (8.98–19.08%) implied the more compact EPS structure as previously it has been suggested that TB-EPS is a well-defined and dense layer firmly connects with the cell surfaces, while LBEPS is a loose structure without an obvious edge (Lin et al., 2014; Sheng et al., 2010). It seemed that the intertwined filamentous EPS structure was seemingly more successful in maintaining the EPS integrity. The increase in the relative percentage of LB-EPS according to the cultivation time likely indicated structural deterioration or cell lysis. Indeed, a high amount of LB-EPS has been reported to have an unfavorable effect on cell flocculation (Li and Yang, 2007). The structure of LB-EPS is not only more diffused, they also tend to be more porous and more loosely connected. Therefore, cells connected by this type of attachment tend to have weaker structure and smaller aggregate size. This result further elucidates the superior FE of the xenic Ettlia culture and explains the greenish tint due to the suspended cells in the supernatant of the axenic culture (Supplementary materials). The predominance of the EPSP fraction in Ettlia exopolymers has also been reported in the previous work of Salim et al. (2014). Due to the reports that most EPSP are hydrophobic and cell surface hydrophobicity is the triggering force for bioflocculation, EPSP fraction has often been reported as vital in maintaining the structure and flocculation ability of aggregates (Basuvaraj et al., 2015; Liu et al., 2004; Qu et al., 2012). Notwithstanding, due to their hydrophilic nature, carbohydrates tend to attract more fluid from the environment, thereby increasing the water content and porosity, and subsequently loosening the linkage strength between adjacent cells (Basuvaraj et al., 2015; Sheng et al., 2010). Therefore, a high content of EPSC, especially in the LB-layer is believed to be detrimental for flocculation. Literature reviews have revealed the connection between relatively high EPSP/EPSC ratio with the high flocculation and settleability in activated sludge (Supplementary materials table). High proportion of EPSP has also seemed to be involved in the intrinsic self-flocculating characteristic of E. texensis and Chlorococcum sp. GD, as well as the colonial growth characteristic of some microalgae/cyanobacteria (Lv et al., 2016, 2018; Salim et al., 2013; Wang et al., 2014). Consequently, a high EPSP/EPSC ratio of both axenic and xenic Ettlia culture, especially at the primary interaction surface of LB-EPS, might explain the self-flocculating characteristic of the Ettlia sp.
Fig. 3. Changes in Ettlia cell suspension ξ-potential (A) and flocculation efficiency (B) during cultivation period. Statistical analysis between axenic and xenic cultures was performed using unpaired Student t-test, where (*) denotes statistical significance (p < 0.05). Data represent means of three biological replicates ± SD.
in the FE between the two cultures. 3.4. Extracellular polymeric substances: quantitative analysis Despite the unique EPS structures discovered previously, there was no statistical difference in the EPS abundance between the axenic and xenic Ettlia cultures. The variation in the EPS profile of the two cultures (Fig. 4) followed a similar trend. In general, the total B-EPS content in the xenic Ettlia was 82–17% higher than that in the pure culture, and an identical trend was also observed for the TB-fraction (Fig. 4A). In the axenic culture, despite a modest content, EPS were steadily produced with a consistent increase throughout the cultivation (Supplementary materials table, r = 0.99). Conversely, there was no significant change in the EPS amount at the p < 0.05 level in the xenic culture. The axenic culture seemed to have a higher proportion of the easily extractable LBfraction, with the absolute abundance roughly 1.5 times higher than that in the xenic culture, although the difference was not statistically significant. During the 46-day cultivation, the EPSP content showed a gradual drop in both cultures, while the EPSC profile showed a similar tendency to the EPS (Fig. 4B and C). In addition, the relatively high mass ratio between EPSP and EPSC (Fig. 4D) has implied the predominance of EPSP in the Ettlia EPS. Microbes can release EPS either actively or passively as the products of metabolic activities and cell lysis. Autotrophic microorganisms can release some of their photosynthates into the phycosphere to attract beneficial bacteria or initiate partner attachment. Bacteria can also interact with EPS in diverse ways by secreting special molecules to
3.5. Extracellular protein: SDS-PAGE analysis SDS-PAGE was conducted for a deeper analysis of the EPSP component in the LB- (Fig. 5A) and TB-fractions (Fig. 5B) of the native and bacteria-associated Ettlia cultures. Both gels repeatedly showed similar protein bands at a high MW range of 180–245 kDa, although the TBEPSP bands intensity were much darker. A thick band also developed exclusively around 135 kDa during the mid-exponential growth phase. 61
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Fig. 4. Changes in EPS content (A) and EPS composition (B–D) of axenic and xenic Ettlia cultures in different growth phases. Statistical analysis was performed using unpaired Student t-test, where (*) denotes statistical significance (p < 0.05). Data represent means of three biological replicates ± SD.
the protein bands with the highest relative abundance. For the LB-EPS, the protein bands with the highest relative abundance appeared in the 63–75 kDa range during the mid-exponential growth phase and then gradually moved to a lower MW range of 48–63 and 35–48 kDa as the cultivation continued. The TB-proteins with the highest relative
Most of the LB-EPSP bands were located in the range of 25–100 kDa with a uniquely thick band appearing around 75 kDa at day 10. The protein bands in the TB-EPSP gel were mainly located in the lower range of 20–48 kDa at day 10, then additional bands appeared in the range of 11–20 kDa at day 26 and 46. Of interest was also the size transition of 62
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Fig. 5. Analysis of compositional (A) LB- and (B) TB-EPSP by SDS-PAGE in different growth phases (D10: mid-exponential phase; D26: stationary phase; and D46: decline phase). Three sample lanes on left were collected from axenic Ettlia (A-), while three lanes on right were from xenic Ettlia (X-). Corresponding heatmap represents relative abundance of proteins from different size fractions. Relative percentage of proteins is color-coded as shown in scale.
appeared in one culture (bands annotated with arrows). While SDS-PAGE profiles of activated sludge have been frequently reported, the current study would seem to be the first SDS-PAGE profile for an isolated microalgae culture at different growth stages (Zhang
abundance were located in the MW range of 35–48 kDa and remained unchanged in the xenic culture, whereas the axenic culture showed a transition to a lower range of 25–35 kDa. Most proteins were found to appear in both the axenic and the xenic cultures, yet certain bands only 63
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et al., 2007; Zhu et al., 2015). When compared with the profiles of activated sludge samples, the bands in the Ettlia EPSP profile were less smeared and more well-defined, probably due to their “purer” constitution and/or the utilization of a more sensitive silver staining protocol. As a result, more noteworthy conclusions could be drawn. There are currently two hypotheses about the origin of LB-EPS: (1) they belong to a higher polymer size fraction that is more prone to be extracted under mild conditions or (2) they result from the shedding of TB-EPS after certain degradation over time (Basuvaraj et al., 2015). Thus, the distinctive higher protein size of EPSP in this study confirmed the accuracy of the first hypothesis and explained why LB-EPS are normally described to have a more diffused and protruded structure. In addition, the dynamic transition of the proteins to a smaller size, especially in the more exposed LB-layer, indicated signs of polymeric degradation by an extracellular enzyme or some kind of transformation in the EPS structure.
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4. Conclusions Self-flocculating Ettlia sp. possesses a distinctly high EPSP/EPSC. A bacterial community is not mandatory, yet a facilitating factor for Ettlia sp. flocculation. In this study, the flocculation of the axenic Ettlia occurred via a patching mechanism mediated by small, dust-like EPS particles. The exclusive long filamentous EPS structure established by the bacterial community further increased the flocculation efficiency via an additional bridging mechanism. The bacterial community also strengthened the aggregate integrity by maintaining a more compact EPS structure. Acknowledgements This work was supported by the Advanced Biomass R&D Center (ABC), a Global Frontier Program funded by the Korean Ministry of Science and ICT (2010-0029723). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.biortech.2019.02.062. References Alam, M.A., Vandamme, D., Chun, W., Zhao, X., Foubert, I., Wang, Z., Muylaert, K., Yuan, Z., 2016. Bioflocculation as an innovative harvesting strategy for microalgae. Rev. Environ. Sci. Biotechnol. 15, 573–583. Alam, M.A., Wan, C., Guo, S.-L., Zhao, X.-Q., Huang, Z.-Y., Yang, Y.-L., Chang, J.-S., Bai, F.-W., 2014. Characterization of the flocculating agent from the spontaneously flocculating microalga Chlorella vulgaris JSC-7. J. Biosci. Bioeng. 118, 29–33. Amin, S.A., Parker, M.S., Armbrust, E.V., 2012. Interactions between diatoms and bacteria. Microbiol. Mol. Biol. Rev. 76, 667–684. Basuvaraj, M., Fein, J., Liss, S.N., 2015. Protein and polysaccharide content of tightly and loosely bound extracellular polymeric substances and the development of a granular activated sludge floc. Water Res. 82, 104–117. Bloemberg, G.V., Lugtenberg, B.J.J., 2001. Molecular basis of plant growth promotion and biocontrol by rhizobacteria. Curr. Opin. Plant Biol. 4, 343–350. Bruckner, C.G., Rehm, C., Grossart, H.-P., Kroth, P.G., 2011. Growth and release of extracellular organic compounds by benthic diatoms depend on interactions with bacteria. Environ. Microbiol. 13, 1052–1063. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294–306. Chun, S.-J., Cui, Y., Ahn, C.-Y., Oh, H.-M., 2018. Improving water quality using settleable microalga Ettlia sp. and the bacterial community in freshwater recirculating aquaculture system of Danio rerio. Water Res. 135, 112–121. Do Nascimento, M., Dublan, M.d.l.A., Ortiz-Marquez, J.C.F., Curatti, L., 2013. High lipid productivity of an Ankistrodesmus-Rhizobium artificial consortium. Bioresour. Technol. 146, 400–407. Grossart, H.-P., 1999. Interactions between marine bacteria and axenic diatoms (Cylindrotheca fusiformis, Nitzschia laevis, and Thalassiosira weissflogii) incubated under various conditions in the lab. Aquat. Microb. Ecol. 19, 1–11. Guo, S.-L., Zhao, X.-Q., Wan, C., Huang, Z.-Y., Yang, Y.-L., Alam, M.A., Ho, S.-H., Bai, F.W., Chang, J.-S., 2013. Characterization of flocculating agent from the self-flocculating microalga Scenedesmus obliquus AS-6-1 for efficient biomass harvest. Bioresour. Technol. 145, 285–289. Heijstra, B.D., Pichler, F.B., Liang, Q., Blaza, R.G., Turner, S.J., 2009. Extracellular DNA
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