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Enhanced microalgae growth through stimulated secretion of indole acetic acid by symbiotic bacteria
Algal Research 33 (2018) 345–351
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Enhanced microalgae growth through stimulated secretion of indole acetic acid by symbiotic bacteria
T
Guo-Hua Daoa, Guang-Xue Wub, Xiao-Xiong Wanga,c, Tian-Yuan Zhanga, Xin-Min Zhanc, ⁎ Hong-Ying Hua,c, a
Environmental Simulation and Pollution Control State Key Joint Laboratory, State Environmental Protection Key Laboratory of Microorganism Application and Risk Control (SMARC), School of Environment, Tsinghua University, Beijing 100084, PR China b Key Laboratory of Microorganism Application and Risk Control (MARC) of Shenzhen, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, Guangdong, China c Shenzhen Environmental Science and New Energy Technology Engineering Laboratory, Tsinghua-Berkeley Shenzhen Institute, Shenzhen 518055, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Microalgae Bacteria Synergistic mutualism Indole acetic acid Soluble algal products
In many microalgal cultivation systems, microalgae co-exist with bacteria, while little is known about the characteristics of their symbiotic relationships. In this study, twenty-six microalgae growth-promoting bacteria were isolated from a culture system of Scenedesmus sp. LX1 cultivated in the secondary effluent from domestic wastewater by using the high-throughput multiple well plate screening method. Ten strains were found to produce and secrete indole acetic acid (IAA), promoting the growth of microalgae. Meanwhile, the microalgae might have secreted signal substances to induce IAA production in bacteria, which was amplified in the tryptophan abundant environment. This indicates that bacteria may mainly promote the growth of the co-existing microalgae through secreting IAA, and microalgae would selectively enhance IAA secretion in turn. Microalgae cultured with microalgal growth-promoting bacteria would be a new potential strategy for improving large-scale microalgal cultivation in an economic and environmentally-friendly way.
1. Introduction In the commonly used open ponds and closed photobioreactors for the large-scale culture of microalgae, bacterial contamination often occurs. This may accompany the entire microalgal culture process and influence microalgae growth [1–3]. The possible interactions between bacteria and microalgae are synergistic, competitive, parasitic and horizontal gene transfer in the culture system [4,5]. Among these, synergistic mutualism can promote the growth of microalgae. Thus, the synergistic mutualism in microalgae culturing systems may be used to improve biomass productivity and reduce the culturing cost for largescale microalgal biomass production [6–8]. There are two types of synergistic mutualism between microalgae and bacteria. One is by exchanging materials and resources, and the other is by signal communication. Early studies were mainly focused on material and resource exchange and obtained some important results. Foster et al. [9] showed that nitrogen-fixing cyanobacteria could fix nitrogen for microalgae, likely in the form of ammonia or dissolved organic nitrogen. In addition to cyanobacteria, Azospirillum, which is a common N2-fixing bacterium, has been implicated in promoting the
growth of Chlorella vulgaris [6]. Some microalgae lack a gene encoding vitamin-B12 independent methionine synthase (MetE) and thus require an exogenous source of vitamin-B12 to synthesize essential methionine. In the aquatic environment, many bacteria are able to produce vitaminB12, thus providing for microalgae [10]. In return, the microalgal dissolved organic matter (DOM) can serve as the carbon, nitrogen and energy sources for the co-existing bacteria. In these cases, the growth of microalgae and bacteria could be significantly enhanced. However, the synergistic mutualism of exchanging materials and resources is a nonspecific interaction. Signal exchange is another important method of synergistic mutualism between microalgae and bacteria. The substances are used for communication, not as nutrients. They can activate or inhibit gene expression or biological activity, resulting in changes in the growth and metabolism of cells. Over the past few years, the signal interactions between microalgae and bacteria have received increasing attention. A symbiotic relationship between a marine bacterium (Phaeobacter inhibens) and marine microalgae (Emiliania huxleyi) was reported whereby the bacteria produced growth hormones and antibiotics to influence the growth of microalgae [11–13]. Chlorella vulgaris secretes a
⁎ Corresponding author at: Environmental Simulation and Pollution Control State Key Joint Laboratory, State Environmental Protection Key Laboratory of Microorganism Application and Risk Control (SMARC), School of Environment, Tsinghua University, Beijing 100084, PR China. E-mail address: [email protected] (H.-Y. Hu).
https://doi.org/10.1016/j.algal.2018.06.006 Received 26 February 2018; Received in revised form 3 June 2018; Accepted 9 June 2018 2211-9264/ © 2018 Elsevier B.V. All rights reserved.
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microalgae culture (a microalgae density of 1 × 106 cell mL−1) and 2 μL of various bacterial colony cultures (the bacteria density was OD600 = 1.0) were added to a new transparent 96-well plate together. Each well was filled with mBG11 medium to 100 μL. In addition, two types of control cultures were used for each batch of 96-well plates, i.e., non-bacterial culture and only culture medium, respectively. The transparent 96-well plate was sealed with a breathable sealing film and rotationally cultured at 25 °C with a 14 h/10 h light and dark cycle. The density of the microalgae was measured at 650 nm using a microplate reader (SpectraMax M5, Molecular Devices, USA).
certain signal substance that inactivates the signal substances of the bacterial acyl-homoserine lactones (AHLs), thereby inhibiting the production of bacterial toxins [14], while Azospirillum could promote the growth of microalgae by secreting some hormones, such as IAA [6,15]. The study of signal exchange between microalgae and bacteria was mainly focused on the ecological environment. However, little attention has been paid to the process of microalgae production. Moreover, the early study of bacterial isolates was limited to traditional methods that required tedious work and were less efficient [1]. There is less understanding of bacterial species and the signal exchange form in this process, especially regarding how microalgae affect bacteria based on their own growth. The aim of this study was to investigate the relationship between microalgae and symbiotic bacteria by signal exchange in the microalgae culture process. The method for highly efficient microalgae growth promoting bacteria screening was established. The characteristics of the isolated bacteria were studied, especially their secretion of small molecules. The hypothesis that IAA played an important role as a signaling molecule of the interaction between bacteria and microalgae was proposed and proved. The results provided a strategy to improve the productivity of microalgal biomass in large-scale microalgae cultures.
2.3. Analytical methods 2.3.1. Microalgae/bacteria growth property The microalgal density was determined by microscopic counting using hemocytometer and absorbance. The bacterial density was determined by the plate counting method. 2.3.2. Identification of bacteria and the phylogenetic relationship Genome extraction from the isolated bacteria was carried out using the TIANamp Bacteria DNA Kit (TIANGEN, China). The 16 s rDNA genes of the extracted genome were amplified by PCR with the primers 27F (5′-AGAGTTTGATCCTGGCTCGA-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The amplified products were sequenced and then BLAST (Basic Local Alignment Search Tool) was used to compare to the NCBI 16S ribosomal RNA sequences database. The phylogenetic trees of the isolates were reconstructed using neighbor joining with MEGA 5.0 [17,18]. Bootstrap support for the neighbor joining tree was determined using 1000.
2. Materials and methods 2.1. Culturing conditions and medium Scenedesmus sp. LX1 (Collection No. CGMCC 3036 at the China General Microbiological Culture Collection Center) isolated from tap water was used as the microalgae inoculum. Scenedesmus sp. LX1 was grown in modified BG11 (mBG11) medium [16] at 25 °C under a light intensity of 6000 lx, with light/dark periods of 14 h/10 h. Bacterial colonies were obtained using the gradient dilution plate method. First, Scenedesmus sp. LX1 was cultivated in the secondary effluent until the end of logarithmic growth. Then, the microalgaebacteria mixed culture was diluted in a 10 times gradient using phosphate buffer saline (PBS) (pH = 7.3). Next, the diluted solution was spread on Reasoner's 2A (R2A) medium (0.5 g starch, 0.5 g yeast extract, 0.5 g tryptic peptone, 0.5 g glucose, 0.3 g K2HPO4, 0.05 g MgSO4, 0.25 g succinate, and 0.5 g casamino acids in 1000 mL distilled water) agar plates and cultured at 25 °C until single colonies appeared. For detecting IAA secretion ability, bacteria were cultured under six conditions. Two conditions with microalgae cells (containing 10 mg L−1 L-tryptophan (Trp) and Trp free, respectively) were used to examine the effect of microalgae cells. Two conditions with microalgal soluble algal products (SAP) which removed microalgae cells (containing 10 mg L−1 Trp and Trp free) were used to investigate the effect of SAP. Two conditions with R2A medium (containing 10 mg L−1 Trp and Trp free) were used as the control. The same dissolved organic carbon (DOC) concentration of 52 ± 2 mg L−1 was used in all six conditions before inoculating the bacteria. Bacteria were inoculated in the six conditions with the inoculation 10 times that of microalgal cells (1 × 106 cells mL−1). There were three replicates for each culture condition. The mixture was incubated for 48 h at 25 °C and 180 rpm (ZQZY-70CS, Zhichu, China).
2.3.3. Qualitative and quantitative analysis of IAA molecules The production of IAA by the isolates was determined according to Glickman and Dessaux [19]. First, the isolates were cultured in 50-mL flasks containing 20 mL R2A supplemented with L-Trp (200 mg L−1) for 48 h on a rotary shaker at 120 rpm and 28 °C. Then, 50 μL of Salkowsky reagent (50 mL 35% HClO4 + 1 mL 0.5 M FeCl3) was added to 50 μL of culture, and was allowed to react under dark conditions for 30 min at room temperature. For the positive control, 50 mg L−1 of IAA solution was added instead of cultures. Finally, a red color appeared indicating the presence of IAA and confirming that the bacteria could secrete IAA. An HPLC (LC-20 AT, Shimadzu, Japan) tandem with a photodiode detector (Shimadzu, Japan) was used to measure the IAA concentration. The samples were filtered using a syringe filter with a pore size of 0.22 μm. The filtrate was collected in a 1.5 mL vial. An ODS-C18 column (5 μm particle size, 150 × 4.2 mm, J&K Chemical Co., China) was used to quantify IAA concentration with an elution phase of methanol:water:acetic acid (550:450:1 v/v). The wavelength for detection was 280 nm. The elution rate of the mobile phase was 1.0 mL min−1. The injection volume was 10 μL for the IAA detection calibrated with standard IAA (Sigma, USA).
2.2. High throughput screening method
2.3.4. Extraction of microalgal soluble algal products The microalgae culture solution (at the end of logarithmic growth) was filtered using a 0.45-μm filtration membrane. The microalgal cells were held on the 0.45-μm filtration membrane and the soluble algal products (SAP) were obtained in the liquid phase.
In this study, the high-throughput multiple-well plate method was developed for efficient isolation of bacteria from microalgae (Scenedesmus sp. LX1) cultivation (Fig. 1). First, the mixed culture of microalgae-bacteria was spread on R2A agar plates for picking up single colonies of bacteria. Single colonies were picked up with sterile toothpicks and then inoculated in 2-mL 96well plates containing the R2A liquid medium. The 2-mL 96-well plates were sealed with a breathable sealing film and cultured at 25 °C and 180 rpm until the OD600 value reached 1.0. Then, 20 μL of the fresh
2.3.5. SEM observation of symbiotic distribution of microalgae and bacteria The sample were harvested by centrifugation at 12000 g for 10 min at 15 °C and the supernatants were removed. The sample were fixed 12 h (4 °C) in the fixative containing 0.1 M phosphate-buffered solution (pH = 7.3), 2% glutaraldehyde, and 4% paraformaldehyde and then were washed with deionized water. Sample was then dehydrated with increasing concentrations of an ethanol solution (50%, 70%, 90% and 100%) and dried in 100% tert-butyl alcohol. Finally, the microcosmic structure of microalgae-bacteria symbiosis was observed using scanning 346
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Fig. 1. High-throughput multiple-well plate screening method.
sp. LX1 cultivated in secondary effluent systems. Compared to the control (the bacteria-free systems), most bacteria not only promoted the microalgal biomass yield but also the growth rate (Fig. S1). Twenty-six bacteria (marked as A-1–26) were isolated that were excellent at promoting the growth of microalgae. The microalgae growth quantity and specific growth rate were > 0.15 (Abs650) and 0.19 (d−1), respectively. Compared with the traditional screening method (using a conical bottle culture mixture of microalgae and bacteria), the high-throughput multiple-well plate screening method improved the efficiency of screening. At low microalgae/bacteria ratio (1:0–1:100) condition, the Abs650 can be good detecting the microalgae growth (Fig. S2). This was the first time the high-throughput method was used to screen microalgae-promoting bacteria, which provides a new method in the field of microalgae research. The 16 s ribosomal DNA phylogenetic analysis showed that the twenty-six bacteria were from four phyla and seven orders (Fig. 2). At
electron microscopy (JSM-7001F, JEOL, Japan). 2.4. Statistical analysis Each experiment consisted of three replicates. Independent-Sample t-test was used for significant difference analysis, using the SPSS (version 22.0) statistical software. 3. Results and discussion 3.1. Isolation of microalgae growth promoting bacteria In this study, the high-throughput multiple well plate screening method was developed (Fig. 1). Then, microalgae growth-promoting bacteria were isolated from the whole culturable bacteria community (including 400 strains in total) that was separated from the Scenedesmus 347
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Fig. 2. Phylogenetic tree of isolated bacterial strains from the phycosphere of Scenedesmus sp. LX1. Table 1 Summary of IAA secretion bacteria. Strain no.
Genus
Order
Phylum
Characteristics
Reference
A-1 A-3 A-4 A-5 A-10 A-12 A-15 A-16 A-19 A-22
Pseudomonas Pseudomonas Pseudomonas Pseudomonas Stenotrophomonas Achromobacter Achromobacter Stenotrophomonas Bacillus Acinetobacter
Pseudomonadales Pseudomonadales Pseudomonadales Pseudomonadales Xanthomonadales Burkholderiales Burkholderiales Xanthomonadales Bacillales Pseudomonadales
Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Firmicutes Proteobacteria
PGPB PGPB PGPB PGPB Sewage treatment bacteria – – Sewage treatment bacteria PGPB –
[21,24] [21,24] [21,24] [21,24] [41] – – [41] [22,25] –
the phyla level, isolated bacteria belonged to β-proteobacteria, γ-proteobacteria, actinomycetes and firmicutes. Cho et al. [1] also reported that fourteen microbes were isolated from xenic Chlorella vulgaris culture, belonging to actinomycetes, firmicutes, proteobacteria and bacteroidetes. At the genus level, the isolates were mainly Pseudomonas, Bacillus, Acinetobacter and Exiguobacterium (Fig. 2). Among those, twelve strains were Pseudomonas, which is a proteobacteria widely found in soil and water. Bacillus, which is a firmicutes, also exists widely in the environment. The previous studies showed that Pseudomonas and Bacillus could significantly enhance the growth of C. calcitrans and N. oculata [20]. Moreover, the previous study has proved that Pseudomonas and Bacillus enhanced plant growth by secrete IAA [21,22]. Therefore, in addition to carbon resource (CO2) supply [23],
the bacteria may stimulate microalgal growth by secretion IAA. Pseudomonas and Bacillus could also act as a general plant growth promoting bacteria (PGPB) for numerous plant species [24,25]. Therefore, they are often used for enhancing plant growth to increase productivity.
3.2. Identification of bacterial IAA secretion ability PGPB could secrete small molecular substances, such as phytohormones, to regulate the growth of plants [26,27]. IAA is one of the most common phytohormones. Previous studies showed that IAA could also enhance the growth and the lipid accumulation of microalgae [28–32]. Therefore, twenty-six isolated bacteria were further investigated for their ability to secrete small molecules (IAA), which 348
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of bacteria increased in all tryptophan overdose conditions (both R2A medium systems and microalgae culture systems) (Fig. 3b). In addition, bacterial culture with microalgae showed a higher IAA production, with an increase of 5.3–89.8 times compared to the bacteria cultured in the R2A medium (p < 0.05). Bacteria cultured with microalgae could also promote the production of IAA. It was inferred that microalgae might have some mechanisms to promote the bacterial ability of synthesizing and secreting IAA, and this promotion mechanism was amplified with the overdose of tryptophan. In addition to the direct exchange of nutrients, signal transduction is another pathway for synergistic mutualism between the microalgae and bacteria [5]. Previous research showed that small molecular exchange between microalgae and bacteria might occur in the culture environment and is not limited by direct contact between them. Kazamia et al. [38] found that M. loti, which could secrete small molecules (VB12), could support the growth of L. rostrate without direct physical contact. The microalgae secreted soluble algal products (SAP) into the culture medium, which were around microalgal cells and formed a region rich in organic matter. This region is considered to be for microbe communications. Fig. 4a shows that the isolated bacteria cultured in the microalgal SAP medium could secrete more IAA than bacteria cultured in the R2A medium. These results were consistent with the previous conjecture. The microalgal SAP was rich in organic matter, with an area containing signal molecules and the IAA synthetic substrate (tryptophan). Among those, the signaling molecules may stimulate the expression of IAA synthetic genes, leading to an increase in IAA secretion. If the signal molecules were contained the SAP, it could explain the previous results showing the obvious promotion ability of the microalgae in the tryptophan-abundant environment. To further determine whether SAP contained the signal molecules, tryptophan (10 mg L−1) overdose in the SAP medium was applied to reduce the effect of tryptophan concentration in different systems. The results showed that bacteria cultured in the SAP medium could secrete more IAA than bacteria cultured in the R2A medium (Fig. 4b). More than 2.1–41.5 times the IAA concentration were secreted by bacteria cultured under the SAP medium versus the R2A medium. This further indicated that microalgae might secrete some kind of signal molecules to affect bacterial metabolism. In our study, the microalgae might secrete signal molecules, which could promote bacterial IAA production. It was further confirmed that the promotion ability was more pronounced with sufficient tryptophan. The SAP had a complex material composition. Yu et al. [39] analyzed the main components of SAP secreted by Scenedesmus sp. LX1. The results showed that the small molecular fraction (< 1 kDa) was 57.8%, the middle molecular fraction (1–10 kDa) was 34.8% and the large molecular fraction (> 10 kDa) was 7.3%. The hydrophobic/hydrophilic property analysis showed that the hydrophilic fractions were the main organic matter in small molecular weight SAPs, while the hydrophobic matter was the larger portion in the large molecular weight SAPs [40]. A previous study showed that the marine P. multiseries-Sulfitobacter model system had a complex microbe interaction potentially occurring within the phycosphere. This area is filled with a certain number of hydrophobic signaling molecules and persisted despite seawater turbulence. Through analysis of the SAP secreted by Scenedesmus sp. LX1, the hydrophilic matter was the largest portion that might contain some signal molecules and stimulate the bacteria to synthesize and secrete IAA. IAA, an important signal substance in plants and bacteria, is also an important signal substance in microalgae and bacteria. It was secreted by bacteria and enhanced the growth of microalgae. At the same time, the microalgae also secreted some other signal molecules that could stimulate bacteria to secrete IAA. This relationship formed a feedback mechanism of signal exchange between the microalgae and bacteria, and it made the relationship closer. These signal molecules deserve further study to fully clarify their underlying mechanisms. This
Fig. 3. The effect of the microalgae system on the secretion of IAA by bacteria. Error bars represent the standard deviation. Note: Trp = tryptophan.
might directly affect the growth of microalgae [28,31,32]. Ten strains were identified with the ability to secrete IAA (Table 1), which was 39% of the total isolates. The results indicated that there was a certain proportion of IAA secreting bacteria in the microalgae culture system. Microalgae might have a screening ability for the enrichment of these IAA secreting bacteria. From the phylogenetic analysis, they were mainly five orders Pseudomonas, Bacillus, Acinetobacter, Achromobacter and Stenotrophomonas (Table 1). Among those, Pseudomonas, Bacillus, Acinetobacter and Stenotrophomonas had been reported with the ability to secrete IAA for symbiotic co-existence with plants [33–36]. However, Achromobacter was rarely found to secrete IAA, especially in a microalgae-bacteria symbiotic system. 3.3. The IAA secretion of symbiotic bacteria enhanced by microalgae Compared with culture in the R2A medium, bacteria cultured with microalgae had a stronger ability to secrete IAA (Fig. 3a). The unit bacteria secretion increased by 113%–265% (p < 0.05), indicating that microalgae culture conditions were more conducive to bacteria to produce IAA. Amin [37] showed that some diatom symbiotic bacteria could secrete growth promoters to influence the growth of microalgae. For example, in the P. multiseries-Sulfitobacter system, Sulfitobacter could stimulate P. multiseries cell division by secreting IAA. However, IAA synthesis also depended on the production of tryptophan by diatoms. At the same time, tryptophan secreted by diatoms might attract a large number of bacteria with the ability of converting tryptophan to IAA. This established a positive feedback mechanism for the growth of diatoms. Similar results were shown by Segev, the IAA production of Phaeobacter inhibens was significantly increased by tryptophan that secreted by Emiliania huxleyi [13]. Therefore, tryptophan provided by microalgae may be the cause of the increase in IAA production by bacteria. To determine whether the mechanism of microalgae-bacteria interaction was similar to the P. multiseries-Sulfitobacter system and E huxleyi-P inhibens system in our study, 10 mg L−1 of tryptophan was added to ensure the overdose of tryptophan in the culture systems. The 10 mg L−1 tryptophan had no obvious effect on the growth of bacteria (Table 2). The results showed that the amount of IAA secretion per unit 349
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Table 2 The bacterial number of different culture systems. Bacterial density was determined by the plate count method. Each sample makes five repetitions. Bacterial density was calculated using the five sample average of single colony number. Not: Trp = tryptophan; SAP = soluble algal products. Strain no.
Microalgae + Trp system (cells mL−1)
Microalgae system (cells mL−1)
SAP + Trp system (cells mL−1)
SAP system (cells mL−1)
R2A + Trp system (cells mL−1)
R2A system (cells mL−1)
A-1 A-3 A-4 A-5 A-10 A-12 A-15 A-16 A-19 A-22
4.85 × 107 6.16 × 108 8.00 × 107 6.40 × 107 2.60 × 107 2.48 × 108 4.10 × 108 3.83 × 108 3.64 × 108 4.48 × 108
4.20 × 107 5.96 × 108 6.55 × 107 7.20 × 107 2.55 × 107 2.71 × 108 3.99 × 108 3.89 × 108 3.45 × 108 3.84 × 108
1.33 × 108 7.46 × 108 9.10 × 107 3.60 × 107 8.50 × 106 4.62 × 108 4.17 × 108 4.63 × 108 4.44 × 108 5.11 × 108
1.56 × 108 6.75 × 108 1.04 × 108 3.45 × 107 1.40 × 107 4.32 × 108 4.31 × 108 4.43 × 108 4.34 × 108 4.90 × 108
1.20 × 108 6.46 × 108 1.42 × 108 1.20 × 108 1.49 × 108 4.39 × 108 4.23 × 108 4.14 × 108 4.27 × 108 5.88 × 108
9.90 × 107 6.90 × 108 1.06 × 108 1.13 × 108 1.26 × 108 4.21 × 108 4.68 × 108 4.28 × 108 4.45 × 108 6.18 × 108
knowledge will help to further promote the growth of microalgae, which is of great significance for large-scale microalgae culture. 3.4. The symbiotic distribution of microalgae and bacteria Scanning electron microscopy (SEM) is a common tool for examining the microstructure of organisms. In this study, the symbiotic microalgae-bacteria system was observed by SEM. From the micrographs, microalgae and bacteria formed a stable co-existence system, in which the bacteria were present on the microalgae cell surface and the microalgal extracellular polymeric substances (EPS) (Fig. 5). The same results was also get in confocal laser scanning microscopy (CLSM) micrographs (Fig. S3). This distribution not only benefits material and gas exchange but is also conducive to the exchange of signal materials. 4. Conclusions Twenty-six excellent microalgae growth-promoting bacteria were isolated from Scenedesmus sp. LX1 cultivated in secondary effluent systems by using the high-throughput multiple-well plate screening method. Of these, ten had the ability to secrete IAA to promote the growth of microalgae and the microalgae selectively enhanced the bacterial IAA secretion in turn. This may be a new microalgae-bacteria interaction mechanism that is of great ecological significance. At the same time, it would be a new potential green and economic strategy for improving large-scale microalgal cultivation.
Fig. 4. The effect of microalgal SAP on the secretion of IAA by bacteria. Error bars represent the standard deviation. Note: Trp = tryptophan.
Acknowledgements This work was supported by the Key Program of the National Natural Science Foundation of China (No. 51738005) and the Collaborative Innovation Center for Regional Environmental Quality of China.
Bacteria Microalgae
Microalgae
Fig. 5. SEM observation of co-cultivation of Scenedesmus sp. LX1 with bacteria (A-12). 350
EPS+Bacteria
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Conflict of interest statement [17]
The authors declare no conflict of interest.
[18]
Author contributions [19]
In this study, Xiao-Xiong Wang and Tian-Yuan Zhang participated in experimental design. Assoc. Prof. Wu, Prof Zhan and Prof. Hu gave general and strategic suggestions in this study. All authors agree to the authorship and submission of the manuscript to Algal research for peer review.
[20]
[21] [22]
Statement of informed consent, human/animal rights
[23]
No conflicts, informed consent, human or animal rights applicable. Appendix A. Supplementary data
[24] [25]
Supplementary data to this article can be found online at https:// doi.org/10.1016/j.algal.2018.06.006.
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Enhanced microalgae growth through stimulated secretion of indole acetic acid by symbiotic bacteria
T
Guo-Hua Daoa, Guang-Xue Wub, Xiao-Xiong Wanga,c, Tian-Yuan Zhanga, Xin-Min Zhanc, ⁎ Hong-Ying Hua,c, a
Environmental Simulation and Pollution Control State Key Joint Laboratory, State Environmental Protection Key Laboratory of Microorganism Application and Risk Control (SMARC), School of Environment, Tsinghua University, Beijing 100084, PR China b Key Laboratory of Microorganism Application and Risk Control (MARC) of Shenzhen, Graduate School at Shenzhen, Tsinghua University, Shenzhen 518055, Guangdong, China c Shenzhen Environmental Science and New Energy Technology Engineering Laboratory, Tsinghua-Berkeley Shenzhen Institute, Shenzhen 518055, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Microalgae Bacteria Synergistic mutualism Indole acetic acid Soluble algal products
In many microalgal cultivation systems, microalgae co-exist with bacteria, while little is known about the characteristics of their symbiotic relationships. In this study, twenty-six microalgae growth-promoting bacteria were isolated from a culture system of Scenedesmus sp. LX1 cultivated in the secondary effluent from domestic wastewater by using the high-throughput multiple well plate screening method. Ten strains were found to produce and secrete indole acetic acid (IAA), promoting the growth of microalgae. Meanwhile, the microalgae might have secreted signal substances to induce IAA production in bacteria, which was amplified in the tryptophan abundant environment. This indicates that bacteria may mainly promote the growth of the co-existing microalgae through secreting IAA, and microalgae would selectively enhance IAA secretion in turn. Microalgae cultured with microalgal growth-promoting bacteria would be a new potential strategy for improving large-scale microalgal cultivation in an economic and environmentally-friendly way.
1. Introduction In the commonly used open ponds and closed photobioreactors for the large-scale culture of microalgae, bacterial contamination often occurs. This may accompany the entire microalgal culture process and influence microalgae growth [1–3]. The possible interactions between bacteria and microalgae are synergistic, competitive, parasitic and horizontal gene transfer in the culture system [4,5]. Among these, synergistic mutualism can promote the growth of microalgae. Thus, the synergistic mutualism in microalgae culturing systems may be used to improve biomass productivity and reduce the culturing cost for largescale microalgal biomass production [6–8]. There are two types of synergistic mutualism between microalgae and bacteria. One is by exchanging materials and resources, and the other is by signal communication. Early studies were mainly focused on material and resource exchange and obtained some important results. Foster et al. [9] showed that nitrogen-fixing cyanobacteria could fix nitrogen for microalgae, likely in the form of ammonia or dissolved organic nitrogen. In addition to cyanobacteria, Azospirillum, which is a common N2-fixing bacterium, has been implicated in promoting the
growth of Chlorella vulgaris [6]. Some microalgae lack a gene encoding vitamin-B12 independent methionine synthase (MetE) and thus require an exogenous source of vitamin-B12 to synthesize essential methionine. In the aquatic environment, many bacteria are able to produce vitaminB12, thus providing for microalgae [10]. In return, the microalgal dissolved organic matter (DOM) can serve as the carbon, nitrogen and energy sources for the co-existing bacteria. In these cases, the growth of microalgae and bacteria could be significantly enhanced. However, the synergistic mutualism of exchanging materials and resources is a nonspecific interaction. Signal exchange is another important method of synergistic mutualism between microalgae and bacteria. The substances are used for communication, not as nutrients. They can activate or inhibit gene expression or biological activity, resulting in changes in the growth and metabolism of cells. Over the past few years, the signal interactions between microalgae and bacteria have received increasing attention. A symbiotic relationship between a marine bacterium (Phaeobacter inhibens) and marine microalgae (Emiliania huxleyi) was reported whereby the bacteria produced growth hormones and antibiotics to influence the growth of microalgae [11–13]. Chlorella vulgaris secretes a
⁎ Corresponding author at: Environmental Simulation and Pollution Control State Key Joint Laboratory, State Environmental Protection Key Laboratory of Microorganism Application and Risk Control (SMARC), School of Environment, Tsinghua University, Beijing 100084, PR China. E-mail address: [email protected] (H.-Y. Hu).
https://doi.org/10.1016/j.algal.2018.06.006 Received 26 February 2018; Received in revised form 3 June 2018; Accepted 9 June 2018 2211-9264/ © 2018 Elsevier B.V. All rights reserved.
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microalgae culture (a microalgae density of 1 × 106 cell mL−1) and 2 μL of various bacterial colony cultures (the bacteria density was OD600 = 1.0) were added to a new transparent 96-well plate together. Each well was filled with mBG11 medium to 100 μL. In addition, two types of control cultures were used for each batch of 96-well plates, i.e., non-bacterial culture and only culture medium, respectively. The transparent 96-well plate was sealed with a breathable sealing film and rotationally cultured at 25 °C with a 14 h/10 h light and dark cycle. The density of the microalgae was measured at 650 nm using a microplate reader (SpectraMax M5, Molecular Devices, USA).
certain signal substance that inactivates the signal substances of the bacterial acyl-homoserine lactones (AHLs), thereby inhibiting the production of bacterial toxins [14], while Azospirillum could promote the growth of microalgae by secreting some hormones, such as IAA [6,15]. The study of signal exchange between microalgae and bacteria was mainly focused on the ecological environment. However, little attention has been paid to the process of microalgae production. Moreover, the early study of bacterial isolates was limited to traditional methods that required tedious work and were less efficient [1]. There is less understanding of bacterial species and the signal exchange form in this process, especially regarding how microalgae affect bacteria based on their own growth. The aim of this study was to investigate the relationship between microalgae and symbiotic bacteria by signal exchange in the microalgae culture process. The method for highly efficient microalgae growth promoting bacteria screening was established. The characteristics of the isolated bacteria were studied, especially their secretion of small molecules. The hypothesis that IAA played an important role as a signaling molecule of the interaction between bacteria and microalgae was proposed and proved. The results provided a strategy to improve the productivity of microalgal biomass in large-scale microalgae cultures.
2.3. Analytical methods 2.3.1. Microalgae/bacteria growth property The microalgal density was determined by microscopic counting using hemocytometer and absorbance. The bacterial density was determined by the plate counting method. 2.3.2. Identification of bacteria and the phylogenetic relationship Genome extraction from the isolated bacteria was carried out using the TIANamp Bacteria DNA Kit (TIANGEN, China). The 16 s rDNA genes of the extracted genome were amplified by PCR with the primers 27F (5′-AGAGTTTGATCCTGGCTCGA-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The amplified products were sequenced and then BLAST (Basic Local Alignment Search Tool) was used to compare to the NCBI 16S ribosomal RNA sequences database. The phylogenetic trees of the isolates were reconstructed using neighbor joining with MEGA 5.0 [17,18]. Bootstrap support for the neighbor joining tree was determined using 1000.
2. Materials and methods 2.1. Culturing conditions and medium Scenedesmus sp. LX1 (Collection No. CGMCC 3036 at the China General Microbiological Culture Collection Center) isolated from tap water was used as the microalgae inoculum. Scenedesmus sp. LX1 was grown in modified BG11 (mBG11) medium [16] at 25 °C under a light intensity of 6000 lx, with light/dark periods of 14 h/10 h. Bacterial colonies were obtained using the gradient dilution plate method. First, Scenedesmus sp. LX1 was cultivated in the secondary effluent until the end of logarithmic growth. Then, the microalgaebacteria mixed culture was diluted in a 10 times gradient using phosphate buffer saline (PBS) (pH = 7.3). Next, the diluted solution was spread on Reasoner's 2A (R2A) medium (0.5 g starch, 0.5 g yeast extract, 0.5 g tryptic peptone, 0.5 g glucose, 0.3 g K2HPO4, 0.05 g MgSO4, 0.25 g succinate, and 0.5 g casamino acids in 1000 mL distilled water) agar plates and cultured at 25 °C until single colonies appeared. For detecting IAA secretion ability, bacteria were cultured under six conditions. Two conditions with microalgae cells (containing 10 mg L−1 L-tryptophan (Trp) and Trp free, respectively) were used to examine the effect of microalgae cells. Two conditions with microalgal soluble algal products (SAP) which removed microalgae cells (containing 10 mg L−1 Trp and Trp free) were used to investigate the effect of SAP. Two conditions with R2A medium (containing 10 mg L−1 Trp and Trp free) were used as the control. The same dissolved organic carbon (DOC) concentration of 52 ± 2 mg L−1 was used in all six conditions before inoculating the bacteria. Bacteria were inoculated in the six conditions with the inoculation 10 times that of microalgal cells (1 × 106 cells mL−1). There were three replicates for each culture condition. The mixture was incubated for 48 h at 25 °C and 180 rpm (ZQZY-70CS, Zhichu, China).
2.3.3. Qualitative and quantitative analysis of IAA molecules The production of IAA by the isolates was determined according to Glickman and Dessaux [19]. First, the isolates were cultured in 50-mL flasks containing 20 mL R2A supplemented with L-Trp (200 mg L−1) for 48 h on a rotary shaker at 120 rpm and 28 °C. Then, 50 μL of Salkowsky reagent (50 mL 35% HClO4 + 1 mL 0.5 M FeCl3) was added to 50 μL of culture, and was allowed to react under dark conditions for 30 min at room temperature. For the positive control, 50 mg L−1 of IAA solution was added instead of cultures. Finally, a red color appeared indicating the presence of IAA and confirming that the bacteria could secrete IAA. An HPLC (LC-20 AT, Shimadzu, Japan) tandem with a photodiode detector (Shimadzu, Japan) was used to measure the IAA concentration. The samples were filtered using a syringe filter with a pore size of 0.22 μm. The filtrate was collected in a 1.5 mL vial. An ODS-C18 column (5 μm particle size, 150 × 4.2 mm, J&K Chemical Co., China) was used to quantify IAA concentration with an elution phase of methanol:water:acetic acid (550:450:1 v/v). The wavelength for detection was 280 nm. The elution rate of the mobile phase was 1.0 mL min−1. The injection volume was 10 μL for the IAA detection calibrated with standard IAA (Sigma, USA).
2.2. High throughput screening method
2.3.4. Extraction of microalgal soluble algal products The microalgae culture solution (at the end of logarithmic growth) was filtered using a 0.45-μm filtration membrane. The microalgal cells were held on the 0.45-μm filtration membrane and the soluble algal products (SAP) were obtained in the liquid phase.
In this study, the high-throughput multiple-well plate method was developed for efficient isolation of bacteria from microalgae (Scenedesmus sp. LX1) cultivation (Fig. 1). First, the mixed culture of microalgae-bacteria was spread on R2A agar plates for picking up single colonies of bacteria. Single colonies were picked up with sterile toothpicks and then inoculated in 2-mL 96well plates containing the R2A liquid medium. The 2-mL 96-well plates were sealed with a breathable sealing film and cultured at 25 °C and 180 rpm until the OD600 value reached 1.0. Then, 20 μL of the fresh
2.3.5. SEM observation of symbiotic distribution of microalgae and bacteria The sample were harvested by centrifugation at 12000 g for 10 min at 15 °C and the supernatants were removed. The sample were fixed 12 h (4 °C) in the fixative containing 0.1 M phosphate-buffered solution (pH = 7.3), 2% glutaraldehyde, and 4% paraformaldehyde and then were washed with deionized water. Sample was then dehydrated with increasing concentrations of an ethanol solution (50%, 70%, 90% and 100%) and dried in 100% tert-butyl alcohol. Finally, the microcosmic structure of microalgae-bacteria symbiosis was observed using scanning 346
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Fig. 1. High-throughput multiple-well plate screening method.
sp. LX1 cultivated in secondary effluent systems. Compared to the control (the bacteria-free systems), most bacteria not only promoted the microalgal biomass yield but also the growth rate (Fig. S1). Twenty-six bacteria (marked as A-1–26) were isolated that were excellent at promoting the growth of microalgae. The microalgae growth quantity and specific growth rate were > 0.15 (Abs650) and 0.19 (d−1), respectively. Compared with the traditional screening method (using a conical bottle culture mixture of microalgae and bacteria), the high-throughput multiple-well plate screening method improved the efficiency of screening. At low microalgae/bacteria ratio (1:0–1:100) condition, the Abs650 can be good detecting the microalgae growth (Fig. S2). This was the first time the high-throughput method was used to screen microalgae-promoting bacteria, which provides a new method in the field of microalgae research. The 16 s ribosomal DNA phylogenetic analysis showed that the twenty-six bacteria were from four phyla and seven orders (Fig. 2). At
electron microscopy (JSM-7001F, JEOL, Japan). 2.4. Statistical analysis Each experiment consisted of three replicates. Independent-Sample t-test was used for significant difference analysis, using the SPSS (version 22.0) statistical software. 3. Results and discussion 3.1. Isolation of microalgae growth promoting bacteria In this study, the high-throughput multiple well plate screening method was developed (Fig. 1). Then, microalgae growth-promoting bacteria were isolated from the whole culturable bacteria community (including 400 strains in total) that was separated from the Scenedesmus 347
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Fig. 2. Phylogenetic tree of isolated bacterial strains from the phycosphere of Scenedesmus sp. LX1. Table 1 Summary of IAA secretion bacteria. Strain no.
Genus
Order
Phylum
Characteristics
Reference
A-1 A-3 A-4 A-5 A-10 A-12 A-15 A-16 A-19 A-22
Pseudomonas Pseudomonas Pseudomonas Pseudomonas Stenotrophomonas Achromobacter Achromobacter Stenotrophomonas Bacillus Acinetobacter
Pseudomonadales Pseudomonadales Pseudomonadales Pseudomonadales Xanthomonadales Burkholderiales Burkholderiales Xanthomonadales Bacillales Pseudomonadales
Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Proteobacteria Firmicutes Proteobacteria
PGPB PGPB PGPB PGPB Sewage treatment bacteria – – Sewage treatment bacteria PGPB –
[21,24] [21,24] [21,24] [21,24] [41] – – [41] [22,25] –
the phyla level, isolated bacteria belonged to β-proteobacteria, γ-proteobacteria, actinomycetes and firmicutes. Cho et al. [1] also reported that fourteen microbes were isolated from xenic Chlorella vulgaris culture, belonging to actinomycetes, firmicutes, proteobacteria and bacteroidetes. At the genus level, the isolates were mainly Pseudomonas, Bacillus, Acinetobacter and Exiguobacterium (Fig. 2). Among those, twelve strains were Pseudomonas, which is a proteobacteria widely found in soil and water. Bacillus, which is a firmicutes, also exists widely in the environment. The previous studies showed that Pseudomonas and Bacillus could significantly enhance the growth of C. calcitrans and N. oculata [20]. Moreover, the previous study has proved that Pseudomonas and Bacillus enhanced plant growth by secrete IAA [21,22]. Therefore, in addition to carbon resource (CO2) supply [23],
the bacteria may stimulate microalgal growth by secretion IAA. Pseudomonas and Bacillus could also act as a general plant growth promoting bacteria (PGPB) for numerous plant species [24,25]. Therefore, they are often used for enhancing plant growth to increase productivity.
3.2. Identification of bacterial IAA secretion ability PGPB could secrete small molecular substances, such as phytohormones, to regulate the growth of plants [26,27]. IAA is one of the most common phytohormones. Previous studies showed that IAA could also enhance the growth and the lipid accumulation of microalgae [28–32]. Therefore, twenty-six isolated bacteria were further investigated for their ability to secrete small molecules (IAA), which 348
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of bacteria increased in all tryptophan overdose conditions (both R2A medium systems and microalgae culture systems) (Fig. 3b). In addition, bacterial culture with microalgae showed a higher IAA production, with an increase of 5.3–89.8 times compared to the bacteria cultured in the R2A medium (p < 0.05). Bacteria cultured with microalgae could also promote the production of IAA. It was inferred that microalgae might have some mechanisms to promote the bacterial ability of synthesizing and secreting IAA, and this promotion mechanism was amplified with the overdose of tryptophan. In addition to the direct exchange of nutrients, signal transduction is another pathway for synergistic mutualism between the microalgae and bacteria [5]. Previous research showed that small molecular exchange between microalgae and bacteria might occur in the culture environment and is not limited by direct contact between them. Kazamia et al. [38] found that M. loti, which could secrete small molecules (VB12), could support the growth of L. rostrate without direct physical contact. The microalgae secreted soluble algal products (SAP) into the culture medium, which were around microalgal cells and formed a region rich in organic matter. This region is considered to be for microbe communications. Fig. 4a shows that the isolated bacteria cultured in the microalgal SAP medium could secrete more IAA than bacteria cultured in the R2A medium. These results were consistent with the previous conjecture. The microalgal SAP was rich in organic matter, with an area containing signal molecules and the IAA synthetic substrate (tryptophan). Among those, the signaling molecules may stimulate the expression of IAA synthetic genes, leading to an increase in IAA secretion. If the signal molecules were contained the SAP, it could explain the previous results showing the obvious promotion ability of the microalgae in the tryptophan-abundant environment. To further determine whether SAP contained the signal molecules, tryptophan (10 mg L−1) overdose in the SAP medium was applied to reduce the effect of tryptophan concentration in different systems. The results showed that bacteria cultured in the SAP medium could secrete more IAA than bacteria cultured in the R2A medium (Fig. 4b). More than 2.1–41.5 times the IAA concentration were secreted by bacteria cultured under the SAP medium versus the R2A medium. This further indicated that microalgae might secrete some kind of signal molecules to affect bacterial metabolism. In our study, the microalgae might secrete signal molecules, which could promote bacterial IAA production. It was further confirmed that the promotion ability was more pronounced with sufficient tryptophan. The SAP had a complex material composition. Yu et al. [39] analyzed the main components of SAP secreted by Scenedesmus sp. LX1. The results showed that the small molecular fraction (< 1 kDa) was 57.8%, the middle molecular fraction (1–10 kDa) was 34.8% and the large molecular fraction (> 10 kDa) was 7.3%. The hydrophobic/hydrophilic property analysis showed that the hydrophilic fractions were the main organic matter in small molecular weight SAPs, while the hydrophobic matter was the larger portion in the large molecular weight SAPs [40]. A previous study showed that the marine P. multiseries-Sulfitobacter model system had a complex microbe interaction potentially occurring within the phycosphere. This area is filled with a certain number of hydrophobic signaling molecules and persisted despite seawater turbulence. Through analysis of the SAP secreted by Scenedesmus sp. LX1, the hydrophilic matter was the largest portion that might contain some signal molecules and stimulate the bacteria to synthesize and secrete IAA. IAA, an important signal substance in plants and bacteria, is also an important signal substance in microalgae and bacteria. It was secreted by bacteria and enhanced the growth of microalgae. At the same time, the microalgae also secreted some other signal molecules that could stimulate bacteria to secrete IAA. This relationship formed a feedback mechanism of signal exchange between the microalgae and bacteria, and it made the relationship closer. These signal molecules deserve further study to fully clarify their underlying mechanisms. This
Fig. 3. The effect of the microalgae system on the secretion of IAA by bacteria. Error bars represent the standard deviation. Note: Trp = tryptophan.
might directly affect the growth of microalgae [28,31,32]. Ten strains were identified with the ability to secrete IAA (Table 1), which was 39% of the total isolates. The results indicated that there was a certain proportion of IAA secreting bacteria in the microalgae culture system. Microalgae might have a screening ability for the enrichment of these IAA secreting bacteria. From the phylogenetic analysis, they were mainly five orders Pseudomonas, Bacillus, Acinetobacter, Achromobacter and Stenotrophomonas (Table 1). Among those, Pseudomonas, Bacillus, Acinetobacter and Stenotrophomonas had been reported with the ability to secrete IAA for symbiotic co-existence with plants [33–36]. However, Achromobacter was rarely found to secrete IAA, especially in a microalgae-bacteria symbiotic system. 3.3. The IAA secretion of symbiotic bacteria enhanced by microalgae Compared with culture in the R2A medium, bacteria cultured with microalgae had a stronger ability to secrete IAA (Fig. 3a). The unit bacteria secretion increased by 113%–265% (p < 0.05), indicating that microalgae culture conditions were more conducive to bacteria to produce IAA. Amin [37] showed that some diatom symbiotic bacteria could secrete growth promoters to influence the growth of microalgae. For example, in the P. multiseries-Sulfitobacter system, Sulfitobacter could stimulate P. multiseries cell division by secreting IAA. However, IAA synthesis also depended on the production of tryptophan by diatoms. At the same time, tryptophan secreted by diatoms might attract a large number of bacteria with the ability of converting tryptophan to IAA. This established a positive feedback mechanism for the growth of diatoms. Similar results were shown by Segev, the IAA production of Phaeobacter inhibens was significantly increased by tryptophan that secreted by Emiliania huxleyi [13]. Therefore, tryptophan provided by microalgae may be the cause of the increase in IAA production by bacteria. To determine whether the mechanism of microalgae-bacteria interaction was similar to the P. multiseries-Sulfitobacter system and E huxleyi-P inhibens system in our study, 10 mg L−1 of tryptophan was added to ensure the overdose of tryptophan in the culture systems. The 10 mg L−1 tryptophan had no obvious effect on the growth of bacteria (Table 2). The results showed that the amount of IAA secretion per unit 349
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Table 2 The bacterial number of different culture systems. Bacterial density was determined by the plate count method. Each sample makes five repetitions. Bacterial density was calculated using the five sample average of single colony number. Not: Trp = tryptophan; SAP = soluble algal products. Strain no.
Microalgae + Trp system (cells mL−1)
Microalgae system (cells mL−1)
SAP + Trp system (cells mL−1)
SAP system (cells mL−1)
R2A + Trp system (cells mL−1)
R2A system (cells mL−1)
A-1 A-3 A-4 A-5 A-10 A-12 A-15 A-16 A-19 A-22
4.85 × 107 6.16 × 108 8.00 × 107 6.40 × 107 2.60 × 107 2.48 × 108 4.10 × 108 3.83 × 108 3.64 × 108 4.48 × 108
4.20 × 107 5.96 × 108 6.55 × 107 7.20 × 107 2.55 × 107 2.71 × 108 3.99 × 108 3.89 × 108 3.45 × 108 3.84 × 108
1.33 × 108 7.46 × 108 9.10 × 107 3.60 × 107 8.50 × 106 4.62 × 108 4.17 × 108 4.63 × 108 4.44 × 108 5.11 × 108
1.56 × 108 6.75 × 108 1.04 × 108 3.45 × 107 1.40 × 107 4.32 × 108 4.31 × 108 4.43 × 108 4.34 × 108 4.90 × 108
1.20 × 108 6.46 × 108 1.42 × 108 1.20 × 108 1.49 × 108 4.39 × 108 4.23 × 108 4.14 × 108 4.27 × 108 5.88 × 108
9.90 × 107 6.90 × 108 1.06 × 108 1.13 × 108 1.26 × 108 4.21 × 108 4.68 × 108 4.28 × 108 4.45 × 108 6.18 × 108
knowledge will help to further promote the growth of microalgae, which is of great significance for large-scale microalgae culture. 3.4. The symbiotic distribution of microalgae and bacteria Scanning electron microscopy (SEM) is a common tool for examining the microstructure of organisms. In this study, the symbiotic microalgae-bacteria system was observed by SEM. From the micrographs, microalgae and bacteria formed a stable co-existence system, in which the bacteria were present on the microalgae cell surface and the microalgal extracellular polymeric substances (EPS) (Fig. 5). The same results was also get in confocal laser scanning microscopy (CLSM) micrographs (Fig. S3). This distribution not only benefits material and gas exchange but is also conducive to the exchange of signal materials. 4. Conclusions Twenty-six excellent microalgae growth-promoting bacteria were isolated from Scenedesmus sp. LX1 cultivated in secondary effluent systems by using the high-throughput multiple-well plate screening method. Of these, ten had the ability to secrete IAA to promote the growth of microalgae and the microalgae selectively enhanced the bacterial IAA secretion in turn. This may be a new microalgae-bacteria interaction mechanism that is of great ecological significance. At the same time, it would be a new potential green and economic strategy for improving large-scale microalgal cultivation.
Fig. 4. The effect of microalgal SAP on the secretion of IAA by bacteria. Error bars represent the standard deviation. Note: Trp = tryptophan.
Acknowledgements This work was supported by the Key Program of the National Natural Science Foundation of China (No. 51738005) and the Collaborative Innovation Center for Regional Environmental Quality of China.
Bacteria Microalgae
Microalgae
Fig. 5. SEM observation of co-cultivation of Scenedesmus sp. LX1 with bacteria (A-12). 350
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Conflict of interest statement [17]
The authors declare no conflict of interest.
[18]
Author contributions [19]
In this study, Xiao-Xiong Wang and Tian-Yuan Zhang participated in experimental design. Assoc. Prof. Wu, Prof Zhan and Prof. Hu gave general and strategic suggestions in this study. All authors agree to the authorship and submission of the manuscript to Algal research for peer review.
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[21] [22]
Statement of informed consent, human/animal rights
[23]
No conflicts, informed consent, human or animal rights applicable. Appendix A. Supplementary data
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Supplementary data to this article can be found online at https:// doi.org/10.1016/j.algal.2018.06.006.
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