Nitrogen supplemented by symbiotic Rhizobium stimulates fatty-acid oxidation in Chlorella variabilis

Algal Research 44 (2019) 101692

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Nitrogen supplemented by symbiotic Rhizobium stimulates fatty-acid oxidation in Chlorella variabilis

T

Cong Fei, Tong Wang, Abeselom Woldemicael, Meilin He, Shanmei Zou, Changhai Wang* Jiangsu Provincial Key Laboratory of Marine Biology, College of Resources and Environmental Science, Nanjing Agricultural University, Nanjing, 210095, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Rhizobium Chlorella Fatty-acid oxidation Nitrogen repletion Biodiesel

Large-scale cultivation of microalgae benefits from co-cultivation with model growth-promoting microorganisms to increase algal biomass and lipids production. Nevertheless, the effect of natural symbiotic bacteria on microalgae cultivation and lipid production is still unclear. Here, the effects of nitrogen-fixing, symbiotic Rhizobium on nitrogen content and algal growth, lipid accumulation, and gene expression were investigated in the green microalga Chlorella variabilis. The results demonstrated that compared to axenic C. variabilis cultures, the bacterial-derived nitrogen supply and algal growth were enhanced by 32.8% and 27% in cocultures, respectively. Transcriptome analyses revealed that C. variabilis growing with R. radiobacter TH729 upregulated genes involved in fatty acid oxidation and downregulated genes related to nitrogen metabolism. Our findings indicated that bacterial fixed nitrogen reduces lipid accumulation in microalgae, which is not beneficial for biodiesel production. Further studies on the effects of other bacteria on microalgae are needed to assess most optimal practices for biodiesel production.

1. Introduction Microalgae are considered promising candidates for commercial lipid production due to their rapid growth rate, high oleaginicity, and easy cultivation [1]. To further enhance the growth and lipid production of microalgae, various strategies were used utilizing physics, chemistry and biology to optimize cultivation conditions. Besides required conditions for microalgal growth (i.e., sunlight, water, carbon dioxide, and nutrients), temperature, diurnal temperature fluctuations and light flux play major roles in regulating biomass and lipid accumulation in microalgae [2,3]. Photoautotrophic, heterotrophic and mixotrophic cultivation also influence the biomass and lipid productivities of Chlorella by different carbon sources [4]. Furthermore, other nutrients variations such as nitrogen deprivation (N-deprivation), phosphate limitation, silicon deficiency and iron supplementation have been shown to increase the lipid content in cells [5]. In particular, Ndeprivation is the most studied factor. Griffiths, van Hille and Harrison [6] found that nitrogen-limited (N-limited) cultures showed higher lipid production per cell than nitrogen-replete (N-replete) cultures in ten of eleven microalgal species. In addition, a ∼20-fold increase in oil accumulation was achieved in Chlorella sorokiniana under N-limited growth conditions [7]. Transcriptome analyses of Phaeodactylum tricornutum and Scenedesmus acutus revealed that during nitrogen

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limitation (N-limitation), most nitrogen assimilation, amino acid degradation and central carbon metabolism-related transcripts were upregulated, while triacylglycerol (TAG) degradation was down-regulated [8,9]. These studies imply that a shift of carbon flux towards fatty acid and TAG biosynthesis, and the downregulation of TAG lipase genes may contribute to TAG accumulation under low nitrogen conditions. These results of transcriptomic analyses are consistent with a previous study which concluded that the starch is a primary carbon and energy storage product in microalgae and carbon is shifted into lipid as a secondary storage product under N-limitation [10]. Except for laboratory-controlled cultures, microalgae always coexist with bacterial consortia that often alter the physiological changes of their hosts. In the aquatic environment, microalgae can produce phytohormones, nutrients (e.g., iron), dissolved organic carbon (DOC) and reduced nitrogen [11] in the region immediately surrounding microalgal cells, known as the phycosphere [12], which presumably contains a higher density of bacteria than bulk water. These molecular changes occurring in the phycosphere play significant roles in the physiology and metabolism of both algal and bacterial partners. Kim, Ramanan, Cho, Oh and Kim [13] revealed that the dominant phycosphere bacterial orders found in three green algae in freshwater were Rhizobiales, Flavobacteriales and Pseudomonadales. One particular bacterium belonging to the order Rhizobiales, a Rhizobium sp., constituted a

Corresponding author at: 1 Tongwei Road, Nanjing, Jiangsu, PR China. E-mail address: [email protected] (C. Wang).

https://doi.org/10.1016/j.algal.2019.101692 Received 19 July 2019; Received in revised form 1 October 2019; Accepted 3 October 2019 2211-9264/ © 2019 Elsevier B.V. All rights reserved.

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Shanghai), 0.5 μM primers, 2 μL bacterial culture and DNA-free water. The PCR reaction was performed as follows: an initial denaturation at 94 °C for 8 min followed by denaturation at 94 °C for 1 min, primer annealing at 55 °C for 1 min, primer extension at 72 °C for 1 min for 30 cycles, and a final extension at 72 °C for 8 min. The PCR amplicons were checked by agarose gel (1.5% w/v) electrophoresis (P&Q, Shanghai) and then the 16S rDNA region was sequenced by Sanger sequencing (Sangon Biotech, Shanghai). The 16S rDNA sequences were aligned with sequences from the GeneBank database using ClustalW Multiple Alignment in BioEdit. A 16S rDNA phylogenetic tree was constructed by both the neighbor-joining method and Maximum Likelihood method using MEGA6 with 100 bootstraps.

significant proportion of the bacterial community of the green alga C. vulgaris and increased algal cell count by ∼72%. Similarly, the biomass of C. vulgaris increased by 20% when co-cultured with this bacterium, in addition to significant changes to lipid accumulation and fatty acid components [14]. Since the heterotrophic bacteria utilize DOC produced by microalgae to survive, the symbiosis always occurs in the bacteria-algae interaction which evolves interaction or closes living relationship between organisms from different species, usually with benefits to at least one of the individuals [15]. The symbiotic relations include mutualism, commensalism, parasitism, depending on which species benefit. For example, the typical mutualism of diatoms and bacteria is the Sulfitobacter promotes diatom growth via secretion of the hormone indole-3acetic acid, synthesized by the bacterium using diatom-derived tryptophan [16]. In return, Sulfitobacter uses the DOC produced by diatom for surviving. The similar mutualism has also been proved in Azospirillum and Chlorella co-cultures [17]. Also, some plant growth-promoting bacteria (PGPB) (e.g., Azospirillum, Brevundimonas and Rhizobium species) show promotion for algal growth [18,19]. Transcriptome analyses help to identify the differentially expressed genes (DEGs), declaring observed differences or changes in read counts or expression levels between two experimental conditions is statistically significant. DEGs always show up in the microalgae when co-cultured with bacteria. 2143 and 492 DEGs were annotated in P. multiseries and T. pseudonana respectively when co-cultured with bacteria [16,20]. Although previous reports have shown that bacteria synthesize substances that may stimulate algal growth, it is still unclear whether these exudates can concurrently stimulate lipid production. Moreover, the natural symbiotic bacterial influences on lipid accumulation and whether these bacteria need to be removed before algal cultivation remains unclear as well. This study aims to examine the effects of extracellular secretions from natural symbiotic bacteria on microalgal cultivation, lipid content and biodiesel production, and whether such bacteria should be retained or removed for biodiesel production. Here, we conduct transcriptome and inorganic nitrogen analyses on C. variabilis F275 and R. radiobacter TH729 co-cultures to investigate the bacterial influence: 1) on the inorganic nitrogen content in substrates, 2) the growth and gene regulations in C. variabilis, and 3) the lipid content and biodiesel production in F275 that is influenced by nitrogen content changes caused by bacteria.

2.3. Experimental design for different cultures 2.3.1. Co-culture of R. radiobacter TH729 with Chlorella species Except for F275, C. vulgaris 1, C. vulgaris 5 and Chlorella sp. 484 were acquired from (Freshwater Algae Culture Collection at the Institute of Hydrobiology, FACHB). After purifying the single algal colonies on BB agar plate, all four axenic Chlorella strains were cultured in 500 mL flasks containing BB medium with an initial density of 4000 cells/mL in triplicates. Cultures were grown as described above. R. radiobacter TH729 (TH729) stocks were recovered on the LB agar plate and a single colony was cultured in BB medium overnight (28℃, 180 r.p.m.). Cells were centrifuged at 2000xg for 10 min, followed by washing twice in BB medium. The initial bacterial density in co-culture was 1 × 105 cells per milliliter. Chlorella growth was monitored by measuring the total chlorophyll content using a PHYTO‐PAM phytoplankton analyzer (Heinz Walz GmbH Effeltrich, Germany) because of a linear relationship between cell number and in vivo chlorophyll a fluorescence. Specific growth rates (μ) were calculated from the linear regression of the natural log of in vivo fluorescence versus time during the exponential growth phase of cultures. 2.3.2. C. variabilis F275 culture under different nitrogen concentrations All axenic F275 were incubated in 200 mL BB medium with an initial density of 5 × 106 cell/mL. For control cultures, the total nitrogen content was 2.94 mM (250 mg/L) NaNO3 in BB medium. 0.37 mM, 0.74 mM and 1.47 mM of NaNO3 were set for nitrogen limitation while the concentrations of NaNO3 were set 5.88 mM, 8.82 mM and 11.76 mM for nitrogen repletion. All cultures were grown in triplicates for 14 days and algal cells were counted by a hemocytometer at that time.

2. Materials and methods 2.1. Bacterial and algal isolation

2.4. RNA extraction and transcriptome analysis of C. variabilis F275 Five hundred milliliters water samples were collected at a depth of 0.5 m at three locations, which were selected for sampling in Lake Taihu, China. Manual cell picking micromanipulators were used to pick a single algal cell with associated bacteria under the inverted microscope by a micro-pipette. C. variabilis F275 (F275) was selected and cultured in 250 mL Bold's Basal (BB) medium at an irradiance of 40 μmol m−2 s−1 with 14:10 h light: dark cycle at 25 ℃. Bacteria were isolated from the non-axenic F275 culture at the initial stationary phase by serially diluting 1000 times into BB medium. After that, aliquots were spread onto LB agar plates and incubated at 25 ℃ in the dark for 3 to 7 days. A single colony with unique morphology was further purified three times before being stored at −80 ℃ in 15% glycerol stocks.

Axenic F275 and TH729-F275 co-cultures were grown as previously described and filtered onto sterilized polycarbonate membrane filters (47 mm diameter, 0.45 μm, Jinlong company, China). Total RNA was isolated with an easy-spin™ IIp Plant RNA Extraction Kit (Biomiga, Inc) and the RNA concentration and quality was assessed using the NanoDrop 2000 (Thermo), the Qubit® 2.0 Fluorometer (Life Technologies) and an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). cDNA was synthesized using random hexamers or oligo(dT) primers and sequencing was performed using Illumina HiSeq™ 2000 at Novogene Bioinformatics Technology Company (Beijing, China). High-quality reads were obtained by removing low quality and adapter related reads from raw reads. These reads were then mapped to the reference transcriptome using the Bowtie2 alignment program in RSEM [21]. Gene function was annotated according to NCBI non-redundant protein (nr) sequences, NCBI non-redundant nucleotide sequences, Protein family (Pfam), Clusters of Orthologous Groups/EuKaryotic Orthologous Groups (COG/KOG), SWISS-PROT, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO). The gene expression levels were evaluated based on fragments per kilobase of transcript per million mapped fragments (FPKM). The

2.2. Bacterial identification and phylogenetic analysis Direct PCR method was used in bacterial identification. Purified strains were incubated in 3 mL LB broth at 28 ℃ overnight and 2 μL of each culture was used as DNA template in PCR. 16S rDNA from all the bacteria was amplified by primers (27 F 5′-AGAGTTTGATCCTGGCT CAG-3′; 1492R 5′-GGTTACCTTGTTACGACTT-3′) with reaction mixtures (25 μL) consisting of 12.5 μL PCR Master Mix 2x (Sangon Biotech, 2

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(Fig. 1). All five isolated strains belonged to the Firmicutes and Proteobacteria phyla, an observation that partially differs from bacteria found in bulk Lake Taihu water, where dominant bacterial phyla were Actinobacteria, Bacteroidetes and Proteobacteria [27,28]. The fact that Proteobacterial species were isolated from our culture and were also found commonly in Lake Taihu samples highlights the importance of Proteobacteria in this environment and to green algae, consistent with previous studies on Proteobacteria and microalgae in general [29,30]. Compared with bulk water, the concentration of organic molecules released by algal cells is higher in phycosphere [11]. This specialized habitat leads to different bacterial compositions in the aquatic environment and phycosphere [31]. Furthermore, current evidence suggests that microalgal species possess unique microbial communities that are consistent across strains and temporal scales [30]. Previous studies have shown that the Pseudomonas and Bacillus genera always cooperate with Chlorella to remove nutrients (e.g., ammonium, phosphate) in wastewater [32,33], while Methylobacterium does not show any relation with Chlorella. As plant growth-promoting bacteria, Rhizobium has been shown to enhance Chlorella biomass [13] and bacteria within this genus have usually been observed in Chlorella cultures [34]. Our findings provide further evidence that this genus is closely associated with Chlorella.

differential expression analysis was performed using DEseq with qvalue < 0.005 and |log2FoldChange| > 1. The Illumina sequencing data were deposited into the NCBI Sequence Read Archive (SRA) database as the BioProject ID PRJNA550224. 2.5. Analysis procedures 2.5.1. Nitrate and ammonium analysis Axenic F275 cultures, F275-TH729 co-cultures and TH729 cultures supplemented with 10 μM glucose were filtered through 0.22-μm syringe filters (Jinlong company, China) after 10 days of incubation at the same condition as mentioned in 2.3.1. All filtrates were used for nitrate and ammonium analyses, which were performed on a Seal Analytical continuous-flow AutoAnalyzer 3 (AA3). Final concentrations of each sample were calculated using SEAL Analytical AACE 6.07 software. 2.5.2. Chlorophyll, protein and soluble sugar content After 14 days of growth of F275 culture under nitrogen stress, Chlorophyll was extracted using a methanol extraction overnight at 4℃ in the dark, followed by spectrophotometric analysis [22]. The soluble sugar fraction was measured by the phenol-sulfuric acid spectrophotometric method [23], while the measurement of protein was performed using the bicinchoninic acid (BCA) assay [24].

3.2. Growth of Chlorella species with R. radiobacter TH729 in co-cultures 2.5.3. Total lipid and neutral lipid analysis A rapid neutral lipid measurement was performed by the Nile red method [25]. Briefly, F275 samples were pretreated with 20% DMSO (v/v) for 20 min at 40℃. A stock solution of Nile red was prepared in acetone (0.1 mg/mL) and 15 μL Nile red dye was added onto 1 mL microalgal suspension and stained for 5 min. The fluorescence intensity was measured using a BioTek Synergy H1 Hybrid plate reader (Ex. 480 nm, Em. 575 nm). Total lipid content was determined as described by Jones, Manning, Montoya, Keller and Poenie [26].

Unless otherwise indicated, all experiments were conducted in triplicates. The mean values along with standard deviations were reported. The significant differences were assessed by one-way analysis of variance (ANOVA) with SPSS statistical software, followed by Duncan tests. Different letters indicate significant differences between treatments (p < 0.05).

Axenic F275, C. vulgaris 1, C. vulgaris 5 and Chlorella sp. 484 were used for co-culture experiments with TH729. While the growth of C. vulgaris 5 and Chlorella sp. 484 were not influenced by the bacterium, TH729 promoted the growth of C. vulgaris 1 and F275 after14 days (Fig. 2). Concomitantly, the growth rates for C. vulgaris 5 and Chlorella sp. 484 growing with TH729 did not show significant differences compared to axenic culture while specific growth rates of F275 and C. vulgaris 1 significantly increased in the presence of TH729 by 27.3% and 16.3%, respectively (Table 1). Interestingly, the growth of F275 increased more than that of the other Chlorella species when cultured with TH729, which was isolated from F275 phycosphere. These findings suggest that bacterial strains can influence the growth of their natural algal partner more than that of foreign algal strains. Rhizobium has previously been shown to enhance the growth of a variety of green algae [13] and in our study, Rhizobium sp. TH729 also promoted the growth of some Chlorella species, providing further evidence that the Rhizobium genus is growth-promoting towards microalgae. Rhizobiaceae increase the biomass of plant by nitrogen fixation and also phytohormones production [35], which may be the main reason that TH729 increased the biomass of F275. It has been reported that symbiotic bacteria could promote algal growth in different ways. First, microalgae could use the CO2 for the photosynthesis process, which was produced by bacterial respiration [36]. The growth of C. vulgaris 1 was significantly increased after 10 days when co-cultured with bacteria, which may be caused by the CO2 produced by bacteria. Furthermore, plant hormones produced in the symbiotic relationship also promote the growth of microalgae. Indole‐3‐acetic acid (IAA) has shown to promote algal growth in the presence of the IAA‐producing bacterium [16,17]. As a result, the growth of F275 that increased in F275-TH729 coculture was not influenced by a single factor.

3. Results and discussion

3.3. Nitrogen changes in co-culture

3.1. Identification of bacteria isolated from C. variabilis F275

To investigate the nutrient exchanges in the TH729- F275 interaction, the nitrate and ammonium contents in axenic cultures and cocultures of F275 and TH729 were measured to explore the dynamics of nitrogen speciation (Table 2). Compared with blank controls, NO3− concentrations showed a decrease of 12.3% in axenic F275 culture, an increase of 5.5% in TH729 axenic culture, and 16.43% increase in cocultures of TH729 and F275 (Fig. 3). These observations suggested that the concentration of NO3− increased by 32.8% in cocultures compared

2.5.4. Fatty acid compositions Fatty acids analysis were performed as described previously [25]. Briefly, after 25 mg freeze-dried microalgal powder was converted into fatty acid methyl esters (FAMEs) by adding 2% H2SO4–methanol solution, 2 μL of each sample were injected in the splitless injection mode for fatty acids analyses that were performed on gas chromatography with mass selective detector (GC–MS, Agilent 7890A), equipped with a HP-5msi column (30 m, 0.25 mm, 0.25 lm; Agilent Technologies). The injection and MS detector temperatures were set at 250 ℃ and 280 ℃, respectively. Fatty acids were identified by comparison of their retention times with Supelco 37 Component FAME Mix (Sigma). Heptadecanoic acid (C17:0) was used as the internal standard. 2.6. Statistical analysis

In our study, five bacterial species were isolated from a C. variabilis F275(F275) culture, which was sampled in Lake Taihu, China. Subsequently, 16S rDNA sequences were amplified and sequenced from these strains and were blasted against GenBank. The bacterial isolates were identified as Bacillus sp. TH2, R. radiobacter TH729, Methylorubrum sp. TH1, Pseudomonas sp. TH3 and Pseudomonas sp. TH7 3

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Fig. 1. 16 s rDNA Neighbor-Joining (NJ) and Maximum-Likelihood (ML) trees of bacteria strains isolated from C. variabilis F275. Node values indicate ML and NJ bootstrap supports of 100 replications respectively. Bacteria isolated from C. variabilis F275 are highlighted in bold type. Accession numbers of GenBank sequences are included in parentheses.

with axenic F275 cultures (Table 2). The NH4+ concentration in all three types of cultures increased by 12.9–19.2% compared with blank controls, but did not show significant variations among cultures. Nitrate and nitrite assimilation is a crucial process for nitrogen acquisition in green microalgae [37]. Nitrate reduction in algae occurs in two steps: first, the reduction of nitrate to nitrite, and then the reduction of nitrite to ammonia [38]. Under nitrogen replete condition, ammonia is used for amino acid synthesis and/or released out of the cells as ammonium [39]. This behavior explains why nitrate appears to decline in axenic F275 whereas ammonia slightly increases. Our results showed that the total nitrogen content increased in axenic TH729 cultures, suggesting that TH729 could fix N2 from the atmosphere. However, we did not observe any remarkable increase in the ammonium content in axenic TH729 culture, while the nitrate content did show significant enhancement. These results are different from the typical way of Rhizobium to fix nitrogen which turns N2 to NH3 [40]. Further studies are still needed to investigate the function of nitrogen fixation and nitrification of Rhizobium in aquatic environments. Because of nitrogen fixation, the nitrogen content in coculture was

observed to be significantly higher than in axenic algal culture, and as it has already been proven that the biomass of Chlorella gets reduced under N-limitation but gets enhanced under N-replete condition [7], we can conclude that one possible reason that the F275 growth rate increased in cocultures is that the biomass of F275 was enhanced by bacterial-derived nitrogen. Besides, the F275 also provided DOC for TH729 surviving; as a result, both partners benefited from this relationship and generated a stable symbiotic relationship (i.e., mutualism). 3.4. Transcriptome analysis 3.4.1. Overview of transcriptome sequencing and annotation Recent studies have demonstrated that bacteria play major roles in regulating transcriptional responses of microalgae [16,41]. To further explore the metabolic responses of F275 that are induced by TH729 and the potential role nitrogen fixation plays in these interactions, the transcriptome of axenic and F275 cocultures with TH729 were analyzed after 10 days of growth. After data filtering and quality assessment,

Fig. 2. Cocultures of R. radiobacter TH729 with four Chlorella species. Coculture of TH729 with C. vulgaris 1(a), C. vulgaris 5(b), Chlorella sp. 484(c) and C. variabilis F275(d). Error bars represent SD of three cultures. 4

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Table 1 The specific growth rates of different Chlorella species cocultured with R. radiobacterTH729.

−1

Specific Growth rates (Axenic, d ) Specific Growth rates (Co-culture, d−1) Growth changes (%)

C. vulgaris 5

Chlorella sp. 484

C. vulgaris 1

C. variabilis F275

1.19 ± 0.23 1.20 ± 0.16 0.8

1.01 ± 0.09 1.01 ± 0.05 0

0.92 ± 0.03 1.07 ± 0.01* 16.3

1.43 ± 0.18 1.82 ± 0.09* 27.3

Values are given as mean ± standard deviation from three samples. Superscripts across columns indicate significant difference (p < 0.05) between treatments.

47,823,550 and 521,66,136 reads were generated by cDNA libraries of axenic F275 and non-axenic F275, respectively. In total, 711 genes with at least 2-fold change were considered DEGs, of which 499 were upregulated genes and 698 were downregulated genes (p < 0.05) (Fig. 4a). For all DEGs, the top three identified Gene Ontology (GO) subcategories were oxidoreductase activity, oxidation-reduction process and carbohydrate metabolic process, which included 100, 102 and 63 regulated genes, respectively. These findings suggest F275 majorly modifies its metabolism in response to the presence of TH729. Our findings are comparable to previous work on bacterial influence on diatoms, where 2143 genes were differentially expressed in the diatom P. multiseries when co-cultured with Sulfitobacter sp. relative to axenic controls [16]. Similarly, after adding Ruegeria pomeroyi DSS-3, 492 genes were differentially regulated in T. pseudonana after 56 h [20]. Cumulatively, these findings show that ‘symbiotic’ bacteria can regulate gene expressions of host microalgae.

Fig. 3. The nitrate and ammonia relative content in cultures. All the relative contents are compared with blank medium control. Bar charts represent means ± SD of triplicate samples (n = 3). Different alphabets above the bar charts indicate significant differences (p < 0.05) between F275, TH729 and F275-TH729 cultures.

3.4.2. DEGs for enzymes involved in nitrogen metabolism In both terrestrial plant-microbe and microalgae-bacteria interactions, a commonly exchanged nutrient is nitrogen [16,42]. Since the nitrate concentration significantly increased in our cocultures, transcript abundances of the nitrogen metabolism pathway were analyzed (Table 3). Compared with axenic F275, both nitrate transporter (NRT) and nitrate reductase (NR) genes were downregulated in cocultured F275. Presumably, the observation that nitrate assimilation and reduction by F275 is downregulated in coculture indicates that F275 is highly attuned to nitrogen fixed and released by TH279. A similar observation has been reported between the coastal diatom, Pseudo-nitzschia multiseries, and a Sulfitobacter sp. [16]. In the algal nitrogen assimilation, the first step is nitrogen transportation. After nitrate is transported into the cell with the help of NRT, NR in cytoplasm catalyzes the reduction of nitrate into nitrite, which is subsequently transported into the chloroplast [43]. NR activity is limited by the nitrate supply and is approximately equal to the nitrate assimilation rates during growth [44]. In addition, the up-regulation of NR and NRT genes were observed in microalgae during N-limitation compared with N-replete conditions [8,9]. It is reasonable to assume that F275 is growing under N-replete conditions in both axenic and coculture experiments. However, given that TH729 fixes nitrogen and may provide some of this nitrogen to F275, leading to a change in nitrogen speciation and concentration in the coculture experiments. This scenario would lead to a reduction in transcripts responsible for transporting and reducing nitrate, as observed in the transcriptome and as has been reported for several microalgae growing in the presence of ammonia and nitrate [45]. Ammonium is incorporated into carbon skeletons by rendering

glutamate, through the glutamine synthetase/glutamine oxoglutarate amino transferase or glutamate synthase (GS/GOGAT) cycle [37]. The ammonia assimilation pathway is repressed in F275 growing with TH279 relative to axenic cultures as evidenced by downregulation of glutamine synthase (GS), that converts ammonia to glutamine, and the upregulation of glutamate dehydrogenase (GDH), that catalyzes the reversible inter-conversion of glutamate to α-ketoglutarate and ammonia in cocultured F275 (Table 3). Collectively, the nitrogen fixed by TH729 increased the concentration of ammonia in cocultures and caused the downregulation of nitrogen metabolism pathways in general (Table 4). 3.4.3. DEGs for enzymes involved in fatty acid metabolism The basic unit of lipids is TAG, which consists of glycerol and fatty acids. The fatty acid accumulation in microalgal cells is controlled by Fatty acid biosynthesis and metabolism pathways. Our results did not show DEGs involved in fatty acid biosynthesis; instead, several fatty acid degradation genes were upregulated that stimulate the fatty acid βoxidation (Table 3). Firstly, in the pre-step reaction for β-oxidation, fatty acids are converted to fatty acyl-CoA by long‐chain acyl‐CoA synthetase (LACS). This step was upregulated as shown by the upregulation of LACS genes in cocultured F275 compared with axenic F275. Secondly, acyl‐CoA oxidase(ACOX) is responsible for the first step of the peroxisomal fatty acid β‐oxidation spiral that catalyzes the desaturation of acyl-CoAs to 2-trans-enoyl-CoAs [46]. As ACOX gene lacking

Table 2 Nitrate and ammonia content in different cultures.

NO3-(mg/L) NH4+(mg/L)

Blank control

F275

TH729

F275-TH729

243.3 ± 5.5c 17.7 ± 0.0a

213.3 ± 11.5d 20.0 ± 0.9b

256.7 ± 5.8b 21.1 ± 0.1b

283.3 ± 11.6a 20.3 ± 0.7b

BB medium (blank control), axenic F275 cultures, axenic TH729 cultures and F275−TH729 cocultures were cultivated 10 days for measuring concentration of NO3− and NH4+. Values are given as mean ± standard deviation from three samples. Different superscripts across rows indicate significant difference (p < 0.05) between treatments. 5

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Fig. 4. Transcriptome sequencing and annotation of cocultured F275 compared with axenic F275. (a) volcano plot of unigenes with significantly different expressions. Significantly upregulated and downregulated genes are shown as red and green dots, respectively. Genes with no significant changes are shown as gray spots. (b) gene ontology (GO) classification of differentially expressed genes. Green, blue and red color present three categories: molecular function, cellular component and biological process respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 3 Annotation and expression changes of significantly different unigenes related to nitrogen metabolism and fatty acid degradation pathways of F275 in coculture. EC No.

KO entry

KO name

KO definition

KEGG term: Nitrogen metabolism pathway (ko00910) – K02527 NRT Nitrate/nitrite tranporter 1.7.1.1 K10534 NR nitrate reductase (NAD(P)H) 1.7.1.1 K10534 NR nitrate reductase (NAD(P)H) 1.4.1.4 K00262 gdhA glutamate dehydrogenase (NADP+) 1.4.1.4 K00262 gdhA glutamate dehydrogenase (NADP+) 6.3.1.2 K01915 glnA glutamine synthetase KEGG term: Fatty acid degradation pathway (ko00071) 6.2.1.3 K01897 fadD long-chain acyl-CoA synthetase 6.2.1.3 K01897 fadD long-chain acyl-CoA synthetase 1.3.3.6 K00232 ACOX1 acyl-CoA oxidase 1.3.3.6 K00232 ACOX1 acyl-CoA oxidase 1.1.1.35/ K10527 HADH enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase 4.2.1.17 1.2.3.7 K11817 AA01-2 indole-3-acetaldehyde oxidase

6

Gene name

log2(Fold change)

q-value

Cluster-13874.13784 Cluster-13874.16143 Cluster-13874.14821 Cluster-13874.13145 Cluster-13874.17542 Cluster-10724.1

−3.46 −1.35 −3.06 1.93 1.51 −2.71

3.33E-31 3.89E-07 9.29E-163 5.54E-08 4.94E-03 2.37E-03

Cluster-13874.14251 Cluster-13874.12585 Cluster-13874.13493 Cluster-13874.13578 Cluster-13874.16440

1.03 1.49 2.01 1.48 1.19

2.20E-11 1.64E-11 4.29E-17 3.50E-04 2.25E-13

Cluster-13874.11819

1.43

2.25E-04

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Table 4 The fatty acids compositions (% of total fatty acids) of C. variabilis F275 cultured under different nitrogen concentration. Fatty acid SFA C16:0 C18:0 sum MUFA C16:1 C18:1n9t C18:1n9c C20:1 C20:1n9 sum PUFA C18:2n6t C18:2n6c C18:3n3 sum UFA/SFA

0.37 mmol·L−1

0.74 mmol·L−1

1.47 mmol·L−1

2.94 mmol·L−1

5.88 mmol·L−1

8.82 mmol·L−1

11.76 mmol·L−1

20.65 ± 1.46 4.51 ± 0.55 25.16 ± 0.38

28.81 ± 0.51 2.09 ± 0.75 30.90 ± 0.42

20.74 ± 2.52 3.92 ± 0.79 24.66 ± 1.67

31.4 ± 0.43 0.63 ± 0.05 32.03 ± 0.09

29.34 ± 0.77 1.91 ± 0.21 31.25 ± 0.18

16.08 ± 0.07 10.18 ± 0.67 26.26 ± 0.75

17.91 ± 2.14 14.27 ± 3.21 25.06 ± 6.81

1.29 ± 0.16 10.08 ± 0.90 12.26 ± 0.52 3.99 ± 0.90 18.19 ± 1.01 45.84 ± 1.05

0.85 ± 0.07 26.70 ± 1.05 2.66 ± 1.14 1.63 ± 0.25 12.12 ± 1.55 43.96 ± 1.36

1.27 ± 0.07 30.31 ± 0.59 5.48 ± 1.08 5.52 ± 1.48 9.64 ± 1.03 52.22 ± 0.70

2.50 ± 0.32 18.37 ± 1.06 1.07 ± 0.19 1.76 ± 0.48 17.52 ± 0.57 41.22 ± 3.28

0.59 ± 0.15 22.72 ± 1.28 1.16 ± 0.10 2.09 ± 0.90 19.26 ± 1.01 45.82 ± 1.56

6.28 ± 0.53 0.45 ± 0.07 19.15 ± 0.38 28.14 ± 0.75 3.80 ± 0.54 57.82 ± 0.07

6.66 ± 0.87 0.67 ± 0.42 17.31 ± 1.56 19.64 ± 0.74 3.91 ± 0.63 55.29 ± 7.56

5.95 ± 1.69 18.61 ± 1.38 4.24 ± 0.99 28.81 ± 0.65 2.97

1.81 ± 0.88 21.78 ± 1.88 1.55 ± 0.39 25.58 ± 1.45 2.25

3.73 ± 1.08 15.93 ± 1.60 3.46 ± 0.38 23.12 ± 1.45 3.06

0.77 ± 0.19 25.38 ± 1.08 0.60 ± 0.09 26.75 ± 2.35 2.12

2.70 ± 0.96 18.93 ± 1.66 1.31 ± 0.07 22.93 ± 0.78 2.2

15.92 ± 0.73 nd nd 15.92 ± 0.74 2.81

nd nd nd 19.65 ± 0.74 2.99

The data are expressed as mean (standard deviation) of three replicate samples.

fatty acid degradation pathway in F275 by the upregulation of ACOX, HADH and LACS coding genes that catalyze multiple steps of fatty acid β-oxidation in microalgae. Interestingly, N-replete conditions led to an increased β-oxidation for rapid lipid degradation and also increased the abundance of five acyl-CoA-oxidases in Chlamydomonas reinhardtii [48], indicating that the upregulation of fatty acid oxidation for rapid lipid degradation in cocultured F275 might be affected by TH729-derived nitrogen. 3.4.4. DEGs involved in the indoleacetic acid synthesis The indole-3-acetaldehyde oxidase gene of F275, involved in the final step of indoleacetic acid (IAA) synthesis that converts indole-3acetaldehyde (IAAld) to indoleacetic acid (IAA), was upregulated in our cocultures (Table 3). Since microalgae more or less lack the genes involved in IAA biosynthesis, the endogenous production of IAA by microalgae remains contentious [49]. Consequently, the upregulation of indole-3-acetaldehyde oxidase gene indicates that F275 may synthesize IAA using various precursors of IAA (e.g., IAAld) produced by TH729. In addition, IAA could enhance the growth of multiple microalgal species [50], which suggests that TH729 promoted the growth of F275 by secreting IAA precursors for F275. First, TH729 secretes IAA precursors that cause upregulations of indole-3-acetaldehyde oxidase gene expressions in F275, and then upregulates the IAA biosynthesis pathway and produces IAA. Even though IAA may enhance the growth of phytoplankton, the algal growth influenced by bacteria relates to multiple reasons. For example, Sulfitobacter produces indole-3-acetic acid by using diatom-derived tryptophan to promotes diatom growth. However the diatom growth enhanced by Sulfitobacter SA11 (19–35%) compared with IAA (10%) suggests that SA11 produces other molecules that further enhance diatom growth [16]. Fig. 5. The changes of chlorophyll and protein concentration (a), soluble sugar and neutral lipid concentration (b) of C. variabilis F275 cultured under different nitrogen concentration. The bar charts represent means ± SD of triplicate samples (n = 3). Different alphabets above the bar charts indicate significant differences (p < 0.05) between different nitrogen concentration.

3.5. Effect of nitrogen concentration on chlorophyll, protein and soluble sugar content Since TH729 significantly enhanced the nitrogen concentration in cocultures, we examined the effects of different concentrations of nitrogen on axenic F275 to study the influence of bacteria-derived nitrogen. The biochemical composition, lipid content and fatty acid composition of axenic F275 were all measured on the 14th day, the end of exponential growth. With the increase of nitrogen concentrations, the chlorophyll content increased from 0.09 pg·cell−1 (0.37 mM NaNO3) to 0.57 pg·cell−1 (11.76 mM NaNO3) in axenic F275, and the protein content changed from 2.02 pg·cell-1 to 6.06 pg·cell-1(Fig. 5a). From the N-limitation (0.37 mM NaNO3) to N-replete (11.76 mM NaNO3), the chlorophyll and

strain of Chlamydomonas has already been shown to be strongly impaired in oil remobilization [47], it suggests that the inactivation of ACOX might help the lipid accumulation in microalgae. In our results, the ACOX gene was upregulated in F275 cocultures, potentially leading to a decrease in lipid concentration of F275. Lastly, 3-hydroxyacyl-CoA dehydrogenase (HADH) catalyzes the third step of β-oxidation. This enzyme was upregulated in F275 when cultured with TH729. As a result, TH729 promoted the upregulation of 7

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coupled with low contents of PUFA are required [25]. Even though the fatty acid compositions were influenced by nitrogen, F275 cells still produced lower than 12% of C18:3 (linolenic acid) contents under different nitrogen concentrations, which meets the requirements of the European Standard EN14214 for biodiesel production. Hence, we can infer that nitrogen does not influence the quality of biodiesel produced by F275, which indicates that the nitrogen provided by TH729 did not affect the fatty acid quality of F275 to produce biodiesel. The lipid content and productivity of F275 declined with the increase of nitrogen concentrations (Fig. 6). Compared with N-repletion (11.76 Mm of NaNO3), the lipid content and productivity reached up to 1.6 folds and 2.2 folds, respectively, at 0.37 mM NaNO3. Lipid content and productivity were both diminished in response to increases in nitrogen availability. Since nitrogen availability increased in TH729-F275 cocultures due to nitrogen fixation by the bacterium, lipid content and productivity would also be expected to be lower in F275 under coculturing conditions. Previous studies have reported the exact mechanism [8,9] and the promotional effect of N-deprivation on lipid accumulation in different microalgal strains [7,52,54]. N-limitation causes an upregulation of carbon flux toward fatty acid and TAG biosynthesis, whereas the downregulation of TAG lipase genes may contribute to TAG accumulation. In our study, N-replete conditions cause a reduction of lipid accumulation. This conclusion is consistent with the enhanced nitrogen in cocultures and the upregulations of F275 genes in fatty acid oxidation which causes fatty acid degradations. Our study suggests that the nitrogen fixed by TH729 in F275-TH729 cocultures increases the nitrogen concentration, which downregulates nitrogen metabolism pathway and upregulates fatty acid oxidation pathway in F275, thus causing a decrease in lipid accumulation of microalgae. Consequently, removing TH729 before large-scale cultivation of F275 could enhance the production of biodiesel.

Fig. 6. Biomass productivity, lipid productivity and lipid content of C. variabilis F275 cultured under different concentration of nitrogen. The bar charts and points represent means ± SD of triplicate samples (n = 3).

protein contents increased by 6.5-fold and 3.0-fold, respectively. This chlorophyll content result was consistent with the degradation of chlorophyll that was induced by N-deprivation in Chlorella protothecoides [51]. Moreover, the protein content changes were consistent with previous work [52], where the lack of NaNO3 limited protein biosynthesis. However, the transcriptome analysis did not show significant DEGs involved in the amino acid synthesis, indicating some unclear effects weakened the influence of nitrogen on protein production. The soluble sugar content of F275 did not show variable trends vis-à-vis N-limitation and N-replete (Fig. 5b). The control group (2.94 mM NaNO3) showed the highest soluble sugar content as compared to other concentrations, but the soluble sugar content did not show a significant difference between N-limitation and N-replete conditions. Soluble sugar is a nutrient produced by algae and is also highly sensitive to environmental stresses [25]. The soluble sugar contents were lower in both N-limitation and N-replete conditions as compared to control conditions, which suggested that the changes in nitrogen concentration raise environmental stress for F275 that caused the soluble sugar decrease.

4. Conclusions Symbiotic Rhizobium of C. variabilis supplemented nitrogen in the coculture system to the alga. The increased nitrogen concentration influenced the growth, lipid accumulation and gene expression of metabolic networks in Chlorella. These results demonstrate that Rhizobium derived nitrogen increases fatty-acid oxidation in C. variabilis, which could reduce biodiesel accumulation. Overall, this is the first study to evaluate the effects of natural symbiotic bacteria on algal cultivation by extracellular inorganic nitrogen analysis and gene expression.

3.6. Effect of Nitrogen stress on lipid content and fatty acid compositions Authors’ contributions

In response to N-limitation and N-replete conditions in F275, neutral lipid accumulated in response to the N-limitation (Fig. 5b). For Nreplete experiments, the neutral lipid contents were higher than the control group only at 8.82 mM and 11.76 mM of NaNO3. This result is consistent with other reports that show microalgae accumulate higher levels of neutral lipids under N-limitation than grown under N-replete conditions [6] The fatty acid composition is one of the standards used to evaluate the quality of biodiesel. Total fatty acids are divided into three classes: saturated fatty acid (SFA), monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA). Compared with the control group (2.94 mM NaNO3), SFA was significantly reduced while MUFA increased in F275 under both N-limited and N-replete conditions. PUFA increased under all N-replete conditions but decreased under two Nlimited conditions (0.37 and 0.74 mmol/L NaNO3). Furthermore, the ratios of unsaturated fatty acids to saturated fatty acids (UFA/SFA) in F275 were higher than control under all nitrogen concentrations, suggesting that the changes of nitrogen concentrations provide a stress condition for F275. The UFA/SFA ratio is considered essential in stress resistance, since those unsaturated fatty acids are more easily oxidized than saturated fatty acids by oxygen radicals [53]. For efficient biodiesel production, high amounts of SFA and MUFA

CF and CHW conceived and designed the study. CF and MLH obtained the samples. CF, TW and AW. performed the experiments. CF and SMZ analyzed the data. CF and TW wrote the paper. CF, MLH and CHW reviewed and edited the manuscript. All authors read and approved the manuscript. Declaration of Competing Interest No conflicts, informed consent, or human or animal rights are applicable to this study. Acknowledgments The financial support from National key R&D program of China (NO. 2018YFD0901605), National Natural Science Foundation of China (NO. 31770436), Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization and Co-Innovation Center for Jiangsu Marine Bio-Industry Technology. We wish to thank Dr. Shady A. Amin and Mr. Muhammad Zain Tariq for providing language help and writing assistance. 8

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