Introduction

Microalgae, the primary producers of aquatic food web have a wide range of mutualistic interactions with bacteria (Cooper and Smith 2015). In addition to nutrient exchange, these interactions might have a dynamic effect on biogeochemical cycling (Ramanan et al. 2015). Thus, this mutualistic interaction in which both the symbionts benefit play an important role in natural ecosystems and contribute to the growth and health of algal biomass (Watanabe et al. 2005; Fuentes et al. 2016). However, due to the complexity of microalgal-bacterial interactions, knowledge is limited and utilisation is still based on empirical knowledge (Guo and Tong 2014). Therefore, a better understanding of these symbiotic mechanisms is crucial to enhance their combined biotechnological potential (Watanabe et al. 2005; Ramanan et al. 2016).

The bacterial communities associated with microalgal cultures play a ubiquitous role in algal growth and survival (Amin et al. 2015; Ramanan et al. 2016; Biondi et al. 2017). They may have a positive effect on algal growth through breakdown of organic metabolites or through the production of various algal growth-promoting substances (Salvesen et al. 2000; Ferro et al. 2019). The algal growth stimulatory compounds produced by the bacteria include vitamin like cobalamin, indole-3-acetic acid and siderophores. (Cole 1982; Croft et al. 2005; de-Bashan and Bashan 2008). Moreover, many aerobic bacteria provide favourable environmental condition (e.g. reduced oxygen tension) for microalgal growth (Mouget et al. 1995). The stimulatory effect of associated bacteria on physiological state of microalgae can be manifested by increased chlorophyll content, higher biomass production and more stable microalgal culture with delayed death phase (Natrah et al. 2014). The bacterial counterpart in turn may benefit by the uptake of extracellular organic carbon (EOC) released during algal photosynthesis (Watanabe et al. 2005). Thus, they can survive in microalgal habitat without the need of additional carbon source (Natrah et al. 2014). Furthermore, it has been reported that many nutrients released by microalgae can act as chemoattractants for marine heterotrophic bacteria which enable them to move towards their phytoplankton host (Miller et al. 2004).

Overall, it is known that microalgae-bacteria interaction can improve and enrich algal biomass production. Nevertheless, in aquaculture, very little attention has been paid to microalgal-bacterial symbiotic associations. Many previous investigators considered these bacteria as mere contamination of algal cultures (Watanabe et al. 2005; Fuentes et al. 2016). In this background, the objective of the present study was to explore symbiotic interaction between the marine microalga, Isochrysis galbana MBTDCMFRI S002 and its bacterial associates. The study evaluates effect of associated bacteria on microalgal growth, potential of bacterial associates to release algal growth stimulatory substances such as antioxidants, siderophores and indole-3-acetic acid (IAA) and growth of bacterial symbionts on algal EOC.

Materials and methods

Bacterial symbionts

Two bacterial symbionts which were isolated from Isochrysis galbana (MBTDCMFRI S002) culture and preserved as glycerol stocks at microbial culture collection of the Marine Biotechnology Division, Central Marine Fisheries Research Institute (CMFRI), Cochin (Kerala, India) were used for this study. They were Alteromonas sp. MBTDCMFRI Mab 25 (GenBank Accession No KR004801) and Labrenzia sp. MBTDCMFRI Mab 26 (GenBank Accession No KR004822) (Sandhya et al. 2017).

Axenisation of I. galbana culture

Isochrysis galbana MBTDCMFRIS002 maintained at the microalgae culture collection of the Marine Biotechnology Division, Central Marine Fisheries Research Institute (CMFRI), Cochin (Kerala, India) was used for the study. Before axenisation, the associated bacterial strains were screened for their sensitivity towards commercially used antibiotics such as penicillin (P-10 U), streptomycin (S-10 mcg), gentamicin (G-10 mcg) and kanamycin (K-10 mcg) (Himedia, India) by standard disc diffusion method (Bauer et al. 1966). The axenisation of I. galbana was carried out by using a cocktail of above antibiotics at four different concentrations as described by Droop (1967) and Guillard (2005). The final ratios of antibiotic concentration in treated culture were 1000:5:5:2.5, 500:2.5:2.5:1.25, 250:1.25:1.25:0.6 and 125:0.6:0.6:0.3 μg mL−1 of penicillin, streptomycin, gentamicin and kanamycin, respectively. After 48 h of antibiotic treatment, the algal culture was inoculated to fresh F/2 medium and incubated under optimum photoautotrophic conditions for 25 days. After incubation, the culture broth was inoculated on Zobell Marine Agar (ZMA) (Himedia, India) to confirm that bacterial symbionts had been removed from I. galbana strain. The obtained axenic culture of I. galbana was maintained at microalgae culture collection of the Marine Biotechnology Division, Central Marine Fisheries Research Institute (CMFRI), Cochin in sterilised sea water (33 ± 1 ppt) enriched with F/2 medium (Andersen et al. 2005) at 22 ± 1 °C under light-dark conditions (16:8 h, 40–50 μmol photons m−2 s−1).

Effect of bacterial symbionts on algal growth

To study the effect of bacterial symbionts on microalgal growth, each bacterial strain was pre-cultured as mono cultures on heterotrophic liquid medium (Zobell Marine Broth, Himedia, india). After 48 h of incubation (200 rpm, 30 °C), bacterial culture broth was centrifuged at 8000 rpm for 15 min, washed three times and finally resuspended in sterile F/2 medium. One milliliter of each symbiont solution (1 × 108 cells mL−1) was mixed with 10 mL of axenic I. galbana culture in 250 mL Erlenmayer flask containing 100 mL sterile F/2 medium. Effect of mixed culture of associated bacteria (with both symbionts) was also investigated. Axenic and non-axenic I. galbana cultures were used as control (Watanabe et al. 2005). The experiment was carried out in triplicate, and the growth of microalgae was monitored by taking cell count at regular time interval using a haemocytometer till day 24. The specific growth rate was calculated according to the equation (Guillard 1973):

$$ \mathrm{Specific}\ \mathrm{growth}\ \mathrm{rate},\mu =\frac{\ln \left(\raisebox{1ex}{${N}_{\mathrm{t}}$}\!\left/ \!\raisebox{-1ex}{${N}_0$}\right.\right)}{t_1-{t}_0.} $$

where Nt is cell density at the end of time interval, No is cell density at the beginning of time interval, tt − t0 is length of time interval

Statistical analysis

The mean growth rate of I. galbana in the presence and absence of bacterial symbionts were examined for significance (p < 0.01) using R software (Version 2.3-0). The results were subjected to normality testing (Kolmogorov–Smimov test) and were further analysed by a one-way ANOVA at 99% confidence level interval.

Release of algal growth promoters by bacterial symbionts

Antioxidant activity

Primary screening of antioxidant potential of selected bacterial strains was done using 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Sigma, USA) as described in Pawar et al. (2015). In brief, pure culture of Alteromonas sp. Mab 25 and Labrenzia sp. Mab 26 were spotted on Zobell Marine Agar (ZMA). The plates were incubated at 30 °C for 24 h. A sterilised Whatman no 1 filter paper was then placed on the agar plates and the plates were further incubated for another 24 h. After incubation, the filter paper was taken out and dried. It was then sprayed with DPPH solution prepared in methanol (80 μg mL−1). The bacterial strains showing white on purple spot were considered as positive, and the antioxidant activity was measured as zones of decolourisation around spotted colonies.

After primary screening, to determine the antioxidant activity of the crude extract of the extracellular metabolites, the bacterial strains were inoculated in 100 mL sterile ZMB (Himedia, India) and incubated at 30 °C at 200 rpm for 72 h. The culture broth was centrifuged and the supernatant was extracted by using same volume of ethyl acetate, for three times. The organic phase was then concentrated in rotary evaporator at 40 °C. The bacterial crude extract dissolved in methanol to prepare different concentrations (2.5, 2.0, 1.5, 1.0, 0.5 mg mL−1) and the antioxidant activity was estimated by DPPH radical scavenging method as described in Brand-Williams et al. (1995).

Siderophore production

For the screening of siderophore production potential, pure culture of Alteromonas sp. Mab 25 and Labrenzia sp. Mab 26 were spotted on chrome azurol S (CAS) agar with 2% NaCl (Lacava et al. 2008; Chaitanya et al. 2014). The plates were incubated at room temperature for 7–10 days and observed for a visual change in colour from dark blue to orange around the colonies.

Indole-3-acetic acid (IAA) production

The IAA production potential of Alteromonas sp. Mab 25 and Labrenzia sp. Mab 26 was determined by the method as described in Vijayan et al. (2012). The bacterial strains were inoculated to sterilised nutrient broth (with 2% sea salt) supplemented with tryptophan (10 μg mL−1) and incubated at room temperature for 3 days at 200 rpm. After incubation, bacterial culture broth was centrifuged at 10000 rpm for 10 min. To the culture supernatant (2 mL), two drops of orthophosphoric acid was added and incubated at room temperature for 10 min followed by addition of 4 mL of Salkowski reagent (1 mL 0.5 M FeCl3, 50 mL 35% HClO4). Development of pink colour indicates the positive result for IAA production.

Growth of bacterial symbionts on algal EOC

The heterotrophic growth of bacterial symbionts on EOC produced by I. galbana was examined as described by Watanabe et al. (2005). The culture broth containing algal EOC as carbon source was prepared by cultivating the axenic I. galbana using modified ASN III medium (without any carbon sources) (Andersen et al. 2005) under photoautotrophic condition for 3 weeks. The culture broth was centrifuged (5000 rpm, 10 min, 4 °C) to remove the cells, and the obtained supernatant was filtered through a sterile 0.2-μm membrane filter (Pall, USA). This filtrate was mixed with an equal volume of the sterile modified ASN III medium for the supplement of inorganic salt. Sterile medium without algal EOC was kept as control. Each bacterial symbiont was inoculated to both test and control cultures and incubated at 30 °C without illumination (200 rpm). The growth of each symbiont was monitored at OD600 (Thermo Scientific, US).

Results

Axenisation of I. galbana culture

Our previous study clearly demonstrated the existence of a strong and close association between I. galbana MBTDCMFRI S002 and bacterial strains such as Alteromonas sp. MBTDCMFRI Mab 25 (2.5 × 103 CFU mL−1) and Labrenzia sp. MBTDCMFRI Mab 26 (1 × 103 CFU mL−1), even under artificial conditions (Sandhya et al. 2017). These bacterial strains are the dominant cultivable bacteria associated with the selected microalgal strain. At the same time, the metagenomic approach has revealed the occurrence of 44 different known bacterial genera and numerous unknown bacterial groups in the phycosphere of same microalgal strain (Unpublished data). The focus of the present study was to explore the symbiotic interaction among associated bacterial groups and I. galbana which require cultivable microorganisms. Thus, the bacterial strains Alteromonas sp. and Labrenzia sp. are of special interest, even if they represent a subset of larger symbiotic consortium exist in the microalgal habitat. Since, the aforementioned study required axenic culture of microalgae which are devoid of these bacterial symbionts, I. galbana culture was axenised using a cocktail of four antibiotics (penicillin, streptomycin, gentamicin and kanamycin) to which the associated bacterial strains were susceptible. The antibiotic concentration of 1000:5:5:2.5 μg mL−1 of penicillin, streptomycin, gentamicin and kanamycin, respectively, for 48 h was found to be most effective in inhibiting bacterial growth in the microalgal culture.

Effect of bacterial symbionts on algal growth

The axenic strain of I. galbana was cultivated along with bacterial symbionts to investigate the effect of associated bacteria on microalgal growth. Time course of cell count of test and control cultures are shown in Fig. 1. Effect of bacterial addition on biomass accumulation and growth rate of I. galbana at day 24 are shown in Fig. 2. There was an increase in both biomass accumulation and growth rate of axenic I. galbana culture in the presence of bacterial symbionts. The biomass of axenic culture of I. galbana was increased from 2.54 ± 0.36 × 106 to 3.87 ± 0.47 × 106 cells mL−1 in the presence of Alteromonas sp. Mab 25. Similarly, in the presence of Labrenzia sp. Mab 26, the biomass increased up to 4.35 ± 0.32 × 106 cells mL−1. Interestingly, it was noticed that even in the presence of added bacterial symbionts, biomass accumulation of axenic culture was less than that of the non-axenic culture (8.55 ± 1.17 × 106 cells mL−1). The enhanced growth of non-axenic culture might be due to the undetectable symbionts in the culture. The growth rate of axenic culture of I. galbana was 0.093 ± 0.006 divisions day−1 whereas that of non-axenic culture was 0.15 ± 0.006 divisions day−1. Concurrently, there was an increase in the growth rate of axenic I. galbana culture when cultivated along with bacterial symbionts (Fig. 2). Analysis of variance showed that the effect of bacterial symbionts on microalgal growth rate was significant (F(4, 10) = 54.95, p < 0.01). The Bonferroni post-hoc test revealed that there was no significant difference in the growth promoting effect of bacterial symbionts when they were inoculated individually and as a consortium (p = 1.00).

Fig. 1
figure 1

Growth curves of I. galbana MBTDCMFRI S002 in the presence and absence of associated bacteria

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Fig. 2
figure 2

Effect of bacterial addition in biomass accumulation (a) and growth rate (b) of I. galbana MBTDCMFRI S002 (A- Non-axenic; B- Axenic; C- Axenic + Mab 26; D- Axenic + Mab 25; E- Axenic + Mab 26 + Mab 25). The error bars indicate the standard deviation)

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Release of algal growth promoters by bacterial symbionts

Both Alteromonas sp. Mab 25 and Labrenzia sp. Mab 26 associated with I. galbana showed white on purple spot when sprayed with DPPH solution which indicated that both bacterial strains are able to produce extracellular antioxidants (Fig. 3). The decolourisation zone for Alteromonas sp. was 70 mm and that of Labrenzia sp. was 20 mm. The DPPH scavenging ability was increased with increase in concentration of bacterial crude extract (Table 1). The scavenging effect of Alteromonas sp. Mab 25 was 41% at a concentration of 2.5 mg mL−1 whereas that of Labrenzia sp. Mab 26 was 25.3% at the same concentration. The IC50 of the two bacterial isolates were 6.38 and 9.55 mg mL−1 respectively.

Fig. 3
figure 3

Antioxidant activity of Alteromonas sp. (MBTDCMFRI Mab 25) and Labrenzia sp. (MBTDCMFRI Mab 26) by DPPH assay

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Table 1 Antioxidant activity of crude extract of bacteria associated with I. galbana using DPPH assay. (Strain code of both isolates start with MBTDCMFRI)
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In the present study, CAS assay was performed to evaluate the production of siderophores by Alteromonas sp. Mab 25 and Labrenzia sp. Mab 26 associated with I. galbana. Both these bacterial strains were CAS positive for siderophore production as indicated by a orange halo around the colonies after incubation (Fig. 4). The zone diameter for Alteromonas sp. Mab 25 was 14 mm and that for Labrenzia sp. Mab 26 was 12 mm. On the addition of orthophosphoric acid and Salkowski reagent, Labrenzia sp. Mab 26 produced slight pink colouration indicating the production of IAA in their culture filtrates. Concurrently, Alteromonas sp. Mab 25 was found to be negative for IAA production (Fig. 5).

Fig. 4
figure 4

Siderophore production potential of Alteromonas sp. (MBTDCMFRI Mab 25) and Labrenzia sp. (MBTDCMFRI Mab 26) by CAS assay

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Fig. 5
figure 5

Screening of Alteromonas sp. (MBTDCMFRI Mab 25) and Labrenzia sp. (MBTDCMFRI Mab 26) for IAA production

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Growth of bacterial symbionts on algal EOC

Both bacterial strains were able to grow in culture broth containing algal EOC as carbon source. Concurrently, they showed no growth in the absence of EOC in the medium (control). Alteromonas sp. Mab 25 reached at the stationary phase of growth on day 4 of inoculation whereas Labrenzia sp. Mab 26 reached stationary phase on day 6 (Fig. 6). These results indicate that the phytoplankton host can provide organic carbon source for heterotrophic growth of both bacteria under photoautotrophic conditions.

Fig. 6
figure 6

Heterotrophic growth of bacterial symbionts on algal EOC

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Discussion

Aquaculture is the fastest growing food producing sector in the world, and microalgae form an important part of diet of many aquaculture organisms, especially in the larval rearing systems (Banerjee et al. 2010; FAO 2014; Natrah et al. 2014). The improved quality of these live feeds will increase the growth and health of aquatic organisms. In this study, growth enhancement of I. galbana was observed when co-cultured with its bacterial symbionts, highlighting the role of bacteria in algal growth. This indicates the potential to use a well-selected consortium of phycosphere bacteria to improve the productivity, efficiency and sustainability of aquaculture. In addition, a suitable combination of microalgae and beneficial bacteria might also lead to better shellfish larval settlement.

The stimulatory effects of bacteria on microalgal growth have been widely studied. Mouget et al. (1995) reported that there was an increase in the maximum cell density of Scenedesmus bicellularis associated with strains of Brevundimonas diminuta. Le Chevanton et al. (2013) observed that strains of bacteria affiliated to Alteromonas sp. and Muricauda sp. can help nitrogen accumulation in Dunaliella sp. and can enhance biomass accumulation. However, the exact mechanisms of these positive effects bought by associated bacteria are largely unknown (Natrah et al. 2014). However, it has been reported that bacteria can produce various growth stimulatory compounds, especially in symbiotic situations (Cole 1982; Amin et al. 2015). For example, Guo and Tong (2014) have reported on bacterial production of vitamins required by microalgae (Guo and Tong 2014). Durham et al. (2015) showed that vitamin B12 produced by Ruegeria pomeroyi have a positive effect on the growth of Thalassiosira pseudonana. Bacteria can secrete hormones like indole-3-acetic acid (IAA) which can stimulate algal metabolism (Natrah et al. 2014). Similarly, siderophores synthesised by bacterial counterparts can provide a supply of soluble iron to algal cells (Cooper and Smith 2015; Rajapitamahuni et al. 2019). These algal growth promoters produced by associated bacteria might have a significant positive impact on algal growth. Thus, in order to unravel the mechanisms of these positive impacts exerted by bacterial associates, they were screened for release of various algal growth promoters viz. antioxidants, siderophores and indole-3-acetic acid.

In general, antioxidant compounds scavenge free radicals and other reactive oxygen species and thus provide protection against cellular damage and oxidative stress induced by these chemical species (Valko et al. 2007; Balakrishnan et al. 2014; Rubavathi and Ramya 2016). Though the antioxidant potential of microalgae is being explored globally, the knowledge on antioxidant activity of its associated bacteria is rather limited. The results of the present study clearly indicate the antioxidant property of the bacterial strains associated with I. galbana, a well explored source of natural antioxidants (Saranya et al. 2014; Sun et al. 2014; Rubavathi and Ramya 2016). Through the production of these bioactive molecules, these microbial partners construct a chemical microenvironment and hence maintain a close cross-relationship with their eukaryotic hosts (Penesyan et al. 2010). The associated bacteria often produce such bioactive compounds to inhibit competing organisms and microbial pathogens (Balakrishnan et al. 2014). Thus, owing to the antioxidant potential, these bacterial strains could play a role in microlagae defence mechanism. Priyanka et al. (2014) reported the antioxidant activity of Labrenzia sp. isolated from deep seawater from offshore of Cochin (India). Similarly, Yeo et al. (2006) isolated antioxidant producing Alteromonas macleodii strain from the coast of Korea. Compared to previous studies, these bacterial strains exhibited lesser antioxidant activity. However, it is assumed that they can boost the antioxidant potential of their phytoplankton counterpart synergistically. Thus, this microbial consortium can complement the live feed which in turn exerts an overall positive effect to the aquaculture rearing system.

Iron is an essential element for virtually all living cells owing to its ability to catalyse redox reactions, transfer electrons and transport ligands such as dioxygen (Litwin and Calderwood 1993; Amin et al. 2012; Chaitanya et al. 2014). The scarcity of dissolved iron in the aerobic marine environment has driven microorganisms to adopt a way for iron acquisition by producing iron-chelating molecule i.e. siderophore (Amin et al. 2012; Ali and Vidhale 2013). These low-molecular-compounds can bind with ferric ion with high affinity (Lacava et al. 2008). A universal siderophore assay using chrome azurol S (CAS) and hexa decyl trimethyl ammonium bromide (HDTMA) as indicators was developed by Schwyn and Neilands (1987). These bacterial siderophores are known to be involved in enhancing the growth of their phytoplankton hosts (Natrah et al. 2014; Fuentes et al. 2016; Rajapitamahuni et al. 2019). They provide a steady supply of Fe (III) to algal cells, potentially in exchange for organic carbon (Amin et al. 2009). As an example, majority of the Marinobacter spp. which are consistently associated with diverse algal cultures produce the siderophore vibrioferrin, which binds Fe (III), making it available for microalgae and bacteria (Amin et al. 2012; Fuentes et al. 2016). Similarly, the microalgae Scrippsiella trochoidea makes use of siderophores produced by bacteria present in its environment (Amin et al. 2009). In addition to the algal growth promoting role, these microbial siderophores have wide applications in various field such as agriculture to improve soil fertility and biocontrol, environmental application and medicinal application (Ali and Vidhale 2013).

It has been reported that bacteria can stimulate the metabolism of microalgae by secreting hormones like indole-3-acetic acid (Natrah et al. 2014; Fuentes et al. 2016). There was a threefold increase in the growth of Chlorella vulgaris in the presence of the IAA-producing bacterium Azospirillum brasiliense (Gonzalez and Bashan 2000; de-Bashan et al. 2008). Furthermore, the production of IAA by Labrenzia sp. Mab 26 may offer a chance to use this phycosphere bacterium as a biofertilizer to improve the growth and yield of agricultural crops in coastal saline influenced lands (Vijayan et al. 2012). Our study clearly shows that associated bacteria can secrete various growth stimulatory substances which can improve the physiological state of phytoplankton host.

The growth of bacterial symbionts on algal EOC clearly suggests that bacteria and microalgae form mutualistic relationship in which algal growth is stimulated by various bacterial products whereas bacteria in turn benefit from phytoplankton exudates. In the ‘phycosphere’ microbial activity is stimulated by phytoplanktonic release of extracellular products (Grossart 1999). Moreover, our previous study showed that these bacterial symbionts were capable of producing hydrolytic exoenzymes such as lipase, amylase, gelatinase and urease (Sandhya et al. 2017). These exoenzyme activities enhance bacterial remineralisation of N- and P- rich lysed algal cellular components which sustain the microalgae–bacteria equilibrium in aquatic ecosystems (Grossart 1999; Fuentes et al. 2016). Thus, these foremost functional entities together drive oceanic biogeochemical cycles and thereby ensure a balance in the nutrient cycles and energy flow (Natrah et al. 2014).

In conclusion, there exists a symbiotic relationship between I. galbana and associated bacteria, which has been proved in the present study. Both Alteromonas sp. Mab 25 and Labrenzia sp. Mab 26 exhibit mutualism, receiving nutrients from the microalgae while promoting its growth. This report is the first observation on symbiotic association of bacteria in I. galbana culture. These findings give a greater insight on microalgal-bacterial interactions which can be further explored to improve productivity and sustainability in aquaculture rearing systems.