Iron-dependent mutualism between Chlorella sorokiniana and Ralstonia pickettii forms the basis for a sustainable bioremediation system

Iron and carbon dependent mutualism between Chlorella sorokiniana and Ralstonia pickettii forms a synthetic phototrophic community

The synthetic microalgal-bacterial community based on the active exchange of iron and carbon was developed by screening multiple siderophore producer bacteria and dye decolorizer algae (Fig. 1; refer to Supplementary Data S1 for detailed results). Out of seven bacterial isolates obtained from untreated textile wastewater, five showed relatively high siderophore production in CAS agar plates and broth (Fig. S1). In broth, Serratia plymuthica PW1, Serratia liquefaciens PW71, and Ralstonia pickettii PW2 produced siderophores in decreasing order of concentration, i.e., 15.26 ± 1.3 > 13.28 ± 0.9 > 10.85 ± 0.7 µMmL⁻¹ (Table 1). Arnow’s assay confirmed that S. plymuthica PW1 (81.10 ± 9.8 µMmL⁻¹), R. pickettii PW2 (97.43 ± 16.8 µMmL⁻¹), and S. liquefaciens PW71 (103.1 ± 8.3 µMmL⁻¹) produced catecholate-type siderophores. On the other hand, Csaky’s assay confirmed that Stenotrophomonas maltophilia PW5 (37.86 ± 0.4 µMmL⁻¹) and Stenotrophomonas maltophilia PW6 (17.73 ± 0.2 µMmL⁻¹) produced hydroxamate-type of siderophores. Out of the five algal species, only freshwater microalgae Chlorella sorokiniana and Scenedesmus sp. showed the highest dye degradation potential; therefore, they were selected for further experiments (Data S1).

After that, the sterile exudates from C. sorokiniana and Scenedesmus sp. were used as the sole source of dissolved organic matter for bacterial growth and selection of appropriate microalgal-bacterial partners comprising the phototrophic community (Fig. 1E; Data S2). All five bacterial isolates grew well on the exudate of C. sorokiniana as a sole source of carbon. On the contrary, on exudates of Scenedesmus sp., S. plymuthica PW1 showed moderate growth in 20 h, while the growth of R. pickettii PW2 and S. liquefaciens PW71 remained insignificant. S. maltophilia PW5 and PW6 failed to grow in the exudate of Scenedesmus sp. (Fig. S2B).

Finally, the compatibility between the phototrophic community of selected microalgae (C. sorokiniana/ Scenedesmus sp.) and siderophore-producer bacteria (S. plymuthica PW1/ R. pickettii PW2/ S. liquefaciens PW71) was tested by co-culturing them in iron limiting BBM media (BBM-Fe; without EDTA) (Fig. 1F). In the absence of EDTA, Fe precipitates rapidly as iron oxyhydroxides and becomes unavailable to microbes. Microalgal growth curves in co-culture assays were used to measure and compare population characteristics such as carrying capacity ‘k’, growth rate ‘r’, etc., in axenic and consortium setups. Algal growth parameters in co-culture with a bacterial partner were used to categorize their interaction as putative mutualistic, antagonistic, and neutral (Data S1, Tables S1 and S2) [42]. Under iron-limiting conditions, axenic C. sorokiniana experienced iron stress as the cell growth was 4.2 ± 0.4 × 10⁶ cells mL⁻¹ after 200 h incubation. On the other hand, axenic Scenedesmus sp. showed a significantly higher growth (11.3 ± 1.2 × 10⁶ cells mL⁻¹) than C. sorokiniana suggesting an effective iron uptake mechanism under iron-limiting conditions (k; t-test, p = 0.001) (Table S1). In contrast to the axenic microalgal culture, C. sorokiniana in co-culture with R. pickettii PW2 showed a significant increase in cell count at 200 h (6.2 ± 0.85 × 10⁶ cells mL⁻¹) (auc; p = 0.000). However, S. plymuthica PW1 exerted a negative effect on C. sorokiniana (Fig. 2A), as indicated by its significant increase in doubling time (p = 0.009) and reduction in auc (p = 0.001) (Fig. 3A). While S. liquefaciens PW71 remained neutral to C. sorokiniana (auc; p = 0.430) (Fig. 2A, Table 2). On the other hand, the interaction of Scenedesmus sp. with both R. pickettii PW2 and S. liquefaciens PW71 was neutral, while S. plymuthica PW1 showed a negative effect (Figs. 2A and 3A).

As indicated by a steeper slope of the log-phase in the growth curve (Fig. 3A), the growth rate ‘r’ of C. sorokiniana in the consortium with R. pickettii PW2 (5.02 ± 1.0 × 10⁻² h⁻¹) remained significantly higher than that of axenically grown microalgae (1.95 ± 0.3 × 10⁻² h⁻¹) (p = 0.000). In the consortium, C. sorokiniana showed a higher population turnover during the early log-phase and reached the stationary phase earlier (at 100 h) than cells grown axenically (~270 h), although the carrying capacity remained similar (Fig. 3A), suggesting that algae grew faster under iron-limiting conditions in the consortium. In addition, the NO₃-N drawdown in media under axenic and consortium setups was monitored to assess whether the difference in N-uptake led to an increase in algal growth in consortium setup. After 310 h, the nitrate concentration dropped from 123 to 74.75 ± 3.15 and 80.25 ± 1.85 mgL⁻¹ in axenic and consortium setups, respectively, suggesting only a slight difference between the two setups. The higher growth of C. sorokiniana in consortium setup was not because of the difference in N-uptake, but because of the difference in iron bioavailability (Fig. S3C). Thus, iron was the only growth-limiting factor in the BBM. The lower bioavailability of iron because of the absence of chelating agents like EDTA in iron-deficient BBM reduced algal growth. However, the presence of siderophore producer R. pickettii in co-culture increased the growth rate of C. sorokiniana under iron-limiting conditions [43].

The principal component analyses (PCA) biplot further explained the difference in the growth of C. sorokiniana and Scenedesmus sp. in axenic and co-culture setups (Fig. 3B; refer to Supplementary Data S2 for detailed analysis). Grouping of Scenedesmus sp. grown axenically (SS), in consortium with R. pickettii PW2 (SSPW2) and S. liquefaciens PW71 (SSPW71) due to similar area under curve ‘auc’ and carrying capacity ‘k’ parameters, suggested a neutral interaction. Both ‘auc’ and ‘k’ contributed to PC1 by 39.17% and 24.39%, respectively (Fig. S2D). The separation based on higher ‘auc’ of Scenedesmus than Chlorella was due to their different growth responses under iron limitation (Fig. 2A), which might govern their interactions with bacteria (Fig. 3A). In contrast with Scenedesmus, C. sorokiniana grown in consortium with R. pickettii PW2 (CSPW2) was separated from axenically grown algae (CS) based on higher growth rate ‘r’ (Fig. 3B). PC2 explains 37.7% of the grouping of different variables, which had ‘r’ as the dominant metric (42.25%). On the contrary, CSPW1 co-culture was separated from axenic C. sorokiniana due to a higher doubling time ‘Dt’, indicating a negative effect of S. plymuthica on the growth of Chlorella (Fig. 2A).

Such an iron-dependent mutualism has been previously reported between alga Dunaliella bardawil and Halomonas sp [44]., diatom Navicula pelliculosa and Cupriavidus necator [45], marine alga Scrippsiella trochoidea and Marinobacter sp [37]., freshwater alga Chlorella variabilis and Idiomarina loihiensis [46]. Previously, a commensal association between R. pickettii and C. sorokiniana has been reported under nutrient-sufficient photoautotrophic conditions [47]. In this study, compared with axenic culture, R. pickettii PW2 showed a higher growth when supplemented with exudate of Chlorella than Scenedesmus, indicating the use of Chlorella-derived organic matter as a preferred substrate for growth (Fig. S2B). R. pickettii PW2 also showed enhanced growth in co-culture with Chlorella compared to axenic culture (Fig. 2A), which suggested a mutualistic association between algae and bacteria.

Algal exopolysaccharides (EPS) serve as a carbon source for bacteria and influence microbial interactions [48, 49]. The HPAEC analyses of EPS of C. sorokiniana detected commonly reported galactose (0.03 ± 0.0 g gcell⁻¹; 52%, relative percentage) as the dominant monosaccharide besides glucose (20%), mannose (20%), arabinose (4%), and rhamnose (4%). The EPS of Scenedesmus sp. had glucose (0.31 ± 0.0 g gcell⁻¹, 37%) as the dominant monosaccharide, followed by mannose (32%), rhamnose (12%), galactose (10%), and arabinose (9%) (Figs. 2B and S3A). In a bacterial growth assay performed on the 5 monosaccharides, mutualistic bacterium R. pickettii PW2 showed significantly higher growth in galactose (auc; p = 0.001) (Fig. 2B, C and Table S15). The higher presence of galactose in EPS has been hypothesized to have a role in maintaining an extended stationary phase in green algae [50]. The putative antagonistic S. plymuthica PW1 showed ~10 times higher growth than R. pickettii PW2 when grown with C. sorokiniana (Fig. 2A). S. plymuthica PW1 also grew well on supplementing with any of the five monosaccharides, with galactose being the preferred carbon source (Fig. 2C). Thus, the negative effect of S. plymuthica PW1 on C. sorokiniana could have been due to its aggressive growth and generalist behaviors. Therefore, the composition of algal EPS and its metabolism by bacteria could have influenced the nature of algal-bacterial interactions [51, 52]. However, further studies at the molecular level will ascertain the influence of EPS composition to initiate and maintain such associations. Apart from the tested monosaccharides, algae produce several organic compounds that could also act as a substrate and influence such associations [53]. Thus, our study posits that Chlorella EPS can serve as a source of DOM for R. pickettii PW2 to form a mutualistic association in exchange for bioavailable iron. Consequently, these algal-bacterial partners were selected to form the phototrophic community.

Bioavailable iron influences dye degradation of phototrophic community

To ascertain the significance of biologically available iron in bioremediation, the dye degradation potential of C. sorokiniana was analyzed in axenic and consortium cultures in iron-deficient (without EDTA) and -sufficient (with EDTA) conditions. In axenic culture, dye degradation rate was 0.005 ± 0.000 h⁻¹ under iron-deficient conditions, which increased to 0.033 ± 0.003 h⁻¹ when iron was more bioavailable (Fig. 4A). In contrast, the dye degradation rate was higher in consortium setups under iron-deficient conditions (0.009 ± 0.000 h⁻¹), which increased to 0.049 ± 0.008 h⁻¹, when iron was more bioavailable.

Under iron-deficient conditions, the difference in the dye degradation rate between axenic and consortium setups was significant (p = 0.000) (Tables S3 and S4). The presence of siderophore-producing R. pickettii significantly increased the dye degradation potential of C. sorokiniana, whereas, the axenic bacteria lacked detectable dye degradation suggesting a significant contribution of a microalgal partner in dye degradation in the consortium. Although, in iron-sufficient conditions, the dye decolorization by C. sorokiniana in both axenic alga and consortium increased, but the difference between them was not statistically significant (p = 0.126) (Fig. 4A).

In the iron-deficient conditions, the dye degradation followed Simple First Order (SFO) kinetics (χ² value, axenic alga: 6.02%; consortium: 8.56%), as also reported in other microbial dye degradation studies (Table S3) [54,55,56]. In SFO kinetics, the rate of degradation depends on the concentration of reactant. In contrast, dye degradation followed a First Order Multi-Compartment (FOMC) kinetics in iron-sufficient conditions, which is characterized by a biphasic degradation indicating an initial steep decline in dye concentration followed by a relatively slower degradation (χ² value, axenic alga: 4.20%; consortium: 2.86%) (Fig. 4A). C. sorokiniana in co-culture with R. pickettii showed a significantly high dye degradation only in the iron-deficient condition (Fig. 4A). Under iron-deficient conditions, bacteria produce siderophores to chelate iron and make it bioavailable to both bacteria and algae [57]; however, because EDTA is a strong chelator, bioavailable iron significantly increased (Fig. 4A and Table S4). Thus, in iron-sufficient conditions, bacterial presence lacks any significant effect on algal dye degradation due to the high bioavailability of iron because of EDTA (p = 0.126). Thus, R. pickettii PW2 increased the dye degradation potential of C. sorokiniana only under iron-limiting conditions.

L₁₆ (4³) design was used to compare the effect of varying Fe³⁺, dye concentration, and pH level on microalgal dye remediation potential in axenic and consortium setups (Fig. S5, Table S5). The Fe³⁺ concentration was kept lower or higher than 1 × 10⁻⁶M, a concentration known to induce iron-starvation due to variation in the equilibrium between intra- and extracellular iron, thus, necessitating bacterial siderophore production [58, 59]. Iron precipitation increases with increasing pH, reducing its solubility and bioavailability [60]. Since the pH of the textile wastewater varies from 6 to 10, the effect of varying pH on dye degradation was tested [35]. The effect of varying AB1 dye (substrate) concentration was tested because it affects the enzymatic activity that determines the rate of dye degradation.

Results of the multiple regression model suggested Fe³⁺ concentration (delta value; axenic alga = 0.02, consortium = 0.03) as a primary factor governing the rate of dye degradation, followed by the azo dye concentration (delta value; axenic alga = 0.01, consortium = 0.02) (Fig. 4B, Tables S6 and S7). The delta value in Taguchi’s orthogonal design takes all the factors individually to determine the difference between the highest and lowest values of the average response variable. Therefore, a higher delta value of a particular factor represents a significant effect of variation in the level of the factor. Changing the concentration of Fe³⁺ led to a considerable variation in the rate of AB1 degradation in both axenic algal (p = 0.001) and consortium (p = 0.002) setups (Fig. 4B). Further analysis using Partial Least Squares Path (PLSP) modeling suggested that the rate of AB1 degradation was inversely proportional to Fe concentration; the effect was prominent in consortium setup (Fig. S4B, Table S16). The consortium setup showed an enhanced average rate of AB1 degradation (0.04 h⁻¹) as compared to axenic cultures (0.03 h⁻¹) (Fig. 4B). Siderophore-producer bacteria increased the dye degradation potential of microalga at 1 × 10⁻⁷ and 1 × 10⁻⁶M Fe, whereas at a higher Fe³⁺ concentration, the bacterial effect remained neutral.

The dye concentration also showed an inverse relationship with the rate of dye degradation in both axenic algal and consortium setups, but the effect was prominent in consortium setup (Fig. 4B, Table S7), as suggested by PLSP analysis (Fig. S4B, C, Table S16). Although the rate of dye degradation decreased with increasing dye concentration, the algal-bacterial consortium could degrade up to ~60% dye even at higher dye concentrations (Fig. 4B, Table S5). At a high concentration, the dye molecules compete for electrons generated by the azoreductase-mediated enzymatic mechanism at the microbial membrane [61], thus reducing the dye degradation rate. Similarly, the siderophore-producer bacteria only increased the dye degradation rate at low dye concentrations (Fig. 4B, Tables S6 and S7). Dye degradation in bacteria is a non-growth associated extracellular process driven by membrane-bound azoreductase, a highly diverse and non-specific oxidoreductase. Azoreductases have been widely reported in bacteria, such as P. aeruginosa, E. coli, and Bacillus sp [62, 63]., but not from algae. Bacterial azoreductase facilitates electron transfer from cells to electron-deficient azo bond (-N = N-), which reduces azo dyes into colorless aromatic amines via a two-cycle transfer of electrons following a ping-pong bi-bi mechanism [18, 61]. Azoreductases use extracellular mediators such as flavins which transfer electrons from within the microbial cell to outside for azo dye reduction. Therefore, the optimal microalgal cell-to-dye ratio significantly influences catalytic efficiency and enzyme turnover for azo dye degradation. On the contrary, pH lacked any significant effect (p > 0.1) on rates of AB1 degradation in both consortium and axenic algal setups (delta value; axenic alga = 0.01, consortium = 0.01), although, the rate of degradation was higher at the lower pH in the consortium setup (Fig. 4B, Table S6). The least impact of the varying pH on the rate of AB1 degradation could also have been due to pH regulation of the culture media by algal photosynthesis and N uptake. C. sorokiniana changed the pH of the neutral BBM to 7.9, suggesting a pH regulation due to algal photosynthesis. This increase in pH has been linked to the consumption of CO₂ and the nitrate metabolism in closed and stationery phototrophic culture conditions [64].

The PLSP model revealed the positive effect of interactions between factors Fe*pH and Fe*Dye on the rate of AB1 degradation in the consortium setup (Fig. S4B). The results suggest that variation in Fe positively influenced the effect pH and Dye concentration had on the rate of dye degradation. However, the interaction effect was significant only in consortium setups, thus, suggesting the presence of bacteria plays a key role in ensuring Fe availability for algae and also the dye degradation potential. However, further analysis of the interactions between abiotic factors in a full factorial design will help determine the influence of factors on each other.

Plasma membrane-bound ferrireductase enzyme influences algal dye degradation

We hypothesize that the increased rate of azo dye degradation with increased bioavailable iron was associated with higher azoreductase activity per cell or was due to increased algal cell numbers. Therefore, the effect of varying iron concentrations on the algal cell growth, enzymatic activities, and iron uptake was investigated in axenic algal and algal-bacterial consortium setups. With the increase in iron concentration, the algal cell growth was increased in both axenic and consortium setups; however, the difference in algal growth in the two setups was significant only in lower iron concentrations (Fig. 5A). In axenic cultures, C. sorokiniana growth varied with Fe concentration in experimental setups, 2 × 10⁻⁶M Fe: 8 × 10⁷ cells mL⁻¹, 1 × 10⁻⁶M Fe: 6 × 10⁷ cells mL⁻¹, and 1 × 10⁻⁷M Fe: 1.4  × 10⁷ cells mL⁻¹. At lower Fe concentrations, R. pickettii PW2 enhanced the growth of C. sorokiniana. The positive effect of the siderophore-producing bacteria on algal growth was also significant at lower Fe concentrations, including growth parameters, such as growth rate ‘r’: p = 0.006 for 1 × 10⁻⁷M Fe, p = 0.028 for 1 × 10⁻⁶M Fe; carrying capacity ‘k’: p = 0.025 for 1 × 10⁻⁷M Fe, and p = 0.017 for 1 × 10⁻⁶M Fe; and area under curve ‘auc’: p = 0.003 for 1 × 10⁻⁷M Fe, and p = 0.001 for 1 × 10⁻⁶M Fe (Fig. S6, Tables S8 and S9). However, at 2 × 10⁻⁶M Fe, microalgae that were grown axenically and in the phototrophic community lacked a significant difference in their growth parameters (p = 0.157 for r, p = 0.551 for k, and p = 0.444 for auc).

Although algal cell density increased at high Fe concentration (Fig. 5A), the reductive iron uptake mechanism was inversely proportional to the increase in iron concentration (Fig. 5B). Ferrireductase activity assesses Fe²⁺ on the cell surface. A higher ferrireductase activity indicates the reduction of Fe³⁺ in Fe³⁺-chelates to Fe²⁺ by algae. In both axenic and consortium setups, the membrane-bound ferrireductase activity of C. sorokiniana was reduced with increasing iron concentration (Fig. 5B) [65]. However, siderophore-producer R. pickettii PW2 significantly increased the ferrireductase activity in microalgae but only at lower Fe concentrations (1 × 10⁻⁷M Fe: p = 0.001, and 1 × 10⁻⁶M Fe: p = 0.003) (Table S10). At the same time, there was no observable ferrireductase activity in the axenic bacterial cultures. Therefore, azo dye reduction and iron reduction mechanisms in C. sorokiniana showed an inverse relationship with iron concentration.

Algae do not produce siderophores, however, they accumulate iron in the phycosphere by biosorption and chelation onto extracellular polymeric substances, including mono- and polysaccharides [66]. In contrast to the axenic culture, the EPS from C. sorokiniana co-cultured with bacteria accumulated significantly more iron (t-test; p < 0.05) (Fig. 5C). However, the difference in accumulation was significant only at low Fe concentrations: 1 × 10⁻⁷M and 1 × 10⁻⁶M, indicating iron accumulation was a stress response (Fig. S9) [67]. Iron accumulation at the surface acts as a signal for ferric-assimilating proteins (FEA1 and FEA2) to assimilate the chelated iron for intracellular uptake via a ferrireductase-dependent reductive pathway (Fig. S9). Such an iron uptake pathway has been reported in marine microalga Chromera velia [68] and freshwater alga Chlorella sorokiniana UTEX 1602 (Tables S13 and S14). In marine and freshwater algae such as Scrippsiella trochoidea [37], Phaeodactylum tricornutum [69, 70], and Chlamydomonas reinhardtii [71], plasma membrane-bound ferrireductase reduces Fe³⁺ chelated with siderophore to bioavailable Fe²⁺ (Fig. S9, Table S14). Different reductive pathways of Fe²⁺ transportation inside cells have been reported, such as reductive multicopper ferroxidase (FOX1) in C. reinhardtii [67] or via engulfment through IRT1/2 and NRAMP4 proteins in C. reinhardtii and Ostreococcus tauri [72]. In contrast, an endocytosis-mediated non-reductive pathway in which the whole Fe³⁺-siderophore is engulfed has also been reported in a marine alga Phaeodactylum tricornutum [37, 73]. Therefore, in this work, despite a considerable increase in microalgal growth in the consortium at a high Fe concentration, the ferrireductase activity lacked any significant increase (Fig. 5B), indicating a potential shift in an iron-uptake mechanism. At higher Fe concentrations, bacteria use ferric iron via direct diffusion across the cell membrane than high-affinity iron uptake by siderophore production and iron chelation [58, 74]. Like bacteria at high iron availability, microalgae uptake iron directly by a non-reductive direct iron-uptake pathway triggered due to the difference in intracellular and extracellular iron concentration (Fig. S9) [67, 75]. Thus, the selection of reductive or nonreductive pathways depends on both the external concentration of iron and its bioavailability [70].

To further examine the effect of ferrireductase in bioremediation, microalgal cells were pretreated with 50 µM Diphenyleneiodonium (DPI), a known inhibitor of ferrireductase. DPI inhibits ferrireductase activity by preventing the transfer of an electron from ferrireductase (flavohemoproteins; Fre1) to the Fe-chelate, which obstructs the reduction of Fe³⁺ to Fe²⁺. Pretreatment of algal cells with 50 µM DPI significantly reduced the ferrireductase activity (p = 0.00) (Fig. 5D, Table S11) [75,76,77]. To further assess the linkage between ferrireductase and azo dye degradation, C. sorokiniana was treated with DPI and azoreductase activity was monitored. DPI (50 µM) treatment significantly inhibited azoreductase activities in algal cells both in the presence (Fe + DPI + ) (p = 0.008) and absence (Fe-DPI + ) (p = 0.035) of supplemented iron (Table S12). However, C. sorokiniana not pretreated with DPI (Fe-DPI-) retained significant azoreductase activity, which even increased when media was supplemented with iron (Fe + DPI-) (p = 0.012) (Fig. 5D). UPLC, FTIR, and LC-MS analyses further confirmed azoreductase-mediated degradation of AB1 dye by the microalgal-bacterial phototrophic community (Fig. 6A). In chromatograms, the peak at retention time (RT) of 2.617 corresponded to the reduction of AB1 dye. Similarly, in the FTIR analysis, the disappearance of vibrational bands at 1488 cm⁻¹ and 1282 cm⁻¹ suggested azoreductase-mediated cleavage of azo bond (-N = N-) in AB1 dye (Fig. 6B) [13]. The LC-MS confirmed the azoreductase-mediated AB1 dye degradation into 4-Nitroaniline (RT 2.7 min, m/z 138.06) and naphthalene-1,2,8-triol (RT 2.45 min, m/z 176.1) via symmetrical reductive cleavage of azo bonds (Fig. 6C, and S7, S8). Also, the presence of catechol (RT 2.3 min, m/z 110.07), a central intermediate of aerobic biodegradation of benzene derivatives, suggested mineralization of AB1 by-products via meta- or ortho-cleavage degradation pathways [78].

Azoreductases belong to oxidoreductases encompassing a diverse group of NADH or NAD(P)H cofactor-dependent flavoenzymes [18], which are involved in azo dye degradation as reported in numerous bacteria, fungi, and yeast [18, 79]. Recently, the dye decolorization potential of algae has been reported [80], but the involvement of azoreductase is not yet clear. We report a NAD(P)H mediated azoreductase activity in C. sorokiniana that is involved in azo dye degradation in completely photoautotrophic culture conditions. In our study, the ferrireductase-mediated reductive iron uptake mechanism in C. sorokiniana also influenced the azoreductase activity (Fig. 5D). A link between azoreductase and ferrireductase has previously been demonstrated in Saccharomyces cerevisiae [79]. In eukaryotes, three types of ferric reductases have been reported, i.e., NADPH oxidases (NOX; cytochrome b₅₅₈), cytochrome b₅ reductases, and cytochrome b₅₆₁ [67]. The Fre1p (Ferric reductase 1) NOX enzyme encoded by the metalloregulator ferrireductase FRE1 gene in S. cerevisiae is responsible for 80–98% extracellular ferric reduction. The authors reported that the FRE1-dependent ferric reduction, inversely regulated by extracellular iron concentration, also participated in extracellular azo dye reduction. A gene knockout study in S. cerevisiae suggested the dependency of azoreductase on ferrireductase since deletion of FRE1 gene resulted in decreased dye degradation, which was restored when S. cerevisiae mutant cells were transformed with plasmid pSP3. Such transmembrane ferric-chelate reductases (FRE1) have also been reported from green microalga Chlamydomonas reinhardtii [71], which share high sequence similarity with ferric-chelate reductase protein from another microalga C. sorokiniana UTEX 1602 (Fig. S10, Table S13 and S14).

Although iron capture and transport mechanisms have been investigated in several single-cell eukaryotes [67, 70], the role of iron bioavailability in biotechnological applications has been largely unexplored. We demonstrate that when C. sorokiniana cells were treated with ferrireductase inhibitor DPI, the azoreductase-mediated dye degradation was inhibited (Fig. 5D). Since both enzymes function on an externally directed plasma membrane redox system, we propose that the ferrireductase-mediated reductive iron uptake mechanism in C. sorokiniana is also vital for azo dye degradation (Table S13). Like azoreductase, the ferric reductase pathway uses cytosolic NAD(P)H as an electron donor to transfer an electron to Fe³⁺ in ligand-bound complexes to release bioavailable Fe²⁺ [67]. Thus, highly diverse ferrireductase in algae can also reduce azo dyes externally. Additionally, iron acts as a redox catalyst in algae and mediates the electron transport reactions. Iron limitation can suppress the photosynthetic electron transfer in algae, reducing NAD(P)H formation [81]. Since the azoreductase mechanism is also a redox reaction mediated by the transport of electrons via NAD(P)H, thus, the reduced dye degradation activity, as observed in our study, could be due to the suppressed electron transport mechanism because of low iron. In contrast, the siderophore-mediated increase in iron bioavailability may have led to an increase in an electron transport mechanism and, consequently, azo reduction. However, further investigations using radiolabeling, omics, and expression-based studies of the oxidoreductases enzymatic machinery in Chlorella reveal the exact role of extracellular iron concentration in driving iron uptake via ferrireductase and dye degradation. Unlike bacteria, azoreductase in algae is relatively unexplored and requires further investigation.

A poor understanding of the extracellular environment, especially the role of micronutrients, has posed a major limitation in the industrial translation of single-species bioremediation designs. A synthetic community of algae and bacteria can be employed to understand several environmental interactions and overcome the challenges of conventional bioremediation designs. This work demonstrated that extracellular iron concentration influenced the ferrireductase-mediated iron uptake in C. sorokiniana, which also affected the dye reduction pathway. Bacterial siderophores have a major influence on iron cycling and shaping phototrophic communities in aquatic ecosystems [45]. In the high-nutrient, low-carbon (HNLC) regions of open oceans, bacterial siderophores have been known to alleviate iron limitation, which benefits algae [69, 82]. Similarly, regulation of iron bioavailability by bacterial siderophores in industrial setups can be used as a strategy to enhance algal growth and enzymatic activities. Synthetic algal-bacterial community designs can be replicated in various wastewater treatment processes or industrial bioprocessing setups characterized by iron and carbon limitations. Algal-bacterial consortium has been used to produce high-value bioproducts like lipids, proteins, vitamins, etc. [83], suggesting the benefit of synthetic community designs over single-species designs. Consortium design has been suggested to improve the growth of algal biomass and bring down operational costs [84]. Algal-bacterial designs will also help overcome the requirement of external chelators, such as EDTA.

Besides iron, external oxygen concentration also influences dye degradation, especially in conventional bacterial processes used in the textile industry. Azoreductase-mediated dye degradation in bacteria is a two-step redox process [13]. The primary reduction step requires oxygen-limiting conditions to reduce azo dyes via the azoreductase pathway. Following this, the secondary oxidation step requires well-oxygenated conditions to degrade by-products via oxygenases enzymes. Unlike bacteria, Chlorella can modify extracellular oxygen concentration in contrasting dark and light cycles to accelerate dye degradation [29]. It has been reported that dark cycles due to oxygen deprivation favor azoreductase, and light cycles due to oxygenated conditions favor degradation of dye by-products. Oxygenation by Chlorella also favors bacterial growth and ensures BOD/ COD reduction [85]. In closed photobioreactor-based designs, several studies on algal-bacterial respiration, COD removal, and nitrification have already highlighted the potential of such inter-kingdom symbionts in replacing monoculture processes in the treatment of industrial wastewater [85]. However, in an open system design, the stability of such symbionts can be challenged by co-existing microbes competing for resources. The stability of synthetic community designs can be ensured by investigating the ecological dynamics of phototrophic communities and identifying key factors responsible for associations, such as a preferred monosaccharide, chemotactic and signaling molecules, quorum sensing, vitamins, and C/N ratio [86, 87]. Regulation and monitoring of such key factors in a bioremediation setup will ensure an exclusive metabolic niche of the synthetic community and provide tolerance to environmental perturbation. Therefore, in addition to iron and carbon-assisted mutualism in C. sorokiniana and R. pickettii, further analysis of the underlying factors influencing microbial interactions would determine the stability of the synthetic community. The use of the systems biology approach would also enhance our understanding of abiotic and biotic interactions for designing synthetic communities for specific biotechnological applications [88].

In conclusion, the phototrophic community of Chlorella sorokiniana and Ralstonia pickettii represents a mutualistic association based on the exchange of specific limiting nutrients (Fig. 7). Our results form a basis for investigating a barter system between algae and bacteria, relying on an iron exchange from R. pickettii to C. sorokiniana and dissolved organic matter from C. sorokiniana to R. pickettii. Under iron stress, bacterial siderophore ensures iron availability for the algal partners, promoting the algal growth rate and potential to degrade dye. Therefore, the bacterial-algal association has the potential to treat industrial wastewater having carbon and iron limitation. We report that the transmembrane ferrireductase activity in C. sorokiniana plays a crucial role in the reductive iron-uptake mechanism that triggers azoreductase activity. Bioavailable iron regulates the activity of both oxidoreductase enzymes, enhancing dye degradation by Chlorella. Therefore, a microalgal-bacterial consortium working under photoautotrophic conditions could provide a self-sustainable alternative to current monoculture remediation processes. It would be worth investigating whether increased iron bioavailability also improves the ability of the microalgal-bacterial consortium to remediate other organic pollutants, which require extracellular reductive cleavage.

Source: Ecology - nature.com