The Cd tolerance of an E. mutabilis natural co-culture with unknown fungi and bacteria was investigated. The culture bank from which the E. mutabilis isolate was obtained had been unable to create an axenic E. mutabilis culture from any isolate. Therefore, we used an axenic E. gracilis culture as a control to determine the comparative impact of treatments on the E. mutabilis co-culture. E. gracilis is the model euglenoid and has been the focus of most the antibiotic and HM experiments using a euglenoid [48, 49, 54, 55]. Recent investigation revealed E. mutabilis had greater tolerance to Cd than E. gracilis, and that it responded differently to pre-growth under nutritional conditions [56]. The current study is the first assessment of the impact of antibiotics on E. mutabilis. Rifampicin had a very toxic effect on both Euglena species in the presence and absence of CdCl2. In all other antibiotic or antimycotic treatments, the combined exposure to CdCl2 significantly decreased the number of viable E. mutabilis cells (Fig. 1). In contrast, the impact of the combined treatments on E. gracilis was either not significant or the reduction was substantially less than noted for E. mutabilis (Fig. 1). The assessment of antibiotic and CdCl2 by determining chlorophyll concentrations was consistent between Euglena species showing that cellular chlorophyll content generally increases after cells have been exposed to CdCl2 - with the exceptions of rifampicin and chloramphenicol where results varied (Fig. 2). The viability of E. mutabilis growing heterotrophically after antibiotic and CdCl2 treatments was substantially decreased, whereas the decrease in E. gracilis was lower or non-existent. However, the fungus in E. mutabilis cultures remained viable and grew to cover the plates after these treatments (Table 1). Since fungal viability was significantly less following phototrophic growth of the culture, it is possible that suppression of bacterial growth enabled the fungus to survive and proliferate. Combined with the negative impact on CdCl2 tolerance in E. mutabilis following amphotericin B treatment, these results reveal several combinatorial interactions between the bacteria and E. mutabilis, the bacteria and the fungus, and the fungus and E. mutabilis. Consistent with synthetic co-culture work [57, 58, 59, 60] these results indicate that the fungus and bacteria assist in the resistance of E. mutabilis to Cd. Further, they reveal multiple organismal interactions influence the response of the co-culture to HM challenge. This knowledge provides the necessary base for subsequent investigations into the mechanisms fungal-E. mutabilis-bacterial consortia resistance to HMs and provides a model for FAB consortia resistance to HMs.
Current methods for treating waterbodies that have been polluted with HMs include neutralization processes, chemical precipitation, coagulation/flocculation, and adsorption; however, these processes suffer from high costs, inefficient HM removal, and may produce secondary pollution [28, 61, 63, 63]. As a result, biotechnological methods are being employed to enhance the bioremediation potential of microbes [59, 64, 65, 66, 67, 68]. Several algae have been investigated for bioremediation of textiles, organic pollutants, and HMs [69, 70, 71, 72], and recent studies have suggested that co-culturing algae with bacteria or fungi provides benefits that include increased flocculation efficiency, increased biomass, independent nutrient exchange, and enhanced tolerance to extreme environments [7, 67, 73, 74, 75, 76]. The model euglenoid E. gracilis has also been co-cultured with other microbes [21, 77], however the discoveries here show that E. mutabilis and its naturally associated microbial partners offer substantial insight into how organisms interact in a co-culture and provides a model to use for developing enhanced technological applications and potential use in bioremediation.
Co-culturing algae with bacteria or fungi has several advantages, including enhanced nutrient exchange or protection factors for the alga [2, 4, 78]. It is widely known that many algae rely on exogenous vitamin B12 (cobalamin) and nitrogen, and that these can be supplied by bacteria [2, 79, 80] in exchange for the bacteria receiving a carbon source from the algae [81, 82]. As examples, the growth of marine diatom Ditylum brightwellii in nitrogen limiting media and a symbiotic bacterium in carbon limited media are both lower relative to co-cultures [83]. Similarly, an investigation of a symbiotic culture of Chlorella vulgaris and Bacillus subtilis revealed that co-culturing resulted in increased growth, photosynthetic activity, carbon fixation, and vitamin B12 content of the alga [84]. Alternatively, fungi that are cultured with algae provide nutrients to the alga as well as mechanisms for protection [78, 85, 86]. When Chlamydomonas reindhardtii was cultured with Aspergillus nidulans and exposed to the algicide azalomycin F, algal cells grew within fungal hyphae avoiding contact with the algicide [78]. Additionally, the presence of azalomycin F prompted A. nidulans to produce polar lipids which attract the algicide and effectively neutralize it [78]. Fungi also provide protection from reactive oxygen species (ROS) generated during HM exposure and produce extracellular polymeric substances (EPS) and organic acids that can bind HMs reducing their toxicity when co-cultured with algae [87, 88, 89]. Our study found that suppression of constituent bacterial and fungal organisms naturally associated in the E. mutabilis ‘FAB’ co-culture led to significantly lower numbers of viable Euglena cells after CdCl2 exposure. This is consistent with previous studies showing the importance of co-cultured organisms with algae; however, we did not have to screen for compatible interactions since we had a naturally compatible and efficient association that evolved in a toxic AMD environment. Further, the majority of research has used cultures of only two organisms: algae and bacteria or algae and fungi. Apart from lichen studies, FAB consortia have rarely been investigated for use in bioremediation despite reports of this tripartite interaction being considered a self-sustaining and cost-conscious system that exhibits superior HM removal efficiency [58]. Our results underscore the innate interaction of FAB co-cultures in extreme environments and establishes a foundation for investigating the association between natural FABs to inform future developments in biotechnology.
Although we observed a decrease in the number of viable Euglena cells following treatment with antibiotics and CdCl2, there was an increase in chlorophyll production by Euglena in the presence of Cd (Fig. 2). This was unexpected because Cd is known to disrupt physiological and metabolic processes of phototrophic organisms including algae, cyanobacteria, and plants primarily by reducing photosynthetic rate and chlorophyll concentration [90, 91, 92, 93, 94]. Both E. mutabilis and E. gracilis display significantly greater chlorophyll per 100,000 cells under Cd exposure. Furthermore, the amount of chlorophyll being produced in the presence of Cd appears unaffected by the addition of most antibiotics suggesting that the impact does not involve the associated bacteria. This phenomenon has been reported in higher plants that have taken up Cd from their surroundings [95, 96]. The structural foundation of a chlorophyll molecule is Mg; however, Mg is also essential for several other metabolic processes including enzyme activation, sucrose transport, and energy metabolism [97]. It has been shown that Cd can replace Mg in the central position of the chlorophyll molecule [95]. Further, the Cd hyperaccumulator Sedum alfredii demonstrated no noticeable reduction in photosynthetic activity and simultaneous increase in chlorophyll content following Cd treatments, which negatively impacted leaf and root growth [96]. A similar result was observed in Chlamydomonas reinhardtii under excess Cu exposure where the correlation between increased chlorophyll and cell survivability suggested to be the result of chlorophyll accumulation in cells that do not divide [98]. Here we showed that Cd treatment of Euglena led to a reduction in cell viability and an increase in the amount of chlorophyll produced per cell. This could indicate that although exposure to divalent HMs debilitates overall culture health, Cd may be able to replace the Mg in chlorophyll leaving a less efficient but more plentiful photosynthetic apparatus, which, although less effective, still allows the Euglena to survive some HM toxicity.
Despite the increasing chlorophyll content per cell, E. mutabilis is unable to recover from antibiotic and Cd exposure during subsequent heterotrophic growth (Table 1). In contrast the fungi and bacteria in the co-culture adapt to the switch to heterotrophic growth and often grow to take over the entire plate. The effects of antibiotics on E. gracilis have been extensively studied [48, 50, 52, 53, 99, 100] and the results here are consitent with previous findings; however, there has been no work on the impact of antibiotics on E. mutabilis. Based on CFU comparisons, E. mutabilis recovers after antibiotic exposure better than E. gracilis, but E. gracilis shows better recovery following treatment with antibiotic and Cd (Table 1). This is notable because a synergistic effect can occur where toxicity is increased through the formation of antibiotic and di-valent HM complexes [101, 102]. The increased chlorophyll content in the presence of Cd suggests that both Euglena species are capable of generating energy and surviving the treatment. Then, upon transfer to nutrient rich media, they could both shift to heterotrophic growth and employ major facilitator superfamily (MFS) transporters, transmembrane (TrkA) transporters, HM pumps (P1 B ATPase) and other means to recover from the Cd stress [103, 104]. We postulate that while both Euglena species can recover, the recovery by the fungi and bacteria co-cultured with E. mutabilis occurs more quickly than that of E. mutabilis; consequently, substantial fungal and bacterial growth in heterotrophic conditions results in fewer E. mutabilis colonies being formed. Related to this are the observations that fungal growth is only visible following heterotrophic growth after antibiotic exposure; whereas, with antibiotic treatment under the phototrophic growth in MAM at pH 4.3, the fungus is not even microscopically observable. It is only upon introduction to nutrient rich media following antibiotic or Cd treatment that fungal colonies can be observed. This may indicate that bacteria interact with, or rely on, the fungus. Our results show that the number of fungal CFUs decreases with the concentration of antibiotics (Table S3) suggesting that the fungal-bacterial interaction is not simply a matter of presence or absence. In heterotrophic cultures containing fungi and microalgae, a similar over-growth by the fungus was observed [105, 106, 107, 108]. Together these results suggest that co-cultures, which lack a readily available carbon source, require a specific light regimen and a specific organism ratio to ensure the fungus does not overwhelm the photobiont.
Sequence analysis of DNA extracted from the E. mutabilis FAB co-culture was used to identify the prominent constituent organisms in the co-culture. The fungus was determined to belong to the genus Talaromyces, and with a possible species identification being T. amestolkiae, while the bacteria were determined to belong to the genus Acidiphilium, and with a probable species identification being A. acidophilum (Fig. 3). Consistent with the fungus belonging to Talaromyces was the observation of yellow and red fungal colonies on PDA plates following amphotericin B and cycloheximide exposure (Figure S3) [109]. Sequence analysis also detected a low-level sequence with similarity to Exophiala oligosperma; however, this may have been an artifact since E. oligosperma colonies are black in colour and there is no evidence that E. oligosperma can tolerate HMs [110, 111]. Talaromyces sp., on the other hand, have been characterized by their ability to produce different coloured pigments based on its carbon source and in response to stress or predators [109, 112, 113]. Furthermore, they have been isolated from HM polluted areas, and can withstand high concentrations of Cr, As, Pb, Ni, Cu, and Cd [114, 115, 116]. Red pigment is produced by fungi that are susceptible to cycloheximide and carry the recessive ade2 gene which, when repressed, results in red pigmentation from the accumulation of phosphoribosylaminoimidazole, an intermediate in the biosynthesis of adenine [117, 118, 119, 120, 121]. The yellow pigmentation following amphotericin B and cycloheximide exposure is most likely comprised of products of the azaphilone family, namely mitorubrinol and mitorubrinic acid [109, 122, 123]. These compounds are found in numerous Talaromyces sp. and are proposed virulence factors regulated by polyketide synthesis genes pks11 and pks12 which become activated under stress [109, 122, 123, 124]. This cumulative information, in combination with confirmatory Sanger sequencing results, indicates that the predominant fungus present in the E. mutabilis co-culture is a Talaromyces sp. Furthermore, the production of pigment by Talaromyces sp only when stressed or acting as a pathogen indicates that the lack of colour observed during control co-culturing conditions suggested the fungus is neither acting as a pathogen nor stressed. This would be consistent with it having a positive role in the FAB co-culture. Talaromyces sp. are known to produce a plant growth promoting hormone, indole-3-acetic acid (IAA), and they display tolerance to Cd when growing in soil [125]. As such, when this fungus was cultured with Arabidopsis thaliana it reduced the amount of Cd in the soil and underground plant tissues, and increased plant growth. It was postulated that the fungus promoted nutrient uptake and IAA production to promote plant development [126] and provided protection by increasing the essential nutrient bioavailability under low Cd concentrations, thereby effectively diluting the presence of Cd and enhancing plant HM tolerance [125]. These mechanisms are prevalent during other plant-fungal interactions [127, 128, 129] and may be fundamental to stress response in algal-fungal symbiosis [60, 87, 129, 130] as well as being present in the Euglena-Talaromyces interaction.
Finding that the predominant bacterial species in the FAB co-culture is an Acidiphilium species is consistent with this species being found in acidic environments with high HM concentrations [131, 132, 133]. A. acidophilum could act as a nutrient source for other organisms in the FAB co-culture as it is a facultative, sulfur-reducing mixotroph [134, 135]; however, there is only evidence of heterotrophic growth in AMD [136, 137]. When growing heterotrophically the main energy source for A. acidophilum is ferrous iron, which it can reduce to generate a usable form [135, 137] and is abundant in AMD (28). The characteristics of Talaromyces sp. and A. acidophilum are consistent with their persistence in the FAB co-culture and suggest they have a role in the stress protection of, and nutrient exchange with, E. mutabilis.