Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Bioremediation is the capacity of organisms to degrade contaminants. Here, we describe methodologies to isolate and test microbes presenting bioremediation ability and potential probiotic characteristics for corals.
Pollution affects all biomes. Marine environments have been particularly impacted, especially coral reefs, one of the most sensitive ecosystems on Earth. Globally, 4.5 billion people are economically dependent on the sea, where most of their livelihood is provided by coral reefs. Corals are of great importance and therefore their extinction leads to catastrophic consequences. There are several possible solutions to remediate marine pollutants and local contamination, including bioremediation. Bioremediation is the capacity of organisms to degrade contaminants. The approach presents several advantages, such as sustainability, relatively low cost, and the fact that it can be applied in different ecosystems, causing minimal impacts to the environment. As an extra advantage, the manipulation of endogenous microbiomes, including putative beneficial microorganisms for corals (pBMCs), may have probiotic effects for marine animals. In this context, the use of the two approaches, bioremediation and pBMC inoculation combined, could be promising. This strategy would promote the degradation of specific pollutants that can be harmful to corals and other metaorganisms while also increasing host resistance and resilience to deal with pollution and other threats. This method focuses on the selection of pBMCs to degrade two contaminants: the synthetic estrogen 17a-ethinylestradiol (EE2) and crude oil. Both have been reported to negatively impact marine animals, including corals, and humans. The protocol describes how to isolate and test bacteria capable of degrading the specific contaminants, followed by a description of how to detect some putative beneficial characteristics of these associated microbes to their coral host. The methodologies described here are relatively cheap, easy to perform, and highly adaptable. Almost any kind of soluble target compound can be used instead of EE2 and oil.
Pollution is a major issue affecting human, animal, and plant health worldwide. Although pollution can be natural, such as volcanic ashes1, human activities are the primary cause of most pollution. Anthropogenic activities are contaminating soil, water, and air, which directly or indirectly lead to almost 20 million premature human deaths2 and decimate billions of other forms of life annually. Pollutants are present even in the most remote areas of the planet. For instance, heavy metals and persistent organic compounds have been detected in deep sea invertebrates and polar mammals, respectively3,4.
Marine environments have been especially impacted by pollution. For a long time, it was assumed that the ocean would remain unaffected and supply an endless source of goods because of its massive volume of water5. For this reason, all types of industry and institutions freely released waste in water bodies for centuries6,7. Several contaminants of all types, such as plastic8, synthetic hormones9, pesticides10, oil11, nutrients12, heavy metals3, and radioactive waste13 have been reported as impacting ocean ecosystems. In this context, coral reefs are among the most important and sensitive ecosystems in marine environments14. Reefs are coastal protectors, crucial to the development of thousands of marine species by playing essential roles in nutrients cycling and climate control. Reefs also contribute to the economy by providing fish, goods, and tourism, among others15. For instance, 4.5 billion people depend on ocean fish as their main food source16, which are greatly supported by coral reefs.
Regardless of their ecological, social, and economic importance, coral reefs are being decimated17,18. Anthropogenic activities are primarily responsible for contributing to the three main causes of corals’ death: climate change, overfishing, and water pollution19. Even though it is important to work on the mitigation of global warming, it is also important to work on minimizing local contamination, including water pollution, that can critically contribute to coral decline20. Thus, there is an urgent need for the development of strategies to increase corals’ lifetime, which could provide them with extra time to adapt and survive.
In this regard, it is extremely important to find solutions to minimize contamination and to develop strategies to increase the fitness of corals. Strategies to remediate marine pollutants are highly diverse and can be grouped into physical, chemical, and biological approaches. Physical approaches are helpful. However, they are not always efficient. For instance, plastic waste can be minimized by physical removal, while water-soluble compounds need other methodologies to be eliminated. Examples of such compounds are crude oil, released by oil industry activities and spills, as well as other micropollutants, such as synthetic hormones, normally used as the estrogenic component in oral contraceptives and present in sewage21,22. The use of chemical substances to decrease contamination can solve a specific problem, but it may also represent an extra source of pollution. This is the case with chemical dispersants to mitigate oil contamination, which have been described as even more toxic to marine ecosystems than the oil contamination itself23. For these reasons, biological approaches present several advantages when compared to the other methods. Bioremediation is the capacity of living organisms, or their metabolic products, to transform contaminants into less toxic or non-toxic forms24. The main advantages of using biological methods are sustainability, relative low cost, the fact they are ecologically friendly, and that they can be applied in different ecosystems, causing minimal or fewer impacts to the environment21,25,26,27.
Additionally, the manipulation of the microbial community present in an environment allows an extra potential advantage. There are microbiomes that are associated with hosts and are essential to their health. It is well known that these associated symbiotic microbiomes are necessary to maintain host homeostasis19. The manipulation of these associated microorganisms has been well explored for hosts such as plants and mammals28,29, but the use of coral probiotics is still novel15. Corals also host, interact with, and depend on large and specific populations of microorganisms to survive19. The role of these microbial communities in the health and dysbiosis of corals is under active study, but it is still far from being fully understood30. One of the most popular hypotheses is called the coral probiotic hypothesis. It suggests the existence of a dynamic relationship between symbiotic microorganisms and environmental conditions which brings about the selection of the most advantageous coral metaorganisms31. Based on this information, key potential probiotic mechanisms, as well as the strategies for isolation, manipulation, and delivery of beneficial microorganisms for corals (BMCs) for several purposes, were proposed32 and tested33. These potential beneficial characteristics include resistance to temperature increase, protection from reactive oxygen species (ROS), nitrogen fixation, resistance to contaminants, and biological control against pathogens, among others32.
This study focuses on the selection of BMCs and free-living microorganisms presenting the ability to degrade two contaminants commonly found in marine environments: the synthetic estrogen 17a-ethinylestradiol (EE2) and crude oil. Pollutants containing hormone active agents are often present in water bodies34,35,36,37,38,39,40,41,42. Among them, synthetic estrogenic endocrine-disruptor compounds (EDCs) mimic the action of estrogens on target cells, causing several impacts on animals, including breast cancer, infertility, and hermaphroditism9. EE2 is excreted by humans because of the use of oral contraceptives. It is not removed from sewage by traditional wastewater-treatment plants and has negative effects even at very low concentrations (e.g., ng/L or µg/L)43,44,45. Little is known about the effects of estrogens on coral physiology46,47. However, on other marine invertebrates, such as sponges, crustaceans, and mollusks, estrogens were reported to cause several negative effects mainly related to reproduction, such as development and/or stimulation of gametes, alteration in enzymatic and protein actions, problems in embryonic processes, and others48,49,50,51,52. The negative consequences caused by EE2 contamination highlight the necessity to develop sustainable approaches to remove this compound from the environment without impacting marine life.
In parallel, with oil currently accounting for almost 40% of the world’s consumed energy sources53, chronic contamination and oil spills often occur near reef areas11. Oil contamination was reported to cause negative effects in several species of marine animals, birds, plants, and humans54,55,56,57. On corals, it causes bleaching, reduces the resistance of larvae to thermal stress58, disrupts the microbial associated communities21, and causes tissue necrosis. In addition, chemical dispersants, an oil remediation technique commonly used by oil companies to remediate spills, are even more toxic to corals than the oil itself23. Beneficial microorganisms isolated from corals, in contrast, are known for playing crucial roles on host health. However, the manipulation of these potential probiotics must be better explored in order to investigate possible negative side effects and the metabolic capacities that can be screened to improve the fitness of the metaorganism. In this context, characteristics such as the antimicrobial activity against coral pathogens, the production of catalase to fight oxidative stress, the ability to degrade urea (which may have important roles in the calcification process), and the presence of genes that confer potential beneficial characteristics, among others, must be the focus of investigation. Here, we show how bioremediation and probiotics can be used to concomitantly mitigate the impacts of pollution and enhance coral health. The development of innovative approaches that can be used as interventions to increase marine species persistence represent a step towards a more sustainable and healthier planet.
1. Water and coral collection and storage for microbial isolation
NOTE: It is essential to take the coordinates and temperature of the sampling sites. If possible, metadata such as salinity, pH, depth, and light intensity can also help in finding fine-tuned cultivation approaches and future interpretation of data. For reliable results, keep the samples stored for the minimum length of time possible. The water/coral microbiomes may change considerably if the samples are not kept at the right temperature and/or are stored for long periods. If the isolation step is not performed instantly after collection, it is crucial to maintain samples at 4 °C until processing. The longer the samples are stored, even at 4 °C, the more the microbial community will change.
2. Isolation of EE2-degrading bacteria from seawater and/or corals
3. Isolation of oil-degrading bacteria from seawater and/or corals
4. Consortium member selection
5. Detection of putative beneficial characteristics for corals
Based on the methods described here, it was possible to isolate microorganisms from different water sources and coral nubbins presenting putative BMC characteristics and capable to degrade different classes of contaminants (Figure 1). Using water samples collected at a sewage treatment plant, obtained from CESA-UFRJ (Experimental Center of Environmental Sanitation of the Federal University of Rio de Janeiro), and based on the procedure presented here, 33 bacterial strains able to degrade EE2 at a final concentration of 5mg/L were isolated (Figure 2A). Additionally, using the technique for selecting oil-degrading bacteria, 20 strains able to degrade both oWSF (Figure 2B) and oWIF (Figure 2C) were isolated.
Putative BMC characteristics were screened in microorganisms isolated from different coral species under diverse conditions. Among them, a strain presenting strong antagonistic activity against the coral pathogen Vibrio coralliilyticus (Figure 3A), strains able to degrade urea (Figure 3B), a good catalase producer (Figure 3C), and microorganisms presenting potentially beneficial genes (Figure 3D) were found.
Employing the two approaches combined (i.e., bioremediation and BMC inoculation), it was possible to protect corals from oil exposure impacts. For this, an oil bioremediator pBMC consortium, isolated from the coral Mussismilia harttii, was inoculated on coral nubbins exposed to 1% oil in triplicates21. The treatments exposed to oil presented a progressive decrease in Fv/Fm from the fourth day onwards, reaching values close to zero by the tenth day. Variable fluorescence/maximum fluorescence (Fv/Fm) provided a measure of maximal photosystem II (PSII) photochemical efficiency of the zooxanthellae, representing an indirect measurement of coral health. On the other hand, coral nubbins present in the aquariums inoculated with the consortium showed a better-preserved photochemical ability (Figure 4).
Figure 1: Summary of the main steps of a bioremediator-pBMC consortium selection and assembly. Scheme of pollutant-degrading microorganisms selection steps (in gray) and final steps used for the consortium microbial selection (DNA sequencing, growth curve, antagonism test, and consortium assembly in red). Please click here to view a larger version of this figure.
Figure 2: Selection of pollutant-degrading bacteria. (A) Bacterial isolates growing on minimum media plates containing EE2 as the only carbon source. (B) Bacteria colonies growing on minimum media plates containing oWSF as the only carbon source. (C) Bacteria colonies growing on minimum media plates containing oWIF as the only carbon source. Please click here to view a larger version of this figure.
Figure 3: Detection of pBMC characteristics. (A) Spots in triplicates of strain presenting antagonistic activity against the coral pathogen Vibrio coralliilyticus (in black) and a control strain (in green). (B) Strains growing on media containing urea as the only carbon source. (C) Strain producing catalase (+) and a bad catalase producer strain (-). (D) Example of PCR detection of the nirK gene (lane 1 = 1kb ladder; lane 2 = blank DNA extraction negative control; lane 3 = nirK detection; lane 4 = PCR reactions without template DNA). Please click here to view a larger version of this figure.
Figure 4: Fv/Fm measurements of M. harttii nubbins dark-adapted at 5 PM, using a diving-PAM chlorophyll fluorometer. Fv/Fm measurements of the treatments control consortium, oil, and oil with consortium were performed in triplicates every day for 10 days. Standard deviation is shown. Features of the graph were modified with permission from previous results21, available at https://www.nature.com/articles/srep18268 under a Creative Commons Attribution 4.0. Full terms at http://creativecommons.org/licenses/by/4.0/. Please click here to view a larger version of this figure.
MOA | Microorganism | Detection technique | References |
Climate regulation; surfur cycling; antimicrobial compounds; increase antioxidant protection of cells. | Aspergillus sydowii | PCR for dddP gene | 81 |
Pseudovibrio sp. P12 | Culture medium with DMSP | 82 | |
Pseudoalteromonas sp. | PCR for dmdA gene | 33 | |
Biological regulation of pathogens. | Microbiome from Acropora palmata mucus | Clear zone of inhibition | 83 |
Extracts from corals | Growth inhibition assay | 84 | |
Marinobacter sp. | Swarming assays | 85 | |
Pseudoalteromonas sp. | Agar plate cross-streaking | 86 | |
Pseudoalteromonas sp. | Agar-diffusion method | 33 | |
Benefit to the calcification process; source of nitrogen for scleractinian corals. | Symbiodinium spp. | Colorimetric method | 87 |
Microbiome from Stylophora pistillata mucus | ___ | 88 | |
Microbiome from Acropora alciminata | Method by Bolland et al.80 | 89 | |
Nitrogen cycle; increase nitrogen fixation. | Microbial community | qPCR | 90 |
Microbial community | PCR | 91 | |
Microbial community | qPCR | 92 | |
Microbial community | Adapted acetylene (C2H2) reduction technique | 93 | |
Pseudoalteromonas sp. and Halomonas taeanensis | PCR | 33 | |
Nitrogen cycle; decrease of the ammonium concentration. | Pseudoalteromonas sp. | PCR | 33 |
Microbiome from Tubastraea coccinea | PCR | 94 | |
Microbiome from Xestospongia testudinaria | Predictive metagenome analysis | 95 | |
Holobiont protection against reactive oxygen species (ROS). | Pseudoalteromonas sp., Cobetia marina and Halomonas taeanensis | Catalase test | 33 |
Symbiodinium spp. | Amplex red | 96 | |
Vibrio pelagius and Sync- chococcus sp. | Horseradish peroxidase-scopoletin method | 97 | |
Vibrio fischeri | Multiple methods | 98 |
Table 1: Detection of putative BMC characteristics, mechanism of action (MOA), reported microorganisms presenting the potential and technique used to detect the characteristic.
Bioremediation approaches have been massively explored over the past 50 years. For instance, over 200 microbes among bacteria, cyanobacteria, microalgae, and fungi in several different habitats, have been designated as able to indicate the presence and/or degrade oil hydrocarbons62,63,64. Additionally, other classes of compounds that cause impacts to the environment and to humans, such as plastic, bisphenol A, endocrine disruptors, and heavy metals, are targets for bioremediation technique development65,66,67. On the other hand, marine probiotic development has been limited to the fields that have an obvious impact on the economy, such as fish probiotics in aquaculture68,69. However, isolation and characterization of beneficial microorganisms to protect coral reefs, marine ecosystems that support fishery, tourism, and other profitable activities, are starting to be valued15. Here, a cheap, easy, and accessible protocol to select pollutant-degrading microorganisms that can also present potentially beneficial characteristics to local marine ecosystems, especially putative beneficial microorganisms to corals (pBMCs), is described.
Additionally, the method demonstrated here is highly adaptable to several compounds and diverse types of microbial sources. It is possible to target different pollutants by replacing the only carbon source added to the minimum media. For this, instead of oil or EE2, other compounds should be added at the desired concentration. This would be the selective pressure to isolate degraders for the targeted pollutants. For instance, microorganisms capable of degrading other classes of endocrine disruptors have been already selected and tested using the same methodology70. Moreover, other marine and terrestrial organisms, such as sponges and plants71,72, as well as distinct types of environmental samples, such as soil, fuel, and rocks can be used as the degrading-microbial sources25,73,74. For instance, it was possible to detect and isolate hydrocarbon-degrading bacteria from different soil and sediment samples25,54,63,64,75. Finally, performing slight modifications in the media, microorganisms other than bacteria can be easily selected as the degrading-microbes. For instance, a microalgae strain with the ability to efficiently degrade estrogen compounds has been reported76.
Ideally, bioremediation-probiotic consortia must be assembled for each specific compound or area. Microbes that grow in a specific environment may not grow as well in new sites compared to their native conditions. Because researchers have not found a product that can be efficiently applied under all different environmental conditions, new consortia assembly should be performed for each specific situation. This would be akin to personalized medicine for environment-tailored recovery. For this reason, the creation of a central bank of microbial strains with potential probiotic characteristics and degradation capacity is a crucial step for the progress of this field. This initiative would save time and work, contributing to the assembly of new specific consortia worldwide.
Microorganisms associated with corals (i.e., microalgae, bacteria, archaea, fungi, and viruses) have a complex and intricate role in maintaining host homeostasis19. Environmental stressors, such as pollution, can also destabilize the coral microbiome, resulting in dysbiosis, which may cause disease and mortality30. The mechanisms by which the coral microbiome may support coral health are starting to be revealed. These mechanisms are the key to understanding coral resistance and resilience to environmental stressors and, consequently, to promote reef persistence and preservation. Additionally, findings in the field will help to understand general host-microbiome interactions, which may contribute to the development of better probiotics and health-promoting strategies in other areas. It is also important to better investigate how these probiotics inoculations can interfere on the metaorganism's health during stress events. For instance, work showing that the augmentation in coral performance is due to the probiotics and not simply the coral using bacteria as a food source are still needed.
In parallel, the development of new consortia delivery approaches and the improvement of the existing ones are of great importance. Alternative methods for consortium immobilization as well as innovative approaches, such as inoculating coral food (i.e., artemia and rotifers) and using them as vectors, are promising. These delivery systems can also be modified to target other marine organisms and will be essential to the success of the marine probiotics field.
Pollution mitigation and coral reef persistence are currently two of the main topics highlighted in environmental conferences regularly. The Agenda 2030, a document published by the United Nations that describes the global goals society should reach to allow a sustainable future, dedicates specific goals for each issue. While Goal 6 highlights the importance of water quality improvement by reducing pollution, Goal 14 reinforces the relevance of conservation and sustainable use of the oceans, seas, and marine resources77. In this context, coral reef conservation depends on changes that should be achieved in the near future, including pollution mitigation. This is of great importance, because most massive coral losses occurred when other factors were added to climate events, such as local habitat destruction and contamination78,79. This paper demonstrated that it is possible to combine bioremediation and pBMC inoculation to degrade specific pollutants, while it may increase coral's resistance and resilience to deal with pollutants and other issues. The optimization of existent protocols and/or the development of innovative methods, combined or independently applied, will be crucial to determine the future of marine ecosystems.
The authors have nothing to disclose.
This research was carried out in association with the ongoing R&D project registered as ANP 21005-4, “PROBIO-DEEP – Survey of potential impacts caused by oil and gas exploration on deep-sea marine holobionts and selection of potential bioindicators and bioremediation processes for these ecosystems” (UFRJ / Shell Brasil / ANP) – “PROBIO-DEEP – Levantamento de potenciais impactos causados pela exploração de óleo e gás em holobiontes marinhos em mar profundo e seleção de potenciais bioindicadores e processos biorremediadores para esses ecossistemas”, sponsored by Shell Brasil under the ANP R&D levy as “Compromisso de Investimentos com Pesquisa e Desenvolvimento. The authors also thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for financial support, and to Camila Messias, Phillipe Rosado, and Henrique Fragoso dos Santos, for the images provided.
500 mL PYREX Media Storage Bottle | thomas scientific/Corning | 1743E20/1395-500 | Used to sample water. |
500 mL Aspirator Bottles | thomas scientific/Corning | 1234B28/1220-2X | Used to separate the oil fractions. |
6-inch wire cutter plier | thomas scientific/Restek | 1173Y64/23033 | Used to cut coral fragments. |
17a-Ethinylestradiol | LGC Standards | DRE-C13245100 | Used as the only carbon source to make the selective media. |
Agar | Himedia | PCT0901-1KG | Used to make solid media. |
Bushnell Haas Broth | Himedia | M350-500G | Used as minimum media to be supplemented with carbon sources. |
Erlenmeyer Flask | thomas scientific/DWK Life Sciences (Kimble) | 4882H35/26500-125 | Used to incubate coral macerate with glass beads. |
GFX PCR DNA and Gel Band Purification kit | GE Healthcare | 28903470 | Used to purify PCR products before sending them for sequencing. |
Glass Beads | MP Biomedicals | 1177Q81/07DP1070 | Used to detach the microorganisms from coral structures. |
Laminar Flow Hood | Needed to work at sterile conditions. | ||
Luria Bertani Broth, Miller (Miller Luria Bertani Broth) | Himedia | M1245-1KG | Used as rich media to grow bacteria. |
Marine Agar 2216 (Zobell Marine Agar) | Himedia | M384-500G | Used as rich media to grow bacteria. |
Orbital-Shaker Incubator | Used to incubate liquid media and oil. | ||
Plates Incubator | Used to incubate plates. | ||
Porcelain Mortar and Pestle | Thomas scientific/United Scientific Supplies | 1201U69/JMD150 | Used to macerate coral fragments. |
Qubit 2.0 Fluorometer | Invitrogen | Used for nucleic acids quantification of DNA and PCR products. | |
Refrigerated Centrifuge | Used to centrifuge bacterial cultures. | ||
Spectrophotometer | Used to measure optical density of bacterial cultures. | ||
Wizard Genomic DNA Purification kit | Promega | A1120 | Used for microbial strains DNA extraction. |