This research was carried out in association with the ongoing R&#38;D project registered&#160;as ANP 21005-4,&#160;&#8220;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&#8221; (UFRJ / Shell Brasil / ANP) &#8211; &#8220;PROBIO-DEEP - Levantamento de potenciais impactos causados pela explora&#231;&#227;o de &#243;leo e g&#225;s em holobiontes marinhos em mar profundo e sele&#231;&#227;o de potenciais bioindicadores e processos biorremediadores para esses ecossistemas&#8221;, sponsored by Shell Brasil under the ANP R&#38;D levy as &#8220;Compromisso de Investimentos com Pesquisa e Desenvolvimento.&#160;The authors also thank&#160;Conselho Nacional de Desenvolvimento Cient&#237;fico e Tecnol&#243;gico (CNPq)&#160;and&#160;Coordena&#231;&#227;o de Aperfei&#231;oamento de Pessoal de N&#237;vel Superior (CAPES)&#160;for financial support, and to Camila Messias, Phillipe Rosado, and Henrique Fragoso dos Santos, for the images provided.

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 &#176;C until processing. The longer the samples are stored, even at 4 &#176;C, the more the microbial community will change.
<ol>
	<li>Sample and store seawater.
	<ol>
		<li>Collect 500 mL samples of water in at least triplicate from each targeted sampling site. Preferably use sterile bottles with screw caps.</li>
		<li>If processing the water instantly after collection, keep the bottles at RT for a short interval. If sample processing is happening later, keep the bottles at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Sample and store the coral.
	<ol>
		<li>Use a sterile pair of pliers to cut coral fragments from the same sampling site of the water samples. To avoid contamination, touch corals only with sterile gloves.</li>
		<li>Rinse the sampled coral fragment using 20 mL sterile saline solution (3% NaCl in distilled water) or artificial seawater to get rid of the loosely attached free-living bacteria of the seawater.</li>
		<li>Using forceps, place each coral fragment into a sterile 250&#8722;500 mL container with a screw cap containing sterile saline solution.</li>
		<li>In the laboratory, using sterile forceps and pliers, weigh 5 g of coral fragments using sterile 100 mm x 20 mm Petri dishes on a weighing scale.</li>
		<li>Transfer the 5 g of coral sample to a sterile mortar and macerate it using a sterile pestle.</li>
		<li>Using a sterile spatula, transfer the macerated sample to a sterile culture flask containing 45 mL of 3% NaCl sterile solution and 10&#8722;15 glass beads of 5 mm. Use some of the 45 mL sterile saline solution to wash the mortar and recover the maximum amount of the macerate.</li>
		<li>Keep the flasks under constant agitation (150 x g) for 16 h at the water temperature of the sampling site. 
		NOTE: For shallow-water corals the optimum temperature will range from 24&#8722;28 &#176;C. This step will detach microorganisms from different coral compartments, such as the ones attached to the host cells, or the ones living inside the tissue and the skeleton. After this step, the coral macerates should not be stored, and the isolation step must be instantly performed.</li>
	</ol>
	</li>
</ol>
2. Isolation of EE2-degrading bacteria from seawater and/or corals
<ol>
	<li>Select bacteria. 
	NOTE: After steps 1.1.2 and 1.2.7, the concentration of microorganisms in seawater that have detached from the different coral macerates will be unknown and variable. In order to guarantee the isolation of individual microbial colonies in Petri dishes containing agar media, serial dilutions are needed.
	<ol>
		<li>Perform serial dilutions up to 10-9 in sterile saline solution for coral samples and up to 10-6 for water samples. Pipette dilutions up and down 5x before discarding the tip. Vortex samples for 5 s every time before performing the next serial dilution.</li>
		<li>Pipette 100 &#181;L of each dilution on Petri dishes containing 3% NaCl lysogeny broth (LB) agar medium, and plate them. 
		NOTE: Use marine agar (MA) as an alternative medium. Triplicates of each dilution are required for reliable results.</li>
		<li>Incubate the plates for 1&#8722;3 days at the target temperature (e.g., 26 &#176;C). Check the plates once a day.</li>
		<li>Select and isolate the colonies presenting distinct growth morphologies on new plates using the streak plate technique. Repeat this step as many times as needed to have pure colonies growing on the plates.</li>
		<li>If the procedure is not instantly continued to step 2.3.1, store isolates at 4 &#176;C or in glycerol as described in section 2.2.</li>
	</ol>
	</li>
	<li>Prepare glycerol stocks. 
	NOTE: This step is optional and can be used for long-term bacterial stocks storage.
	<ol>
		<li>Pick single colonies from the fresh plate or from the plates stored at 4 &#176;C and independently inoculate them in 2 mL of sterile LB medium.</li>
		<li>Place tubes under constant agitation (150 x g) at 24&#8722;28 &#176;C overnight (ON).</li>
		<li>Add 1 mL of the bacterial cultures from step 2.2.2 and sterile glycerol to a final concentration of 20% to the 2 mL cryovials.</li>
		<li>Leave the cryovials ON at 4 &#176;C.</li>
		<li>Place the glycerol bacterial stocks at -80 &#176;C until needed.</li>
	</ol>
	</li>
	<li>Perform the EE2-degradation ability test.
	<ol>
		<li>Activate the isolates in LB broth or alternative media. For this, pick a single colony from the fresh plates or the plate stored at 4 &#176;C, and inoculate 2 mL of sterile LB medium. In case it is a glycerol stock, first streak it on LB agar plates and incubate at 24&#8722;28 &#176;C ON to have single colonies growing. Place the tube containing LB medium inclined under constant agitation (150 x g) at 24&#8722;28 &#176;C ON.</li>
		<li>After bacterial growth, pellet the cells by centrifuging them at 8,000 x g for 8 min at room temperature (RT). Discard the supernatant and, gently pipetting up and down, resuspend the cells in an equal volume (2 mL) of saline water to wash the remaining LB broth.</li>
		<li>Repeat step 2.3.2 twice to guarantee that there are no traces of carbon source, resuspending the cells in an equal volume of saline solution. For instance, if it was started with a 2 mL culture, resuspend the cells in a final volume of 2 mL saline solution.</li>
		<li>Inoculate the washed and resuspended cells in minimum Bushnell Haas culture medium (BH Broth) containing EE2 as the only carbon source59. 
		NOTE: EE2 is dissolved in ethanol at a final concentration of 5 mg/L in the culture medium. Make changes in the pollutant type and/or concentration if needed.</li>
		<li>Assess bacterial growth by optical density at 600 nm and/or colonies forming units (CFU) on LB agar medium33, for 16&#8722;72 h of incubation. 
		NOTE: Alternatively, the microorganisms can be directly isolated on minimum media containing EE2, or other compounds, as the only carbon source. This step would direct the selection and avoid undesirable growth.</li>
	</ol>
	</li>
</ol>
3. Isolation of oil-degrading bacteria from seawater and/or corals
<ol>
	<li>Prepare minimum media containing an oil water-soluble fraction (oWSF) and oil water-insoluble fraction (oWIF) as the only carbon source21.
	<ol>
		<li>Add 1&#8722;2% crude oil to 500 mL of sterile distilled water. Use a filter flask opened on the bottom to take the soluble fraction out without disturbing the upper layer of the insoluble fraction.</li>
		<li>Keep the mixture under constant agitation at 24&#8722;28 &#176;C at 150 x g for 48 h.</li>
		<li>Place the filter flask containing the crude oil fractions on a stable surface and wait 10&#8722;20 min to allow soluble and insoluble fraction separation.</li>
		<li>Transfer the oWSF to a new sterile flask, saving the oWIF by opening the bottom filter flask and taking out the soluble fraction.</li>
		<li>Using all the oWSF recovered in the previous step (~400 mL), prepare 1 L of BH agar minimum medium containing oWSF as the only carbon source.</li>
		<li>Using the oWIF remaining in the flask from step 3.1.4, prepare 1 L of BH agar minimum medium containing oWIF as the only carbon source.</li>
	</ol>
	</li>
	<li>Isolate oWSF- and oWIF-degrading bacteria.
	<ol>
		<li>Using water and coral macerate from steps 1.1.2 and 1.2.7, respectively, dilute the samples up to 10-6 in sterile saline solution as described in step 2.1.1.</li>
		<li>Pipette 100 &#181;L of each dilution on Petri dishes containing BH-oWSF and BH-oWIF agar media and plate them.</li>
		<li>Repeat the procedures described from step 2.1.3 to step 2.1.5.</li>
	</ol>
	</li>
</ol>
4. Consortium member selection
<ol>
	<li>Extract and sequence the DNA for taxonomic identification.
	<ol>
		<li>Activate the isolates stocked in glycerol as described in step 2.3.1.</li>
		<li>Extract the DNA using a DNA extraction kit (see Table of Materials).</li>
		<li>Use primers 27f (5&#8242;-AGA GTT TGA TCA TGG CTC AG-3&#8242;) and 1492r (5&#8242;-GTT TAC CTT GTT ACG ACT T-3&#8242;) for amplification of the 16S rRNA gene.</li>
		<li>Perform 50 &#181;L PCR reactions according to the following protocol: 5 &#956;L of 10x polymerase buffer, 2 mM MgCl2, 0.2 mM dNTPs, 5 mM of each primer, 10 ng of genomic DNA, and 2.5 U of Taq DNA polymerase. Add negative controls (i.e., blank DNA extractions and PCR reactions without template DNA) to ensure that there is no contamination.</li>
		<li>Set up the following thermal cycling steps: a first denaturation cycle at 94 &#176;C for 4 min; 35 cycles at 94 &#176;C for 1 min, followed by 50 &#176;C for 1 min, and 72 &#176;C for 2.5 min; a final extension cycle for 10 min at 72 &#176;C.</li>
		<li>Check amplicon integrity in 1.2% agarose gel using 80 V.</li>
		<li>Gel purify the samples using a gel purification kit (see Table of Materials).</li>
		<li>Quantify PCR products using a fluorometer.</li>
		<li>Send product for sequencing. 
		NOTE: For better taxonomical classifications, the Sanger method is recommended60, because it provides long sequences. The universal primers 27f and 1492r can be used to amplify nearly the entire length of the 16S rRNA gene61. If contigs cannot be assembled using one pair of primers, an extra pair in the middle of the sequence should be considered.</li>
	</ol>
	</li>
	<li>Determine the growth curve.
	<ol>
		<li>Activate the isolates in glycerol stocks from step 2.2.5 as described in step 2.3.1 but using 5 mL instead of 2 mL.</li>
		<li>Add 1% (v/v) of the grown 5 mL culture from step 4.2.1 in triplicates, to a 250 mL flask containing 100 mL of 3% LB media. Make sure to prepare triplicates of a negative control (no inoculum) as well.</li>
		<li>Place the flasks in an incubator under constant agitation (150 x g) at 24&#8722;28 &#176;C.</li>
		<li>Take 1 mL aliquots every 4 h for 48 h. If the strains present a high growth rate, decrease the 4 h interval.</li>
		<li>Measure the optical density (OD) estimation at 600 nm wavelength and colony forming units (CFU) counted from serial dilutions plated on rich media plates (100 &#181;L should be inoculated in each plate and normalized to the volume of 1 mL).</li>
		<li>Plot the OD and CFU curves and analyze the correlation of OD/CFU of each individual strain. From now on, calculate the number of cells based on the OD values.</li>
	</ol>
	</li>
	<li>Perform antagonism test.
	<ol>
		<li>Activate the isolates as described in step 2.3.1.</li>
		<li>Inoculate one strain at a time along the middle of plates containing agar media and the other ones perpendicular to the central one. Repeat until every selected microbial strain is tested against all others.</li>
		<li>Incubate the plates at 24&#8722;28 &#176;C and monitor them daily in order to observe potential halos indicating antagonistic activity. If halos are observed indicating antagonistic activity between two strains, one of them should be excluded.</li>
	</ol>
	</li>
	<li>Perform consortium assembly.
	<ol>
		<li>Activate the isolates as described in step 2.3.1.</li>
		<li>Pellet the cells at 3,500 x g and gently wash them 2x in an equal volume of saline solution. Resuspend the cells in an equal volume (i.e., if the final volume was 2 mL of cells, wash them using 2 mL of saline solution and resuspend them in a final volume of 2 mL).</li>
		<li>Inoculate 1 mL culture in 100 mL 3% NaCl LB medium and incubate ON at 150 x g.</li>
		<li>Repeat step 4.5.2, inoculate the 100 mL grown, washed, and resuspended cultures in 10 L cultures. Incubate ON in sterile air-lift bioreactors, receiving filtered air from a pump. If more culture is needed, always inoculate 1% of grown culture in fresh media.</li>
		<li>Centrifuge grown cultures at 8,000 x g for 8 min at 4 &#176;C. Discard supernatant after centrifugation.</li>
		<li>Wash the pellet, gently resuspending the cells in 500 mL of sterile saline solution.</li>
		<li>Repeat steps 4.5.5 and 4.5.6.</li>
		<li>Resuspend each individual culture in 100 mL of saline solution and measure the OD to estimate the number of cells.</li>
		<li>Calculate the volume of each culture necessary to reach a final concentration of 107 cells mL-1.</li>
		<li>Perform CFU counts on rich media for a final confirmation of cell viability and concentration.</li>
		<li>Mix an equal volume of each individual culture in sterile flasks and aliquot the consortium into 50 mL sterile tubes.</li>
		<li>Keep at 4 &#176;C until inoculation. 
		NOTE: Prepare the consortium assembly as fresh as possible to guarantee cells will still be viable. Alternatively, CFU counts can be performed before inoculation. To assemble an ideal consortium, it is necessary to use isolates presenting different and complementary metabolic capacities. Usually, 6&#8722;10 isolates are used to form consortia, but this number will vary depending on the members&#8217; characteristics and purposes.</li>
	</ol>
	</li>
</ol>
5. Detection of putative beneficial characteristics for corals
<ol>
	<li>Activate the isolates from the glycerol stocks by streaking them on LB agar plates and incubating them at 24&#8722;28 &#176;C ON to have single colonies growing. Pick a single colony from the plates and inoculate it in 2 mL of sterile LB medium. Place tube containing LB medium inclined under constant agitation (150 x g) at 24&#8722;28 &#176;C ON.</li>
	<li>Perform DNA extraction as described in section 4.1 for detection of potentially beneficial genes by PCR. 
	NOTE: PCR reaction conditions and primers will depend on the targeted gene. Methodologies to test BMC characteristics of individual strains and PCR detection for potentially beneficial genes are described in Table 1.</li>
</ol>

The authors have nothing to disclose.

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&#39;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&#39;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.

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&#8217; 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&#8217; 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 &#181;g/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&#8217;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.

<table>
	<tbody>
		<tr>
			<td>500 mL PYREX Media Storage Bottle</td>
			<td>thomas scientific/Corning</td>
			<td>1743E20/1395-500</td>
			<td>Used to sample water.</td>
		</tr>
		<tr>
			<td>500 mL Aspirator Bottles</td>
			<td>thomas scientific/Corning</td>
			<td>1234B28/1220-2X</td>
			<td>Used to separate the oil fractions.</td>
		</tr>
		<tr>
			<td>6-inch wire cutter plier</td>
			<td>thomas scientific/Restek</td>
			<td>1173Y64/23033</td>
			<td>Used to cut coral fragments.</td>
		</tr>
		<tr>
			<td>17a-Ethinylestradiol</td>
			<td>LGC Standards</td>
			<td>DRE-C13245100</td>
			<td>Used as the only carbon source to make the selective media.</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Himedia</td>
			<td>PCT0901-1KG</td>
			<td>Used to make solid media.</td>
		</tr>
		<tr>
			<td>Bushnell Haas Broth</td>
			<td>Himedia</td>
			<td>M350-500G</td>
			<td>Used as minimum media to be supplemented with carbon sources.</td>
		</tr>
		<tr>
			<td>Erlenmeyer Flask</td>
			<td>thomas scientific/DWK Life Sciences (Kimble)</td>
			<td>4882H35/26500-125</td>
			<td>Used to incubate coral macerate with glass beads.</td>
		</tr>
		<tr>
			<td>GFX PCR DNA and Gel Band Purification kit</td>
			<td>GE Healthcare</td>
			<td>28903470</td>
			<td>Used to purify PCR products before sending them for sequencing.</td>
		</tr>
		<tr>
			<td>Glass Beads</td>
			<td>MP Biomedicals</td>
			<td>1177Q81/07DP1070</td>
			<td>Used to detach the microorganisms from coral structures.</td>
		</tr>
		<tr>
			<td>Laminar Flow Hood</td>
			<td></td>
			<td></td>
			<td>Needed to work at sterile conditions.</td>
		</tr>
		<tr>
			<td>Luria Bertani Broth, Miller (Miller Luria Bertani Broth)</td>
			<td>Himedia</td>
			<td>M1245-1KG</td>
			<td>Used as rich media to grow bacteria.</td>
		</tr>
		<tr>
			<td>Marine Agar 2216 (Zobell Marine Agar)</td>
			<td>Himedia</td>
			<td>M384-500G</td>
			<td>Used as rich media to grow bacteria.</td>
		</tr>
		<tr>
			<td>Orbital-Shaker Incubator</td>
			<td></td>
			<td></td>
			<td>Used to incubate liquid media and oil.</td>
		</tr>
		<tr>
			<td>Plates Incubator</td>
			<td></td>
			<td></td>
			<td>Used to incubate plates.</td>
		</tr>
		<tr>
			<td>Porcelain Mortar and Pestle</td>
			<td>Thomas scientific/United Scientific Supplies</td>
			<td>1201U69/JMD150</td>
			<td>Used to macerate coral fragments.</td>
		</tr>
		<tr>
			<td>Qubit 2.0 Fluorometer</td>
			<td>Invitrogen</td>
			<td></td>
			<td>Used for nucleic acids quantification of DNA and PCR products.</td>
		</tr>
		<tr>
			<td>Refrigerated Centrifuge</td>
			<td></td>
			<td></td>
			<td>Used to centrifuge bacterial cultures.</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td></td>
			<td></td>
			<td>Used to measure optical density of bacterial cultures.</td>
		</tr>
		<tr>
			<td>Wizard Genomic DNA Purification kit</td>
			<td>Promega</td>
			<td>A1120</td>
			<td>Used for microbial strains DNA extraction.</td>
		</tr>
	</tbody>
</table>

prospecting microbial strains for bioremediation and probiotics development for metaorganism research and preservation

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.

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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60238/60238fig01.jpg" /> 
Figure 1: Summary of the main steps of a bioremediator-pBMC consortium selection and assembly.&#160;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). <a href="https://www.jove.com/files/ftp_upload/60238/60238fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60238/60238fig02.jpg" /> 
Figure 2: Selection of pollutant-degrading bacteria.&#160;(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. <a href="https://www.jove.com/files/ftp_upload/60238/60238fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60238/60238fig03.jpg" /> 
Figure 3: Detection of pBMC characteristics.&#160;(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). <a href="https://www.jove.com/files/ftp_upload/60238/60238fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60238/60238fig04.jpg" /> 
Figure 4: Fv/Fm measurements of M. harttii nubbins dark-adapted at 5 PM, using a diving-PAM chlorophyll fluorometer.&#160;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/. <a href="https://www.jove.com/files/ftp_upload/60238/60238fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>MOA</td>
			<td>Microorganism</td>
			<td>Detection technique</td>
			<td>References</td>
		</tr>
		<tr>
			<td rowspan="3">Climate regulation; surfur cycling; antimicrobial compounds; increase antioxidant protection of cells.</td>
			<td>Aspergillus sydowii</td>
			<td>PCR for dddP gene</td>
			<td>81</td>
		</tr>
		<tr>
			<td>Pseudovibrio sp. P12</td>
			<td>Culture medium with DMSP</td>
			<td>82</td>
		</tr>
		<tr>
			<td>Pseudoalteromonas sp.</td>
			<td>PCR for dmdA gene</td>
			<td>33</td>
		</tr>
		<tr>
			<td rowspan="5">Biological regulation of pathogens.</td>
			<td>Microbiome from Acropora palmata mucus</td>
			<td>Clear zone of inhibition</td>
			<td>83</td>
		</tr>
		<tr>
			<td>Extracts from corals</td>
			<td>Growth inhibition assay</td>
			<td>84</td>
		</tr>
		<tr>
			<td>Marinobacter sp.</td>
			<td>Swarming assays</td>
			<td>85</td>
		</tr>
		<tr>
			<td>Pseudoalteromonas sp.</td>
			<td>Agar plate cross-streaking</td>
			<td>86</td>
		</tr>
		<tr>
			<td>Pseudoalteromonas sp.</td>
			<td>Agar-diffusion method</td>
			<td>33</td>
		</tr>
		<tr>
			<td rowspan="3">Benefit to the calcification process; source of nitrogen for scleractinian corals.</td>
			<td>Symbiodinium spp.</td>
			<td>Colorimetric method</td>
			<td>87</td>
		</tr>
		<tr>
			<td>Microbiome from Stylophora pistillata mucus</td>
			<td>___</td>
			<td>88</td>
		</tr>
		<tr>
			<td>Microbiome from Acropora alciminata</td>
			<td>Method by Bolland et al.80</td>
			<td>89</td>
		</tr>
		<tr>
			<td rowspan="5">Nitrogen cycle; increase nitrogen fixation.</td>
			<td>Microbial community</td>
			<td>qPCR</td>
			<td>90</td>
		</tr>
		<tr>
			<td>Microbial community</td>
			<td>PCR</td>
			<td>91</td>
		</tr>
		<tr>
			<td>Microbial community</td>
			<td>qPCR</td>
			<td>92</td>
		</tr>
		<tr>
			<td>Microbial community</td>
			<td>Adapted acetylene (C2H2) reduction technique</td>
			<td>93</td>
		</tr>
		<tr>
			<td>Pseudoalteromonas sp. and Halomonas taeanensis</td>
			<td>PCR</td>
			<td>33</td>
		</tr>
		<tr>
			<td rowspan="3">Nitrogen cycle; decrease of the ammonium concentration.</td>
			<td>Pseudoalteromonas sp.</td>
			<td>PCR</td>
			<td>33</td>
		</tr>
		<tr>
			<td>Microbiome from Tubastraea coccinea</td>
			<td>PCR</td>
			<td>94</td>
		</tr>
		<tr>
			<td>Microbiome from Xestospongia testudinaria</td>
			<td>Predictive metagenome analysis</td>
			<td>95</td>
		</tr>
		<tr>
			<td rowspan="4">Holobiont protection against reactive oxygen species (ROS).</td>
			<td>Pseudoalteromonas sp., Cobetia marina and Halomonas taeanensis</td>
			<td>Catalase test</td>
			<td>33</td>
		</tr>
		<tr>
			<td>Symbiodinium spp.</td>
			<td>Amplex red</td>
			<td>96</td>
		</tr>
		<tr>
			<td>Vibrio pelagius and Sync- chococcus sp.</td>
			<td>Horseradish peroxidase-scopoletin method</td>
			<td>97</td>
		</tr>
		<tr>
			<td>Vibrio fischeri</td>
			<td>Multiple methods</td>
			<td>98</td>
		</tr>
	</tbody>
</table>
Table 1: Detection of putative BMC characteristics, mechanism of action (MOA), reported microorganisms presenting the potential and technique used to detect the characteristic.

Watch this Scientific Journal Video about Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation at JoVE.com

Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation

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. ...

prospecting-microbial-strains-for-bioremediation-probiotics

Research

JoVE Journal

Bioengineering

21.8K Views.  Federal University of Rio de Janeiro (UFRJ). 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.

Video: Prospecting Microbial Strains for Bioremediation and Probiotics Development for Metaorganism Research and Preservation

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Harmonic Nanoparticles for Regenerative Research

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation nanoparticles (HNPs). The latter are a new family of probes recently introduced for labeling biological samples for multi-photon imaging. HNPs are capable of doubling the frequency of excitation light by the nonlinear optical process of second harmonic generation with no restriction on the excitation wavelength.
Multi-photon based methodologies for hESC differentiation into cardiac clusters (maintained as long term air-liquid cultures) are presented in detail. In particular, evidence on how to maximize the intense second harmonic (SH) emission of isolated HNPs during 3D monitoring of beating cardiac tissue in 3D is shown. The analysis of the resulting images to retrieve 3D displacement patterns is also detailed.

Protocol details are provided for in vitro labeling human embryonic stem cells with second harmonic generating nanoparticles. Methodologies for hESC investigation by multi-photon microscopy and their differentiation into cardiac clusters are also presented.

In this visualized experiment, protocol details are provided for in vitro labeling of human embryonic stem cells (hESC) with second harmonic generation ...

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is needed to open up the structure of the plant cell wall, increasing the accessibility of the cell wall carbohydrates. Lignin, a polyphenolic material present in many cell wall types, is known to be a significant hindrance to enzyme access. Reduction in lignin content to a level that does not interfere with the structural integrity and defense system of the plant might be a valuable step to reduce the costs of bioethanol production. In this study, we have genetically down-regulated one of the lignin biosynthesis-related genes, cinnamoyl-CoA reductase (ZmCCR1) via a double stranded RNA interference technique. The ZmCCR1_RNAi construct was integrated into the maize genome using the particle bombardment method. Transgenic maize plants grew normally as compared to the wild-type control plants without interfering with biomass growth or defense mechanisms, with the exception of displaying of brown-coloration in transgenic plants leaf mid-ribs, husks, and stems. The microscopic analyses, in conjunction with the histological assay, revealed that the leaf sclerenchyma fibers were thinned but the structure and size of other major vascular system components was not altered. The lignin content in the transgenic maize was reduced by 7-8.7%, the crystalline cellulose content was increased in response to lignin reduction, and hemicelluloses remained unchanged. The analyses may indicate that carbon flow might have been shifted from lignin biosynthesis to cellulose biosynthesis. This article delineates the procedures used to down-regulate the lignin content in maize via RNAi technology, and the cell wall compositional analyses used to verify the effect of the modifications on the cell wall structure.

Lignin Down-regulation of Zea mays via dsRNAi and Klason Lignin Analysis

A double stranded RNA interference (dsRNAi) technique is employed to down-regulate the maize cinnamoyl coenzyme A reductase (ZmCCR1) gene to lower plant lignin content. Lignin down-regulation from the cell wall is visualized by microscopic analyses and quantified by the Klason method. Compositional changes in hemicellulose and crystalline cellulose are analyzed.

To facilitate the use of lignocellulosic biomass as an alternative bioenergy resource, during biological conversion processes, a pretreatment step is ...

Development of a Multicellular Three-dimensional Organotypic Model of the Human Intestinal Mucosa Grown Under Microgravity

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., flasks or dishes). In fact, it is widely recognized that cells grown in flasks or dishes tend to de-differentiate and lose specialized features of the tissues from which they were derived. Currently, there are mainly two types of 3-D culture systems where the cells are seeded into scaffolds mimicking the native extracellular matrix (ECM): (a) static models and (b) models using bioreactors. The first breakthrough was the static 3-D models. 3-D models using bioreactors such as the rotating-wall-vessel (RWV) bioreactors are a more recent development. The original concept of the RWV bioreactors was developed at NASA&#39;s Johnson Space Center in the early 1990s and is believed to overcome the limitations of static models such as the development of hypoxic, necrotic cores. The RWV bioreactors might circumvent this problem by providing fluid dynamics that allow the efficient diffusion of nutrients and oxygen. These bioreactors consist of a rotator base that serves to support and rotate two different formats of culture vessels that differ by their aeration source type: (1) Slow Turning Lateral Vessels (STLVs) with a co-axial oxygenator in the center, or (2) High Aspect Ratio Vessels (HARVs) with oxygenation via a flat, silicone rubber gas transfer membrane. These vessels allow efficient gas transfer while avoiding bubble formation and consequent turbulence. These conditions result in laminar flow and minimal shear force that models reduced gravity (microgravity) inside the culture vessel. Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa composed of an intestinal epithelial cell line and primary human lymphocytes, endothelial cells and fibroblasts cultured under microgravity provided by the RWV bioreactor.

Cells growing in a three-dimensional (3-D) environment represent a marked improvement over cell cultivation in 2-D environments (e.g., flasks or dishes). Here we describe the development of a multicellular 3-D organotypic model of the human intestinal mucosa cultured under microgravity provided by rotating-wall-vessel (RWV) bioreactors.

Because cells growing in a three-dimensional (3-D) environment have the potential to bridge many gaps of cell cultivation in 2-D environments (e.g., ...

Design and Implementation of an Automated Illuminating, Culturing, and Sampling System for Microbial Optogenetic Applications

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular processes. There is a need for culturing and measuring systems that incorporate programmed illumination and stimulation of optogenetic systems. We present a protocol for building and using a continuous culturing apparatus to illuminate microbial cells with programmed doses of light, and automatically acquire and analyze images of cells in the effluent. The operation of this apparatus as a chemostat allows the growth rate and the cellular environment to be tightly controlled. The effluent of the continuous cell culture is regularly sampled and the cells are imaged by multi-channel microscopy. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses in the fluorescence intensity and cellular morphology of cells sampled from the culture effluent are measured over multiple days without user input. We demonstrate the utility of this culturing apparatus by dynamically inducing protein production in a strain of Saccharomyces cerevisiae engineered with an optogenetic system that activates transcription.

We designed a continuous culturing apparatus for use with optogenetic systems to illuminate cultures of microbes and regularly image cells in the effluent with an inverted microscope. The culturing, sampling, imaging, and image analysis are fully automated so that dynamic responses to illumination can be measured over several days.

Optogenetic systems utilize genetically-encoded proteins that change conformation in response to specific wavelengths of light to alter cellular ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Bulk Droplet Vitrification for Primary Hepatocyte Preservation

Vitrification is a promising ice-free alternative for classic slow-freezing (at approximately 1 &#176;C/min) cryopreservation of biological samples. Vitrification requires extremely fast cooling rates to achieve transition of water into the glass phase while avoiding injurious ice formation. Although pre-incubation with cryoprotective agents (CPA) can reduce the critical cooling rate of biological samples, high concentrations are generally needed to enable ice-free cryopreservation by vitrification. As a result, vitrification is hampered by CPA toxicity and restricted to small samples that can be cooled fast. It was recently demonstrated that these inherent limitations can be overcome by bulk droplet vitrification. Using this novel method, cells are first pre-incubated with a low intracellular CPA concentration. Leveraging rapid osmotic dehydration, the intracellular CPA is concentrated directly ahead of vitrification, without the need to fully equilibrate toxic CPA concentrations. The cellular dehydration is performed in a fluidic device and integrated with continuous high throughput generation of large sized droplets that are vitrified in liquid nitrogen. This ice-free cryopreservation method with minimal CPA toxicity is suitable for large cell quantities and results in increased hepatocyte viability and metabolic function as compared to classical slow-freezing cryopreservation. This manuscript describes the methods for successful bulk droplet vitrification in detail.

This manuscript describes an ice-free cryopreservation method for large quantities of rat hepatocytes whereby primary cells are pre-incubated with cryoprotective agents at a low concentration and vitrified in large droplets.

Vitrification is a promising ice-free alternative for classic slow-freezing (at approximately 1 &#176;C/min) cryopreservation of biological samples. ...

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the microscale&#8213;to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert (&#39;caged&#39;) by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...

Metabolic Profiling to Determine Bactericidal or Bacteriostatic Effects of New Natural Products using Isothermal Microcalorimetry

Due to the global threat of rising antimicrobial resistance, novel antibiotics are urgently needed. We investigate natural products from Myxobacteria as an&#160;innovative source of such new compounds. One bottleneck in the process is typically the elucidation of their mode-of-action. We recently established isothermal microcalorimetry as part of a routine profiling pipeline. This technology allows for investigating the effect of antibiotic exposure on the total bacterial metabolic response, including processes that are decoupled from biomass formation. Importantly, bacteriostatic and bactericidal effects are easily distinguishable without any user intervention during the measurements. However, isothermal microcalorimetry is a rather new approach and applying this method to different bacterial species usually requires pre-evaluation of suitable measurement conditions. There are some reference thermograms available of certain bacteria, greatly facilitating interpretation of results. As the pool of reference data is steadily growing, we expect the methodology to have increasing impact in the future and expect it to allow for in-depth fingerprint analyses enabling the differentiation of antibiotic classes.

The elucidation of the mode-of-action of a novel antibiotic is a challenging task in the drug discovery process. The goal of the method described here is the application of isothermal microcalorimetry using calScreener in antibacterial profiling to provide additional insight into drug-microbe interactions.

Due to the global threat of rising antimicrobial resistance, novel antibiotics are urgently needed. We investigate natural products from Myxobacteria as ...

Establishing an Octopus Ecosystem for Biomedical and Bioengineering Research

Many developments in biomedical research have been inspired by discovering anatomical and cellular mechanisms that support specific functions in different species. The octopus is one of these exceptional animals that has given scientists new insights into the fields of neuroscience, robotics, regenerative medicine, and prosthetics. Research with this species of cephalopods requires the set-up of complex facilities and intensive care for both the octopus and its ecosystem that is critical for the project&#39;s success. This system requires multiple mechanical and biological filtering systems to provide a safe and clean environment for the animal. Along with the control system, specialized routine maintenance and cleaning are required to effectively keep the facility operating long term. It is advised to provide an enriched environment to these intelligent animals by changing the tank&#39;s landscape, incorporating a variety of prey, and introducing challenging tasks for them to work through. Our results include MRI and a whole-body autofluorescence imaging as well as behavioral studies to better understand their nervous system. Octopuses possess unique physiology that can impact many areas of biomedical research. Providing them with a sustainable ecosystem is the first crucial step in uncovering their distinct capabilities.

Understanding the unique physiological and anatomical structures of octopuses can greatly impact biomedical research. This guide demonstrates how to set-up and maintain a marine environment to accommodate this species and includes state-of-the-art imaging and analytical approaches to visualize octopus' nervous system anatomy and function.

Many developments in biomedical research have been inspired by discovering anatomical and cellular mechanisms that support specific functions in different ...