SEA
I. Executive Summary
-Title: Bioremediation of oil pollutants by fungi associated with coastal mangroves in Puerto Rico
-Date: 26 February 2020
-Project number: R/23-1-17
-Name and affiliation of researchers
Dr. Matias J. Cafaro, Professor and Principal Investigator, Department of Biology, University of Puerto Rico-Mayagüez
Dr.KristinR.Peterson,Lecturer andCo-PrincipalInvestigator,DepartmentofBiology,University of Puerto Rico-Mayagüez
-Dates Covered: May 22, 2017 – January 31, 2020
-Objectives
1. Isolate microalgae and potential members of the Desulfobulbaceae from mangrove habitats for inclusion in our fungal petroleum hydrocarbons (PHC)-degrading consortia. We partially accomplished this objective since many of the microorganisms were not recovered in our first attempts. Culturing microalgae under unstable conditions proved extremely difficult. Nonetheless, we managed to isolate other microbes such as bacteria and yeast, which showed promising abilities as degraders of PHC.
2. Culture all consortia members ex situ under oxic and anoxic conditions, in seawater and in sediments, in order to ascertain the optimal assemblage for degrading PHCs. We accomplished cultures of consortia of fungi under oxigenic conditions in the laboratory. Several different filamentous fungi (15 strains) and yeast (64 strains) were isolates. Among the isolated yeast, 26 (40%) strains are promising hydrocarbon degraders that need further analysis. Four different consortia were tested for used motor oil degradation in the laboratory. Each one was composed of 4 fungal strains obtained from the mangrove environment. In three different experiments, we showed that the consortia produced more biomass and used more oil than each fungus growing alone; hence, supporting our initial hypothesis about improving degradation with multiple fungal isolates rather than using one at a time.
3. Characterize all consortia components with respect to degradation of PHCs, selected metabolites, molecular sequences, and ability to be cultured en masse for use in the field
Our lab experiments allowed to grow significant biomass of fungal strains to perform our studies, but mass production of consortia was not possible under the current conditions. Nonetheless, all fungal and yeast strains were sequenced and tested for degradation capacities including PHCs, complex carbohydrates, cellulose and azole dyes.
4. Develop and test mass production of consortia on wood chips, in pellets of agar, agarose, or alginate for introduction into different mangrove sedimentary depths. – not accomplished due to lack of time for the project.
5. Present our final products to the Departamento de Recursos Naturales y Ambientales del Estado Libre Asociado de Puerto Rico (DRNA) to convince them to permit us to conduct field trials and
collect data over a period of years to document beneficial and problematic outcomes. – not accomplished due to lack of time for the project.
-Advancement of the field
The survey of fungi and yeast conducted revealed several new organisms for science and new records for Puerto Rico in general. Very little is known about yeast and fungi in mangrove environments;hence, the cultureswenowhavein the labcanbefurther described and incorporated into the record. In addition, the characterization of species that were not previously described as PHCs degraders opens opportunities for applications of these fungi not anticipated. Consortia of fungi are a novel way of bioremediation allowing faster processes and improve conditions to address environmental cleanups.
-Problems encountered
Hurricanes: Fungi were previously isolated from the red mangrove (Rhizophora mangle) and later grown in vitro conditions in different substrate experiments. Four different fungi were selected based on their strong capacity to degrade polycyclic aromatic hydrocarbons, Congo Red and naphthalene: Purpureocillium lilacinum, Fusarium solani, Aspergillus niger, and Aspergillus caelatus. All these isolates were lost during Hurricane Maria. New fieldtrip collections were necessary to carry on with the proposed work. This fieldwork was not part of the original proposal; thus, the project was significantly delayed.
Infrastructure problems: The Biology building had been without power for over two weeks after hurricane Maria. All reagents in freezers and fridges were lost. In addition, the air conditioning system in the building collapsed after September 2017 and since then, it had been inconsistent up until October 2018 causing extremely difficult conditions to conduct microbiological work in the laboratory. For example, our incubators did not work because room temperatures were above 32C and incubation conditions of 25C or less were unachievable during that time. Many microorganisms require low temperatures to grow and develop; thus, growing conditions were not ideal in the laboratory.
Personnel issues: Since working conditions were suboptimal, many workers, faculty and students did not return to the university until after January 2018. Many lost their houses and were unable to perform consistently. Students were the most reliable, but some left the island. Specifically, for this project we were further delayed due to lack of technicians and support personnel.
Technical issues: Aside from temperature control issues mentioned before, many pieces of equipment were damaged after the hurricanes. In our case, GC-MS available in Chemistry Department was seriously damaged beyond repair. The University has not been able to replace it due to slow insurance claim processing. FEMA funds have been released to Puerto Rico only recently, after our project ended. We have attempted other techniques such as IR and Raman spectroscopy, but proved unsuccessful.
-List PIs supported
R. Peterson
-List students supported Undergraduate students
1. Shekina Gonzalez Ferrer, shekina.gonzalez@upr.edu, BS Biology, no SG support, research credits as matching.
2. Michael Hernandez Lamberty, michael.hernandez15@upr.edu, BS Mechanical Engineering, no SG support, research credits as matching.
3. Aslin Vázquez Nieves, aslin.vazquez@upr.edu, BS Biology, no SG support, research credits as matching.
4. Rafael Rivera Sotomayor, rafael.rivera40@upr.edu, BS Biology, no SG support, research credits as matching.
5. Shelsy Torres, shelsy.torres@upr.edu, BS Biology, no SGsupport, research credits as matching.
Graduate students
1. Carla Aponte Lopez, carla.aponte@upr.edu, MS Biology, $1,500 SG summer stipend.
2. Yoana Guzman Salgado, yoana.guzman@upr.edu, MS Biology, $1,500 SG summer stipend.
3. Kathleen Matías Caban, kathleen.matias1@upr.edu, MS Biology, $1,500 SG summer stipend.
4. Carla Colon Rosario, carla.colon3@upr.edu, MS Biology, $1,500 SG summer stipend.
-List thesis and dissertations from students supported by the project.
• 2020 Matías-Cabán, Kathleen. Bioremediation of oil pollutants by fungi associated with coastal mangroves in Puerto Rico. Not defended yet.
• 2020 Colón-Rosario, Carla. Survey of marine yeasts associated with mangroves in Puerto Rico. Not defended yet.
-List presentations, technical reports and special awards
• 2019 Bioremediation of oil pollutants by fungi associated with coastal mangroves in Puerto Rico. Matías-Cabán, Kathleen N., Vázquez-Nieves, Aslin, Cafaro, MJ. MSA Annual Meeting, Minneapolis, MN 10-15 August
• 2019 Survey of Marine Yeasts associated with Mangroves in Puerto Rico. Colón-Rosario, Carla F., Rivera-Sotomayor, Rafael, Torres-Nieves, Shelsy M., Cafaro, MJ MSA Annual Meeting, Minneapolis, MN 10-15 August.
• 2019 Survey of Marine Yeasts associated with Mangroves in Puerto Rico. Colón-Rosario, Carla F., Rivera-Sotomayor, Rafael, Torres-Nieves, Shelsy M., Cafaro, MJ. 38th Puerto Rico Interdisciplinary Scientific Meeting/53rd ACS Junior Technical Meeting (PRISM/JTM) May 4th.
• 2019 Survey of Marine Yeasts associated with Mangroves in Puerto Rico. Carla F. Colón Rosario, Rafael E. Rivera Sotomayor, Shelsy M. Torres Nieves, Cafaro, MJ. XII Symposium Frontiers in Environmental Microbiology: interconnections for progress. February 25.
• 2019 Bioremediation of oil pollutants by fungi associated with coastal mangroves in Puerto Rico. Kathleen N. Matías Cabán, Aslin Vázquez Nieves, and Cafaro, MJ. XII Symposium Frontiers in Environmental Microbiology: interconnections for progress. February 25.
• 2018 Gonzalez-Ferrer, S, Hernandez-Lamberty, M, Peterson, KR & Cafaro, MJ. Bioremediation of petroleum products in coastal mangroves by lignicolous fungal consortia. 65th Caribbean Tribeta Convention, UPR-Rio Piedras, 24 March 2018.
• 2017 Gonzalez-Ferrer, S, Hernandez-Lamberty, M, Peterson, KR & Cafaro, MJ. Bioremediation of petroleum products in coastal mangroves by lignicolous fungal consortia. IX Congreso Latinoamericano de Micologia. Lima, Peru. 22-25 August.
-List references for, books, chapters, and peer reviewed publications, in press, and submittals. None yet.
-Matching funds: University of Puerto Rico, Mayaguez Campus, $11,860 (as release time for PI).
-New extramural funding
• August 2019 – July 2020 Rafael Rivera Sotomayor. Mangrove Marine Yeast Metabolites Against Common Pathogenic Bacteria. PRIMBRE Undergraduate student research associate program. Successful proposal. $9,420 for student support.
• August 2019 – May 2020 Rafael Rivera Sotomayor. Mangrove Marine Yeast Metabolites Against Common Pathogenic Bacteria. Undergraduate Research 2019-2020 program of Puerto Rico Louis Stokes Alliance for Minority Participation (PR-LSAMP). $1,500 for student support plus travel expenses.
-Impact Statement
1. RECAP - Use of autochthonous microbial consortia of fungi and bacteria selected for degradation efficacy of petroleum hydrocarbons in highly vulnerable mangrove sediments through biofiltration bags at the sea surface and pellets to be introduced directly onto and into coastal mangrove sediments for more efficient, and cost-effective bioremediation methods.
2. RELEVANCE ‐ Puerto Rico has a 12% coastal mangrove cover around the island occurring in areas of low-oxygen soil, where slow-moving waters allow fine sediments to accumulate. Coastal mangroves play a large ecological role by stabilizing the coastline, reducing erosion from storm surges, currents, waves, and tides. Mangrove forests are highly vulnerable to pollution regularly exposed to petroleum hydrocarbons from motorboats, such as gasoline, diesel, and motor oil leakage, and can be further damaged by many types of cleanup activities.
3.RESPONSE-Thisresearchwillhavean anticipatedmeasurableimpactonPHCbioremediation by augmenting mangrove sediments with mass produced microorganism consortia specially selected for their efficiency and ability to work together as a team in reducing sedimentary contaminants. Once optimized, anyone (DRNA, researchers, NGOs) with permission from appropriate governmental and regulatory agencies will be able to put our system to use.
4. RESULTS - Culture isolation was restarted after hurricane Maria and PHC degradation assays were performed. Laboratory conditions affected growth of microorganisms due to infrastructure problems.Nonetheless,biologicaldegradationof wastedmotoroilwasachievedby isolatedfungal strains and the consortia derived from them. Also, marine yeasts were isolated as part of the same project. The yeasts were able to degrade different sugars, starch and cellulose found in the
mangrove environments. Molecular characterization of microbial strains was managed. Two MS thesis will result from this work.
II. Final Report Narrative
Problem: Puerto Rico has 12% mangrove cover around the island occurring in areas of lowoxygen soil, where slow-moving waters allow fine sediments to accumulate. Mangroves play a large ecological role by stabilizing the coastline, reducing erosion from storm surges, currents, waves, and tides. Mangrove forests are highly vulnerable to pollution regularly exposed to petroleum hydrocarbons from motorboats, such as gasoline, diesel, and motor oil leakage and can be further damaged by many types of cleanup activities. Conventional cleanup methods include removal, alterationor isolationof the pollutant, but thesetechniques canbeexpensive and,in many cases, transfer the pollutant from one phase to another. Microorganisms have an important role in the recycling of organic matter in mangrove ecosystems. More than 200 species of fungi and bacteria have been shown to be capable of degrading hydrocarbons. Adding laboratory grown microbial cultures obtained from autochthonous microorganisms originally isolated from the environment in need of bioremediation, in a process called bioaugmentation, and selected for their potential services is one of the most efficient, and cost-effective methods utilized today, particularly via biodegradation.
To promote sustainability of local mangrove biomes, we grew strains in the laboratory in an aged seawater-sodium nitrate-waste motor oil liquid medium to replicate conditions involving the release of PAHs and other hydrocarbons frommotorboats, terrestrial runoff, and spills. With waste motor oil as their only carbon source, the fungi are able to utilize the oil for growth both singly and as a consortium with potentially synergistic capabilities for degrading the hydrocarbons. In bioaugmentation applications, microbial consortia, often composed of both fungi and bacteria, are consistently demonstrated to perform more efficiently than applications employing only single cultures, presumably due to synergistic effects of the microbes employed, and complementing and increasing populationnumbers of in situ microbial communities(Zhu et al.2001;Tyagietal.2011; Ameen 2015; McGenity et al. 2012; Bell et al. 2016; Dellagnezze et al. 2016; Angelim et al. 2013; Winquist et al. 2014).
Methods
Use of microbial consortia in biodegradation of PHCs
Because autochthonous assemblages of fungi and bacteria selected for degradation efficacy and returned to the environment en masse can be quite effective (Balba et al. 1998; Zhu et al. 2001; Ameen et al. 2015; Priya et al. 2015), we added to our original consortia of biodegrading microorganisms, Aspergillus caelatus, Aspergillus niger, Fusarium solani, Purpureocillium lilacinum. Unfortunately, all samples were lost after hurricane Maria and new fieldwork was necessary to isolate new fungi.
Fieldwork. Soil and submerged decaying leaves samples were collected manually from red mangrove, Rhizophora mangle, located around the Buyé, Boquerón, Playuela and Punta Guaniquilla in Cabo Rojo and La Parguera, Lajas with a disposable spatula and placed in sterile bags. Samples were processed by dilution techniques with the use of a phosphate buffer. Twigs werealso collectedintobags andincubatedatroomtemperatureand examined untilfruitingbodies were observed (Kohlmeyer and Kohlmeyer, 1979). These were later transferred into Petri dishes with appropriate culture media
Fungal and yeast isolation. For isolation of mangrove fungi, different culture media were used. Potato Dextrose Agar (potato extract: 4.0g, dextrose: 20g, agar: 20g and dH2O: 1L) media was used for overall isolation. In addition, Nutrient Agar (beef extract: 3.0g, peptone: 5.0g, agar: 15.0g and dH2O: 1L). Yeast Maltose Extract Agar (dextrose: 10.0g, peptone: 5.0g, malt extract: 3.0g, yeast extract: 3.0g, agar: 20.0g and dH2O: 1L) media was also used for isolation of fungi. Cultures were incubated at 25 - 30˚C for a period of five days. For marine yeast, collected leaves were cleansed with aged seawater or artificial seawater [18g of NaCl, 1.8g MgSO4, 7H2O, 0.18 g of KCl, 0.055g of Tris 1M, 0.072g of NaBr, 0.018g of NaHCO2, and 0.045g of CaCl in 1L of distilled water] Decaying areas were cut with sterile scissors into small pieces (approx. 0.5cm in diameter) and then were directly placed into seawater agar medium (SWA) [5g of dextrose, 1g of peptone, 1g of yeast extract, 10g of agar, 500ml of artificial or aged seawater and 500ml of distilled water at pH 7.0 (Difco Laboratories, Detroit, MI), supplemented with 1g/100mL of penicillinstreptomycin to avoid bacterial growth]. Natural seawater was aged and stored for at least one week before use. Plates were incubated at 25° C for 48 hours. After this time, colonies were transferred into new SWA plates for purification.
Molecular characterization of microorganisms DNA sequencing of appropriate rDNA genes was performed for all new isolates. Samples were extracted and amplified using PCR standard methods and sent out for cleaning and sequencing to Molecular Cloning Laboratories (MCLAB, South San Francisco, CA).
Laboratory PHC degradation studies. As a first approach, all isolates were tested for their capacity to degrade Congo Red (azo dye). Fungi isolates were inoculated in a medium containing Congo Redassolecarbonsource (MgSO4.7H2O:0.5g,HK2PO4:0.6g,H2KPO4:0.5g,NANO3 [10mg/ml]: 200ml and dH2O: 800ml containing 0.04mg/ml of Congo Red) (Kelly, 2016). These were incubated for a period of one week at 25C. Daily observations were made in order to measure the possible degradation of the Congo Red. Fungi that showed degradation were selected for further PHC degradation studies. Microbial consortia using aged seawater-sodium nitrate-waste motor oil liquid medium to replicate conditions involving the release of hydrocarbons from motorboats, terrestrial runoff, and spills were tested. We evaluated growth of strains under different carbon sources derived from PHC. The media contained two Master Mixes (MM): the first (MM1) containing 30ml of used motor oil and the second Master Mix (MM2) using 100ml of NaNO3 in 400ml of aged sea water. This medium was prepared using a concentration of 10% waste motor oil.Eachfungal isolatewas inoculated in aflaskcontaining1mlof MM1and9mlof MM2.Isolates were then incubated at 25C on a shaker for approximately 20 days. Each isolate was inoculated in 4 different flasks in order to measure the biomass every 5 days as a checkpoint. Upon each checkpoint, one flask of each isolate was filtrated using Whatman Filter Papers (0.45m) and dried at45C.Afterfiltering,biomasswasmeasuredforeachsampleusing thefollowingequation: Final Filter Weight – Initial Filter Weight = Biomass of sample. Same procedure was performed for 4 consortia containing different set of fungi.
Enzyme assays for yeasts. Assayforamylaseproductionbymarineyeastswasperformed onStarch Agar plates. This assay served as a good indicator of amylolytic activity of microorganisms. The composition of the media consisted of 3.0 g of Malt Extract, 6.0 g of NaCl, 2.0 g of Starch, and 12.0 g of Agar in 1L distilled water. First, yeasts were inoculated in triplicates and then were incubated for 48 to 72 hours at 25°C until growth was observed. After this period, all plates were stained with Lugol solution (Gram iodine solution: 0.1% I2 and 1% KI) for 2 to 4 minutes to observe the degradation zone (clearing) around the colony. Carboxymethylcellulose (CMC) assay served as an indicator of cellulolytic activity of microorganisms. CMC is a substrate for the
detection of endo-β-1,4-glucanase activity and β-glucosidase activity. The assay for determining cellulase production by marine yeasts was performed on CMC agar plates (NH4H2PO4 1.0 g, KCl 0.2 g, MgSO4.7H2O 1.0 g, Yeast Extract 1.0 g, Carboxymethylcellulose 26.0 g, Agar 1.0 g in 1L dH2O; adjust pH 7.0+/-2). First, yeasts were inoculated in triplicates on CMC agar and then incubated at 25° C for 72 hours, until growth was observed. Subsequently, all plates were flooded with Congo Red solution (1% aqueous) for 15 to 20 minutes. The Congo red was poured out and unbound dye removed via two washes with 1 M NaCl. Regions of the agar where the hydrolysis of CMC occurred were clear, whereas intact CMC retained the Congo Red dye.
-Results and findings
During the re-isolation of new microbes from mangroves we managed to have in pure culture 15 unknown filamentous fungi and 64 yeast strains. Molecular work indicate we managed to recover at least two strains from the original set of fungi. Blastn searches of our new mangrove isolates indicate we have filamentous fungi: Aspergillus aculeatus, Aspergillus nidulans, Diaporthe sp., Emericella sp., Penicillium paxillii, Purpureocillium lilacinum, Tallaromyces ruber, Trichoderma reseei, Thrichosporon asahii and basidiomycete yeasts: Cystobasidium calyptogenae, Cryptococcus sp., Diutina catenulate, Jaminaea sp., Kwoniella mangrovensis, Meyerozyma guillermondii, Oberwinklerozyma yarrowii, Sympodiomycopsis sp., Vishniacozyma sp. and Rhodotorula mucilaginosa.
Consortia characterization included various experimentsforbiomassproductionwithdifferent fungal composition. Almost all the consortia tested presented augmented production compared to the fungal strains growing alone. Single isolates and consortia produced more biomass in higher concentrations of waste motor oil (10%) as the sole carbon source versus a mixture of wood mimic and oil (5% lignin plus cellulose and 5% oil), suggesting that wood is not required for the qualitative expression of lignolytic enzymes involved in the degradation of waste petroleum products.
Consortium Experiment
This graph represents an example of the type of experiments that were performed where 4 consortia with different fungi were grown. CB1CB4 represent consortia. Note that they reach 400mg/ml biomass production while single isolates do not pass 300 mg/ml.
Table. Results from the enzymatic assays of isolated yeast for starch and cellulose.
Starch Hydrolysis
Cellulose Hydrolysis
#2 N/A N/A N/A N/A 42 Pb-t N/A N/A
N/A S5A N/A N/A N/A N/A S5B N/A N/A N/A N/A
5.00 9.00 N/A N/A
N/A N/A N/A N/A
4.00
N/A N/A
PR#20 N/A N/A N/A N/A
PRT1 N/A N/A N/A N/A PRT3 N/A N/A N/A N/A PRT4 N/A N/A N/A N/A
PRT5 N/A N/A N/A N/A
PRT6 N/A N/A N/A N/A
TL#8 N/A N/A N/A N/A
TL#9 N/A N/A N/A N/A
TL#10 N/A N/A N/A N/A
TL#11 N/A N/A N/A N/A TL#12A N/A N/A N/A N/A
TL#15 N/A N/A N/A N/A
TL#21 N/A N/A N/A N/A #8 N/A N/A N/A N/A
N/A N/A N/A N/A
5.00
N/A N/A
N/A N/A N/A N/A Y9 N/A N/A N/A N/A Y10 5.00 8.00 N/A N/A Y12 N/A N/A N/A N/A Z3 4.00 8.00 N/A N/A Z4 3.00 5.00 N/A N/A
Z7 4.00 6.00 N/A N/A
Z8 4.00 6.00 N/A N/A
Z9 4.00 6.00 N/A N/A
Z10 3.00 6.00 N/A N/A
Z14 3.00 6.00 N/A N/A
Z15 3.00 7.00 N/A N/A
Z16 N/A N/A N/A N/A
Z17 N/A N/A N/A N/A
Z18 N/A N/A N/A N/A
Z20 N/A N/A N/A N/A
Z21 3.00 7.00 N/A N/A
PG #3A N/A N/A N/A N/A
PG #3B 4.00 9.00 N/A N/A
PG #4A N/A N/A N/A N/A
PG #8A N/A N/A N/A N/A
PST #2A N/A N/A N/A N/A
PST #7 N/A N/A N/A N/A
PST #7 I N/A N/A N/A N/A PST #7 II 5.00 10.00 5.00 12.00
#8 4.00 7.00 N/A N/A PST #9A 4.00 9.00 N/A N/A
PST #9B N/A N/A N/A N/A
PSM #15 N/A N/A N/A N/A
PSM #18 N/A N/A N/A N/A
Marine yeasts have unique properties such as high osmotic tolerance, higher special enzyme productivity and industrial enzyme production (Zaky et al., 2014). Their environmental role is similar to many other fungi, acting as saprophytes on plant or animal materials, where they preferentially catabolize sugars but can also utilize alcohols, polyols, organic acids, and amino acids as carbon and sources of energy converting these in biomass and by-products, which may be of commercial importance. Amylases are enzymes which hydrolyze starch molecules to give diverse products including dextrins, and progressively smaller polymers composed of glucose units. Of all studied yeasts, 25 were producing enough amylases to be detected in plates. TL#12B2 is an undescribed yeast producing white colonies with mucoroid texture that has the most production of amylases in our study, which needs further characterization Cellulases are the enzymes which degrade crystalline cellulose to glucose with diverse applications in fuel, leather, textile, food, medical, pulp, environmental and agricultural industries The cellulases used in industries require higher activity and stability in order to endure diverse harsh conditions. Marine yeasts have the ability to produce theses enzymes, but apparently not in abundance from our results. Only 7 isolates presented the enzymatic activity in our experiments. Sympodiomycopsis sp. (42 Pb-t) showed promising results with good degradation activity. The other isolates are
unidentified, but also present degradation halos. These yeast isolates need further characterization and a better analysis of enzyme production and kinetics. Several parts of this project have been presented at local and national meetings. We presented results at the XII Symposium Frontiers in Environmental Microbiology: interconnections for progress in February 2019 at Universidad Ana G. Mendez, at the 38th Puerto Rico Interdisciplinary Scientific Meeting/53rd ACS Junior Technical Meeting (PRISM/JTM) in May 2019 and in August 2019at theMycologicalSociety ofAmerica inMinneapolis. Ourfindingscoveredmostlyisolation results and degradation assays of different substrates (see abstracts).
-Objectives accomplished
1. Isolate microalgae and potential members of the Desulfobulbaceae from mangrove habitats for inclusion in our fungal PHC-degrading consortia
2. Culture all consortia members ex situ under oxic and anoxic conditions, in seawater and in sediments, in order to ascertain the optimal assemblage for degrading PHCs
3. Characterize all consortia components with respect to degradation of PHCs, selected metabolites, molecular sequences, and ability to be cultured en masse for use in the field
4. Develop and test mass production of consortia on wood chips, in pellets of agar, agarose, or alginate for introduction into different mangrove sedimentary depths
5. Present our final products to the Departamento de Recursos Naturales y Ambientales del Estado Libre Asociado de Puerto Rico (DRNA) to convince them to permit us to conduct field trials and collect data over a period of years to document beneficial and problematic outcomes
6. Encourage undergraduates currently working in our laboratory on this project to apply to REU and summer internships that can possibly give them the skills needed for future experiments using methods new to our laboratory.
-Discussion of project impacts and products
1%
40%
Very little of this objective was achieved due to hard conditions in the lab. Only a few bacteria were isolated from sediments.
Consortia were tested for oxic conditions in the lab with good results. All tested consortia outperformed individual strains at degrading waste motor oil and biomass production.
80% We managed to isolate and characterized an acceptable amount of filamentous fungi from the mangrove environment. In addition, 64 yeast strains were characterized as well, which are new data for Puerto Rico.
0%
0%
This goal was not achieved due to lack of time.
This goal was not achieved due to lack of time.
100% Three students participated in summer internships. One graduated and is pursuing a PhD degree. One undergraduate managed to submit proposals and secured 2 scholarships.
The main products of this project are the cultures that were obtained in the new sampling of mangrove environments with the additional information processed in the laboratory. Fungi are gooddegradersofwaste motor oil andcanbe easilygrowninthe laboratoryforfurther applications in bioremediation. The experiments presented here serve as proof-of-concept evidence to apply in field bioaugmentation tests. We were unable to reach this part of the project due to lack of time and infrastructure problems, but the initial part of the work is done. Isolates will be deposited in resilient facilities at the Puerto Rico Science and Technology Trust to guarantee their preservation and availability for future studies.
-Recommendations
1. Now that the microbes have been secured and characterized, the main recommendation is to follow through the experiments for identifying which compounds are selectively consumed by the consortia.
2. Select consortia to produce en masse for application in mock set-up experiments with sediments.
3. Determine efficiency of each consortia and scale up experiments.
4. Design a delivery system for the bioremediation process.
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