Item No. 1 of 1

INVESTIGATOR: Mitra, M.

PERFORMING INSTITUTION:
University of Maryland Eastern Shore
11868 College Backborne Road
Princess Anne, MARYLAND 21853

PROFESSIONAL DEVELOPMENT: GRACILARIA (RED MACROALGA) BIOREFINERY SYSTEM: AN INTEGRATED APPROACH FOR BIOETHANOL PRODUCTION

NON-TECHNICAL SUMMARY: The professional development project will provide essential support for the investigator to conduct sabbatical research on the efficient methodologies in integrated aquaculture and for the generation of sustainable bioethanol from a red macroalgal feedstock, Gracilaria, at a pioneer institute of science, engineering, and technology in the USA. The overall goal of this professional development project is to address some of the critical needs of sustainable renewable energy generation and mitigating the harmful effects of the environment through the implementation of a seaweed/macroalgal biorefinery with co-cultures of fish-plant species. The specific objectives are: 1) to implement and maintain an integrated aquaculture system with species of Gracilaria, and edible white shrimp, Litopenaeus vannamei for evaluating the bioremediation potential of the macroalga in a biorefinery setting; 2) to utilize the macroalga, Gracilaria for the production of sustainable bioenergy in the form of bioethanol; and 3) to enhance professional training, networking, and strengthening collaborations between two minority serving institutions. The professional development will facilitate the University of Maryland Eastern Shore (UMES) to have the capability for implementing bioenergy research in education (courses and experiential learning activities), student and faculty training (workshops), and outreach activities to the community in the areas of sustainable aquaculture, bioenergy, and bioremediation. In addition, research collaborations with another minority institution, California State University at Fresno, CA, will be expanded and strengthened, establishing a platform for future student exchange and faculty visits.

OBJECTIVES: The overall goal of this professional development project is to address some of the critical needs of sustainable renewable energy generation and mitigating the harmful effects of the environment through the implementation of a seaweed/macroalgal biorefinery with co-cultures of fish-plant species. The specific objectives are: 1) to implement and maintain an integrated aquaculture system with species of Gracilaria, and edible white shrimp, Litopenaeus vannamei for evaluating the bioremediation potential of the macroalga in a biorefinery setting; 2) to utilize the macroalga, Gracilaria for the production of sustainable bioenergy in the form of bioethanol; and 3) to enhance professional training, networking, and strengthening collaborations between two minority serving institutions. The professional development will facilitate the University of Maryland Eastern Shore (UMES) to have the capability for implementing bioenergy research in education (courses and experiential learning activities), student and faculty training (workshops), and outreach activities to the community in the areas of sustainable aquaculture, bioenergy, and bioremediation. In addition, research collaborations with another minority institution, California State University at Fresno, CA, will be expanded and strengthened, establishing a platform for future student exchange and faculty visits.

APPROACH: FIRST PHASE--- During the first phase, cultures of Litopenaeus vannamei post larvae will be obtained from Miami Aquaculture and stocked in temporary stock tanks with sterilized seawater and constant aeration will be provided for a period of two weeks. The shrimp will be fed with harvested Spirulina and soy bean meal obtained from a screw press extrudate. A weekly 50% water change will also be undertaken during the acclimation process. Gracilaria tikvahiae will be harvested from the coastal bays of the Delmarva Peninsula. The macroalgal samples will be cleaned and maintained in sterilized seawater for two weeks prior to the trials with constant aeration to prevent frond deterioration, and artificial light. Samples will be fertilized weekly with Micro Algae Grow to main the cultures. Water quality parameters including ammonium, nitrate, nitrite, and phosphate levels will be recorded at least once per week for the duration of each trial. The following trials/cultures will be maintained and compared: monocultures of Gracilaria; monocultures of shrimp; and integrated cultures of Gracilaria and shrimp. The principal performance metric for Gracilaria and shrimp performance for the study will be the specific growth rate. In addition to the growth rate, the mass, Gracilaria tip elongation, shrimp body length, shrimp mortality, and nutrient levels (nitrate, nitrite, phosphate, and ammonium) will be analyzed using an ANOVA and Tukey's separation of means to ascertain differences between culturing conditions (mono vs. integrated). SECOND PHASE--Sabbatical Research on Bioethanol Production at California State University Fresno--During the three month period, Dr. Mitra will collaborate with Dr. Santanu Maitra on the following: a) Agar Extraction Procedure from Gracilaria tikvahiae--following the extraction; the agar pulp will be analyzed for the available total sugar, total reducing sugar content; and glucose based on the modified Miller (1959) method; b) Enzymatic hydrolysis of Gracilaria pulp-Experiments will be set up to determine the optimum saccharification conditions and enzyme efficiency and compared with the saccharification efficiency of other seaweeds as reported in documented studies (Yanagisawa et al. 2011; Khambhaty et al. 2011); c) Fermentation of enzymatic hydrolysates with strains of Saccharomyces cerevisiae will be conducted for obtaining ethanol; and d) Residue Analysis-following the fermentation, the residue pulp will be used for analyzing the organic matter content for the purpose of utilizing it for a biofertilizer.

PROGRESS: 2019/04 TO 2020/03
Target Audience:Target audiences--Undergraduate students in the Marine and Estuarine Ecology (BIOL 600) at University of Maryland Eastern Shore course that was offered in Spring of 2020, learned about various marine feedstock and how they can be used for the generation of biofuels. They also learned about Gracilaria's efficacy as a biofuel feedstock and how to turn a nuisance alga into an investment in cleaner energy. They also learned about culturing marine microalgae and how these could be used for the generation of biodiesel. Such an approach will provide avenues for sustainable aquaculture and energy systems via production of economically valuable and renewable products. The undergraduate and graduate students from the collaborators' labs at California State University at Fresno, CA, were also trained in usage of macroalgae for the purpose of generating biofuels during the investigator's visit in early fall of 2019 . More than 50 percent of these students from the above-mentioned universities were from the minority and educationally-disadvantaged populations. Efforts- 1) Increased of content knowledge in the areas of Integrated MultiTrophic Aquaculture; bioenergy from feedstock like micro and macro-algae; 2) Hands-on laboratory and field experiences with respect to utilizing macroalgae for the purpose of producing biofuels were replaced with virtual experiences, as the university was shut down due to the current pandemic of Covid-19. These activires were included in the Marine and Estuarine Ecology course, and also a mini online workshop was conducted for the collaborators and their students at California State University, Fresno, CA in Fall of 2019. Changes/Problems:The major problems encountered by the Principal Investigator (Dr. Mitra) and her collaborators were connected to the closure of the universities due to the current pandemic of Covid-19. The University System of Maryland required all universities to teach and conduct research (as much as possible) on the virtual platform. Professional travel is also restricted. The University System of California also adopted similar measures of teaching courses online and restricting research and travel as well as hosting collaborators/scholars. The last component of the research for training on the generation of ethanol from seaweeds could not be continued due to the current situation. It is anticipated that Dr. Mitra will be requesting an extension of the project beyond November 2020, to accomplish the final objective of the project. What opportunities for training and professional development has the project provided?The project has served as an initiative for faculty professional development and collaborative research in applied agricultural and natural sciences at the University of Maryland Eastern Shore (UMES). The research program will enhance the opportunity for sustainable aquaculture and bioethanol production at the university, facilitating UMES to have the capability for implementing education, student training, and outreach activities in the areas of sustainable bioenergy, bioproducts, and bioremediation. Already some of the activities have been infused in two courses: Marine and Estuarine Ecology and Introduction to Bioenergy and Bioproducts. In addition, research collaborations with another minority institution, California State University, Fresno, CA, (Hispanic serving institution) is expanded and strengthened through this professional development project leading to publishing book chapters and the book on Bioenergy; and also establishing a platform for future student exchange and faculty visits. The students and faculty in the Chemistry and Biology departments at Cal State Fresno learned about the importance of feedstock like macroalgae and their importance in marine aquaculture along with generation of bioethanol and bioproducts. Due to the pandemic of COVID-19, the visit to California State University, Fresno has been postponed until next year, as the university will continue to operate on the virtual platform. How have the results been disseminated to communities of interest?An invited panel presentation on Bioenergy, Bioproducts, and Sustainability was led by Dr. Mitra at the American Society of Engineering Education's annual conferences in Tampa Florida, in June of 2019. The panel reached more than 100 people who attended the presentations. Dr. Mitra also shared the results with the collaborators and their students in Chemistry and Biology courses along with university faculty at California State University, Fresno, during her visit in summer/spring of 2019. What do you plan to do during the next reporting period to accomplish the goals?Dr. Mitra will be trained on the approaches to generate bioethanol from potential seaweeds in the department of Chemistry at the California State University Fresno, CA. She will be trained on the various techniques for enzymatic hydrolysis of algal pulp along with chemical analysis; and ethanol fermentation with microbes at Dr. Maitra's laboratory. Part of the training was scheduled for summer of 2020, and the remaining training for October through November of 2020. Due to the pandemic of Covid-19 with restricted travel and universities going online/virtual platforms for teaching, research, and collaborations, it is anticipated that Dr. Mitra can only travel to Fresno when the restrictions due to travel and research are eased at both the universities. So the expected dates will be in Spring or Summer of 2021 after the request for no-cost extension is granted under special conditions.

IMPACT: 2019/04 TO 2020/03
What was accomplished under these goals? During the first phases of the project, the construction and installation of the core components of a prototype Integrated MultiTrophic Aquaculture system has been completed. The tanks have been constructed from lumber recycled from shipping crates, then insulated with construction 1 inch foam, and finally lined with 6 mm construction plastic. The necessary pumps and plumbing have also been installed and the generator has been acquired. More efforts have been focused on racewaytype growth which has shown much promise for the intensive culture of shrimp and may benefit from the integrated remediation abilities of integrated algaculture. A hybridized closed photobioreactor has been constructed to culture the blue green microalga, Spirulina, for carbon capture as well as a source of shrimp nutrition. During the first phase, cultures of Litopenaeus vannamei post larvae have been obtained from Miami Aquaculture and stocked in temporary stock tanks with sterilized seawater and constant aeration have been provided for a period of two weeks. The shrimp was fed with harvested Spirulina and soy bean meal obtained from a screw press extrudate. A weekly 50% water change was also undertaken during the acclimation process. Gracilaria tikvahiae was harvested from Assawoman and Chincoteague bays of the Delmarva Peninsula. The macroalgal samples were cleaned and maintained in sterilized seawater for two weeks prior to the trials with constant aeration to prevent frond deterioration, and artificial light. Samples were fertilized weekly with Micro Algae Grow to maintain the cultures. Water quality parameters including ammonium, nitrate, nitrite, and phosphate levels were recorded once per week for the duration of each trial. The following trials/cultures were maintained and compared: monocultures of Gracilaria; monocultures of shrimp; and integrated cultures of Gracilaria and shrimp. The principal performance metric for Gracilaria and shrimp performance for the study was the specific growth rate. In addition to the growth rate, the mass, Gracilaria tip elongation, shrimp body length, shrimp mortality, and nutrient levels (nitrate, nitrite, phosphate, and ammonium) are being analyzed using an ANOVA and Tukey's separation of means to ascertain differences between culturing conditions (mono vs. integrated). During the visit to California State University, Fresno, in summer to fall of 2019, Dr. Mitra received training on the various techniques of algal culturing and extractions. She was trained on the the Algem HT24, a high throughput, compact, computer-controlled photobioreactor for cultivation of various microalgae such as Chlorella vulgaris, Arthrospira platensis, Haematococcus pluvialis, and Dunaliela salina.

PUBLICATIONS (not previously reported): 2019/04 TO 2020/03
1. Type: Books Status: Published Year Published: 2020 Citation: Mitra M, and Nagchaudhuri A. 2020. Practices and Perspectives in Sustainable Bioenergy. A Systems Thinking Approach. Springer Publishers ISSN 1865-3529.
2. Type: Book Chapters Status: Published Year Published: 2020 Citation: Nagchaudhuri A, and Mitra M. 2020. Bioenergy Systems-A Wholistic Approach. In Mitra M Nagchaudhuri A (eds). Practices and Perspectives in Sustainable Bioenergy. A Systems Thinking Approach. Springer Publishers ISSN 1865-3529. pgs 1-12.
3. Type: Book Chapters Status: Published Year Published: 2020 Citation: Mitra M, Henry X, Nagchaudhuri A, and Maitra K. 2020. Photobioreactors for Bioenergy Systems and Lipid Extraction Methods from Microalgae. In Mitra M Nagchaudhuri A (eds). Practices and Perspectives in Sustainable Bioenergy. A Systems Thinking Approach. Springer Publishers ISSN 1865-3529. pgs 131-157.