Progress 09/01/13 to 08/31/17

Outputs
Target Audience:Target Audiences Included: 1. Students at Cal Poly Pomona and Cal Poly San Luis Obispo, commonly first generation college students, and some graduate students, and mostly Hispanic, were recruited to work on the algal research project. 2. Academia, various government departments (including USDA and EPA), NGOs, dairy farmers, industry, algal conferences [e.g., International Conference of Algal Biomass, Biofuels and Bioproducts (ABBB) and Algal Biomass Organization (ABO)], book publishers (Elsevier), and peer reviewed scientific journals (PLOSone), researchers working with algae, and commercial algae producers. Changes/Problems:Delays with Starting Project: Our pond research started 6 months later, about March 2014, after the 09/01/2013 project award date. Research in ponds was initiated several months later after Cal Poly San Luis Obispo (CPSLO) acquired its sub award from Cal Poly Pomona (CPP). Progress at CPP and USDA-ARS depended on algae samples supplied from the ponds at CPSLO. We were not able to collect samples in Winter and Spring 2014 since our newly established ponds had not attained steady-state growth conditions that allowed drawing out of algal biomass from ponds without negatively impacting growth. This was mostly ascribed to the cold weather that discouraged luxuriant growth of algae. From summer 2014, we successfully collected seasonal algae samples from the CPSLO ponds. We had problems associated with uncontrollable changes in the weather at the CPSLO pond bioreactors, which included, very heavy rains, persistent droughts (2013-2016) and cracking of our open raceway, paddle-wheeled ponds, and subsequent repairs that affected steady progress in sample collection from the ponds. Bacteria and fungi that invariably grow faster than algae at times overwhelmed algal growth and hindered isolation of target algae strains in pure cultures. This drawback was often encountered in the lab. In 2016 (CPP) we had serious fungal contamination issues with our algal cultures and this halted our DNA sequencing efforts (where pure single-culture DNA is mandated). Several fungicidal and fungi-static agents were screened for efficacy before we could progress. Cyanobacteria and Cyanotoxin Detection: Other setbacks pertained to method development, for example the difficulties in acquiring relevant or appropriate quality control cyanobacterial algae strains for routine use in calibration of genotype or toxin tests. Some cyanobacterial strains are not well-characterized for toxins they produce as they are only indicated as "toxin-producing" and there is severe reluctance by other researchers to provide the strains that produce toxins of interest to our study that they reported in peer-reviewed literature or other reputable forums. We herefore characterized strains that have been "suspected" to produce toxins to see if they produced the toxins of interest. Algae cultures were prone to contamination and/or predation by other organisms while in culture. We succesfully developed end-point PCR protocols to reliably detect cyanobacteria, and the cyanobacteria toxins microcystin/nodularin and anatoxin-a. We were not been able to identify quality control strains for the other target toxins, i.e., nodularin and cylindrospermopsin. Commercial labs were able to test for (presence/absence and quantitatiion) all 4 toxins in our algae samples. We had originally proposed use of the TC20 Cell Counter and cultured mamaliuan cells for toxicity studies, however we opted to use toxin-specific ELISAs and GC-MS toxin detection and quantitation.The TC-20 counter was not able to read/count trypan blue stained cells hence it could not be used with cultured cells to assess toxicity. Metagenomic Studies: For metagenomic studies Pyrosequenceng was replaced with Illumina's Miseq sequencing platform which yields more in-depth data. Pyrosequencing has been surpassed by the latter method. Problems with Bioreactors/Ponds: Good nutrient uptake rates were measured during this research while moderate algae biomass production rates (g algae/m2/d) and DLE treatment capacities (L DLE/m2/d) were measured. There are still two major challenges for this treatment system to overcome, to become a viable option in the future. The first challenge centers around the treatment process being algal vs. bacterial. If this process is to be a truly algal based treatment system then there needs to be a DLE pretreatment step that can remove a large fraction of the suspended solids in the DLE. This will remove a large fraction of the organic nutrients (those not immediately available to the algae) and it will reduce some of the dark color, which prevents light penetration into the algae raceways and thus reduces algal productivity. This step may lead to a 50% improvement in algal productivity, but may be very costly. The second problem pertains to the cost of harvesting the algae biomass. Current industry harvesting utilizes chemical and mechanical harvesting strategies or a combination of both, including; centrifugation, dissolved air floatation, and chemical flocculation, among others. A biological method could reduce these costs. The bioflocculation strategy utilized in these studies worked very well. One challenge on scale up is the requirement of a raceway that does not have any quiescent zones or the algae will settle in those areas and senesce. These types of quiescent zones are typically found around the 180º bends. A continuous biological harvest strategy could be applied but might require a mechanical method following the biological process to thicken the biomass to the desired concentration. Even with this mechanical process, costs would be less due to the biological thickening before the mechanical thickening step. Problems with Final Scale-up Using Well-characterized Algal Strains (Goal #3): Algal species from samples of the outdoor raceways at CPSLO were isolated and cultured on plates at CPP. The isolates were to be amplified and sent back to CPSLO for culture in sequential 2.5 L PBRs, 150 L indoor raceway and then to the 971 L outdoor raceway ponds. The scale up units at CPSLO were tested with an indoor monoculture to evaluate the scale up process. However, problems were encountered with the amplification process. Some of the challenges encountered with this process resulted from the nature of moving the outdoor culture to the indoor environment and contamination during the amplification process. What opportunities for training and professional development has the project provided?Student Training: At least 20 undergraduate students and 6 MS students worked on this project, some for multiple years. Some will become algae experts of the future. New tools were used and new technics learned by both students and research scientists. Students learned and acquired research experiences and earned internship or research units that contributed to their graduation, including acquisition of life-long skills that enhanced their resumes and employability, and competitive preparation for graduate school admission. One student (Amera Kmech) enrolled on an MS program and worked with algae and successfully completed thesis research focusing on lipid analysis of algae isolated seasionally from the ponds. Two students are currently enrolled on MS programs with research focusing on further characterization of our algae isolates and method development . All students working on the algal project attained greater proficiency in ability to conduct research by interacting with their mentors (PDs and co-PDs). The students also developed advanced professional skills. They improved skills in use of cutting edge instrumentation, data collection, storage, analysis and interpretation, as well as data dissemination at conferences (local and international) via poster and oral presentations. Some of the students had never prepared a poster or presented at a professional conference, thus they were mentored to develop and refine these skills. Professional development activities resulted in increased knowledge and skills that were acquired in the lab training environment through targeted reading and training materials (e.g., journal publications, reputable websites and YouTube instructional videos). One MS student received a PPOHA-MENTORES Fellowship for $7,500 from the PPOHA-MENTORES grant for Energy- or Water-related projects for his work on our algae project. Senior Personnel: Faculty and other senior researchers working on the project also learned new cutting edge technics working with algae.Data and Research Materials: We have significantly enhanced the quantity and quality of data collected during the second year of this project. Attainment of steady-state in the algal bioreactors/ponds has enabled continuous manipulation of experimental variables and data collection. Instruments or Equipment: Two new ponds (bioreactors, identical to the four we started with on this project) were installed (with funds from a non-USDA source) at the dairy lagoon site at Cal Poly San Luis Obispo (CPSLO) in 2015. The new ponds were seeded with algae (starting August, 2015). The additional ponds enhanced degrees of freedom, improved the quality of data collected, and enabled better controls for the experiments we conducted, as well as enhanced parameters pertinent to our model development using data generated from the 4 paddle-wheeled algal ponds (CPSLO) and feedback from Phenometrics bioreactors (CPP). Research Protocols: Standard operating research protocols were developed and refined for conducting routine analyses: e.g., pond analysis (pH, BOD, N, P), algal biomass compositional analysis (N, P, amino acid and fatty acid profiles), and detection of cyanobacteria and their toxins, and DNA extraction for use in metagenomics/microbial community studies using MiSeq or Shotgun sequencing. Databases: Extensive databases were initiated and developed for pond data, compositional analysis, and metagenomic analysis of pond microbiota. For example, the databases developed using MiSeq Illumina NGS were analyzed enabled genus and species identification of bacterial (including pathogens) and algal species, virulence encoding genes etc., in the ponds in different seasons. Physical Collections: We isolated, purified and cryopreserved (at -80ºC) algal species isolated from the bioreactors/ponds that were/are being further characterized for nutritional profiles and future use in algal biomass or feed manufacture. Seasonal samples from the ponds were preserved for future analyses of toxins and pathogens. How have the results been disseminated to communities of interest?Dissemination venues for our research project included: authored book chapters, peer-reviewed journal articles, published conferences abstracts, and attending various conferences/symposiums (local/international. including ASM, ABBB, ABO, SCCUR, etc.), and USDA Annual Project Directors' meetings. The USDA Project Directors Meeting Abstract Booklet and oral presentations are available online and are accessible to diverse constituents or stakeholders (USDA, Academia, Students, Farmers, Industry, NGOs). Other conference presentations (oral/posters) are available as hard copy proceedings, as well on-line at conference websites. Additional disseminatiion forums included USDA annual reports and the final USDA report, including one project-related MS thesis. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? IMPACT: Removal of greater than 90% soluble nitrogen (N) and 50% soluble phosphate (P) was achieved. This translates to 8-16 acres of algae ponds treating dairy lagoon effluent (DLE) from a 1000 cow dairy generating 100-650 kg of algae biomass dry weight/day (wt./d). Seasonally-dominant algal strains were isolated, identified then characterized for growth and nutritional profiles and were frozen stored for future use in production of safe algae feeds. Routine methods for detection of cyanobacteria and their toxins were developed. Cyanotoxins and pathogens were not found in most algae pond samples. GOAL #1: GENERATE EXPERIMENTAL PILOT PLANT DATA AND CALIBRATE OPTIMIZATION MODELS Accomplishments: >90% soluble N and 50% soluble P removal could be achieved at DLE feed rates of up to 6 L/m2 of algae reactor/d. A 1,000 cow dairy would require 8-16 acres of algae ponds to treat DLE resulting in 100-650 kg of algae biomass dry wt./d. Bioflocculation was achieved and it could result in significant biomass harvesting cost savings. Experimental Overview and Operation: Four outdoor pilot reactors (30-cm deep, 971-L, paddle wheel-mixed raceway ponds) were operated for 2.5 years and seasonal data was collected. Ponds were fed DLE and operated in semi-batch mode. Water exchanges occurred once/d, 7d/week. DLE was added daily at the time of exchange at 40% or 80-100% of the predicted N needs, the remainder came from synthetic fertilizer. Experiments were conducted targeting addition of 1-2.8g of N/m2/d depending on season. A model was developed based on multiple variables: primary nutrient source, hydraulic residence time (HRTs), solar radiation, water temperature, available nutrient concentration, etc. Samples of DLE and reactor water were characterized for nutrient content. The data were analyzed to determine seasonal nutrient uptake rates at HRTs ranging from 2.5-10.5 d. Units fed 40% DLE vs. 80-100% DLE had slightly higher nutrient uptake rates. A CO2-based pH control system was incorporated. A bioflocculating algal community was successfully developed. Harvesting of algal biomass was achieved via gravity settling; 60%-90% of volatile suspended solids could be settled within 15 min. Model Development: An empirical model was developed to predict land requirements for DLE treatment using the outdoor reactors. Best uptake rates were in summer and fall mostly due to longer daylight periods and higher temperatures; nutrient uptake rates reached 1.2 g N/m2/d and 0.3 g P/m2/d. Winter and spring rates reached 0.5 g N/m2/d and 0.15 g P/m2/d. The model suggested that ~6 L/d of DLE/m2 of reactor, or 3% DLE addition with the daily water exchange could be treated. When feeding higher DLE concentrations bacterial treatment predominated algal treatment. Estimates indicated 3-12g of algae biomass could be produced/m2 of reactor/d. GOAL 2: MAXIMIZE THE NUTRITIONAL VALUE OF PRODUCED ALGAE FOR ANIMAL FEED Accomplishments: Seasonally-dominant algal strains were isolated, identified then characterized for growth and nutritional profiles. Microscopic data correlated with DNA sequence analysis data (Goal 4). Isolation and Identification of Algae and Growth Modelling in Bioreactors: Seasonal pond sample sets (n=7) were processed to assess algal community diversities. Seasonally dominant strains were isolated (simple plating or single-cell manipulator). Axenic isolates were cultured and sequenced targeting the ITS 4-5 intergenic region and identified species (Scenedesmus, Chlorella, Desmodesmus, etc.). The mostly axenic isolates (n=128) were characterized for growth patterns, heterotophy, and C and N preferences that support rapid growth and lipid biosynthesis under diurnal light cycles. Stock cultures were cryopreserved. Experiments were conducted in benchtop Phenometrics bioreactors to determine optimal diurnal cycles of light, temperature and light intensity as a function of stirring, cell density and CO2 enrichment on growth. Harvested biomass was analyzed to assess chlorophyll:dry wt. ratios to determine the proportion of algae. Biomass in the control ponds (inorganic NPK) in summer and winter had ~ 95% chlorophyll/biomass vs. axenic cultures. This ratio dropped to 79% in summer and 53% in winter in DLE ponds indicating a higher proportion of heterotrophs:algae biomass in the C-rich DLE. Compositional Analysis: Promising strains harvested at various stages of growth under controlled bioreactor conditions and pond biomass harvested seasonally were used for proximate analysis. Total lipids were determined gravimetrically and correlated with Bodipy fluorescent staining. Triacyglycerides were profiled and quantified using GC-FID and GC-MS. Fatty acid methyl esters ranged from C10-C22. A majority were C16 and C18, with varying degrees of saturation. Soluble proteins (micro-Bradford method) and nucleic acids (propidium iodide fluorescent assay) were quantified. Soluble/insoluble carbohydrates and proteins were hydrolyzed and the monomers are currently being identified and quantified using NMR. GOAL 3: OPTIMIZE PATHOGEN INACTIVATION METHODS Accomplishments: We commenced and are continuing studies employing optimized model conditions for algal biomass production using well-characterized seasonally-dominant strains. There are indications pathogens die-off in the ponds (Goal 4). GOAL 4: QUANTIFY AND CONTROL ANY CYANOBACTERIAL TOXINS Accomplishments: We developed routine methods for cyanobacteria and cyanobacterial toxin detection and detected and quantified toxins in pond samples as well as characterized species diversity in seasonal pond samples using MiSeq and Shotgun sequencing. Cyanobacteria and Cyanotoxin Detection: We successfully generated standard curves for quantification of cyanobacteria using 16S rRNA and rpoC1 gene sequences and cyanotoxins targeting mcyE and anaC gene sequences. Toxin analysis targeted microcystins/nodularins (MCs/NODs), cylindrospermopsin (CYN), anatoxin-a (ANTX-A), and paralytic shellfish toxins [PSTs/saxitoxins](STX). For analysis of MCs/NODs and STX toxin-specific ELISA kits were utilized (Abraxis, Warminster, PA). ANTX-A and CYN detection was conducted using LC-MS/GS. MCs/NODs were detected above the detection limit for a treatment algae paste sample (43 ng/ml), whereas, ATX, CYN and STX were not detected. MCs/NODs, ATX, CYN and STX were not detected in the Control Unit algae paste. Seasonal pond samples (controls and treatments units) were negative for MCs/NODs, ATX, CYN and STX (Greenwater Laboratories, Palatka, FL). Microbial and Cyanobacterial Community Structures in Pond Samples: DNA extracted from seasonal samples was used for analysis of total bacteria and cyanobacteria using Illumina's MiSeq next generation sequencing platform (Illumina Inc., San Diego, CA). The microbiome profiles of the DLE and bioreactors formed distinct clusters. Preliminary V4 16S rDNA analysis and bioinformatics enabled identification of dominant microalgae and cyanobacteria in the samples. Shotgun sequencing, conducted to relate composition of bacterial community to functions generated 1579 and 4999 bacterial genera and species, 2458 virulence-associated genes, 289 and 512 fungal genera and species, and 72 and 184 protist genera and species (Cosmos ID, Rockville MD). Pond Pathogen Characterization and Quantification: DNA extracted from seasonal samples was tested for presence of select pathogens. Shiga toxin-producing E. coli O157 (STEC O157) screening was conducted using multiplex qPCR and digital droplet PCR (ddPCR) targeting stx1, stx2, eae, and rfbO157 genes associated with STEC O157. A few samples were positive for these genes suggesting this pathogen dies out in the ponds. Detection and quantification for qPCR ranged 103-107 CFU/ml, whereas, ddPCR ranged 1-104 CFU/ml. Representative seasonal samples (n=12) tested negative for Salmonella spp. and Listeria monocytogenes. DLE samples (n=4) tested positive for nonpathogenic Listeria innocua.

Publications

• Type: Book Chapters Status: Awaiting Publication Year Published: 2017 Citation: M. Murry, S. Murinda,S. Hung, G. Schwartz, A.M. Ibekwe, T. Lundquist. 2017. Bioconversion of agricultural wastes from the livestock industry for biofuel and feed production. In: Handbook of Biotechnology for Renewable Fuels: Technology Assessments, Emerging Industrial Applications, and Future Outlooks; Majid Hosseini, Editor. Elsevier Publishing Company. Chapter 12. 30 pages.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Ibekwe, A. Mark, Shelton E. Murinda, Marcia A. Murry, Gregory Schwartz, Trygve Lundquist. 2017. Microbial Community Structures in Algae Cultivation Ponds for Bioconversion of Agricultural Wastes from Livestock Industry for Feed Production. Science of the Total Environment. 580:1185-1196.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Joe McHugh, Isis Janikarn-Urena, Natalie Euloglio, Amera Kchech, Shelton E. Murinda, Gregory Schwartz, Marcia Murry, Mark Ibekwe, Trygve Lundquist. Production of Algae Feeds from Dairy Waste. Inaugural Annual Agricultural Research Institute (ARI) Principal Investigator Meeting. Sacramento, CA. September 7, 2017.
• Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Amera Kmech. The Quantification of Fatty acids in Microalgae Using GC-FID and GC-MS Chemistry (MS Thesis)
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Lari Z., N. Moradi-Kheibari, H. Ahmadzadeh, P. Abrishamch, N. R. Moheimani and M.A. Murry Bioprocess engineering of microalgae to optimize lipid production through nutrient management. J Appl. Phycol. Published online: 10 June 2016. doi:10.1007/s10811-016-0884-6.
• Type: Book Chapters Status: Awaiting Publication Year Published: 2017 Citation: Moradi-Kheibari, N, H. Ahmadzadeh, A.F. Talebi, M. Hosseini, M. A. Murry. Recent Advances in Lipid Extraction for Biodiesel Production. In Press: Handbook of Biotechnology for Renewable Fuels: Technology Assessments, Emerging Industrial Applications, and Future Outlooks, 2017. Elsevier. ed. Majid Hosseini.
• Type: Book Chapters Status: Awaiting Publication Year Published: 2017 Citation: Moradi, KN, Ahmadzadeh H, Murry M.A, Hui Ying Liang,, Majid Hosseini. Fatty acid profiling of biofuels produced from microalgae, vegetable oil, and waste vegetable oil. In Press 2017: Handbook of Biotechnology for Renewable Fuels: Technology Assessments, Emerging Industrial Applications, and Future Outlooks. Elsevier. ed. Majid Hosseini.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Marcia Murry, Shelton Murinda. Bioremediation: A Practical Approach to Algae Biomass Production. Provosts Symposium on Faculty Scholarship, December 12, 2015. California State Polytechnic University, Pomona, CA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Tim Yoon, Julia Gonzalez, Roger Lee, Moon Seo. Mentor: Marcia A. Murry. A rapid sampling technique for isolating lipid-rich algae strains from environmental samples. 22nd Southern California Conferences for Undergraduate Research (SCURR). November 22, 2014. California State University, Fullerton, CA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Joseph McHugh, Kaylynn Atkinson, Shelton Murinda, Marcia Murry. Universal detection of cyanobacteria and their toxins using PCR/qPCR. 22nd Annual Southern California Conference on Undergraduate Research. November 22, 2014. California State University Fullerton, CA
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Alyssa Sancio, Joseph McHugh, Kaylynn Atkinson, Shelton Murinda, Marcia Murry. Universal detection of cyanobacteria and their toxins using PCR/qPCR. 10th Annual Research Symposium. May 29th, 2015. California State Polytechnic University, Pomona, CA
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Mark Ibekwe, Shelton Murinda, Marcia Murry, Gregory Schwartz, Trygve Lundquist. Impact of Different DNA Extraction Methods on Total Bacterial and Cyanobacterial Community Structure in Algae Cultivation Reactors. American Society for Microbiology, 115th Annual General Meeting. May 30-June 2, 2015. New Orleans, Louisiana
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Shelton Murinda, Marcia Murry, Mark Ibekwe, Gregory Schwartz, Trygve Lundquist. Algae for conversion of manure nutrients to animal feed: Evaluation of advanced nutritional value, toxicity, and zoonotic pathogens. USDA-NIFA NIWQP and AFRI Project Directors Annual Meeting. July 26-29, 2015, Greensboro, NC.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Isis Janilkarn-Urena. Algal bioremediation of organic wastes coupled to biomass production for feed and biofuels. RISE Symposium September 4th, 2015. California State Polytechnic University, Pomona, CA. Mentor: Marcia A. Murry.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Natalie Eulogio. Can algae save the dairy industry? RISE symposium September 4th, 2015. California State Polytechnic University, Pomona,CA. Mentor: Marcia A. Murry.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Janilkarn-Urena, N. Eulogio, M.A. Murry, S.E. Murinda, A.M. Ibekwe, G. Schwartz, T. Lundquist. Bioprocess Engineering of Native Algae Strains to Optimize Bioremediation coupled to Feed Production. Southern California Conference for Undergraduate Research (SCCUR). UC Riverside, CA. 11/12/16.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: S. Wu, Joe McHugh, A. Sancio, I. Janilkarn-Urena, N. Euloglio, A. Kchech, S.E. Murinda, G. Schwartz, M. Murry, A. M. Ibekwe, T. Lundquist. Production of Algae Feeds from Dairy Waste. 16th Annual ARI Showcase. Cal Poly Pomona, CA. 11/3/16.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Janilkarn-Urena, N. Eulogio, M.A. Murry, S.E. Murinda, A.M. Ibekwe, G. Schwartz, T. Lundquist. Bioconversion of agricultural wastes from the livestock industry for biofuel and feed production. 1st International Conference: Bioresource Technology for Bioenergy, Bioproducts and Environmental Sustainability. 10/23-10/26/16. Sitges, Spain.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: S. Murinda, M. Murry, G. Schwartz, T. Lundquist, A. M. Ibekwe. Algae for Conversion of Manure Nutrients to Animal Feed: Evaluation of advanced nutritional value, toxicity, and zoonotic pathogens. USDA Annual Project Directors Meeting. Washington DC. 10/12-13/16.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: J. McHugh, A. Sancio, S.E. Murinda, G. Schwartz, M. Murry, A.M. Ibekwe, T. Lundquist. Detection of cyanobacteria and their toxins for safe algae-based feed production. 6th Intnl. Conference on Algal Biomass, Biofuels and Bioproducts. 6/26  6/29/16. Paradise Point, San Diego. CA
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: J. McHugh, A. Sancio, S. E. Murinda, G. Schwartz, M. Murry, A. M. Ibekwe, T. Lundquist. Detection of cyanobacteria and their toxins for safe algae-based feed production. American Society for Engineering Education PSW Conference. 4/21-4/23/16. Cal Poly Pomona, CA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: W. Shires, S. Atmadja, G. Schwartz, S. Murinda, M. Murry, A.M. Ibekwe, T. Lundquist. Algae for conversion of manure nutrients to animal feed: Development of an algae production model. 6th Intnl. Conference on Algal Biomass, Biofuels and Bioproducts. 6/26  6/29/16. Paradise Point, San Diego, CA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: I. Janilkarn-Urena, N. Eulogio, M.A. Murry, S.E. Murinda, A.M. Ibekwe, G. Schwartz, T. Lundquist. Bioprocess Engineering of Native Algae Strains to Optimize Bioremediation coupled to Feed Production. 6th Intnl. Conference on Algal Biomass, Biofuels and Bioproducts. 6/26  6/29/16. Paradise Point, San Diego, CA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: A. M. Ibekwe, S. Murinda, M. Murry, G. Schwartz, T. Lundquist. Cyanobacterial and Microalgae Community Structures in Algae. Cultivation Reactors for Biofuel Production. 6th Intnl. Conference on Algal Biomass, Biofuels and Bioproducts. 6/26  6/29/16. Paradise Point, San Diego, CA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: A. Sancio, S. Wu, Y. Patel, S. Murinda. Universal Detection of Cyanobacteria and their Toxins Using PCR for Safe Algae-based Feed Production. 4th Annual Cal Poly Pomona Student Research Scholarship and Creative Activity (RSCA)Conference. 3/4/16. Cal Poly Pomona, CA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: N. Eulogio, I. Janilkarn-Urena, M. Murry-Ewers, S. Murinda. Characterization of Native Microalgae for Bioremediation coupled to Feed Production. 3/4/16. Cal Poly Pomona, CA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: J. McHugh, A. Sancio, N. Euloglio, S. E. Murinda, G. Schwartz, M. Murry, M. Ibekwe, T. Lundquist. Universal Detection of Cyanobacteria and Their Toxins Using PCR. 28th Annual CSU Biotechnology Symposium. California State University Program on Education and Research in Biotechnology (CSUPERB). 11/7-11/9/16. Garden Grove, CA.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: A. Sancio, J. McHugh, S. E. Murinda, G. Schwartz, M. Murry, A. M. Ibekwe. Production of Safe Algae Animal Feed from Dairy Waste Nutrients: Cyanobacteria and Cyanotoxin Detection. Southern California Conference for Undergraduate Research (SCCUR). Harvey Mudd College, Claremont, CA. 11/21/15.
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: J. McHugh, A. Sancio, S. E. Murinda, G. Schwartz, M. Murry, A. M. Ibekwe, T. Lundquist. Production of Algae Feeds from Dairy Waste. 16th Annual Agricultural Research Institute Showcase. Cal Poly Pomona, CA. 11/3/15. 15th Annual ARI Showcase. Cal Poly Pomona, CA
• Type: Journal Articles Status: Submitted Year Published: 2017 Citation: Su Ting Huang, Jo Lynn Goh, H. Ahmadzadeh, M.A. Murry. A Rapid Sampling Technique for Isolating Highly Productive Lipid-rich Algae Strains from Environmental Samples. Algae Research (submitted).
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Takia M, H. Ahmadzadeh, MA Murry. Growth of Chlorella vulgaris in high concentrations of nitrate and nitrite for wastewater treatment. Current Biotechnology. 4:441-447.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Takia M, H. Ahmadzadeh, S. Lyon, M Murry. 2015. Nitrate and nitrite removal from wastewater using algae. Current Biotechnology. 4(4).DOI: 10.2174/2211550104666150828193607.
• Type: Book Chapters Status: Awaiting Publication Year Published: 2017 Citation: Narges Moradi-kheibari, Hossein Ahmadzadeh, Ahmad Farhad Talebi, Majid Hosseini, Marcia A. Murry. Recent Advances in Lipid Extraction for Biodiesel Production. Handbook of Biotechnology for Renewable Fuels. ed. Majid Hosseini.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Lari, Z., Moradi-kheibari, N., Ahmadzadeh, H. Abrishamchi P. Moheimani N.R , M. A. Murry. Bioprocess engineering of microalgae to optimize lipid production through nutrient management. Journal of Applied Phycology. 2016, 28:3235 https://doi.org/10.1007/s10811-016-0884-6.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Fatimah Ansari, Joe McHugh, Amera Kchech, Shelton E. Murinda, Marcia Murry, Gregory Schwartz, Mark Ibekwe, and Trygve Lundquist. Universal Detection of Cyanobacteria and Their Toxins Using PCR for Safe Algae-based Feed Production. Southern California Conference for Undergraduate Research (SCCUR). California State Polytechnic University, Pomona. November 18, 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Marcia A. Murry, Lesly M. Palacios Castillo, Kristen M. Bush, Alejandra Avila, Greg Barding, Shelton Murinda. Proximate Composition of Algae Biomass to Access Nutritional Value as a Feed Supplement. Southern California Conference for Undergraduate Research (SCCUR). California State Polytechnic University, Pomona. November 18, 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Fatimah Ansari, Joe McHugh, Amera Kchech, Shelton E. Murinda, Marcia Murry, Gregory Schwartz, Mark Ibekwe, and Trygve Lundquist. Universal Detection of Cyanobacteria and Their Toxins Using PCR for Safe Algae-based Feed Production. 17th Annual Agricultural Research Institute (ARI) Showcase. California State Polytechnic University, Pomona. December 1, 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Joe McHugh, Fatimah Ansari, Amera Kchech, Shelton E. Murinda, Marcia Murry, Gregory Schwartz, Trygve Lundquist, A. Mark Ibekwe. Production of Algae Feeds from Dairy Waste. 17th Annual Agricultural Research Institute (ARI) Showcase. California State Polytechnic University, Pomona. December 1, 2017.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Shelton Murinda, Marcia Murry, Mark Ibekwe, Gregory Schwartz, Trygve Lundquist. Algae for conversion of manure nutrients to animal feed: Evaluation of advanced nutritional value, toxicity, and zoonotic pathogens. USDA-NIFA NIWQP and AFRI Project Directors Annual Meeting. October 28-29, 2014. Washington, DC.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Shelton Murinda, Marcia Murry, Mark Ibekwe, Gregory Schwartz, Trygve Lundquist. Algae for conversion of manure nutrients to animal feed: Evaluation of advanced nutritional value, toxicity, and zoonotic pathogens. USDA-NIFA NIWQP and AFRI Project Directors Annual Meeting. October 12-13, 2016. Washington, DC.

Progress 09/01/15 to 08/31/16

Outputs
Target Audience: Students at Cal Poly Pomona and Cal Poly San Luis Obispo, commonly first generation college students, mostly Hispanics, are recruited to work on the algal research project. Academia, various government departments (including USDA), NGOs, farmers and industry. Changes/Problems:Problems Encountered: Our pond research started 6 months later, about March 2014, after the 09/01/2013 project award date. Research in ponds was initiated several months later after Cal Poly San Luis Obispo (CPSLO) acquired its subaward from Cal Poly Pomona (CPP). Progress at CPP and USDA-ARS depends on algae samples supplied from the ponds at CPSLO. We were not able to collect samples in Winter and Spring 2014 since our newly established ponds had not attained steady-state growth conditions that allowed drawing out of algal biomass from ponds. This was mostly ascribed to the cold weather that discouraged luxuriant growth of algae. Starting in summer 2014 we have successfully collected seasonal algae samples from the CPSLO ponds. We sampled the ponds in Summer and Fall 2016 and also collected biomass (compositional analysis with NGS, dominant species and nutritional composition plus modeling in photobioreactors mimicking pond conditions). We have also had problems associated with uncontrollable changes in the weather at the CPSLO pond bioreactors which include, very heavy rains, persistent droughts (2013-2016) and cracking of our open raceway, paddle-wheeled ponds, and subsequent repairs that affected steady progress in sample collection from the ponds. Bacteria and fungi that can grow faster than algae can to can overwhelm growth and isolation of target algae strains in pure cultures.This drawback was often encountered in the lab. In recent months (CPP) we have had serious fungal contamination issues with our algal cultures and this halted our DNA sequencing efforts (where pure single-culture DNA is mandated). Several fungicidal and fungi-static agents were screened for efficacy. Other setbacks pertain to method development, for example the difficulties in acquiring relevant or appropriate quality control cyanobacterial algae strains for routine use in calibration of genotype or toxin tests. Some cyanobacterial strains are not well-characterized for toxins they produce as they are only indicated as "toxin-producing" and there is severe reluctance by other researchers to provide the strains that produce toxins of interest to our study that they reported in peer-reviewed literature or other reputable forums. We are therefore characterizing strains that have been "suspected" to produce toxins to see if they produce the toxins of interest for use in study (i.e., cyanotoxins, namely, anatoxin-a, nodularin, microcystin, and cylindrospermopsin). Due to the inherent slow growth and contamination of fungal stocks, it can take 8 weeks or more to obtain quality control algae strains from commercial suppliers (e.g., UTEX and ATCC). Furthermore, the algae are prone to contamination and/or predation by other organisms while in culture. And after they are finally shipped to our labs they take just as long to grow and use in subsequent tests. While we successfully developed end-point PCR protocols to reliably detect cyanobacteria, we have not been able to identify quality control strains for the other toxins (nodularin, and cylindrospermopsin). We have additionally encountered problems confirming toxin production (using ELISA technics) by the strains that are positive for the microcystin and anatoxin-a toxin-encoding pathways (and are working on optimizing these technics) and complementing them with cell toxicity assays that we initiated in Spring 2016. No Cost Extension: Persuant to the problems outlined above, we applied for a No Cost Extension to the project and this was granted to 8/31/2017. The no cost extension plan adheres to previously approved objectives of the project and will enable model testing with well-characterized algae monocultures. No new goals or objectives were proposed. What opportunities for training and professional development has the project provided?New tools were used and new technics learned in our 4 labs by both undergraduate and graduate research assistants and research scientists (PD And Co-PDs). Students learned and acquired research experiences and earned internship or research units that contributed to graduation, including acquisation of life-long skills that enhanced their resumes regards graduate school admission or employability. One student research assistant graduated in spring 2016 and got a job with Cedars Sinai Medical Center, CA, with a prestigious biotechnology company. Her experiences as an intern and research assistant on the algae project enabled this opportunity. All students working on the algal project attained greater proficiency in ability to conduct research by interacting with their mentors (PDs and co-PDs) and attending workshops and conferences. The students also developed advanced professional skills in use of cutting edge instrumentation, data collection, storage, analysis and interpretation, as well as data dissemination via poster and oral presentations. Several students had never prepared a poster or presented at a professional conference, thus they were mentored to develop and refine these fundamental skills. Professional development activities resulted in increased knowledge and skills that were acquired in the lab training environment through targeted reading and training materials (e.g., journal publications, reputable websites and YouTube instructional videos). Students also attended various conferences [local and international; e.g., Southern California Conference on Undergraduate Research (SCCUR), Algal Biomass, Biofuels and Bioproducts (ABBB), Agricultural Research Institute (ARI) Showcase] where they presented and tapped from others research experiences. Two students from Cal Poly Pomona working on the algae project were seconded to our Co-PD's lab (Dr. Mark Ibekwe, USDA-ARS), in summer 2016, to hone skills in DNA sequencing technics we are using for characterization (i.e., speciation) of fungal isolates. In April 2016, one MS student working in my lab (Algae Project) received $7,500 award for a PPOHA-MENTORES Fellowship for Energy- or Water-related projects. How have the results been disseminated to communities of interest?Dissemination venues for our research project included: authoring a book chapter, peer-reviweed journal article, and attending various conferences/symposiums (local/international., including ASM, ABBB, ABO, SCCUR) and USDA Annual Project Directors meeting. The USDA Project Directors Meeting Abstract Booklet and oral presentations are available online at the NIFA website: http://www.cpe.vt.edu/nifa/index.html and https://nifa.usda.gov/2016-afri-and-niwqp-project-directors-meeting where it will be available to diverse constituents or stakeholders (USDA, Academia, Students, Farmers, Industry, NGOs). Other conference presentations (oral/posters) are available as hard copy proceedings, as well on-line on conference websites. What do you plan to do during the next reporting period to accomplish the goals?Pond samples collected from all seasons since 2014 were cryopreserved and are awaiting further analysis or more in-depth testing: i.e., isolation of microbes, isolation of DNA and RNA, toxin isolation and detection, and quantitation using PCR/qPCR and ELISA technics, etc. In the previous phases of the study (2014-2016) we relied on adventitious (natural) contamination of our algae ponds/bioreactors. This enabled us to isolate and characterize strains that are seasonally dominant, as well as characterize their growth and nutritional profiles. Furthermore, the isolates were genetically identified (CPP and USDA-ARS) to genus and/or species level. In the final phase of our study, which started in Fall 2016, we will inoculate the ponds with known species of well-characterized seasonally dominant strains (originally isolated from the CPSLO ponds) that were characterized for growth and nutritional profiles, and monitor for maximum biomass production of safe algal feeds. We have initiated scale-up procedures to intermediate-sized indoor reactors (150-L capacity) to eventually inoculate the outdoor reactors (~970-L capacity) at CPSLO to accomplish our model development.

Impacts
What was accomplished under these goals? GOAL #1: GENERATE EXPERIMENTAL PILOT PLANT DATA AND CALIBRATE OPTIMIZATION MODELS We operated the four bioreactors in steady-state and continued to collect seasonal data for our database to accomplish each of the specified tasks. We monitored seasonal nutrient (N and P) uptake rates, identified seasonally dominant algal species, initiated bioflocculation selection, and established routine laboratory protocols (SOPs). Dairy lagoon effluent (DLE) nutrient characteristics were quantified. Data collected to help develop a productivity model based on multiple variables includes: hydraulic residence time (HRT), solar radiation, water temperature, available nutrient concentration, and primary nutrient source. A pH control system was installed to introduce carbon dioxide as needed, ensuring carbon limitation, which might retard biomass production, does not occur. The systems typically removed 50-60% of the fed N in spring, summer and fall (short HRT) and 20-40% in winter (long HRT). Experimental Overview: Nutrient additions as well as a fraction of nutrients from DLE were adjusted to meet growth needs of algae enabling development of a productivity model based on multiple variables indicated above. Nutrient concentrations (total N, total inorganic N, nitrate, total P) in the DLE were changing throughout the trial, as a function of dairy operation and weather. Apparatus and Operations: Progress was made in developing a bioflocculating algal community in the bioreactors. Flocc (clump) settling times, volatile suspended solids (VSS) concentration and percent of the VSS that settled over time were monitored. Harvesting of algal biomass was achieved via gravity settling. Media: Algae ponds were fed dairy flush water from DLE. A water quality analysis laboratory (set up in 2015) was used and enabled routine analysis of N and P, solids, chemical oxygen demand (COD) and alkalinity, to accurately analyze DLE. Due to elevated nutrient levels in the DLE the DLE additions to ponds were diluted to 3-7% of the daily refill volume depending on the season. The deep brown color of the DLE was diluted to promote a green water or algae-based treatment system as opposed to a brown water bacteria-based treatment system. Model Development: Seasonal data were verified or enhanced with more data, to continue to compile data for the development of a predictive model for nutrient uptake and biomass production potential. We have been collecting various data sets, including: solar radiation, temperature, residence time, nutrient and biomass concentration data, which will be used to build and calibrate the model. To complement this pond model being developed (*CPSLO), axenic algae isolates are being cultured in PBR 101 Phenometrics Bioreactors (**CPP) to simulate the environmental parameters at CPSLO. Experiments, Variables, Sampling and Analysis: Water quality data were collected and sorted by season to examine pond operational variables. Data were analyzed to determine seasonal algal productivity and nutrient uptake at HRTs ranging from 2.5-10.5 days. Nutrient uptake values ranged 0.4-1.4 g N/m2/day and 0.08-0.17 g P/m2/day and were higher in summer and fall. Biomass productivities ranged 4-26 g VSS/m2/d. GOAL #2: Maximize the Nutritional Value of Produced Algae for Animal Feed Isolation and Identification of algae and compositional analysis: To date we have processed 7 seasonal samples from each of the ponds. We isolated dominant strains and sequenced them targeting the ITS 4-5 intergenic region and identified species of Scenedesmus, Chlorella, Desmodesmus and Chlorophyta. We have >100 mostly axenic isolates that are being characterized for growth patterns, heterotophy and carbon and N preferences that support rapid growth and lipid production under diurnal light cycles in PBR bioreactors. Growth and proximate analysis of 9 strains was tested. Samples were harvested at log and stationary phases. Lipid analysis was conducted using GC-FID and GC-MS. Cells were cryopreserved for analysis of protein, fiber and CHO content. Aliquots of each strain were collected at various stages of growth for dry weight and proximate analysis including elemental N to assess protein levels, lipid quantification and fatty acid profiling (fatty acid methylesters), soluble carbohydrate and fiber content. We developed routine assays for biochemical composition based on NREL protocols. Harvested biomass was analyzed to assess the chlorophyll:dry weight ratio to determine the proportion of algae biomass in the harvest. GOAL #3 Optimize Pathogen Inactivation Methods We continue to employ optimized model conditions under steady state bioreactor conditions for algal biomass production. This will enable pathogen inactivation methods to be tested in the final phase of the study when we grow selected well-characterized seasonally-dominant strains. GOAL #4 Quantify and Control any Cyanobacterial Toxins Culturing of Quality Control (QC) Strains: New species of cyanobacteria have been added to our culture collection and are prospective toxin producers. QC cyanobacterial strains were acquired from UTEX (Culture Collection of Algae). Routine detection of cyanobacteria and cyanotoxins has been achieved using end-point PCR that targets unique DNA sequences. We also identified cyanobacteria strains positive for microcystin and anatoxin-a synthesis pathways. Quantitative PCR: We successfully generated standard curves for quantification of cyanobacteria using 16S rRNA, and microcystin synthesis-associated mcyE gene sequences. Multiplex qPCR protocols will be implemented with three targets (16S rRNA, microcystin and anatoxin-a) that can be used for testing pond samples and algal biomass. Toxin Detection via ELISA: Peptide extraction was performed on algal samples that were analyzed by ELISA kits. Standard curves were generated for each toxins, however, ELISA test results on culture extracts yielded false positives. We are refining our toxin isolation and detection technics. Analysis of Microbial and Cyanobacterial Community Structures in Ponds: DNA extracted from pond samples was used for analysis of total bacteria (targeting 16S rRNA-encoding genes), cyanobacteria and algae using Illumina MiSeq's next generation sequencing platform. Pathogens will be identified and quantified using PCR (real-time or digital droplet). The MP Biomedicals, MoBio and Zymo DNA extraction kits yielded 302, 187, 280, operational taxonomic units, respectively. The data suggested that the MPBio kit could be most suitable for analyzing 16S rDNA, while the Zymo kit may be better for analyzing microalgae. Total bacterial and cyanobacterial community composition will be tested in the pond water samples. Preliminary V4 16S rDNA analysis including bioinformatics work have been conducted on the first year samples. From this analysis, we were able to identify the dominant microalgae and cyanobacteria in our samples. We will conduct shotgun sequencing to relate composition of bacterial community to functions on all samples. Pond Pathogen Characterization and Quantification: We collected seasonal pond samples from 2014-2016 for quantification of pathogens using real-time PCR and total bacterial counts (USDA-ARS). We have extracted DNA and cryopreserved the pond samples. We commenced screening for E. coli O157 using multiplex qPCR targeting stx1, stx2, eae, and rfbO157 genes associated with Shiga toxin-producing E. coli O157 (STEC O157). Only a few samples were positive for these genes suggesting that the STEC O157 pathogen dies out in the ponds. Another quadriplex quantitative-PCR (qPCR) assay capable of detecting and quantifying toxin genes from the microcystin, nodularin, cylindrospermopsin and saxitoxin biosynthesis pathways will be used to quantify cyanobacterial toxins. These assays have all been optimized and are ready to be deployed. [*CPSLO; Cal Poly San Luis Obispo. **CPP; Cal Poly Pomona]

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: I. Janilkarn-Urena, N. Eulogio, M.A. Murry, S.E. Murinda, A.M. Ibekwe, G. Schwartz, T. Lundquist. Bioprocess Engineering of Native Algae Strains to Optimize Bioremediation coupled to Feed Production. Southern California Conference for Undergraduate Research (SCCUR). UC Riverside, CA. 11/12/16. S. Wu, Joe McHugh, A. Sancio, I. Janilkarn-Urena, N. Eugenio, A. Kchech, S.E. Murinda, G. Schwartz, M. Murry, A. M. Ibekwe, T. Lundquist. Production of Algae Feeds from Dairy Waste. 16th Annual ARI Showcase. Cal Poly Pomona, CA. 11/3/16. I. Janilkarn-Urena, N. Eulogio, M.A. Murry, S.E. Murinda, A.M. Ibekwe, G. Schwartz, T. Lundquist. Bioconversion of agricultural wastes from the livestock industry for biofuel and feed production. 1st International Conference: Bioresource Technology for Bioenergy, Bioproducts and Environmental Sustainability. 10/23-10/26/16. Sitges, Spain. S. Murinda, M. Murry, G. Schwartz, T. Lundquist, A. M. Ibekwe. Algae for Conversion of Manure Nutrients to Animal Feed: Evaluation of advanced nutritional value, toxicity, and zoonotic pathogens. USDA Annual Project Directors Meeting. Washington DC. 10/12-13/16. J. McHugh, A. Sancio, S.E. Murinda, G. Schwartz, M. Murry, A.M. Ibekwe, T. Lundquist. Detection of cyanobacteria and their toxins for safe algae-based feed production. 6th Intnl. Conference on Algal Biomass, Biofuels and Bioproducts. 6/26  6/29/16. Paradise Point, San Diego. CA. W. Shires, S. Atmadja, G. Schwartz, S. Murinda, M. Murry, A.M. Ibekwe, T. Lundquist. Algae for conversion of manure nutrients to animal feed: Development of an algae production model. 6th Intnl. Conference on Algal Biomass, Biofuels and Bioproducts. 6/26  6/29/16. Paradise Point, San Diego, CA. I. Janilkarn-Urena, N. Eulogio, M.A. Murry, S.E. Murinda, A.M. Ibekwe, G. Schwartz, T. Lundquist. Bioprocess Engineering of Native Algae Strains to Optimize Bioremediation coupled to Feed Production. 6th Intnl. Conference on Algal Biomass, Biofuels and Bioproducts. 6/26  6/29/16. Paradise Point, San Diego, CA. A. M. Ibekwe, S. Murinda, M. Murry, G. Schwartz, T. Lundquist. Cyanobacterial and Microalgae Community Structures in Algae. Cultivation Reactors for Biofuel Production. 6th Intnl. Conference on Algal Biomass, Biofuels and Bioproducts. 6/26  6/29/16. Paradise Point, San Diego, CA. J. McHugh, A. Sancio, S. E. Murinda, G. Schwartz, M. Murry, A. M. Ibekwe, T. Lundquist. Detection of cyanobacteria and their toxins for safe algae-based feed production. American Society for Engineering Education PSW Conference. 4/21-4/23/16. Cal Poly Pomona, CA. A. Sancio, S. Wu, Y. Patel, S. Murinda. Universal Detection of Cyanobacteria and Their Toxins Using PCR for Safe Algae-based Feed Production. 4th Annual CPP Student RSCA Conference. 3/4/16. Cal Poly Pomona, CA. N. Eulogio, I. Janilkarn-Urena, M. Murry-Ewers, Murinda. Characterization of Native Microalgae for Bioremediation coupled to Feed Production. 3/4/16. Cal Poly Pomona, CA. J. McHugh, A. Sancio, N. Eugenio, S. E. Murinda, G. Schwartz, M. Murry, M. Ibekwe, T. Lundquist. Universal Detection of Cyanobacteria and Their Toxins Using PCR. 28th Annual CSU Biotechnology Symposium. California State University Program on Education and Research in Biotechnology (CSUPERB). 11/7-11/9/16. Garden Grove, CA. A. Sancio, J. McHugh, S. E. Murinda, G. Schwartz, M. Murry, A. M. Ibekwe. Production of Safe Algae Animal Feed from Dairy Waste Nutrients: Cyanobacteria and Cyanotoxin Detection. Southern California Conference for Undergraduate Research (SCCUR). Harvey Mudd College, Claremont, CA. 11/21/15. J. McHugh, A. Sancio, S. E. Murinda, G. Schwartz, M. Murry, A. M. Ibekwe, T. Lundquist. Production of Algae Feeds from Dairy Waste. 16th Annual ARI Showcase. Cal Poly Pomona, CA. 11/3/15. 15th Annual ARI Showcase. Cal Poly Pomona, CA.
• Type: Journal Articles Status: Under Review Year Published: 2016 Citation: A. Mark Ibekwe, Shelton E. Murinda, Marcia A. Murry, Gregory Schwartz, Trygve Lundquist. Microbial Community Structures in Algae Cultivation Ponds for Bioconversion of Agricultural Wastes from Livestock Industry for Feed Production. Science of the Total Environment (In Review).
• Type: Book Chapters Status: Under Review Year Published: 2016 Citation: M. Murry, S. Hung, S. Murinda, G. Schwartz, A.M. Ibekwe, T. Lundquist. 2016. Bioconversion of agricultural wastes from the livestock industry for biofuel and feed production. In: Handbook of Biotechnology for Renewable Fuels: Technology Assessments, Emerging Industrial Applications, and Future Outlooks; Majid Hosseini, Editor. Elsevier Publishing Company. (Invited Review: Manuscript submitted September 2016).

Progress 09/01/14 to 08/31/15

Outputs
Target Audience:1) Students: At Cal Poly Pomona, commonly first generation college students, mostly Hispanics, are recruitred to work on the algal research project. 2) Government, Academia, NGOs, Industry, Farmers: Members of Soil & Water Conservation Society, academia, and various govenment departments, NGOs, including some farmers and representatives from industry were in attendance at some of the conferences (e.g., ASM, USDA). Changes/Problems:1) For cytotoxicity studies, we plan to focus on using washed red blood cells (sheep/goat or cattle) that we will bombard with cell-free extracts and monitor cytotoxicity indices. Traditional cell-free cutures were found to be cumbersome and costly, and require highly specialized laboratories dedicated to cell cultures. 2) We have also encountered perennial problems with acquisition of standard quality control (QC) cyanobaterial strains that produce the toxins of interest. Invariably, scientists that have reported work with toxin-producing cyanobacteria are not willing to share their isolates/strains thus we rely on cyanobacteria culture collections or respositories such as UTEX (University of Texas) Culture Collection to provide quality control cyanobacteria. UTEX strains are not characterized for toxin production. A major problem encountered with UTEX is that it can take 8 weeks or more for UTEX to prepare requested cultures. Once received it can take an additional 3-4 weeks or more to propagate these cultures before they can be used routinely. The slow rate of cyanobacterial growth can lead to contamination by faster growing bacterial and fungal contaminants. 3) Due to the delayed start of our project and slow growth and establishment of the ponds (as reported lin 2014), in 2016 we will be officially requesting for a no-cost extension of this project into 2017. What opportunities for training and professional development has the project provided?New tools were used and new technics learned by both students and research scientists. Students learned and are acquiring research experiences and earning internship or research units units that contribute to graduation, including acquisation of life-long skills that enhance their resumes and employability, as well as focused and competitive preparation for graduate school admission. For example, one student research assistant was accepted on an MS program in Biological Sciences (CPP) solely because of the research experiences acquired working on the algae project. All students working on the algal project attained greater proficiency in ability to conduct research by interacting with their mentors (PDs and co-PDs). The students also developed advanced professional skills. They improved skills in use of cutting edge instrumentation, data collection, storage, analysis and interpreataion, as well as data dissemination via poster and oral presentations. Some of the students had never prepared a poster or presented at a professional conference, thus they were mentored to develop and refine these skills. Professional development activities resulted in increased knowledge and skills that were acquired in the lab training environement through targeted reading and training materials (e.g., journal publications, reputable websites and YouTube instructional videos). Students also attended various conferences where they presented and learned from others research experiences. How have the results been disseminated to communities of interest?The USDA Project Directors Meeting Abstract Booklet is available online @ https://drive.google.com/file/d/0ByBTot97JBKNQUtfQ2JtSl9yclk/view?pli=1 where it will be available to diverse constituents (USDA, Academia, Students, Farmers, Industry, NGOs). Other conference presentations (oral/posters) are available as hard copy proceedings, as well on-line on conference websites. What do you plan to do during the next reporting period to accomplish the goals?Goal #1: Generate Experimental Pilot Plant Data and Calibrate Optimization Models: TASK 1: Studies on generating experimental field data, calibration and optimization of algae pond culture models will be continued. Our next phase will focus on continued data collection and algal identification, development of biomass settling as well as harvesting bioflocculated algal biomass. We will focus on harvesting this biomass through gravity settling. We will also evaluate different techniques to decrease the required dilution of the DLE to promote optimum algae production. TASK 2: Using a computer-aided micromanipulator, we will focus on isolating several seasonally dominant strains not obtainable by dilution plating (CPP). TASK 3: After steady state is attained in the 2 new ponds, further tests will be conducted employing all 6 ponds, using optimized hydraulic and organic loading rates and other model parameters. Experiments will be conducted to produce algal biomass (g/m2/day) and standing crop (VSS; mg/L) including nutrient assimilation rates (i.e., g N/m2/day and g P/m2/day). Water and biomass samples will continue to be collected from CPSLO and delivered to co-investigators (at CPP and USDA-ARS) for microbial community characterization, toxicity, pathogen and compositional analysis, consitent with Goal #2, 3 and 4. TASK 4: Observations will be made to determine the impact of hydraulic and organic loading on the development of bio-flocculant consortia of algae and other organisms to assist with the harvesting of biomass. TASK 5: Microscopy and the hemocytometer will continue to be used for cell observations with respect to species, counts and size evaluation. Counts will be performed as operating parameters are adjusted to associate species with season and nutrient uptake kinetics. GOAL #2: Maximize the Nutritional Value of Produced Algae for Animal Feed: TASK 1: Studies will be continued to maximize the nutritional value of algae produced for animal feed. The pond cultures will be optimized to produce biomass at a high rate while also having the highest value composition for feed. Investigations of feed potential quality will focus on determining digestibility, concentration of lipids, essential fatty and amino acid profiles, as well as balanced protein and carbohydrate concentrations. Methods for these determinations were investigated and refined. Amino acid and fatty acid composition, nucleic acid and carbohydrate content and digestibility are key to developing feed supplements for specific target animals, thus, analysis of these parameters will be carried out using axenic strains grown (CPP) under controlled lab conditions in and samples harvested from CPSLO ponds. The goal is to determine proximate composition relative to nutritional value and identify strain-specific physiological responses to environmental variables in order to select strains favorable for feed production and/or to determine cultivation conditions that lead to highest value algae biomass. TASK 2: Cells will be harvested and disrupted and amino acid and fatty acid profiles and other components will be analyzed. Assays of biochemical composition (soluble protein, CHO, nucleic acids and lipids), that were developed based on NREL protocols will be used to characterize proximate composition of promising strains in exponential growth phase. Due to high costs of algal production, especially monocultures, and temporal variations in proximate composition which pose problems for feed operations, our approach will be to characterize the dominant strains individually then in polyculture. Combinations of algal species, each rich in specific nutrients which others may lack, would allow formulation of a balanced diet for the animal reminiscent of the way animal production facilities blend different feed sources to meet the specific nutritional requirements of the target animal species. GOAL #3: Optimize Pathogen Inactivation Methods: Since steady state conditions have been achieved in the bioreactors, pathogen inactivation methods will be investigated and optimized. Pathogens from dairy flush water will be expected to die-off over time in the ponds and during disinfection processing of the harvested algae biomass. We are at a stage were biomass can be collected after optimizing bioflocculation technics. Inactivation rates for representative pathogen indicators will be determined under various algae cultivation conditions and during trials with several biomass disinfection techniques. We will determine optimal combinations of pond conditions and biomass processing to achieve needed log inactivation of pathogens. GOAL #4: Quantify and Control any Cyanobacterial Toxins: TASK 1: Our DNA extraction data using commercial kits (MoBio Power water extraction kit, MP Biomedicals FastDNA spin kit, and Zymo fungi/bacterial extraction kit) indicated that care must be taken in DNA extraction procedures used to evaluate specific groups of bacteria for specific functions. The MiSeq sequencing data further suggested that the MoBio kit may be the best extraction kit for analyzing 16S rDNA from the algae cultivation ponds, while the Zymo kit may be better for analyzing microalgae. We are currently refining the technics for DNA extraction using these kits. Once this goal is achieved, previously collected and cryopreserved samples and subsequently samples that will be collected will be analyzed according to the standard protocols developed. The extensive data sets that will be generated from this study using MiSeq NGS will be used for monitoring the ponds as well as for correlating the abundance and diversity of microbial communities (algae/bacteria), including target pathogens. TASK 2: We will continue to optimize PCR/qPCR assays for detection of cyanobacteria and cyanobacterial toxins (microcystin, nodularin, saxitoxin,anatoxin-a and cylindrospermopsin). Quantitation protocols for cyanobacteria and cyanotoxins will be developed. The assays will be used for interrogating the algae biomass harvested from bioreactors at CPSLO. Measures will be taken to monitor and control invasion of the ponds by toxin-producing cyanobacterial strains. TASK 3: Mammalian cells will be used to screen for global toxicity, and a TC 20 Cell Counter (Bio-Rad Laboratories, Hercules, CA) will enable counting of dead and live cells, after bombardment with cell-free extracts of test cyanobacterial isolates. The target toxins will be detected and quantitated using ELISA technics. Commercially available ELISA kits (Abraxis, LLC, and Beacon Analytical Systems, Inc.) will be used for cyanotoxin detection. The toxin data will be correlated with DNA data on presence of genes associated with cyanotoxin synthesis.

Impacts
What was accomplished under these goals? GOAL #1: Generate Experimental Pilot Plant Data and Calibrate Optimization Models: Appreciable progress was made. Seasonal data was collected for our database to accomplish each of the specified tasks. Four outdoor pilot reactors were operated for one year and we collected seasonal data. Seasonal nutrient uptake rates were monitored and dominant algal species dentified. For harvesting of algal biomass, bioflocculation selection was initiated. Routine laboratory standard operating protocols were developed. 1.1 Experimental Overview: Data were collected from the outdoor pilot scale algae cultivation reactors under steady-state conditions. Nutrient additions as well as a fraction of nutrients from dairy lagoon effluent (DLE) were adjusted to meet the growth needs of the algae enabling development of a productivity model based on multiple variables, including: hydraulic residence time (HRT), solar radiation, water temperature, available nutrient concentration, and primary nutrient source. As a function of dairy operation and weather, the nutrient concentrations (total nitrogen, total inorganic nitrogen, nitrate, total phosphate) in the DLE were changing throughout the trials. Samples of the DLE were characterized to gain an understanding of the types of nutrients present. 1.2 Apparatus and operations: Four identical algae production tanks were operated adjacent to the waste lagoons at the 250-head Cal Poly San Luis Obispo (CPSLO) campus dairy. The algae tanks are 30-cm deep, paddle wheel-mixed raceways that simulate standard algae production ponds. Progress was made in developing a bioflocculating algal community in the pilot ponds. Patterns of how the algae floccs settle over time, volatile suspended solids (VSS) concentration and the percent of the VSS that settles over time have been monitored. The next phase of the project will focus on harvesting of this algal biomass through gravity settling. 1.3 Media: The algae ponds were fed dairy flush water from the storage lagoons. A secure water quality analysis laboratory was set up. This allows for analysis of nitrogen and phosphorus nutrients, solids, chemical oxygen demand (COD) and alkalinity to accurately analyze the DLE. Due to the elevated nutrient levels in the DLE the DLE additions to ponds were diluted to 3-7% of the daily refill volume depending on the season. The deep brown color of the DLE was diluted to promote a green water or algae-based treatment system as opposed to a brown water or bacteria-based treatment system. 1.4 Algal Strains: Algae strains are being identified using microscopy (plus a digital camera); this predominantly identifies Scenedesmus species. Fifty one strains were isolated from 4 seasonal samples by simple-plating and cultured axenically (Cal Poly Pomona/CPP). Isolates were sequenced targeting the ITS 4-5 intergenic region and this identified species of Scenedesmus (dominant), Chlorella, Desmodesmus and a variety of small Chlorophyta. Using a microcapillary-guided, single-cell manipulator sytem, we are now trying to isolate several seasonally dominant strains not obtainable by dilution plating. 1.5 Model Development: As seasonal data is verified or enhanced with more data, work for this task will begin during this next period (Fall). We have been collecting various data sets, including: solar radiation, temperature, residence time, nutrient and biomass concentration data, all of which will be used to build and calibrate the model (CPSLO). To complement this pond model being developed at CPSLO for dairy effluents, axenic isolates are being cultured in Phenometrics Bioreactors (PBR 101 lab-based bioreactor systems; CPP) to simulate the environmental parameters (diurnal solar radiation and pond temperature, residence times, mixing rates and concentrations of biomass, carbon, N, P loads and pH, under well controlled conditions. The PBR 101 bioreactors can accurately mimic production conditions at CPSLO through programmable temperature, diurnal light cycles, light intensity, real time pH monitoring, variable mixing, continuous-flow turbidistatic cultivation and real-time growth monitoring. Furthermore, we have developed assays of biochemical composition (soluble protein, CHO, nucleic acids and lipids) based on National Renewable Energy Laboratory (NREL) protocols and will characterize proximate composition of promising strains in exponential growth phase. 1.6 Experiments, Variables, Sampling and Analysis: Data has been collected thus far examining pond operational variables that include hydraulic and organic loading rates. The four algae production reactors were operated and utilized for data collection. Water quality data that that was collected and sorted by season includes: total suspended solids (TSS), VSS, total nitrogen concentration, total inorganic nitrogen concentration, total phosphorus, alkalinity, Secchi disk visibility, oxygen concentration, pH, and temperature. The data were analyzed to determine algal productivity and nutrient uptake rates for each season at HRTs of 4 vs. 6 days. Algal productivity values ranged 6.5-13.9 g/m2/day, whereas, nutrient uptake values ranged 0.8-1.4 g N/m2/day and 0.08-0.17 g P/m2/day and were all higher at HRTs of 4 days in all seasons. Seasonal data for the other parameters including DLE analysis were collected. GOAL #2: Maximize the Nutritional Value of Produced Algae for Animal Feed: We have developed assays of biochemical composition (soluble protein, CHO, nucleic acids and lipids) based on NREL protocols and will characterize proximate composition of promising strains in exponential growth phase (CPP). GOAL #3: Optimize Pathogen Inactivation Methods: We continue to employ optimized model conditions under steady state bioreactor conditions for algal biomass production. This will enable pathogen inactivation methods to be tested in the next phase. GOAL #4: Quantify and Control any Cyanobacterial Toxins: 4.1 Quality Control Bacterial Strains: Quality control (QC) cyanobacterial strains (toxin and non-toxin-producers) were acquired from The Culture Collection of Algae (University of Texas, Austin, TX). Routine detection of cyanobacteria and cyanotoxins has been achieved using PCR protocols that target unique DNA sequences present in cyanobacteria. The tests were validated using the QC strains. 4.2 Sample Analysis: Seasonal samples were collected from the 4 algae bioreactors and from the dairy lagoon at the CPSLO dairy. DNA was extracted from these samples for analysis of total bacteria (targeting 16S rRNA-encoding genes), cyanobacteria and algae using Illumina's MiSeq next generation sequencing (NGS) platform (Illumina Inc., San Diego, CA). Pathogens will be identified and quantified using PCR (real-time or digital droplet). 4.3. Impact of Different Commercially Available DNA Extraction Kits On Bioreactor Bacterial Community Structures: DNA extraction was undertaken using the MoBio Power water extraction kit, Zymo fungi/bacterial extraction kit, and MP Biomedicals FastDNA spin kit for the analysis of total bacteria and cyanobacteria using MiSeq Illumina sequencing. The microbiome profile from the lagoon effluent and bioreactors formed distinct clusters. The MP Biomedicals kit yielded 302 taxa, MoBio 187, and Zymo 280. However, DNA samples isolated from the Zymo kit had a higher proportional abundance of Chlamydomonadaceae, a family of microalgae. Our data suggest that care must be taken in DNA extraction procedures used to evaluate specific groups of bacteria for specific functions. The kits wiil be further evaluated. 4.3. Determination of Pathogens in Bioreactor Samples: The MiSeq sequencing data suggested that the MPBio kit could be the best extraction kit for analyzing 16S rDNA from the algae cultivation ponds, while the Zymo kit may be better for analyzing microalgae. Refinement of the prrotocols and deeper analysisi of the data will lead to identification of specific organisms including pathogens.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: 1). Marcia Murry, Shelton Murinda. Bioremediation: A Practical Approach to Algae Biomass Production. Provosts Symposium on Faculty Scholarship, December 12, 2015. California State Polytechnic University, Pomona, CA. 2). Tim Yoon, Julia Gonzalez, Roger Lee, Moon Seo. Mentor: Marcia A. Murry. A rapid sampling technique for isolating lipid-rich algae strains from environmental samples. 22nd Southern California Conferences for Undergraduate Research (SCURR). November 22, 2014. California State University, Fullerton, CA. 3). Joseph McHugh, Kaylynn Atkinson, Shelton Murinda, Marcia Murry. Universal detection of cyanobacteria and their toxins using PCR/qPCR. 22nd Annual Southern California Conference on Undergraduate Research. November 22, 2014. California State University Fullerton, CA. 4). Alyssa Sancio, Joseph McHugh, Kaylynn Atkinson, Shelton Murinda, Marcia Murry. Universal detection of cyanobacteria and their toxins using PCR/qPCR. 10th Annual Research Symposium. May 29th, 2015. California State Polytechnic University, Pomona, CA. 5). Mark Ibekwe, Shelton Murinda, Marcia Murry, Gregory Schwartz, Trygve Lundquist. Impact of Different DNA Extraction Methods on Total Bacterial and Cyanobacterial Community Structure in Algae Cultivation Reactors. American Society for Microbiology, 115th Annual General Meeting. May 30-June 2, 2015. New Orleans, Louisiana. 6). Shelton Murinda, Marcia Murry, Mark Ibekwe, Gregory Schwartz, Trygve Lundquist. Algae for conversion of manure nutrients to animal feed: Evaluation of advanced nutritional value, toxicity, and zoonotic pathogens. USDA-NIFA NIWQP and AFRI Project Directors Annual Meeting. July 26-29, 2015, Greensboro, NC. 7). Isis Janilkarn-Urena. Algal bioremediation of organic wastes coupled to biomass production for feed and biofuels. RISE Symposium September 4th, 2015. California State Polytechnic University, Pomona, CA. Mentor: Marcia A. Murry. 8). Natalie Eulogio. Can algae save the dairy industry? RISE symposium September 4th, 2015. California State Polytechnic University, Pomona,CA. Mentor: Marcia A. Murry.

Progress 09/01/13 to 08/31/14

Outputs
Target Audience: Students that were recruited to work on this project gained experiences in laboratory methods for isolation, culture, identification, and DNA isolation and are practicing PCR and qPCR technics for detection and quantitation of cyanobacteria and their toxins. They also learned and developed expetise in designing experiments, collecting data and maintaining accurate lab notebooks. Student research assistants acquired skills in techniques used in culture, monitoring and harvesting of algae where they measured various parameters and collected bio-reactor water samples for lab analysis. Changes/Problems: Major delays in disbursement of subawards to SLO and USDA-ARS Riverside, including slow rate of growth in the established bonds inhibited an earlier start for this project. Subawards were made available February, 2014. Initial tasks conducted at CPP and USDA depend on progress at CPSLO. No major changes are perceived except that progress is lagging since CPP and USDA depend on product from CPSLO to accomplish tasks in GOAL # 2, 3 and 4 with relevance to microbial communities and pathogen identification, quantitation and toxin analyses. Since writing our proposal in 2013, 454 Pyrosequencing has become outmoded by other NGS platforms.In our NGS analyses, we will be substituting/replacing Pyrosequencing with Illumina's MiSeq NGS. What opportunities for training and professional development has the project provided? New tools were used and new technics learned by both students and research scientists. Students are acquiring research experiences and earning internship units for graduation, including life-long skills that enhance their resumes and employability, including preparation for graduate school. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Goal #1: TASK 1. Studies on generating experimental field data and calibration and optimization of algae pond culture models will be continued. Since our preliminary data shows very low N:P ratio of the influent as well as a high COD (weekly and biweekly measurements), ammonia fertilizer will be supplemented to achieve an N:P ratio of 8:1. The COD will be addressed through either aeration in a pretreatment step or through anaerobic digestion. Aerobic degradation can occur quickly, but will increase costs and complexity of any treatment process. Our preliminary results show good COD reduction through aerobic digestion. Anaerobic digesters will be set up in the laboratory (CPSLO) as either a complete mixed reactor or a fixed film reactor due to the very low solids content of the lagoon influent. Nutrient data will be analyzed to determine the impact of both pre-treatment processes on constituents other than COD. TASK 2. The 20-liter digesters (n = 4) that were set up (CPSLO lab) will be used to determine an anaerobic digestion pretreatment step that will both destroy COD and increase the N:P ratio. The units are mesophillic and are quipped with temperature control and gas flow monitoring. Two of the units are complete mix and the other 2 are fixed film reactors. TASK 3. Since steady state was attained further tests will be conducted for each hydraulic and organic loading rate. Measurements will be taken of the entire water sample as well as a filtered sample to determine the fraction of constituents trapped or assimilated in the algae biomass. Assimilation rates will be calculated for nutrients including total N, and total N as well as different N species including total inorganic fraction, ammonia and nitrate. Although algal productivity and standing crop for the units have been monitored, steady state was achieved late (July/August). HRT was 8.5 days. Experiments will be conducted at various HRT’s to measure algal productivity (g/m2/day) and standing crop (VSS/L) including nutrient assimilation rates. Water and biomass samples will be collected and delivered from CPSLO to co-investigators (at Cal Poly Pomona, CA and USDA ARS , Riverside, CA) for microbial community characterization, toxicity, pathogen and compositional analysis, consitent with Goal #2, 3 and 4. TASK 4. Observations will be made to determine the impact of hydraulic and organic loading on the development of bio-flocculant consortia of algae and other organisms to assist with the harvesting of biomass. Additionally, a pH control system will be installed in the coming period to ensure no carbon limitation for growth as well as assess its impact on the development of bio-flocculating algae species. TASK 5. Microscopy and the hemocytometer will continue to be used for cell observations with respect to species, counts and size evaluation. Counts will be performed as operating parameters are adjusted to associate species with season and nutrient uptake kinetics. GOAL #2: TASK 1. Studies will be initiated to maximize the nutritional value of algae produced for animal feed. The pond cultures will be optimized to produce biomass at a high rate while also having the highest value composition for feed. Investigations of feed potential quality will focus on determining digestibility, concentration of lipids, essential fatty and amino acid profiles, as well as balanced protein and carbohydrate concentrations. Methods for these determinations are being investigated and refined. Amino acid and fatty acid composition, nucleic acid and carbohydrate content and digestibility are key to developing feed supplements for specific target animals, thus, analysis of these parameters will be carried out using axenic strains grown (CPP) under controlled lab conditions and samples harvested from CPSLO ponds. The goal is to determine proximate composition relative to nutritional value and identify strain specific physiological responses to environmental variables in order to identify strains favorable for feed and fuel, and/or to determine cultivation conditions that lead to highest value algae biomass. TASK 2. Cells will be harvested and disrupted and amino acid and lipid profiles determined in log and late stationary phases under N-replete and N-limited conditions. Aliquots of each sample will be subjected to lipid analysis including total lipid content as a percentage of algal dry weight. Profile analysis of fatty acid methyl esters (FAMEs) will be conducted using GC-MS. FAME analysis will determine whether this is a stable characteristic of each strain. Due to high costs of algal production, especially monocultures, and temporal variations in proximate composition which pose problems for feed operations, our approach will be to characterize the dominant strains individually then in polyculture. Several algal species, each rich in specific nutrients others may lack would allow formulation of a balanced diet for the animal reminiscent of the way animal production facilities blend different feed sources to meet the specific nutritional requirements of the target animal species. GOAL #3: Pathogen inactivation methods will be investigated and optimized under steady state conditions in the bio-reactors. Pathogens from dairy flush water will be expected to die-off over time in the ponds and during disinfection processing of the harvested algae biomass. Inactivation rates for representative pathogen indicators will be determined under various algae cultivation conditions and during trials with several biomass disinfection techniques. The optimal combination of pond conditions and biomass processing will be determined to achieve needed log inactivation of pathogens. GOAL #4: TASK 1: We are currently working on identifying the best commercially available DNA extraction kit for our genomic analyses (algae and bacteria). Once this is achieved, subsequent samples will be analyzed according to the standard protocol developed. This will be written as a standard operating procedure for the rest of the project, and information will be published for the scientific community to use. Computer software and bioinformatics analyses will facilitate identification of pathogens and non-pathogens from NGS. The very large data sets that will be generated from this study will be used for monitoring and correlating the abundance and diversity of pathogens to different environmental factors. TASK 2: PCR/qPCR assays will be developed and optimized for detection of cyanobacteria and cyanobacterial toxins (targeting genes associated with toxin biosynthesis pathways). Once this goal is achieved, quantitation protocols for cyanobacteria and cyanotoxins will be developed. The assays will be used for detection of cyanobacteria and cyanotoxins in algae biomass harvested from bio-reactors at CPSLO, measures will be taken to control invasion of the ponds by toxin-producing cyanobacteria strains. Cytotoxicity studies will be initiated for detection of toxins in harvested algae biomass. Cultured mammalian cells (ideally hepatocytes and neurocytes) will be used to screen for global toxicity, after which selected major target toxins (microcystin, nodularin, saxitoxin and cylindrospermopsin) will be detected and quantitated using optimized qPCR protocols or ELISA technics. A TC 20 Cell Counter (Bio-Rad Laboratories, Hercules, CA) was acquired for cytotoxicity studies, and it will enable counting of dead and live mammalian cells, after bombardment with cell-free extracts of test cyanobacteria isolates.

Impacts
What was accomplished under these goals? GOAL #1: Generate Experimental Pilot Plant Data and Calibrate Optimization Models: 1.1 Experimental Overview: Both pilot plant and laboratory algae cultivation bio-reactors were used during this period (starting March 2014 when sub-award funds were made available to Cal Poly San Luis Obispo/CPSLO). Permits were obtained to install 4 identical outdoor 3.2 m2 bio-reactors adjacent to the 300-head CPSLO campus dairy. The paddle wheeled reactors are operated by one variable speed motor that enables any desired water velocity. Preliminary experiments are using a water velocity in the medium speed range of 0.35 - 0.5 ft/sec. Currently, there is no supplemental carbon dioxide addition for pH control. A control system will be installed to allow operator control of pH, thus enhancing productivity and possibly facilitating algae biomass harvestability. In the laboratory, 4 indoor photo bio-reactors were set up to operate with dairy lagoon water as their only nutrient source. After attaining steady state, the units are being used for experiments to determine the impact of hydraulic residence time (HRT) on nutrient uptake rate, to monitor impacts of carbon dioxide supplementation and its effect on algae growth and nutrient uptake. 1.2 Apparatus and operations: The 4 identical algae production tanks at the CPSLO dairy simulate standard 30-cm deep algae production ponds. Physical parameters of the units, such as, volume and the relationship between paddle wheel RPM and channel water velocity as well as cross sectional flow patterns that will be critical for developing bio flocculating algal communities, were analyzed. 1.3 Media: The algae ponds were fed dairy flush water from the CPSLO dairy storage lagoons. Lagoon water was characterized for total nitrogen, N (291 mg/L, SD ±18 mg/L), phosphate, P (99 mg/L, SD ±25 mg/L) and water quality characteristics (chemical oxygen demand, COD: 4,495 mg/L, SD ±1,123 mg/L). A secure water quality laboratory was set up that allows for analysis of N and P nutrients, solids, COD and various metals, to accurately analyze the lagoon effluent. From weekly and biweekly measurements, our preliminary data shows very low N:P ratios of the influent as well as a high COD values. Preliminary results show good COD reduction through aerobic digestion (~5,500 to 2,000 mg/L). 1.4 Algal Strains: A microscope is used for algae species determinations and a hemocytometer for cell counting. These tools are being evaluated and will be extensively deployed in the next period for cell observations with respect to species, counts and size evaluation as well as correlating resident algal species with season and nutrient uptake kinetics. As steady state was approached during (July/August 2014), Scenedesmus, particularly the 4 cell strain, became the dominant algal species. Other species that were observed included Closterium, Chlamydemonus, Stichococus, and different varieties of pennate diatoms. 1.5 Model Development: No work was done on this task during this period since steady state in the bio-reactors was only achieved recently (July/August). 1.6 Experiments and Variables: The major pond operation variables being evaluated during these studies are hydraulic and organic loading rate. Currently the hydraulic loading rate or hydraulic residence time (HRT) is on the high end at an exchange rate of 17%/day, 5 days/week (average HRT = 8.5 days). The organic loading rate is on the low end of our goal of 10 to 50g biological oxygen demand (BOD)/m2/day, at a calculated 7.5g chemical oxygen demand (COD)/m2/day. Since steady state was reached at these loading rates, the HRT has been adjusted to 4 and 5.5 days and subsequently the organic loading rate increased to ~25g COD/m2/day. 1.7 Sampling and Analysis: Sampling and analysis were initiated during this period utilizing a Hach DR 5000 spectrophotometer and Hach DR 890 colorimeter. Nutrient, COD, solids and alkalinity are being monitored either weekly or as needed per experimental test. The laboratory is equipped with analytical scales, a microscope, reactor heating blocks, vacuum pump, and an oven and furnace for solids determinations. Algae productivity measurements conducted recently ranged 2.8-7.1 and 4.5-8.4 algae/m2/day in July and August, whereas, biomass concentration ranged 79-195 and 126-235 mg of volatile suspended solids (VSS)/L. GOAL#2: Maximize the Nutritional Value of Produced Algae for Animal Feed: Goal will be addressed in the next phase. GOAL#3: Optimize Pathogen Inactivation Methods: Goal will be addressed in the next phase under steady state bio-reactor conditions. GOAL#4: Quantify and Control any Cyanobacterial Toxins: 4.1 Quality Control (QC) Bacterial Strains: Cyanobacteria strains (toxin and non-toxin-producers) were acquired from The Culture Collection of Algae (University of Texas, Austin, TX). After culture of the QC strains, DNA was extracted and preliminary detection of QC cyanobacteria has been achieved using PCR targeting unique cyanobacteral sequences of the rpoC1 gene that encodes the gamma subunit of the cyanobacterial RNA polymerases (Glowacka et al., 2011), and 16s rRNA gene (Nubel et al., 1997). 4.2 Sample Analysis: On our first sampling visit for DNA studies (6/25/14), water samples were collected from the 4 algae bio-reactors and from the dairy lagoon at the CPSLO dairy. DNA was extracted from these samples for analysis of total bacteria (targeting 16S rRNA-encoding genes), cyanobacteria and algae using MiSeq Illumina's (Illumina Inc., San Diego, CA) next generation sequencing (NGS) platform. Pathogens will also be quantified using real-time PCR or digital PCR. 4.3. Impact of Different Commercially Available DNA Extraction Kits On Bio-reactor Bacterial Community Structures: We are currently evaluating the best commercially available kits for our DNA analyses. DNA extraction from bio-reactor samples was conducted using the Mo Bio Power water extraction kit, (Mo Bio Laboratories, Inc., Carlsbad, CA), Zymo fungi/bacterial extraction kit (Zymo Research Corp., Irvine, CA), and MP Biomedicals FastDNA Spin Kit (MP Biomedics, Santa Ana, CA). Concentration and quality of DNA from the three kits will be compared. Work on PCR amplification for MiSeq of bacterial 16S rRNA genes is currently being performed on the samples (n=4) collected from the algae bio-reactors at CPSLO. Hierarchical clustering of sequencing data will be conducted using the Yue and Clayton similarity coefficient. Linear modelling on relative abundance of common bacterial families from the three kits will be compared. Furthermore, the abundance of algae and cyanobacteria from the three kits will be determined. 4.3. Determination of Pathogens in Bio-reactor Samples: Detection and quantification of microbial pathogens using NGS is providing the fastest method for culture-free identification of different bacterial pathogens. This is significant because of the complexity of different contaminants, low-abundance of species, including difficulties associated with culturing all organisms of interest. Three commercially available DNA extraction methods are being tested for DNA extraction for use in identification and quantification of human bacterial pathogens (including E. coli O157) and other microbial genera for water quality assessment using MiSeq, Illumina’s NGS platform. For quantitation, PCR or droplet digital PCR will be evaluated after the optimization of DNA extraction technics. Optimization of this assay will provide the first step in preparing high-quality DNA for pathogen identification using NGS. Computer software and bioinformatics analyses will facilitate identification of pathogens and non-pathogens. The experiment will produce very large data sets for monitoring and correlating the abundance and diversity of pathogens to different environmental factors.

Publications