<p>1. Generate experimental field data and calibrate optimization models. For treatment, expected removals are 85-95% biochemical oxygen demand and soluble Nitrogen (N) and 40-80% solublePhosphate (P) removal, depending on culturing technique and season.</p>
<p>2. Maximize the nutritional value of produced algae for animal feed. The cultures will be optimized to produce biomass at a high rate while also having the highest value composition for feed (in terms of lipids, digestibility, essential fatty and amino acid profiles, including balanced protein and carbohydrate concentrations).</p>
<p>3. Optimize pathogen inactivation methods. Pathogens will die-off in the ponds and during disinfection processing of the harvested 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 (e.g., high pH) and biomass processing (e.g., pasteurization) will be determined to achieve needed log inactivation of pathogens, which is typically 1- >4 log10 reduction (Sobsey et al., Available Online).</p>
<p>4. Quantify and control any cyanobacterial toxins. qPCR assays described by Al-Tarineh et al. (2012 a and b) will be used and optimized to reliably determine the copy number of cyanotoxin biosynthesis genes, as well as an internal cyanobacteria 16S rDNA control, in a single reaction. The latter detects for presence of cyanobacteria. If toxins are detected, measures will be taken to control invasion of the ponds by cyanotoxin-producing cyanobacteria strains.</p>
<p>Overall Goal Benefit agriculture and the environment by introducing microalgae, a fast-growing livestock feed crop.</p>
<p>NON-TECHNICAL SUMMARY:<br/> Rationale The need to control manure-derived nutrient pollution is straining the confined animal production industry. California is the top milk producing state and has some of the strictest nutrient regulations. But in the San Joaquin Valley, many dairies do not have affordable access to more land for manure application. A highly productive crop is needed that will convert manure nitrogen (N) and phosphate (P) into feed but in smaller land areas than crops such as corn. Algae are a candidate feed with annual yields typically 7-13 times greater than soy or corn. Beyond 40-50% protein, algae also contain fatty acids, amino acids, pigments, and vitamins that are valuable in animal feeds, especially for adding value to milk. Advances in molecular biology allow us to gather needed information on the risks and benefits of algae-based animal feeds.
Overall goal Benefit animal agriculture and the environment by introducing microalgae as a fast-growing livestock feed crop. Aim 1 Cultivate algae in dairy freestall barn flush water, treating this wastewater, while producing algae feedstock at a high annual rate, at least 10-times greater than corn. Algae will be cultivated in 30-cm deep raceway ponds at the 300-head Cal Poly campus dairy farm where extensive manure management research already occurs under USDA and USEPA sponsorship. Aim 2 Produce algae with favorable nutritional characteristics (high digestibility, valuable fatty and amino acid profiles, balanced protein and carbohydrate concentration, etc.) by adjusting the treated-water recycling into the ponds to optimize the N concentration in the growth medium. Aim 3 Test pathogen survival in algae feeds prepared by pasteurization and/or drying and heating. A trend in municipal
wastewater treatment is pasteurization of treated effluent using waste heat from natural gas electrical generator. Large dairies with digesters will have waste heat available for pasteurization and drying. High-protein algae will be pelletized with high carbohydrate feeds to create a balanced feed. The heat of pelletization also contributes to pasteurization. Cal Poly has a research feed mill for producing such blended feeds. Aim 4 Monitor contamination by cyanobacteria and any cyanobacterial toxins. Approach Removal of N, P, and other constituents will be optimized in influent and effluent of identical ponds. Algal biomass (harvested by bioflocculation+settling) will be analyzed for N, P, protein, carbohydrates, and profiles of fatty and amino acids. Pathogen and algal communities extant in raw and feed-processed algal biomass will be analyzed using metagenomics and pyrosequencing.
Potential toxicity of algal biomass will be studied using toxicity evaluation of cell-free extracts on cultured mammalian cells. A TC 20 Cell counter (BioRad Laboratories) will be used to monitor toxicity events on treated cells using trypan blue staining. Cytotoxic positive samples will be tested for both presence and concentration of known cyanobacterial toxins. The researchers have decades' experience in algae production, wastewater treatment, and food safety. Expected outcomes Starting with dairy, the project will lead the way towards an algae feed industry based on advanced nutritional features to enhance agricultural products (e.g., milk protein, poultry pigment) while assisting farmers to meet manure management challenges. We will address topics rarely covered in the algae field: potential toxicity and zoonotic pathogens. Our approach is unique in that it integrates and
addresses a triad of issues, namely, food safety issues along with algae production techniques and waste management.<p>
APPROACH: <br/>Task 1: Generate experimental pilot plant data and calibrate optimization models Laboratory and pilot plant algae cultivation will be used to develop cultivation and bioflocculation harvesting methods and to identify preferred algal strains for testing in ponds. Four identical 30-cm deep, paddle wheel-mixed raceway tanks will be installed adjacent to dairy waste lagoons at Cal Poly San Louis Obispo (CPSLO) and will be operated as two sets of duplicates. Influent to algae ponds will be flush water storage lagoons at CPSLO dairy. N concentrations can be decreased by dilution with well water or clarified pond effluent, and increased by addition of Nitrogen (N fertilizer). Bottled CO2 will be bubbled into the ponds for some experiments to eliminate any inorganic C limitation on algae growth. Initially indigenous strains from five main genera will be used. Any
exceptional strains or cultivation methods developed in Cal Poly Pomona (CPP) lab studies will be implemented in the ponds. JMP software will be used to optimize wastewater treatment performance, feed value and safety. Multivariate modeling will be used to account for uncontrollable environmental variables and to determine statistically significant growth or treatment parameters. During the first 18 months green algae polycultures will be grown with a focus on protein production. In the final 18 months, diatom algae will be grown with a focus on nutritional lipid production. pH control via CO2 addition will be a main factor in experiments on pathogen inactivation. Task 2: Laboratory Culturing: Rapidly identify and test strains and cultivation methods Lab cultures will be optimized to produce algae at a high rate with corresponding attempts to have highest value algae composition for
feed (in terms of lipids, carbohydrates, proteins, digestibility, valuable fatty and amino acid profiles, balanced protein and carbohydrate concentrations). Biomass and species composition, characterization of proximate composition, including photosynthetic function using pulse amplitude modulated (PAM) fluorometry will be monitored routinely. Cultivation methods developed at CPP and CPSLO will be evaluated at pilot scale (Task 1). Our goal is to produce algae with favorable nutritional characteristics, high digestibility, valuable fatty and amino acid profiles, balanced protein and carbohydrate concentration by developing modern analytical methods to study community structure (Task 3) and rapid fluorometric techniques for pond management. Strains will be cultured in well-controlled bioreactors sparged with air/CO2 mixes, and will be screened for patterns of lipid, protein and nucleic
acid biosynthesis as a function of growth phase under varying light and nutrient regimes. Since protein and lipid biosynthesis are fed by photosynthetic carbon fixation, key questions to be addressed by this work are to determine if light saturation levels, and N-deprivation influence photosynthetic efficiency, lipid and protein biosynthesis, cell viability and growth. Task 3: Maximize the compositional value of the produced algae for animal feed 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 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. 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. Task 4: Optimize pathogen inactivation methods Several options for pelletization (pasteurization) and drying will be evaluated for their effectiveness in reducing bacterial pathogen loads. Thermophilic processes, such as pasteurization, thermophilic digestion and composting, can inactivate pathogens (>4 log10). Treated residuals are likely to contain low pathogen concentrations. Liquid samples will be collected from algae raceway ponds at CPSLO. Survival of pathogens (e.g.. E. coli O157:H7, Listeria monocytogenes, Campylobacter jejuni and Salmonella spp.) will be studied using plate count, real-time PCR (qPCR) and reverse transcriptase
real-time PCR. qPCR will be used to quantify total bacterial counts in pond samples targeting universal bacterial 16S rRNA. Bacterial cell counts will be estimated by comparisons of threshold cycle (Ct) values to an E. coli O157:H7 genomic DNA standard curve. Ct levels are inversely proportional to the amount of target nucleic acid and correlates to numbers of organisms. High-protein algae will be pelletized with high carbohydrate feeds to create a balanced feed (CPSLO research feed mill). The heat of pelletization contributes to pasteurization. Autoclave and drying parameters will be evaluated to determine time-temperature relationships needed for sterilization. Task 5. Analysis of Microbial and Cyanobacterial Community Structure in Ponds Fragments of the 16S rRNA-encoding region of DNA from bacteria isolated from the ponds will be amplified and subjected to 454 pyrosequencing analysis.
Comparison of bacterial communities among different pond treatments will reveal degree of specialization of different pond biota, and provide insights into the identity of potential agents of negative and positive feedback on algal bloom. This will be done in parallel with GeoChip-based metagenomic studies whereby fluorescently-stained hybridized DNA will be scanned using an MS 200 Microarray Scanner. For the analysis of cyanobacterial and algal populations in ponds the large subunit of the rRNA-encoding gene will be targeted. Sequencing of 23S rRNA genes will be performed at the Research and Testing Laboratory (Lubbock, TX). Bacterial pyrosequencing population data will be analyzed using multiple sequence alignment techniques in MOTHUR, version 1.9. For the GeoChip data Vegan package R 2.9.1 and the pipeline developed at University of Oklahoma (http://ieg.ou.edu) will be used to assess
overall functional compositions of pond communities. Task 6: Toxicity Assessment of Algal Biomass Cytotoxicity assessments will be conducted before qPCR because they can pick additional toxicities encoded by unknown genes. Potential toxicity of algal biomass and stockfeed will be evaluated on cell-free extracts that will be tested on cultured mammalian cell lines (hepatocyte and neurocyte). Cytotoxic positive samples will be subjected to qPCR. A quadriplex quantitative-PCR (qPCR) assay capable of detecting and quantifying toxin genes for microcystin, nodularin, cylindrospermopsin and saxitoxin biosynthesis will be used. The assay targets hepato- and neuro-toxigenic cyanobacteria of global significance.</p>