Sustainable Cultivation of Microalgae using Diluted Anaerobic Digestate as a Nutrient Medium

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1 Sustainable Cultivation of Microalgae using Diluted Anaerobic Digestate as a Nutrient Medium Husam A. Abu Hajar, The University of Jordan, Amman, Jordan R. Guy Riefler, Ohio University, Athens, Ohio Ben J. Stuart, Old Dominion University, Norfolk, Virginia Corresponding Author: Ben Stuart, Old Dominion University In this study, the cultivation of two microalgal species using anaerobic digestate as a nutrient medium was investigated. Several pretreatment methods were applied to the anaerobic digestate including hydrogen peroxide treatment, filtration using polyester filter bags, and supernatant extraction; the latter proved to be the simplest and most effective method in reducing the turbidity and COD of the diluted digestate while maintaining sufficient nutrients for the cultivation of microalgae. Initially, the microalga Neochloris oleoabundans was cultivated using diluted anaerobic digestate supernatant on the bench-scale; however, when scaled up to 100 L raceway ponds, the culture was contaminated with other algal species. The microalga Scenedesmus dimorphus was then cultivated using anaerobic digestate supernatant on the bench-scale. It was found that mg N/L dilutions were sufficient to maximize the specific growth rates while still producing relatively high biomass concentrations. The microalgae cultivation was scaled up to 100 L raceway ponds using 100 mg N/L dilution at 454 and 317 µmol/m2/s light intensities and 50 mg N/L dilution at 384 and 234 µmol/m2/s light intensities. The maximum biomass concentration attained was 432 mg/l in the 100 mg N/L and 454 µmol/m2/s culture. Furthermore, nitrogen was removed at 65-72% efficiency with complete ammonia removal. Phosphorus removal efficiencies were in the % range whereas the presence of bacteria in the unsterilized nutrient media resulted in COD removal efficiencies in the 78-82% range. Finally, the effect of mixing on the growth of the microalga S. dimorphus was assessed by cultivating this microalga in a raceway pond at 0.1, 0.2, and 0.3 m/s water surface velocities. It was concluded that increasing the mixing velocity yielded higher biomass concentrations and growth rates. However, by balancing the power required to operate the

2 pond at different velocities with the potential energy yield from biodiesel synthesis, it was concluded that running the pond at 0.1 m/s surface velocity was the only case with a positive net energy yield.

3 Use of Urea and Ammonium Chloride to Limit Zooplankton Grazers in Scenedesmus Cultures Sandeep Kumar, Old Dominion University, Caleb Talbot, Old Dominion University; Siobhan McFarlane, Old Dominion University; Blake Steiner, Old Dominion University. Corresponding Author: Sandeep Kumar, Old Dominion University With the growing need of renewable energy, algae have researched as a potential biomass source for biofuel and bioproduct development. However, zooplankton grazers continue to be an obstacle to maintaining open ponds, as they reduced yields and help cause culture crashes. Recently at Old Dominion University s open pond, it was observed that zooplankton grazers were limited via a ph>9.5, with a total nitrogen concentration of 30mg/L using urea. Similar results were found in the literature, but specifically for rotifers and did not look into how algae were affected. Furthermore, the majority of literature was published in the 1980 s. Thus, this study was conducted to understand the phenomenon. In this experiment, 2.5 L cultures of Scenedesmus with zooplankton were inoculated in bench scale photobioreactors with 640 mg/l urea. This was first done with 4 replicates of urea and 8 replicates of control cultures. Then a 500 mg/l spike of NH4Cl was used in addition to the 640 mg/l urea on day 0; this was done to see if grazers may be limited sooner, with 4 replicate cultures. Cell counts were performed with the hemocytometer, then with a Gridded Sedgewick Rafter 1 mm2 for the spike experiment for count comparisons. Indeed, ph was not controlled for in this experiment, but was recorded. In addition, temperature and ambient light were also measured. Total Suspended Solids (TSS) was implemented to obtain a liquid sample. This allowed for the determination of ionized ammonium concentration, which was then converted to free ammonia. The specific amounts were determined by a cation chromatograph. In the end, it was found that urea (640 mg/l) seems to have an impact on zooplankton by day 8 out of 14, with free ammonia concentration peaking by day 6, indicating a hour lag time. This was confirmed with the NH4Cl spike (500 mg/l). If this continues to stand, then use of free ammonia hydrothermal liquefaction processes may be an option to promote sustainability.

4 Biofilm Microalgae Treats Municipal Wastewater with High Ammonia Concentration Celeste Hancock, Utah State University, Ronald Sims, Utah State University Corresponding Author: Celeste Hancock, Utah State University High ammonia concentrations that can be present in the filtrate of biosolids from anaerobic digesters may present a challenge for treatment and removal for water reclamation facilities working to meet newer and more stringent discharge standards. The rotating algal biofilm reactor (RABR) system has previously demonstrated significant nitrogen removal at ammonia concentrations from 10 to 100 mg/l. The effectiveness of these reactors in terms of nitrogen remediation and algal biomass production are being tested with wastewater with ammonia concentrations greater than 500 ppm at the Central Valley Water Reclamation Facility, the largest municipal wastewater treatment plant in the State of Utah. Biomass produced from treating the wastewater can be used in anaerobic digesters to produce biogas or can be added to compost to increase its fertilizer value. In order to optimize biomass growth, a variety of growth substrata, consisting of different plastics and polystyrene as a control, were tested. Water samples from the anaerobic biosolids filter pressate and microalgae from the trickling filters were collected at the CVWRF and are being tested using laboratory scale RABRs in batch mode. Nitrogen concentration and biomass production were monitored over time. Initial studies using polystyrene as the growth substrata demonstrated a 78% removal rate after 18 days. Preliminary results showed that polypropylene is a potential growth substrata producing 0.7 g/m2/day when compared to the polystyrene control which produced 0.5 g/m2/day. Other prospective materials include virgin UHMW (0.5 g/m2/day), CPVC (0.5 g/m2/day), and PVC (0.4 g/m2/day).

5 An Algal Revolving Biofilm (RAB) system for microalgal cultivation with enhanced biomass production and reduced footprint Zhiyou Wen, Iowa State University, Martin Gross, Iowa State University Corresponding Author: Zhiyou Wen, Iowa State University Microalgae have been widely studied to remove nutrients from wastewater due to their rapid absorption of those nutrients for their growth. However, commercial implementation of open pond- or enclosed photobioreactors-based microalgae cultivation as a treatment system has not happened due to high operation cost and the large footprint of these systems. Dr. Wen s group recently developed a unique algal culture system so called revolving algal biofilm (RAB) for solving the above problems. The RAB system drastically increases the algae growing surface area by using a vertical orientation of algal biofilm to capture sunlight and CO2, thus requiring a much smaller footprint. Also, the biomass can be retained on the RAB system s attachment material for a much longer solids retention time than the hydraulic retention time of the liquid, which is crucial for highly efficient nutrient removal from wastewater. The RAB system has been used for treating effluent from various food industrial operations, including meat processing effluent, yeast fermentation based broth; and animal feed production, with high removal efficiency of nitrogen, phosphorus and special chemicals such as selenium.

6 Hazardous Algal Blooms: Cyanobacterial Microcystin Degradation through Anaerobic Digestion Corresponding Author: Kyle Hillman, Utah State University Kyle Hillman, Utah State University Utah Lake, a major source of recreation and farmland irrigation in the Provo area, was closed to all use and access in the summer of There was a toxic blue-green algae (cyanobacteria) outbreak, which resulted in microcystin concentrations over eight times the recreational water threshold limit (20 ug/l threshold and 176 ug/l detection). The ability to harvest and dispose of these toxic algae before contamination of the lake occurs is important to ensure protection of human and animal health and the environment, and more hazardous algae blooms (HABs) are anticipated in the future. The Central Valley Water Reclamation Facility (CVWRF), a local municipal wastewater treatment facility, utilizes anaerobic digesters for wastewater treatment. A preliminary experiment was conducted to test cyanobacteria microcystin degradation via anaerobic digestion using anaerobic digester sludge obtained from this facility. The microcystin concentration readings were conducted using ABRAXIS Recreational Water Microcystin Dipstick ELISA Test and AbraScan II Dipstick Reader. This test is semi-quantitative, especially regarding the approximate microcystin concentration. Preliminary experimentation shows a trend of microcystin degradation over time in laboratory-scale anaerobic digesters. Microcystin concentration decreased from approximately 10 ug/l to approximately 2.5 ug/l, a 75% reduction, in diluted samples through the thirty-six day treatment test. Average microcystin concentration was plotted vs time, resulting in a first-order decay constant of 0.03/day. Based on this trend, an estimated 75% reduction in microcystin can occur within approximately forty-six days. The change in microcystin concentration was approximately zero in control groups consisting of microcystin in distilled deionized water. Further testing is needed, but this data indicates a possibility of harvesting HABs from Utah Lake for disposal in the CVWRF digesters.

7 Lipid Extraction from Microalgae Using Supercritical CO2 with Methanol Shihong Liu, Ohio University; Guy Riefler, Ohio University Corresponding Author: Shihong Liu, Ohio University Microalgae are considered to be the most promising feedstocks for biodiesel production. The main drawback of microalgae as feedstocks, however, is their energy intensive and costly production process, especially the lipid extraction step. In this research, the lipid extraction from microalgae Spirulina and Schizochytrium was investigated. For both microalgae, supercritical CO2 was used for extraction. In addition, the effect of methanol as a polar modifier in supercritical CO2 lipid extraction was also investigated. For comparison, lipid extraction using different organic solvents with Soxhlet device was also investigated. The results showed that the addition of methanol in supercritical CO2 can increase the lipid extraction yield significantly. Finally, the extracted lipids were analyzed with gas chromatograph to study the fatty acids profile in the lipids.

8 In vitro investigation to explore use of post-extracted microalgal residue as a sustainable source of dietary protein for ruminants Jong-Su Eun, Animal, Dairy, and Veterinary Sci. Dept., Utah State University, S. Y. Yang, Department of Animal, Dairy, and Veterinary Sciences, Utah State University, J. M. Yang, Department of Animal, Dairy, and Veterinary Sciences, Utah State University, J.-S. Eun, Department of Animal, Dairy, and Veterinary Sciences, Utah State University, R. C. Sims, Department of Biological Engineering, Utah State University, and R. C. Anderson, USDA-ARS, Southern Plains Agricultural Research Center, Food and Feed Safety Research Unit Corresponding Author: Jong-Su Eun, Animal, Dairy, and Veterinary Sci. Dept., Utah State University The fat-extracted microalgal biomass derived from the biofuel production has been suggested as a promising source of protein supplement. In addition, the recently developed wet lipid extraction procedure (WLEP) allows an opportunity to harvest a solid phase biomaterial containing a high concentration of protein material (microalgal protein precipitate material; APP). Consequently, we performed a series of in vitro experiments to test the effects of supplementing APP in dairy diets on ruminal fermentation using continuous cultures. In experiment 1, microalgae was obtained from the Logan City, Utah Wastewater Treatment Plant (Logan, UT), and APP was generated using dilute acid and base hydrolysis of the microalgal biomass based on the WLEP. The experiment was conducted as a completely randomized design with 2 (high-forage (HF) vs. low-forage diet (LF)) 2 (without vs. with APP) factorial arrangement of treatments. Total volatile fatty acid (VFA) concentration was not affected due to forage concentration in the diets, whereas APP supplementation increased total VFA concentration in HF diet (P < 0.05). Decreasing forage in the diets as well as APP supplementation reduced culture ammonia-nitrogen concentration (P < 0.01). Supplementing APP elevated C18:1 trans-11 concentration (P < 0.01), regardless of forage concentration in the diets. However, methane production in the culture was not affected by dietary treatments. Supplementation of APP successfully replaced soybean meal and canola meal mixture, typical sources of protein supplement, without any negative effect on microbial fermentation profiles. Increased total VFA concentration coupled with decreased ammonia-n concentration in HF diet indicates that APP used in the present study exerted its potential effects in a diet-dependent manner. In experiment 2, APP was obtained using the same WLEP, but the source of microalgae was dairy waste from Caine Dairy Research Center (Wellsville, UT). The APP used in the experiment 2 had low bacterial counts on blood agar, approximately CFU/g. In addition, in a 24-h incubation assay with a suspension of bovine fecal contents, we did found neither an inhibitory nor a stimulatory effect of the APP on growth or survivability of Salmonella typhimurium or Escherichia coli O157:H7. The APP used in the experiment 2

9 contained lessen crude protein concentration compared with the one used in the experiment 1 (30.6 vs. 58.7%). The experiment was performed as a completely randomized design to test 4 dietary treatments in a typical lactation dairy diet: 1) no APP (control), 2) 1.2% APP, 3) 2.4% APP, and 3.6% APP in a dry matter basis. Although increasing APP in the diet tended to linearly decreased culture ph (P = 0.08), the culture ph was maintained at least 6.0 throughout culture incubation. Dietary treatments did not affect culture ammonia-nitrogen concentration as well as methane production. Thus, the APP from dairy waste can be supplemented in dairy diets up to 3.6% dry matter without any negative effect on ruminal fermentation. Overall results on the two in vitro experiments indicate that APP can have a potential to be used as a sustainable source of dietary protein for ruminants.

10 A Case Study of Pilot-Scale Continuous Flow Reactor for Hydrothermal Liquefaction of Algae Feng Cheng, Travis Le-Doux, Juanita Miller, Catherine E. Brewer, Umakanta Jena Department of Chemical and Materials Engineering, New Mexico State University Corresponding Author: Umakanta Jena, New Mexico State University Algae-based biofuels have attracted significant research interest due to their advantages of not competing with land for food production, abilities to grow in low-quality water, higher growth rates, and strong CO2-mitigation abilities. In recent years, research has focused on hydrothermal liquefaction (HTL) of whole, wet algae biomass. HTL uses hot, compressed water ( C and 8-18 MPa) to convert the organic constituents into an energy-dense biocrude oil that can be upgraded to liquid transportation fuels. Most HTL research studies to date have reported results from batch reactors of ml sizes operated at 5-20% algal solids load. In order to develop commercial scale operations, future HTL systems would need a significant technological shift from batch processes to development of continuous flow reactor (CFR) systems. The CFR systems suffer from several challenges including smooth flow of biomass slurry through pumping/preheating unit/reactor units, clogging of solids, solid-liquid-gas separation/ filtration, involvement of large number of unit operations and the safety and control issues. Unlike the batch systems, maintaining a high solids loads (>5%) pose a significant challenge for CFR systems. The current study presents New Mexico State University s experience in development and modification of a pilot-scale CFR for HTL of fresh water microalgae. The goal of the reactor is to be able to perform continuous HTL on slurries with solid algae contents of 5-10 wt.%, and to produce char-free bio-oils. The presentation provides an overview of the operational issues with a continuous HTL reactor and suitable modifications made to address some of the above challenges.

11 Two-stage Hydrothermal Processing of Algae and Aqueous co-product (ACP) Recycling Umakanta Jena, New Mexico State University, S. Kent Hoekman, Desert Research Institute Corresponding Author: Umakanta Jena, New Mexico State University Hydrothermal processing of algae into a liquid hydrocarbon fuel has received considerable attention in the past few years due to the inherent ability of the process to convert wet biomass without energy intensive pretreatments such as drying. The present study investigates a two-step hydrothermal conversion of algae: a low temperature hydrothermal liquefaction (HTL) process for removal of N and O from algae for subsequent conversion by a second stage HTL at more severe conditions. The specific objectives under the above broad goals were to evaluate a first-stage (low temperature) HTL process at ?C temperature and evaluate the recycling of nitrogen and phosphorous in the aqueous phase co-products (ACP) for growth of mixed consortia diatom and cyanobacteria algae. First-stage HTL experiments were conducted using a custom designed two-chamber reactor (TCR) system at different conditions of reaction temperature (175, 200, 225, and 250?C) and time (1, 2.5, and 5 min). Treated solids and aqueous co-products (ACP) were analyzed by standard laboratory methods including mass yields, elemental C, H, N, and O contents, higher heating value (HHV), removal of N and O from the solids, and total nitrogen, ammonia nitrogen, total phosphorous, total organic carbon, ph, and non-volatile residues for the aqueous co-products. Statistical analyses were performed to evaluate solid yields in the first-stage HTL experiments. Solid yields were higher at less severe reaction conditions (lower temperature and shorter time) than at more severe conditions (higher temperature and longer time). The highest solid yield (~60%) was obtained at 170?C and 1 min; the lowest solid yield (~35%) was obtained at 250?C and 5 min HTL conditions. Recycling of ACP and further growth of algae was tested in batch cultivation of diatom-cyanobacteria mixed consortia algae and compared with growth using WC standard medium. Results showed that use of ACP medium, combined with phosphorous-depleted WC medium, provided significant growth.

12 Pressure Cycles-Assisted Ozonation Treatment of Produced Water Before and After Algae Cultivation Xinyue Zhao, Ching-Chieh Lin, Andy Hong Civil & Environ. Eng., University of Utah, Salt Lake City, UT Corresponding Author: Andy Hong, University of Utah On average, seven barrels of produced water are brought to the surface with every one barrel of crude oil that is produced. This water cannot be used without treatment, which is expensive and often simply too difficult to approach cost-effectively. Algal biomass can be energy-rich and its lipid content has potential to be a significant source of renewable biofuel. Microalgae cultivated by produced water are seen as desirable because they produce high lipid yields and require no freshwater or pesticides often associated with environmental pollution. With these underlying opportunities, this paper employs a unique ozonation technique to treat raw produced water and produced water after its use in cultivation of algae. The technique involves expanding ozone bubble created by pressurizing and depressurizing of the water being treated, which allows ozone to react effectively with hydrocarbon at the gas-liquid interface, thus converting the dissolved and suspended oil to substances retainable by sand filtration. The ozonated water was filtered through a sand bed and then contacted with activated sludge in attached mode. The results showed that organic contents in the effluent following ozonation and sand filtration treatments were significantly reduced and the biodegradability of the residual compounds was significantly increased. The technique holds promise for treatment of raw produced water and as a polishing step for algae-cultivated produced water, which potentially enables subsequent membrane processes for beneficial uses of the treated water.