(218a) Equipment and Method for Batch Screening of Methanotroph-Microalgae Cocultures for Waste-to-Value Conversion

In the US, the biogas produced from anaerobic digestion (AD) of industrial, municipal and agricultural waste streams has immense potential as a renewable feedstock, with more than 7.8 million metric tons of CH₄ and over 14.4 million metric tons of CO₂ per year. However, the utilization of biogas is limited to heat and electricity production due to the presence of contaminants such as H₂S, NH₃, and volatile organic compounds (VOCs). At the same time, the digestate/wastewater from AD is rich in nitrogen, phosphorous and other contaminants. If not properly treated before released into waterways, digestate/wastewater can have detrimental impacts on the local community and environment. In fact, the excessive amount of nitrogen and phosphorus in released wastewater has caused increasingly negative consequences to our ecosystems and public health, including worsening of the greenhouse effect, reduction of the protective ozone layer, adding to smog, contributing to acid rain, and contaminating drinking water^1,2.

If biogas valorization and wastewater treatment can be integrated together through biological conversion, it will not only reduce the detrimental environmental and social impact of wastewater, but also generate revenue to offset the cost of wastewater treatment and even make the process profitable. Motivated by this idea, we have demonstrated that the methanotroph-microalgae coculture offers a highly effective platform for biogas valorization and nutrient recovery from wastewater^3,4. This is largely attributed to the metabolic coupling of methane oxidation and photosynthesis oxygenation. In order to develop a robust and effective waste-to-value biotechnology based on the methanotroph-microalgae coculture, selection of the microbial strains that are fast growing and exhibit robust growth on wastewater would be highly desirable. To this end, we have developed a parallel screening equipment that allow us to test multiple strains simultaneously, as shown in Figure 1.

Temperature, pH, gas feed composition, and agitation affect the growth rate of microbes, so controlling these parameters is essential for screening. In addition, light intensity must be regulated for photoautotrophs. The temperature of the liquid phase was maintained at 30°C using a Type-K thermocouple connected to a heating plate. While water baths ensure a more uniform temperature distribution, the temperature gradient throughout the well-mixed solution is negligible. For pH control, most wastewater plants operate at neutral conditions. Methanotrophs produce CO­₂ which decreases the pH in the liquid phase, so the addition of 1M NaOH was used to maintain a pH of 7.0 using an automated pH controller. For feed composition, each vessel was provided the same, continuous rate of feed gas throughout the experiment; methanotrophs were fed the same ratio of CH₄ and O₂ while microalgae samples were fed the same ratio of CO₂ and N₂. Agitation is necessary for a well-mixed solution, but too much agitation can disrupt optimal growth of certain cultures. The stir plates have a large stirring error of ±50 rpm. Therefore, each stir rate was chosen so that an external laser tachometer read the same stir rate for every stir plate. Although the feed gas causes uncontrollable agitation when bubbled through the liquid, the predominate source of agitation is stirring. Finally, the light intensity was consistent across all tests by using one LED light sheet set at a single intensity.

Table 1 enumerates the species of interest, which are selected based on available literature. The selected methanotrophs and microalgae species all grow readily at neutral pH conditions^5-8, which is desirable since many wastewater streams are fairly neutral. Each species will be tested in triplicate. During testing, anaerobic digestate provided by Columbus Water Works (which has AD installed) will be diluted and used for culture medium.

The efficacy of each strain was determined in three ways. The first metric was growth rate, which was calculated based on the change in optical density (OD) of the microbial solution. The optimal OD wavelength was selected based upon previous literature studies for the microbes tested. The OD was determined using ultraviolet-visible spectroscopy (UV-VIS). The second metric was to determine the extracellular biomass in the liquid phase. The extracellular polysaccharides (EPS) can be utilized as biofuel and bioplastic precursors once separated from the solid biomass. After removing cells using centrifugation, a Shimadzu total organic carbon analyzer (TOC-L) determined the concentration of EPS in the liquid phase. The last metric focuses on how effectively the microbes utilized the ammonia and phosphate present in the medium. The total nitrogen and phosphorous concentrations in the liquid phase were recorded every 24 hours.

Once the individual methanotroph and microalgae species are tested, top ranked species in terms of biomass yield and nutrient recovery performance will then be paired together and screened for synergism and biomass production. The screening results obtained from this work will provide important information future experiments, such as the choice of the coculture model systems for additional investigation, as well as for scaling up and continuous operations.

Works Cited

[1] Driscoll, C., Whitall, D., Aber, J., Boyer, E., Castro, M., Cronan, C., et al. (2003). Nitrogen pollution: sources and consequences in the U.S. northeast. Environment 45, 8–22. doi:10.1080/00139150309604553

[2] Galloway, J. N., Dentener, F. J., Capone, D. G., Boyer, E. W., Howarth, R. W., Seitzinger, S. P., et al. (2004). Nitrogen cycles: past, present, and future. Biogeochemistry 70, 153–226. doi:10.1007/s10533-004-0370-0

[3] Roberts, N., Hilliard, M., He, Q. P., & Wang, J. (2020). A Microalgae-Methanotroph Coculture is a promising platform for fuels and chemical production from wastewater. Frontiers in Energy Research, 8. doi:10.3389/fenrg.2020.563352

[4] Badr, K., Whalen, W., He, Q. P., & Wang, J. (2020). Fast and easy quantitative characterization of methanotroph-photoautotroph cocultures. Biotech and Bioeng, 118(2), 703-714. doi:10.22541/au.159708707.70834744

[5] AlSayed, A., Fergala, A., & Eldyasti, A. (2018). Sustainable biogas mitigation and value-added resources recovery using methanotrophs intergrated into wastewater treatment plants. Reviews in Environmental Science and Bio/Technology, 17(2), 351-393. doi:10.1007/s11157-018-9464-3

[6] Christenson, L., & Sims, R. (2011). Production and harvesting of microalgae for wastewater treatment, biofuels, and bioproducts. Biotechnology Advances, 29(6), 686-702. doi:10.1016/j.biotechadv.2011.05.015

[7] Kadir, W. N., Lam, M. K., Uemura, Y., Lim, J. W., & Lee, K. T. (2018). Harvesting and pre-treatment of microalgae cultivated in wastewater for biodiesel production: A review. Energy Conversion and Management, 171, 1416-1429. doi:10.1016/j.enconman.2018.06.074

[8] Zhao, W., Sun, H., Ren, Y., Wu, T., He, Y., & Chen, F. (2018). Chlorella zofingiensis as a promising strain in wastewater treatment. Bioresource Technology, 268, 286-291. doi:10.1016/j.biortech.2018.07.144