(263e) Expeditious Screening of Gas-Dependent Biological Systems Using Adaptable Fed-Batch Bioreactors

Biological systems that sequester greenhouse gases, like methanotrophs and algae, are showing increased promise for ameliorating climate change. Elucidating the most promising biological systems for large-scale integration for industrial applications requires adequate screening of different microorganisms. Screening requires ample time and scrutiny to assess the performance of biological systems both accurately and unbiasedly. Typically, screening is performed in batch with volumes less than 50mL that do not mimic large-scale operations. These trials allow the biological systems to reach the stationary phase without replenishing feed or adjusting pH, and these limited results influence the direction of posterior research. For gas-dependent biological species, batch screening is complicated by the exhaustion of headspace gas and the poor solubility of gases in aqueous media. Discerning the true cause of inhibition usually involves invasive sampling, reducing the working volume with each disturbance.

A screening system to overcome these barriers is needed for investigating methanotroph-photoautotroph cocultures (MPC’s). Anaerobic digestion (AD) is a prevalent technology in the wastewater, agricultural, and food industries that catabolizes large waste products into biogas and liquid effluent. Valorizing the methane of biogas is hindered by corrosive and volatile contaminants, so this methane is predominantly flared for its low return on investment. The MPC can dually utilize CH₄ and CO₂ from the biogas, as well as eutrophication nutrients from the AD liquid effluent, without any pretreatment at near ambient conditions [1]. The MPC valorizes this waste by producing biomass for single-cell protein and value-added chemicals for bioplastic synthesis. Given the novelty of the MPC, many cocultures should be screened to find promising pairs for large-scale AD biogas and effluent remediation.

To address these issues, a fed-batch screening system (Species Screening Station (S3)) was developed to greatly expedite the screening processes [2]. Shown in Figure 1, the S3 consists of nine parallel reactors (three sets of triplicates) with working volumes of roughly 250mL that regulate six abiotic factors: temperature, agitation rate, pH, light intensity, gas feed composition, and aeration rate. The continuous feed gas relieves gas-dependent microorganisms of carbon limitations, so each species’ growth will be hindered by deficiencies in the liquid medium. In this work, different methanotrophs, algae, and their cocultures have been screened not only to highlight the capabilities of the S3, but also to highlight promising biological systems for greenhouse gas remediation.

Before any testing, the S3 was validated by culturing Chlorella sorokiniana, a heavily studied microalga, on the same liquid medium across all nine vessels set at the same six abiotic conditions. The tight standard deviations at each time point (<5%) and estimated maximum growth rate (0.178 ± 0.005 1/hr) validated that the vessel location does not significantly impact growth performance. With this lack of bias, three sets of triplicate biological trials can occur simultaneously, greatly reducing the amount of time required for lab-scale screening.

The first set of experiments investigated a synergistic MPC – Methylotuvimicrobium buryatense 5GB1 (5GB1) and Arthrospira platensis (Spirulina) – grown on defined media and synthetic biogas. Previously, this MPC exhibited accelerated growth rates and higher methane and carbon dioxide uptake rates than their respective monocultures [3]. To further validate these results, the monocultures and coculture were cultured on the S3 at the same abiotic conditions on synthetic biogas. For the 5GB1 monoculture, oxygen was supplemented as 5GB1 is an aerobe. On the other hand, oxygen was not supplemented to the MPC since Spirulina provides oxygen for 5GB1. After 150 hours of screening, the biomass of the MPC exceeded that of both monocultures. Unlike the monocultures, the coculture had not entered the stationary phase, continuing to accumulate biomass. Synonymous with these results, the MPC exhibited higher total nitrogen and phosphorous recovery rates (4.92 mg/L/hr and 1.60 mg/L/hr, respectively) than the monocultures (the greatest of each being 3.76 mg/L/hr from Spirulina and 1.14 mg/L/hr from 5GB1, respectively).

Next, a variety of methanotroph and microalga species were screened for MPC evaluation on diluted AD effluent and synthetic biogas. Both microalgae and methanotrophs have been studied separately for effective wastewater remediation [4,5]. The MPC can simultaneously remove methane and carbon dioxide from biogas and valorize eutrophication nutrients from the liquid effluent into biomass [1]. The S3 was used to determine what methanotrophs and microalgae could best serve for dual biogas and wastewater remediation as MPC’s. Five microalgae and seven methanotrophs were screened on diluted AD at abiotic conditions. Each monoculture was allowed to grow until it reached the stationary phase. The growth performance was evaluated by analyzing growth data with a hybrid Gompertz and exponential growth model. The top monocultures determined from this analysis were screened in full as MPC’s on diluted AD and synthetic biogas. The most promising MPC was Methylosarcina fibrata and Chlorella sorokiniana, which produced more than double the biomass of its monocultures, unlike the other MPC’s. In addition, this MPC experienced a second growth phase after 40 hours, suggesting that this MPC possessed synergistic traits not seen by the other MPC’s. While the reason for this synergism is unclear, this MPC appears to be a promising biological system for wastewater remediation.

Lastly, the S3 was slightly modified to screen a mutant of 5GB1 for its potential to remediate low concentrations of methane. M. buryatense 5GB1C (5GB1C) is a mutant of 5GB1 that lacks a plasmid to make the strain easier to genetically manipulate. Recently, this strain has demonstrated its potential for growth on methane concentrations in air as low as 200ppm at working volumes of 10mL [6]. These results indicate that 5GB1C could be useful for remediating methane emissions from different agricultural sectors, such as rice fields or livestock farms [6]. The S3 was modified to determine if 5GB1C could grow on 500ppm methane at larger working volumes at different aeration rates. In one test, the S3 revealed that 5GB1C could grow on 500ppm methane at aeration rates of 0.4, 0.8, and 1.6 vvm with a working volume of 260mL. In addition, exponential growth models indicated that aeration rate scaled linearly with the growth rate. This result is due to 5GB1C’s limited access to methane given the poor solubility of methane.

My current work continues to investigate the synergistic interactions of 5GB1 and Spirulina by integrating experimental, computational, and -omics data. The exact metabolite exchange that promotes accelerated growth and nutrient uptake rates is currently unknown. Understanding this exchange would expedite the development of genetically modified MPC’s that could further accelerate dual AD biogas and effluent conversion and valorization. Recent genome-scale modeling results have suggested that light attenuation and oxygen concentration are the respective inhibitors of the photoautotroph and methanotroph in the MPC when grown on synthetic biogas. My upcoming experiments will first analyze each monoculture in a chemostat by introducing a disturbance in light intensity or oxygen concentration. The gas effluent, liquid effluent, and biomass will be sampled dynamically after the disturbance to elucidate the phenotype shift in each monoculture. Samples from this dynamic shift will be frozen for transcriptomic analysis to investigate the genotypic response. This experiment will be repeated using the MPC, taking samples at the same time intervals as the monocultures. The effluent streams, biomass, and transcriptomic data, compared to the results from the monocultures, should reveal the metabolic interlinks between the MPC. These results will be incorporated into the pre-existing, semi-structured kinetic model coupled with genome-scale modeling [2] to paint a clearer picture of how the carbon and redox flow adapts in this MPC.

References

[1] Roberts, N, Hilliard, M, He, QP & Wang, J. (2020). A Microalgae-Methanotroph Coculture is a Promising Platform for Fuels and Chemical Production from Wastewater. Front Energy Res, 8, 563352. https://doi.org/10.3389/fenrg.2020.563352.

[2] Murphy, L, Badr, K, He, QP & Wang, J. (2022, Nov 15). Fed-Batch Screening of Methanotroph-Algae Cocultures on Anaerobic Digester Effluent for Larger-Scale Wastewater Treatment and Valorization. AIChE 2022 Annual Conference, Phoenix, AZ, USA.

[3] Badr, K, He, QP & Wang, J. (2022). Identifying interspecies interactions within a model methanotroph-photoautotroph coculture using semi-structured and structured modeling. IFAC-PapersOnLine, 55(7), 106-111. https://doi.org/10.1016/j.ifacol.2022.07.429.

[4] Mahari, WAW, Razali WAW, Manan, H, Hersi, MA, Ishak, SD, Cheah, W, Chan, DJC, Sonne, C, Show, PL & Lam SS. (2022). Recent advances on microalgae cultivation for simultaneous biomass production and removal of wastewater pollutants to achieve circular economy. Bioresource Technol, 364, 128085. https://doi.org/10.1016/j.biortech.2022.128085.

[5] AlSayed, A, Fergala, A & Eldyasti A. (2018). Sustainable biogas mitigation and value-added resources recovery using methanotrophs integrated into wastewater treatment plants. Rev Environ Sci Biotechnol, 17, 351-393. https://doi.org/10.1007/s11157-018-9464-3.

[6] He, L, Groom, JD, Wilson, EH, Fernandez, J, Konopka, MC, Beck, DAC, and Lidstrom, ME. (2023). A methanotrophic bacterium to enable methane removal from climate mitigation. PNAS, 120(35), e2310046120. https://doi.org/10.1073/pnas.2310046120.