Introduction
Biogas is comprised primarily of methane (CH4, 50%~70%) and carbon dioxide (CO2, 30% ~50%). It can be produced through anaerobic digestion (AD) of various organic waste sources, including landfill waste; animal manure; wastewater sludge; and industrial, institutional, and commercial organic wastes. CO2 and CH4 are the two leading greenhouse gases (GHGs) that cause many detrimental effects to our ecosystem, including climate change. On the other hand, CH4 is also a valuable fuel. It is estimated that currently US biogas production potential is 654 billion cubic feet per year, which could displace 7.5 billion gallon of gasoline (AgSTAR, 2018). Although waste-derived biogas has immense potential as a renewable feedstock for producing high-density fuels and commodity chemicals, the contaminants (e.g., H2S, NH3, and volatile organic carbon (VOC) compounds) present significant challenges to biogas utilization. Currently the AD-derived biogas is primarily used for heating/cooking or flared, with only a small fraction for electricity generation due the cost associated with biogas cleanup (AgSTAR, 2018). To tap into this immense potential, effective technologies that can co-utilize both CO2 and CH4 without costly biogas cleanup are needed.
Recent studies have demonstrated that natural microbial communities have developed a highly efficient way to recover the energy and capture carbon from natural biogas streams through interspecies metabolic coupling of methane oxidation to oxygenic photosynthesis (Kip et al., 2010; Milucka et al., 2015; Raghoebarsing et al., 2005). Figure 1(a) illustrates the key synergistic interactions within the methanotroph-photoautotroph coculture: the photoautotroph converts CO2 into biomass while producing O2 via photosynthesis and the methanotroph utilizes the in situ produced O2 to convert CH4 into biomass while producing CO2 for the photoautotroph. Figure 1(b) depicts the total mass balance and key substrate exchanges in the coculture.
Following the principles that drive the natural consortia, different synthetic methanotroph-photoautotroph (e.g., cyanobacteria or microalgae) cocultures have been demonstrated to simultaneously convert both CH4 and CO2 into microbial biomass without external oxygen supply (Badr, Hilliard, Roberts, He, & Wang, 2019; Hill, Chrisler, Beliaev, & Bernstein, 2017; Rasouli, Valverde-Pérez, D’Este, De Francisci, & Angelidaki, 2018; van der Ha et al., 2012; Wang & He, 2018). The biogas-derived coculture biomass could be further processed to produce biofuels (such as biodiesel), directly used as single cell protein for animal feed supplement, or serves as feedstock to produce bioplastics. In addition, the coculture could be engineered to produce other value-added chemicals (such as succinate or lactic acid) using biogas as feedstock. Therefore, the methanotroph-photoautotroph coculture offers a highly promising biological platform for waste-to-value conversion.
In order to develop methanotroph-photoautotroph based biotechnology for biogas conversion, a key prerequisite is an effective tool to enable fast, easy and accurate characterization of each organism in the coculture in terms of biomass growth and biogas conversion performance. However, currently no such tool is available. In fact, one major challenge associated with characterizing any mixed culture is the accurate determination of the individual biomass concentration for each microorganism. Existing approaches to quantify individual biomass concentration in mixed culture include molecular biological, biochemical, and microbiological method (Sabra, Dietz, Tjahjasari, & Zeng, 2010; Spiegelman, Whissell, & Greer, 2005). However, these methods require either expensive equipment such as flow cytometry, community genome sequencing, or time-consuming and challenging techniques, such as RNA/DNA extraction, isolation, or amplification. Therefore, these approaches are suitable for off-line, infrequent characterization of mixed culture, and cannot provide the frequent or real-time measurements desired for dynamic modelling of the coculture systems. As a result, among the published methanotroph-photoautotroph research, only Hill et al. (2017) tracked the individual biomass concentration over time through cell counting using flow cytometry, while others just reported the total optical density of the coculture over time without differentiating the contribution from the methanotroph and the photoautotroph (Rasouli et al., 2018; van der Ha et al., 2012).
Besides individual biomass concentration, the individual substrate consumption rates and product excretion rates of each organism are needed in order to develop a kinetic model for the coculture. However, when there is cross-feeding in the coculture (i.e., any exchange of metabolite(s) between different organisms), it is highly challenging to obtain the individual consumption/production rates because they cannot be measured directly. For the case of methanotroph-photoautotroph coculture, as shown in Figure 1(b), both O2 and CO2 are cross-feeding metabolites: O2 is produced by the photoautotroph while consumed by the methanotroph, while CO2 is produced by the methanotroph and consumed by the photoautotroph. However, what can be directly measured are the overall or total consumption/production rates of O2 and CO2 by the coculture, not individual rates by each organism. Currently how to use the measured overall rate to infer or estimate the individual consumption/production rates remains an unsolved problem. It is worth noting that in our experiments, oftentimes no oxygen was detectable in the gas phase or liquid phase, as all the oxygen produced by the photoautotroph was consumed by the methanotrophin situ .
To address the above mentioned challenges, we have developed an experimental-computational (E-C) protocol to fully characterize the synthetic methanotroph-photoautotroph coculture based on the overall mass balance and each organism’s growth stoichiometry. Besides tracking the biomass concentration of each organism in the coculture over time, the E-C protocol also obtains estimates on the substrate consumption rates (CH4 and O2 uptake rates for the methanotroph and CO2 uptake rate for the photoautotroph) and product secretion rates (CO2 for the methanotroph and O2 for the photoautotroph). Such quantitative characterizations will enable better understanding of the coculture growth kinetics, and will lay the foundation for the development of the coculture-based biotechnology to convert biogas into valuable products. The E-C protocol only requires the commonly measured variables including total optical density for the coculture (UV/Vis spectroscopy), gas phase composition (GC), dissolved CO2 in the culture broth (total carbon analyser). Therefore, the E-C protocol does not require any special equipment, and it does not require any special sample preparation such as DNA/RNA extraction or cell fixation in order to achieve the above-mentioned characterizations.
In this work, we use one methanotroph-cyanobacteria pair and one methanotroph-microalgae pair to demonstrate the performance of the developed protocol; To validate its accuracy, we compared the individual biomass concentrations obtained by the E-C protocol with cell counting results obtained using flow cytometry. In this work, the methanotroph-cyanobacteria coculture pair is Methylomicrobium alcaliphilum 20ZR - Synechococcus sp. PCC7002 , which prefers high salt high pH medium and has demonstrated robust growth on different concentrations of biogas (Hill et al., 2017). The methanotroph-microalgae coculture pair is Methylococcus capsulatus - Chlorella sorokiniana , which prefers low salt and neutral pH medium and has been used for wastewater treatment (Rasouli et al., 2018).
Materials and Methods