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