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Methanotrophic bacteria can use methane as their only energy and carbon source, and they can be deployed to manufacture a broad range of value-added materials, from single cell protein (SCP) for feed and food applications over biopolymers such as polyhydroxybutyrate (PHB) to value-added building blocks and chemicals. SCP can replace fish meal and soy for fish (aquacultures), chicken and other feed applications, and also become a replacement of meat after suitable treatment, as a sustainable alternative protein. Polyhydroxyalkanoates (PHA) like PHB are a possible alternative to fossil-based thermoplastics. With ongoing and increasing pressure towards decarbonization in many industries, one can assume that natural gas consumption for combustion will decline. Methanotrophic upgrading of natural gas to valuable products is poised to become a very attractive option for owners of natural gas resources, regardless of whether they are connected to the gas grids. If all required protein, (bio)plastics and chemicals were made from natural gas, only 7, 12, 16–32%, and in total only 35–51%, respectively, of the annual production volume would be required. Also, that volume of methane could be sourced from renewable resources. Scalability will be the decisive factor in the circular and biobased economy transition, and it is methanotrophic fermentation that can close that gap.
Federal Research Center “Pushchino Scientific Center for Biological Research of the Russian Academy of Sciences”, G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Russian Federation
*Address all correspondence to: m.lackner@circe.at
1. Introduction
While most petrochemicals are made from crude oil, they are also accessible through natural gas or coal. For instance, synthesis gas can be converted to various hydrocarbons through Fischer Tropsch synthesis [1], which was performed on a large scale in economies without access to oil in times of war (Germany) or Apartheid (South Africa). Over the last 50 years, petrochemicals have seen stronger growth than other materials such as steel or aluminum, because of cheap crude oil and the versatility of the products. It was particularly the plastics that have experienced a tremendous increase in use. The importance of oil and gas for various industries is depicted in Figure 1.
Figure 1.
How crude oil (left) and natural gas (right) are used. Source: [2].
As one can see from Figure 1, approx. 14% of crude oil (left) and 8% of natural gas (right) are deployed to produce petrochemicals. These are first some standard “base chemicals” or building blocks, from which a huge variety of materials can be derived at low-cost in large quantities.
From natural gas, today chiefly hydrogen, methanol and ammonia are made. Ammonia is used for urea and fertilizer production, for instance. The importance of natural gas as feedstock [3] varies with geography and is shown in Figure 2.
Figure 2.
Feedstocks and primary products. COG = coke oven gas; HVC = high value chemicals; Mtoe = million tons of oil equivalent. Source: [2].
The key question, in times of an impending huge energy transition towards renewables, is which trajectory petrochemicals will take in the two or three decades to come, whether crude oil will continue to dominate or whether, e.g., coal or natural gas will be brought in as main feedstocks, or possibly even biomass, in the medium turn. In the long run, i.e., 2050 and beyond, it can be assumed that biomass will have become the dominant feedstock.
The world population is growing [4], and industrialization and economic development are moving ahead. Hence, the demand for petrochemicals is bound to further increase. For plastics (thermoplastics), it can be expected that the historic growth rate of ~6%/year will go down significantly, and that more recycling will be implemented. Other products, such as fertilizers, cannot be turned into a circular economy concept as easily as for instance polymers, so with them, demand most probably will continue to rise.
The energy consumption and its source in different industries is another important aspect. Let us take a look at Figure 3, the energy demand and the CO2 emissions (footprint) of 5 vital sectors.
Figure 3.
Source: CO2 emissions and energy demand for materials in 2017. The final energy demand is given in Mtoe (million tons of oil equivalent). Source: [2].
Iron and steel, as well as cement, today are very carbon-intense industries, due to the processes which are being used. Global cement production consumes on the order of 6% of global primary energy demand, mainly by combustion of fossil fuels in the rotary kiln reactors and due to the large production volumes on the order of 4.1 billion tons/year [5]. Iron and steel still rely to a large extent of the blast furnace, being fuelled by coke, but less carbon-emitting reducing agents are being tested and implemented, such as CH4 or H2, as well as more electric arc furnaces particularly for the increasing share of recycled steel (scrap smelting). Chemicals account for significant energy demand, too, from various processes. Figure 4 looks at the mass flow in the chemical sector.
Figure 4.
Part of a Sankey diagram in the chemical sector: Approx. 200 million tons/year of natural gas and NGL (natural gas liquids) are converted into NH3 (which is then processed to fertilizer) and other products. Source: [6].
As Figure 4 shows, coal plays a minor role today, and crude oil dominates. Natural gas has a significant share, though. Decarbonization by moving to a less carbon-intense feedstock such as CH4 will be a key driver in the industry in the decades to come. Natural gas is less carbon-intense than crude oil and coal, so one might see more natural gas conversion to chemicals.
1.1 Natural gas today
Natural gas is an established fossil fuel, with existing infrastructure in many parts of the world. It is considered as a “clean” fuel, which burns without ash or significant soot production, and releases less CO2 per unit of energy delivered than do oil-derived fuels or particularly coal-based heating materials. The main constituent of natural gas, methane (CH4), can also be sourced and be distributed through the grid from alternative sources such as landfills (“landfill gas”) or anaerobic digestion facilities (“biogas”). There is also a strong link towards renewables, though the option of storing (excess) wind energy or (excess) photovoltaic energy in the huge natural gas network under the P2G (power2gas) [7] concept, as hydrogen or as methane (which can be made from H2 in the Sabatier process). The market size of natural gas is on the order of 1000 billion USD per year, and it amounts to approx. 4000 billion m3/year [8]. 24% of the global energy mix comes from natural gas, which is double the contribution from renewables today. For electricity, the share of natural gas is even higher, where it lies at 55.7% (and at 38.1% for renewables, for comparison). Almost all countries rely on natural gas to some extent. It is the UAE, Russia, Iran, Qatar, Oman and Algeria that are gas-based economies [9]. Methane emissions from industrial operations (production, distribution and use) have lately been identified as a significant and previously underestimated contribution to climate change, and can be addressed by organizational and technological means, see later.
Pipeline transport offers the lowest unit cost for short distances for natural gas. Other methods exist, too, e.g., LNG (liquefied natural gas) [10], particularly for economic long-haul transportation.
1.2 Projection on natural gas utilization
The IEA World Energy Outlook [9] is an authoritative reference work, updated annually to provide trustful projections of global energy developments. According to the latest 2021 report by IEA (WEO-2021), the natural gas demand is believed to increase over the next 5 years, which is the case for all scenarios considered. After that period, one can see sharp divergences in the different scenarios. The report states that “Today, natural gas is the largest source of electricity in advanced economies and its level of use remains broadly stable in those economies over the next decade, while it increases by about one-third in the emerging market and developing economies, helping to moderate the use of coal”, according to IEA [9]. Figure 5 shows the scenarios.
Figure 5.
Demand for oil, natural gas and coal according to IEA, in the STEPS (stated policies scenario) in the reports of 2016 (blue), 2020 (green) and 2021 (red). One can see that the new report is much less in favor of fossil fuels than previous projections. Source: [9].
For their latest report WEO-2021, IEA has modeled four scenarios:
Announced Pledges Scenario (APS)
Net Zero Emissions by 2050 Scenario (NZE)
Stated Policies Scenario (STEPS)
Sustainable Development Scenario (SDS)
“The NZE is normative, in that it is designed to achieve specific outcomes – an emissions trajectory consistent with limiting the global temperature rise to 1.5 °C without a temperature overshoot (with a 50% probability), universal access to modern energy services and major improvements in air quality – and shows a pathway to reach it. APS and STEPS are exploratory, in that they define a set of starting conditions, such as policies and targets, and then see where they lead based on model representations of energy systems, including market dynamics and technological progress. The SDS is also normative, mapping out a pathway consistent with the “well below 2 °C” goal of the Paris Agreement, while achieving universal access and improving air quality” [11].
The historic price development of the fossil fuels natural gas, coal and oil is depicted in Figure 6 [9].
Figure 6.
Regional oil, coal and natural gas prices from 2010 to 2021. MBtu = million British thermal units. Source: [9].
One can infer from Figure 6 that natural gas is less volatile than oil, and can be on the lowest unit costs level.
In the STEPS scenario, the demand for natural gas will increase to approx. 4500 billion m3 by 2030 (which is 15% higher than in 2020) and to 5100 billion m3 in 2050. According to IEA, there will be a rise in natural gas utilization both in industry and in the power sector, and it will remain the standard for space heating.
By contrast, the APS scenario predicts a peak of natural gas use shortly after 2025, followed by a decrease to 3850 billion m3 by 2050. “Countries with net zero pledges move away from the use of gas in buildings, and see a near 25% decrease in consumption in the power sector to 2030”.
In the NZE scenario, the demand for natural gas is forecast to drop even more sharply from 2025 onwards and will fall to 1750 billion m3 in 2050. “By 2050, more than 50% of natural gas consumed is used to produce low-carbon hydrogen, and 70% of gas use is in facilities equipped with CCUS”. (CCUS = carbon capture, utilisation and storage). Likewise, we can expect more natural gas being deployed for the production of additional materials.
Figure 7 offers a visual depiction of the scenario projections.
Figure 7.
Left: Timeline of natural gas use; right: Supply of low emissions gasses in 2030. Bcm = billion m3. Note: Hydrogen gases are inclusive of low-carbon gaseous hydrogen and synthetic methane with 1 EJ = 29 bcm. Source: [9].
An interesting aspect worth noting from Figure 7 is that biogas is forecast to have a relevant role in all scenarios, on the order of 5% of natural gas volumes, being at ~double the level of 2020 production. It is complemented by hydrogen to various degrees. Natural gas demand changes per scenario are detailed in Figure 8.
Figure 8.
Development of natural gas demand from 2020 to 2030. Bcm = billion m3. Source: [9].
Only emerging markets and developing economies are expected to use more natural gas, as do industry and hydrogen production. Details on the projected natural gas consumption per region are shown in Figure 9.
Figure 9.
How natural gas production is expected to change by region and scenario from 2020 to 2050. Bcm = billion m3; C & S America = central and South America. Source: [9].
One can observe from Figure 9 that in all 3 scenarios, up to 2050, natural gas use in Europe will decline.
Table 1 shows the remaining natural gas resources in terms of “technically recoverable natural gas”.
Natural gas (trillion m3)
Proven reserves
Resources
Conventional gas
Tight gas
Shale gas
Coalbed methane
North America
17
149
50
10
81
7
Central and South America
8
84
28
15
41
—
Europe
5
46
18
5
18
5
Africa
19
101
51
10
40
0
Middle East
81
121
101
9
11
—
Eurasia
70
169
131
10
10
17
Asia Pacific
21
139
45
21
53
2ß
World
221
809
425
80
253
49
Table 1.
The remaining “technically recoverable” resources of natural gas. Source: [9].
As Table 1 demonstrates, it is in the Middle East and Eurasia where most of the proven reserves of natural gas are sitting. All regions have significant proven reserves and even more resources, so that availability is not the determining factor for natural gas use but rather other factors such as climate change considerations.
1.3 Methane emissions from the gas industry
The debate of climate change has shifted from merely considering CO2 to also methane, where a significant fraction of the emissions is associated with oil and gas production. These methane emissions can reduce or even completely offset the greenhouse gas benefits of using gas instead of the more carbon-intense fuels coal and oil. The “2021 Oil & Gas Benchmarking Report “[12] with a focus on the USA reads: “EPA-estimated methane emissions from crude oil and refined oil product systems decreased 28% from 1990 to 2015. However, emissions estimates remain uncertain” [13], see also Figure 10.
Figure 10.
Sources of methane emissions in the USA in 2015, as estimated by the Environmental Protection Agency (EPA). Source: [13].
In the US, it is estimated that enteric fermentation, e.g., from cattle, accounts for ¼ of anthropogenic methane emissions. Landfills are responsible for 1/5 of the emissions, on the same level as manure. Approx. 1/3 of man-made methane emissions are attributed to natural gas and petroleum systems, with high uncertainty (and recent evidence that this fraction will be higher).
Recent work has assessed the well-to-city-gate greenhouse gas intensities of natural gas in a Chinese setting, see Figure 11.
Figure 11.
“Well-to-city-gate” greenhouse gas intensities of natural gas supplies to China from >100 different fields. In the depiction, the colors represent emissions by individual processes. The confidence intervals are 90% (error bars). X-axis: Number of gas field; Y-axis: kg of CO2 equivalent per MJ [14].
We can see from that study that there are huge differences in the negative climate effects of natural gas. Domestic natural gas from domestic sources is slightly better, on average, than overseas LNG, but can be a factor of 10 better than international pipeline-derived natural gas. Natural gas can be lost in pumping stations or at the end user, e.g., as unburnt fuel, which all adds up to the radiative forcing.
1.4 The future of natural gas
The world is hungry for energy. On the one hand, there is a need for cheap and increasing energy, on the other hand, fossil fuels are seeing more and more resistance, and the energy sector has started its transformation.
“Industry discussions about the future of gas in North America are polarizing. On [the] one hand, the shale revolution keeps delivering, displacing liquefied natural gas (LNG) imports since the late 2000s, as abundant gas resources and technological innovation drove costs down… On the other hand, as state-level decarbonization policies ramp up, the demand for natural gas in key segments such as power generation and local distribution companies (LDC) is expected to decline” [15].
Natural gas can bring supply and demand of electricity into balance, by fuelling dispatchable on-demand power generation (as huge energy storage devices are lacking). There will probably be more volatility in demand for that natural gas with an increasing share of wind power and photovoltaics, which are difficult to predict in their actual production and which cannot be tuned to demand. Also, it is not yet clear how fast and how effectively the gas industry can curb its fugitive methane emissions, and which policies different countries will enact.
In any case, we are on a path of decarbonization, and times have become unsecure for natural gas owners and industry incumbents. This is not a convenient situation, and energy-related investments are typically characterized by very long (several decades) investment times.
In the near future, we might see
natural gas to replace the carbon-intense coal
more co-generation and possibly polygeneration
more use of methane as feedstock for chemicals
more coal gasification and biomass gasification to produce chemicals (due to the price volatility of natural gas)
SNG (synthetic natural gas) [16], where coal, waste or biomass is the feedstock
more combinations with carbon capture and storage [17]
What can be taken for granted, owners of natural gas will be more inclined to produce value-added materials from natural gas, rather than betting on their resource being continuously used as fuel in the next decades.
Also, owners of natural gas resources that have not yet been connected to the grid might turn towards materials production, as the corresponding production plants can be economic also at smaller scale than mere natural gas extraction for energy. Depending on the value of the products, units of 10,000 tons/year of materials or less can be viable. In the transition to the bioeconomy, biorefineries face the challenge of scalability. Larger scale translates into lower unit costs, however, at the same time, raw material transport over longer distances will increase the costs, so the optimum scale is narrower than with a natural-gas operated plant.
What’s more, there has been a strong technology push for gas fermentation technology from biogas gasification (to synthesis gas), which generates technology spill-over potential for natural gas fermentation. Biogas production by anaerobic digestion of various substrates [18] is an established technology.
These 3 aspects make natural gas fermentation a highly attractive route for methane valorization.
2. Natural gas fermentation to value-added products
Fermentation processes are ubiquitous in nature. They can be aerobic (see e.g. [19, 20] for methane) or anaerobic (see e.g. [21, 22, 23, 24] for methane) and they have been used by mankind for thousands of years [25, 26, 27]. Methane is produced by anerobic fermentation, as it is well known from and deployed in biogas plants [28] or happening in landfills [29]. Also, methane is released from waste-water treatment plants, manure storage pits, ruminants (e.g., cattle) or natural soil such as wetlands. It is in these habitats where also methane-consuming microorganisms, so-called methanotrophic bacteria [30, 31, 32, 33] can be found. They can be isolated [34] and be deployed for various target products. The methanome contains methanogenic and methanotrophic bacteria. Basically, methanotrophs are found in all habitats where methane is available [30, 35]. However, methane-oxidizing bacteria only constitute a fraction of the microbial community in most soils, in general <4% of all bacteria [36]. Anaerobic methane fermentation constitutes an important process in the deep subsurface, e.g., in marine sediments [37], as a biogeochemical process that limits the release of methane into the atmosphere. For industrial use, aerobic methanotrophs are more relevant. There are also other bacteria which feed on (higher) hydrocarbons, e.g., as they are found in contaminated land [38].
Methanotrophic bacteria (methanotrophs, methane-oxidizing bacteria (MOB)) [39] belong to the wider “methylotrophic” group that subsumes microorganisms which can use C1 substrates like methane or methanol [32, 40, 41]. Methane is found in nature; In the atmosphere, its concentration is slightly below 2 ppm. Biogenic methane is emitted from rice fields, various wetlands, termites and herbivores. Also, methanogenic archaea liberate methane from organic matter decomposition [42]. Aerobic methanotrophs are estimated to consume 10–90% of the methane that comes out of the deep anoxic layers of wetlands before it reaches the atmosphere [43], hence they have a strong impact on the climate, as methane is a very potent greenhouse gas with a greenhouse warming potential (GWP) of 24 relative to CO2 [44].
Said methanotrophs are gram-negative bacteria that use methane as their only source of carbon and energy [31], making them unique and interesting for biotechnological applications. Apart from being hosts for biotechnological production with native and engineered microorganisms in industry, they could be used for geoengineering purposes to fight global warming by methane, e.g., from thawing permafrost soil, fugitive anthropogenic emissions or direct air capturing of methane. Methanotrophs have also been described to co-metabolize toxic compounds [31]. Table 2 lists several methane-utilizing bacteria.
Methanotrophs are an important biological sink for methane, and thereby are very relevant for the global carbon cycle [46]. The very first methanotrophic bacterium, bacillus methanicum, was discovered in the year 1906. To date, more than 100 different methanotrophs are known. Aerobic methanotrophs comprise >20 [47] recognized genera that belong to three major phylogenetic groups (type I, type II and type X). That classification is done on the microorganisms’ characteristics such as morphology or carbon assimilation pathways [31]. Apart from the described bacteria, also some yeasts were found to feed on methane [48, 49, 50].
2.1 Motivation for microbial methane fermentation
Biocatalytic conversion of methane [51] is a promising route for both commodities and for specialty materials. There is consensus on the need for alternative proteins other than meat. Projections say that alternatives for meat can account for up to 60% of the market in 2040 [52]. Also, there is significant consumer interest in such alternatives [53].
Agriculturally-produced alt protein, e.g., from peas or soy, is one option, non-crop-based proteins are another one. However, there are significant cost hurdles when non-agricultural protein is to be made. For insect protein [54], the price (2021 level) is between 4250 and 6066 USD/ton [55]. Single cell protein (SCP) from side streams of lignocellulose was estimated at requiring a minimum selling price of 5160–9007 €/ton to be economically viable [56]. A ubiquitous raw material is methane, but its use is not straightforward either; one of the most invidious molecules for the organic chemist is CO2. Almost next in line comes CH4. One requires 438.8 kJ/mol [30] to activate the C▬H bond. As a result, thermochemical processes with methane are energy-intensive, requiring high temperatures and pressures, and/or costly catalysts, see Table 3.
Methylomonas methanica
Methylococcus capsulatus
Methylococcus vinelandii
Methylomonas carbonatophila
Methylococcus capsulatus strain Bath
Methylosinus sporium
Methylomonas rubrum
Methylococcus ucrainicus
Methylosinus trichosporium
Methylomonas rosaceus
Methylococcus fulvus
Methylosinus trichosporium TG
Methylomonas agile
Methylococcus thermophilus
Methylocystis parvus
Methylomonas albus
Methylococcus albus
Methylobacterium organophilum
Methylomonas streptobacterium
Methylococcus minimus
Mycobacterium methanicum
Methylomonas methanooxidans
Methylococcus luteus
Mycobacterium cuneatum
Methylomonas methanitrificans
Methylococcus bovis
Nocardia rhodochrous
Pseudomonas strain L-8
Methylococcus chroococcus
Nocardia ucrainica
Pseudomonas strain L-47
Methylococcus whittenburii
Rhodopseudomonas gelatinosa
Pseudomonas strain L-49
Strain TM-10
Table 2.
Selection of methane-oxidizing bacteria. Source: [45].
Catalyst
Pressure [atm]
Temperature [°C]
Oxidant
Methane conversion [%]
Chemical conversion
Hg(II)
—
180
O2
50
Au + additives (H2SeO4/SeO4/O2)
27 bar
180
SO3
3–28
PMo11V
1 atm
700–750
O2
3–13
PMo11Fe
4–23
SiMo11Fe
4–32
ZSM-5
30.5 bar
50
H2O2
0.3
Bioconversion
M. trichosporium OB3b
1
30
O2
64
Methanotrophic consortium
1
30
O2
43–80
M. trichosporium OB3b
1
30
O2
73.8–75.2
Table 3.
Comparison of process conditions and carbon conversion efficiencies of chemical and biochemical catalysts. Source: [30].
By contrast, biocatalysts operate at mild conditions. Also, their carbon conversion efficiency tends to be higher. Therefore, biotechnological methane oxidation using methanotrophic bacteria offers an interesting route.
2.2 Methanotroph product range and cultivation
When we look at the products accessible through methanotrophic bacteria, wild types can be used to obtain compounds from the natural microorganisms’ metabolic pathways. Generic engineering allows to expand that range, see Figure 12.
Figure 12.
Potential fuels and chemicals accessible through methane fermentation. Methanotrophic bacteria can generate all relevant 1-, 2- and 3-carbon intermediate compounds. “Virtually all biosynthetic modules for the production of advanced fuels or chemicals, developed for glucose-based fermentation in E. coli, could potentially be implemented in methane-utilizing strains”. PHB = polyhydroxybutyrate; PHV = polyhydroxyvalerate; FAEEs = fatty acid ethyl esters; FAMEs = fatty acid methyl esters; source: [57].
Genetic engineering is useful when the target products are chemicals. Obtaining approval for feed and food made with GMO (genetically modified organisms) will be significantly harder and is therefore discouraged. To date, most metabolic engineering was achieved with type I methanotrophs [58]. Synthetic methanotrophs [59, 60] offer the potential for improved product range, yields and productivities [61]. Recently, the engineering of type II methanotrophs to produce 3-hydroxypropionic acid (3-HP), cadaverine and lysine [62, 63, 64] was reported. They are of particularly interest, because type II methanotrophs (e.g., M. trichosporium OB3b) can co-metabolize methane and CO2 [58]. Also, they were described to consume C1, C2 and C3 compounds and to be able to fix nitrogen [58]. Whilst most methanotrophs are mesophiles, extremophile methanotrophs are known, e.g., alkaliphiles, acidophiles, thermophiles [65], psychrophiles (cryophiles) and halophiles [31]. Obligate methanotrophs (most known methanotrophs) can only use methane as their carbon source, whereas the limited facultative ones (Methylocapsa aurea, Methylocystis spp.) can also grow on acetate and ethanol. Facultative methanotrophs (Methylocella silvestris) are also able to utilize higher hydrocarbons. Methylocella silvestris was found to consume acetone, acetol, 2-propanol, 1,2-propanediol, methylacetate, glycerol, gluconate, propionate, tetrahydrofurane, ethane and propane [66].
Methanotrophs were shown to produce methanol, ectoine [67], polyhydroxy butyrate (PHB, more specifically P3HB), 2,3-butanediol, single cell protein (SCP) [68], carotenoids, vitamin B12 [69] and lactic acid [47, 70]. Basically, production can be intracellularly or extracellularly, where the latter offers advantages in downstream processing through avoided need for cell lysis, provided that no cell inhibition [71] occurs by the excreted compounds as was shown e.g., for methanol.
For methane-utilizing Pseudomoms sp., it was found that Hyphomicrobium sp., when added to a mixed culture fermentation regime, could eliminate any inhibitory methanol [72].
Methanol may be used as biofuel [73]. It can be burnt directly, be used in fuel cells or utilized for transesterification of various plant oils to fatty acid methyl esters (FAME, aka biodiesel).
The methanotrophs, when deployed in a bioreactor, are typically grown as free cells. Using immobilized cells was found to increase productivity [74, 75] for methanol.
Production can be carried out with pure cultures or with mixed cultures [76, 77, 78] with the latter being more robust for industrial production, e.g., of single cell protein (SCP) [72, 79, 80].
In [81] a mixed culture of type I methanotrophs that had been obtained from waste activated sludge was tested. The key genera were Methylococcus, Methylobacter, Methaylocaldum, Methylomicrobium, Methylomonas, Methylosarcina and Methylosphaera. The maximum specific growth rate (μmax) was determined as 0.358 h−1 (8.59 per day), and the maximum specific methane biodegradation rate (qmax) was 14.52 g CH4 per total g(DCW) and per hour reported in mixed cultures (DCW = dry cell weight).
In [72], a culture of Pseudomonas sp. and Hyphomicrobium sp. could be stabilized by the addition of Acinetobacter sp. and/or Flavobacterium sp.
Co-cultures of methanotrophs have been described, too, e.g., the syntrophic co-culturing of a methanotroph and heterotroph to obtain mevalonate from methane [82]. Syntrophy (symbiosis) describes a system where one species lives off the products of another one. M. capsulatus bath was combined with E. coli SBA01 in that study. Mevalonic acid (MVA) is a precursor molecule for terpenes and steroids. In another study, co-culturing methanotrophs with microalgae was suggested [83, 84], or with hydrogen-oxidizing bacteria [85].
Many industrial fermentation processes rely on (sterile) monocultures. However, one has to bear in mind that in nature, mixed cultures are the norm. For traditional fermentation processes such as cheese, yoghurt, soy sauce and sauerkraut, it has also been mixed cultures that have been used.
Harrison [78] coined the term of “structured mixed cultures” to describe cultures which were obtained by a combination of well-defined microorganisms instead of enriched undefined natural and thus “uncontrolled” mixtures [45]. Table 4 gives some examples.
Raw material
Organisms
Benefits of culture
Methane
A methane oxidizer + a methanol-utilizer + a citrate-utilizing bacterium
“structured mixed cultures” and their benefits exemplified. Source: [45].
The advantages of these “structured” mixed cultures, compared to monocultures, are summarized as follows [45]:
Improved utilization of a mixture of carbon sources or a complex carbon source.
Targeted protein composition can be obtained.
Organic carbon that is secreted by one microorganism of the consortium is removed by another one:
removal of toxic substances (which can affect the microbiome)
Higher total biomass yield from the primary carbon source
Elimination of foam
Higher growth rate
Possibility for water recycling
Better tolerance of perturbations
Resistance against contamination by fungi, yeasts, bacteria and phages [45].
Picking up the point of extracellular products, these two disadvantages might arise next to inhibition:
Risk for contamination with other microorganisms feeding on these compounds and being unable to utilize the primary substrate. That risk can be alleviated by sterile operation, which however is difficult to maintain over several weeks of production [45]. Secondly, more effort to purify the fermentation medium for reuse. Therefore, mixed cultures were proposed to avoid the need for aseptic operation with the minor partner removing unwanted compounds from the fermentation broth. Hamer et al. [86] reported an excellent mixed culture. It contained 4 bacteria:
Hyphomicrobium sp.—a bacterium that can grow on methanol, but not on methane.
two Gram-negative bacteria, incapable of growth on either methane or methanol, but growing on a broad variety of organic molecules (Acinetobacter sp., Flavobacterium sp.)
In the EU feed catalog EU No. 68/2013 [87], this mixed culture is listed for protein production from methane:
Methylococcus capsulatus [88, 89, 90] is the most-studied host for SCP. Other strains are Pseudomonas methanica [91], Methylosinus trichosporium (OB3B) [92] and methanotroph Methylomicrobium buryatense5GB1 [93].
As a feedstock for the fermentation process, pure methane, pure [94] or mixed C1 feedstock [95], natural gas, biogas or hythane (a mixture of CH4 and H2) can be used [96, 97].
While methanotrophs favor CH4 as their source of carbon and energy, some of them can also use methanol in its absence. Methylocella tundrae was found to prefer methanol over methane [98]. Methanol has a better solubility in the growth medium than methane, but is more costly. The Pruteen™ [99] process was based on methanol, and the strain Methylophilus methylotrophus.
All type II methanotrophs, some type I methanotrophs (Methylococcus, Methylosoma, Methyloglobulus, Methyloprofundus, and selected strains within Methyomonas and Methylobacter) can fix atmospheric nitrogen [98]. Therefore, type II methanotrophs are generally dominant under N-limiting conditions or high carbon to nitrogen (C/N) ratios, while type I methanotrophs generally need a high nitrogen content, i.e., lower value of the C/N ratio [98].
Nitrogen addition was found to decrease methane uptake in a natural environment containing methanotrophs and methanogens [100]. For the effect of ammonia on methanotrophs growth, see [101].
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