*Value-Added Products from Natural Gas Using Fermentation Processes: Fermentation of Natural… DOI: http://dx.doi.org/10.5772/intechopen.103813*

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 [62–64] 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 [76–78] 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 coculturing 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


#### **Table 4.**

*"structured mixed cultures" and their benefits exemplified. Source: [45].*

instead of enriched undefined natural and thus "uncontrolled" mixtures [45]. **Table 4** gives some examples.

The advantages of these "structured" mixed cultures, compared to monocultures, are summarized as follows [45]:


Organic carbon that is secreted by one microorganism of the consortium is removed by another one:

a. removal of toxic substances (which can affect the microbiome)

b.Higher total biomass yield from the primary carbon source


g.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

*Value-Added Products from Natural Gas Using Fermentation Processes: Fermentation of Natural… DOI: http://dx.doi.org/10.5772/intechopen.103813*

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:


In the EU feed catalog EU No. 68/2013 [87], this mixed culture is listed for protein production from methane:

1.*Methylococcus capsulatus (Bath)* (NCIMB strain 11,132).

2.*Alcaligenes acidovorans* (NCIMB strain 12,387).

3.*Bacillus brevis* (NCIMB strain 13,288).

4.*Bacillus firmus* (NCIMB strain 13,280).

*Methylococcus capsulatus* [88–90] 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].
