Analysis of Methanotroph Populations from Various Sources for Production of High-Value Products ^†

Abstract

Methanotrophs are bacteria that can consume methane as their sole carbon and energy source to produce a wide variety of high-value products such as lipids, biopolymers, ectoine, and single cell proteins (SCPs). Collected samples from various sources were subjected to DNA extraction followed by 16S rRNA analysis to determine the identity and relative abundance of their microbial population. Several taxa of methanotrophs were detected in the samples including Type I (Methylobacter), Type X (Methylocaldum), Type II (Methylocystis, Methylosinus, and Beijerinckia), and Type III (Verrucomicrobium). This paper expounds the effects of environmental/cultivation conditions on the growth and population of different types of methanotrophs. The results could be used to systematically identify source(s) of natural consortia that can be enriched and developed to produce specific target product(s) under a given cultivation conditions/limitations.

1. Introduction

In recent years, the increase in greenhouse gas (GHG) emissions, causing global warming, has been a pressing issue due to its evident harmful environmental effects. Methane (CH₄) is considered as the second most prominent GHG produced next to carbon dioxide (CO₂) and has a global warming potential of 27–30 times higher than CO₂ over 100 years [1]. Methane is the key component of natural gas, which is typically used for power, fuel, and heat. The advancement in shale gas production has resulted in the instability of natural gas prices in the past decade, e.g., $8.86 per million British thermal unit (MMBtu) in 2008, $2.05/MMBtu in 2020, and $7.88/MMBtu in September 2022 [2]. As a result, large volumes of CH₄ are vented and flared into the atmosphere mainly due to unprofitability, operational safety, and costly connection to the pipeline [3]. In 2021 alone, the US flared about 8764 million cubic meters (MCM) of natural gas with ~23 million metric ton of carbon dioxide equivalent (MMT CO₂e) emission and with an equivalent economic value of over 1 billion dollars [4], causing significant negative environmental impacts and lost revenues. The growing concerns toward climate change mitigation led to the continuous quest for economically viable technologies to reduce these GHG emissions. Hence, there is an opportunity to develop processes to economically convert CH₄ to high-value products. One such process is the utilization of CH₄ as substrate for microbial bioconversion instead of using expensive sugar-based feedstocks [5,6,7].

Methanotrophs are gaining interests because of their ability to utilize CH₄ as their sole carbon and energy source [8]. They play a vital role in carbon cycling as they can convert CH₄ into a wide variety of valuable bioproducts such as lipids, biopolymers, ectoine, and single cell proteins (SCPs) [9,10,11,12]. These lipids can be used to produce renewable diesel/green energy or as feedstock for oleochemical manufacturing. Biopolymers such as polyhydroxyalkanoates (PHAs) are biodegradable, non-toxic, and thermoplastic molecules which can be applied in various energy and environmental applications as well as a potential replacement of conventional plastics [12,13,14]. On the other hand, ectoine is broadly employed in cosmetics industry, dermatology, and it is also an effective stabilizer for nucleic acids, enzymes, and DNA-protein complexes applied in pharmaceutical industries [12,14,15,16], while SCPs can be used as an alternative protein source that has the advantage of being independent of agricultural products (e.g., soybean) as a staring material [7,14].

Methanotrophs are gram-negative members of Proteobacteria that are ubiquitous in nature commonly found in soil, natural gas fields, wetlands, sewage sludges, and waste treatment facilities [9,16,17,18]. They are classified into taxonomic groups based on their 16S rRNA gene sequence, their cell morphology, ultra-structure, phylogeny, and metabolic pathways [8,11,16] as shown in Figure 1.

Distinct types of methanotrophs react differently to changing environmental conditions. The knowledge on the behavior of methanotrophs under different conditions is critical for choosing the suitable type of methanotrophs for culture enrichment and cultivation optimization tailored for producing a certain bio-product. In this work, natural microbial consortia present in samples collected from various sources (particularly low and high O₂ levels) were tested for the presence of methanotrophs. The results could be used to identify possible sources of seed-stocks that contain certain type(s) of methanotrophs for further studies.

2. Materials and Methods

Samples were collected from various sites including sediments from three open drainage ditches and sludges from different wastewater treatment plants (WWTPs) located in Lafayette, LA, USA—South WWTP (activated sludge and aerobic digester), East WWTP (activated sludge) and Ambassador Caffery WWTP (activated sludge). The samples were immediately subjected to DNA extraction using DNeasy^® Powersoil^® Pro Kit (Qiagen, Germany) and the extracts were subjected to full length 16S rRNA gene diversity analysis using bTEFAP^® technology (Mr. DNA Molecular Research LP, Shallowater, TX, USA). Sequencing was performed on a MiSeq following the manufacturer’s protocols and sequence data were processed using ribosomal and functional gene analysis pipeline. The final zero-radius operational taxonomic units (zOTUs) were classified using BLASTn against a curated database derived from the National Center for Biotechnology Information (NCBI).

3. Results and Discussion

In all the samples, low but detectable levels of methanotrophs were identified including Type I (Methylobacter), Type X (Methylocaldum), Type II (Methylocystis, Methylosinus, and Beijerinckia), and Type III (Verrucomicrobium) (see

Table 1. Methanotroph composition from various sources.

Type	Species	Abundance
		DS1 a	DS2 b	DS3 b	EWAS c	SWAS d	SWDS e	AWAS f
Type I	Methylobacter spp.	-	-	-	0.0042	0.0215	0.0231	0.0082
TypeX	Methylocaldum spp.	0.0086	0.0832	0.2370	0.0084	-	-	0.0123
γ-Proteobacteria	Methylocaldum sp.	-	-	-	0.1256	0.0086	0.0185	0.1809
Type II α-Proteobacteria	Methylocystis spp.	-	-	-	0.0126	0.0258	0.0507	-
	Methylocystis aldrichii	0.2395	0.0832	0.0421	0.0586	0.0129	0.0554	-
	Methylocystis echinoides	-	0.0059	0.0158	0.0419	0.0215	0.0369	0.0041
	Methylosinus trichosporium	0.1796	0.1605	0.0474	0.1298	-	0.0046	0.0041
	Methylosinus sporium	0.0941	0.0416	0.0316	0.0209	0.0043	0.0185	0.0452
	Beijerinckia spp.	0.0941	0.0535	0.0632	0.0670	0.4782	0.3829	0.0946
Type III Verrucomicrobium	Verrucomicrobium spp.	1.2061	0.4755	0.3160	0.0712	0.0646	0.4613	0.2591

^a DS1—drainage sediment from a cow farm; ^b DS2 and DS3—sediments from storm drainage; ^c EWAS—east WWTP activated sludge; ^d SWAS—South WWTP activated sludge; ^e SWDS—South WWTP digester sludge; ^f AWAS—Ambassador Caffery WWTP activated sludge.

).

Intensive studies involving methanotrophs has revealed that changing parameters such as CH₄ and O₂ concentrations, nitrogen sources, copper content, pH, and temperature promote the growth and enhance the population of particular types of methanotrophs. In general, Type I methanotrophs prefer low CH₄ and high O₂ concentrations while Type II methanotrophs favor high CH₄ and low O₂ concentrations [7,20]. This is evident from the results in Table 1, showing that Type I methanotrophs were not detected in samples collected from drainage sediments (DS1, DS2, and DS3). These samples were collected in low O₂ environments (i.e., under <1 foot of stagnant muddy water), and thus, favored Type II methanotrophs. In contrast, samples collected from WWTPs (EWAS, SWAS, SWDS, and AWAS) contain Types I, X and II. These samples were collected from aerobic treatment units (i.e., high O₂ environments) that favor Type I. Nevertheless, localized low O₂ regions within these treatment units might have allowed the proliferations of Type II methanotrophs as well.

Among the parameters that affect methanotrophs growth, only the CH₄ and O₂ levels do not require chemical analyses of the growth environment. In particular, the level of O₂ can be easily speculated as illustrated above. In this work, only the level of O₂ was used as parameter for choosing the source of consortia. Nevertheless, whenever chemical assays are feasible, the following can be used in deciding the source of seed consortium. In terms of nitrogen, Type I methanotrophs preferred an environment with high nitrogen content or lower carbon to nitrogen (C/N) ratio while Type II methanotrophs are more common in N-limited (or high C/N ratio) conditions [21]. Type II methanotrophs and some strains of Methylobacter (Type I) can fix atmospheric N₂ because they possess the nitrogenase enzyme. Moreover, studies revealed that methanotrophs grow better on inorganic nitrogen sources (nitrate or ammonia) than atmospheric N₂ [7,21]. Copper content, on the other hand, greatly influences the growth of methanotrophs that have the particulate monooxygenase (pMMO) since copper regulates the expression of this enzyme [7,21].

Methanotrophs are not known to produce neutral lipids (e.g., triglycerides, waxes). Membrane lipids in the form of phospholipids are the only class of lipids typically found in these microbes. As such, the amount of phospholipids that can be obtained from methanotrophs is directly proportional to the biomass produced during cultivation. However, the type of phospholipids is dependent on the type of methanotrophs. For example, phosphatidyl dimethyl ethanolamine and phosphatidyl methyl ethanolamine are found in Type I (Methylobacter), Type II (Methylocystis and Methylosinus), and Type X (Methylocaldum) methanotrophs [12]. Therefore, if these types of phospholipids are the target products, the consortium in EWAS is the most suitable seed for cultivation. The most studied species for ectoine production is Methylomicrobium alcaliphilum 20Z (Type I), but ectoine can also be synthesized by Methylosinus sporium (Type II) and Methylobacter marinus 7C (Type I) [14]. Any of the samples collected can be used as seed for ectoine production because Methylosinus sporium was detected in all of them. However, the most suitable might be DS1 as it contains the highest concentration of this species (Table 1). Favorable characteristics of methanotrophs that can produce SCP should have a rapid growth rate, easy to cultivate, and with high protein production capacity [16]. Methylocystis sp. (Type II) is one of the methanotrophs species that had been used for SCP production [14] at broad pH and temperature ranges. Although any of the samples can be used as seed consortium for SCP production, DS1 is the best choice if abundance is required. Otherwise, if abundance and species diversity is sought, SWDS should be chosen.

Poly(3-hydroxybutyrate) or PHB, which is a member of the PHA family, is another potential high-value product from methanotrophs. The PHBs are accumulated in all Type II methanotrophs as a survival mechanism under nutrients starvation [16,21]. The results (Table 1) suggest that any of the samples could be used as seed for PHB production. The final choice comes down to whether abundance or diversity or both is required by the cultivation. For PHB or for any of the target products, the ultimate choice of which seed to use will also depend on the cultivation conditions. Generally, the growth conditions that lead to PHB accumulation include: (i) low N level (ammonia or nitrate), (ii) copper deficiency, and (iii) fed-batch cultivation [16]. Additionally, AlSayed et al. [21] reported that most PHB accumulation studies were conducted at temperature from 20 to 40 °C and pH of 6–7. Some reports suggest that increasing the medium acidity also increased PHB accumulation in Type II methanotrophs [21]. The PHB accumulation in Methylocystis sp. GB25 DSM 7674 was successfully enhanced under N-limited condition during fed-batch cultivation [16]. In any case, the cultivation temperature and pH should be considered, noting that some species are more tolerant to drastic conditions than others (

Table 2. Temperature and pH growth conditions of different types of methanotrophs.

Type	Species	Temperature (°C)	pH	Reference
Type I	Methylobacter spp.	20–62	6.0–8.5	[7]
Type X	Methylocaldum spp.	0–40	5.5–9.5	[7]
γ-Proteobacteria
Type II α-Proteobacteria	Methylocystis spp.	10–40	6.0–9.0	[22]
	Methylosinus spp.	10–40	5.5–9.0	[22]
	Beijerinckia spp.	10–35	3.0–10.0	[22]
Type III Verrucomicrobium	Verrucomicrobium spp.	37–65	0.8–6.0	[7]

) and might necessitate species diversity over abundance. Additionally, the composition of feed gas should also be considered. As indicated in Figure 1, Types II and III methanotrophs can simultaneously consume CO₂ and CH₄ and should preferably be used for cultivation involving biogases (a mixture mainly composed of CH₄ and CO₂).

4. Conclusions

In this work, samples from several locations, including sediments from three open drainage ditches and sludges from different WWTPs, were collected to identify and quantitate different types of methanotrophs. Based on the 16S rRNA analysis, the samples from each location were composed of diverse types of methanotrophs including Type I (Methylobacter), Type X (Methylocaldum), Type II (Methylocystis, Methylosinus, and Beijerinckia), and Type III (Verrucomicrobium). Although different parameters could affect growth and proliferation of methanotrophs, this work focused mainly on O₂ levels. As anticipated, samples collected from locations with low O₂ levels (i.e., drainage ditches) contained non-detectable levels of Type I methanotrophs. The results of this work emphasize the importance of environmental conditions on the choice of a natural source of methanotrophic consortium. It should be noted, however, that other parameters might still need to be considered, along with target product(s) and cultivation conditions, to identify the most suitable natural consortium source for further studies.