Single-Cell Protein Using an Indigenously Isolated Methanotroph Methylomagnum ishizawai, Using Biogas

Abstract

The use of methane as a carbon source for producing bacterial single-cell protein (SCP) has been one of the most interesting developments in recent years. Most of these upcoming industries are using a methanotroph, Methylococcus capsulatus Bath, for SCP production using natural gas as the substrate. In the present study, we have explored the possibility of using an indigenously isolated methanotroph from a rice field in India, Methylomagnum ishizawai strain KRF4, for producing SCP from biogas [derived from cow dung]. The process was eco-friendly, required minimal instruments and chemicals, and was carried out under semi-sterile conditions in a tabletop fish tank. As the name suggests, Methylomagnum is a genus of large methanotrophs, and the strain KRF4 had elliptical to rectangular size and dimensions of ~4–5 µm × 1–2 µm. In static cultures, when biogas and air were supplied in the upper part of the growing tank, the culture grew as a thick pellicle/biofilm that could be easily scooped. The grown culture was mostly pure, from the microscopic observations where the large size of the cells, with rectangular-shaped cells and dark granules, could easily help identify any smaller contaminants. Additionally, the large cell size could be advantageous for separating biomass during downstream processing. The amino acid composition of the lyophilized biomass was analyzed using HPLC, and it was seen that the amino acid composition was comparable to commercial fish meal, soymeal, Pruteen, and the methanotroph-derived SCP-UniProtein^®. The only difference was that a slightly lower percentage of lysine, tryptophan, and methionine was observed in Methylomagnum-derived SCP. Methylomagnum ishizawai could be looked at as an alternative for SCP derived from methane or biogas due to the comparable SCP produced, on the qualitative level. Further intensive research is needed to develop a continuous, sustainable, and economical process to maximize biomass production and downstream processing.

1. Introduction

A significant challenge facing the 21st century is striking a balance between the growing need for quality protein and minimizing the adverse impacts on the environment [1,2]. Single-cell protein (SCP) production from microbes such as yeasts, fungi, algae, and bacteria surged in the 1950s and 1960s [3]. Single-cell protein (SCP) derived from microbes has been proposed as an alternative to animal products to meet global protein demand [4]. Methylotrophs such as Methylophilus were one of the successful examples where bacteria were used in the production of SCPs, and a product known as ‘Pruteen’ was developed [5]. Methane, the simplest hydrocarbon, is the main component of natural gas and is available in a renewable form in the form of biogas. Currently, methane is mainly used as a fuel; however, in the last forty years, methane has also been used for the production of valuable chemicals like single-cell protein, and recently for the production of other valuable chemicals [3,6]. In the late 1980s, natural gas was seen to be an abundant substrate, mostly flared. The commercial production of methanotrophic SCP using natural gas and a methanotroph, Methylococcus capsulatus Bath, was started initially in Norway. Early industrial applications included the Norferm plant in Norway, which used methane to cultivate M. capsulatus for animal feed [6]. The bacterium gained renewed attention with the rise in natural gas availability through hydraulic fracturing, prompting companies like UniBio (Denmark) and Calysta Inc. (located in the UK and the US) to explore methane-based SCP production at scale. Unibio, founded in 2001 and headquartered in Denmark, uses U-loop^® fermentation technology to convert methane into UniProtein^®, a sustainable, high-protein feed alternative to fishmeal and soymeal (https://www.unibio.dk accessed on 25 July 2025). With growing global demand for eco-friendly protein, Unibio’s SCP is approved for use in animal feed across Europe [7]. The Unibio company had developed a U-loop^® technology to convert natural gas into a protein product that could be used as a direct supplement in animal feed (fish meal). Recently, Calysta has also developed its plants, and Feedkind^® is a similar product derived from Methylococcus capsulatus biomass [8]. Methane-derived SCP is one of the most advanced and accessible SCP production technologies [4] and has high protein completeness because its essential amino acid content is similar to, or higher than, the FAO guidelines [9,10,11].

The availability and abundance of methane drives a growing interest in methanotrophic bio-catalysis, methane being the main component of natural gas. The production of SCP using waste materials and biogas as a substrate provides an economically feasible and valuable source of protein in animal feed [12]. The bioconversion of methane to value-added products using methanotrophs has been performed using the following species: Methylococcus capsulatus, Methylomonas sp., Methylosinus trichosporium OB3b, and Methylocystis parvus OBBP [13,14,15,16].

Our goal in the experimental study was to explore an alternative methanotroph that could be used for SCP production using methane and to see whether the amino acid composition was comparable to fish feed or the methanotroph-derived SCP-UniProtein^®. As we routinely used biogas produced from cow dung to feed methanotroph cultures in our laboratory, biogas was explored as an alternative substrate to pure methane for increased sustainability.

2. Materials and Methods

2.1. Primary Optimization for Growth of the Methanotroph

We used the indigenously isolated methanotroph Methylomagnum ishizawai KRF4, a rice field isolate from our earlier studies [17]. The optimization experiments were set to maximize the growth of M. ishizawai strain KRF4. As stated in our earlier studies, M. ishizawai strain KRF4 was routinely grown in the presence of a methane:air (20:80) gas mixture at 25–28 °C [17]. The first parameter analyzed was temperature. All the experiments were performed in 125 mL serum bottles filled with sterile 30 mL NMS medium, followed by the additions of filter-sterilized phosphate buffer and vitamin solutions [17]. All bottles were inoculated with 2 mL of a grown culture of M. ishizawai strain KRF4 (OD600 ~0.3–0.4) and were incubated in the presence of a methane:air (20:80) gas mixture at temperatures of 15, 20, 25, 30, 35, 37, and 40 °C. The decline in methane concentration and increase in OD were checked periodically using gas chromatography (Chemito 8610, Chemito instruments Pvt. Ltd., Mumbai, India). The OD was monitored by using a spectrophotometer.

A small-scale cow dung-based biogas reactor (20 L) was run to obtain biogas, and its setup and handling were explained in our earlier studies [18]. The biogas produced usually contained ~48–49% methane and 46–47% CO₂, and the composition was routinely checked using gas chromatography as described [18]. A second optimization experiment was set as explained previously in the presence of different concentrations of methane:air (10:90, 20:80, and 50:50) and biogas:air (20:80, 40:60, and 80:20) mixtures under static and shaking incubation conditions. The experimental bottles were incubated at an optimized temperature.

2.2. Growing Methanotrophs in a Simple and Eco-Friendly Way in a Fish Tank

For further scale-up, a plexiglass fish tank with a lid (dimensions of 60 cm × 45 cm × 30 cm and a capacity of 80 L), which was locally available in the market, was used to grow M. ishizawai strain KRF4 at a larger volume. Twenty liters of semi-sterile NMS medium was directly prepared in the fish tank using the following method: Firstly, the tank was thoroughly cleaned, followed by the addition of autoclaved concentrated stock of the media components, and further dilution by using distilled water in the tank. A filter-sterilized 100 mL 10× phosphate buffer and 2 mL 100× vitamin solutions were added later. Three hundred milliliters of a grown culture of M. ishizawai strain KRF4 (OD₆₀₀ ~1.0) was added as an inoculum in the fish tank. The fish tank was initially flushed with ~10–12 L of biogas through a small tubing fitted at the back slit opening of the tank, and the lid was immediately closed. The small openings of the lid were covered with plastic wrap to avoid a loss of gases and to minimize the chances of contamination. Ultimately, the headspace in the fish tank was filled with a ~1:4 biogas:air mixture (optimized gas mixture concentration).

The 16S rRNA gene sequence was used for the construction of a phylogenetic tree of Methylomagnum ishizawi KRF4 with its closest members, which was performed using MEGA XI [19]. The biomass was developed at the gas-water interphase in ten days with daily ~10–12 L of biogas flushing in the tank’s headspace. One liter of grown biomass (cell paste) from the water–gas interphase was collected using sterile beakers. The cells from the biomass were microscopically observed to check the presence of grown M. ishizawai strain KRF4 cells. The biomass was centrifuged (Kubota centrifuge, model 7780, Tokyo, Japan) at 7000 rpm for 20 min at 4 °C. The supernatant was discarded, and the pellet was collected in a Petri dish and stored at 4 °C. Furthermore, the lid of the tank was closed, and the tank was flushed with biogas again and then sealed. Three biomass harvesting cycles were carried out without adding medium components or inoculum. Collectively, the biomass was weighed and lyophilized at −70 °C for 6 h to make a dry powder using a Scanvac, Coolsafe55-4 Pro lyophilizer (Labor gene ApS, Lynge, Denmark). This dried powder was further used to analyze amino acid composition.

2.3. Amino Acid Analysis of the Biomass

The amino acid composition of the lyophilized dry biomass of M. ishizawai strain KRF4 was estimated by TUV, Nord, Laboratory, Pune, India, using high-performance liquid chromatography (HPLC). There, the lyophilized powder was weighed and dissolved in an appropriate solvent to extract the amino acids. A set of calibration standards containing known concentrations of individual amino acids was prepared to quantify the amino acids present in the sample. The sample was injected into the HPLC system along with the calibration standards. The HPLC system had a suitable detector to monitor the eluted amino acids. Total elemental nitrogen analysis was performed using the Kjeldahl method to determine the protein content of the biomass derived from M. ishizawai strain KRF4. Total nitrogen estimation was carried out using the Kjeldahl method, using the Kjeltec™ and Foss DU 2100 (Thermo Fisher Scientific, MA, USA) and standard protocol. Briefly, 0.5 g of sample was added to one ml of sterile filter-sterilized water and added to the digestion tube. Six ml concentrated H₂SO₄ and one catalyst tablet (HgSO₄ + K₂SO₄) were added to the same tube. The digestion was performed at 400 °C for 1.5 h, followed by cooling of the sample. After cooling, 38 mL of filter-sterilized water and 25 mL NaOH were added manually. We took 30 mL mixture indicator-boric acid solution in a flask and collected the solution up to 150 mL from the distillation unit. The collected solution was titrated against 0.1 N HCl. Total elemental nitrogen was converted into percent protein using the following equation: %Protein = %Nitrogen × Nf%, where Nf is the nitrogen factor.

3. Results and Discussion

M. ishizawai strain KRF4, isolated in our earlier studies, is a large-sized methanotroph (4–5 µm × 1–2 µm) (Figure 1A), and the 16S rRNA is sequenced with an accession number OR473174. It shows 99.87% 16S rRNA gene similarity and groups closely with Methylomagnum ishizawai RS11Dr-P^T within the Type Ib methanotrophs (Supplementary Figure S1). The other closest relative is Methylococcus capsulatus Bath, the methanotroph used for commercial SCP production, with 88.4% 16S rRNA gene similarity [20]. Strain KRF4 forms white to cream colonies of 2–3 mm diameter in two weeks of incubation in the presence of a methane:air mixture 20:80 at 30 °C on NMS agarose medium plates incubated in a desiccator (Figure 1B). Furthermore, under a phase-contrast microscope, large rectangular to elliptical cells of 4–5 μm in length and 1–2 µm are seen. Due to its relatively large size, it is one of the largest methanotrophs and is named Methylomagnum (a large-sized methanotroph) (Figure 1A) [20]. The cells are Gram-negative and do not form chains but occur singly or in pairs (Figure 1A).

The strain KRF4 grew in the range of temperatures 15–30 °C with optimum growth in the 25–30 °C range (OD values between 0.3 and 0.5), showing that the culture was mesophilic. The optimum temperature was found to be 30 °C. In this case, the headspace was composed of 20% methane and 80% air in the headspace. In further experiments, where biogas was supplied as an alternative to pure methane, it was seen that a biogas: air at 20:80 gas mixture concentration, under static incubation conditions at 30 °C, was ideal, and OD~1 was achieved; hence, these conditions were used for further experiments (Supplementary Table S1).

The scale-up of strain KRF4 at 20 L NMS medium volume was successfully performed using a fish tank, under static conditions (Figure 2A). Biogas and air (20:80) mixture in the headspace was favorable for the growth of the strain KRF4, and the culture grew as a ~2–3 cm thick white creamy layer at the water–gas interphase (Figure 2B). After one week, the thick, grown biomass layer was scooped using a sterile beaker. The optical density of the collected biomass at 600 nm was ~1.8–1.9, checked using a spectrophotometer. In spite of the semi-sterile conditions used for scaling up the culture growth, the cells of M. ishizawai KRF4 grew without contamination with other bacteria. The purity of the developed biomass culture was confirmed by phase contrast microscopy (Figure 2C). This biomass slurry was centrifuged and concentrated as a thick paste (~30–35 g paste accumulated in one week). After three consecutive weeks, ~90–100 g of wet biomass paste was collectively obtained and lyophilized (Figure 2D,E). After lyophilization, this resulted in ~10 g of dried powder after about three to four weeks of growth in the tank.

The specific nitrogen factor for M. capsulatus was assumed to be the traditional Kjeldahl conversion factor of 6.25, and the same factor was used for KRF4 [21]. The %Nitrogen was found to be 10. Hence, it could be deduced that Methylomagnum ishizawai KRF4 produced ~62.5% protein of its dry weight, a little less than Methylococcus-derived protein. The amino acid composition of the biomass produced from Methylomagnum ishizawai KRF4 was compared with UniProtein^®, fish meal [12], soybean meal, and Pruteen (Methylophilus-derived SCP) [5] (

Table 1. Amino acid composition of Methylomagnum ishizawai KRF4 grown on biogas in comparison with UniProtein^® (https://www.unibio.dk/uniprotein accessed 25 July 2025), fish meal, soybean meal, and Pruteen-Methylophilus-derived SCP [5,12].

Quantity (g/100 g) Dry Weight of Biomass	Methylomagnum ishizawai KRF4	UniProtein®	Fish Meal	Soybean Meal	Pruteen
Essential amino acid
Arginine	3.77	4.45	3.49	4.6	2.87
Histidine	1.44	1.62	2.34	1.58	1.18
Isoleucine	2.34	3.17	2.69	2.93	2.68
Leucine	4.47	5.45	4.54	4.9	4.3
Lysine	2.68	4.07	4.97	5.1	3.75
Methionine	0.28	1.94	1.63	1.8	1.5
Phenylalanine	2.75	3.06	2.50	2.56	2.5
Threonine	3.06	3.23	2.62	2.5	2.8
Tryptophan	0.30	1.89	0.65	0.5	0.56
Valine	3.60	4.22	2.93	3.3	3.5
Non-essential amino acid
Alanine	3.80	5.13	3.83	2.62	4.4
Glycine	2.38	3.63	3.82	2.6	3.56
Proline	2.67	2.86	2.54	3.1	1.81
Serine	1.96	2.59	2.16	3.2	2.06
Tyrosine	2.29	2.39	1.88	2.3	2.12
Cysteine	2.05	0.46	-	0.93	0.43
Aspartic acid	5.41	6.18	-	7.0	5.5
Glutamic acid	6.99	7.57	-	11.3	6.6
Glutamine	0.77	-	6.92	-	-
Cystine	0.44	-	0.84	-	-

). The KRF4-derived biomass showed comparable values of the following essential amino acids (g/100 g of dry biomass): arginine, histidine, isoleucine, leucine, phenylalanine, threonine, and valine (Table 1) [12]. Lysine, methionine, and tryptophan were slightly less compared to the other commercial products (Uniprotein, fish meal, and soybean meal), Table 1. All of the non-essential amino acids were present in Methylomagnum strain KRF4-derived biomass and were generally comparable to the commercially available fish meal, soymeal, as well as UniProtein^®.

The type Ib or type X methanotroph, Methylococcus capsulatus, has been the best-studied methanotroph, which has been used on a commercial scale, especially for the production of SCP. Our present study highlights the potential of Methylomagnum ishizawai, another Type X/Type Ib methanotroph, in SCP production from biogas or natural gas. Unlike typical bacteria with small cell sizes (~1 µm size) that require intensive concentration and drying steps, M. ishizawai stands out due to its larger size, flocculating nature, and ability to form surface biofilms. These properties could simplify downstream processing, allowing biomass to be scooped and dried directly, potentially reducing processing cost and complexity. Though a preliminary study, it offers promising insights into scalable SCP applications using an alternative methanotroph, awaiting further studies. Another interesting feature of M. ishizawai strain KRF4 was that the culture grew under stationary conditions and better with biogas and air in the headspace. This could make the process more sustainable, where medium-sized biogas reactors can be used to prepare SCP for animal feed, allowing decentralized production of SCP. Strain KRF4 grew best at mesophilic temperatures (25–30 °C) and therefore would not need any heating jackets. Another advantage of this cultivation process is that it runs under semi-sterile conditions, and the grown biomass is still in pure form, requiring low maintenance. In downstream processing, auto-flocculation and microbial morphology engineering offer potential solutions, at least to overcome the inefficient harvesting of small-sized microalgae and bacteria [22].

4. Conclusions

Methylomagnum ishizawai, a Type Ib/Type X methanotroph, presents a promising candidate for single-cell protein (SCP) production using waste biogas or methane. Further research focused on biomass optimization and scale-up strategies would be instrumental in unlocking its full potential. In terms of quality, the SCP derived from M. ishizawai is comparable to the existing protein sources such as Uni-protein^®, fish meal, and soymeal. The process outlined in this study is both sustainable and low-tech, making it a viable green alternative. However, transitioning to industrial-scale applications would require additional standardization across multiple stages.