Single Cell Protein is the dried cells of microorganisms consumed as a protein supplement by humans or animals.

Autor: Georgii Feodoridi

            Kseniia Feodoridi

Place: HYDITEX CORPORATION, Port Vila

Date: October 2025

Abstract: This article explores and analyzes the production of single cell protein (SCP) from methane and hydrogen using various types of bioreactors. It examines the biochemical basis of the metabolism of methane-producing and hydrogen-oxidizing bacteria, various microbial cultivation strategies, and historical and current industrial experience with their application. Key technological aspects of cultivation, including bioreactor design, are discussed, with a particular focus on the use of membrane and U-loop fermenter. The article also addresses the legal application of microorganisms for food, the use of methane and hydrogen as feedstocks for microbial cultivation, issues of production scaling, explosion hazards, gas mass transfer, and regulatory requirements. The advantages and disadvantages of pure and mixed cultures are also discussed, as well as the potential for using mixed microbial communities to increase protein yield and improve its quality. The article provides examples of successful research and industrial projects demonstrating the potential of SCP as a sustainable protein source for human and animal nutrition. The article emphasizes that SCP represents a promising technology for creating sustainable and environmentally friendly protein sources by reducing reliance on traditional agriculture and mitigating the effects of climate change. 

Key Words: Protein production, Protein, Microorganisms, Bacteria, Cellular agriculture, Alternative protein, Single Cell Protein (SCP), Methane, Methane-oxidizing bacteria (MOB), Hydrogen, Carbon dioxide, Hydrogen-oxidizing bacteria (HOB), Bioreactor, Membrane bioreactor, U-loop fermenter. 

DOI: 10.5281/zenodo.17253356

Food Protein from methane and/or hydrogen. Bioreactors. Cultivation.

Introduction

The modern food system faces challenges related to the need to provide the planet with nutritious food in the face of limited resources, climate change, increasing environmental pressure, and population growth. Traditional livestock farming, the primary source of protein such as meat and fish, is associated with significant greenhouse gas emissions and high water and land consumption, making it one of the least sustainable industries. Therefore, the search for and development of alternative, environmentally friendly, and resource-efficient protein sources has become a pressing task for scientists and industry. 

One of the most promising areas is the production of single cell protein (SCP) — microbial biomass rich in protein and suitable for use in human food and animal feed. Historically, SCP has been produced from a variety of substrates, including organic waste. However, the use of inexpensive and readily available carbon and energy sources, such as methane and hydrogen, opens up new opportunities for creating closed-loop production systems independent of agricultural land. 

Despite significant potential, industrial production of SCP from gases faces a number of technological and economic challenges, including the low solubility of gases in liquids, the explosive nature of methane/hydrogen mixtures with oxygen, high energy costs for aeration and mass transfer, and regulatory barriers related to product safety. 

Problem and relevance

Transforming the existing food system to ensure healthy diets and environmental sustainability is one of the most important challenges of our time. Achieving these goals is a prerequisite for achieving the UN Sustainable Development Goals (SDGs) [1]. 

To improve the sustainability of food systems, it is necessary to reduce the consumption of animal products, and new food production technologies are being developed to achieve this goal. One such technology is the emerging field of cellular agriculture, which uses cell culture technologies from animals, microorganisms, or plants to produce food products. This technology has the potential to contribute to the creation of sustainable alternatives to animal products [1, 2, 3]. 

While some may find the use of microbes as a food source unacceptable, the idea of consuming microbes for both humans and animals is undoubtedly an innovative solution to the global food crisis. For millennia, humans have intentionally or accidentally consumed foods such as alcoholic beverages, cheese, yogurt, and soy sauce, along with the biomass needed to produce them [4]. The first written record of the use of microorganisms dates back to 2600 BC and was found in Babylon in the form of traces of bread [5]. 

Yeast was the first microorganism whose importance as an animal feed additive was recognized nearly a century ago. During World War I, Germany replaced half of its imported protein sources with yeast [6]. 

Food products based on microbial cells can be called microbial food cultures (MFCs). 

It is noteworthy that MFCs do not have a legal definition. To address this situation, the European Starter Culture Association proposed the following definition: "Microbiological starters are live bacteria, yeasts, or molds used in food production." MFCs are formulations consisting of one or more species and/or strains of microorganisms, including the components of the nutrient medium remaining after fermentation and the components necessary for their survival, storage, standardization, and facilitation of their use in food production [7]. 

However, when speaking of food products based on microorganism cells, the term SCP (Single Cell Protein) is most often encountered. 

Single cell proteins are cultured microbial cells that are used as protein supplements in human food or animal feed. Microorganisms such as algae, fungi, yeast, and bacteria use inexpensive raw materials and waste as sources of carbon and energy for growth to produce biomass, protein concentrate, or amino acids. Since protein constitutes a quantitatively important part of microbial cells, these microorganisms, also called single cell protein, are a natural protein concentrate [6].

List of abbreviations

MFC (microbial food cultures);

SCP (Single Cell Protein);

DAC (direct air capture);

MBR (membrane bioreactors);

dMBfR (dual-membrane biofilm reactor);

HFM (incorporating hollow fiber membranes);

MOB (Methane-oxidizing bacteria);

HOB (Hydrogen-oxidizing bacteria);

FaldDH (formaldehyde dehydrogenases);

FateDH (formate dehydrogenases);

MMOs (methane mono-oxygenases);

MDH (methanol dehydrogenase);

pMMO (particulate MMO);

sMMO (soluble MMO);

CytC (cytochrome complex);

NAD (nicotinamide adenine dinucleotide);

NAD+ (nicotinamide adenine dinucleotide (oxidized form);

NADH (nicotinamide adenine dinucleotide (reduced form);

PQQ (pyrroloquinoline quinone). 

Purpose and objectives

The aim of this paper is to investigate the production of single cell protein from methane and hydrogen using methanotrophic (MOB) and hydrogen-oxidizing (HOB) bacteria. 

In this study, we set the following objectives: 

1. Analyze the historical and industrial context of SCP production, identifying the reasons for the successes and failures of past projects; 

2. Consider the biochemical basis of MOB and HOB metabolism, which determine the efficiency of gas-to-biomass conversion; 

3. Consider issues related to the safety of SCP production, including the risks associated with the use of methane and hydrogen, as well as the environmental impact of the process; 

4. Systematize information on modern types of bioreactors and cultivation strategies that determine the efficiency and safety of the process; 

5. Consider regulatory aspects and safety requirements (toxins, nucleic acids) for the final SCP product for its use as feed or food ingredient; 

6. Formulate promising areas for further research and development in the field of gas-based SCP production. 

These objectives aim to provide a comprehensive understanding of the SCP production process and its potential as a sustainable protein source for human and animal nutrition.

Historical and industrial context 

Various types of microorganisms such as algae, fungi, yeast, and bacteria are used as single cell protein and are produced on an industrial scale [6]. These organisms can use a variety of substances as a carbon source. For example, heterotrophic organisms require glucose, which is typically obtained from grain or sugar crops [2], making this option dependent on crop production. The use of autotrophic microbes, which can obtain carbon from carbon dioxide (CO2) or methane (CH4), offers advantages because the production process is completely independent of outdoor farming. Methanotrophic bacteria obtain energy and carbon from methane, while hydrogen-oxidizing bacteria utilize hydrogen and carbon dioxide; therefore, crops are not needed as a carbon source [3].

Protein from methane

Various types of microorganisms such as algae, fungi, yeast, and bacteria are used as single cell protein and are produced on an industrial scale [6].

It's worth noting that methane is a promising option in this case as an inexpensive feedstock for cultivating methane-oxidizing microorganisms (methanotrophs) and producing single cell proteins [8]. This is primarily due to the fact that methane (CH4), a potent greenhouse gas, is available worldwide as both natural gas and biogas for domestic, commercial, and industrial use [8].

Over the past decade, natural gas supplies have reached record levels, and its cost is consistently lower than that of oil. Despite these economic advantages, large volumes of natural gas are flared at wellheads daily to reduce greenhouse gas emissions or are released into the environment through intentional or unintentional leaks during production [6, 9, 10]. Current trends are shifting toward a circular economy. This involves the use of biogas obtained from the anaerobic digestion of organic waste [11] and biogas obtained from the anaerobic digestion of sewage sludge and digested wastewater [12], which leads to a reduction in production costs and an increase in the environmental friendliness of production [11, 13, 14].

Considering that raw materials are the major operating cost item in biochemical production [15], the choice of carbon source can significantly impact profitability [16]. Therefore, from an industrial and economic perspective, methane-oxidizing bacteria are the most advanced and market-ready bacteria for SCP production [12, 17]. 

Protein from hydrogen

Another type of autotrophic bacteria—hydrogen-oxidizing bacteria—can also produce microbial biomass with high protein content. These bacteria, which can use hydrogen as an electron donor and convert carbon dioxide in the presence of oxygen, can do so. To produce SCPs, hydrogen-oxidizing bacteria also require a nitrogen source and, to a lesser extent, certain minerals [18].

Hydrogen-oxidizing bacteria are distinguished by their ability to grow on hydrogen of various origins. There are numerous industrial options for H₂ production, including steam methane reforming, water electrolysis, gasification of solids (e.g., coal or biomass), etc. However, the growing interest in the possibilities of producing SCPs using hydrogen bacteria is primarily due to the possibility of using renewable energy sources, such as hydrogen production from waste or byproducts of chemical and related industries. Wastewater can also serve as a source of hydrogen (e.g., [18, 19, 20, 21]) [19]. Natural hydrogen extracted from the Earth's interior can also be used to produce SCPs.

The production of SCPs from hydrogen requires an external carbon source, which is carbon dioxide. CO2, in turn, can be captured from air or industrial exhaust gases (flue gases) using absorbents followed by desorption [22, 23], thereby reducing CO2 emissions.

CO2 can also be captured during the conversion of biogas to biomethane. In this context, hydrogen-oxidizing bacteria can be considered one of the most powerful microbial activators of the transition to integrated biorefineries [19]. 

Production experience

It's worth noting that methane-based SCPs have been studied and even produced on an industrial scale since the mid-20th century in various countries, but large-scale commercial production ceased due to restrictions [24]. However, the production of SCPs by methanotrophs is currently again attracting public attention due to the growing demand for protein and concerns about climate change caused by greenhouse gas emissions [12].

In the USSR, in 1983, the Protein-Vitamin Concentrate (BVK) Plant launched production of gaprin, a feed additive made from bacteria grown on methane. The producer was the bacterium Methylococcus capsulatus. This feed additive was recognized as suitable for all types of farm animals, poultry, fur-bearing animals, and fish. Production capacity was 1,000 tons of protein per month. In 1989, the USSR had eight plants producing protein on various substrates, including methane [25, 26]. 

Since 1976, Dansk Bioprotein, a Danish company, had been developing a process for producing microbial protein from gas using the methane-oxidizing bacteria Methylomonas methylosinus. This company was later acquired by the Norwegian company Statoil (now Equinor), which has large gas fields on the Norwegian continental shelf and is constantly exploring opportunities to develop an onshore industry based on natural gas. This began the production of the feed product Bioprotein, which is produced by the bacteria Methylococcus capsulatus. In 1995, Bioprotein received EU approval for use in Atlantic salmon and pet food [26, 27]. 

In the 1970s, Imperial Chemical Industries (ICI) in the UK developed the microbial protein Pruteen, produced by growing Methylophilus methylotrophus bacteria in methanol, which is derived from methane or natural gas. Pruteen contained up to 70% protein and was used in pig feed [28]. 

In the second half of the 20th century, a technology for producing bacterial biomass using hydrogen was also developed in the USSR. Initially, this biotechnological process was aimed at solving space problems—providing astronauts with protein products during long-duration missions and purifying the atmosphere of space stations of carbon dioxide.

In recent decades [30, 31], the production of single cell protein (SCP) from hydrogen suitable for human consumption has attracted increasing interest. Several companies in various countries are already pursuing this goal, including [18, 31, 32, 33]. 

In Finland, Solar Foods has launched production of a protein called Solein. This involves cultivating a strain of Xanthobacter tagetidis, or VTT-E-193585, in continuous culture using hydrogen as an energy source and an inorganic carbon source, where the inorganic carbon source is carbon dioxide [33, 34, 35].

In the US, Air Protein Inc. has launched production of its eponymous protein, Air Protein. Air Protein claims to use air, water, and energy to grow its protein. Air includes carbon dioxide, oxygen, and nitrogen, while water and energy are represented by renewable H2 produced through electrolysis. Chemotrophic microorganisms are used for production [33, 36, 37].

Another US company, NovoNutrients, has found a way to recycle industrial carbon dioxide waste and convert it into high-quality protein for humans and animals. The process utilizes hydrogen and natural microorganisms that convert CO2 into alternative protein products. NovoNutrients has expressed interest in building a plant to produce SCP from hydrogen with a capacity of approximately 100,000 tons per year [18, 33, 38, 39].

Deep Branch Biotechnology Ltd. of Nottingham, UK, has developed a technology for converting carbon dioxide into an environmentally friendly protein called Proton. The technology has now been acquired by Aerbio. Proton production requires not only carbon dioxide, but also hydrogen and oxygen [33].

Lessons from past projects: economic and technological barriers

The production of microbial feed additives from HOB and MOB, which was attempted to be established in the second half of the 20th century, encountered a number of problems.

For example, in the USSR, a few years after the launch of bacterial feed additive production, all factories were closed due to an outbreak of allergic diseases associated with human exposure to protein dust generated during BVK production [25, 26].

In 1989, an analytical report, "Production of Microbial Feed Protein. Environmental Problems," was published, detailing the effects of paprin (a Candida yeast biomass, n-paraffins, a petroleum product) on humans and the results of long-term experiments. The author concluded that paprin is a safe additive, but only if all conditions are met during both concentrate production and animal feeding. If production requirements were violated, people more often suffered from asthma, bronchitis and allergies due to factory emissions, and animals overfed with paprin were retarded in development and became ill [25, 26].

In 1979, ICI invested a huge amount (around £40 million) in the production of Pruteen, an animal feed made from methanol, installing a continuous culture system for single cell protein production. It was the largest continuous fermenter in the world. Unfortunately, due to economic reasons, the plant did not operate for long [26, 29]. 

For example, in 1984, the price of soybean meal was approximately $125–$200 per ton, while Pruteen was selling for $600 per ton! This was primarily due to the high cost of methanol, which accounts for approximately half of production costs. Therefore, Pruteen could not compete with cheaper animal feeds that emerged in the late 1970s, and production was discontinued [29, 40].

Despite the difficulties that arose in the early days of SCP production, the problem of protein deficiency is now more acute than it was in the early days of the microbiological industry. Our planet is already experiencing a significant shortage of food in general, and protein foods in particular. Therefore, interest in SCP production is growing.

The global, large-scale development of SCP processes is making a significant contribution to the advancement of modern biotechnology. Research and development of SCP processes has included work in the fields of microbiology, biochemistry, genetics, chemical and process engineering, food technology, agriculture, animal nutrition, ecology, toxicology, medicine, veterinary science, and economics. The development of SCP processes also leads to the development of new technical solutions for other related technologies in wastewater treatment, alcohol production, enzyme technology, and nutritional science. The future of SCP will depend largely on reduced production costs and improved quality through fermentation, downstream processing, and improvement of producer organisms through traditional applied genetics combined with recombinant DNA technologies [6, 41]. 

Biochemical foundations 

Bacteria possess a variety of metabolic processes, including enzymatic assimilation (the uptake and utilization of organic and inorganic chemicals necessary for cell growth and maintenance) and dissimilation reactions (substrate oxidation and breakdown). Assimilation reactions require energy, while dissimilation reactions generate energy. These reactions underlie bacterial cell self-reproduction and are involved in essential cellular functions [42, 43]. 

In other words, bacteria are cells that can convert energy obtained from the environment to perform their basic functions [43, 44]. 

Chemical energy is stored in adenosine triphosphate (ATP), adenosine diphosphate (ADP), and/or molecules with a thioether linkage (e.g., succinyl-CoA and acetyl-CoA). These compounds contain high-energy phosphate bonds that are used by enzymatic systems to synthesize new compounds necessary for the existence and development of cells [43]. 

Bacterial enzyme systems include B vitamins as functional coenzymes that participate in cell growth and energy conversion processes, catalyzing numerous redox reactions [43, 45]. SCP metabolism involves the biological oxidation of organic compounds and results in the formation of simple organic and/or inorganic compounds, as well as ATP. Bacterial cells require these compounds for anabolic processes. 

Within the framework of heterotrophic metabolism, bacteria have two known energy production pathways: anaerobic respiration (fermentation) and aerobic respiration [43]. 

Fermentation is an anaerobic process, meaning that the final electron acceptor is not O2 (e.g., SO42, NO3–, or fumarate). During glycolysis, glucose is broken down to pyruvate with the formation of ATP and NADH (via the conversion of NAD). Pyruvate, in the presence of NADH, forms the end products of fermentation. Aerobic respiration involves the catabolism of glucose. Pyruvate, formed as a result of glucose breakdown, is converted into acetic acid, carbon dioxide, and NADH through mechanisms involving electron transfer and chemiosmosis. Acetic acid, together with coenzyme A, forms acetyl-CoA, after which the acetyl radical enters the reaction cycle (Krebs or glyoxylate) after separation from CoA [43]. 

Aerobic respiration is an exothermic process in which 1 mole of glucose is completely oxidized to form carbon dioxide and H2O, releasing approximately 38 moles of ATP [43]. 

Thus, the metabolic process differs among different bacterial species; let's examine it in more detail for HOB and MOB.

Metabolism of methane-oxidizing bacteria

Methane is an inexpensive source of energy and carbon for methane-producing bacteria [11, 46], and they can use methane as the sole source of carbon and energy [17, 47, 48]. The metabolic pathway of methane-oxidizing bacteria in a fermenter is shown in Figure 1 [11]. 

The first step in methane catabolism is oxidation to methanol by methane monooxygenase [49]. Methanol is then oxidized to formaldehyde [16, 50, 51].

These reactions involve various enzymes, including methane mono-oxygenases (MMOs), methanol dehydrogenase (MDH), formaldehyde dehydrogenases (FaldDH), and formate dehydrogenases (FateDH). Two forms of MMO exist: particulate MMO (pMMO), a membrane-bound enzyme, and soluble MMO (sMMO), a cytoplasmic enzyme. After MOB takes up methane, MMO catalyzes the oxidation of methane to methanol. Depending on whether pMMO or sMMO is employed, MMOs utilize reducing equivalents like cytochrome complex (CytC) or NADH2 to cleave the O-O bonds in molecular oxygen (O2) into monovalent oxygen atoms. One oxygen atom is reduced to produce H2O, while the other is incorporated into methane, leading to methanol formation. Methanol is further oxidized to formaldehyde by MDH, a pyrroloquinoline quinone (PQQ)-dependent enzyme [11].

Formaldehyde is further oxidized to formic acid and then to CO2 for energy by the enzymes FaldDH and FateDH, respectively [11], producing reducing equivalents that can be converted to ATP via the electron transport chain and ATP synthase [16, 52] while the other part is metabolized via the serine pathway or the ribulose monophosphate pathway (RuMP) pathway for biomass synthesis [11]. The ribulose monophosphate pathway is used by group I methanotrophs belonging to the Gammaproteobacteria. The RuMP pathway uses a five-carbon sugar as a substrate for the assimilation of formaldehyde and the production of a six-carbon sugar [50, 51, [52]. Every three turns of these cycles result in the formation of one dihydroxyacetone phosphate [16]. In contrast, the serine pathway, common among alphaproteobacteria, involves the assimilation of formaldehyde by reaction with glycine to form serine [49, 52]. Every two turns of this cycle result in the assimilation of two formaldehydes and one carbon dioxide to form 2-phosphoglycerate (the main cycle can be supplemented by other reactions to produce acetyl-CoA and intermediates of the Krebs cycle, with additional cycles and CO2 assimilation). Overall, the RuMP pathway is considered the most efficient pathway in terms of energy yield [16].

There is a third type of bacteria that uses the Calvin–Benson–Bassam (CBB) cycle [11, 53]. This pathway is inefficient and is not common in nature [16]. 

Metabolism of hydrogen-oxidizing bacteria

The metabolism of hydrogen-oxidizing bacteria is determined by the metabolic type of the organism [54].

Hydrogen-oxidizing bacteria are divided into two groups. The first group includes bacteria that utilize hydrogen under aerobic conditions. These are chemolithoautotrophic aerobic bacteria that oxidize hydrogen (e.g., Alcaligenes eutrophus) [54].

The second group, capable of utilizing hydrogen only under anaerobic conditions, includes both chemotrophic and phototrophic bacteria. Chemotrophic bacteria that utilize hydrogen include methanogenic bacteria (e.g., Methanobacterium barkeri) and acetogenic bacteria (e.g., Clostridium aceticum and Acetobacterium woodii). Sulfate-reducing bacteria (e.g., Desul[ovibrio vulgaris), nitrate-reducing, denitrifying bacteria (e.g., Paracoccus denitri[icans), and fumarate-reducing bacteria (e.g., Escherichia coli). Among phototrophic bacteria, there are at least some species capable of growing with hydrogen as an electron donor in each of the major families: Chromatiaceae (e.g., Chromatium vinosum, Thiocapsa roseopersicina), Rhodospirillaceae (e.g., Rhodospirillum rubrum, Rhodopseudomonas capsulata), and Chlorobiaceae (e.g., Chlorobium limicola var. thiosalphatophilum). Some cyanobacteria and several species of green algae can be adapted to use hydrogen for anoxygenic photosynthesis. The carbon source used together with hydrogen is carbon dioxide, at least in most bacteria that use H2 as a source of reducing power and energy; only sulfate- and fumarate-reducing bacteria do not have the ability to fix CO2 autotrophically [54].

Anaerobic metabolism fueled by H2 and CO2 is only feasible if 70–80% of the carbon is converted to reduced products such as acetate, and approximately 20% to MCTs. From a protein production perspective, this is inefficient unless the resulting acetate is used to fuel the second-stage aerobic process, where acetate is used as a carbon source. This has recently been demonstrated in vitro [30, 55].

H2-driven aerobic autotrophy is capable of producing cellular biomass without any significant byproducts. This is due to the energy released during the reaction of H2 with oxygen (O2) [30].

For this reason, the first group of hydrogen-producing bacteria is of interest for SCP production. These bacteria constitute a physiological group; they are facultative autotrophs and have the ability to grow with hydrogen as an electron donor and carbon dioxide as the sole carbon source [54]. Hydrogenases, metalloenzymes that catalyze the oxidation of hydrogen, play a key role in the metabolism of aerobic hydrogen bacteria. The function of hydrogenases is to split molecular hydrogen and direct its electrons into the electron transport chain, where they are transferred to the final acceptor, O2, extracting energy in the process. Ultimately, hydrogen is oxidized to water, the end product [56].

There are two types of hydrogenases in hydrogen-oxidizing bacteria. The first type is soluble and reduces NAD (H2:NAD oxidoreductases), while the second type is membrane-bound and does not reduce pyridine nucleotides [54]. Based on the distribution of hydrogenases, hydrogen-oxidizing bacteria can be divided into three groups depending on whether they contain (i) both types of enzymes (Alcaligenes eutrophus), (ii) only the soluble enzyme (Nocardia opaca lb), or (iii) only the membrane-bound enzyme (most genera and species) [54].

If hydrogen-oxidizing bacteria contain both forms of hydrogenase, the functions between them are clearly separated. Membrane-bound hydrogenase, which catalyzes the H2 uptake reaction, transfers electrons to the respiratory chain at the level of cytochrome b and is thus directly related to energy processes. Water-soluble hydrogenase transfers electrons to NAD+ molecules, which are then involved in various biosynthetic reactions. It has been shown that bacteria possessing both types of hydrogenases have a higher biomass yield from hydrogen oxidation than microorganisms containing only the membrane-bound enzyme.

Hydrogen bacteria containing only membrane-bound hydrogenase reduce NAD+ via reverse electron transport, which costs energy.

However, if hydrogen bacteria contain only water-soluble hydrogenase, it performs both functions: some of the reducing equivalents from NADH enter the respiratory chain, while the rest are consumed through constructive metabolism.

Thus, of all chemolithoautotrophic prokaryotes, only hydrogen bacteria, using soluble hydrogenase, can directly reduce NAD+ by oxidizing an inorganic substrate. In all other groups, NADH is formed using the reverse electron transport mechanism.

CO2 assimilation in most hydrogen bacteria occurs via the reductive pentose phosphate cycle (the Calvin cycle). An exception to this rule are bacteria of the species Hydrogenobacter thermophilus and Aquifex pyrophilus, which assimilate CO2 through the reductive cycle of tricarboxylic acids (Arnon cycle) [57].

Raw materials and safety

The widespread use of SCPs based on methane-oxidizing and hydrogen-oxidizing bacteria is associated with a number of difficulties, such as slow growth of microorganisms and their increased oxygen demand (for methane it is 5 times higher than on carbohydrates and 2–2.5 times higher than on paraffins); low solubility of methane, hydrogen and oxygen in the culture liquid (for methane no more than 0.02 g/l at atmospheric pressure [58], hydrogen 18.8 ml/l under normal conditions, oxygen 4.9 ml/100 g at 0°C, 2.09 ml/100 g at +50°C); explosion hazard (methane forms explosive mixtures with air in the concentration range of 5–15% by volume [58], for hydrogen there is an increased risk of explosion at an oxygen concentration exceeding 4% by volume). Let us consider them in more detail.

Regulation of the use of SCPs

Like any food or feed product, SCPs must be safe to produce and use. Most regions have regulations to ensure that food and feed are safe for human consumption [29, 59]. These typically distinguish not only between food (for humans) and feed (for animals), but also between food (providing nutrition and possibly flavor) and food additives (preservatives, colors, texture modifiers, etc.), and between feed and feed additives. Precise definitions may vary by region, but international standards governed by the Joint FAO/WHO Expert Committee on Food Additives apply to products marketed internationally [29, 60].

In the United States, foods and substances used in food production are regulated by the Food, Drug, and Cosmetic Act (1958), which established the "generally recognized as safe" (GRAS) designation [61]. Accordingly, a substance with GRAS status is generally recognized by qualified experts as safe for its intended use. A substance recognized for such use before 1958 is, by default, considered safe for food consumption (as are foods used in the EU before 15 May 1997 and that are not novel foods) [7, 62, 63].

In the European Union, microorganisms used in food production are considered ingredients and must comply with Regulation (EC) No. 178/2002 [64]. Therefore, food manufacturers are responsible for the safe use of microorganisms in food production. In 2007, the European Food Safety Authority (EFSA) introduced the concept of "Qualified Presumption of Safety" (QPS) to assess the safety of microorganisms used in food and feed production before placing them on the market. QPS applies to food and feed additives, food enzymes, and plant protection products [7, 65].

A joint project between the International Dairy Federation (IDF) and the European Food and Feed Cultures Association (EFCA) has resulted in the compilation of an authoritative list of microorganisms whose use in food products has been documented. This list was published in 2002 [7, 66, 67].

In 2012, this list was updated. It now covers a wide range of food products (including dairy products, fish, meat, beverages, and vinegar) and contains an updated taxonomy of microorganisms [7].

It is worth noting that the conditions for using microorganisms as novel food on the EU market require that the protein be obtained from a pure culture of microorganisms. This is in contrast to a mixed culture, which is more efficient and cheaper to produce, as we discuss below in our article [68]. 

Product biosafety

Toxins from single cell protein products

One of the key concerns is the presence of toxins produced by microorganisms (production hosts or contaminants), which can cause potential allergy symptoms. 

Bacteria can also produce toxins, which limits their use as SCPs. Toxins can be extracellular (exotoxins) or intracellular (endotoxins). For example, both Pseudomonas spp. and Methylomonas methanica produce large amounts of protein and are suitable for use as SCPs. Both species also produce endotoxins that cause febrile reactions [69]. They can be destroyed by heating. Furthermore, a study of the immunogenicity of SCPs from M. capsulatus showed that an acellular preparation (i.e., a preparation without the cell wall) did not elicit immune responses in mice, unlike whole-cell preparations [29, 70]. 

Gram-negative bacteria like Cupriavidus necator appear to contain endotoxins integratedь in the cell walls. Endotoxins could cause inflammatory reactions 1 [71, 72]. To inactivate endotoxins, hydrogen peroxide is used for biosynthesis of poly (3-hydroxybutyrateco-4-hydroxybutyrate) produced by Cupriavidus necator [32, 68]. 

Thus, the problem of toxins is solved by careful selection of the producing organism, process conditions and product composition [29]. 

Nucleic acids from single cell protein products 

Rapidly reproducing bacterial species contain high amounts of nucleic acids (RNA). RNA content and its degradation are influenced by growth conditions, growth rate, and the carbon-to-nitrogen ratio [73]. In the production of SCPs for human consumption, high nucleic acid content is a concern because purine compounds formed during RNA degradation increase plasma uric acid concentrations, which can lead to gout and kidney stones [74]. SCPs with high nucleic acid content intended for animal feed are recommended for use only in feeding short-lived animals [75, 76, 77]. Methods for measuring the nucleotide content of complex SCPs have recently been described [29]. 

Various methods for reducing the RNA content of SCPs have been developed [78] and continue to be used. RNA degradation can be achieved by using endogenous RNA-degrading enzymes (ribonucleases) after their activation by heat treatment (60–70 °C), as is done in the production of Quorn™ [79]. Ribonucleases can also be added to the reaction mixture or used as immobilized enzymes [80, 81]. Degraded RNA components diffuse from the cells, but biomass loss occurs (35–38%). The process can be improved by using higher temperatures (72–74 °C) for 30–45 min, resulting in lower biomass loss (30–33% [82]). Increasing the temperature requires the addition of steam, which is a cost factor, but heat is also required for the final treatment of the biomass at 90 °C after RNase activation [29, 83]. 

Alkaline hydrolysis and chemical extraction methods have also been studied. The authors [84] used alkaline treatment to reduce the RNA content of P. varioti biomass used in the Pequilo process to below 2%. Treatment at 65 °C and pH 7.5–8.5 to activate endogenous ribonuclease also reduced the RNA content to <2%, while the protein content remained at 50% [29]. 

Carbon sources

Purification of raw materials

The use of various types of waste as feedstock for SCP production is attractive from a cost and environmental perspective, but may pose safety challenges, and the origin of the feedstock must be carefully considered [29]. 

For example, the use of organic waste for SCP production carries certain risks, such as the presence of pathogens, harmful substances, and heavy metals in the feedstock [85]. 

Studies conducted on crude biogas containing H2S reported lower yields compared to pure methane, as the presence of sulfides in the medium reduced the amino acid content [86, 87]. Better results can be achieved with enhanced biogas, as shown by [88], who achieved nearly 70% protein content in a process conducted on enriched methanotrophic and hydrogenotrophic cultures [89]. 

Natural gas requires pre-treatment with monozantholamine to remove impurities such as mercaptans, hydrogen sulfide, and water before feeding to the fermenter. This process is necessary to ensure that natural gas meets the requirements of the microbiological industry and can be used for the cultivation of microorganisms [58]. 

Composition of purified natural gas [58]:

• Methane: no less than 95% 

• Ethane homologues: no more than 2% 

• Propane: no more than 1% 

• Sum of the rest: no more than 1% 

• Carbon dioxide: no more than 1% 

• Hydrogen sulfide: no more than 0.01% 

• Sulfur-containing compounds: no more than 0.001%

A unique aspect of culturing methane-oxidizing bacteria on natural gas is that the primary nutrients are gases (methane and oxygen), which must be supplied in sufficient quantities to the cell walls. Bacteria require 2-3 times more oxygen than methane. However, this stoichiometric methane:air ratio lies within the explosive concentration range, and to account for this, the cultivation process is carried out with a gas phase composition that deviates from the optimal range, with excess methane and a corresponding oxygen limit [58]. 

Furthermore, it has been noted that the presence of carbon dioxide in the gaseous mixture at concentrations of 3 to 10% (by volume) improves bacterial growth, while oxygen levels above 18% (by volume) cause cell death. Methane-oxidizing bacteria grow at a pH of 6.5-7.1 and a temperature of 34-38°C. To ensure efficient cell growth, fermenters with intensive heat and mass transfer are usually used, which is achieved by increasing the gas flow rate, improving mixing and increasing the working pressure in the apparatus to increase gas solubility [58]. 

The dangers of producing methane from biogas

More specifically, this hazard is associated with the fermentation of biogas to methanol, including toxic gas emissions, explosion hazards, and fire hazards [11].

Anaerobic digestion of organic waste produces biogas consisting primarily of CO2, methane, H2S, and ammonia (NH3), each of which poses specific hazards. Methane is explosive at certain concentrations and is lighter than air, while CO2, which is heavier than air, can accumulate near the floor, affecting heart rate and respiratory rate. H2S, highly toxic and odorless at hazardous concentrations, poses a serious health hazard because it has no characteristic odor. NH3, with its pungent odor, can irritate the eyes and respiratory tract and displace oxygen from the bloodstream. Furthermore, the digestion of organic waste poses risks of contamination by pathogenic microorganisms [11].

Addressing safety concerns is paramount to reducing the risks associated with the fermentation of biogas to methanol, including toxic gas emissions, explosion hazards, and fire hazards [90, 91, 92]. Proper handling of flammable biogas is essential, requiring the design and operation of bioreactors and associated equipment to minimize the risk of explosion. Implementation of safety protocols, such as proper gas ventilation, explosion-proof equipment, and detection systems, is essential to prevent accidents [93]. Preventive measures, including gas detection systems, proper ventilation, and personal protective equipment (PPE), such as self-contained breathing apparatus (SCBA), must be implemented to prevent exposure to these gases [11, 87, 94]. 

The dangers of carbon dioxide capture

CO2 is required as a carbon source for bacterial growth. CO2 supplied to the reactor can come from a variety of sources. Flue gases from combustion [95], fermentation byproducts, or air are just a few examples of CO2 sources that can be separated from atmospheric air using adsorbents. In this case, DAC, a new technology for collecting CO2 directly from the atmosphere, is used [21]. However, the technology itself requires significant energy inputs to capture carbon dioxide from the atmosphere. This may lead to increased greenhouse gas emissions [21]. This technology is also unlikely to be economically viable in the short term, as large-scale cultivation requires addressing numerous issues, but it may be profitable if it generates revenue through the green premium.

Explosive hazard due to oxygen compound.

Furthermore, there are difficulties in scaling up microbial production using methane as a feedstock. Efficient methane assimilation requires both methane and oxygen. This gas combination can be explosive, posing a safety hazard when used in industrial-scale reactors.

Conventional fermenters use sparging to supply oxygen and methane, which leads to the formation of an explosive atmosphere [96, 97]. When the methane level in the gas phase exceeds the lower explosion limit (5%, [98]), reactor operation is stopped, limiting protein production [99].

One solution to methane explosion safety in SCP production is the use of modern membrane reactors, which we will discuss later in this article. This safety principle relies on separate supply of methane and oxygen to the bioreactor using membranes, which prevents the formation of explosive bubbles.

Hydrogen also exhibits an increased risk of explosion at oxygen concentrations exceeding 4% by volume, while bacteria grow optimally within the explosion range of 6% by volume [100]. However, studies also report optimal growth conditions at 4% by volume oxygen, which is within the explosion limit [101]. Gas distribution and composition are significant obstacles that must be overcome to achieve high biomass yields [68, 102]. 

One solution is to cultivate at higher pressures, thereby increasing the oxygen concentration in the liquid while maintaining the gas ratio at the same level [68].

Gas diffusion into aqueous solutions

Methane is a water-insoluble (hydrophobic) gas, or more accurately, sparingly or slowly soluble [16, 49]. Under normal conditions, methane is slightly soluble in water (∼20 mg CH4/L), so the efficiency of methane use for production is reduced by its availability to cells. Methane can be oxidized to methanol, which is highly soluble in water. However, chemical synthesis methods require significant energy expenditure, and biotechnical methods are underdeveloped. When using methane as a substrate, efficient mass transfer from gas to liquid must be ensured. Therefore, continuous methane supply to the reaction zone is required during cultivation. This approach significantly increases the volumetric methane flow rate. These features are taken into account when designing modern laboratory bioreactors. Modifying the geometry of the reaction zone and adding gas-permeable membranes and agitators can ensure recirculation and homogeneity of the culture medium [47].

Hydrogen and oxygen, as well as methane, are poorly soluble in water: hydrogen 18.8 ml/l under normal conditions, oxygen 4.9 ml/100 g at 0 °C, 2.09 ml/100 g at +50 °C.

Gaseous nutrition plays a crucial role in the vital functions of hydrogen bacteria, as they obtain the primary substrates for their energy and metabolic functions—hydrogen, oxygen, and carbon—from the gas phase, through dissolution in the pericellular environment. Their dissolved state in the culture fluid lasts only a fraction of a second. Therefore, culturing these microorganisms can only occur under conditions of intensive artificial ventilation, and even a brief interruption in the gas supply to the cells will damage cellular structures. This determines the specific methods for culturing hydrogen bacteria and managing this process [103].

Design and types of bioreactors

SCP production occurs in closed bioreactors, where carefully selected microorganisms are cultivated in the presence of biogas in a sterile environment containing the necessary nutrients for their growth [11].

Despite the wide variety of bioreactor designs used in the microbiological industry, all fermentation devices are equipped with standard design elements designed to ensure optimal conditions for the biochemical process, as well as to optimize the underlying physical processes (hydrodynamic, thermal, and mass transfer) [58].

Basic fermentation plant

The main apparatus in a bioreactor is the fermenter, a complete mixing apparatus that ensures:

– growth and development of microorganism populations within the liquid phase;

– transport of nutrients to microorganism cells;

– removal of metabolic waste products from microbial cells;

– removal of heat generated by cells as a result of their vital activity from the environment. 

To perform these functions, each fermenter must be equipped with the following elements or systems of elements: [58].

- supply of liquid (or bulk) streams into the apparatus;

- input and output of gas streams;

- aeration of the fermentation medium;

- mixing of the fermentation medium;

- defoaming of the fermentation medium;

- thermostatting of the fermentation volume;

- sterilization of the enzyme and fermentation medium;

- output of liquid (or bulk) streams from the apparatus; 

- control and regulation of the specified process parameters.

Although the design of fermentation apparatuses is based on standard design solutions, it is necessary to consider the specific characteristics of the organisms being cultured and the method of energy supply to the apparatus. Delivery options include gas-phase, liquid-phase, and combined energy delivery in both the liquid and gas phases [104, 105]. 

The fermenter has a capacity that provides the working volume for microorganism growth; the capacity of the fermenter can range from several tens of liters for experimental setups to tens of tons for industrial production. 

The entire fermentation unit can consist of several fermenters connected in series or in parallel (some sources refer to them as a "fermentor battery" or "fermentor cascade"). 

Several types of fermenters have become widely used in industry. They differ in their design, operational complexity, the type of feedstock used (liquid, gaseous, water-soluble), and the amount of biomass produced, and are linked into various process flow diagrams [106]:

• A battery of fermenters operating in parallel allows for greater productivity of a plant using a monosubstrate as feedstock [106];

• A battery of fermenters operating in series is used when using a complex substrate, the components of which are assimilated by the producer strain at different rates, to ensure the most complete utilization of all carbon-containing components [106];

• A battery of fermenters operating in series with an additional intermediate supply of nutrient medium (with refilling) is usually necessary when a high substrate concentration inhibits or suppresses the growth and development of the microbial population, and a low concentration does not allow achieving an economically justified concentration of biomass in the suspension going to the thickening stage [106];

• A battery of fermenters operating in series with recirculation of a portion of the microorganism suspension is used when using a complex substrate that is difficult to utilize, when microorganisms require a long time to adapt to one or more components of the feedstock; In this case, the introduction of a certain number of cells already adapted to a given substrate allows for a significant reduction in the residence time of microorganisms in the fermenter, an increase in the flow rate, and an increase in the degree of substrate utilization [106];

• A battery of sequentially connected fermenters with feeding and recirculation of the microorganism suspension allows for the successful solution of the problem of the most complete utilization of a multi-component, difficult-to-digest substrate that inhibits microbial cells in high concentrations [106]. 

Examples of the use of multiple fermenter arrays include Solar Foods' Patent No. AU2022247353B2, which describes a cascade of two sequential fermenters for cultivating at least one isolated microbe in continuous culture with hydrogen as an energy source and carbon dioxide as an inorganic carbon source [107]. 

The first reactor receives and processes collected biological waste and forms a nutrient medium for transfer to the second reactor. The second reactor, located downstream of the first reactor, receives nutrient medium from the first reactor for growing microbial mass [107].

A cascade of two sequential fermenters is also described in Solar Foods' Patent No. AU2020317552B2 [108]. The patent describes a bioreactor for growing microorganisms, which consists of two reaction chambers. The first chamber contains a smaller number of microorganisms and serves to feed the reaction mixture and remove excess gases. The second chamber, located downstream, contains a larger number of microorganisms and is designed to supply gases and remove the reaction mixture. The two chambers are connected by a means that ensures the flow of the reaction mixture from the first chamber to the second and the flow of gases in the opposite direction. This means is the only entry point for gases into the first chamber and for the reaction mixture into the second chamber.

Solar Foods has launched production of a protein called Solein in Finland. It involves cultivating a bacterial strain in continuous culture using hydrogen as an energy source and carbon dioxide as an inorganic carbon source. 

Aeration and mixing

To grow aerobic microorganisms by submerged cultivation in a bioreactor, it is first necessary to ensure intensive oxygen transfer from the gas phase to the cells, which can only be achieved with active aeration and mixing of the medium [58].

Aeration is necessary to saturate the fermentation medium with oxygen and then supply dissolved oxygen to the microorganism cells, as two interrelated processes continuously occur in the culture fluid: oxygen absorption from the gas by the liquid and oxygen consumption from the liquid by the microorganism cells [58]. 

Mixing in fermenters is necessary to solve the following problems [58]: 

• intensification of gas-liquid and liquid-cell mass transfer; 

• intensification of heat transfer during medium thermostatting; 

• dispersion of liquid droplets and gas bubbles. 

• equalization of temperature within the stirred medium; 

• equalization of nutrient concentrations within the medium.

In some devices, the functions of aeration and mixing of the fermentation medium are combined in a single device [58].

Mixing of the fermentation medium is accomplished in several ways:

Pneumatic mixing with compressed gas

For this purpose, the apparatus is equipped with a special device (a bubbler) located at the bottom of the apparatus (suitable for supplying energy in the gas phase) [58, 104]. 

Some varieties of bubble fermenters include bubble-airlift fermenters, which utilize the airlift principle of aeration and mixing of the medium, and column fermenters equipped with various contact devices and external circulation loops [58].

This type of fermenter is the basis for the modern U-loop fermenter currently used by UniBio A/S, which converts natural gas into protein for animal feed using methanotrophic bacteria.

Loop (airlift) fermenters have some disadvantages. For example, to overcome the hydrostatic pressure at the bottom of the reactor, gas must be supplied under high pressure. If air is used as the gas, this requires the use of compressors. Furthermore, airlift reactors make relatively poor use of the supplied gas. Typically, only 20–40% of the gas is used. Furthermore, it is difficult to achieve good and rapid release of gas bubbles from the fermenting liquid at the top of the reactor and separation of the resulting gas phase (which can foam heavily) from the liquid phase before the fermenting liquid descends to the bottom of the reactor [109].

Devices with self-priming mixers

They provide both aeration and mixing at the same time (suitable for supplying energy in the liquid phase) [58]. 

Devices equipped with ejectors

These are so-called jet fermenters, in which aeration and mixing are accomplished by ejectors (suitable for supplying energy in the liquid phase) [58, 104]. 

An example of a fermenter with a stirred ejector is patent No. US10696941B2 [109] of Kiverdi Inc. In the US, Air Protein Inc (a subsidiary of Kiverdi Inc) has launched production of the eponymous Air Protein, produced from carbon dioxide, oxygen, nitrogen, water, and H2, obtained through electrolysis.

Mechanical mixing 

These units are equipped with specialized stirrers of various configurations (suitable for combined energy delivery in both the liquid and gas phases) [58, 104].

Mechanically stirred fermenters have some drawbacks; for example, they are not particularly suitable for scaling up, as it is difficult to achieve the same uniform mixing and mass transfer in large reactors as in small laboratory and pilot reactors. Furthermore, intensive mixing leads to significant heating of the fermentation liquid [110].

Various mixing device configurations are also possible, such as using a mechanical stirrer and a bubbler in a single unit [58]. Thus, fermenters share many common components; the differences lie in the design of the aerating and mixing devices.

Currently, a wide variety of bioreactor designs for aerobic cultivation using traditional mixers, ejectors, circulation pumps, and bubblers are available for cultivating methane-oxidizing microorganisms. However, in most cases, their design characteristics and energy consumption make this process ineffective. These shortcomings make the development of a new apparatus for the aerobic cultivation of methane-oxidizing microorganisms both urgent and practically significant. Currently, two types of fermenters—membrane fermenters and U-loop fermenters—have gained the most popularity for producing biomass from natural gas. Let's examine them in more detail. 

Loop bioreactors or so-called U-loop fermenter

This type of fermenter is used by UniBio A/S, which holds a patent [110] for this development. U-loop and/or loop-shaped fermenters of the above type are also described in Danish patent No. 163066 (EP-B-0 418 187). A typical diagram of a U-loop fermenter is shown in Figure 2 [105, 111, 112].

The difference is that liquid circulation is achieved using one or more built-in pumps. The loop bioreactor consists of two vertical pipelines for the ascending and descending flows of culture fluid, connected at the bottom by a U-loop pipe. At the top, the pipelines are connected to a degassing unit. The descending pipeline contains a gas inlet. The degassing unit has a gas outlet. The pump is installed in one of the pipeline sections. Static mixers, to ensure uniform gas distribution in the culture fluid, are located in the vertical sections of the bioreactor's loop.

The difference is that the fluid is circulated using one or more built-in pumps. The loop bioreactor consists of two vertical pipelines for the ascending and descending flows of culture fluid, connected at the bottom by a U-loop pipe. At the top, the pipelines are connected to a degassing unit. The descending pipeline contains a gas inlet. The degassing unit has a gas outlet. The pump is installed in one of the pipeline sections. Static mixers, to ensure uniform gas distribution in the culture fluid, are located in the vertical sections of the loop portion of the bioreactor. 

Applications of U-loop fermenters

There are numerous studies devoted to the use of U-loop reactors. For example, study [111] is devoted to a U-loop fermenter equipped with static mixers. In this study, the mixing time and mass transfer coefficients were determined in a U-loop forced-flow fermenter with a volume of 0.15 m3 of this design. The effect of gas supply to the system on the impeller power consumption was also investigated. And the article [113] presents a mathematical model describing the dynamics of SCP production in a U-loop reactor, for calculating the optimal start-up profile for stable isotope production in a U- loop reactor. Study [114] presents a dynamic model of SCP production in a U- loop reactor, taking into account both the hydrodynamics in the U-loop reactor and the kinetics of microbial reactions of Methylococcus capsulatus. 

U-loop fermentation units also have some disadvantages [115]: 

- variable concentrations of liquid nutrient medium components and introduced gas components (methane and oxygen) along the length of the reactor, which significantly reduces the efficiency of the fermentation process [115]. 

- the inability to ensure a controlled quantity and optimal size of gas bubbles with a diameter of 2-5 mm in the liquid phase, which reduces mass transfer from gas to liquid relative to mass transfer with bubble diameters greater than 2-5 mm [115].

Membrane fermenter

In recent years, membrane bioreactors (MBRs) have begun to be widely used. They are a fundamentally new hybrid system consisting of two main elements: fermenters and membrane modules, integrated into a single system. The main advantage of this design is the use of membrane tubes for substrate delivery, preventing the formation of explosive gas bubbles. Studies have shown that macroporous membranes are quite effective compared to bubble columns.

Today, membrane bioreactors are relatively simple in technical design and offer promise for expanding the possibilities for research and control of fermentation processes, in which population development occurs in a constantly renewed environment. This renewal is achieved through the continuous supply of substrate and the continuous removal of metabolites (all or part) through a semipermeable membrane. Depending on the membrane type, three operating modes of membrane reactors are distinguished: dialysis mode, ultrafiltration mode, and microfiltration mode [58].

In recent years, membrane bioreactors (MBRs) have begun to be widely used. They are a fundamentally new hybrid system consisting of two main elements: fermenters and membrane modules, integrated into a single system. The main advantage of this design is the use of membrane tubes for substrate delivery, preventing the formation of explosive gas bubbles. Studies have shown that macroporous membranes are quite effective compared to bubble columns.

Today, membrane bioreactors are relatively simple in technical design and offer promise for expanding the possibilities for research and control of fermentation processes, in which population development occurs in a constantly renewed environment. This renewal is achieved through the continuous supply of substrate and the continuous removal of metabolites (all or part) through a semipermeable membrane. Depending on the membrane type, three operating modes of membrane reactors are distinguished: dialysis mode, ultrafiltration mode, and microfiltration mode [58].

Types of membranes

The type of membrane selected is determined by the culture method. For dialysis, conventional dialysis membranes made of regenerated cellulose are used. They effectively retain high-molecular compounds such as enzymes and toxins, but allow sugars and salts to pass through. Microporous membranes with pore sizes up to 25 nm can be used for dialysis. They are made of cellulose acetate, ceramics, porous metal and glass, and asbestos. Finally, purely diffusion-resistant nonporous membranes made of silicone rubber or Teflon can be used in dialysis [58].

For ultrafiltration, membranes made of cellulose acetate, polyamide, polysulfonide, or ethylcellulose are used [58].

For microfiltration, membranes made of cellulose nitrate, polyvinyl chloride, photoreactive plastic, and polycarbonate can be used [58]. 

Membranes are designed to separate components or cells in a liquid mixture by filtration. The separation of mixed compounds in a liquid is usually achieved by applying pressure or vacuum to a porous permeable selective membrane, but can also be achieved by a concentration gradient [116, 117]. The membrane forms a barrier allowing some components to pass through it more easily than others, and this selectivity is mainly determined by the pore size of the membranes [118], but other membrane characteristics such as hydrophilicity can also influence the choice by preventing hydrophilic (or hydrophobic) components from entering the membrane [119]. Membranes are usually specified according to their separation mode, i.e., microfiltration, ultrafiltration, nanofiltration, and reverse osmosis [120]. 

A fundamentally different application of membrane technology and the microbiological synthesis process is the immobilization of cells in porous membranes, primarily hollow fibers. The method involves the adsorption of producer cells within the pores of anisotropic hollow fibers and the pumping of a nutrient mixture through the fibers. As the substrate passes along the fiber, it undergoes microbiological transformation, and a product-enriched stream is discharged from the other side. Oxygen is supplied by pre-saturating the nutrient medium with it [58].

Design of membrane bioreactors

Two different membrane bioreactor designs are currently in use. The membrane can be placed either in an external loop, shown in Figure 3, or immersed in the reactor, shown in Figure 4 [120, 121]. 

The membrane, placed in an external loop, operates under direct pressure; a pump forces the bioreactor broth into the membrane module and passes it through the membrane [117]. External MBRs operate in a cross-flow mode, where the fluid being filtered flows at high velocity parallel to the membrane surface. This design prevents the formation of deposits on the membrane surface and reduces the fouling potential of external cross-flow membranes [122] and increases the flux through the membrane. Disadvantages of external cross-flow MBRs include the significant amounts of energy required to maintain continuous flow through the membrane [118] and the complex reactor design [115].

The design with the membrane submerged in the reactor is operated using a vacuum pump. The advantages of submerged MBRs are that they typically require less energy to operate compared to external cross-flow MBRs [117, 118]. However, the operation of a submerged MBR can be problematic at high solids or cell concentrations due to fouling. Typically, a larger membrane surface area is required in submerged MBRs than in cross-flow MBRs [118]. A method to prevent fouling and fouling is to vigorously blow gas through the submerged membrane surface [115, 117].

Application of membrane bioreactors

The industrial application of membrane technology has grown rapidly in recent decades. Currently, most MBRs are used in water supply or wastewater treatment. MBR for SCP production is a new concept that requires further development for larger-scale use. Membranes are made from materials such as cellulose, acetate, nitrate, polyvinylidene fluoride, polysulfone, polypropylene, polytetrafluoroethylene, and polyacrylonitrile [123]. Interest in the use of MBRs has increased due to the constant emergence of new filtration materials with unique properties—hollow fibers, ceramic and metal-ceramic filters, sterilizable polymer membranes, ion-exchange membranes, silicone rubber membranes, etc.

To date, several studies have been conducted on the use of MBRs for SCP production; we will examine them in more detail. In this [124] study, a double membrane biofilm reactor (dMBfR) was developed for the production of CH-based SCPs, which utilizes HFM for separate feeding of methane and oxygen.

The study focuses on a dual-membrane biofilm reactor (dMBfR), incorporating hollow fiber membranes (HFM) to enhance the delivery of methane and oxygen. The hollow fiber membrane efficiently delivers gases to microorganisms without creating bubbles, so it is used as a diffuser to improve methane utilization efficiency and reduce energy costs [125, 126]. According to [127, 128], HFM can increase gas transfer efficiency by 262% compared to conventional bubbling and increase biomass growth rate by 67% [127, 128]. Methane can be pressurized in the lumen of the HFM and diffuse into the liquid phase. Since no bubbles are formed in this process, gas diffusion can be minimized [129]. On the other hand, gas supply in HFM is achieved by means of a concentration gradient between the gas and liquid phases, which is considered a more economical solution than the use of mechanical static mixers [130]. ​​In this study, biomass was cultured in biofilms [124].

The reactor described in the study [124] was equipped with eight bundles of non-porous HFM fibers. Three bundles were for methane and five bundles were for air, as the theoretical methane to oxygen consumption ratio is 1 V/1.4 V [131]. Each bundle of hollow fibers was 30 cm long and had a surface area of 0.039 m2, providing an A/V ratio of 1950 m-1. For methane supply, one end of the fiber was connected to a methane cylinder, and the other end was sealed (i.e., methane did not escape from the distal ends). For air supply, one end of the fibers was connected to an air blower, and the other end was not sealed (i.e., it was open and the supplied air was continuously released), except during certain stages of the study [124]. The culture medium was supplied using a pump. The reactor was continuously stirred by recirculating the medium between the main reactor and the overflow tank using a pump.

The initial culture medium consisted of 0.2 g/L MgSO4×7H2O, 0.03 g/L CaCl2×2H2O, 0.35 g/L KNO3, 0.36 g/L Na2HPO4×12H2O, 0.136 g/L KH2PO4, 0.2 mL/L alkaline trace solution, and 0.5 mL/L acidic trace solution. Methylococcus capsulatus Bath (ATCC 33 009) was used as an inoculum at the beginning of the experiment [124].

Methane utilization efficiency is an important factor in evaluating process performance. In this reactor, the methane utilization efficiency was nearly 100% throughout the entire operation, indicating that using HFM as a gas diffuser can effectively reduce gas dispersion. This high efficiency may also be due to the interaction between methanotrophic and non-methanotrophic bacteria grown in our reactor, as discussed in the section on mixed culture cultivation [124].

This study also determined [124] the maximum oxygen transfer rate in the open-end aeration mode to be 65.1 mg/L/h, and in the closed-end mode to be 20.5 mg/L/h. These results indicate that open-end aeration provided a higher oxygen delivery rate than closed-end aeration, which promoted microbial growth and metabolism and had a positive effect on methane conversion to SCP [124]. This study also demonstrated that the open-end aeration reactor achieved higher protein content.

The authors of this study previously conducted a similar study [128], but in which a nonporous hollow fiber membrane was used only for methane delivery. HFM was also used as a diffuser to more efficiently transfer methane from the gas to the liquid phase. Oxygen was supplied from the other side of the reactor using a dedicated oxygen supply system. In this study, the biomass was cultured in suspension in the main reactor.

The nutrient medium consisted of 0.2 g/L MgSO4 × 7H2O, 0.03 g/L CaCl2 × 2H2O, 0.14 g/L NH4Cl, 0.36 g/L Na2HPO4 × 12H2O, and 0.136 g/L KH2PO4. Activated sludge collected from a wastewater treatment plant was used as the inoculum [128].

The results of this study [128] also highlight that HFM offers a more cost-effective solution with superior carbon efficiency for the production of SCP.

A membrane reactor for producing SCPs from methane was also investigated in [99]. The reactor consisted of a chamber sealed with a lid and an attached magnetic stirrer. The medium was pumped. Gas was supplied through hydrophobic hollow fiber membranes. Dead-end membranes were used to deliver oxygen and methane as pure gases. When air was used as the oxygen source, a flow-through membrane system was used for gas delivery. This ensured that methane and oxygen were dissolved only upon contact in the liquid phase, avoiding the formation of an explosive atmosphere inside the reactor [99].

The inoculum consisted of the previously used mixed methanotrophic culture grown on synthetic medium. Ammonium chloride (Sigma Aldrich, USA) was used as the nitrogen source instead of nitrate. Before the experiments, the nutrient medium was autoclaved. Hydrophobic membranes provided safe gas supply to bioreactors growing aerobic methanotrophs for microbial protein production. No explosive atmosphere was created during reactor operation. Biomass yield was lower than in bioreactors using bubbling, but research [99] showed that by increasing the membrane surface area in continuous membranes or the feed rate in dead-end membranes, SCP yield could potentially be increased.

Overall, these studies demonstrate that membrane bioreactors are a very promising approach to producing SCPs from methane. This technology requires further research to scale up such reactors for production capacity. Developing an optimal bioreactor capable of ensuring more efficient mass transfer between the gas and liquid phases is a critical technological challenge for achieving cost-effective production [11, 47, 132]. 

Cultivation: Strategies and Communities

A variety of microorganisms grown on various carbon sources are used to produce single cell proteins. The producer microorganism and the raw material base have the greatest impact on the final product. 

For bacterial strains to be considered suitable for SCP production, they must meet several criteria: reaction conditions (temperature and oxygen requirements during fermentation and foam formation); productivity (yield, growth rate, pH, and thermal stability); behavior during fermentation (growth morphology and genetic stability); and the final product (in terms of bacterial protein composition and structure, purification yield, and recovery rate) [43, 133]. 

The nutritional and nutritive value of SCPs depends on the microorganisms used. Microorganisms used for SCP production must be non-pathogenic, toxin-free, easy to handle and separate from the substrate, and capable of process scaling. High-volume production (biomass weight produced per unit of time) requires fast-growing microorganisms. However, at high productivity, more RNA is produced in the cells, which is undesirable because it acts as an anti-nutritional factor in the final product. The method of collection, drying, and processing affects the nutritional value of the finished product [43, 134, 135, 136, 137, 138]. 

Currently, SCP is produced from a limited number of microbial species, especially for human consumption [29]. 

Pure cultures 

To be sold in the European Union, a bacterial protein must be approved by the European Food Safety Authority. One of the requirements is that the bacteria must grow in pure culture [68]. 

However, pure culture is commercially unattractive because it is not inexpensive enough to compete with other alternative protein sources [68, 139]. Isolating a pure culture of seed material used for seeding, overseeding, and subculture of industrial primary fermenters is a labor-intensive process. Furthermore, the correct selection of the producer is essential for ensuring a high yield of the final product. Insufficient maintenance of culture purity can significantly reduce the technological and economic performance of the protein, or make further industrial exploitation of the strain impossible. 

Among HOBs, Cupriavidus necator is the most well-described and studied organism in the literature for SCP production from pure HOB cultures [68]. Among MOBs, Methylococcus capsulatus has been considered the most frequently considered methanotroph for SCP production for many years [89, 140].

Cultivation of mixed crops

Methanotrophic bacteria are part of the larger methylotrophic group, which includes microorganisms capable of utilizing substrates such as methane, methanol, and other methylated compounds and metabolizing the resulting formaldehyde as a source of cellular carbon [141]. The ability to utilize methane as a sole source of carbon and energy, directly oxidize it, and convert it into other products within the cell is a unique characteristic of methanotrophs that distinguishes them from other methylotrophs [89].

Despite the wide diversity of methanotrophic bacteria, the general metabolic pathway of methane oxidation to carbon dioxide via formaldehyde as an intermediate remains the same for all members of this microbial group [89, 142].

The biotechnological potential of methanotrophic bacteria has been the subject of research for many years and is used in industry for the commercial production of several products, including biopolymers and methanol, as well as single-cell proteins [89].

For many years, Methylococcus capsulatus was considered the most frequently studied methanotroph for SCP production [89, 140].

However, implementing the process of microbial protein production in mixed microbial cultures has been a major focus of research over the past few years. Although the symbiosis of a methanotroph with another microorganism does not always positively affect protein accumulation in the culture, as in the case of co-cultivation of Methylococcus capsulatus and Chlorella sorokiniana [97], the cultivation of mixed methanotrophic cultures usually yields better or comparable results to SCP production by pure strains. The content of the resulting protein ranged from 40% to 70%, and in most cases it exceeded 50%. In addition to differences in protein content, the amino acid profile can also vary depending on the microorganism used and the process conditions, but in most cases the achieved profiles were suitable for replacing traditional protein sources [89].

A number of studies have shown that the cultivation of mixed microbial communities is the most effective. Let's take a closer look at them:

In a study [124] using a pure culture of Methylococcus capsulatus Bath as an inoculum, the biofilm community diversified during long-term operation. Overall, Proteobacteria dominated the microbial community in the reactor, accounting for >35% of the total community. Cercozoa, a major group of eukaryotes, represented the second most abundant population, with a relative abundance of 10% to 20%. In contrast, Cercozoa accounted for only 0.74%, followed by Chlorflexi and Bacteroidetes, each accounting for approximately 6% of the community [124].

The key methane-oxidizing genus Methylococcus was consistently present and increased in abundance throughout the study. As discussed previously, this genus is considered one of the most commercially successful SCP producers, using methane as the sole carbon and energy source [111, 143]. The study noted that stages with higher Methylococcus abundance also had higher protein content, highlighting the important role of Methylococcus in SCP production [124].

In addition to methanotrophs, the reactor harbored a variety of non-methanotrophic bacteria capable of utilizing CO2 or byproducts of methanotrophic metabolism. These included species from families such as Rhodobacterales and Devosiaceae, which are involved in CO2 fixation, as well as Methylophilaceae, Rhodocyclaceae, Comamonadaceae, Rubinisphaeraceae, and Gemmataceae, which can metabolize methanol or organic acids released by methanotrophs. These microorganisms not only increased the efficiency of carbon use in the reactor [144] but also contributed to SCP production [124, 145].

The community structure of non-methanotrophic bacteria varied throughout the study. Under open-end aeration conditions, the growth of anaerobic microorganisms (e.g., Caldilineaceae and Pseudomonas families) was suppressed [146, 147], as open-end aeration provided higher oxygen levels. Additionally, increased oxygen levels may have promoted the growth of aerobic microorganisms (e.g., Simkaniaceae family) [124, 148].

This study [124] did not aim to maintain bacterial community purity, as mixed culture systems are generally less expensive to operate, which also indicates greater environmental resilience and carbon use efficiency, but it did find that the use of mixed cultures not only did not reduce protein yield, but actually enhanced it. In an earlier study [128] by the same authors, a mixed culture was also used due to its lower cost and greater environmental tolerance than pure culture, with activated sludge collected from a local full-scale wastewater treatment plant being used as the inoculum [128].

Overall, Proteobacteria, Chloroflexi, Bacteroides, and Planctomycetes dominated the reactor, accounting for over 60% of the total community [128].

Two key families of methane-oxidizing bacteria, Methylomonadaceae and Methylococcaceae, were enriched and present at all stages of the study. These families are widespread in natural environments where methane serves as the sole source of carbon and energy [143]. The authors [128] indicate that they have been used in previous studies to produce SCPs with protein contents exceeding 50% [97, 149]. This study reports that the total abundance of aerobic methanotrophs was highest in the first stage (5%) due to the processing of biomass, which promotes their enrichment. In subsequent stages, the abundance of aerobic methanotrophs decreased to, which led to an increase in the abundance of fast-growing microorganisms such as Roseiflexaceae and Comamonadaceae during these periods [150]. Despite the lower abundance of methanotrophs, no decrease in SCP productivity and quality was observed in some stages. It is also noted that the stage characterized by the second highest total methanotroph abundance achieved the highest SCP yield and content. This suggests that SCP productivity and quality may depend not only on the total abundance of methanotrophs but also on the interactions between methanotrophic and non-methanotrophic microbes [128].

At the stage of this study, characterized by the lowest O2 loading level, a high number of anaerobic microorganisms, such as methanogens (e.g., [151] Methanobacteriaceae, Methanospirillaceae, and Methanosarcinaceae) and Anaerolineaceae, were observed. Since anaerobic bacteria typically contain less protein and grow more slowly than aerobic bacteria, the productivity and quality of SCP at this stage are lower, [128], indicating less tolerance to declining oxygen levels [152, 153, 154]. From a reactor operational perspective, sufficient aeration is necessary to maintain high SCP production in the reactor [128].

Another possible reason for the higher SCP quality is related to the mixed culture used in this study. A culture with higher microbial diversity can yield more protein and produce a wider range of amino acids, thereby improving protein quality [144, 155]. This study, however, highlights that whether mixed SCP culture is superior to monoculture remains controversial. For example, some previous studies have shown that protein content is higher in monoculture systems than in mixed systems [156]. Overall, high-methane gas supply and suspended mixed biomass cultivation contributed to increased SCP productivity and quality [128].

The use of a mixed culture of microbial communities was also investigated in [12]. This study is also referenced by the authors [11].

It states that a mixed methanotrophic culture was also cultivated using synthetic methane and diluted ammonium mineral salt in sterilized serum-containing vials. The results showed that a mixed methanotrophic culture of Methylomonas (56.26%) and Methylophilus (24.60%) could thrive when provided with pasteurized AD supernatant and biogas, resulting in the production of SCPs with a protein content of over 41%. The mixed culture demonstrated robust growth on pasteurized AD supernatant and biogas, resulting in a promising dry weight yield of 0.66 ± 0.01 g dry weight/g methane. This SCP also contains various essential amino acids, such as histidine, which can replace fishmeal and soy as a protein source in animal feed [11].

Although pure cultures have previously been used for commercial SCP production, potential risks (e.g., contamination, low resistance to inhibitors) limit commercialization. The above studies confirm that biomass obtained from mixed culture has the potential to be used as an inoculum for SCP production. A stable bacterial consortium can maintain high levels of biomass growth for a long time [157]. A stable bacterial consortium may not require strictly sterile conditions, which reduces operating costs [86]. Furthermore, the nutrient and feed requirements of mixed cultures are correspondingly reduced [12, 158]. Another important factor in using mixed cultures is that, in addition to differences in protein content, the amino acid profile may also vary depending on the microorganism used [89]. This makes it possible to adjust the amino acid profile of the produced SCP to replace traditional protein sources.

Co-cultivation of methane-oxidizing and hydrogen-oxidizing bacteria

In our work, we would also like to highlight a study [85] that not only cultivated a mixed culture but also investigated the co-cultivation of methane-oxidizing and hydrogen-oxidizing bacteria.

The goal of co-cultivating methane-oxidizing and hydrogen-oxidizing bacteria was to investigate the use of organic waste as a substrate for SCP production. The use of organic waste streams for SCP production carries certain risks, as mentioned earlier in our article, such as the presence of pathogens or toxic substances. These risks can be mitigated through anaerobic digestion and subsequent aerobic assimilation of (sterilized by filtration) biogas. Co-cultivation can provide clear advantages in terms of metabolism, carbon utilization and recovery, and potentially system stability. The combination of MOB and HOB, in addition to the CH4 and CO2 contained in biogas, can also provide valorization of CO2, which is formed as a result of the oxidation of methane by MOB [85].

Also of particular interest in the study of the co-cultivation of methane-oxidizing and hydrogen-oxidizing bacteria are carbon sources such as natural gas, which consists of a mixture of methane and hydrogen, which has been recorded in various seeps, such as the Neoproterozoic Amadeus Basin in Australia [159], fluid seeps in New Caledonia (N2-H2-CH4-rich gases show N2 contents from 50 to 62%, H2 from 26 to 36% and CH4 from 11 to 16%) [160] and the Rainbow Field located on the Mid-Atlantic Ridge (end-member concentrations of H2 (16 mmol/kg), CH4 (2.5 mmol/kg) and CO (5 μmol/kg)) [161]. As well as hydrogen, which mixes with methane during transport and storage. Natural hydrogen extracted from the earth or excess hydrogen produced by water electrolysis can be stored in a variety of underground reservoirs, including saline aquifers, permeable rocks, and depleted oil and gas reservoirs. These underground geological features offer suitable properties for efficient hydrogen storage, such as large storage volumes and long-term stability [162]. Another possible solution is to transport hydrogen through pipelines, as was the case with natural gas. This would allow the transport and distribution of cleanly produced hydrogen from large production sites throughout the relevant supply chain. The hydrogen transported through these pipelines can be pure or blended with natural gas to utilize the existing network [163]. Thus, natural gas, consisting of a mixture of methane and hydrogen, can be used for the production of SCPs using HOB and MOB.

For the study [85], ten different combinations of MOB and HOB were selected and grown in serum flasks to determine the most effective. All bacterial combinations showed a synergistic effect, with growth in cocultures outperforming growth in axenic culture in six of the ten combinations. Two combinations exhibited both higher protein concentrations and amino acid contents than expected based on pure cultures. The combination of MOB and HOB resulted in a 3.8-fold increase in protein concentration and a 6.1-fold increase in essential amino acid content compared to pure cultures, while the essential amino acid profile of the (co)cultures was comparable to common food ingredients. The most promising combination in terms of protein concentration and essential amino acid profile was Methyloparacoccus murrelli LMG 27482 and Cupriavidus necator LMG 1201 [85].

Increased protein production during co-culture (as indicated by higher protein concentration) is a result of the higher cell density achieved during co-culture [85].

It is worth noting that these two groups of bacteria have different properties. For example, MOB contain unsaturated acids, while HOB can accumulate very large amounts of PHB [164]. Thus, the combination of MOB and HOB can significantly increase the value of the final product [85].

Co-cultivation of MOB and HOB has a positive effect on their nutritional properties. Thus, the combination of MOB and HOB can ensure more efficient carbon extraction from biogas and contribute to the development of safer and more environmentally sustainable food/feed production systems [85].

The authors [103] also note another advantage: cultivating hydrogen bacteria together with methane-oxidizing bacteria on a mixture of dioxide gas and hydrogen may prove economically viable, since hydrogen bacteria will perform the function of purifying the culture medium from the metabolites of their heterotrophic companions—carbon dioxide and organic acids, which inhibit the growth of microorganisms on dioxide gas [103]. 

Directions for further research

The main area for further research is translating SCP production technology from laboratory to industrial scale. Despite promising results demonstrating high biomass yield, excellent protein content, and a balanced amino acid profile in small (laboratory) bioreactors, scaling up is fraught with a host of engineering, biological, and economic challenges.

Scaling up the production process could follow several of the most promising approaches or combine them:

1. Improving the design of bioreactors to ensure efficient mass transfer of poorly soluble gases (CH4, H2, O2) and maintaining medium homogeneity in large volumes. A promising approach is the development of hybrid solutions; for example, the integration of membrane modules for separate and safe gas supply to high-performance U-loop fermenters could be considered. This would combine the advantages of both types of apparatus: high productivity and the elimination of the risk of explosive gas mixtures.

2. Developing methods for monitoring and maintaining optimal species composition under continuous cultivation conditions. To reduce costs and improve process sustainability, a transition from pure cultures to mixed communities is needed.

3. Assessing the life cycle of the technology—from the source of raw materials (natural gas, biogas, "green" hydrogen) to the finished product. Research should provide clear answers about the final cost of protein, the energy consumption of the process, and its actual environmental footprint compared to traditional protein sources (soy, fishmeal) and other alternatives.

4. Developing a regulatory framework and standardization for assessing the safety of SCPs from mixed cultures. Creating new standards and food regulations that take into account the specifics of bacterial protein production.

5. Studying and analyzing consumer willingness to accept products based on bacterial biomass. Developing communication strategies that highlight the environmental and nutritional benefits of SCPs. Creating appetizing and convenient food products incorporating SCP ingredients.

These research directions may facilitate further development and implementation of SCP production from methane and hydrogen as a sustainable protein source for human and animal nutrition. 

Reference 

1. Rischer H, Szilvay GR, Oksman-Caldentey KM. Cellular agriculture — industrial biotechnology for food and materials. Curr Opin Biotechnol. 2020;61:128-134. doi:10.1016/j.copbio.2019.12.003.

2. Tuomisto HL. The eco-friendly burger. EMBO Rep. 2019;20:e47395. doi:10.15252/embr.201847395

3. Järviö N, Maljanen NL, Kobayashi Y, Ryynänen T, Tuomisto HL. An attributional life cycle assessment of microbial protein production: a case study on using hydrogen-oxidizing bacteria. Sci Total Environ. 2021;776:145764. doi:10.1016/j.scitotenv.2021.145764.

4. Tusé D, Miller MW. Single cell  protein: current status and future prospects. Crit Rev Food Sci Nutr. 1983;19(4):273-325. doi:10.1080/10408398409527379. 

5. Anupama, Ravindra P. Value-added food: Single cell protein. Biotechnol Adv. 2000;18(6):459–479. doi:10.1016/S0734-9750(00)00045-8

6. Nasseri AT, Rasoul-Amini S, Morowvat MH, Ghasemi Y. Single Cell Protein: production and process. Am J Food Technol. 2011;6(2):103-116. doi:10.3923/ajft.2011.103.116

7. Bourdichon F, Casaregola S, Farrokh C, Frisvad JC, Gerds ML, Walter P. Food fermentations: Microorganisms with technological beneficial use. Int J Food Microbiol. 2012;154(3):87-97. doi:10.1016/j.ijfoodmicro.2011.12.030.

8. Meraz JL, Dubrawski KL, El Abbadi SH, Choo KH, Criddle CS. Membrane and Fluid Contactors for Safe and Efficient Methane Delivery in Methanotrophic Bioreactors. J Environ Eng. 2020;146(6):03120006. doi:10.1061/(ASCE)EE.1943-7870.0001703.

9. Howarth R, Ingraffea A, Engelder T. Should fracking stop? Nature. 2011;477:271-275. doi:10.1038/477271a.

10. Salmon R, Logan A. Flaring up: North Dakota natural gas flaring more than doubles in two years. Ceres. 2013;1.

11. Shahzad HMA, Almomani F, Shahzad A, Mahmoud KA, Rasool K. Challenges and opportunities in biogas conversion to microbial protein: A pathway for sustainable resource recovery from organic waste. Process Saf Environ Prot. 2024;185:644-659. doi:10.1016/j.psep.2024.03.055.

12. Zha X, Tsapekos P, Zhu X, Khoshnevisan B, Lu X, Angelidaki I. Bioconversion of wastewater to single cell protein by methanotrophic bacteria. Bioresour Technol. 2021;320(Pt A):124351. doi: 10.1016/j.biortech.2020.124351.

13. Khoshnevisan B, Tsapekos P, Zhang Y, Valverde-Pérez B, Angelidaki I. Urban biowaste valorization by coupling anaerobic digestion and single cell protein production. Bioresour Technol. 2019;290:121743. doi:10.1016/j.biortech.2019.121743.

14. Khoshnevisan B, He L, Xu M, et al. From renewable energy to sustainable protein sources: Advancement, challenges, and future roadmaps. Renew Sustain Energy Rev. 2022;157:112041. doi:10.1016/j.rser.2021.112041.

15. Klein-Marcuschamer D, Simmons BA, Blanch HW. Techno-economic analysis of a lignocellulosic ethanol biorefinery with ionic liquid pre-treatment. Biofuels Bioprod Biorefining. 2011;5:562-569. doi:10.1002/bbb.303.

16. Comer AD, Long MR, Reed JL, Pfleger BF. Flux balance analysis indicates that methane is the lowest cost feedstock for microbial cell factories. Metab Eng Commun. 2017;5:26-33. doi:10.1016/j.meteno.2017.07.002.

17. Strong PJ, Kalyuzhnaya M, Silverman J, Clarke WP. A methanotroph-based biorefinery: Potential scenarios for generating multiple products from a single fermentation. Bioresour Technol. 2016;215:314-323. doi:10.1016/j.biortech.2016.04.099.

18. García Martínez JB, Egbejimba J, Throup J, Matassa S, Pearce JM, Denkenberger DC. Potential of microbial protein from hydrogen for preventing mass starvation in catastrophic scenarios. Sustain Prod Consum. 2021;25:234-247. doi:10.1016/j.spc.2020.08.011.

19. Matassa S, Boon N, Verstraete W. Resource recovery from used water: the manufacturing abilities of hydrogen-oxidizing bacteria. Water Res. 2015;68(1):467-478. doi:10.1016/j.watres.2014.10.028.

20. Liu C, Colón BC, Ziesack M, Silver PA, Nocera DG. Water splitting-biosynthetic system with CO2 reduction efficiencies exceeding photosynthesis. Science. 2016;352(6290):1210-1213. doi:10.1126/science.aaf5039.

21. Sillman J, Nygren L, Kahiluoto H, et al. Bacterial protein for food and feed generated via renewable energy and direct air capture of CO2: Can it reduce land and water use? Glob Food Secur. 2019;22:25-32. doi:10.1016/j.gfs.2019.09.007.

22. Tan HSG, Verbaan YT, Stieger M. How will better products improve the sensory-liking and willingness to buy insect-based foods? Food Res Int. 2017;92:95-105. doi:10.1016/j.foodres.2016.12.021.

23. Ma Z, Mondo M, Valencia FG, Hernández-Álvarez AJ. Current state of insect proteins: extraction technologies, bioactive peptides and allergenicity of edible insect proteins. Food Funct. 2023;14:8129-8156. doi:10.1039/D3FO02865H. 

24. Øverland M, Tauson AH, Shearer K, Skrede A. Evaluation of methane-utilising bacteria products as feed ingredients for monogastric animals. Arch Anim Nutr. 2010;64(3):171-189. doi: 10.1080/17450391003691534.

25. Neft.media. How the USSR created proteins from oil and gas and how it is useful for KMAO [Kak v SSSR sozdali belki iz nefti i gaza i chem e`to polezno XMAO]. Available from: https://neft.media/vse-regiony/materials/paprin-i-gaprin. [Accessed 24 September 2025].

26. Feodoridi G. Production of food protein by microorganisms from methane. 2024. doi:10.13140/RG.2.2.27668.31364.

27. Moen E, Norway S. Oil & gas – feed for fish? Bioprotein, a new high quality single cell protein based on natural gas. 2002. Available from: https://www.researchgate.net/publication/235955564_OIL_GAS_-_FEED_FOR_FISH_BIOPROTEIN_A_NEW_HIGH_QUALITY_SINGLE_CELL_PROTEIN_BASED_ON_NATURAL

28. Xu J, Wang J, Ma C, Wei Z, Zhai Y, Tian N, et al. Embracing a low-carbon future by the production and marketing of C1 gas protein. Biotechnol Adv. 2023;63:108096. doi:10.1016/j.biotechadv.2023.108096.

29. Ritala A, Häkkinen ST, Toivari M, Wiebe MG. Single Cell Protein-State-of-the-Art, Industrial Landscape and Patents 2001-2016. Front Microbiol. 2017;8:2009. doi:10.3389/fmicb.2017.02009.

30. Pander B, Mortimer Z, Woods C, McGregor C, Dempster A, Thomas L, Maliepaard J, Mansfield R, Rowe P, Krabben P. Hydrogen oxidising bacteria for production of single cell  protein and other food and feed ingredients. Eng Biol. 2020;4(2):21–24. doi:10.1049/enb.2020.0005.

31. Nyyssölä A, Ojala L, Wuokko M, Peddinti G, Tamminen A, Tsitko I, Nordlund E, Lienemann M. Production of Endotoxin-Free Microbial Biomass for Food Applications by Gas Fermentation of Gram-Positive H2-Oxidizing Bacteria. ACS Food Sci Technol. 2021;1(3):470-479. doi:10.1021/acsfoodscitech.0c00129.

32. Shepon A, Eshel G, Noor E, Milo R. Energy and protein feed-to-food conversion efficiencies in the US and potential food security gains from dietary changes. Environ Res Lett. 2016;11(10):105002. doi:10.1088/1748-9326/11/10/105002.

33. Feodoridi G, Feodoridi K. Food protein produced by microorganisms using hydrogen and carbon dioxide as a carbon source. 2025. doi:10.13140/RG.2.2.10503.92321.

34. Holmström S, Pitkänen JP. Strains and processes for single cell protein or biomass production. United States Patent US20240093141A1. 2024 Mar 21.

35. Jämsä T, Pitkänen JP, Salusjärvi L. Variant bacterial strains and processes for protein or biomass production. Australian Patent AU2022263641A1. 2022 Nov 03.

36. Reed J, Robertson D. Microorganism-Derived Protein Hydrolysates and Methods of Preparation and Use Thereof. United States Patent US20210395677A1. 2021 Dec 23.

37. Dyson L, Rao K, Reed J. High protein food compositions. Patent WO2023278301A1. 2023 Jan 05. 

38. Rosenberry B. NovoNutrients: Making Aquatic Feeds from Waste Products [Internet]. Shrimpnews.com. 2020 [cited 2023 May 23]. Available from: https://www.shrimpnews.com/FreeReportsFolder/SpecialReports/NovoNutrientsShrimpFeedsFromWasteProducts

39. Sefton B. NovoNutrients: Food from CO2 [Internet]. 2018 [cited 2024 Jun 20]. Available from: http://nas-sites.org/dels/files/2018/02/2-2-SEFTON-NovoNutrients-NAS.pdf

40. Nadkishor JL. Production of Single cell  Protein and Mushrooms [Internet]. Biologydiscussion.com. [cited 2024 Jun 20]. Available from: https://www.biologydiscussion.com/single cell -protein/production-of-single cell -protein-and-mushrooms/10392

41. Omar S, Sabry S. Microbial biomass and protein production from whey. J Islamic World Acad Sci. 1991;4:170-172.

42. Jurtshuk P. Bacterial metabolism. In: Baron S, editor. Medical Microbiology. 4th ed. Galveston (TX): University of Texas Medical Branch at Galveston; 1996. Chapter 4. Available from: https://www.ncbi.nlm.nih.gov/books/NBK7919/ 

43. Bratosin BC, Darjan S, Vodnar DC. Single Cell Protein: A Potential Substitute in Human and Animal Nutrition. Sustainability. 2021;13(16):9284. doi:10.3390/su13169284. 

44. Vasdekis AE, Stephanopoulos G. Review of methods to probe single cell metabolism and bioenergetics. Metab Eng. 2015;27:115-135. doi:10.1016/j.ymben.2014.10.006. 

45. Müller WEG, Schröder HC, Wang X. Inorganic Polyphosphates As Storage for and Generator of Metabolic Energy in the Extracellular Matrix. Chem Rev. 2019 Dec 26;119(24):12337-12374. doi:10.1021/acs.chemrev.9b00460.

46. Li R, Fan X, Jiang Y, Wang R, Guo R, Zhang Y, et al. From anaerobic digestion to single cell protein synthesis: A promising route beyond biogas utilization. Water Res. 2023;120417. doi:10.1016/j.watres.2023.120417.

47. Tikhomirova TS, But SY. Laboratory scale bioreactor designs in the processes of methane bioconversion: Mini-review. Biotechnol Adv. 2021;47:107709. doi:10.1016/j.biotechadv.2021.107709.

48. Fei Q, Guarnieri MT, Tao L, Laurens LML, Dowe N, Pienkos PT. Bioconversion of natural gas to liquid fuel: Opportunities and challenges. Biotechnol Adv. 2014 May-Jun;32(3):596-614. doi:10.1016/j.biotechadv.2014.03.011.

49. Hwang IY, Lee SH, Choi YS, Park SJ, Na JG, Chang IS, et al. Biocatalytic conversion of methane to methanol as a key step for development of methane-based biorefineries. J Microbiol Biotechnol. 2014;24(12):1597-1605. doi:10.4014/jmb.1407.07070.

50. Müller JEN, Meyer F, Litsanov B, Kiefer P, Potthoff E, Heux S, et al. Engineering Escherichia coli for methanol conversion. Metab Eng. 2015 Mar;28:190-201. doi:10.1016/j.ymben.2014.12.008.

51. Whitaker WB, Sandoval NR, Bennett RK, Fast AG, Papoutsakis ET. Synthetic methylotrophy: engineering the production of biofuels and chemicals based on the biology of aerobic methanol utilization. Curr Opin Biotechnol. 2015 Jun;33:165-175. doi:10.1016/j.copbio.2015.01.007.

52. Yurimoto H, Kato N, Sakai Y. Assimilation, dissimilation, and detoxification of formaldehyde, a central metabolic intermediate of methylotrophic metabolism. Chem Rec. 2005;5(6):367-375. doi:10.1002/tcr.20056.

53. Carere CR, McDonald B, Peach HA, Greening C, Gapes DJ, Collet C, et al. Hydrogen oxidation influences glycogen accumulation in a verrucomicrobial methanotroph. Front Microbiol. 2019;10:1873. doi:10.3389/fmicb.2019.01873.

54. Schink B, Schlegel HG. Hydrogen metabolism in aerobic hydrogen-oxidizing bacteria. Biochimie. 1978;60(3):297-305. doi:10.1016/S0300-9084(78)80826-8.

55. Molitor B, Mishra A, Angenent LT. Power-to-protein: converting renewable electric power and carbon dioxide into single cell protein with a two-stage bioprocess. Energy Environ Sci. 2019;12(12):3515-3521. doi:10.1039/c9ee02381j.

56. Bowien B, Schlegel HG. Physiology and biochemistry of aerobic hydrogen-oxidizing bacteria. Annu Rev Microbiol. 1981;35:405-452. doi:10.1146/annurev.mi.35.100181.002201.

57. Pinevich AV. Hydrogen chemolithotrophy [Vodorodnaia khemolitotrofiia]. In: Mikrobiologiia. Biologiia prokariotov [Microbiology. Biology of Prokaryotes]. St. Petersburg: SPbGU; 2007. Vol. 2. p. 153-157. Russian.

58. Vinarov AY, Gordeev LS, Kukharenko AA, Panfilov VI. Fermentatsionnye apparaty dlya protsessov mikrobiologicheskogo sinteza [Fermentation Apparatus for Microbiological Processes]. Moscow: DeLi Print; 2005. 278 p. Russian.

59. Bagchi D. Nutraceuticals and functional foods regulations in the United States and around the world. Toxicology. 2006;221(1):1-3. doi:10.1016/j.tox.2006.01.001.

60. WHO (2017). Avaiable online at: http://www.who.int/mediacentre/factsheets/food-additives/en/

61. U.S. Food and Drug Administration. Generally Recognized as Safe (GRAS) Notifications [Internet]. Silver Spring (MD): FDA; 2010 [cited 2024 Month Day]. Available from: http://www.fda.gov/AnimalVeterinary/Products/AnimalFoodFeeds/GenerallyRecognizedasSafeGRASNotifications/default.htm

62. Foedevarestyrelsen (Danish Veterinary and Food Administration). Liste over anmeldte mikrobielle kulturer [List of notified microbial cultures] [Internet]. Denmark; 2009 [cited 2024 Month Day]. Available from: http://www.foedevarestyrelsen.dk/Foedevarer/Tilsaetningsstoffer_og_teknologi/mikrobielle_kulturer/Anmeldelsesordning/Sider/Anmeldelsesordning.aspx

63. ILSI Europe Novel Food Task Force. The safety assessment of novel foods and concepts to determine their safety in use. Int J Food Sci Nutr. 2003;54 Suppl:S1-32.

64. European Parliament, Council of the European Union. Regulation (EC) No 178/2002 of the European Parliament and of the Council of 28 January 2002 laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety. Off J Eur Union. 2002 Feb 1;L31:1-24

65. European Food Safety Authority. EFSA Scientific Colloquium — Microorganisms in Food and Feed: Qualified Presumption of Safety — 13–14 December 2004, Brussels, Belgium [Internet]. Parma: EFSA; 2005 [cited 2024 Month Day]. Available from: http://www.efsa.europa.eu/en/supporting/pub/109e.htm

66. Mogensen G, Salminen S, O'Brien J, Ouwehand A, Holzapfel W, Shortt C, et al. Food microorganisms — health benefits, safety evaluation and strains with documented history of use in foods. Bull Int Dairy Fed. 2002;377:4-9.

67. Mogensen G, Salminen S, O'Brien J, Ouwehand A, Holzapfel W, Shortt C, et al. Inventory of microorganisms with a documented history of use in food. Bull Int Dairy Fed. 2002;377:10-19

68. Angenent SC, Schuttinga JH, van Efferen MFH, Kuizenga B, van Bree B, van der Krieken RO, et al. Hydrogen oxidizing bacteria as novel protein source for human consumption: an overview. Open Microbiol J. 2022 Nov 8;16:e18742858V16E2207270. doi:10.2174/18742858-v16-e2207270.

69. Rudravaram R, Chandel AK, Rao LV, Hui YZ, Ravindra P. Bio (Single Cell) protein: issues of production, toxins and commercialisation status. In: Ashworth GS, Azevedo PA, editors. Agricultural wastes. New York, NY: Hauppage; 2009. p. 129-153.

70. Steinmann J, Wottge HU, Müller-Ruchholtz W. Immunogenicity testing of food proteins: in vitro and in vivo trials in rats. Int Arch Allergy Immunol. 1990;91:62-65. doi:10.1159/000235091.

71. McAllister CH, Beatty PH, Good AG. Engineering nitrogen use efficient crop plants: the current status. Plant Biotechnol J. 2012;10(9):1011-1025. doi:10.1111/j.1467-7652.2012.00700.x 

72. Jiang G, et al. Biomass extraction using non-chlorinated solvents for biocompatibility improvement of polyhydroxyalkanoates. Polymers (Basel). 2018;10(7):731. doi:10.3390/polym10070731

73. Trevelyan WE. Chemical methods for the reduction of the purine content of the baker's yeast, a form of single cell protein. J Sci Food Agric. 1976;27:225-230. doi:10.1002/jsfa.2740270305.

74. Edelman J, Fewell A, Solomons GL. Myco-protein – a new food. Nutr Abstr Rev Clin Nutr. 1983;53:471-480.

75. Strong PJ, Xie S, Clarke WP. Methane as a resource: can the methanotrophs add value? Environ Sci Technol. 2015;49(7):4001-4018. doi:10.1021/es504242n.

76. Gao H, Xu JG. Method for Measuring Total Nucleotide in Protein Products. China Patent CN104316621A. 2015 Nov 4.

77. Xu JG. Method for Evaluating Destroy Degree on Nucleotide of Determination Method and Production Technology of Free Nucleic Acid Hydrolysate in Protein Product. China Patent CN104515817A. 2015 Oct 22.

78. Sinskey AJ, Tannenbaum SR. Removal of nucleic acids in SCP. In: Tannenbaum SR, Wang DIC, editors. Single cell  Protein II. Cambridge, MA: MIT Press; 1975. p. 158-178.

79. Anderson C, Solomons GL. Primary metabolism and biomass production from Fusarium. In: Moss MO, Smith JE, editors. The Applied Mycology of Fusarium. Cambridge: Cambridge University Press; 1984. p. 231-250.

80. Martinez MC, Sanchez-Montero JM, Sinisterra JV, Ballesteros A. New insolubilized derivatives of ribonuclease and endonuclease for elimination of nucleic acids in single cell protein concentrates. Biotechnol Appl Biochem. 1990;12:643-652.

81. Hameş EE, Demir T. Microbial ribonucleases (RNases): production and application potential. World J Microbiol Biotechnol. 2015;31(11):1853-1862. doi:10.1007/s11274-015-1945-8.

82. Ward PN. Production of Food. United States Patent US5739030A. 1998 Apr 14.

83. Knight N, Roberts G, Shelton D. The thermal stability of Quorn™ pieces. Int J Food Sci Technol. 2001;36(1):47-52. doi:10.1046/j.1365-2621.2001.00424.x.

84. Viikari L, Linko M. Reduction of nucleic acid content of SCP. Process Biochem. 1977;12:35

85. Kerckhof F-M, Sakarika M, Van Giel M, Muys M, Vermeir P, De Vrieze J, et al. From biogas and hydrogen to microbial protein through co-cultivation of methane and hydrogen oxidizing bacteria. Front Bioeng Biotechnol. 2021;9:733753. doi:10.3389/fbioe.2021.733753.

86. Tsapekos P, Khoshnevisan B, Zhu X, Zha X, Angelidaki I. Methane oxidising bacteria to upcycle effluent streams from anaerobic digestion of municipal biowaste. J Environ Manage. 2019;251:109590. doi:10.1016/j.jenvman.2019.109590.

87. Xu M, Zhou H, Yang X, Angelidaki I, Zhang Y. Sulfide restrains the growth of Methylocapsa acidiphila converting renewable biogas to single cell protein. Water Res. 2020;184:116138. doi:10.1016/j.watres.2020.116138.

88. Acosta N, Sakarika M, Kerckhof FM, Law CKY, De Vrieze J, Rabaey K. Microbial protein production from methane via electrochemical biogas upgrading. Chem Eng J. 2020;391:123625. doi:10.1016/j.cej.2019.123625.

89. Gęsicka A, Oleskowicz-Popiel P, Łężyk M. Recent trends in methane to bioproduct conversion by methanotrophs. Biotechnol Adv. 2021;53:107861. doi:10.1016/j.biotechadv.2021.107861.

90. Carboni M, Pio G, Vianello C, Salzano E. Safety distances for the sour biogas in digestion plants. Process Saf Environ Prot. 2021;147:1-7. doi:10.1016/j.psep.2020.09.025.

91. Dee SJ, Hietala DC, Sulmonetti TP. Process hazard considerations for utilization of renewable methane from biogas. Process Saf Prog. 2022;41(4):670-677. doi:10.1002/prs.12389.

92. Salehi R, Chaiprapat S. Conversion of biogas from anaerobic digestion to single cell protein and bio-methanol: mechanism, microorganisms and key factors-A review. Environ Eng Res. 2022;27(4):XXX-YYY. doi:10.4491/eer.2022.092.

93. Erndwein P. Sludge gas leaks: a novel approach for preventing explosions. Prof Saf. 2012;57(8):44-47.

94. Tayou LN, Lauri R, Incocciati E, Pietrangeli B, Majone M, Gottardo M, et al. Acidogenic fermentation of food waste and sewage sludge mixture: effect of operating parameters on process performance and safety aspects. Process Saf Environ Prot. 2022;163:158-166. doi:10.1016/j.psep.2022.05.019.

95. Leung DYC, Caramanna G, Maroto-Valer MM. An overview of current status of carbon dioxide capture and storage technologies. Renew Sustain Energy Rev. 2014;39:426-443. doi:10.1016/j.rser.2014.07.093.

96. Hamer G. Methanotrophy: from the environment to industry and back. Chem Eng J. 2010;160(2):391-397. doi:10.1016/j.cej.2010.04.008.

97. Rasouli Z, Valverde-Pérez B, D’Este M, De Francisci D, Angelidaki I. Nutrient recovery from industrial wastewater as single cell protein by a co-culture of green microalgae and methanotrophs. Biochem Eng J. 2018;134:129-135. doi:10.1016/j.bej.2018.03.010.

98. Leis J, Buttsworth D, Snook C, Holmes G. Detection of potentially explosive methane levels using a solid-state infrared source. IEEE Trans Instrum Meas. 2014;63(12):3088-3095. doi:10.1109/TIM.2014.2327457.

99. Valverde-Pérez B, Xing W, Zachariae AA, Skadborg MM, Kjeldgaard AF, Palomo A, et al. Cultivation of methanotrophic bacteria in a novel bubble-free membrane bioreactor for microbial protein production. Bioresour Technol. 2020;310:123388. doi:10.1016/j.biortech.2020.123388. 

100. Tanaka K, Ishizaki A, Kanamaru T, Kawano T. Production of poly(D-3-hydroxybutyrate) from CO₂, H₂, and O₂ by high cell density autotrophic cultivation of Alcaligenes eutrophus. Biotechnol Bioeng. 1995;45(3):268-275. doi:10.1002/bit.260450312.

101. Ishizaki A, Tanaka K. Batch culture of Alcaligenes eutrophus ATCC 17697T using recycled gas closed circuit culture system. J Ferment Bioeng. 1990;69(3):170-174.

102. Yu J, Munasinghe P. Gas fermentation enhancement for chemolithotrophic growth of Cupriavidus necator on carbon dioxide. Fermentation. 2018;4(3):63. doi:10.3390/fermentation4030063.

103. Volova TG, Okladnikov YuN, Sid'ko FYa, Terskov IA, Trubachev IN, Fedorova YaV. Proizvodstvo belka na vodorode [Production of protein on hydrogen]. Novosibirsk: Nauka; 1981. 168 p.

104. Blinov NP. Osnovy biotekhnologii [Fundamentals of Biotechnology]. St Petersburg: Nauka; 1995. 600 p.

105. Kochetkov VM, Gaganov IS, Kochetkov VV, Nyunkov PA. Technology and implementation of fermentative units for bioprotein production from natural gas. Russian Journal of Biopharmaceuticals. 2023;18(3):230-242. doi:10.32362/2410-6593-2023-18-3-230-242.

106. Bykov VA, Manakov MN, Panfilov VI, Svittsov AA, Tarasova NV. Proizvodstvo belkovykh veshchestv: Biotekhnologiya. V 8 kn. Kn. 1 [Production of protein substances: Biotechnology. In 8 books. Book 1] / Pod red. N.S. Egorova, V.D. Samuilova. Moscow: Vysshaya shkola; 1987. 

107. Pitkänen J-P, Vainikka P. Methods and systems for growing microbial mass. Australian Patent AU2022247353B2. 2023 Oct 12.

108. Pitkänen J, Vainikka P. Bioreactors for growing micro-organisms. Australian Patent AU2020317552B2. 2022 Dec 22.

109. Dalla-Betta P, Reed JS. Method and apparatus for growing microbial cultures that require gaseous electron donors, electron acceptors, carbon sources, or other nutrients. United States Patent US10696941B2. 2020 Jun 30. 

110. Larsen EB. U-shape and/or nozzle u-loop fermentor and method of carrying out a fermentation process. United States Patent US6492135B1. 2002 Dec 10.

111. Petersen LAH, Villadsen J, Jørgensen SB, Gernaey KV. Mixing and mass transfer in a pilot scale U-loop bioreactor. Biotechnol Bioeng. 2017;114(2):344-354. doi:10.1002/bit.26084.

112. Xu F, Guan X, Yang N, Lackner M. Influence of Liquid Level Height on Flow Characteristics and Mixing Efficiency in a Loop Bioreactor. J Chem Eng Jpn. 2025;58(1). doi:10.1080/00219592.2025.2525541.

113. Drejer A, Ritschel T, Jørgensen SB, Jørgensen JB. Economic Optimizing Control for Single cell  Protein Production in a U-Loop Reactor. In: Espuña A, Graells M, Puigjaner L, editors. Computer Aided Chemical Engineering. Vol. 40. Elsevier; 2017. p. 1759-1764. doi:10.1016/B978-0-444-63965-3.50295-6.

114. Petersen LAH, Bequette BW, Jørgensen SB, Villadsen J, Christensen I, Gernaey KV. Modeling and system identification of an unconventional bioreactor used for single cell protein production. Chem Eng J. 2020;390:124438. doi:10.1016/j.cej.2020.124438.

115. Lalova MV, Mirkhin MG, Naidin AV, Safonov AI, Baburchenkova OA. Fermentation installation for methane-assimilating microorganisms. Russian Patent RU2580646C1. 2016 Apr 10.

116. Carstensen F, Apel A, Wessling M. In situ product recovery: submerged membranes vs. external loop membranes. J Memb Sci. 2012;394-395:1-36. doi:10.1016/j.memsci.2011.11.029.

117. Liao BQ, Kraemer JT, Bagley DM. Anaerobic Membrane Bioreactors: Applications and Research Directions. Crit Rev Environ Sci Technol. 2006;36(6):489-530. doi:10.1080/10643380600678146.

118. Judd S, Judd C. The MBR Book: Principles and Applications of Membrane Bioreactors for Water and Wastewater Treatment. 2nd ed. Oxford: Butterworth-Heinemann; 2011.

119. Pourbafrani M, Talebnia F, Niklasson C, Taherzadeh MJ. Protective Effect of Encapsulation in Fermentation of Limonene-contained Media and Orange Peel Hydrolyzate. Int J Mol Sci. 2007;8(8):777-787.doi:10.3390/i8080777.

120. Ylitervo P, Akinbomi J, Taherzadeh MJ. Membrane bioreactors’ potential for ethanol and biogas production: a review. Environ Technol. 2013;34(13-14):1711-1723. doi:10.1080/09593330.2013.813559.

121. Sárvári Horváth I, Tabatabaei M, Karimi K, Kumar R. Recent updates on biogas production - a review. Biofuel Res J. 2016;3(2):394-402. doi:10.18331/BRJ2016.3.2.4.

122. Chang HN, Yoo IK, Kim BS. High density cell culture by membrane-based cell recycle. Biotechnol Adv. 1994;12(3):467-487. doi:10.1016/0734-9750(94)90020-5.

123. Jaibiba P, Vignesh SN, Hariharan S. Chapter 10 - Working principle of typical bioreactors. In: Singh L, Yousuf A, Mahapatra DM, editors. Bioreactors. Elsevier; 2020. p. 145-173. doi:10.1016/B978-0-12-821264-6.00010-3.

124. Ma Y, Liu T, Yuan Z, Guo J. Microbial conversion of methane into single cell protein in a dual-membrane biofilm reactor. Water Res. 2025;283:123838. doi:10.1016/j.watres.2025.123838.

125. Rittmann BE. The membrane biofilm reactor: the natural partnership of membranes and biofilm. Water Sci Technol. 2006;53(3):219-225. doi:10.2166/wst.2006.096.

126. Chen H, Zhao L, Hu S, Yuan Z, Guo J. High-rate production of short-chain fatty acids from methane in a mixed-culture membrane biofilm reactor. Environ Sci Technol Lett. 2018;5(11):662-667. doi:10.1021/acs.estlett.8b00460.

127. Kim KJ, Choi SW, Kim MY, Lee MS, Kim YS, Kim HS. Fabrication characteristics of SOFC single cell with thin LSGM electrolyte via tape-casting and co-sintering. J Ind Eng Chem. 2016;42:69-74. doi:10.1016/j.jiec.2016.07.041.

128. Ma Y, Liu T, Yuan Z, Guo J. Single cell protein production from methane in a gas-delivery membrane bioreactor. Water Res. 2024;259:121820. doi:10.1016/j.watres.2024.121820.

129. Chen H, Luo J, Liu S, Yuan Z, Guo J. Microbial Methane Conversion to Short-Chain Fatty Acids Using Various Electron Acceptors in Membrane Biofilm Reactors. Environ Sci Technol. 2019;53(21):12846-12855. doi:10.1021/acs.est.8b06767. 

130. Orgill JJ, Atiyeh HK, Devarapalli M, Phillips JR, Lewis RS, Huhnke RL. A comparison of mass transfer coefficients between trickle-bed, hollow fiber membrane and stirred tank reactors. Bioresour Technol. 2013;133:340-346. doi:10.1016/j.biortech.2013.01.124.

131. Villadsen J, Nielsen J, Lidén G. Bioreaction Engineering Principles. 3rd ed. New York: Springer; 2011. doi:10.1007/978-1-4419-9688-6.

132. Yasin M, Jeong Y, Park S, Jeong J, Lee EY, Lovitt RW, et al. Microbial synthesis gas utilization and ways to resolve kinetic and mass-transfer limitations. Bioresour Technol. 2015;177:361-374. doi:10.1016/j.biortech.2014.11.022.

133. Kelechi M, Ukaegbu-Obi KM. Single Cell Protein: A Resort to Global Protein Challenge and Waste Management. J Microbiol Microb Technol. 2016;1:5.

134. Bhalla T, Sharma NN, Sharma M. Production of Metabolites, Industrial enzymes, Amino acid, Organic acids, Antibiotics, Vitamins and Single Cell Proteins. J Environ. 2007;6:34-78.

135. Jamal P, Alam M, Salleh N. Media optimization for bioproteins productions from cheaper carbon source. J Eng Sci Technol. 2008;3(2):124-130.

136. Mahajan A, Dua S. A perspective on biotechnological potential. J Food Sci Technol. 1995;32:162-165.

137. McEvoy LA, Navarro JC, Hontoria F, Amat F, Sargent JR. Two novel Artemia enrichment diets containing polar lipid. Aquaculture. 1996;144(4):339-352. doi:10.1016/0044-8486(96)01325-7.

138. Olvera-Novoa MA, Martínez-Palacios CA, Olivera-Castillo L. Utilization of torula yeast (Candida utilis) as a protein source in diets for tilapia (Oreochromis mossambicus Peters) fry. Aquac Nutr. 2002;8(4):257-264.

139. Zhang F, Zhang W, Qian DK, Dai K, van Loosdrecht MCM, Zeng RJ. Synergetic alginate conversion by a microbial consortium of hydrolytic bacteria and methanogens. Water Res. 2019;163:114892. doi:10.1016/j.watres.2019.114892.

140. Pieja AJ, Morse MC, Cal AJ. Methane to bioproducts: the future of the bioeconomy? Curr Opin Chem Biol. 2017;41:123-131. doi:10.1016/j.cbpa.2017.10.024.

141. Hanson RS, Hanson TE. Methanotrophic bacteria. Microbiol Mol Biol Rev. 1996;60(2):439-471. doi:10.1128/mmbr.60.2.439-471.1996.

142. Semrau JD, Dispirito AA, Yoon S. Methanotrophs and copper. FEMS Microbiol Rev. 2010;34(4):496-531. doi:10.1111/j.1574-6976.2010.00212.x.

143. . Zheng Y, Wang H, Yu Z, Haroon F, Hernández ME, Chistoserdova L. Metagenomic insight into environmentally challenged methane-fed microbial communities. Microorganisms. 2020;8:1614. doi:10.3390/microorganisms8101614.

144. Areniello M, Matassa S, Esposito G, Lens PNL. Microbial protein production from sulfide-rich biogas through an enrichment of methane- and sulfur-oxidizing bacteria. Bioresour Technol. 2023;383:129237. doi:10.1016/j.biortech.2023.129237.

145. Lee J, Yasin M, Park S, Chang IS, Ha KS, Lee EY, et al. Gas-liquid mass transfer coefficient of methane in a bubble column reactor. Korean J Chem Eng. 2015;32:1060-1063. doi:10.1007/s11814-014-0341-7.

146. Dolan SK, Kohlstedt M, Trigg S, Ramirez PV, Kaminski CF, Wittmann C, et al. Contextual flexibility in Pseudomonas aeruginosa central carbon metabolism during growth in single carbon sources. mBio. 2020;11(2):e02684-19. doi:10.1128/mbio.02684-19.

147. Sekiguchi Y, Hanada S. Caldilineaceae. In: Bergey’s Manual of Systematics of Archaea and Bacteria. John Wiley & Sons, Ltd; 2020. p. 1-3. doi:10.1002/9781118960608.fbm00320.

148. Maire J, Collingro A, Horn M, van Oppen MJH. Chlamydiae in corals: shared functional potential despite broad taxonomic diversity. ISME Commun. 2024;4(1):ycae054. doi:10.1093/ismeco/ycae054.

149. Khoshnevisan B, Dodds M, Tsapekos P, Torresi E, Smets BF, Angelidaki I, et al. Coupling electrochemical ammonia extraction and cultivation of methane oxidizing bacteria for production of microbial protein. J Environ Manage. 2020;265:110560. doi:10.1016/j.jenvman.2020.110560.

150. Zheng R, Cai R, Wang C, Liu R, Sun C. Characterization of the first cultured representative of "Candidatus Thermofonsia" Clade 2 within Chloroflexi reveals its phototrophic lifestyle. mBio. 2022;13(2):e00287-22. doi:10.1128/mbio.00287-22.

151. Sigalevich P, Meshorer E, Helman Y, Cohen Y. Transition from anaerobic to aerobic growth conditions for the sulfate-reducing bacterium Desulfovibrio oxyclinae results in flocculation. Appl Environ Microbiol. 2000;66(11):5005-5012. doi:10.1128/AEM.66.11.5005-5012.2000.

152. Arcangeli JP, Arvin E. Modelling the growth of a methanotrophic biofilm: estimation of parameters and variability. Biodegradation. 1999;10:177-191. doi:10.1023/A:1008317906069.

153. Modin O, Fukushi K, Nakajima F, Yamamoto K. Aerobic methane oxidation coupled to denitrification: kinetics and effect of oxygen supply. J Environ Eng. 2010;136(2):211-219. doi:10.1061/(ASCE)EE.1943-7870.0000134.

154. van Bodegom P, Stams F, Mollema L, Boeke S, Leffelaar P. Methane oxidation and the competition for oxygen in the rice rhizosphere. Appl Environ Microbiol. 2001;67(8):3586-3597. doi:10.1128/AEM.67.8.3586-3597.2001.

155. Rajoka MI, Ahmed S, Hashmi AS, et al. Production of microbial biomass protein from mixed substrates by sequential culture fermentation of Candida utilis and Brevibacterium lactofermentum. Ann Microbiol. 2012;62:1173-1179. doi:10.1007/s13213-011-0357-8.

156. Matassa S, Verstraete W, Pikaar I, Boon N. Autotrophic nitrogen assimilation and carbon capture for microbial protein production by a novel enrichment of hydrogen-oxidizing bacteria. Water Res. 2016;101:137-146. doi:10.1016/j.watres.2016.05.077.

157. Bothe H, Møller Jensen K, Mergel A, et al. Heterotrophic bacteria growing in association with Methylococcus capsulatus (Bath) in a single cell protein production process. Appl Microbiol Biotechnol. 2002;59:33-39. doi:10.1007/s00253-002-0964-1.

158. Nunes JJ, et al. Enhanced production of single cell protein from M. capsulatus (Bath) growing in mixed culture. J Microbiol Biotechnol Food Sci. 2016;6(3):894-899. doi:10.15414/jmbfs.2016/17.6.3.894-899. 

159. Leila M, Loiseau K, Moretti I. Controls on generation and accumulation of blended gases (CH4/H2/He) in the Neoproterozoic Amadeus Basin, Australia. Mar Pet Geol. 2022;140:105643. doi:10.1016/j.marpetgeo.2022.105643.

160. Deville E, Prinzhofer A. The origin of N2-H2-CH4-rich natural gas seepages in ophiolitic context: A major and noble gases study of fluid seepages in New Caledonia. Chem Geol. 2016;440:139-147. doi:10.1016/j.chemgeo.2016.06.011.

161. Charlou JL, Donval JP, Fouquet Y, Jean-Baptiste P, Holm N. Geochemistry of high H2 and CH4 vent fluids issuing from ultramafic rocks at the Rainbow hydrothermal field (36°14'N, MAR). Chem Geol. 2002;191(4):345-359. doi:10.1016/S0009-2541(02)00134-1.

162. Altalbawy FMA, Al-saray MJ, Vaghela K, Alruwaili M, Aftab A, Mahmoud M, et al. Machine-learning based prediction of hydrogen/methane mixture solubility in brine. Sci Rep. 2024;14:30227. doi:10.1038/s41598-024-80959-1.

163. Zanella E, Longhi M, Tondelli G, Minguzzi A, Comite A, Vertova A, et al. Separation of hydrogen-methane mixtures by electrochemical hydrogen compressor: Experimental and modelling investigation on the influence of different Nafion membranes and operative conditions. Int J Hydrogen Energy. 2025;126:439-449. doi:10.1016/j.ijhydene.2025.04.014.

164. Khosravi-Darani K, Mokhtari ZB, Amai T, Tanaka K. Microbial production of poly(hydroxybutyrate) from C1 carbon sources. Appl Microbiol Biotechnol. 2013;97:1407-1424. doi:10.1007/s00253-012-4649-0.