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, North Cyprus

Date: January 2025

Abstract: Currently, there is a growing consumer demand for alternative protein that is not of animal origin but has similar properties. This demand is due to the fact that agriculture cannot fully meet the needs of the ever-growing world population with sufficient protein, and the fact that the expansion of agricultural land through deforestation has a harmful impact on the environment.

Today, alternative protein is produced from plants, fungi, yeast, algae and even insects, but the most promising solution is the production of protein products by microorganisms from carbon dioxide and hydrogen. This method of protein production does not require a large area and special conditions for farming, does not require large amounts of water for irrigation and pesticide treatment, thus avoiding environmental damage in the form of wastewater pollution or greenhouse gas emissions.

Protein obtained in this way is called single-cell protein (SCP) and has great advantages compared to proteins of plant origin. The protein content in the biomass of microorganisms reaches 70%, and the composition of amino acids is comparable to chicken eggs, in addition to protein it contains other nutrients, vitamins, minerals and fatty acids.

Today SCP is produced by different groups of microorganisms on different substrates. In this article, we have considered microorganisms that synthesise protein from carbon dioxide and hydrogen, which can be obtained by various means such as water electrolysis, natural gas, biogas, waste gas from industrial processes or landfill gas, etc.

Key Words: Protein production, Protein, Single-cell protein (SCP), Hydrogen, Carbon dioxide, Microorganisms, Bacteria, Cellular agriculture, Alternative protein

DOI: 10.13140/RG.2.2.10503.92321

Food Protein produced by microorganisms using hydrogen and carbon dioxide as a carbon source

Introduction

According to FAO estimates, by 2050, per capita food demand will increase by more than 60% compared to the current situation. The increase in demand is due to demographic changes and rising global wealth. Increased demand for food leads to a deficit in livestock products. Also, the deficit in livestock products is associated with other factors, such as land degradation, lack of fresh water, overfishing and global warming. The expansion of agricultural land to eliminate the deficit has a harmful effect on the environment due to deforestation and pollution of land and water resources with mineral fertilizers. Reducing or eliminating human consumption of animal products leads to a shortage of vital elements in the diet, the main ones of which are proteins.

Proteins consist of 20 amino acids, they are called proteinogenic. Combinations of these amino acids determine a wide variety of protein properties. These amino acids can enter the human body only with the consumption of protein consisting of these amino acids, some of which are essential. Essential amino acids are not synthesized in the human body, or are synthesized at an insufficient rate. Essential amino acids perform a number of important functions in the human body [1]: 

• Participate in the formation of other amino acids [1];

• Are part of various natural compounds - coenzymes, bile acids, antibiotics [1];

• Participate in the formation of hormones, mediators and neurotransmitters [1]

• Are sources of metabolites involved in metabolism [1]. 

Animal proteins contain essential amino acids in full. It is currently impossible to fully replenish their deficiency using proteins of other origins.

Thus, in order to support the ever-growing population of the planet and reduce the impact of humanity on the environment, alternative and more environmentally friendly methods of obtaining food products are needed [2], namely proteins that in their characteristics fully correspond to proteins of animal origin and are suitable for consumption in the human diet. This is one of the main tasks of technical progress. 

Cellular agriculture

Cellular agriculture is the production of agricultural products using animal or plant cell cultures or microbes instead of using animals or plants. The term cellular agriculture can be confusing because it does not refer to agriculture, but rather to an alternative to agriculture [3]. In other words, cellular agriculture is the production of alternative proteins, i.e. proteins that are not of animal origin. Alternative protein is already present in the human diet and is obtained from a variety of sources.

The most popular protein today is plant protein, which is obtained from plants (legumes, cereals, oilseeds). Plant protein is a copy of meat and dairy products created from plant-based ingredients [4]. Plant protein is a good alternative in terms of nutritional value, but has a lower degree of absorption than animal proteins and requires arable land and water.

Another alternative protein source is insects, a protein derived from insects called Entomoprotein. Insects are widely consumed by people of all ages in about 80 countries in Asia, Africa, and the Americas. Insects are nutritious, with the average protein content of insects being about 40% [5, 6, 7]. Insect protein is more digestible than plant proteins, but slightly less digestible than animal protein [8, 9]. It is important to note that insect proteins contain potential allergens, which is an important factor in the rejection of the widespread use of Entomoprotein.

Another source of alternative protein is cultured meat, i.e. meat that has been artificially grown in a test tube from several cells of “classic” meat. Cultured meat has high nutritional value, but today, mass production of meat requires complex technical solutions, such as reproducing the exact structure, texture, color, taste, simulating the role of blood in delivering oxygen and nutrients, as well as co-cultivation of fat, muscle and connective tissues, which have not yet been developed. Another disadvantage of this technology is that animals that undergo biopsy are used to produce cultured meat, which causes them certain suffering and causes discontent among animal rights activists [10, 11].

Alternative proteins also include single-cell protein, which is produced by single-celled microorganisms, including bacteria, fungi, and yeast, as well as algae.

The protein from fungi is called mycoprotein, which is incorrectly classified as a plant protein, which is by definition incorrect since the kingdom of fungi is separate from the animal and plant kingdoms. Mycoprotein has an excellent amino acid profile and digestibility comparable to beef and soy [12]. Disadvantages include a slower growth rate and lower protein content [13]. These production organisms are grown using starch-derived glucose as a carbon source, and therefore ultimately rely on agriculture for their microbial biomass production [9, 14]

Another disadvantage of mycoprotein is that some fungi produce mycotoxins [15, 16]. The effects of fungal toxins range from allergic reactions to carcinogenesis and death [10].

Yeast protein, also known as feed yeast. Some sources also classify it as mycoprotein, since the term “yeast” does not have a taxonomic status and is classified as a fungi [17]. The disadvantages of yeast protein, as well as mycoprotein, include a lower growth rate and lower protein content (from 45 to 65%), as well as a lower methionine content, compared to protein produced from bacteria [13].

Proteins derived from algae, which include both macroalgae (e.g. seaweed) and microalgae (e.g. spirulina and chlorella), are included in the human diet. Algae are a good alternative to animal protein in terms of amino acid composition. However, algae production is mainly carried out outdoors in open ponds, which exposes them to pollution (not only biological but also mineral, which affects the quality of the final product) and makes production dependent on weather conditions [18]. Algae cultivation is based on photosynthesis, whether in open ponds or bioreactors, consists of limited light penetration into the growing medium, which requires large surface areas to effectively capture light energy [9, 19]. Another disadvantage is that algae accumulate heavy metals [13].

Single-cell protein obtained from bacterial biomass has a number of advantages over other proteins produced by single-cell microorganisms. Various raw materials can be used to grow bacteria, such as by-products and waste from agriculture and the food industry, as well as abundantly available compounds such as methane or carbon dioxide [3]. Bacterial SCP contains up to 80% protein, as well as essential vitamins, amino acids, minerals and lipids [20, 21]. It is worth noting that the amino acid composition is the main advantage of SCP produced using bacteria.

In our paper, we would like to take a detailed look at the production of alternative proteins from hydrogen-oxidizing bacterial biomass, which can use hydrogen as an electron donor and convert carbon dioxide into high-protein microbial biomass [22].

The prospects for the development of hydrogen-oxidizing bacteria compared to other known protein producers are due to their autotrophy, i.e. independence from scarce organic resources, rapid growth (doubling time 2.0–2.5 h), high content of complete amino acid protein (up to 60–70%), the absence of extracellular intermediate products of organic metabolism (the only by-product of hydrogen oxidation is water), high environmental friendliness of the product production process, as well as the ability to grow on hydrogen of various origins, including on products of carbon fuel processing [23]. Also, hydrogen-oxidizing bacteria that assimilate carbon dioxide simultaneously with aerobic oxidation of hydrogen are of considerable interest for the production of bacterial food, since carbon dioxide can be captured from the air or industrial exhaust gases (flue gases) using absorbents with subsequent desorption [9, 24] and thereby reduce carbon dioxide emissions

World experience in producing SCP from hydrogen 

In recent decades the production of single-cell protein from hydrogen suitable for human consumption has attracted increasing interest [25, 26]. Today, several companies in different countries are already engaged in this, for example [22, 26, 27]:

In Finland, SolarFoods has launched the production of a protein called Solein. It is the cultivation of a strain of bacteria of the genus Xanthobacter tagetidis or VTT-E-193585 (type strain Xanthobacter tagetidis TagT2C) in continuous culture using hydrogen as an energy source and an inorganic carbon source, where the inorganic carbon source is carbon dioxide [28, 29].

In the US, Air Protein Inc. has launched production of the eponymous Air Protein protein. Air Protein states that the elements of air, water, and energy are used to grow the protein. The elements of air include carbon dioxide, oxygen, and nitrogen, water, and energy is renewable hydrogen obtained through electrolysis. Chemotrophic microorganisms are used for production [30, 31].

Another US company, NovoNutrients, has found a way to recycle industrial carbon dioxide waste and turn it into high-quality protein for humans and animals. The process uses hydrogen and natural microorganisms that convert carbon dioxide into alternative protein products. NovoNutrients has expressed interest in building a plant to produce SCP from hydrogen with a capacity of about 100,000 tons per [22, 32, 33].

Deep Branch Biotechnology Ltd. of Nottingham, UK, developed a technology that converts carbon dioxide into an eco-friendly protein called Proton. The technology has now been acquired by Aerbio. Proton requires not only carbon dioxide, but also hydrogen and oxygen.

The single-cell protein, produced using hydrogen-oxidising bacteria, could potentially be used as an ingredient in products such as bread, pasta, meat and plant-based dairy products, as well as a protein supplement similar to whey protein, meaning it could be consumed in a variety of forms, such as meat products and drinks or broths [22]. The widespread use of such a protein is due to its high biological value.

Biological value of SCP from hydrogen and carbon dioxide

One of the main indicators of the biological value of microbial biomass is the total protein and amino acid content, including essential amino acids [23]. The protein content of hydrogen-oxidizing bacteria biomass and its quality in terms of essential amino acid content vary and depend not only on the microbial species and substrate type, but also on the cell growth stage, nutrient sources and environmental conditions [34, 35, 36]. In general, microbes are considered as sources of high-quality protein, as they are able to produce essential amino acids in amounts close to the FAO/WHO reference value of 40% [37].

In the work [23] the amino acid composition of the biomass of three hydrogen-oxidizing bacteria Alcaligenes eutrophus Z1 (type strain Wautersia eutropha 335) and Ralstonia eutropha B5786 (type strain Wautersia eutropha 335) and the CO-resistant strain of carboxydobacterium Seliberia carboxydohydrogena Z1062 was investigated. The bacteria were cultivated under strictly sterile conditions using an automatic fermentation system.

A similar study was conducted in another paper [26], which investigated the microbial production of food products by gas fermentation of two Gram-positive autotrophic strains of hydrogen-oxidizing bacteria Nocardioides nitrophenolicus KGS-27 (type strain Nocardioides nitrophenolicus DSM 15529) and Rhodococcus opacus DSM 43205 (type strain Rhodococcus opacus 1b) bacteria. The study used two cultivation modes in a bioelectrochemical system (BES) or a shake flask (SF). The cells grown in the two modes were very similar in terms of protein content. In both strains studied, the differences in the relative amounts of amino acids between the two cultivation modes were mostly minor (less than 10%) [26].

Figure 1 shows a comparison of the amino acid composition of proteins of the above strains with traditional food animal proteins (casein), plant proteins (soy protein) and protein from Сhlorella algae [38]. For the strains of hydrogen-oxidizing bacteria Nocardioides nitrophenolicus KGS-27 (type strain Nocardioides nitrophenolicus DSM 15529) and Rhodococcus opacus DSM 43205 (type strain Rhodococcus opacus 1b), data obtained during cultivation in a shaker flask (SF) are presented [26]. Figure 1 also shows the provisional amino acids pattern recommended by FAO/WHO.

This study showed that the proteins of all the strains mentioned have a complete balanced amino acid composition, and it is also clear that the proteins of hydrogen-oxidizing bacteria are close in composition and distribution of amino acids to casein, a protein of animal origin [23, 26].

This indicates a high biological value of the proteins studied and indicates the potential food suitability of protein from the biomass of hydrogen-oxidizing bacteria as a main source of protein and essential amino acids.

Carbon and energy sources of SCP microorganisms

Autotrophic cultivation of hydrogen-oxidizing bacteria requires an energy source and a carbon source, the energy source is hydrogen, and the carbon source is carbon dioxide [26]. The scheme of cultivation of hydrogen-oxidizing bacteria is shown in Figure 2, which shows the energy source and the carbon source. 

Carbon dioxide supplied to the bioreactor can come from a variety of sources, including: 

Flue gases from large point sources such as power plants and energy-intensive emission sources such as cement kilns. In this approach, carbon dioxide is first captured from the flue/fuel gases, separated from the sorbent, transported, and then can be used in industry [39, 40].

Fermentation by-products, such as in the case of hydrogen production from gasification, sufficient carbon dioxide produced from [22, 41].

Atmospheric air: Ambient air is passed through a solid or liquid medium in which carbon dioxide molecules are trapped. when energy is supplied to the medium, concentrated carbon dioxide is released, allowing it to be collected, stored, and used [40]. According [42], the efficiency of converting electricity into biomass decreased from 54% to 20% when atmospheric air was supplied to the reactor instead of 100% carbon dioxide [40]. However, it has recently been shown that SCP production using photovoltaics from carbon dioxide captured from the air is superior to crop cultivation in terms of protein yield per unit area [3, 43]

The latter source of carbon dioxide production is of particular interest because it can reduce greenhouse gas emissions throughout the entire production process. Since carbon dioxide capture and storage is considered a key strategy for achieving zero carbon dioxide emissions [39].

The hydrogen needed to reduce carbon dioxide emissions can be produced by electrolysis of water [3, 40]. It should be noted that the production of hydrogen by electrolysis, whether on-site or in a separate installation, requires electricity to power the metabolism of hydrogen-oxidizing bacteria[22], which is relatively energy intensive (Approximately 90% of the energy consumed in SCP production is spent on the electrolysis process [40, 44]. It has been calculated that if all the hydrogen needed by bacteria were produced by electrolysis using polymer electrolyte membranes, 537–902 GW of energy would be required to produce 175–307 million tons of bacterial mass with 70% protein content. This indicates that renewable energy, such as wind or solar power, is needed to ensure sustainability of such processes [26, 45]. Energy costs can also be reduced by using other technologies or another source of hydrogen, such as natural hydrogen extracted from the earth.

There are several other ways to obtain hydrogen, in particular: steam reforming of methane, coal gasification, decomposition of organic matter, etc. You can read more about them in the article [46].

In the production of hydrogen based on coal gasification, a significant portion of the resulting carbon dioxide can be used as a carbon source for the production of SCP [22].

Steam methane reforming is not a viable option as a hydrogen source for SCP production due to the existence of microbes known as methane-oxidizing bacteria that can produce SCP directly from methane without the need to convert it to hydrogen or supply an external carbon source, as discussed in the paper [22].

Limitations in the use of hydrogen-oxidizing bacteria

Despite the high growth rate, low resource requirements, high protein content, and complete composition of essential amino acids, the bacterial biomass of hydrogen-oxidizing microorganisms still has some drawbacks that limit its wider application. These limiting factors are related to the subsequent processes after cultivation. For example, it is necessary to ensure that the selected bacteria are not pathogenic to humans or animals and do not contain toxic compounds. The cytotoxic and genotoxic properties of the cell masses should be studied before use as food [26]. Another challenge is to ensure sterile production conditions, since contaminating microorganisms usually grow well in the nutrient medium [3, 40, 47, 48, 49].

Another common problem associated with edible microbial biomass is its high nucleic acid content (7–12%) [3], due to the high rate of cell proliferation. Nucleic acids consist of pyrimidine and purine nucleotides. The metabolic product of purines contained in food is uric acid, the accumulation of which can eventually lead to gout or kidney stones. However, the nucleic acid content can be effectively reduced, for example, by heat treatment [3, 26].

Some hydrogen-oxidizing bacteria species have a Gram-negative cell wall that typically contains lipopolysaccharide endotoxins as major components [50]. Bacterial lipopolysaccharides have been linked to diabetes, liver damage, neurological disorders, and chronic intestinal inflammation [51]. Exposure to lipopolysaccharides is also considered an occupational hazard in the production of Gram-negative bacterial feed [52] Gram-positive hydrogen-oxidizing bacteria appear to be more suitable production organisms because they are naturally lipopolysaccharide-free and therefore potentially more suitable for food production [26].

Discussion

Currently, the alternative protein from hydrogen-oxidizing bacteria biomass is not widely used, but the research conducted to date and the production experience of some companies around the world indicate its great potential. This technology should be considered as a promising tool for the sustainable development of cellular agriculture. For further advancement, several issues need to be explored:

• Conduct research to ensure the safety of the food product in the human diet, taking into account the nutritional value and post-processing requirements of alternative sources of feed protein.

• Conduct a comprehensive environmental assessment. Although one of the methods of producing protein involves capturing carbon dioxide from the air, which helps to reduce it in the atmosphere, it is also of interest to study greenhouse gas emissions during the entire production process.

• Assess the extent to which bacterial protein can replace animal protein and the most common plant-based alternative protein today.

Conclusion

Growing microbial biomass rich in proteins and other nutrients can play an important role in ensuring food security while reducing the negative impact of agriculture on the environment. Microorganism products can significantly help in providing food for a growing population and allocating limited land resources in the future, because microorganisms are able to assimilate large amounts of chemicals and synthesize biomass of high biological value [23].

In our work, we have considered various sources of alternative protein that are produced today. We have considered the advantages of protein production technology using hydrogen-oxidizing bacteria. Currently, there is a significant increase in activity in this direction. Various hydrogen-oxidizing bacteria are intensively studied as protein producers, such as Alcaligenes eutrophus (type strain Wautersia eutropha 335), Seliberia carboxydohydrogena, Ralstonia eutropha (type strain Wautersia eutropha 335), Rhodococcus opacus (type strain Rhodococcus opacus 1b) and Nocardioides nitrophenolicus (type strain Nocardioides nitrophenolicus DSM 15529). In our article, using two studies conducted earlier as an example, we have considered the composition of essential amino acids for various strains of hydrogen-oxidizing bacteria and compared it with the amino acid composition of other types of proteins.

We have looked at the energy sources and carbon sources needed to grow hydrogen-oxidizing bacteria. We have provided an overview of companies from around the world that are currently producing protein from hydrogen and carbon dioxide.

We reviewed some factors that impose certain limitations on the widespread use of hydrogen-oxidizing bacteria and which require further research.

Overall, it can be concluded that the technology of producing single-cell protein using hydrogen and carbon dioxide should be considered a promising tool for cellular agriculture. The unique ability of autotrophic hydrogen-oxidizing bacteria to grow rapidly on various substrates and the possibility of reducing environmental impact by capturing carbon dioxide from the air and restoring valuable resources is an urgent and necessary task for sustainable development.

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