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

Autor: Georgii Feodoridi

Place: HYDITEX CORPORATION, North Cyprus

Date: December 2024

Abstract: In the last few decades, the world has been facing food shortage for the ever increasing world population. To solve this problem, various biotechnological methods have been developed to produce alternative food protein of non-animal origin. The alternative protein is suitable for human and animal consumption and exactly mimics the properties of conventional protein.

There are several ways to produce alternative food protein, among which it is worth highlighting the protein produced by microorganisms that feed on hydrocarbons, this protein is also called single-cell protein (SCP).  The advantages of this method of protein production are enormous: it is an environmentally friendly product, its production does not require large areas and does not depend on climatic conditions, microorganisms can be grown all year round, which eliminates problems with storage and spoilage of the product. According to some data, the share of protein in microbial biomass is about 70%, its composition of amino acids is close to milk, it is enriched with vitamins and microelements, easily and completely digested.

In this article we have considered microorganisms that use methane as a carbon source. Methane as a carbon source has a number of advantages, such as large reserves of natural gas, good transportability, and the possibility of obtaining a finished product without additional purification from the substrate. 

 The protein produced from methane is also called Bioprotein or Gaprin. This paper also gives an overview of the different sources of methane that can be used for protein production such as associated petroleum gas, coal mine methane and biogas.

Key Words: Protein production, Protein, Single-cell protein (SCP), Bioprotein, Natural gas, Methane, Microorganisms, Bacteria

DOI: 10.13140/RG.2.2.27668.31364

Production of food protein by microorganisms from methane.

Introduction

Food shortages are the most important problem that humanity will face in the 21st century due to the constant growth of the population and the resulting increase in the consumption of animal products. The expansion of agriculture cannot be a solution to food problems, since agricultural lands already occupy more than a third of the land surface [1]. and have a significant impact on the environment, since traditional agriculture pollutes land and water resources with mineral fertilizers.

The shortage of meat and dairy protein requires changes in the food system that go beyond traditional agriculture, namely the search for new sources of dietary protein that are fully consistent in their characteristics with animal protein.

Protein is the main building material for the human body, without it growth, development and life activity in general are impossible. The nutritional value of protein is determined by its amino acid composition, it usually contains 20 amino acids, some of them, for example, phenylalanine, valine, threonine, tryptophan, methionine, leucine, isoleucine, lysine and histidine, also useful arginine, cysteine, glycine, glutamine, proline and tyrosine cannot be synthesized by humans or animals, therefore they are essential and must be supplied to the body with food [2, 3].

Today, the main sources of protein are meat, fish, dairy products and eggs. But there are also alternative sources of protein, or in other words, alternative protein. The term “alternative protein” does not yet have a precise universal definition, but in the source [4] it is defined as “Alternative proteins are produced from sources that have low environmental impact to replace established protein sources. They can also be obtained from animal husbandry with good animal welfare.” 

Alternative protein sources

• Today, alternative protein sources can be divided into 4 groups: 

Plants, especially the protein-rich legume family (soybeans, peas, chickpeas, beans), cereals (oats, rice, wheat, corn) and oilseeds (peanuts, flaxseed, sesame, sunflower). These produce so-called plant proteins, which are copies of meat and dairy products created from plant-based ingredients [5]. 

Plant sources of protein are valuable from a nutritional standpoint, but they require arable land and water, which will become scarce as we strive to meet global demand for protein. 

• Meat grown in vitro, or cultured meat, is a product grown from cells in a laboratory. 

The method is as follows: animal cells are selected, which are then combined with a special nutrient medium containing certain growth factors. The cells are then placed in a bioreactor, where they begin to grow. Then, to give the artificial meat the volume and structure of muscle tissue, special frames are used, which can also be eaten [6, 7, 8, 9, 10]. 

Cultured meat has a high nutritional value and has a number of advantages over “classic” meat, namely, a reduced content of hormonal drugs used in raising farm animals and the absence of pathogenic microorganisms. But the downside of this alternative protein is that animals that undergo biopsy are used to produce cultured meat, which causes them some suffering.

Another disadvantage of this technology is that it is almost impossible to achieve completely sterile conditions for the production of cultured meat, so antibiotics will most likely be required to suppress the growth of bacterial pathogens in the nutrient medium [11, 12].

But the main difficulty in producing cultured meat is reproducing the exact structure, texture, color, taste and nutritional value of meat grown on farms. This requires, among other things, simulating the role of blood in delivering oxygen and nutrients to thicker areas of tissue, as well as co-cultivation of fat, muscle and connective tissue; such complex technical solutions have not yet been developed to date [13, 14].

Insects, a protein obtained from insects, is called Entomoprotein. Today, insects are widely consumed as food by people of different populations in about 80 countries in Asia, Africa and America. Protein obtained from insects as food ingredients can be an attractive way to consume insects as food [15]. The amino acid profile of entomoprotein is similar to meat protein. The protein content in insects is about 40%, and in some species up to 89.05%, and depends on the insect species or individual differences observed in the same insect species [15, 16, 17, 18, 19].

An important factor in refusing to use Entomoprotein on a large scale is the high allergenicity of insects.

Biomass or protein extract from pure or mixed cultures of algae, yeast, fungi or bacteria. Such protein is called single-cell protein (SCP), i.e. protein produced by single-cell microorganisms and algae (despite the fact that some microorganisms can be multicellular).

The protein from mushrooms is called Mycoprotein, it is mistakenly classified as a plant protein, which is incorrect by definition, since the kingdom of fungi is separated from the kingdom of animals and plants.

Due to the root-like structure of the mycelium, the texture of mycoprotein allows the creation of products with a fibrous texture of meat. Mycoprotein has an excellent set of amino acids and digestibility comparable to beef and soy [20]. The disadvantages include a slower growth rate and lower protein content [21].

Another disadvantage of mycoprotein is that some fungi produce mycotoxins [3, 22]. The effects of fungal toxins range from allergic reactions to carcinogenesis and death [13].

Yeast protein, otherwise known as fodder yeast. Some sources also classify it as mycoprotein, since the term “yeast” does not have a taxonomic status and is classified as a fungi [23].

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 [21].

Both macro- and microalgae are included in the human diet. In microalgae biomass, the crude protein content varies from 30 to 80% [24], which is higher than in dry skim milk (36%), soy flour (37%), chicken meat (24%), fish (24%) and peanuts (26%) [25, 26].

However, there is a practical obstacle to extracting proteins from microalgae. The fact is that microalgae have a complex cell wall structure that is difficult to destroy, which makes it difficult to access the proteins and extract them efficiently [27]. Thus, protein extraction is expensive and energy-intensive, which reduces the economic feasibility of large-scale protein production from microalgae [26, 28].

Humans lack the enzyme cellulase, so they cannot digest cellulose, which makes up the cell wall of algae [22]. This enzyme is present in ruminants.

Another obstacle is that 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 [29]. Another disadvantage is that algae accumulate heavy metals [21].

One of the promising directions for obtaining SCP is production using bacteria. Bacterial biomass has advantages over other microorganisms, bacteria use feedstock more flexibly and have a higher protein content.

Protein production from bacteria mainly consists of fermentation of feedstock, which can be wood, straw, sugar, starch, various by-products and waste, such as fruit waste, molasses, canning and food industry waste, alcohol production residues, combustible waste, and hydrocarbons.

Bacterial SCP typically contains 50–80% protein on a dry weight basis [22], and also contains essential vitamins, amino acids, minerals, and lipids, making them versatile for a variety of applications, from human food and animal feed production to additional applications in other industries, such as the paper industry [26, 30].

SCP made from methane 

In this work, we have considered the use of an alternative protein produced by bacteria through the oxidation of methane. Methane is of particular interest as a substrate because it is one of the simplest and most abundant organic compounds, it is formed as a result of both geological and biological processes, methane is a by-product of animal husbandry, and is also formed as a result of waste processing and disposal and in the production of biogas, natural gas mainly consists of methane. Excess methane is currently flared [3]. In addition, various microorganisms such as methanotrophs, cyanobacteria and acetogens can grow on methane as the only substrate [31].

Another advantage of using methane as a carbon source is that it can use existing facilities for natural gas production [32]. This will allow for a fairly rapid increase in production and the launch of the finished product, as well as prompt compensation for agricultural losses. The use of existing infrastructure will also help reduce the cost of producing such SCP and determine its availability for the most economically vulnerable segments of the population [32].

Methanotrophs are the main producers of methane-based SCPs, in which amino acids can be synthesized from various carbon metabolic pathways, including the ribulose monophosphate (RMP) cycle, serine cycle, and tricarboxylic acid (TCA) cycle. For example, two essential amino acids, valine and leucine, are converted from pyruvate, a product of both the RMP and serine cycles [33, 34, 35, 36]. Therefore, methanotrophic bacterial biomass contains a wide range of essential amino acids, making it comparable to traditional protein sources [36].

The essential amino acid content of methane-based SCPs ranges from 16.2 to 37.7% [37, 38, 39, 40]. In a study [36], a special bioreactor configuration for SCP production allowed the achievement of a relative content of essential amino acids (EAA) above 42%, which is the highest content reported [36].

The composition of methane-oxidizing amino acids of proteins is shown in Figure 1 [41] in a decrease with the addition of amino acids to casein and chlorella hydrous acid, as well as to soy protein [42]. The figure also shows a diagram of the content of essential amino acids pattern recomended FAO/WHO. 

Methane protein was developed in the mid-20th century and was considered as a protein component in animal feed and human food in different countries of the world. 

So in the USSR In 1983, the protein-vitamin concentrate (PVC) plant launched the 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 animals and fish. The production capacity was 1 thousand tons of protein per month. In 1989, there were eight plants in the USSR that produced protein on various substrates, including methane. But after a few years, all of these plants were closed due to an outbreak of allergic diseases associated with the effects on the human body of protein dust formed during the production of PVC [43]. Currently, several companies are trying to revive similar production in the territory of the former USSR. 

In Denmark, since 1976, the company Dansk Bioprotein has been developing a process for producing microbial protein from gas using the methane-oxidising 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 looking for opportunities to create a land-based industry based on natural gas. This is how the production of the feed product Bioprotein began, the producers of this protein are the bacteria Methylococcus capsulatus. In 1995, Bioprotein received EU approval for use in Atlantic salmon and pet food [44].

In the 1970s, Imperial Chemical Industries (ICI) in the UK developed a microbial protein called Pruteen, produced by growing Methylophilus methylotrophus bacteria in methanol, which is produced from methane or natural gas. Pruteen contained up to 70% protein and was used in pig feed [31]. The company invested over £100 million in the initiative, but high production costs and falling prices of competing products led to the project being abandoned despite its high nutrient content [3].

VTT Ltd. is currently investigating reactor design and options for combining on-farm methane production with the production of microbial oil and feed protein from methanotrophic bacteria Methylococcus capsulatus (Group I), Methylosinus trichosporium (Group II) and Methylocystis parvus (Group II) [3].

Calysta Inc. opened a manufacturing facility for its alternative microbial protein in the UK in 2016. To date, three products have been launched [3]: FeedKind - for fish and livestock nutrition, FeedKind Net Zero - for pet nutrition, and Positive Protein - food ingredients for human nutrition.

In 2001, UniBio A/S (using knowledge from Dansk BioProtein A/S) and Calysta Inc. developed a fermentation technology to convert natural gas into protein for animal feed using methanotrophic bacteria. UniBio A/S uses a U-shaped fermenter to achieve a capacity of 4 kg m−3 h−1 and produce UniProtein® with a protein content of ~70%, which is approved for use in animal feed [3]

It should be noted that SCPs have been a stable part of the diet of farm animals and fish for several decades, but SCPs are still rarely used in human food. The growing global demand for protein is likely to increase the importance of SCPs in the human diet.

Already, many companies are announcing the development of SCP suitable for human consumption. The problem is that bacterial SCP contains a high amount of nucleic acids (8–12%), which is a problem for human consumption, since the consumption of purine compounds formed during the breakdown of RNA increases the concentration of uric acid in the plasma, which can cause gout and kidney stones [45]. SCP with a high content of nucleic acids intended for animal feed is recommended for use only in feeding short-lived animals [3, 46]. 

Various methods for reducing the RNA content of SCP have been developed [47] and continue to be used. Endogenous enzymes that cleave RNA (ribonucleases) can be used to cleave RNA after activation by heat treatment (60–70 °C), as in the production of Quorn™ [48]. Ribonucleases can also be added to the process or used as immobilized enzymes [49, 50]. The degraded RNA components are released from the cells, but there is a loss of biomass (35–38%). The process can be improved by using higher temperatures (72–74°C) for 30–45 min with less biomass loss [51]. Increasing the temperature requires the addition of steam, which is a cost factor, but heat is also needed for the final treatment of the biomass at 90°C after RNase activation [3, 52].

Alkaline hydrolysis and chemical extraction methods have also been studied [53]. used alkaline treatment to reduce the RNA content of P. varioti biomass used in the Pequila process to less than 2%. Treatment at 65 °C, pH 7.5–8.5 to activate endogenous ribonuclease also reduced the RNA content to less than 2%, while the protein content remained at 50% [3]. 

One of the challenges in SCP production is the content of harmful substances in the raw materials, such as heavy metals. Using various types of waste as raw materials for SCP production is attractive from a cost and environmental perspective, but can be challenging from a safety perspective, and the origin of the raw materials must be carefully considered [3].

Also, one of the key issues is the toxins produced by bacteria. Toxins can be extracellular (exotoxins) or cell-bound (endotoxins). For example, Pseudomonas spp. and Methylomonas methanica produce large amounts of protein and have been evaluated for use as SCPs. Both species also produce endotoxins that cause febrile reactions [54]. They can be destroyed by heating. In addition, an immunogenicity study of SCPs from Methylococcus capsulatus showed that the acellular preparation (i.e., without the cell wall) did not elicit immune responses in mice, unlike whole-cell preparations [55]. Thus, the toxin issue is addressed by careful selection of the producing organism, process conditions, and product composition [3].

The bank of methanoremos bacteria currently contains several hundred strains, including many bacteria producing SCP. The most frequently encountered bacterium in literature and patents is Methylococcus capsulatus, such strains as GBS-15, BF19-07, MC19, VKPM B-13479, VSB-874, ACR-22, ATCC33009, BKM B-2116, etc. (type strain Methylococcus capsulatus ATCC 19069). Other protein producers include bacteria Methylocystis parva BKM B-2129 (type strain Methylocystis parva IMET 10483); Methylosinus sporium BKM B-2123 (type strain Methylosinus sporium 5); Methylosinus trichosporium BKM B-2117 (type strain Methylosinus trichosporium OB3b); Methylomonas methanica (type strain Methylomonas methanica NCIB 11130); Methylophilus methylotrophus (type strain Methylophilus methylotrophus DSM 5691); Acidomonas methanolica BF 21-05M (type strain Acidomonas methanolica MB 58); Methylomonas koyamae VKM B-3802D (type strain Methylomonas koyamae Fw12E-Y; Other protein-producing bacteria are also known, such as: Pseudomonas methanica, Pseudomonas spp., Methylobacter acidophilus, Methylomonas rubra VSB-90, Methylococcus sp. ЧМ-9, Methylococcus minimus, Methylomonas agile, Methylomonas methylosinus and others. Microorganisms producing SCP differ in the rate of fermentation and protein content in the biomass, this difference can even be among different strains of the same microorganism. 

SCP Production

Depending on the substrate material, the producing organism and the intended use as animal feed or food, different production steps are required to produce SCP from methane. However, there are some common and most important steps to obtain SCP, such as:

1. Isolation of methanotrophic microorganisms. They can be found in nature, for example, in swamps, tundra, soil, water, and animals can also be sources.

2. Growing microorganisms in a special nutrient medium containing minerals, vitamins, carbons, and nitrogen.

3. Transferring microorganisms to a reactor, where the protein production process itself begins. Methane enters the reactor, microorganisms feed on it and convert it into protein. The output is the so-called “fermentation broth,” which contains a biomass concentration of 1–3% of dry weight (i.e. bacterial cells) [32].

4. The fermentation broth is then purified and dewatered to obtain the final product. Purification may involve various steps such as filtration, ultrafiltration, dialysis, and chromatography. Water is removed by mechanical dehydration and drying.

Discussion

Alternative proteins are of great interest in the development of new food products that will help feed the growing world population. Despite the long history of using methane-based alternative proteins, replacing animal proteins with alternative proteins requires further study.

Much of the existing research on alternative proteins is conducted by companies developing or promoting these products. Work in the field of alternative proteins should be directed at assessing the safety of such proteins for use in the human diet. 

It is also necessary to develop forms in which SCP can be consumed as food and improve the organoleptic properties of SCP, in other words, so that SCP is similar to animal protein not only in the composition of amino acids and other beneficial micronutrients, but also in taste, color, smell and shape. This will increase the positive perception of alternative proteins by people, which should contribute to further market expansion. 

Conclusion

Currently, alternative protein accounts for a relatively small share of the modern human diet, but the growing global demand for protein is likely to increase its importance. Alternative protein can solve not only the food problem, but also the problem of environmental pollution, since traditional agriculture has a negative impact on land and water resources.

Single-cell protein (SCP), i.e. protein produced by single-cell microorganisms, is a promising option. High growth rates or the ability to use unique substrates lead to processes that are much more efficient and environmentally friendly than traditional agriculture. SCP, obtained by fermentation of methane using a bacterial culture containing mainly methanotrophic microorganisms, is characterized by a high protein content in the final product, a composition of essential amino acids, and digestibility for humans.

Methane is quite common and therefore of particular interest as a substrate, methane is a by-product of animal husbandry, and is also formed as a result of waste processing and disposal and in the production of biogas, methane is a component of natural gas. Also, one of the advantages of using methane as a carbon source is that it is possible to use existing facilities for the extraction of natural gas.

A big advantage of using alternative protein sources is that in this case there is no need to use a huge number of animals to meet your own needs.

When switching to a new food, you also need to overcome a psychological barrier, because some people may find it unacceptable to use microbes as a food source, but for thousands of years, humans have intentionally or unintentionally consumed products such as alcoholic beverages, cheese, yogurt, soy sauce, and along with them, the biomass needed to produce them.

The alternative protein industry is developing dynamically. Scientists are developing increasingly original and unusual ways to obtain proteins and products from them, and are also developing ways to give them all the characteristics inherent in animal proteins, such as smell, color, taste and consistency, and most importantly, all the beneficial properties of animal protein.

Public perception and approval from the point of view of safety of products obtained from microorganisms is a key factor in the mass production and use of SCPs in the diet. 

Reference 

1. Foley JA, Ramankutty N, Brauman KA, et al. Solutions for a cultivated planet. Nature. 2011;478(7369):337-342. Available from: doi.org/10.1038/nature10452

2. Wu G. Amino acids: metabolism, functions, and nutrition. Amino Acids. 2009;37:1–17. Available from: doi.org/10.1007/s00726-009-0269-0

3. 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. Published 2017 Oct 13. Available from: doi.org/10.3389/fmicb.2017.02009 

4. Grossmann L, Weiss J. Alternative Protein Sources as Technofunctional Food Ingredients. Annu Rev Food Sci Technol. 2021;12:93-117. Available from: doi.org/10.1146/annurev-food-062520-093642 

5. Chandran AS, Suri S, Choudhary P. Sustainable plant protein: an up-to-date overview of sources, extraction techniques and utilization. Sustain Food Technol. 2023;1:466–483. Available from: doi.org/10.1039/d3fb00003f

6. Benjaminson M, Gilchriest J, Lorenz M. In vitro edible muscle protein production system (mpps): stage 1, fish. Acta Astronaut. 2002;51:879-889. Available from: doi.org/10.1016/S0094-5765(02)00033-4

7. Kazama T, Fujie M, Endo T, Kano K. Mature adipocyte-derived dedifferentiated fat cells can transdifferentiate into skeletal myocytes in vitro. Biochem Biophys Res Commun. 2008;377(3):780–785. Available from: doi.org/10.1016/j.bbrc.2008.10.046

8. Edelman P, McFarland D, Mironov V, Matheny J. Commentary: In Vitro-Cultured Meat Production. Tissue Eng. 2005;11(5-6):659–662. Available from: doi.org/10.1089/ten.2005.11.659

9. ‌Post MJ. Cultured meat from stem cells: Challenges and prospects. Meat Sci. 2012;92(3):297–301. Available from: doi.org/10.1016/j.meatsci.2012.04.008

10. Post MJ. Cultured beef: medical technology to produce food. J Agric Food Sci. 2013;94(6):1039–41. Available from: doi.org/10.1002/jsfa.6474 

11. Thorrez, L., Vandenburgh, H. Challenges in the quest for “clean meat”. Nat Biotechnol. 2019;37:215–216. Available from: doi.org/10.1038/s41587-019-0043-0

12. Stephens N, Di Silvio L, Dunsford I, Ellis M, Glencross A, Sexton A. Bringing cultured meat to market: Technical, socio-political, and regulatory challenges in cellular agriculture. Trends Food Sci Technol. 2018;78(0924-2244):155–166. Available from: doi.org/10.1016/j.tifs.2018.04.010

13. ‌Fraeye I, Kratka M, Vandenburgh H, Thorrez L. Sensorial and Nutritional Aspects of Cultured Meat in Comparison to Traditional Meat: Much to Be Inferred. Front Nutr. 2020;7(35). Available from: doi.org/10.3389/fnut.2020.00035

14. ‌Santo RE, Kim BF, Goldman SE, Dutkiewicz J, Biehl EMB, Bloem MW, et al. Considering Plant-Based Meat Substitutes and Cell-Based Meats: A Public Health and Food Systems Perspective. Front Sustain Food Syst. 2020;4(4). Available from: doi.org/10.3389/fsufs.2020.00134

15. ‌Sosa DAT, Fogliano V. Potential of Insect-Derived Ingredients for Food Applications [Internet]. Insect Physiology and Ecology. InTech. 2017;215–231. Available from: doi.org/10.5772/67318

16. Psarianos M, Dimopoulos G, Ojha S, Cavini ACM, Bußler S, Taoukis P. Effect of pulsed electric fields on cricket (Acheta domesticus) flour: Extraction yield (protein, fat and chitin) and techno-functional properties. Innov Food Sci Emerg Technol. 2022;76:102908. Available from: doi.org/10.1016/j.ifset.2021.102908

17. ‌Laroche M, Perreault V, MarciniakA, Gravel A, Chamberland J, Doyen A. Comparison of Conventional and Sustainable Lipid Extraction Methods for the Production of Oil and Protein Isolate from Edible Insect Meal. Foods. 2019;8(11):572. Available from: doi.org/10.3390/foods8110572

18. ‌Yi L, Lakemond CM, Sagis LM, Eisner-Schadler V, van Huis A, van Boekel MAJS. Extraction and characterisation of protein fractions from five insect species. Food Chem. 2013;141(4):3341–3348. Available from: doi.org/10.1016/j.foodchem.2013.05.115

19. ‌Ma Z, Mondor M, Goycoolea Valencia F, Javier Hernández-Álvarez A. Current state of insect proteins: extraction technologies, bioactive peptides and allergenicity of edible insect proteins. Food Funct. 2023;14:8129-8156 Available from: doi.org/10.1039/D3FO02865H

20. ‌Miller S, Dwyer J. Evaluating the Safety and Nutritional Value of Mycoprotein. Food Technol. 2001;55(7):42-47.

21. Nasseri AT, Rasoul-Ami S, Morowvat MH, Ghasemi Y. Single Cell Protein: Production and Process. Am J Food Technol. 2011;6(2):103-116. Available from: doi.org/10.3923/ajft.2011.103.116

22. Anupama, Ravindra P. Value-added food: Single cell protein. Biotechnol Adv. 2000;18(6):459–79. Available from: doi.org/10.1016/S0734-9750(00)00045-8

23. ‌Belik S, Morgul E, Kruchkova V, Avetisyan Z. Produkts of microbial synthesis in solving protein deficiency. East Eur Sci J. 2016;7(1):122-129.

24. Kumar D, Tewary T. Techno-economic assessment and optimization of a standalone residential hybrid energy system for sustainable energy utilization. Int. J. Energy Res. 2021;46:10020–10039. Available from: doi.org/10.1002/er.6389

25. Haroun AA, Matazu IK, Abdulhamid Y, Sani J. Molecular Identification of Green Algae, Spirogyra Porticalis, along Parts of River Kaduna and its Potential for Singlecell Protein (SCP) Production. Niger. Ann Pure Appl Sci. 2019;1:38–43. Available from: doi.org/10.46912/napas.61

26. ‌Li YP, Ahmadi F, Kariman K, Lackner M. Recent advances and challenges in single cell protein (SCP) technologies for food and feed production. NPJ Sci Food. 2024;8:1-25. Available from: doi.org/10.1038/s41538-024-00299-2

27. Hülsen T, Hsieh K, Lu Y, Tait S, Batstone DJ. Simultaneous treatment and single cell protein production from agri-industrial wastewaters using purple phototrophic bacteria or microalgae – A comparison. Bioresour. Technol. 2018;254:214–23. Available from: doi.org/10.1016/j.biortech.2018.01.032

28. Hülsen T, Sander EM, Jensen PD, Batstone DJ. Application of purple phototrophic bacteria in a biofilm photobioreactor for single cell protein production: Biofilm vs suspended growth. Water Res. 2020;181:115909. Available from: doi.org/10.1016/j.watres.2020.115909

29. ‌Harun R, Singh M, Forde GM, Danquah MK. Bioprocess engineering of microalgae to produce a variety of consumer products. Renew. Sust. Energy Rev. 2010;14(3):1037–1047. Available from: doi.org/10.1016/j.rser.2009.11.004

30. Ding W, Zhang Y, Shi S. Development and Application of CRISPR/Cas in Microbial Biotechnology. Front. Bioeng. Biotechnol. 2020;8:711. Available from: doi.org/10.3389/fbioe.2020.00711

31. 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. Available from: doi.org/10.1016/j.biotechadv.2023.108096.

32. García Martínez JB, Pearce JM, Throup J, Cates J, Lackner M, Denkenberger DC. Methane Single Cell Protein: Potential to Secure a Global Protein Supply Against Catastrophic Food Shocks. Front. Bioeng. Biotechnol. 2022;10:906704. Available from: doi.org/10.3389/fbioe.2022.906704

33. ‌Fisher M. Lehninger Principles of Biochemistry, 3rd edition; By David L. Nelson and Michael M. Cox. Chem. Educator. 2001;6(1):69–70. Available from: doi.org/10.1007/s00897000455a

34. ‌Shapiro BM, Stadtman ER. The regulation of glutamine synthesis in microorganisms. Annu Rev Microbiol. 1970;24:501-524. Available from: doi.org/10.1146/annurev.mi.24.100170.002441

35. Umbarger HE. Amino Acid Biosynthesis and its Regulation. Annu Rev Biochem. 1978;47(1):533–606. Available from: doi.org/10.1146/annurev.bi.47.070178.002533.

36. ‌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. Available from: doi.org/10.1016/j.watres.2024.121820

37. Khoshnevisan B, Dodds MH, 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. Available from: doi.org/10.1016/j.jenvman.2020.110560

38. ‌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. Available from: doi.org/10.1016/j.biortech.2019.121743

39. Xu X, Zhu J, Thies JE, Wu W. Methanol-linked synergy between aerobic methanotrophs and denitrifiers enhanced nitrate removal efficiency in a membrane biofilm reactor under a low O2:CH4 ratio. Water Res. 2020;174:115595. Available from: doi.org/10.1016/j.watres.2020.115595

40. ‌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;320:124351. Available from: doi.org/10.1016/j.biortech.2020.124351

41. ‌Mosichev M, Skladnev A, Kotov B. General technology of microbiological production. Moscow: Light and Food Industry. 1982.108 p. [Mosichev M, Skladnev A, Kotov B. Obshhaya texnologiya mikrobiologicheskix proizvodstv. Moskva: Legkaya i pishhevaya promy`shlennost. 1982. 108 str] 

42. Hulmi JJ, Lockwood C, Stout JR. Effect of protein/essential amino acids and resistance training on skeletal muscle hypertrophy: A case for whey protein. Nutr Metab. 2010;7(1):51. Available from: doi.org/10.1186/1743-7075-7-51 

43. 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 27 November 2024]. 

44. 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

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

46. Strong PJ, Xie S, Clarke WP. Methane as a Resource: Can the Methanotrophs Add Value? Environ. Sci Technol. 2015;49(7):4001–4018. Available from: doi.org/10.1021/es504242n

47. ‌Sinskey A, Tannenbaum S. Removal of nucleic acids in SCP. In: Tannenbaum S, Wang DI, editors. Single-Cell Protein II. Cambridge: MIT Press; 1975. p. 158–178.

48. Anderson C, Solomons G. Primary metabolism and biomass production from Fusarium. In: Moss M, Smith J, editors. The Applied Mycology of Fusarium. Cambridge: Cambridge University Press; 1984. p. 231–250.

49. ‌Martinez M, Sanchez-Montero J, Sinisterra J, Ballesteros A. New insolubilized derivatives of ribonuclease and endonuclease for elimination of nucleic acids in single cell protein concentrates. Biotechnol Appl Biochem. 1990;12(6):643–652.

50. Hameş E, Demir T. Microbial ribonucleases (RNases): production and application potential. World J Microbiol Biotechnol. 2015;31(12):1853–1862.

51. ‌Ward P. Production of Food. United States patent 5,739,030. 14 April 1998

52. Knight N, Roberts G, Shelton D. The thermal stability of Quorn™ pieces. Int J Food Sci Technol. 2001;36(1):47-52. Available from: doi.org/10.1111/j.1365-2621.2001.00424.x 

53. Viikari L, Linko M. Reduction of nucleic acid content of SCP. Process Biochem. 1977;12(4), 17–35.

54. Rudravaram R, Chandel A, Rao L, Hui Y, Ravindra P. Bio (Single Cell) protein: issues of production, toxins and commercialisation status. In: Ashworth G, Azevedo P, editors. Agricultural wastes. New York: Hauppage; 2009, p.129–153.