It is estimated that by 2050, the world’s population will exceed 9 billion people [1]. Based on current consumption trends, the world will need to produce 1,250 million tonnes of meat and dairy products yearly to meet the global demand for animal-derived proteins with this increased population size [2]. However, given the current rate of food production, this increased demand cannot be met with conventional agriculture approaches currently in use. Thus, there is a need for alternate approaches to achieve sustainable development goals focused on food security. A promising approach is the use of single-cell proteins.

Single-cell proteins (SCP) generally refer to the edible biomass of unicellular microorganisms. This can be the whole biomass or protein extracts from single or mixed cultures of various microbial groups, including algae, bacteria, yeasts/fungi and others [3]. Single-cell proteins can be utilised as animal feeds or human foods and consumed whole or applied as supplements. There is growing interest in the utilisation of SCP to meet the global demand for nutritious food as they have various advantages over conventional plant and animal proteins [4]. These benefits include; their ease of production, which requires a very short period of time, compared to the time interval of weeks, months or years required for crop farming and animal husbandry. In addition, SCPs are cost-effective as they can be grown in bioreactors. Therefore, they do not require extensive land use or high-water demand that is synonymous with conventional agriculture [5]. Furthermore, the industrial production of SCP in bioreactors ensures uniformity of products and high yields because the product does not compete with pests and weeds. Importantly, SCPs are not prone to diseases associated with conventional agricultural efforts which are responsible for a significant cost in crop farming and animal production with loss in products. In addition, SCP production does not result in biodiversity loss, environmental degradation or contribute to greenhouse gases and climate change [6].

Agricultural waste is continually generated in the food sector and generally are expensive to dispose of [7]. Single-cell protein can be produced using these wastes. This conversion of wastes to foods not only reduces wastage and pollution but ensures that increased demand for food from the ever-growing world population will be met using SCP which has minimal carbon fingerprint [7]. Furthermore, with the global increase in agricultural wastes, it is imperative to devise innovative approaches to dispose of these waste products to safeguard environmental and public while saving cost. Thus, the ability to convert inorganic substrates such as methane, petroleum waste and CO₂ to biomass offers an incredible sustainability potential that is not currently available using conventional agriculture.

Although the current production and consumption of SCP only account for a small percentage of protein intake for humans, the growing demand for protein is likely to make SCPs increasingly important [8, 9]. For animal feed, there is a greater diversity of microbial species which can be used as SCP, and they can be prepared from varied substrates, including wastes. This is unlike SCP for human consumption, which must be prepared from food-grade substrates. This is because single-cell proteins for animal feed are not held to the same regulatory standards as those destined for human consumption, which requires more expensive substrates, product controls, and monitoring. Because of this factor, it may be more advantageous and cost-effective to produce single-cell proteins from diverse substrates such as inexpensive waste materials from forestry and agricultural sources, food and beverage processing [10] as well as other non-food grade sources and use these as animal feeds. This will reduce demand for foliage and feed formulations from edible foods for animals.

Over the years, research on SCP has grown, with contributions across the globe. This study aims to review the body of research on SCP using a bibliometric and bibliographic approach, highlighting commercialisation efforts in SCP production and industry developments. Bibliometric indicators such as the most cited authors, countries, and institutions are discussed. A critical analysis of the production processes and microorganisms used for SCP production, feedstock, biotechnological improvements and potential uses was undertaken. The information provided in this review will guide researchers on recent developments in SCP production and help in harmonising industry and laboratory research towards the use of SCP for food and feed. It will also support researchers within the subject area in fostering collaborations with more experienced research teams/groups in different countries and institutions.

A total of 425 published articles were retrieved from the Web of Science Database. The first recorded publication in single-cell protein was by Mateles et al. [13], which focused on studying the growth of the thermophilic bacterium Bacillus stearothermophilus on hydrocarbons and the use of this as a new source of SCP. There was a gradual increase in publications over the next two decades, with a peak in 1981 of 27 papers (Fig. 1). This growth was followed by a fluctuation in the number of publications over the next four decades, with a second peak in 2018 when 17 papers were published. Overall, an average number of 7 publications per year was made over the 60 years period. Although a limitation of this study is searching and using only articles in which the selected keywords appeared in the title, this is necessary to screen and obtain only articles related and relevant to SCP research. From this, the data obtained suggests that despite the length of time SCP has been a focus of research, there has been no significant growth in the number of publications over the last six decades. More scientific research is required to improve understanding of growth requirements and process conditions for SCP production and to position SCP as a viable protein source to help in meeting the global demand for protein.

3.1. Categorisation of Research

Research on the production and utilisation of SCP for food and feeds is growing. Retrieved articles highlighted the major themes within SCP research. These include the different microbial species utilised for SCP production, substrates used and process optimisation. The retrieved articles are categorised mainly into the biotechnology and applied microbiology, food science and technology and chemical engineering subject areas, as shown in Fig. (2). The highest number of published articles were in Biotechnology and Applied Microbiology. This category represents the principal research focus directed to discovering suitable microorganisms for use in SCP production, optimisation of fermentation, and growth conditions necessary for the production of high biomass. The second-highest category is Food Science and Technology, with articles published under this category focusing on the nutritional composition and safety of produced SCPs. Furthermore, research under this category investigated the formulation of suitable food products from SCP into edible food and feed for human and animal consumption by the supplementation of existing food products with portions of single-cell proteins.

Fig. (1). Publications by year.

Fig. (2). Categorisation of research on single-cell protein.

3.3. Publication by Countries

A total of 62 countries have participated in SCP based research (Fig. 4). An article is assigned to a country based on the affiliation of the first author. The country with the highest number of publications was the United States of America with 90 publications, followed by India (40) and England (28). Within the last ten years (2010-2020), The United States of America had 20 publications, followed by The Peoples Republic of China (18), Iran and Thailand (8 each). Publications from the United States of America focused mainly on the modelling and validation of the production processes of single-cell proteins for food uses and the genetics of single-cell protein in yeast populations [17-19]. China’s research focus is mainly on the volarization of food wastes for single-cell protein production, making use of indigenous micro-flora [20-22] and the quantification of single-cell protein production [23]. Interestingly, England and Norway were the top publishing countries amongst other European nations. Although in Europe, there is high consumption of proteins at about 70% above recommended levels (Westhoek et al., 2011), there is low research output on alternative protein sources such as SCP, which is more sustainable and economically viable in Europe (Voutilainen et al., 2021). Africa, Asia-Pacific and South American regions have low participation in SCP research.

Fig. (3). SCP producer organisms.

Fig. (4). Country contribution in single-cell protein research.

Table 1. Institution Contribution to Research on Single-cell Protein.

S.No	Institution	Publications	Percent
1	Dalhousie University	10	2.35%
2	Iowa State University	10	2.35%
3	Massachusetts Institute of Technology MIT	9	2.12%
4	Council of Scientific Industrial Research CSIR india	8	1.88%
5	Cornell University	7	1.65%
6	National Research Centre NRC	7	1.65%
7	Ocean University of China	7	1.65%
8	University of California System	7	1.65%
9	Norwegian University of Life Sciences	6	1.41%
10	Punjab Agricultural University	6	1.41%
11	Technical University of Denmark	6	1.41%
12	University of Quebec	5	1.18%

3.7. Keyword Analysis

Keyword analysis shows that a total of 1207 keywords were used to describe research in this field. Expectedly, the keywords; single, cell, and protein were used in all publications. This is followed by production (189) times, yeast (41), waste (35) and whey (27). The spread of keywords used indicates the focus of research within the field. The co-occurrence of keywords that have occurred a minimum of 5 times is visualised using VosViewer in Fig. (5).

Table 3. Highest cited articles in single-cell protein-related research.

S/N	Author	Title	Citations	Journal	Year	Ref
1	Windass JD, Worsey MJ, Pioli EM, Pioli D, Barth PT, et al.	Improved conversion of methanol to single-cell protein by Methylophilus methylotrophus	157	Nature	1980	[24]
2	Chae SR, Hwang EJ, Shin HS	Single cell protein production of Euglena gracilis and carbon dioxide fixation in an innovative photo-bioreactor	117	Bioresource Technology	2006	[15]
3	Gefen O, Gabay C, Mumcuoglu M, Engel G, Balaban NQ	Single-cell protein induction dynamics reveals a period of vulnerability to antibiotics in persister bacteria	96	Proceedings of the National Academy Of Sciences of the United States of America	2008	[25]
4	Cysewski G.R. and Wilke C.R	Utilisation of cellulosic materials through enzymatic hydrolysis. I. Fermentation of hydrolysate to ethanol and single-cell protein	85	Biotechnology and Bioengineering	1976	[26]
5	Papanikolaou S, Chevalot I, Galiotou-Panayotou M, Komaitis M, Marc I, et al.	Industrial derivative of tallow: a promising renewable substrate for microbial lipid, single-cell protein and lipase production by Yarrowia lipolytica	73	Electronic Journal of Biotechnology	2007	[27]
6	Howells ER	Single-cell protein and related technology	68	Chemistry & Industry	1982	[28]
7	Shahi P, Kim SC, Haliburton JR, Gartner ZJ, Abate AR	Abseq: Ultrahigh-throughput single cell protein profiling with droplet microfluidic barcoding	67	Scientific Reports	2017	[29]
8	Albert FW, Treusch S, Shockley AH, Bloom JS, Kruglyak L	Genetics of single-cell protein abundance variation in large yeast populations	64	Nature	2014	[19]
9	Ferrer J, Paez G, Marmol Z, Ramones E, Garcia H, et al.	Acid hydrolysis of shrimp-shell wastes and the production of single cell protein from the hydrolysate	61	Bioresource Technology	1996	[30]
10	Shipman RH, Kao IC, Fan LT	Single-cell protein production by photosynthetic bacteria cultivation in agricultural by-products	56	Biotechnology and Bioengineering	1975	[31]
11	Humphrey AE, Moreira A, Armiger W, Zabriskie D	Production of single cell protein from cellulose wastes	55	Biotechnology and Bioengineering	1977	[32]
12	Moon NJ, Hammond EG, Glatz BA	Conversion of cheese whey and whey permeate to oil and single-cell protein	55	Journal of Dairy Science	1978	[33]
13	Avnimelech Y, Mokady S, Schroeder GL	Circulated ponds as efficient bioreactors for single cell protein production	52	Israeli Journal of Aquaculture-Bamidgeh	1989	[34]
14	Aggelopoulos T, Katsieris K, Bekatorou A, Pandey A, Banat IM, et al.	Solid state fermentation of food waste mixtures for single cell protein, aroma volatiles and fat production	52	Food Chemistry	2014	[35]
15	Revah-moiseev S, Carroad PA	Conversion of the enzymatic hydrolysate of shellfish waste chitin to single-cell protein	50	Biotechnology and Bioengineering	1981	[36]
16	Bothe H, Jensen KM, Mergel A, Larsen J, Jorgensen C, et al.	Heterotrophic bacteria growing in association with Methylococcus capsulatus (Bath) in a single cell protein production process	50	Applied Microbiology and Biotechnology	2002	[37]

Fig. (5). Keyword analysis to describe research in SCP field using VosViewer.

Of all the keywords used, 32 occurred a minimum of 5 times. The highest co-occurrence of keywords was between single-cell protein and fermentation used together in 20 documents. This is followed by yeast and single-cell protein (15) and yeast with fermentation (11), then finally fermentation and cheese whey (8). These keywords can guide researchers to retrieve relevant papers within this field of study.

Microbial genera employed in single-cell protein production include; bacteria, yeasts/moulds, or algae. Although the most investigated genera are fungi, algae are the richest source of protein as they typically contain over 40% of rough protein (dry weight) [38]. The production of SCP by these microbial genera can be achieved using several substrates, including; wood, straw, cannery, spent liquor, whey, hydrocarbons, among others. The traditional raw materials utilised for SCP production are mostly mono and disaccharides, including glucose, other hexose and pentose sugars. Other possible substrates for SCP include; bagasse, citrus squanders, molasses, compost, whey, starch, and sewage [39, 40].

Single cell protein derived from bacteria is desirable because the cells contain about 50-80% of dry protein by mass, are easy to grow using cheap substrates as sources of carbon and energy [2, 41, 104]. (Table 7) shows some examples of bacteria used for SCP production. Generally, the genus Methylotrophus sp. is mainly used by different companies to produce SCP [2, 107]. Bacteria have some advantages over algae and fungi for use in SCP production, not only because of their protein composition and use of cheap substrates but also because of their faster growth rate, which is higher compared to those of algae and fungi. The specific growth rate of bacteria varies from 0.28μ to 0.65μ per hour, while that of algae and fungi varies between 0.60μ to 0.70μ and 0.16μ to 0.39μ per hour [122]. These qualities make bacteria a choice organism for SCP production [2, 104]. However, the use of bacteria SCP production presents some challenges:

1. Bacterial cells have high nucleic acid content, which can be toxic to both man and animals. The removal of nucleic acid materials from the cells introduces additional processing costs, thereby impeding commercial production.

2. Bacterial cell size and low density makes harvesting difficult and expensive from the fermentation medium.

3. Human bias has also made the acceptability of bacteria SCP difficult [41].

4. The presence of toxins produced by some gram-negative bacteria can lead to food poisoning.

Bacteria cells are fermented in bioreactors to produce SCP. Recent developments in technology have focused on improving the processing of SCP from bacteria cells to maximise production [2]. The use of bioreactors for SCP production also reduces the risk of contamination and increases growth efficiency.

Other bacteria used for the production of SCP include; Aeromonas hydrophylla, Acinetobacter calcaoceticus, Alcaligenes eutrophus, Bacillus sp., Cellulomonas sp., Methylomonas sp., Mycobacterium sp., Nocardia sp., Pseudomonas sp., Rhodopseudomonas sp., Brevibacterium sp., Methanomonas methanica, and Methylophilus sp [104].

Proteins derived from microorganisms offer certain advantages compared to animal or plant-derived proteins, and these can be considered in their nutritional quality, ease of propagation and production, resource requirements and the ancillary metabolites it produces.

The qualities and quantities of the various nutrients derived from organisms used for SCP production vary from one organism to another and rely on extrinsic factors such as the nature and quality of the substrate and presence of contaminant [105]. Of the microorganisms used for SCP, bacteria produce the highest quantity of protein by dry mass, but due to their high nucleic acid content and presence of toxins, fungi and algae are preferable sources of SCP [112]. Generally, SCP microbes are primarily needed for their protein content and quality. However, what determines the acceptability of a particular species as food or feed are growth rate, the substrate used, contamination and presence of toxins [39]. Algae are one of the most studied SCP microorganisms, with authors comparing the essential amino acid constituents of algae with some conventional protein sources such as egg and soybean. This comparison shows that microalgae are good surrogates for conventional protein sources [95].

The amino acid composition of microbial proteins and their high protein value makes them potent alternative sources of proteins for human and animal consumption. Furthermore, the cheap propagation method of single-cell proteins gives it a selective advantage over conventional protein sources. This includes high growth rate and low generation time, cheap multiple substrates, less demand for space and the possibility of all year production [2]. Generally, algae have the lowest growth rate, followed by yeast and filamentous fungi, with bacteria having the fastest growth rate of about 20 minutes to two hours [39]. However, the growth rate may vary depending on the growth conditions such as the availability of nutrients in certain forms, which is usually determined by the fermentation technique adopted and environmental factors such as pH, presence of contaminants or absence of competition [104]. This is unlike animal or plant-based proteins which usually take months to years for growth and maturation [2].

Organisms utilised for single-cell protein production have different modes of nutrition, including heterotrophic, chemoautotrophic, photoautotrophic and methylotrophic [132], enabling them to utilise a wide range of substrates and survive in different growth conditions, unlike heterotrophic organisms, which require organic matter as a source of carbon and the photoautotrophs which can only fix carbon using carbon dioxide in the presence of sunlight. The ability of SCP organisms to engage in the dual phototrophic and heterotrophic modes of nutrition is referred to as mixotrophy. This is common in microalgae and confers a unique advantage over other organisms with a single mode of nutrition [132]. Furthermore, this mixotrophic nature of algae makes it possible for integrated propagation of algal single-cell protein with simultaneous treatment of waste [106]. This has further reduced the cost of production of protein, making it commercially feasible [105]. Additionally, single-cell proteins require less space and can be grown in a controlled environment, thus making it possible for an all-year supply of protein [104].

In addition to the viable role for SCP as food and feed, SCP produced by microorganisms contains vitamins and metabolites with diverse health and other benefits. Examples include some single-cell proteins fractions that exhibit foaming and gelation properties and can be used in food to increase palatability [133].

Although SCPs present numerous advantages compared to conventional protein sources, there are some challenges associated with single-cell protein production. These include the cost of production, sensory bias, regulatory challenges, underdevelopment of food technology techniques and safety concerns [2]. Furthermore, the downstream processing of single-cell protein can be capital intensive. The series of processes involved in cell biomass concentration, extraction, and possible purification of proteins demands significant resources. For instance, proteins from bacteria sources need to be purified to remove the nucleic acid content in the cell biomass, as this could have serious adverse health implications. At the same time, algae and fungi derived single-cell proteins needs to undergo some downstream processing to enhance the biological availability of the proteins. These organisms have thick cell walls that cannot be digested by animals and thus need to be removed to increase the protein efficiency ratio (PER) [94]. In some cases, the cost of obtaining purified substrates and the upgrading/valorisation of specific substrates for use by feedstock organisms can also be relatively high, especially for chemoheterotrophic organisms thus, limiting the commercialisation of numerous single-cell protein production [132].

Generally, humans are sceptical about consuming single-cell proteins. This bias can be attributed to safety concerns, organoleptic qualities and concern for the substrate used in production. Single-cell proteins derived from algae have poor organoleptic quality, which makes it difficult for human consumption. Also, safety is a vital aspect of food production and foremost consideration for SCP for human consumption. Several bacteria-derived SCP are yet to be granted the GRAS status because of the risk of intoxication commonly associated with gram-negative bacteria.

An important consideration about single-cell protein consumption is the potential for the bioaccumulation of toxic substances and heavy metals within the cells of the organism from waste effluents from homes or industries used for their production as these can lead to cases of food poisoning [134]. Similarly, fungi derived SCP are also less desired because of toxins in some filamentous fungi [104]. On the other hand, algal cells are relatively more desired than bacteria and fungi because of the low potential of toxin accumulation in algal-derived SCP [104]. Furthermore, bacteria and fungi cells contain high nucleic acid, inimical to health when consumed in high quantities [41]. These safety challenges have elongated the process of certifying some SCP, creating some bottlenecks for them to attain the generally regarded as safe (GRAS) status. The attainment of GRAS status is a requirement for novel foods before they can be approved for consumption. Generally, in the United States and the European Union, novel foods are subjected to long trial periods before being considered safe for consumption. In Europe, Regulation (EU) 2015/2283 governs the development and consumption of novel foods, including SCP [135] and adequate care is taken when the food is from microbial sources to validate the safety status of these foods [136]. Finally, a key challenge in advancing the production and utilisation of SCP is a lag in developing novel food technology equipment that can facilitate the propagation and processing of single-cell proteins on a commercial scale without incurring a high cost of production [137].

In this review, a bibliometric study was used to analyse the body of research on single cell proteins. Our study revealed that although papers in this area have been published over the last 60 years, there has been minimal growth. Thus, investigating the role of microorganisms as a significant source of dietary proteins requires further attention from the scientific research community. Research on SCP is multinational involving several institutions, with major research contributions emanating from the United States of America. With the ever-growing world population and projections on the inability of conventional agriculture to cater to the food needs of the future, especially in Africa and Asia, more collaborative research activities are needed to increase food production using SCP through capacity building and increasing infrastructure for SCP production especially within these regions.

A more in-depth analysis of the original research showed that SCP from fungi is the focus of most research activity in this area, followed by algae and bacteria. Some benefits of SCP identified from our study include the relatively lower cost compared to other dietary protein sources; the possibility of all year-round production of proteins and increased production rate due to the rapid growth rate of microbes. However, this review identified several challenges with SCP production, including the high cost of downstream process, poor yield and safety issues. In order to address these challenges, future research should focus on (1) use of genetic engineering approaches to improve potential strains with desirable characteristics, e.g. removing toxin producing genes (2) understanding consumer perception to the use of Single cell protein, to identify misconceptions and address these (3) simplifying and reducing the downstream processing required to make SCP readily consumable. Overall, SCP has an important role to play in ensuring food security in the coming years and the dedication of scientific effort in improving this aspect of food production will be beneficial in providing nutritious food and feed while ensuring a cleaner and more sustainable environment as SCP production is generally carbon neutral without adverse environmental impact as obtainable in conventional agriculture.