Due to the rapid increase in the world’s population, from 2.6 to 7 billion in last six decades (Upadhyaya et al., 2016) and with an estimation of 9.5 billions by 2050 (Henchion et al., 2017), many developing countries are facing famine, food insecurity, malnutrition and other diseases (Srividya et al., 2013). In particular, deficiency of protein is becoming a major problem for human and animal’s health. Indeed, several amino acids found in proteins are essential and cannot be replaced since they are necessary to build up new structural, functional and essential proteins (Adedayo et al., 2011). Thus, the demand for protein sources has increased (Anupama and Ravindra, 2000) with a protein consumption passing from 65 to 80 g/capita/day between 1960 and 2011 (Henchion et al., 2017). Agriculture and food industries may not be able to meet high protein demands and this is due to the depletion of natural resources, seasonality, plant diseases, climatic contingencies and the low amount of proteins in the available resources (about 25% in milk, 35% in soybean and 45% in meat) (Ukaegbu-Obi, 2016, Younes et al., 2011). Therefore, scientists and industrials seek to supplement available sources with unconventional, novel and alternative protein sources with an attractive amino acids composition combined with a sustainable production (Nasseri et al., 2011).

Thanks to biotechnology developments, microorganisms are exploited as new sources of protein called “Single Cell Protein” (SCP) (Anupama and Ravindra, 2000). This approach has multiple characteristics, including its independence from seasonal and geographic factors, its high productivity and efficiency in substrate conversion as well as the wide variety of raw materials, microorganisms and culture methodologies that can be used for these purposes (Araujo Neto and Ferreira Pinto, 1975). The term “Single Cell Protein” was introduced in the 1960s to replace “microbial protein” and “petro protein”, giving it a more suitable image (Mateles and Tannenbaum, 1968). These are dried cells extracted from microbial cell culture of yeasts, bacteria, filamentous fungi or alga and used as ingredients or rich protein-supplements in human food (as protein sources, auxiliary emulsifiers, flavor enhancers or vitamin sources in soups, ready meals and diet recipes) or animal feed (for fattening calves, poultry and pig food, for fish farming or for pet feed) (Anupama and Ravindra, 2000, Sharif et al., 2021, Srividya et al., 2013).

The microbial biomass is characterized by a large amount of proteins (40-60%DW for algae, 50-65%DW for bacteria, 30-45%DW for fungi and 45-65%DW for yeasts) and a complete amino acid profile that can exhibit excellent protein quality (Adedayo et al., 2011, Nasseri et al., 2011, Suman et al., 2015).

Nowadays, some companies developed their own food and feed products which are based on microbial biomass from cyanobacteria (All-G Rich® using Aurantiochytrium limacinum and Spirulina® using Arthrospira sp.), bacteria (FeedKind® using Methylococcus capsulatus, KnipBio Meal® using Methylobacterium extorquens, Pruteen® using Methylophilus methylotrophus, UniProtein® using Methylococcus capsulatus, etc.), filamentous fungi (Quorn® using Fusarium venenatum) and yeasts (Provesta® using Pichia pastoris) (Jones et al., 2020, Linder, 2019).

For production, many parameters are important and can have an impact on Single Cell Proteins production and their chemical composition. In the literature, SCP production performances are expressed either as biomass production and or/ as total protein production. Studies regarding growth and/or protein production using different environmental parameters (principally temperature and pH) dealt only with some cyanobacteria (Spirulina maxima (Kosaric et al., 1974)), bacteria (Raoutella ornithinolytica (Abdul et al., 2018), Acetobacter diazotrophicus (Emtiazi et al., 2003)), fungi (Aspergillus terreus (Jaganmohan et al., 2013)) and yeasts (Cryptococcus aureus (Gao et al., 2007), Schawanniomyces castellii (Hongpattarakere and Aran, 1995), Rhodotorula glutinis (Kot et al., 2017) and Saccharomyces cerevisae (Putra et al., 2020)). However, it is difficult to extract an overall trend on the impact of the parameters due to the broad experimental conditions and methods. In particular, the choice of the selected strain is very important because the evaluation of a SCP study may depend on it (Ukaegbu-Obi, 2016).

Cupriavidus necator is a gram negative, non-pathogenic, facultative chemo-lithoautotrophic bacterium belonging to the beta-proteobacteria class. It has the ability to grow under both heterotrophic and autotrophic conditions. The phylogenetic classification of this bacterium has been modified many times. Since mid-2004, it was identified as “Cupriavidus necator” (Vandamme and Coenye, 2004). Previously, this bacterium was identified as: Hydrogenomonas eutropha (between 1958 and 1969) (Wittenberger and Respaske, 1958), Alcaligenes eutrophus (between 1969 and 1995) (Davis et al., 1969), Ralstonia eutropha (between 1995 and 2004) (Yabuuchi et al., 1995) and Wautersia eutropha (from the beginning of 2004 to mid-2004) (Vaneechoutte et al., 2004).

This bacterium is known to be able to synthesize bio-plastics which are PolyHydroxyAlkanoates (PHAs) which present an alternative to synthetic plastics due to their durability and thermal stability.

Poly(3-hydroxybutyrate) (PHB) is the most known type of PHA (Chee et al., 2019). Many studies had the objective to investigate the PHB production in C. necator using limitation conditions such as nitrogen, oxygen, substrates and carbon/nitrogen ratio and/or environmental stress factors such as temperature and pH (Aragao, 1996, Aramvash et al., 2015, Gaudin, 1998, Kerketta and Vasanth, 2019, Khanna and Srivastava, 2005, Kim et al., 1994, Kim et al., 1995, Linko et al., 1993, Morinaga et al., 1978, Nowroth et al., 2016, Obruca et al., 2020, Sonnleitner et al., 1979, Yamane, 1993).

In the 1970s, C. necator was found as a good candidate for SCP production for human consumption and animal feed containing at least 50% DW of proteins (Raberg et al., 2018). Foster and Litchfield (Foster and Litchfield, 1964) published the first study of using C. necator as SCP source. Using gaseous substrate, the final biomass contained 74% DW of protein which is a high content compared to other microorganisms (around 50% DW) (Foster and Litchfield, 1964). Most of the older studies focus on the protein production using C. necator grown under autotrophic conditions. Some studies have investigated the effect of different factors, such as strain, dilution rate, H2/O2 ratio, nitrogen form and ammonium concentration on growth and protein production (Schlegel and Lafferty, 1971, Yang et al., 2021). However, no assessment of protein production under environmental stress has been performed using C. necator in the literature. Only the study by Sakarika et al., (Sakarika et al., 2020) gave information on growth and protein production in C. necator in response to two pH values, pH 6 and pH 7.

Regarding protein composition, amino acid profiles suggest that C. necator could potentially be used as a protein supplement (Calloway and Kumar, 1969). Indeed, the amino acid profile was equal or superior to animal and plant proteins. Compared to the other microorganisms, the lysine content was higher between 7 and 9 g per 100 g-of protein (Calloway and Kumar, 1969, Foster and Litchfield, 1964, Volova and Barashkov, 2010). For instance, lysine content was lower for Aspergillus niger (5 g per 100 g of protein), Penicillium notatum (4 g per 100 g of protein), Candida utilis (3.5 g per 100 g of protein), Yarrowia lipolytica (2-3 g per 100 g of protein) and Pichia pinus (4.5 g per 100 g of protein) (Drzymała et al., 2020, Lagos and Stein, 2020, Mateles and Tannenbaum, 1968, Rashad et al., 1990, Righelato et al., 1976). Currently, C. necator biomass is commercialized as a feed for fish, livestock and pets by Novo-Nutrients®, Solar Foods® and Kiverdi®, using gaseous substrates. The final product contains more than 70%DW of proteins (Jones et al., 2020, Linder, 2019).

C. necator shows interesting metabolic flexibility, switching from growth and protein accumulation under nutrient un-limited conditions to PHAs accumulation under nutrient limited conditions. C. necator has been extensively studied for its ability to produce PHAs, in the contrary much less for its ability to produce SCPs. Therefore, the main objective of this study was to evaluate the robustness of C. necator to produce SCPs in conditions of variation of environmental factors. Specifically, the study compared the dynamic of SCP production and the biomass composition in well-controlled batch bioreactors using different temperatures (25, 30, 35 and 40°C) and different pH values (5, 6, 7 and 8) under nutrient un-limited conditions.