Introduction

According to a report of the Food and Agriculture Organization of the United Nations (FAO), the world population is predicted to grow to 9.7 billion by 2050. Food scarcity is a serious problem due to the drastically increasing global population (Abdullahi et al., 2021). More than 1 billion global populations are suffering from malnutrition because of widening gap between food consumption and accessibility, inflation, decline in forest cultivation, global warming, and other factors. Proteins rich in nitrogen are required by humans and animals to produce new structural and functional proteins, which include enzymes and hormones essential for developmental processes and regeneration of cells (Aziz et al., 2024; Benkirin et al., 2024; A. Bacha et al., 2024; Kurek et al., 2022). In the context of the current global protein deficiency, single-cell protein (SCP) derived from microbes is receiving considerable recognition as a significantly secure and cost-effective source of proteins.

Microorganisms have long been utilized for food fermentation and the production of foodstuffs, such as bread, cheese, yogurt, soy sauce beverages, and beverages (Chaudhary et al., 2024; Ejaz et al., 2024; Afsar et al., 2024; Khatoon et al., 2023; Ullah et al., 2023a). However, the utilization of microbes as a substitute source of nutritional components is not an innovative approach (Riesute et al., 2021).

Single-cell protein is described as dead desiccated cells of microorganisms or proteins from single-celled algae and cyanobacteria that are nurtured on different growth media and utilized as an alternative source of proteins. SCP is a rich source of protein constituting 60–80% of total dry weight (Allah and Saleem, 2021). SCP also contains a significant amount of amino acids, such as methionine and lysine, that are deficient in most plant and animal sources. Moreover, other nutritional components include fatty acids, carbohydrates, vitamins, and minerals. Due to its healthy nutrient composition, SCP has very appealing qualities as a food source (Suman et al., 2015). According to Spalvins et al. (2018), the problem of the unavailability of protein sources for animal feed was easily resolved by using SCP in the specific regions of Indonesia where natural sources of proteins, such as fish and soybeans, were insufficient. SCP has gained consideration as an excellent alternative source of proteins because it could be easily produced from inexpensive carbon sources by using organic waste materials from agricultural leftovers and the food manufacturing and processing industry (Bakratsas et al., 2023).

Important factors considered while selecting substrate media for fermentation include accessibility, cost, and expenses of subsequent processing of substrate (Li et al., 2023; Arooj et al., 2023; Shah et al., 2023; Brastosin et al., 2021). Potato peel in bulk is discarded as a waste material of the potato processing industry, although it is an excellent source of protein, lipids, cellulose, starch, ash, glucose, lignin, and non-starch carbohydrates (Chaudhary et al., 2024; Ullah et al., 2023b; This waste potato peel is used as a carbon source for the production of industrial food products (Arifeen et al.,2024; Shehzadi et al., 2024; Aziz et al., 2023; Calcio et al., 2020).

The aim and objective of the current research were to investigate the possibility of bioconversion of potato peel, easily accessible locally, into SCP through the process of submerged fermentation (SmF). Considering this, the present investigation attempted to figure out the potential of harvesting SCP from Rhizopus oligosporus by liquid state fermentation utilizing plenty of locally accessible potato peels as an affordable substitute of carbon sources (Perveen et al., 2023). This study tried to determine an alternative best substrate for SCP production and to enhance the amount of protein generated in potential substrate and reduce cost of production. Furthermore, optimization of fermentation conditions, such as pH (3.5–7.5), temperature (30, 37 40, and 45°C), and duration of fermentation (5, 7, and 9 days), in contrast to the control medium (glucose), was also performed. Multiple methods were employed to conduct a proximate analysis of potato peel. Moreover, mixed cultures of Rhizopus oligosporus and Saccharomyces cerevisiae were used for raising the production of SCP.

Methodology

Source of microorganisms

Saccharomyces cerevisiae and Rhizopus oligosporus were collected from the PCSIR Laboratory in Lahore, Pakistan. Cultured potato dextrose agar medium slants were incubated at 30°C for 7 days. The inoculated slants were stored at 4°C and subcultured once every 2 weeks.

Collection and pretreatment of potato peel

Potato peel was collected in sterilized glass containers from a nearby market in Lahore, Pakistan. Potato peel was washed and cleaned in distilled water, and converted to fine powder in an electric grinder after a 3-day drying at 40°C in an oven. Some of the potato peel was used for analysis of nutrient composition.

Optimization of acid hydrolysis

Different concentrations of sulfuric acid (H₂SO₄) were used to analyze the optimum value which generated maximum glucose content in potato peel slurry. In each test tube, 1-g potato peel powder and 9-mL distilled water were used to make slurry. Six samples were prepared in test tubes with H₂SO₄ concentration of 1–5%; however, the control sample was not treated with H₂SO₄. Test tubes were heated at 90°C in a water bath for 1 h and centrifuged after cooling. The supernatant was used for glucose analysis using the dinitrosalicylic acid (DNS) method.

Glucose analysis

Six test tubes were labeled according to used H₂SO₄ concentrations, and 0.2-mL supernatant of the above-mentioned sample was added to each test tube. Then 0.8 mL of distilled water and 3 mL of DNS solution were added into each test tube. These test tubes were heated for 15 min in a beaker containing boiling water. Glucose absorbance was examined at 540 nm.

Hydrolysate preparation

Potato peel powder was further processed by treating 100-g of it with 1% H₂SO₄. This media was heated for 2 h on a hot plate to form potato peel hydrolysate, which was filtered using a muslin cloth. This supernatant was autoclaved and used as a substrate for submerged fermentation.

Proximate nutritive composition of potato peel

The nutritive composition of potato peel, including moisture, ash, and protein, was examined. For ash determination, five potato peels were taken in a crucible flask and burnt on flame. This burnt potato peel was converted into ash in a furnace at a temperature of 5000°C, and its mass was measured.

The moisture content of potato peel was examined following the standard procedure of the Association of Official Agricultural Chemists (AOAC, 2016). The Kjeldahl method (Bremner,1960) was used to determine the protein content of potato peel.

Production and harvesting of SCP

Inoculum preparation

In a 100-mL conical flask, 50-mL distilled water was taken along with two loops of Rhizopus oligosporus spores. This inoculum was used in the fermentation process for SCP production.

Fermentation at flask level

Screening of nitrogen source

Nitrogen source has a significant effect on SCP yield. Different nitrogen sources were used to analyze their effect on SCP yield. These include inorganic sources of sodium nitrate and organic sources of urea, peptone, and yeast extract.

Optimization of parameters

Optimization of certain parameters was performed by following the “one factor at a time” procedure. These factors include the incubation period (5, 7, and 9 days), temperature (30, 37, 40, and 45°C), and pH (3.5, 4.5, 5.5, 6.5, and 7.5).

Bioreactor scale-up production

Scale-up bioreactor production of SCP was performed by maintaining optimized conditions in a jar fermenter with an inoculum of Rhizopus oligosporus. The biomass was filtered, dried, and weighed. To enhance the yield of SCP, co-culture of Saccharomyces cerevisiae and Rhizopus oligosporus was used in a bioreactor.

Statistical analysis of data

The results were obtained as mean value and standard deviation (SD) of three trials. Statistical Package for Social Sciences (SPSS) was applied for statistical analysis.

Results

Analysis of the proximate nutritive composition of potato peel

The nutritive composition of potato peel, such as nitrogen, protein, moisture, and ash, was examined (Table 1).

Optimization of acid hydrolysis

For hydrolysate preparation, different concentrations of H₂SO₄ were used, with each concentration resulting in different glucose content. Maximum glucose content of 4% was observed by treating potato peel with 1% H₂SO₄. A small increase in the content was observed with other H₂SO₄ concentrations (Table 2). In further experiments, potato peel hydrolysate was prepared with 1% H₂SO₄.

Nitrogen source screening

The effect of different nitrogen sources on SCP production was examined. Results revealed that the inorganic source of sodium nitrate and the organic nitrogen source of yeast extract yielded maximum SCP (12.6333 ± 0.50332 g/L and 12.6667 ± 0.08819 g/L, respectively, Table 3); these were used as an inexpensive and readily available optimum nitrogen source in further experiments.

Effect of temperature and pH on SCP yield

Optimization of temperature was performed by incubating culture media at different temperatures. Results presented in Table 4 revealed that 37°C was the optimum temperature for maximum growth of SCP (11.4667 ± 0.25166 g/L).

Optimization of pH was performed. Maximum SCP yield (11.6667 ± 0.25166) was obtained at an optimum pH of 4.5. Decline in yield was observed at basic pH of 6.5 and 7.5, as shown in Table 5.

Effect of incubation period on SCP

Results of SCP yield at different incubation periods are presented in Table 6. Biomass from each flask was gathered, dried, and weighed after respective periods as shown in table 6. The lowest yield was observed after 9 days of incubation. Maximum yield (11.7000 ± 0.20000 g/L) was obtained after 7 days of incubation, which was the optimum period for production of SCP.

Bioreactor Scale-up production of SCP

A jar fermenter was used for scale-up production of SCP by using Rhizopus oligosporus; 17-g/L SCP was obtained. To increase the yield, co-culture of Rhizopus oligosporus and Saccharomyces cerevisiae was used in a bioreactor, resulting in a yield of 19-g/L SCP.

Discussion

Rhizopus oligosporus was collected from PCSIR Laboratories in Lahore, Pakistan, and was preserved for the production of SCP using potato peel as a substrate by submerged fermentation.

Co-culture of Saccharomyces cerevisiae and Rhizopus oligosporus was also used for higher yield of SCP, compared to Rhizopus oligosporus. Higher production of SCP using potato peel as a substrate could be due to the composition of potato peel, microbial sources, or genetic composition of microbial strains. The composition of substrate has an important supportive role in the biomass production of microbes. Fungus growth is mainly affected by the nutritional composition of waste (Abdel-Rahman, 2016).

In this study, submerged fermentation was selected for SCP production because all the nutrients were thoroughly dissolved in a liquid medium and it was feasible to control parameters, such as temperature and pH. To achieve maximum yield of SCP, we examined optimum fermentation conditions, such as the effect of the incubation period, temperature, pH, and nitrogen source.

Nitrogen acts as a protein-forming element in substrate media. Its structural properties have an important role for microbial growth in substrate media. Usage of peptone urea and sodium nitrate decreased SCP yield, while organic source of nitrogen as yeast extract enhanced it. Yeast extract included different metal ions, vitamins, proteins, and organic compounds, which may achieve the physiological requirements of Rhizopus oligosporus for production of SCP. Results were aligned with the study conducted by Ouedraogo et al. (2017), which obtained C. utilis biomass (3.25 g/L) with 0.75-g peptone. The authors observed that maximum biomass (4.56 g/L) was obtained by using 0.5% yeast extract.

Temperature is an important factor in microbial production of SCP. Biosynthesis of maximum SCP by Rhizopus oligosporus was obtained at 37°C. At higher temperatures, a decrease in growth was observed, which could be due to unfavorable conditions for microbial enzyme activity. Our results aligned with the study conducted by Milala et al. (2018), which reported 37°C as the optimum temperature for obtaining the highest yield of SCP with S. cerevisiae. The findings were comparable to those of Aslam et al. (2020), who presented the maximum production of pectinase from Bacillus licheniformis KIBGE-IB3 at 37°C. pH has a significant role in the biosynthesis of SCP. Rhizopus oligosporus yielded a high production of SCP at pH of 4.5–5.5; maximum SCP yield was obtained at pH = 4.5. The results are in good agreement with a study conducted by Uçkun Kiran et al. (2015), who used S. cerevisiae and virgin grapes as substrate for maximum SCP yield at pH = 5.8. The results agreed with those of Chen et al. (2016), who studied the effect of different pH values (from 3.5 to 5.5) on SCP production from dry yeast, determining 5.0 as the optimum pH (Zhou et al., 2023).

The experiment revealed that an incubation of 7 days was the optimum period. Decrease in SCP yield by increasing fermentation period of more than 7 days could be due to depletion of substrate concentration in growth media or accumulation of byproducts. The results aligned with the findings of Oshoma et al. (2019), who observed that incubation period of 6 days was optimum for SCP production with A. niger by using different fruit wastes as substrates, including pineapple, banana, orange, and watermelon wastes.

In a bioreactor, scale-up fermentation using Rhizopus oligosporus and co-culture of Rhizopus oligosporus and Saccharomyces cerevisiae resulted in 17 g/L and 19 g/L SCP, respectively. Our results were better than those reported by Sun et al. (2017), who obtained 13.5% SCP by using potato peel and wheat bran as a substrate and microbial strains of Bacillus subtilize, Aspergillus niger, and Candida tropica. The higher SCP yield could be due to the utilization of efficient microbial strains and optimized fermentation conditions.

Conclusion

Single-cell protein exhibits excellent qualities over plant- and animal-derived proteins and can be used to resolve universal problems related to protein scarcity, because it can be produced throughout the year and its production is not affected by season or climate changes. Another factor of importance is its healthy nutritional composition, such as phosphorus and potassium as well as lipids and carbohydrates, which establish SCP as a favorable dietary supplement. In this study, potato peel was selected as an inexpensive, readily available, and nutrient-rich substrate that resulted in maximum production of SCP and also reduced expenses of the experiment. This study also revealed an effective and useful solution to reduce environmental pollution caused by waste disposal of potato peel.