Yeast extract was obtained from Thermo Fisher Scientific (Difco^™, Israel); it consisted (w/w) of 34.76% C, 9.30% N, 6.31% H, and 0.54% S with a C:N ratio of 3.74. Red sea salt was obtained from Aquazone Ltd. (Israel) and contained (g kg⁻¹): Na 358.9, Cl 553.9, Mg 37.4, S 25.7, Ca 12.3, K 11.4, Sr. 0.234, B 0.126, F 0.037, I 0.002, and other minor trace elements. The trace elements solution was prepared as follows: (mg L⁻¹) ZnSO₄·7H₂O, 100; H₃BO₃, 300; CoCl₂·6H₂O, 200; CuSO₄, 6; NiCl₂·6H₂O, 20; Na₂MoO₄·2H₂O, 30; MnCl₂·2H₂O, 25 (Koller et al., 2008). The α-amylase enzyme from Bacillus amyloliquefaciens (≥250 U g⁻¹), amyloglucosidase enzyme from Aspergillus niger (≥260 U mL⁻¹), and alcalase® protease enzyme from Bacillus licheniformis Subtilisin A (≥2.972 U mL⁻¹) were purchased from Sigma-Aldrich (Israel). Thirteen different BW samples and their mixtures were collected from local restaurants and bakeries (Kfar Qara’, Israel).

Enzymatic hydrolysis of dry BW samples was carried out in three steps (Hudečková et al., 2017). First, 10 g of BW samples were homogenized with 100 mL of distilled water. The pH of the slurry was adjusted to 6.0; then, 4 mL of thermostable α-amylase (250 U g⁻¹) was added. The mixture was kept at 80°C for 3 h with magnetic stirring at 150 rpm. The liquefaction was curtailed by freezing the mixture at −20°C for 12 h. In the second hydrolysis step, saccharification, the pH of the liquefied suspension was adjusted to 4.2, and the saccharification was performed in liquefied suspension by adding 4 mL amyloglucoamylase (260 U mL⁻¹) at 60°C for 90 min. Heating the enzyme at 80°C for 5 min resulted in its inactivation, followed by subsequent cooling of the mixture to room temperature. Next, 1 mL of endopeptidase alcalase (2.972 U mL⁻¹) was used in the third step after pH adjustment to 8.0, followed by heating at 80°C for 24 h. The enzyme’s activity was nullified by subjecting the suspension to heating at 100°C for 5 min. Finally, the mixture was filtered and stored at 4°C until use.

The concentration of glucose in the hydrolysate was determined using the D-glucose Assay Kit (GOPOD, Megazyme, Ireland), following the manufacturer’s protocol. Briefly, 0.1 mL of the hydrolysate sample was mixed with 3.0 mL of the GOPOD reagent, which contains glucose oxidase and peroxidase. The reaction between D-glucose and the reagents forms a colored compound, which was incubated at 40–50°C for 20 min. The absorbance of the resulting solution was measured at 510 nm using a spectrophotometer (Infinite M Plex Elisa, Tecan, Austria). A reagent blank was used as a reference, and a glucose standard solution was prepared to create a calibration curve for accurate quantification. The glucose concentration in the samples was calculated based on the absorbance relative to the glucose standard.

H. mediterranei (ATCC 33500, CCM 3361) from the DSMZ (DSM 1411) culture collection was used for strain activation and culture. The following medium was employed for all H. mediterranei cultivation experiments under different conditions: 9.0–20.0% w/v of red sea salt powder, 0–24 mL L⁻¹ of trace element solution, 0–0.07% w/v of NH₄Cl, 0–0.06% w/v of KH₂PO₄, 0–5.5% w/v of glucose, 0–0.5% w/v of yeast extract, and 0–5.5% w/v of BW hydrolysate were added to 800 mL deionized water with a stirring rate of 300 rpm at 42°C for 3 h. Next, the solution was microfiltered under vacuum (Corning^® 500 mL, United States), and deionized water was added to complete the volume up to 1,000 mL. In the preparation of large volumes of medium (liters scale), the solution was autoclaved at 121°C for 30 min (Chauhan et al., 2020). Finally, the pH of the solution was adjusted (using 1 M NaOH or 1 M HCL solution) in the range of 2 to 13 and kept at 4°C until use.

Cell pellets obtained from 2 to 24 μL of inoculum solution were resuspended in 176–198 μL of red sea salt medium in a 96-well plate, sealed with an adhesive plate sealer, and cultivated at 42°C for 120 h with shaking at 150 rpm. The culture microplate was shaken for 30 s and then placed into the multi-reader (Infinite M Plex Elisa, Tecan, Austria). The optical density was measured at 600 nm at 25°C at specific times of 0, 24, 48, 72, 96, and 120 h (5 replicates) with a cell-free supernatant serving as a blank. After 120 h, the cultivation solutions were transferred to 2 mL microcentrifuge tubes (Tarasons, India), and the cell biomass was collected by centrifugation (Neofuge 13R high-speed refrigerated benchtop centrifuge, China), operating at 13,000 rpm for 10 min. Next, the biomass was washed with 200 μL deionized water and dried at 60°C for 24 h. The resulting biomass was weighed and analyzed for PHBV and protein content using Fourier transform infrared (FTIR).

Batch cultivation was conducted in culture flasks using a 100 mL solution containing 20 mL of H. mediterranei inoculum, 3 g of BW hydrolysate, and 20 g of red sea salt. The pH of the mixture was adjusted to 7.3, and then the mixture was cultured at 42°C for 72 h with constant shaking at 150 rpm. The culture flasks were sealed with aerated covers that allow oxygen to enter while keeping the environment controlled and preventing contamination. Following the cultivation, the cultures were subjected to centrifugation, washing, and drying at 60°C for 24 h. Finally, the dried biomass was weighed, and the components, including protein and PHBV, were examined using FTIR.

Following the cultivation, the cultures were subjected to centrifugation, washing, and drying at 60°C for 24 h. Finally, the dried biomass was weighed. A dry, fine powder of cell biomass (200 mg) was put in a preweighted crucible and weighed again. The crucible containing the biomass sample was placed in a muffle furnace preheated at 550°C. The sample was kept in the furnace for 5 h. The crucible was carefully removed from the furnace, cooled to 100°C, and then placed in a desiccator to cool further. The crucible with the remaining ash was weighed, and the ash content was calculated.

A 200 mg of H. mediterranei biomass and a 10 mL chloroform were subjected to reflux at 62°C for 12 h. Next, the cooled mixture was filtered using a Whatman filter (no. 4, Macherey-Nagel, Germany). The off-white solid (non-PHBV cell mass) was collected, dried at 60°C for 12 h and weighed. In parallel, the supernatant, i.e., a PHBV/chloroform solution, was gradually poured into 20 mL methanol. Finally, the PHBV precipitate was isolated by centrifugation at 4,000 × g for 30 min, dried at 60°C for 24 h, weighed, and stored at −20°C. Analysis of amino acids in H. mediterranei biomass.

First, 100 mg of H. mediterranei biomass was placed in a glass tube and hydrolyzed with 5 mL of 6 N HCl solution and phenol at 110°C for 22 h. Another 100 mg aliquot of this sample was first oxidized with formic acid and hydrogen peroxide at 2–8°C for 16 h. Next, the oxidized samples were dried under vacuum and then hydrolyzed with 5 mL of 6 N HCl and phenol at 110°C for 22 h. Aliquots of the two hydrolysates were dried by a vacuum centrifuge and dissolved in an amino acid sample buffer. The hydrolysate solutions were sonicated, vortexed, and filtered using a 0.45 μm nylon filter. Next, 20 μL of the hydrolysate solutions were injected into the Biochrom 30+ Amino-Acid-Analyzer (AminoLab, Analytical Laboratory Services, Israel). The amino acids were separated on an ion exchange column (Biochrom H-1552), derivatized with ninhydrin after eluting from the column, detected at 570 and 440 nm, and quantified against a standard.

H. mediterranei’s total protein quality was assessed by evaluating their amino acid composition and in-vitro protein digestibility-corrected amino acid score (PDCAAS) using the Megazyme assay kit (Wicklow, Ireland, https://www.megazyme.com/). To 500 mg of milled biomass sample, 19 mL of 0.06 N HCl was added, and the mixture was incubated at 37°C for 30 min with shaking at 120 rpm. Next, 1 mL of pepsin solution was added, and the sample was incubated at 37°C for 1 h. The pH was brought to 7.4 with 2 mL of 1 M Tris–HCL buffer, followed by the addition of 200 μL of a trypsin-chymotrypsin mixture. The sample was vortexed and incubated at 37°C for 4 h with shaking at 120 rpm, then placed in a boiling water bath for 10 min. Next, the sample was vortexed, cooled to 25°C for 20 min, mixed with 1 mL of 40% trichloroacetic acid solution, incubated at 4°C overnight, and then centrifuged at 25°C for 10 min at 13,800 rpm. A 10-fold dilution in acetate buffer (50 mM, pH 5.5) was performed before the colorimetric assay. PDCAAS values were computed by utilizing the Megazyme Mega-CalcTM program (K-PDCAAS Mega-Calc). A standard curve derived from L-glycine used to plot the absorbance values recorded at 570 nm against L-glycine concentrations spanning from 0 to 1 mM was used to assess the primary amine concentration (CI) in unidentified samples. The concentration of primary amines (in mM) in the unknown samples was determined using Equation 1, in which CI represents the unknown primary amine concentration, Y corresponds to the absorbance, B denotes the y-intercept, and A represents the slope of the line.

Equation 2 was used to calculate the primary amine concentration (C₂) in the initial sample solution. In this equation, CI represents the concentration of primary amines in the samples after dilution, D stands for the dilution factor applied to the samples before amine measurement, 1.25 denotes the dilution factor associated with trichloroacetic acid, W represents the weight of the sample (g), and 0.5 signifies the nominal quantity (g).

Amino acid constants were employed to compute the adjusted primary amine concentration (CN) for the individual amino acids, as indicated in Equation 3. In this equation, C2 represents the adjusted primary amine concentration in the initial sample solution (measured in mM), whereas proline, lysine, histidine, and arginine denote the concentrations of these respective amino acids in the original sample. The constants 2, 0.5, 0.2, and 0.2 are specific values associated with the corresponding amino acids.

In-vitro digestibility was determined utilizing Equation 4, which relies on established literature values for the rat model. To assess the corrected primary amine concentration (CN), the data fits were compared using a linear regression equation. In this equation, X denotes the corrected primary amine concentration for individual samples, M represents the slope of the regression line, B signifies the y-intercept, and 100 is the conversion factor used to convert percentages to grams.

The amino acid ratio and the identification of the limiting amino acids, expressed in grams per 100 grams of protein, were computed using the total crude protein values in the dry cell biomass, as demonstrated in Equation 5. The amino acid ratio within the sample was determined in accordance with the recommended values, as outlined in Equation 6. The in-vitro PDCAAS score was derived by multiplying the in-vitro digestibility obtained from Equation 4 by the limiting amino acid ratio (the smallest value) obtained from Equation 6.

Results were evaluated using a one-way ANOVA for the following variables: dry weight, ash content, and the C:N ratio of BW. Additionally, a two-way ANOVA with repeated measures was performed to investigate several abiotic factors (the pH, BW type, BW concentration, and red sea salt concentration) that affected the growth of H. mediterranei. Statistically significant differences were identified using Tukey multiple comparison tests using GraphPad Prism version 10, with significance indicated when p < 0.05.

Various BW samples (BW-1 to BW-13, Supplementary Figure S1) were collected from local restaurants and bakeries. Their commercial nutritional values, including carbohydrates, lipids, and proteins, are listed in Table 1. The carbohydrate contents in the BW samples ranged from 4.0 to 56.5% (w/w), lipids from 0 to 25.4% (w/w), and proteins from 9.1 to 27.5% (w/w). In addition, all BW samples were analyzed for their dry weight, ash content, C:N ratio, and the N-to-protein conversion factor. The corresponding values for dry weight ranged from 53.8 to 65.9% (w/w), ash content from 1.6 to 7.1% (w/w), C:N ratio from 7.1 to 23.4, and N-to-protein conversion factor from 4.9 to 7.9. The average N-to-protein conversion factor obtained for all BW samples in this study was 6.1, similar to that reported for wheat (5.8) (Fujihara et al., 2008). The protein concentration of BW was estimated by multiplying its nitrogen content, which was determined by elemental analysis, by the average N-to-protein conversion factor (6.1). Among the BW samples analyzed by one-way ANOVA and Tukey’s multiple comparisons test, BW-9 displayed the highest dry weight (65.6 ± 0.6% w/w) (significance, p < 0.0001). BW-2 exhibited the highest ash content (7.1 ± 0.4% w/w) (significance, p < 0.0001). BW-9 exhibited the highest C:N ratio (23.4 ± 0.1 w/w) (significance, p < 0.0001). BW-4 presented the highest N-to-protein conversion factor (7.9 ± 0.1). The large variability between different BW samples regarding the values of carbohydrates, lipids, proteins, dry weight, ash content, and the C:N ratio could result from differences in moisture content, mineral composition, protein composition, and nitrogen content in the BW samples. However, all BW samples were found to be rich in nitrogen, i.e., having a C:N ratio from 7.1 to 23.4, which is an essential factor for the growth of H. mediterranei and is suitable for microbial protein production.

The current study extensively investigated the key factors that impact the growth of H. mediterranei under various challenging conditions (Figure 1; Table 2). Specifically, the effect of the concentration of red sea salt (9–20.0%w/v), trace elements (0–2.4% w/v), NH₄Cl (0–0.7% w/v), KH₂PO₄ (0–0.66% w/v), glucose (0–5.5% w/v), and BW hydrolysate (DW of 0.5–5.5% w/v), as well as pH variations (2–13) were investigated. The enzymatic hydrolysis of BW samples and their mixtures was carried out with liquefaction by α-amylase, saccharification by amyloglucoamylase, and protein hydrolysis by endopeptidase alcalase. H. mediterranei cultivation was carried out in a 96-well plate at 42°C and 150 rpm for 120 h.

In the batch cultivation of H. mediterranei under optimized conditions, i.e., pH 7.3, at 42°C, and 150 rpm for 3 days, a mixture of 3.0% w/v of BW hydrolysate and 20.0% w/v of red sea salt was found to produce a maximum cell biomass concentration of 8.0 ± 0.1 g L⁻¹ with a productivity of 11.1 mg L⁻¹ h⁻¹. The biomass contained 36.0 ± 6.3% (w/w) of PHBV. The stoichiometry for the fermentation of H. mediterranei on BW was obtained by a CHNS elemental mass balance, revealing a chemical formula of [C₃₅₅H₆₆₀N₂₃S_2.6O₂₉₇] for BW and [C₂₄₁H₄₀₈N₂₇S₃O₃₉₀] for the cell biomass, as presented in Equation 7:

During fermentation, H. mediterranei utilizes BW as a carbon and nitrogen source, along with oxygen (O₂), to grow and produce biomass. This metabolic process generates carbon dioxide (CO₂) and water (H₂O) as byproducts. The reaction’s stoichiometry ensures a balanced distribution of all elements (C, H, N, S, and O). CO₂ was determined by comparing the C content in the BW hydrolysate to that in the produced biomass. The difference in C atoms for the CO₂ generated, and when scaled to 1 mole of cell biomass, 174 moles of CO₂ are produced, ensuring a complete C balance. More in detail, the BW hydrolysate has a chemical composition represented by [C₃₅₅H₆₆₀N₂₃S_2.6O₂₉₇], meaning that each unit of BW contains 355 moles of C atoms, 660 moles of H atoms, 23 moles of N atoms, 2.6 moles of S atoms, and 297 moles of O atoms. Similarly, the cell biomass produced from fermentation has the formula [C₂₄₁H₄₀₈N₂₇S₃O₃₉₀]. The total amount of C atoms in the BW hydrolysate is either incorporated into the cell biomass or released as CO₂. Since each mole of BW contains 355 moles of C atoms, and the resulting biomass contains 241 moles, the remaining 114 moles of C are released as CO₂. Therefore, for every 1 mole of BW hydrolysate consumed, 114 moles of CO₂ are produced. The final coefficient of CO₂ (174 moles) is determined in the equation by scaling the balance to 1 mole of cell biomass. The O₂ provided during the fermentation also contributes to the complete oxidation of C into CO₂. O₂ and H₂O are also accounted for in order to balance the equation completely based on H and O conservation.

Examples of cell biomass and PHA production by H. mediterranei utilizing various industrial and agricultural wastes compared to this study are presented in Table 3. These results show that PHA contents range from 6 to 57% (w/w) compared to 36.0% in the current study.

The ash content in the H. mediterranei biomass was 20.2% w/w, which surpasses that of several protein sources, including yeast (5–9.5% w/w), bacteria (3–7% w/w), plants (0.5–1.6% w/w), milk (5.8% w/w), beef (4% w/w), and eggs (3.9% w/w) (Nalage et al., 2016), but it was lower than that of seaweed (30–40% w/w) (Sonchaeng et al., 2023) (Table 4). This notable mineral content of H. mediterranei is due to its high salinity habitat.

The CHNS elemental analysis of the cell biomass revealed 28.9 ± 4.9% carbon, 3.8 ± 0.6% nitrogen, and 0.9 ± 0.2% sulfur. The C:N ratio was 7.6 ± 1.3, and the N content determined by the Kjeldahl method was 4.0 ± 0.3%, implying a protein-rich composition.

The total macroelements in cell biomass were 10.91 ± 0.13 g kg⁻¹ and mainly consisted of Na 7.05 ± 0.36 g kg⁻¹, K 1.04 ± 0.18 g kg⁻¹, P 0.91 ± 0.04 g kg⁻¹, Mg 0.85 ± 0.05 g kg⁻¹, S 0.73 ± 0.10 g kg⁻¹, and Ca 0.34 ± 0.02 g kg⁻¹. The total trace elements in cell biomass were 91.5 ± 0.9 mg kg⁻¹, mainly comprising Fe, Si, Al, Li, B, and Cu.

Archaea cell biomass had a low lipid content of 0.93 ± 0.02 g kg⁻¹; it mainly consisted of palmitic acid, stearic acid, 1-hexadecanol, and 1-tetracosanol, as well as a low level of carbohydrate content, 3.0 ± 0.2 g kg⁻¹; it consisted of arabinose, galactose, and glucose.

The amino acid profile of archaea cell biomass (Table 5) consists of 17 amino acids. Tryptophan remained undetectable under the analysis conditions, whereas both asparagine and glutamine were incorporated within the measured values of aspartic acid and glutamic acid, respectively. The archaea cell biomass comprised 358.0 ± 3.9 g kg⁻¹ of amino acids, 146.7 ± 4.8 g kg⁻¹ of essential amino acids (Hou and Wu, 2018), 211.3 ± 1.0 g kg⁻¹ of non-essential amino acids, and 71.2 ± 0.7 g kg⁻¹ of branch chain amino acids. According to the Food and Agriculture Organization of the United Nations (FAO, 2010), the total amino acids in rolled oats 101 g kg⁻¹, lentils 16.9 g kg⁻¹, wheat 86 g kg⁻¹, peas 170 g kg⁻¹, and kidney beans 165 g kg⁻¹, are lower than those of archaea cell biomass 358 g kg⁻¹, whereas casein 775 g kg⁻¹, has a higher amount of total amino acids. In addition, the essential amino acids in rolled oats 37 g kg⁻¹, lentils 67 g kg⁻¹, wheat 28 g kg⁻¹, peas 68 g kg⁻¹, and kidney beans 70 g kg⁻¹, are lower than those in archaea cell biomass 146.7 g kg⁻¹, whereas casein 347 g kg⁻¹, has a higher amount of essential amino acids.

In summary, the archaea cell biomass exhibits substantial protein content and quality. It consists of 358 g kg⁻¹ of proteins and 360 g kg⁻¹ of PHBV as the major constituents, whereas ash 201.5 g kg⁻¹, carbohydrates 3.0 g kg⁻¹, lipids 0.93 g kg⁻¹, and moisture 76.7 g kg⁻¹, contributed to cell biomass’s diverse composition (Figure 3).

FTIR analysis of the cell biomass revealed the characteristic vibration band of the ester carbonyl bond (C=O) at 1,724–1,734 cm⁻¹ and the stretching band of the C-H bond (CH₃) at 2,930 cm⁻¹, suggesting the presence of PHBV polymer in the cell biomass. The FTIR spectra corresponded to the typical profile of a copolymer PHBV, previously reported in H. mediterranei by Simó Cabrera et al. (2024). The 1,628 cm⁻¹ and 1,527 cm⁻¹ bands correspond to the protein amide I and II vibrations (Figure 4). Other peak frequency assignments, such as 1,179 cm⁻¹ and 978 cm⁻¹ for C-O-C vibration, 1,054 cm⁻¹ for C-O-C and C-C stretching as well as C-O-H bending, were also observed and match the values in the literature (Alsafadi and Al-Mashaqbeh, 2017).

The ¹³C NMR spectra of the pure PHBV polymer (Figure 5) displayed 9 singlet signals as follows: at 169.4 ppm for the two C=O carbons, 72.1 ppm for the -CH-CH₂CH₃ carbon, 67.8 ppm for the -CH-CH₃ carbon, 41.0 ppm and 39.0 ppm for the two -CH₂-C=O carbons, 27.1 ppm for the -CH₂-CH₃ carbon, 20.0 ppm for the -CH-CH₃ carbon, and 9.6 ppm for the -CH₂-CH₃ carbon. The ¹H NMR spectra of the PHBV polymer exhibited seven signals as follows: a sextet and quintet at 5.21–5.26 ppm for the -CH- hydrogens, two doublets of doublet at 2.42–2.61 ppm for the -CH ₂ -CO- hydrogens, a quintet at 1.58 ppm for the -CH ₂ -CH₃ hydrogens, a doublet at 1.24–1.26 ppm for the -CH-CH₃, and a triplet at 0.83 ppm for the -CH₂-CH₃ hydrogens (Ghosh et al., 2019). The ¹H- and ¹³C-NMR spectra of the extracted PHBV polymer (Figure 5) confirmed the structure of a copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate with a composition of 91:9 mol%, as determined by GC–MS.

The GPC analysis conducted on the extracted PHBV polymer from H. mediterranei cell biomass (Figure 6) revealed that the M_w = 3,827 kDa and M_n = 3,140 kDa values of PHBV produced from BW were notably higher than those of PHBV produced from glucose (M_w = 2,564 kDa, M_n = 2,115 kDa) but had a similar polydispersity index (PDI = M_w/M_n) of 1.212 and 1.219, respectively, which suggests the production of homogeneous PHBV polymers in both cases. The high M_w value of PHBV reflects its improved mechanical properties, including increased tensile strength and durability. Sato et al. reported that the M_w, M_n, and PDI values of PHBV produced by H. mediterranei in different media are 1,722–5,280 kDa, 800–3,500 kDa, and 1.6–2.2, respectively (Sato et al., 2021). This study demonstrated that under low concentrations of amino acid sources, 0.1–1 g L⁻¹, the M_w = 5,500 kDA and M_n = 3,500 kDa values of the produced PHBV were relatively high, possibly due to the amount of PHBV synthase produced under such conditions.

The thermal stabilities of cell biomass, isolated PHBV, and isolated protein were investigated by TGA and DSC (Figure 7). PHBV exhibited thermal stability with an initial weight decrease at 258°C, T_d at 277°C and T_m at 274°C. The protein underwent thermal degradation starting at 272°C, with a significant structural change at 312°C, T_d at 349°C, and a cross-linking transition at 370°C. Cell biomass experienced thermal degradation at 244°C, featuring a notable structural alteration at 255°C, and it reached a substantial decomposition at 269°C, along with a T_m value of 258°C and a cross-linking point at 538°C.

Archaea protein quality was assessed by evaluating its composition and in-vitro protein digestibility-corrected amino acid score (PDCAAS). The calculated parameters for H. mediterranei biomass were as follows: the in-vitro digestibility was 0.91, the first limiting amino acid (L-lysine) score was 0.86, the essential amino acid ratios ranged from 0.86 to 2.45, the PDCAAS was 0.78, and the crude protein percentage values were 35.8% (Table 5). According to the FAO recommendation (FAO, 2010), the PDCAAS values of high- and excellent-quality protein should be higher than 0.75 and 1.00, respectively (Zeng et al., 2022). Animal protein sources such as casein have PDCAAS values equal to 1 (Qin et al., 2022). Plant protein has lower PDCAAS values that range from 0.39 to 1.00, for instance, and the soy protein PDCAAS value is 0.92 (Rutherfurd et al., 2015), whereas the almond PDCAAS value is 0.39 (Weindl et al., 2020). Edible mycoproteins have a PDCAAS value of 0.35–0.70 (Ahmad et al., 2022), and yeast protein concentrate ranges from 0.82 to 0.90 (Ariëns et al., 2021). Regarding algae, the PDCAAS values range from 0.08 to 0.69 (Zeng et al., 2022). Therefore, archaea biomass derived from H. mediterranei has a relatively high protein quality, as indicated by its PDCAAS value (0.78).

The carbon footprint of 1,000 g of BW was estimated to be 1,555 g of CO₂ and 350 mL of CH₄ emissions (Carlsson and Uldal, 2009). Using BW (10 million tonnes worldwide) in microbial fermentation by H. mediterranei to produce cell biomass, rather than allowing it to decompose in landfills, could potentially reduce CH₄ emissions by 3.5 million tonnes/year while generating 6.5 million tonnes/year of CO₂. Furthermore, besides the financial and bioresource losses, BW, like any other food waste, causes significant damage to the broader environment by contributing to global warming, acidification, and eutrophication (Ben Rejeb et al., 2022). Therefore, reducing, recycling, or valorizing food waste can potentially save billions of dollars in global economic value, safeguard invaluable bioresources, and prevent the release of millions of tons of greenhouse gasses into the atmosphere.