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Single-cell protein (SCP) derived from microorganisms is widely recognized as a viable alternative protein source for the future. Nevertheless, the commercialization of yeast-based SCP is hampered by its relatively low protein content. Therefore, this study aimed to enhance the protein content of Saccharomyces cerevisiae via random mutagenesis. To achieve this, S. cerevisiae KCCM 51811, which exhibited the highest protein concentration among 20 edible S. cerevisiae strains, was selected as a chassis strain. Subsequently, a KCCM 51811 mutant library was constructed (through UV irradiation) and screened to isolate mutants exhibiting high protein content and/or concentration. Among the 174 mutant strains studied, the #126 mutant exhibited a remarkable 43% and 36% higher protein content and concentration, respectively, compared to the parental strain. Finally, the #126 mutant was cultured in a fed-batch system using molasses and corn-steep liquor, resulting in a protein concentration of 21.6 g/l in 100 h, which was 18% higher than that produced by the parental strain. These findings underscore the potential of our approach for the cost-effective production of foodgrade SCP.

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Nitrate pollution in groundwater is a serious problem worldwide, as its concentration in many areas exceeds the WHO-defined drinking water standard (50 mg/L). Hydrogen-oxidizing bacteria (HOB) are a group of microorganisms capable of producing single-cell protein (SCP) using hydrogen and oxygen. Furthermore, HOB can utilize various nitrogen sources, including nitrate. This study developed a novel hybrid biological-inorganic (HBI) system that coupled a new submersible water electrolysis system driven by renewable electricity with HOB fermentation for in-situ nitrate recovery from polluted groundwater and simultaneously upcycling it together with CO2 into single-cell protein. The performance of the novel HBI system was first evaluated in terms of bacterial growth and nitrate removal efficiency. With 5 V voltage applied and the initial nitrate concentration of 100 mg/L, the nitrate removal efficiency of 85.52 % and raw of 47.71 % (with a broad amino acid spectrum) were obtained. Besides, the HBI system was affected by the applied voltages and initial nitrogen concentrations. The water electrolysis with 3 and 4 V cannot provide sufficient H2 for HOB and the removal of nitrate was 57.12 % and 59.22 % at 180 h, while it reached 65.14 % and 65.42 % at 5 and 6 V, respectively. The nitrate removal efficiency reached 58.40 % and 50.72 % within 180 h with 200 and 300 mg/L initial nitrate concentrations, respectively. Moreover, a larger anion exchange membrane area promoted nitrate removal. The monitored of the determination of different forms of nitrogen indicated that around 60 % of the recovered nitrate was assimilated into cells, and 40 % was bio-converted to N2. The results demonstrate a potentially sustainable method for remediating nitrate contaminant in groundwater, upcycling waste nitrogen, CO2 sequestration and valorization of renewable electricity into food or feed.

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The production of alternative proteins is of great significance in the mitigation of food problems. This study proposes an integrated approach including protein extraction, enzymatic hydrolysis, and fermentation to produce both plant proteins and single-cell proteins as alternative proteins from tobacco leaves, a highly-abundant and protein-rich agricultural waste. Alkaline extraction of proteins before polysaccharide hydrolysis was found to be preferable for increasing the yields of plant proteins and mono-sugars. The combined use of pectinase-rich enzymes from Aspergillus brunneoviolaceus and hemicellulase-rich enzymes from Penicillium oxalicum achieved the release of 80.7 % of the sugars after 72 h. Cutaneotrichosporon cutaneum could simultaneously utilize multiple sugars, including galacturonic acid, in the enzymatic hydrolysate to produce single-cell proteins. Via this approach, 43.54 g crude proteins of high protein contents and rich in essential amino acids can be produced from 100.00 g waste tobacco leaves, providing a promising strategy for its valorization.

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Xanthobacter sp. SoF1 (SoF1) is an autotrophic hydrogen-oxidizing bacteria that produces protein-rich biomass and has potential to be an alternative protein source that is more environmentally sustainable than animal and plant derived proteins. A protein-rich powder derived from SoF1 was the test material in a 90-day repeated-dose oral toxicity study to explore major toxic effects, demonstrate target organs, and provide an estimate of a no-observed-adverse-effect level (NOAEL). Daily doses of 0 (vehicle only), 375, 750, and 1500 mg/kg bw/day of the test material were administered by gavage to 10 Han:WIST rats/sex/group. An additional group was administered 1290 mg/kg bw/day whey protein concentrate as positive control. No treatment-related adverse effects were observed, and no target organs were determined after 90/91 days of consecutive administration of the test item. A NOAEL of 1500 mg/kg bw/day was determined.

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The construction and optimization of microbial cell factories are crucial steps and key technologies in achieving green biomanufacturing. As concern has been aroused regarding the excessive carbon dioxide (CO2) emissions and food security, a new and promising research field, microbial conversion of CO2 into food compounds, has emerged. The research in this field not only holds significant implications for achieving the carbon peaking and carbon neutrality goals but also plays a role in maintaining food security. This paper provides a comprehensive review and outlook of the research on utilizing CO2 and its derived low-carbon chemicals for the production of food compounds, focusing on the production of glucose, sugar derivatives, and single-cell proteins and the development of artificial CO2 fixation pathways.

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The production of single cell protein (SCP) using lignocellulosic materials stands out as a promising route in the circular bioeconomy transition. However, multiple steps are necessary for lignocellulosics-to-SCP processes, involving chemical pretreatments and specific aerobic cultures. Whereas there are no studies that investigated the SCP production from lignocellulosics by using only biological processes and microbial biomass able to work both anaerobically and aerobically. In this view, the valorisation of industrial hemp (Cannabis sativa L.) biomass residues (HBRs), specifically hurds and a mix of leaves and inflorescences, combined with cheese whey (CW) was investigated through a semi-continuous acidogenic co-fermentation process (co-AF). The aim of this study was to maximise HBRs conversion into VFAs to be further used as carbon-rich substrates for SCP production. Different process conditions were tested by either removing CW or increasing the amount of HBRs in terms of VS (i.e., two and four times) to evaluate the performance of the co-AF process. Increasing HBRs resulted in a proportional increase in VFA production up to 3115 mg HAc L-1, with experimental production nearly 40% higher than theoretical predictions. The synergy between HBRs and CW was demonstrated, proving the latter as essential to improve the biodegradability of the former. The produced VFAs were subsequently tested as substrates for SCP synthesis in batch aerobic tests. A biomass concentration of 2.43 g TSS L-1 was achieved with a C/N ratio of 5.0 and a pH of 9.0 after two days of aerobic fermentation, reaching a protein content of 42% (g protein per g TSS). These results demonstrate the overall feasibility of the VFA-mediated HBR-to-SCP valorisation process.

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Today, feeding protein supply according to need in high-yielding lactating cows has become a big challenge. Protein is the most costly bulk constituent of animal diet, and the price of protein sources is increasing steadily, which is different from milk price rising. Therefore, one way for farmers to reduce feed costs is to reduce dietary protein share. Ruminants obtain their amino acids from 2 sources: amino acids from ruminally undegraded protein (RUP) and microbial protein synthesized in the rumen. A key goal in ruminant nutrition strategies, maximizing the use of rumen degradable protein (RDP), is through its efficient conversion into microbial protein. Urea is a supplement and a possible source of non-protein nitrogen (NPN) in ruminants' diets which meets bacteria's ammonia needs. Rumen ammonia sources include protein, peptides, amino acids, and other nitrogen-bearing compounds. As urea, uric acid, nitrate, and possibly nucleic acid are rapidly converted to ammonia, the ammonia reservoir indicates that the ruminal metabolism of ammonia is relatively small. Bacteria in the rumen can obtain between 40 and 95 percent of their nitrogen demand from ammonia, depending on their diet. Using NPN (non-protein nitrogen) as a reliable nitrogen source for ruminants was recognized over 100 years ago. Urea is quickly released in the rumen, its use in the diet is limited due to ammonia toxicity. So, the solution to this problem is that the product in nitrogen release rate from urea changes according to the digestion of fibers in the rumen. In the past, several slow-release products were made and evaluated. Slow-release urea (SRU) sources will also affect microbial growth and livestock performance compared to conventional plant protein sources. Acceptance of SRU sources, depending on their price compared to conventional plant protein ingredients is feasible. Studies has shown that the use of slow-release urea did not have a negative effect on digestibility, rumen parameters, milk production and livestock performance. Single-cell protein (SCP) is an emerging alternative protein source, currently being mainly studied for chicken and aquatic species.Finally, it is concluded that slow release urea can be used in feeding ruminants without any side effects.

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In this study, the characterization of fish oil (FO) emulsion gel (EGEL) containing single cell protein (SCP) produced from Lentinula edodes (L. edodes) and its potential inhibition against Acinetobacter baumannii (A. baumannii) were investigated. Oil extracted from the fish liver was emulsified with tween 80 and water, and then gelled using gelatin with the assistance of an ultrasonic homogenizer. The characteristics and surface analysis of SCP-EGEL were examined using FTIR (Fourier-transform infrared spectroscopy) and SEM (Scanning electron microscope). The particle size distribution and zeta potential of SCP-EGEL were measured using a Malvern Zetasizer. When SCP-EGEL was applied to the surface of the medium inoculated with A. baumannii, the inhibition zone (IZ) was 8.2 mm. An expansion of the IZ was observed (10.2 mm) when SCP-EGEL was applied to a fish skin (FS) surface prepared in the shape of a 6-mm diameter disc. In the SEM images, when SCP was added to lipo gel, the gel structure appeared flattened or swollen in some areas. The appearance of SCP cells being covered with gel gave the impression that they have a secondary wall. Therefore, the resulting complex can potentially be used as an additive in animal and human nutrition, in functional food coatings to suppress A. baumannii, and in fish feed to enrich it with protein.

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The present work examined the production of single-cell protein (SCP) by a newly isolated strain of Kluyveromyces marxianus EXF-5288 under increased lactose concentration of deproteinized cheese whey (DCW) and different temperatures (in °C: 20.0, 25.0, 30.0 and 35.0). To the best of the authors' knowledge, this is the first report examining the ability of Kluyveromyces marxianus species to produce SCP at T = 20.0 °C. Different culture temperatures led to significant differences in the strain's growth, while maximum biomass and SCP production (14.24 ± 0.70 and 6.14 ± 0.66 g/L, respectively) were observed in the cultivation of K. marxianus strain EXF-5288 in shake-flask cultures at T = 20.0 °C. Increased DCW lactose concentrations (35.0-100.0 g/L) led to increased ethanol production (Ethmax = 35.5 ± 0.2 g/L), suggesting that K. marxianus strain EXF-5288 is "Crabtree-positive". Batch-bioreactor trials shifted the strain's metabolism to alcoholic fermentation, favoring ethanol production. Surprisingly, K. marxianus strain EXF-5288 was able to catabolize the produced ethanol under limited carbon presence in the medium. The dominant amino acids in SCP were glutamate (15.5 mg/g), aspartic acid (12.0 mg/g) and valine (9.5 mg/g), representing a balanced nutritional profile.

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Increasing global consumption of protein over the last five decades, coupled with concerns about the impact on emissions of animal-based protein production, has created interest in alternative protein sources. Microbial proteins (MPs), derived through the fermentation of agro-industrial byproducts, present a promising option. This review assesses a century of advancements in this domain. We conducted a comprehensive review and meta-analysis, examining 347 relevant research papers to identify trends, technological advancements, and key influencing factors in the production of MP. The analysis covered the types of feedstocks and microbes, fermentation methods, and the implications of nucleic acid content on the food-grade quality of proteins. A conditional inference tree model and Bayesian factor were used to ascertain the impact of various parameters on protein content. Out of all the studied parameters, such as type of feedstock (lignocellulose, free sugars, gases, and others), type of fermentation (solid, liquid, gas), type of microbe (bacteria, fungi, yeast, and mix), and operating parameters (temperature, time, and pH), the type of fermentation and microbe were identified as the largest influences on protein content. Gas and liquid fermentation demonstrated higher protein content, averaging 52% and 42%, respectively. Among microbes, bacterial species produced a higher protein content of 51%. The suitable operating parameters, such as pH, time, and temperature, were also identified for different microbes. The results point to opportunities for continued innovation in feedstock, microbes, and regulatory alignment to fully realize the potential of MP in contributing to global food security and sustainability goals.

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Single cell protein (SCP, or microbial protein) is one of the emerging alternative protein sources to address the global challenge of food insecurity. Recently, the SCP produced from methane has attracted substantial attention since methane is a renewable resource attainable from anaerobic digestion. However, the supply of methane, an insoluble gas in water, is one of the major challenges in producing methane-based SCP. This work developed a novel bioreactor configuration, in which hollow fiber membrane was used for efficient methane supply while microorganisms were growing in the suspended form favourable for the biomass harvest. Over a 312-day operation, the impacts of three critical parameters on the SCP production were investigated, including the ratio of methane loading to ammonium loading, the ratio of methane loading to oxygen loading, and the sludge retention time (SRT). Under the condition of 4 g CH4/g NH4+, 4 g O2/g CH4, and SRT of 4 days, the highest SCP production yield was observed and determined to be 1.36 g SCP/g CH4 and 5.05 g SCP/g N, respectively. The protein content was up to 67 %, which is higher than the majority of reported values to date. Moreover, the methane and ammonium utilization efficiencies were both close to 100 %, suggesting the highly efficient utilization of substrates in this new bioreactor configuration. A high relative abundance of essential amino acids (EAA) above 42 % was achieved, representing the highest EAA content reported. These findings provide valuable insights into SCP production using methane as a feedstock.

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The global food system is shifting towards cellular agriculture, a second domestication marked by cultivating microorganisms and tissues for sustainable food production. This involves tissue engineering, precision fermentation, and microbial biomass fermentation to establish food value chains independent of traditional agriculture. However, these techniques rely on growth media sourced from agricultural, chemical (fossil fuels), and mining supply chains, raising concerns about land use competition, emissions, and resource depletion. Fermentable sugars, nitrogen, and phosphates are key ingredients derived from starch crops, energy-intensive fossil fuel based processes, and finite phosphorus resources, respectively. This review explores sustainable alternatives to reduce land use and emissions associated with cellular agriculture media ingredients. Sustainable alternatives to first generation sugars (lignocellulosic substrates, sidestreams, and gaseous feedstocks), sustainable nitrogen sources (sidestreams, green ammonia, biological nitrogen fixation), and efficient use of phosphates are reviewed. Especially cellulosic sugars, gaseous chemoautotrophic feedstocks, green ammonia, and phosphate recycling are the most promising technologies but economic constraints hinder large-scale adoption, necessitating more efficient processes and cost reduction. Collaborative efforts are vital for a biotechnological future grounded in sustainable feedstocks, mitigating competition with agricultural land and emissions.

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An experiment was conducted to evaluate the effects of single-cell protein (SCP), as a valuable by-product and substitute for soybean meal, and multistrain probiotics on the performance, blood biochemistry, intestinal histomorphology, ileal microbiota, and immune responses in laying hens. Leghorn laying hens were randomly assigned to nine experimental treatments with eight replicates of five birds each. Dietary treatments consisted of three levels of replacement of soybean meal with SCP (0.0%, 30%, and 60%) and three levels of probiotics (0.0, 50, 100 mg/kg) offered through 70 days of main recording. The result indicated that replacing 30% soybean meal with SCP in the diet significantly increased the eggshell weight, thickness, and strength; however, hens receiving 60% SCP replacement showed lower productive performance. During the second 35-d trial, hens receiving at least 50 mg/kg probiotics produced higher eggshell weight, thickness, and strength than the control hens. Remarkable lower levels of serum cholesterol content were observed in the SCP and probiotic-supplemented diets. The significant reduction in the ileal pH, Escherichia coli and Clostridium spp. enumerations and a remarkable increase in Lactobacillus count was observed after feeding incremental levels of probiotics. The highest villus height to crypt depth ratio, antibody titer against sheep red blood cells (in secondary response) and Newcastle disease vaccine, as well as egg production and egg mass, were found in the hens that received 30% SCP replacement in combination with 50 or 100 mg/kg probiotic (SCP × Probiotic). The present study concluded that feeding SCP, as a cheap protein alternative source, along with probiotics while reducing production costs can support productive performance via modulating the intestinal microbiomes and health indices and fortifying the immune system in laying hens.

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Transformer-based models have revolutionized single cell RNA-seq (scRNA-seq) data analysis. However, their applicability is challenged by the complexity and scale of single-cell multi-omics data. Here a novel single-cell multi-modal/multi-task transformer (scmFormer) is proposed to fill up the existing blank of integrating single-cell proteomics with other omics data. Through systematic benchmarking, it is demonstrated that scmFormer excels in integrating large-scale single-cell multimodal data and heterogeneous multi-batch paired multi-omics data, while preserving shared information across batchs and distinct biological information. scmFormer achieves 54.5% higher average F1 score compared to the second method in transferring cell-type labels from single-cell transcriptomics to proteomics data. Using COVID-19 datasets, it is presented that scmFormer successfully integrates over 1.48 million cells on a personal computer. Moreover, it is also proved that scmFormer performs better than existing methods on generating the unmeasured modality and is well-suited for spatial multi-omic data. Thus, scmFormer is a powerful and comprehensive tool for analyzing single-cell multi-omics data.

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Single cell western blot (scWB) is one of the most important methods for cellular heterogeneity profiling. However, current scWB based on conventional photoactive polyacrylamide hydrogel material suffers from the tradeoff between in-gel probing and separation resolution. Here, a highly sensitive temperature-controlled single-cell western blotting (tc-scWB) method is introduced, which is based on a thermo/photo-dualistic-sensitive polyacrylamide hydrogel, namely acrylic acid-functionalized graphene oxide (AFGO) assisted, N-isopropylacrylamide modified polyacrylamide (ANP) hydrogel. The ANP hydrogel is contracted at high-temperature to constrain protein band diffusion during microchip electrophoretic separation, while the gel aperture is expanded under low-temperature for better antibody penetration into the hydrogel. The tc-scWB method enables the separation and profiling of small-molecule-weight proteins with highly crosslinked gel (12% T) in SDS-PAGE. The tc-scWB is demonstrated on three metabolic and ER stress-specific proteins (CHOP, MDH2 and FH) in four pancreatic cell subtypes, revealing the expression of key enzymes in the Krebs cycle is upregulated with enhanced ER stress. It is found that ER stress can regulate crucial enzyme (MDH2 and FH) activities of metabolic cascade in cancer cells, boosting aerobic respiration to attenuate the Warburg effect and promote cell apoptosis. The tc-scWB is a general toolbox for the analysis of low-abundance small-molecular functional proteins at the single-cell level.

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A recent focus has been on the recovery of single-cell protein and other nutritionally valuable bioproducts, such as Coenzyme Q10 (CoQ10) from purple non-sulfur bacteria (PNSB) biomass following wastewater treatment. However, due to PNSB's peculiar cell envelope (e.g., increased membrane cross-section for energy transduction) and relatively smaller cell size compared to well-studied microbial protein sources like yeast and microalgae, the effectiveness of common cell disruption methods for protein quantification from PNSB may differ. Thus, this study examines the efficiency of selected chemical (NaOH and EDTA), mechanical (homogenization and bead milling), physical (thermal and bath/probe sonication), and combined chemical-mechanical/physical treatment techniques on the PNSB cell lysis. PNSB biomass was recovered from the treatment of gas-to-liquid process water. Biomass protein and CoQ10 contents were quantified based on extraction efficiency. Considering single-treatment techniques, bead milling resulted in the best protein yields (p < 0.001), with the other techniques resulting in poor yields. However, the NaOH-assisted sonication (combined chemical/physical treatment technique) resulted in similar protein recovery (p = 1.00) with bead milling, with the former having a better amino acid profile. For example, close to 50% of the amino acids, such as sensitive ones like tryptophan, threonine, cystine, and methionine, were detected in higher concentrations in NaOH-assisted sonication (>10% relative difference) compared to bead-milling due to its less disruptive nature and improved solubility of amino acids in alkaline conditions. Overall, PNSB required more intensive protein extraction techniques than were reported to be effective on other single-cell organisms. NaOH was the preferred chemical for chemical-aided mechanical/physical extraction as EDTA was observed to interfere with the Lowry protein kit, resulting in significantly lower concentrations. However, EDTA was the preferred chemical agent for CoQ10 extraction and quantification. CoQ10 extraction efficiency was also suspected to be adversely influenced by pH and temperature.

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The polyextremophilic Galdieria sulphuraria is emerging as a promising microalgal species for food applications. This work explores the potential of heterotrophically cultivated G. sulphuraria as a protein producer for human consumption. To this end, the performances of four G. sulphuraria strains grown under the same conditions were compared. Amino acid profiles varied among strains and growth phases, but all samples met FAO dietary requirements for adults. The specific growth rates were between 1.01 and 1.48 day-1. After glucose depletion, all strains showed an increase of 38-49 % in nitrogen content within 48 h, reaching 7.8-12.0 % w/w. An opposite trend was observed in protein bioaccessibility, which decreased on average from 69 % during the exponential phase to a minimum of 32 % 48 h after stationary phase, with significant differences among the strains. Therefore, selecting the appropriate strain and harvesting time is crucial for successful single-cell protein production.

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This study evaluated the nutritional quality of different microbial biomass samples by assessing their protein digestibility and carbohydrate fermentability in the colon using in vitro methods. Four microbial samples were produced: one hydrogen-oxidizing bacterial strain (Nocardioides nitrophenolicus KGS-27), two strains of filamentous fungi (Rhizopus oligosporus and Paecilomyces variotii), and one yeast strain (Rhodotorula babjevae). The microorganisms were grown in bioreactors, harvested and dried before analysis. The commercial fungal product Quorn was used as a reference. The protein digestibility of the microbial samples was analysed using the INFOGEST in vitro model, followed by quantification of N-terminal amine groups. An in vitro faecal fermentation experiment was also performed to evaluate the degradation of carbohydrates in microbial biomass samples and formation of short-chain fatty acids (SCFA). The fungal biomass samples had higher protein hydrolysis (60-75 %) than the bacterial sample (12 %) and Quorn (45 %), while the yeast biomass had the highest protein digestibility (85 %). Heat-treatment of the biomass significantly reduced its protein digestibility. Total dietary fibre (DF) content of fungal biomass was 31 - 43 %(DW), mostly insoluble, whereas the bacterial biomass contained mainly soluble DF (total DF: 25.7 %, of which 23.5 % were soluble and 2.2 % insoluble). After 24 h of colonic in vitro fermentation, SCFA production from the biomass of Paecilomyces, Quorn and Rhodotorula was similar to that of wheat bran, while 17 % and 32 % less SCFA were produced from the biomass of Rhizopus and the bacterial strain, respectively. Further studies are needed to clarify the reasons for the observed differences in protein digestibility and DF fermentability, especially regarding the cell wall structures and role of post-processing.

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Despite the large carbon footprint of livestock production, animal protein consumption has grown over the past several decades, necessitating new approaches to sustainable animal protein production. In this techno-economic analysis, single cell protein (SCP) produced via gas fermentation of carbon dioxide, oxygen, and hydrogen is studied as an animal feed source to replace fishmeal or soybean meal. Using wind-powered water electrolysis to produce hydrogen and oxygen with carbon dioxide captured from corn ethanol, the minimum selling price (MSP) of SCP is determined to be $2070 per metric ton. An emissions comparison between SCP, fishmeal, and soybean meal shows that SCP has a carbon intensity as low as 0.73 kg CO2-equiv/kg protein, while fishmeal and soybean meal have an average carbon intensity of 2.72 kg CO2-equiv/kg protein and 0.85 kg CO2-equiv/kg protein, respectively. Moreover, SCP production would occupy 0.4% of the land per ton of protein produced compared to soybean meal and would disturb less than 0.1% of the marine ecosystem currently disturbed by fishmeal harvesting practices. These results show promise for the future economic viability of SCP as a protein source in animal feed and indicate significant environmental benefits compared to other animal feed protein sources.

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