Single Cell Protein Production Research Articles

Microbial electrosynthesis of single cell protein and methane by coupling fast-growing Methanococcus maripaludis with microbial electrolysis cells

Single cell protein (SCP) is a promising alternative protein source, as its production bypasses the disadvantages of animal protein production in industrial agriculture. Coupling a fast-growing hydrogen consuming organism with microbial electrolysis cells (MECs) could be a viable method for SCP production. In this study, a fast-growing and protein-rich methanogen, Methanococcus maripaludis was selected as the primary SCP source. The inoculation of M. maripaludis in MECs triggered cell synthesis with methane production. The doubling time measured was 11.2 h and the specific growth rate was 0.062 1/h. The highest SCP production rate was 13.7 mg/L/h. In the dried biomass, the weight of protein was over 60 %. Amino acid profiling of the harvested biomass demonstrated high abundance of essential amino acids. The electron flux analysis indicated that 31.3 % electrons in the electrochemical systems were directed into SCP synthesis. These results illustrated the potential for SCP production by coupling a fast-growing methanogen with MECs.

Potential of enriched phototrophic purple bacteria for H2 bioconversion into single cell protein

Single cell protein (SCP) has emerged as an alternative protein source, potentially based on the recovery of carbon and nutrients from waste-derived resources as part of the circular economy. From those resources, gaseous substrates have the advantage of an easy sterilization, allowing the production of pathogen-free SCP. Sterile gaseous substrates allow producing pathogen-free SCP. This study evaluated the use of an enriched phototrophic purple bacteria (PPB) consortium for SCP production using H2 and CO2 as electron and C sources. The influence of pH (6.0–8.5), temperature (15–50 °C) and light intensity (0–50 W·m−2) on the growth kinetics and biomass yields was investigated using batch tests. Optimal conditions were found at pH 7, 25 °C and light intensities over 30 W·m−2. High biomass and protein yields were achieved (~ 1 g CODbiomass·g CODH2consumed−1 and 3.9–4.4 g protein·g H2−1) regardless of the environmental conditions, being amongst the highest values reported from gaseous streams. These high yields were obtained thanks to the use of light as a sole energy source by the PPB consortium, allowing a total utilization of H2 for growth. Hydrogen uptake rates varied considerably, with values up to 61 ± 5 mg COD·d−1 for the overall H2 consumption rates and 2.00 ± 0.14 g COD·g COD−1·d−1 for the maximum specific uptake rates under optimal growth conditions. The latter value was estimated using a mechanistic model able to represent PPB growth on H2. The biomass exhibited high protein contents (>50 % w/w) and adequate amino acid profiles, showing its suitability as SCP for feed. PPB were the dominant bacteria during the experiments (relative abundance over 80 % in most tests), with a stable population dominated by Rhodobacter sp. and Rhodopseudomonas sp. This study demonstrates the potential of enriched PPB cultures for H2 bioconversion into SCP.

Effect of different types of bacterial single cell protein on feed intake, digestibility, growth and body composition of Pacific white shrimp (Penaeus vannamei)

This study assessed the potential of four bacterial (Methylococcus capsulatus) single cell protein (SCP) products as alternative protein sources for Pacific white shrimp (Penaeus vannamei) diets. A growth trial and a digestibility trial were undertaken, during which the bacterial SCP products were compared with a high-quality fishmeal and a soy protein concentrate, regarding their impact on ingredient digestibility, growth, feed intake and whole-body composition of juvenile P. vannamei. Seven diets were formulated; one reference diet (REF) and six test diets. The test diets consisted of 85% of the REF diet and 15% of a test ingredient. Ingredients tested were four bacterial SCP products (SCP1–4), which differed in processing conditions, fishmeal (FM) and soy protein concentrate (SoyProt). Growth and feed utilization were similar for P. vannamei fed either the FM diet or one of the bacterial SCP diets, whilst lowest growth and feed utilization were observed for shrimp fed the SoyProt diet. Final whole-body protein content did not differ between shrimp fed the FM diet or one of the four bacterial SCP diets. However, shrimp fed the SCP diets had a significantly higher final phosphorus body content and a higher phosphorus retention than shrimp fed the FM or SoyProt diets. This indicates a higher phosphorus availability in the bacterial SCP products compared to FM and SoyProt. Protein digestibility of the SCP products was similar to FM, whilst amino acid (AA) digestibility was comparable to FM for three of the four SCP products (SCP1, SCP2 and SCP4). The SCP3 product showed the lowest digestibility for most AA, indicating a possible influence of processing conditions on AA availability of bacterial SCPs. Overall, this study highlights that bacterial SCP originating from M. capsulatus is a viable alternative protein source for Pacific white shrimp diets, but processing conditions should be taken into account.

Rhodotorula paludigena CM33 cultivation process development for high β-carotene single cell protein production

Rhodotorula yeast has potential in terms of biotechnology for the production of various compounds for the environmental, industrial, and medical sectors. It is well known to generate carotenoid pigments that are beneficially distinguished. This investigation aimed to optimize the growth of Rhodotorula paludigena CM33 and its production of β-carotene. Response Surface Methodology was employed to optimize temperature, ammonium sulfate, and ferric chloride concentrations in a 5L-bioreactor batch culture. The optimal conditions of 30 °C, 6.2 g/L of ammonium sulfate, and 75.0 mg/L of ferric chloride provided biomass of 17.71 g/L, a specific growth rate of 0.22 h−1, and β-carotene concentration of 40.29 mg/L. Upon scaling up the cultivation process, the 22L-bioreactor displayed similar growth patterns to those of the 5L-bioreactor. However, the 22L-bioreactor exhibited higher product yields in a shorter time period. The maximum biomass production of 18.96 ± 1.31 g/L and the maximum β-carotene production of 42.81 ± 0.61 mg/L were achieved at 28 h. This scaling-up strategy resulted in improved production efficiency. Additionally, the nutritional value of the cells was evaluated, suggesting their potential as animal feed supplements. R. paludigena CM33 demonstrated a β-carotene single-cell protein content, making it a valuable source for aquaculture and livestock feed. These findings highlight the potential of this yeast strain for large-scale production of β-carotene and single-cell protein, offering nutritional benefits for animal feed.

Reusing wastewater from Coffea arabica processing to produce single-cell protein using Candida sorboxylosa: Optimizing of culture conditions.

Coffee is a crop of significant socioeconomic importance, and the reuse of agri-food by-products and biowaste has great potential across several industries. Coffee wastewater (CWW) is a valuable resource containing essential nutrients that can be utilized by Candida sorboxylosa for single-cell protein (SCP) production. This utilization contributes to mitigating the negative impacts of agro-industrial waste. The optimization of culture conditions using the design of experiments (DoE) technique is crucial in understanding the environmental factors influencing metabolite production. In our study, the DoE technique was employed to analyze culture conditions, including room temperature, pH 8.4, agitation at 200 rpm, a headspace of 60% (v/v), and an inoculum of 0.75 DO600nm over 28-h period. This approach resulted in a remarkable SCP yield of 64.4% and dry cell weight (DCW) of 2.26 g/L. It is noteworthy that there is no literature reporting SCP production under alkaline pH conditions in yeast. Interestingly, our work demonstrated that an alkaline pH of 8.4 significantly influenced SCP production by C. sorboxylosa. The DoE technique proved to be an efficient statistical tool for optimizing culture conditions, offering several advantages, such as: (i) conducting cultures at room temperature to minimize unnecessary energy consumption; (ii) reducing the incubation time from 46 to 28 h, thereby enhancing overall productivity; (iii) achieving 1.7-fold increase in SCP yield compared to previous basal production levels.

Conversion of Orange and Pineapple Fruit Peel Waste into Single Cell Protein Using Saccharomyces Cerevisiae

The biological treatment of fruit wastes into single-cell protein has the potential to address the global protein shortage problem by producing a cost-effective product for food and feed. Pollution may be reduced if the substrate was made from the leftovers from the food processing industry. Herein, the submerged fermentation method was to make single-cell protein from pineapple and orange peels. Results indicated that fruit wastes analysed were highly hydrolysable. Proximate analysis of fruit wastes protein content of orange peel (10.73±0.02%) was significantly higher (P<0.05) than the protein content of pineapple peels (7.50±0.016%). When Saccharomyces cerevisiae was cultivated on fruit hydrolysate medium (FHM) without supplementation, the proportion of protein in single-cell protein was substantially lower, which produced (41.50±0.05% and 37.97±0.006%) total carbohydrate with (29.73±0.031% and 34.50±0.4%) crude protein, in both pineapple and orange medium respectively. The introduction of glucose to the supplemented fruit hydrolysate medium (GSFHM), increased the level of protein (45.50±0.031 and 37.73±0.011%) within the yeast cell, the comparison of treatment showed P<0.05, thus we can conclude that there is significant difference in the overall comparison of treatments in most of the parameters. The ITS2 region sequence analysis of Saccharomyces cerevisiae MA851180, revealed 99% identity with Saccharomyces cerevisiae. Thus, Saccharomyces cerevisiae produces higher amount of Single Cell Protein by submerged fermentation of pineapple peel hydrolysate as compared to orange peel hydrolysate. Hence, the single cell protein production by yeast depends on the growth substrates or media composition. SCP from yeast using fruit waste can serve as cheaper alternative to the conventional protein gotten from plant and animal and by selecting the most suitable prospective microbe and cost-effective technique.

Economical production of Pichia pastoris single cell protein from methanol at industrial pilot scale

BackgroundMethanol, synthesized from CO2, is a potentially sustainable one-carbon (C1) resource for biomanufacturing. The use of methanol as a feedstock to produce single cell protein (SCP) has been investigated for decades as an alternative to alleviate the high global demand for animal-derived proteins. The methylotrophic yeast Pichia pastoris is an ideal host for methanol-based SCP synthesis due to its natural methanol assimilation ability. However, improving methanol utilization, tolerance to higher temperature, and the protein content of P. pastoris are also current challenges, which are of great significance to the economical industrial application using methanol as a feedstock for SCP production.ResultsIn the present work, adaptive laboratory evolution (ALE) has been employed to overcome the low methanol utilization efficiency and intolerance to a higher temperature of 33 °C in P. pastoris, associated with reduced carbon loss due to the lessened detoxification of intracellular formaldehyde through the dissimilation pathway and cell wall rearrangement to temperature stress resistance following long-term evolution as revealed by transcriptomic and phenotypic analysis. By strengthening nitrogen metabolism and impairing cell wall synthesis, metabolic engineering further increased protein content. Finally, the engineered strain via multi-strategy produced high levels of SCP from methanol in a pilot-scale fed-batch culture at 33 °C with a biomass of 63.37 g DCW/L, methanol conversion rate of 0.43 g DCW/g, and protein content of 0.506 g/g DCW. SCP obtained from P. pastoris contains a higher percentage of protein compared to conventional foods like soy, fish, meat, whole milk, and is a source of essential amino acids, including methionine, lysine, and branched-chain amino acids (BCAAs: valine, isoleucine, leucine).ConclusionsThis study clarified the unique mechanism of P. pastoris for efficient methanol utilization, higher temperature resistance, and high protein synthesis, providing a P. pastoris cell factory for SCP production with environmental, economic, and nutritional benefits.

A zero-waste approach towards a sustainable waste management of apple: Extraction of value-added products and their application as edible coating

This study aimed to valorize apple pomace (AP) for the production of value-added products in a zero-waste system. A Box-Behnken design was used to optimize the microwave-assistant extraction factors on simultaneous extraction of pectin (APP) and phenolic compounds (APPC). Under optimum conditions (microwave power of 600 W, irradiation time of 6 min, liquid/solid ratio of 45, and pH of 1.5), the APP and APPC yields were 28.37 ± 1.19% and 3.42 ± 0.48%, respectively. The solid residue after co-extraction was used as a carbon source for microbial production of pullulan and single cell protein (SCP) and the maximum production yields of pullulan (43.7 g/L) and SCP (33.5%) were gained after sequential homogenization, ultrasonication and enzymatic treatment (H + U + E). The molecular weight of APP and pullulan were found to be 690.293 and 366.246 kDa, respectively. Also, the FT-IR and 1H NMR analysis confirmed the presence of pectin and pullulan in the resulting sample and their XRD pattern showed an amorphous structure. The results of DPPH, ABTS and FRAP assays indicated that the antioxidant capacity of apple pomace phenolics was comparable with vitamin C and BHA at high concentrations. Moreover, the coating with 50:50 ratios of APP and pullulan enriched with 15% APPC was selected as the best edible coating for strawberry that prolonged its shelf life until 24 days at 4 °C. The outcomes revealed that the extraction of these valuable compounds from AP in line with the sustainable agriculture development not only reduces environmental problems, but also the obtained products can be used to coat fruits.

Single Cell Protein Production by Isolated And Identified Bacteria Using Crude Oil As A Carbon Source

Single Cell Protein Production by Isolated And Identified Bacteria Using Crude Oil As A Carbon Source

Single-Cell Protein Production from Industrial Off-Gas through Acetate: Techno-Economic Analysis for a Coupled Fermentation Approach

Third-generation (3G) biorefineries harnessing industrial off-gases have received significant attention in the transition towards a sustainable circular economy. However, uncertainties surrounding their techno-economic feasibility are hampering widespread commercialization to date. This study investigates the production of single-cell protein (SCP), a sustainable alternative food and feed protein, from steel mill off-gas through an efficient coupled fermentation approach utilizing acetate as an intermediate. A comprehensive model that comprises both the gas-to-acetate and the acetate-to-SCP fermentation processes, as well as gas pretreatment and downstream processing (DSP) operations, was developed and used to perform a techno-economic analysis (TEA). Sensitivity analyses demonstrated that significant cost reductions can be achieved by the process intensification of the gas-to-acetate fermentation. As such, an increase in the acetate concentration to 45 g/L and productivity to 4 g/L/h could lead to a potential cost reduction from 4.15 to 2.78 USD/kg. In addition, the influence of the production scale and other economic considerations towards the commercialization of off-gas-based SCPs are discussed. Conclusively, this research sheds light on the practical viability of a coupled fermentation process for SCP production by identifying key cost-influencing factors and providing targets for further optimization of the acetate platform, fostering sustainable and economically feasible bio-based innovations.

Single cell protein and oil production from solid cocoa fatty acid distillates co-fed ethanol

The use of solid lipid sidestreams have been overlooked as a feedstock for the production of microbial biomass for food and feed applications and little to no recent work has examined the utilization of solid fatty acid distillates (FADs), which are a significant residue from vegetable oil processing. Yarrowia lipolytica and Rhodosporidium toruloides cultivated on cocoa fatty acid distillates (CFAD) generated final cell dry weight values > 40 g/L, with strong productivity (3.3 g/L·h) and rich protein (>45%) and lipid content (>25%). Interestingly, microbial oils were > 65% unsaturated fatty acids, compared < 20% unsaturated content in FAD. Importantly, to overcome mass-transfer limitations associated with bioconversion of solid lipid residues, ethanol was applied as a co-substrate to solubilize FAD residues. Here, FAD residues from cocoa deodorization have been demonstrated to be high energy feedstocks that represent an attractive substrate for the production of both single cell protein and oil (SCPO).

Construction of Cupriavidus necator displayed with superoxide dismutases for enhanced growth in bioelectrochemical systems

It is of great significance to utilize CO2 as feedstock to synthesize biobased products, particularly single cell protein (SCP) as the alternative food and feed. Bioelectrochemical system (BES) driven by clean electric energy has been regarded as a promising way for Cupriavidus necator to produce SCP from CO2 directly. At present, the key problem of culturing C. necator in BES is that reactive oxygen species (ROS) generated in cathode chamber are harmful to bacterial growth. Therefore, it is necessary to find a solution to mitigate the negative effect of ROS. In this study, we constructed a number of C. necator strains displayed with superoxide dismutase (SOD), which allowed the decomposition of superoxide anion radical. The effects of promoters and signal peptides on the cell surface displayed SOD were analyzed. The proteins displayed on the surface were further verified by the fluorescence experiment. Finally, the growth of C. necator CMS incorporating a pBAD-SOD-E-tag-IgAβ plasmid could achieve 4.9 ± 1.0 of OD600 by 7 days, equivalent to 1.7 ± 0.3 g/L dry cell weight (DCW), and the production rate was 0.24 ± 0.04 g/L/d DCW, around 2.7-fold increase than the original C. necator CMS (1.8 ± 0.3 of OD600). This study can provide an effective and novel strategy of cultivating strains for the production of CO2-derived SCP or other chemicals in BES.Graphical

Bioindustrial applications of thermostable Endoglucanase purified from Trichoderma viride towards the conversion of agrowastes to value-added products

Importance of biocatalytic reactions and biotransformations mediated by fungal enzymes has increased tremendously in various industries. Endoglucanase obtained from Trichoderma viride has been utilized for bioconversion of agrowastes; wheat straw (WS) and corn stover (CS) as biomass into citric acid and single cell protein (SCP) as value-added products. The enzyme was purified to apparent homogeneity with Mr:44.67 kDa; purification-fold, yield, specific activity to be 19.5-, 29.2%, and 150.4 Units.mg−1, respectively, with thermostability up to 70 °C. The enzyme showed a novel N-terminal peptide and its computational analysis revealed a conserved ‘SG’ amino acid sequence alike microbial cellulases. The experimental results have shown the potential of endoglucanase for the conversion of agrowastes; wheat straw (WS) and corn stover (CS) into citric acid, maximum yield (KgM−3) found in submerged (WS:50;CS:45) fermentation process. Single-cell protein (SCP) production in WS (68 KgM−3) hydrolysate was superior to both CS hydrolysate (60 KgM−3) and YEPD (standard medium) (58 KgM−3).

Sequential Bioprocess with Gluconobacter oxydans and Candida tropicalis for Gluconic Acid and Single-Cell Protein Production from Enzymatic Hydrolysate

To meet the growing global demand for gluconic acid as a cement and concrete retarder, inexpensive and abundant lignocellulosic materials are regarded as the most suitable alternatives to starchy materials. However, their enzymatic hydrolysate contains not only glucose but also xylose, which negatively affects the performance of gluconic acid as a retarder. Notably, glucose is preferentially bio-oxidized into gluconic acid by Gluconobacter oxydans, but gluconic acid cannot be metabolized by Candida tropicalis. Given this, an artificially designed biological cascade process, respectively employing Gluconobacter oxydans and Candida tropicalis, was established to successfully carry out glucose conversion into gluconic acid, and xylose into a single-cell protein, using the enzymatic hydrolysate of corncobs as a feedstock. This sequential fermentation process produced 95.8 g/L gluconic acid and 9.0 g/L single-cell protein from one liter of the enzymatic hydrolysate that initially contained 98.1 g/L of glucose and 25.4 g/L of xylose. The mass-balance calculation showed that approximately 280 grams of gluconic acid and 27 grams of the single-cell protein could be harvested from 1000 grams of the corncob feedstock. The results suggest that the above-mentioned two-step bioconversion method is efficient in utilizing glucose and xylose from lignocellulosic hydrolysates.

Inverse metabolic engineering for improving protein content in Saccharomyces cerevisiae.

Production of Saccharomyces cerevisiae-based single cell protein (SCP) has recently received great attention due to the steady increase in the world's population and environmental issues. In this study, an inverse metabolic engineering approach was applied to improve the production of yeast SCP. Specifically, an S. cerevisiae mutant library, generated using UV-random mutagenesis, was screened for three rounds to isolate mutants with improved protein content and/or concentration. The #1021 mutant strain exhibited a respective 31% and 23% higher amino acid content and concentration than the parental S. cerevisiae D452-2 strain. Notably, the content, concentration, and composition of amino acids produced by the PAN2* strain, with a single nucleotide polymorphism in PAN2 coding for a catalytic subunit of the poly(A)-nuclease (PAN) deadenylation complex, were virtually identical to those produced by the #1021 mutant strain. In a glucose-limited fed-batch fermentation, the PAN2* strain produced 19.5g/L amino acids in 89h, which was 16% higher than that produced by the parental D452-2 strain. This study highlights the benefits of inverse metabolic engineering for enhancing the production titer and yield of target molecules without prior knowledge of rate-limiting steps involved in their biosynthetic pathways. This article is protected by copyright. All rights reserved.

Electro-stimulation of tofu wastewater for the production of single cell protein from various microorganisms

Tofu wastewater can be utilized as a substrate for microorganisms that produce single-cell proteins (SCPs). Because different microorganisms have different cellular components, the composition of SCPs varies. Electro-stimulation has the potential to speed up fermentation and increase product yield. The goal of this study was to find the best way to produce SCPs from Aspergillus awamori, Rhizopus oryzae, and Saccharomyces cerevisiae in the tofu wastewater substrate using electro-stimulation. The experimental method was used in the study, the data were analyzed using independent t-test statistical analysis, and the best treatment was identified using the effective index method. This treatment consisted of producing SCP with electro-stimulation of −1.5 V and without electro-stimulation for 72 h for the yeast and 96 h for the mold at 25 °C in tofu wastewater that had already been conditioned to a pH of 5. The parameters measured included measurement of population of microorganism, change in pH, dry biomass weight, carbohydrate content, and protein content. Electro-stimulation reduced the optimum fermentation time of A. awamori SCP from 56 to 32 h, resulting in 0.0406 g/50 mL of dry biomass, 30.09% carbohydrate content, and 6.86% protein content. Meanwhile, the optimal fermentation time on R. oryzae and S. cerevisiae were not accelerated by electro-stimulation. The best treatment was A. awamori without electro-stimulation, which produced 0.0931 g/50 mL of dry biomass, 20.29% carbohydrate, and 7.55% protein.

Sustaining a Mars Colony through Integration of Single-cell Protein and Oil Production in Food Supply Chains

As humanity sets its sights on establishing a sustainable and prosperous colony on Mars, one of the key challenges to overcome is ensuring a reliable and nutritious food supply for settlers. While various solutions for food production on Mars have been proposed, there is a growing interest in the use of microorganisms as a means of producing essential nutrients. This review article highlights the advantages of utilizing single-cell protein and single-cell oil technologies to produce essential amino acids and fatty acids for the food supply chains of a Mars colony. We provide an analysis of the potential benefits, challenges and limitations of these solutions and outline the necessary steps to be taken in order to successfully integrate them into the infrastructure of a Martian settlement.

Sugar beet processing waste as a substrate for yeast protein production for livestock feed

This study investigated the optimal yeast strains for producing single cell protein based on waste sugar beet pulp (SBP) under various biomass loads. All tested strains were capable of growing on the waste biomass. Candida utilis and Saccharomyces cerevisiae Ethanol Red produced the greatest increase in protein on fresh SBP (ΔN 1.84%). Scheffersomyces stipitis (ΔN 2.27%) provided the highest increase on dried SBP. All tested strains showed significant assimilation of nitrogenous compounds. Based on the crude fiber content after fermentation, the largest reduction in fiber occurred with Candida utilis R6 (10.5%) on fresh SBP, and Yarrowia lipolytica (13.1%) on dried SBP. These results demonstrate the potential of SBP as a substrate for the production of single cell protein and highlight the importance of selecting the appropriate strains to optimize the process.

Utilization of Water Hyacinth (Eichhornia crassipes) as a Feasible Substrate for the Production of Single Cell Protein Using Saccharomyces cerevisiae (Baker’s Yeast)

Utilization of Water Hyacinth (Eichhornia crassipes) as a Feasible Substrate for the Production of Single Cell Protein Using Saccharomyces cerevisiae (Baker’s Yeast)

Recovery and upcycling of residual lactic acid and ammonium from biowaste into yeast single cell protein

The yeast Saccharomyces cerevisiae is a potential candidate for converting waste resources into food-grade single cell protein (SCP). Food waste fermentation broth rich in lactic acid was tested as an alternative substrate source for S. cerevisiae in this study. An electrodialysis (ED) system was developed to recover lactate, acetate and ammonium ions (La-, Ac- and NH4+) from the broth. First, we evaluated the system resilience under various volatile fatty acids (VFAs) and pH levels. Initial acetate and lactate concentrations did not affect La- and Ac- recovery efficiencies but affected their ion fluxes and competitive ion transport. The migration of La- was delayed by increasing the initial acetate concentration, while Ac- migration was not affected by lactate concentrations. Increasing the broth pH reduced competitive H+ ions and thus improved the NH4+ recovery efficiency. Further, broth recirculation and pH control were examined to identify potential challenges associated with continuous flow in real-life applications. Recirculation caused a greater loss of recovered Ac- and NH4+ over the extended period. The pH control decreased VFAs recovery efficiencies because VFAs could not fully dissociate in the dilute, and more VFAs were lost in the concentrate. Finally, the recovery from lactate-rich fermentation broth was used as yeast substrate and contributed to a robust SCP yield of 0.62 g/g-C. Manufacturing one kilogram of SCP costs €6-10 in electricity and €3-6 in raw material. This study provides a better understanding of competitive ion transport during ED and uncovers potential biowaste resources for sustainable SCP production.